한국생물공학회, 생물공학의 동향 : 2015.10
Plenary
Lecture S101 Using Fully Defined Activation Protocols and Cell-free Screening to Study [FeFe]hydrogenase Maturation and OxygenTolerance
James SWARTZ1,2, Liliana De La PAZ1, Jamin KOO1, Stacey SHIIGI2, Tim SCHNABEL1 1Dept. of Chemical Engineering, 443 Via Ortega, Stanford University, Stanford, CA 94305-4300, 2Dept. of Bioengineering, 443 Via Ortega, Stanford University, Stanford, CA 94305-4300
In spite of hydrogen’s appeal as a clean and potentially sustainable fuel, we still lack carbon neutral production technology. Photosynthetic biological hydrogen production offers great promise, but has been stymied because a major side product of water splitting, oxygen, potently inactivates nature’s most prolific hydrogen producers, [FeFe]hydrogenases. Undefined hydrogenase maturation requirements pose an additional challenge. This presentation will describe the first fully defined [FeFe]hydrogenase maturation protocol. The procedure identifies all of the activation requirements and facilitates the production of fully active enzymes for more precise evaluation. A new hydrogenase screening technology will also be described. This procedure couples cell-free hydrogenase synthesis and activation with high throughput mutant evaluation. Both directed and random mutagenesis results indicate that oxygen tolerant [FeFe]hydrogenase can be developed and early results suggest mechanistic hypotheses for oxygen tolerance. Together, these advances indicate the feasibility for economical production of hydrogen from sunlight and water as an important weapon in our fight to control global warming.
Keywords : hydrogen, oxygen-tolerant hydrogenase, hydrogenase maturation, hydrogenase maturation S201 Soft, Bioresorbable Optoelectronic Interfaces to the Brain
John A. ROGERS University of Illinois at Urbana Champaign, USA
Biocompatible optoelectronic systems that can intimately integrate with the brain have the potential to accelerate progress in neuroscience research and to spawn new therapies in clinical medicine. Specifically, capabilities for injecting electronics, light sources, photodetectors, multiplexed sensors, programmable microfluidic networks and other components into precise locations of the deep brain and for softly laminating them onto targeted regions of the cortical surface will open up unique and important opportunities in stimulation, inhibition and real-time monitoring. In this talk, we will describe foundational concepts in materials science and assembly processes for these types of technologies, in 1D, 2D and 3D architectures. Examples in system level demonstrations include experiments on freely moving animals with ‘cellular-scale’, injectable optofluidic neural probes for optogenetics research and with bioresorbable, implantable intracranial sensors for treatment of traumatic brain injury. S202 Time to Do Something to Save Us and Our Planet by Industrial Biotechnology
Doyoung SEUNG GS Caltex R&D Center, Daejeon-city 305-380, Korea
Since homo sapiens, referred to as modern humans, appeared on the face of our planet about 200,000 years ago, humans have succeeded as a species and the population has greatly increased to nearly 7.2 billion in 2015. However, the rapid development of science and technology during the 19th and 20th centuries led to the fossil-resources-driven life style that ultimately may destroy us and our planet by climate change, resource depletion, and environmental pollution. As Dr. Albert Einstein said, the world will not be destroyed by those who do evil, but by those who watch them and do nothing. Fortunately, many scientists and engineers from governments, academia, industries, and civil societies have being developed various new technologies to counter the aforementioned problems. Among them, industrial biotechnology seems to gives a great promise to avoid the use of fossil resources because it makes bio-based products from renewable resources by enzymatic or microbial processes. Here, I would like to review what we have learned over the last decade about industrial biotechnology and suggest the way what to do. This work was supported by the Industrial Strategic Technology Program (No. 10050407, MOTIE, Korea).
Keywords : Industrial Biotechnology, Fossil Resources, Renewable Resources, Climate Change
References 1. U.S. and World Population Clock, Unites States Census Bureau, http://www.census.gov/popclock/ S301 Wide-spread Common Biological Nanomotors with Revolution Motion Mechanisms without Rotation
Peixuan GUO Nanobiotechnology Center, Markey Cancer Center, and College of Pharmacy, University of Kentucky, Lexington, USA.
Biological motors are responsible for most forms of biological motion in living beings. They are essential in all aspects of crucial cellular processes critical to replication or survival, such as cell mitosis, bacterial division, genome replication, RNA transcription, ATP synthesis, muscle contraction, DNA repair, homologous recombination, Holliday junction resolution, viral genome packaging and directional motility of cellular components to their destinations. These ubiquitous motors were once classified into two categories: linear and rotation motors. Recently, a third type with a revolution mechanism without rotation has been discovered and is found to be widespread among bacteria, eukaryotic viruses, and dsDNA bacteriophages. Rotation is the turning of an object along its own axis, resembling the Earth rotating one cycle per day; while revolution is the turning of an object around a second object, resembling the Earth revolving around the Sun one cycle per year (http://nanobio.uky.edu/movie.html). The common structural aspects of motor chirality, stoichiometry, entropy, channel size, conformational change, electrostatic interaction, and ATP usage rate were found to relate to the motor mechanism in energy conversion, direction control, or sequential action. Nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation and cell division. Channel chirality is critical in controlling motion direction by one way-traffic. Rotation motors use a right-handed channel to drive the right- handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use a left-handed motor channel to revolve the right-handed dsDNA. Rotation motors use a small channel to ensure the close contact of the channel wall with the dsDNA bolt; revolution motors have a larger channel to provide room for the bolt to revolve. Binding of ATP to the ATPase results in entropy reduction and conformational change of the ATPase, leading to a high affinity for dsDNA. ATP hydrolysis results in entropy increase and a second conformational change to a low DNA-affinity, allowing the release of dsDNA for concomitant transfer to the adjacent subunit. The presentation will also include how the studies on the pRNA of viral DNA packaging motor has led to the emerging field or RNA nanotechnology with applications in cancer therapy, viral treatment, vesicular cell targeting, tissue engineering, electronic and computer device design, controlled drug release, polymer materials application, and disease diagnosis and chemical sensing, and how the motor studies have led to applications in single pore sensing, single diseased molecule fingerprinting, and potential high throughput single pore DNA sequencing.
Keywords : ATPase, DNA packaging motor, RNA nanotechnologies, cancertherapy, viral disease, DNA replication
References 1. Guo P, et al.2014.Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation. Biotechnology Advances. 32:853–872. 2. Wendell, et al. and Guo P. 2009. Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature Nanotechnology. 4:765-72. 3. Guo P. 2010. The emerging field of RNA nanotechnology. Nature Nanotech. 5: 833-842. 4. Jasinski DL, Khisamutdinov EF et al. and Guo P. 2014. Physicochemically tunable poly-functionalized RNA square architecture with fluorogenic and ribozymatic properties. ACS Nano. 8:7620-7629. 5. Haque F, et al. and Guo P. 2012. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today. 7:245-257. 6. Shu D, et al. and Guo P. 2011. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotech. 6:658-667. 7. De-Donatis GM, et al. and Guo P. 2014. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size. Cell & Bioscience. 4:30. 8. Lee T, Yagati AK, Pi F, Sharma A, Choi JW, Guo P. 2015. Construction of RNA-Quantum Dot Chimera for Nanoscale Resistive Biomemory Application. ACS Nano. (in press) 9. ACS News: http://www.acs.org/content/acs/en/pressroom/presspacs/2013/acs-presspac-march-20-2013/discovery-of-first-motor- with-revolution-motion-in-a-virus-killing-bacteria-advances-nanotechnology.html 10. NIH contest: https://www.youtube.com/watch?v=rRGYHFulzkQ 한국생물공학회, 생물공학의 동향 : 2015.10
Nanobiotechnology & Organ on a Chip A101 Nanobioelectronic Device for Bioprocessor: Toward Biocomputing System
Jeong-Woo CHOI Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Korea
Nanobioelectronic devices based on hybrid biomaterial have emerged as a breakthrough with huge potentiality to generate new concepts and technologies for the development of new age electronic devices. The main concept of bioelectronics was generated from the fact that biomaterial, especially metalloproteins, can be used as a functional unit in an electronic device. Major challenges in bioelectronic field include the miniaturization, and the demonstration of various functions implemented in biomaterial to alter silicon-based electronic devices. It has been difficult to demonstrate a single molecular-based computing device in current computing system, since such silicon-based system requires complex functionality to be developed at the single molecular level. In this point of view, metalloprotein-based conceptual biomemory device was developed which demonstrates memory characteristics including ‘read’, ‘write’ and ‘erase’ function. Further, multi-bit memory function and nanoscale memory function are also demonstrated. Afterwards, new hybrid materials including metalloprotein/DNA conjugated materials, protein/inorganic materials and redox enzyme complex have been developed to construct bioelectronic devices. Towards the development of biocomputing system at molecular level, metalloprotein/DNA/nanoparticle hybrid material is developed as bioprocessing device to achieve various functions at the single molecular level. A metalloprotein which exhibits redox property is used as a biomemory signal source. Various nanoparticles with complementary DNA and metal ions are used as input signals to acquire processed output signals. Various functions including ‘information reinforcement’, ‘information regulation’ and ‘information amplification’ are accomplished in this device due to various input signals. This hybrid material-based bioprocessing device by the integration with neural cell can suggest a new type of platform which could be a foundation for development of biomolecular-based biocomputing system. In the near future, biocomputing device with entire processes including information storage, regulation, calculation and read-out in single molecular-based chip will be able to be demonstrated by the development of newly designed and synthesized hybrid biomaterials with genetic and chemical modification, and the integration with neural cell to develop proto type biocomputer.
Acknowledgements This work was supported by Samsung Research Funding Center of Samsung Electronics under Project Number SRFC- MA1401-04.
Keywords : Metalloprotein, Hybrid material, Bioprocessing device, Bioelectronics
References 1. Taek Lee, Sang-Uk Kim, Junhong Min, Jeong-Woo Choi, Multilevel Biomemory Device Consisting of Recombinant Azurin/Cytochrome c (2010), Advanced Materials, 22(4), 510-514 2. Taek Lee, Ajay Kumar Yagati, Junhong Min, Jeong-Woo Choi, Bioprocessing Device Composed of Protein/DNA/Inorganic Material Hybrid (2014), Advanced Functional Materials, 24(12), 1781-1789 3. Taek Lee, Ajay Kumar Yagati, Fengmei Pi, Ashwani Sharma, Jeong-Woo Choi, Peixuan Guo, Construction of RNA-Quantum Dot Chimera for Nanoscale Resistive Biomemory Application (2015), ACS Nano, 9(7), 6675- 6682 A102 A BioSpleen Blood Cleansing Device for Sepsis Therapy
Joo H. KANG, Michael SUPER, Chong YUNG, Ryan COOPER, Karel DOMANSKY, Amanda GRAVELINE, Tadanori MAMMOTO, Julia B. BERTHET, Heather TOBIN, Mark CARTWRIGHT, Alexander WATTERS, Martin ROTTMAN, Anna WATERHOUSE, Akiko MAMMOTO, Nazita GAMINI, Melissa RODAS, Anxhela KOLE, Amanda JIANG, Thomas VALENTIN, Alex DIAZ, Kazue TAKAHASHI, Donald E. INGBER Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA
Sepsis is a major cause of deaths of patients in state-of-the-art hospital intensive care units, affecting about 750,000 Americans each year. Pathogens, including bacteria, fungi, and viruses, which have invaded the bloodstream, activate a systematic immune response that often leads to death when the pathogen load is significantly high or the immune system is depressed. We have previously reported the development of microfluidic blood cleansing devices for use in sepsis therapy1; however, their flow throughput capacity was not practical for clinical use. Here we describe an improved biospleen device that can remove pathogens and endotoxins from septic blood flowing at high rates and clearance efficiencies without knowing the pathogen type2. This was accomplished using magnetic nanoparticles coated with a generic opsonin–Mannose Binding Lectin (MBL)–that binds various bacteria, fungi, viruses and endotoxin, and by designing a microfluidic configuration that mimics the microarchitecture and cleansing function of the human spleen. We engineered the biospleen device using computational simulation programs, which allowed us to optimize flow conditions for magnetic separation of pathogens from blood. The core parts of the microfluidic device were fabricated using hot- embossed medical grade polycarbonate materials; the outer surfaces of the machined parts were then bonded with a thin (125 µm) layer of transparent polycarbonate film. The engineered MBL opsonin was produced and immobilized on magnetic nanoparticles (128 nm) to capture a variety of pathogens and endotoxin without pre-identifying the pathogens. To determine the functionality of the device, we spiked various types of pathogens into human whole blood anticoagulated with heparin (1.5 U/mL), and then flowed the blood samples through the device. Blood-cleansing efficiencies were quantified using blood cultures and ELISA. When anticoagulated human blood was flowed through the biospleen device at 1.25 L/hr for 26 hr, we did not observe any evidence of blood clotting or coagulation. We then confirmed that the biospleen device can remove a variety of pathogens, including C. albicans, E. coli, S. aureus, and mixtures of gut flora isolated from the cecum of rats, with clearance efficiencies consistently over 90% when flowing at rates of 10 to 1000 mL/hr. To provide proof-of-concept for our blood cleansing device therapy, we carried out testing in rats. Blood is drawn from a rat infected with pathogens or endotoxin via a jugular catheter at a flow rate of 10 mL/h, and is continuously mixed with magnetic nanobeads coated with FcMBL. The blood and magnetic opsonins are then flowed through an in-line mixer to promote bead binding, and into millimeter-sized fluidic channels of the magnetic separator unit of the biospleen device. Stationary magnets pull the magnetically opsonized pathogens and toxins from the blood through sinusoid-like slits, and into a parallel saline channel; the cleansed blood is then returned back to the rat. The biospleen treatment significantly reduced pathogen levels in blood and lung of rats infected with S. aureus.
Keywords : sepsis, bacteremia, mannose binding lectin, blood cleansing device, magnetic separation
References 1. Chong W. Yung, et al., “Micromagnetic-microfluidic blood cleansing device”, Lab Chip, (2009) 9 2. Kang, J. H. et al. An extracorporeal blood-cleansing device for sepsis therapy. Nat. Med., (2014) 20
A103 A Ring Shaped Protein as a Flexible Building Block for Novel Nanostructures
Jonathan HEDDLE Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30–387, Krakow, Poland
In recent years much progress has been made in the field of bionanoscience – building small structures and machines using biological molecules. In particular DNA origami has allowed the design and production of programmable nanodevices while exploitation of symmetry matching has also enabled protein structures be designed and built. Designed proteins are particularly exciting as the large number of amino acids allows for a wide repertoire of activities as evidenced by the ubiquity of protein-based enzymes and structural components in life. This however also leads to problems in designing predictable protein structures and activities meaning that the field is still in its infancy. We have found that TRAP protein shows promise as a nanometric building block and here we describe how addition of cysteine residues at particular positions can result in the assembly of the normally ring shaped TRAP into either self assembled protein nanotubes or spherical cage proteins. It is anticipated that such structures may have application in “smart” nanodevices.
Keywords : Bionanoscience, protein engineering, protein cage, nanotube, nanomachine, artificial capsid
References 1. Heddle, J. G., Yokoyama, T., Yamashita, I., Park, S. Y. & Tame, J. R. Rounding up: Engineering 12-membered rings from the cyclic 11-mer TRAP. Structure14, 925-933, (2006). 2. Heddle, J. G. et al. Using the ring-shaped protein TRAP to capture and confine gold nanodotson a surface. Small3, 1950-1956, (2007). 3. Heddle, J. G. et al. Using the ring-shaped protein TRAP to capture and confine gold nanodotson a surface. Small3, 1950-1956, (2007). 4. Malay, A. D. et al. Gold Nanoparticle-Induced Formation of Artificial Protein Capsids. Nano Lett.12, 2056-2059, (2012). 5. Imamura, M. et al. Dynamic Structural Insights Into A Large, Artificial Protein Cage Revealed Using High-Speed AFM Nano Lett.15, 1331-1335, (2015).
A104 Automated Convergence Systems for Drug Screening and Personalized Medicine
Jong Wook HONG Department of Bionano Engineering, Hanyang University
Newly developed tools opened new science in biology and medicine. Automated convergence systems are examples of recently available platforms that could change medicine and biotechnology into another level with accurate, reliable, and convenient systems. With automated convergence systems, ultimate sensitivity could be guaranteed targeting single cells and molecules. In this lecture, we discuss engineered micro-, nanofluidic systems for biopharmaceutical and medical application. The systems and methodologies could be applied to the advancement of biotechnology and medicine including early-stage diagnostics and new target development in drug discovery.
A105 New Concepts for Lab-on-a-chip Systems using Electrospun Nanofibers
A.J. BAEUMNER1,2, L. MATLOCK-COLANGELO2, A. GEORGESCU1,2, M.W. FREY3 1Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstr. 31, Regensburg, Germany, 2Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA, 3Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY, USA
Microfluidic biosensors, labs-on-a-chip and lateral flow assays for the detection of viable organisms, toxins, and clinically relevant markers have been successfully developed in our research group including analytes such as B. anthracis, C. parvum, dengue virus, E. coli, S. pyogenes, cholera toxin, CD4+ T-lymphocytes, thrombin and myoglobin.
Recently, we initiated the study of electrospun nanofibers and their potential to enhance bioassays in paper-based lateral- flow assays (LFA) and in polymer-based microfluidic devices by adding functionalities to the formats otherwise not available. In the case of the LFA format we successfully demonstrated the de novo fabrication of nanofiber-mats as membrane material enabling immobilization of biorecognition elements, adding novel surface chemistries and preventing non-specific binding without the use of blocking reagents.
Nanofiber-enhanced microfluidic devices provide additional degrees of freedom for bioassay designs, as nanofibers with various surface chemistries are electrospun into distinct locations in the microfluidic channels. As the resulting fiber mats can be of varying density and size and hence generate a 3D-structure (Figure 1) within the channels intimate contact with the sample is guaranteed throughout the channel volume. Current investigations study these systems for sample preparation, as mixers, and as concentrators where, for example, a concentration of E. coli cells by a factor of 20,000 has already been demonstrated. We also develop a nanofiber modelling software that enables fluid dynamic studies to investigate mixing capabilities within our microfluidic devices.
References 1. Reinholt, S.; Sonnenfeldt, A.; Naik, A.; Frey, M.; Baeumner, A.J “Developing new materials for paper-based diagnostics using electrospun nanofibers” Anal. Bioanal. Chem. 406 (14) pp. 3297-3304 (2014) 2. Matlock-Colangelo, L., Cho, D., Frey, M.W., Pitner, C.L., Baeumner, A.J. “Functionalized Electrospun Nanofibers as Bioseparators in Microfluidic Systems” Lab Chip, 12 (9), 1696 – 1701 (2012)
A106 Simple Microfluidic Sample Preparation Device for Nucleic Acid based Detection of Pathogen in Complex Samples
Junhong MIN School of Integrative Engineering, Chung-Ang University, Seoul
The nucleic acid based detection of bacteria and virus have been highly required in food, clinical, and environmental fields because frequent outbreak of infectious pathogen in the world. Even though various commercial methods and kits to detect bacteria and virus have been already developed, they have mainly focused on the blood samples. One of the main issues in other sample (food, soil, stool and so on) analysis based on molecular detection is sample preparation. For 20 years, gold standard isolation method of nucleic acid from bacteria/virus in various samples is “boom technology”, which uses high chaotropic salt such as guanidine thiocyanate and organic solvent like ethanol. Even though its powerful extraction capability of DNA or RNA from the pathogen, it has some drawback to be applied to food control field. The enrichment or concentration steps of pathogen are essentially required before getting nucleic acid. Second, it can’t be applied to the pneumatic-type lab on a chip, which is fully automatically operated from a sample to an answer. These drawbacks may induce false positive or negative errors by cross-over contamination. Therefore, in this study, we introduce new simple sample preparation processes, which can be easily combined to current nucleic acid based detection technologies such as target amplification (PCR, NASBA). The four serial main processes (Target concentration, Target lysis, Wash, and Elution) are employed to sample preparation steps. Simple modification of surface and optimization of buffers were performed for relative adsorption of target on the surface. Physical lysis tools were investigated to lyse various pathogens. These technologies were successfully performed in micro chambers with various samples (river, beef, oyster, and soils) and highly sensitive detection results of pathogens were achieved.
Keywords : sample preparation, DNA isolation, Bead Beating
References 1. Sung Hee Chung, Changyoon Baek, Vu Tan Cong, Junhong Min, The microfluidic chip module for the detection of murine norovirus in oysters using charge switchable micro-bead beating (2015), Biosensors and Bioelectronics, 67, 625
A107 Stem Cell Reprogramming using Nanoparticle-mediated Transcription Factors (NanoScript)
KiBum LEE Dept. of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey
Even though stem cells hold great potential to treat many debilitating disease and injuries, several advances in methods to control their differentiation should be made before harnessing therapeutic potential of stem cells. There are two critical aspects need to be addressed towards this challenge: i) efficient delivery of soluble cues such as genetic materials and small molecules, and ii) effective, non-invasive, and real-time tracing of transplanted stem cells.
To this end, we have been developing an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto multifunctional nanoparticles. In addition to regulating genes in a non-viral manner, we designed the NanoScript platform to be interchangeable, and hence applicable for almost any cellular application, especially stem cell differentiation applications. NanoScript, synergistically combined with epigenetic modulators, has been demonstrated to both effectively activate or deactivate specific endogenous genes in stem cells, which could lead to myogenesis, chrondrogenesis, and neurogenesis. In this presentation, a summary of the most updated results from these efforts and future directions will be discussed.
Keywords : Stem cell differentiation, Nanoparticle-based genetic manipulation, Cellular reprogramming, Transcription Factors
References 1. Patel, S.; Jung, D.; Yin, P. T.; Carlton, P.; Yamamoto, M.; Bando, T.; Sugiyama, H.; Lee, K.-B.†, “NanoScript: A nanoparticle-based artificial transcription factor for effective gene expression”, ACS Nano, 2014, 8, 8959-8967. 2. Patel, S.; Pongkulapa, T.; Yin, P.T.; Pandian, G; Rathnam, C.; Bando, T.; Vaijayanthi, T.; Sugiyama, H.†; Lee, K.-B.†, “Integrating Epigenetic Modulators into NanoScript for Enhanced Chondrogenesis of Stem Cells”, J. Am. Chem. Soc., 2015, 137, 4598-4601. 3. Patel, S.; Pongkulapa, T.; Yin, P.T.; Pandian, G; Rathnam, C.; Bando, T.; Vaijayanthi, T.; Sugiyama, H.†; Lee, K.-B.†, “Integrating Epigenetic Modulators into NanoScript for Enhanced Chondrogenesis of Stem Cells”, J. Am. Chem. Soc., 2015, 137, 4598-4601. 4. Patel, S.; Chueng, S. –T.; Yin, P.T.; Dardir, K.; Song, Z.; Pasquale, N.; Kwan, K.; Sugiyama, H.; Lee, K.-B.†, " Induction of stem cell-derived Functional Neurons via NanoScript-based Gene Repression", Angew. Chem. Int. Ed., 2015, In Press. 5. Patel, S..; Lee, K.-B.†, “Probing Stem Cell Behavior using Nanoparticle-based Approaches”, WIREs Nanomedicine & Nanobiotechnology, 2015, DOI: 10.1002/wnan.1346
A108 Formation of Cellular Sticker on Extracellular Matrix Network and its Potential Medical Application
Kwanwoo SHIN1, Yuhwan KIM1, Kiseok KIM1, Seungkuk AHN1,2, Kevin Kit PARKER2 1Dept. of Chemistry and Sogang-Harvard Disease Biophyisics Center, Sogang University, Seoul 121-742, Korea, 2 Sogang- Harvard Disease Biophyisics Center, School of Applied Science and Engineering, Harvard University, USA
Spontaneous, highly ordered large-scale fibronectin networks driven by electrostatic polymer patterns are fabricated, and these precisely controlled protein connections are demonstrated. It is examined whether this scheme, universal to various fibrillar extracellular matrix proteins beyond fibronectin, collagen, and laminin, can be self-organized. These data reveal a novel bottom-up method to form anisotropic, free-standing protein networks to be used as flexible, transferrable substrates for cardiac and neuronal tissue engineering. We further demonstrated applications of these networks to template the formation of ECM composite fibers and cell patches, so-called “cell sticker”. Collectively, our results suggest that proteins that are endowed with β-sheet molecular domains and do not require external crosslinking to form insoluble fibers are capable of self-assembling into networks and that these precisely controlled connections provide a flexible, transferable substrate customized for tissue engineered cardiac and neuronal scaffolds. In this method, one of the most advantageous points is that one can use patient’s own cells to produce the patient-specific patches, while minimizing severe inflammation. Therefore, we will present our recent application, using “cell sticker”, applying self-healing patches for dermatological, and organ-specific application.
Keywords : Extracellular Matrix Protein, protein networks, cell sticker, self-healing patches
References 1. Seungkuk Ahn, Leila F. Deravi, Sung-Jin Park, Borna E. Dabiri, Joon-Seop Kim, Kevin Kit Parker and Kwanwoo Shin, Self-organizing Large-scale Extracellular Matrix Protein Networks, Advanced Materials 27 (18), 2838-2845 (2015) - selected as an front cover article 한국생물공학회, 생물공학의 동향 : 2015.10
Current Issues and Perspectives on Food Biotechnology B101 Understanding of Epigenetic Regulations in Alzheimer’s Disease
Hyemyung SEO Department of Molecular and Life Sciences, Hanyang University, Korea
Alzheimer’s disease (AD) is the most common neurodegenerative disease with cognitive deficits and neuronal degeneration. We previously showed sets of miRNA-mRNA interaction associated in AD at early- or late-symptomatic stages (Noh et al., 2014). We applied the Joint profiling analysis of mRNA and miRNA expression levels in Tg6799 AD model mice. We constructed gene regulatory networks and used the PageRank algorithm to predict significant interactions between miRNA and mRNA. Total 54 miRNAs were identified as being differentially expressed (DE) in AD and 50 significant miRNA- mRNA interactions were predicted. In this study, we detected the expression levels of DE miRNAs including miR139-5p, and miR3460a in Tg6799 AD mice at early- or late-symptomatic stages. For functional validation, we determined that down- regulation of miR-139-5p altered the expression of several target genes from the list of predicted miRNA-mRNA associations. Overexpression of arrestin containing domain 3 (ARRDC3), target gene of the several DE miRNAs, altered cell viability under the treatment of amyloid beta. These data suggest that DE miRNAs and target genes have potential application for future therapeutics of AD.
Keywords : epigenetic regulation, neurodegenerative disease, miRNA-mRNA associations, aging, Alzheimer’s disease model
References 1. Noh, H. et al. (2014) Age-dependent effects of valproic acid in Alzheimer's disease (AD) mice are associated with nerve growth factor (NGF) regulation. Neuroscience., 266:255-65 2. Noh, H., Park, C., Park, S., Lee, Y. S., Cho, S. Y., and Seo, H. (2014) Prediction of miRNA-mRNA associations in Alzheimer’s disease mice using network topology. BMC Genomics., 15:644 3. Kim, W. et al. (2014) MiR-126 Regulates Growth Factor Activities and Vulnerability to Toxic Insult in Neurons. Mol Neurobiol., 4. Kim, W. et al. (2014) miR-126 contributes to Parkinson's disease by dysregulating the insulin-like growth factor/phosphoinositide 3-kinase signaling. Neurobiol Aging., 35(7):1712-21 B102 Improved Production of Megalosaccharides, Namely Linear Isomaltomegalosaccharides, and Their Function
Atsuo KIMURA Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
The scientific term of "megalosaccharide" was proposed in 1959, and it is defined by sugar-size as follows: degree of polymerization (DP) of megalosaccharide ranges between oligosaccharide (DP = 2 to 9) and polysaccharide (DP > 100 or 200). However, a term of megalosaccharide became obsolete, since no efficient production method was available, even though the valuable function of this sugar was expected. Recently, we found that mutant of dextran dextrinase (DDase; an enzyme from Gluconobacter oxydans ATCC 11894) produced the a-1,6-linked linear megalosaccharide (isomaltomegalo- saccharide; IMS), enabling us to analyze the excellent function of megalosaccharide for the first time. IMS exhibited the remarkable action which solubilized compounds belonging to BCS Class II (BCS-II; BCS, Biopharmaceutics Classification System), like flavonoid. From the standpoint of application for food industry, the native DDase-produced IMS is favorable. Very recently, we discover that native enzyme also synthesizes IMS. This presentation notes the improved production of IMS using native DDase.
DDase catalyzes a successive a-1,6-glucosyl transfer reaction to form dextran using maltooligosaccharides (MOSs) as a substrate. Wild-type DDase (or native DDase) synthesized less or no IMS from MOS, and only produced isomaltooligosaccharides and dextran. We constructed the deletion-mutant of C-terminal region of DDase and found that this mutant produced a large amount of IMS, indicating that structural element related to IMS-formation was present in C- terminal region. IMS enhanced the uptake of quercetin-3-O-b-glucoside (Q3G; a flavonoid showing anti-cancer, anti- diabetic and anti-atherogenic actions) in the intestine by increasing solubility of Q3G [1]. IMS also solubilized the water- insoluble ethyl red (ER; azo-benzene dye, an environmental pollutant by less accepting degradation in nature), and assisted azoreductase to degrade ER [2]. Interestingly, both of Q3G and ER are members of BCS-II, which displays the low water- solubility and high membrane-permeability. Most of medicinal drugs and many functional food materials belong to BCS-II, and therefore IMS may have the valuable function to solubilize those BCS-II compounds.
Native DDase generated IMS under quite high concentration of substrate, implying the possibility of native DDase-mediated production of IMS. For this, we had to prepare enzyme from G. oxydans efficiently. It has been long believed that G. oxydans produced two different DDases extracellularly and intracellularly depending on carbon sources in medium, MOS and glucose, respectively. We revealed that both DDases were identical and the intracellular DDase was localized on outer- membrane or periplasmic space [3], suggesting that MOS could release all DDase from cells to medium. Isomaltotriose (IG3) was selected for this use, since dextran-formation of DDase was quite low for IG3 and MOSs longer than IG3 were efficiently converted to viscous dextran. Native DDase recovered by IG3 from cells was utilized for IMS-generation and we succeeded in high-level production of IMS.
Keywords : megalosaccharide, Gluconobacter oxydans, dextran dextrinase
References 1. Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara, A novel mechanism for promotion of quercetin glycoside absorp¬tion by megalo -1,6-glucosaccharide in the rat small intestine (2013), Food Chem, 136, 293- 296 2. Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Janjira Maneesan, Masayuki Okuyama, Hirude Mori, Nobuo Sakairi, Atsuo Kimura, Different molecular complexity of linear isomaltomegalo¬sac¬charides and β- cyclodextrin for enhancing a solubility of azo ethyl red: towards the dye biodegra¬dation (2014), Bioresour Technol, 169, 518-524 3. Juri Sadahiro, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura, Identity of the two dextran dextrinases produced by Gluconobacter oxydans ATCC 11894 and its localiza¬tion change depending on the cell growth (2015), Biochem Biophys Res Commun, 456, 500-505 B103 Stem Cell Aging and Anti-aging Effects of Placenta Stem Cells on Aging
Jisook MOON The Department of Biotechnology, CHA University,463-400, Korea
The aging process is characterized by gradual, cumulative damages to structure and function of stem cells which exist during the lifespan of organisms. Aging can be conceived of as a process that a pool of endogenous stem cells loses progressively its ability to replenish the damaged cells over age. In almost all living organisms, the time-dependent decline in regenerative potential of stem cells is responsible for an increased susceptibility to aging and several age-related diseases. However, many studies with OMICS on aging heavily rely on blood samples which consist of different cell types, thereby placing obstacles in the way of interpretation on the phenomenon. An alternative approach can be to focus on stem cell aging among diverse hallmarks of the process using OMICS technology. In consistent with the strategy, our study investigated gene expression changes of neural stem cells during differentiation into dopaminergic cells and with increasing passages in a proliferation state, both of which can be seen as aging: differentiation as a part of “chronological aging” and increasingpassage as “replicative aging”. Neural stem cells showed cell stage-specific patterns of gene expression during differentiation and specific genes participated in neurogenesis by forming molecular co-expression network. Also, we determined the effects of hypoxia or normoxia on the placenta-derived cells with increasing passage based on the transcriptome data.Genes that are differentially expressed under hypoxia are enriched for nucleosome assembly and chromatin organization, suggesting the involvement in epigenetic regulation. These findings add weight to the notion that study of stem cell aging with OMICS is an efficient means for elucidating the biological basis of the aging process. In this regard, the maintenance of stem cell pool or stem cell rejuvenation holds great therapeutic promise for age-related impairments. Human placenta derived MSCs (hpMSCs) has been used as a candidate for anti-aging treatment. Our animal studies exhibited better cognitive functions measured 12 weeks after hpMSCs injection. Analyses using OMICs technology is ongoing. Taken together, we will discuss here the integrative studies of the stem cell aging and a therapeutic effect of adult stem cells with OMICs technology and the underlying mechanisms of the complex process at diverse molecular levels, with the final goal of practically applying stem cell treatment to the aged for maintaining health over time.
B104 Application of Biotransformation in Cosmetic Industry
Kyeong hwan HWANG, Jun seong PARK AMOREPACIFIC R&D Center, Republic of Korea
There have been attempts to use enzymes as active ingredients in cosmetics, especially for skin exfoliation products since various proteases have been studied extensively. Among those proteases, enzymes with keratinolytic activities hold potential as natural exfoliation agent since keratin is a major constituent in dead skin cells [1]. To explore the keratinolytic enzyme from micro-organism in green tea, a total of 4 strains were isolated from green tea. Among these isolates, AP sulloc 331261 of micro-organism with keratinolytic activities were screened using keratinase assay and identified. The strains named AP sulloc 331261 belonged to the genus Lactobacillus planetarium. The enzymes extracted from Lactobacillus planetarium AP sulloc 331261 were found to have stronger keratinolytic activity compared to papain which is a popular protease used in exfoliation products in cosmetic industry. Ginseng has been used as a traditional medicine in Asian countries, to strengthen immunity, supply nutrition, and decrease fatigue. These beneficial functions are attributed mainly to the ginsenosides in ginseng. The ginsenoside compound K has attracted attention in recent years because of its pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects [2-4]. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf) were mixed with the β-D-glucopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bglu). Under optimum conditions, ginsenosides Rb1, Rb2, Rc, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 18hr via biotransformation pathway Rb1, Rb2 or Rc → Rd → F2 → compound K.
Keywords : Biotransformation, enzyme, microbial, fermentation, cosmetic
References 1. T. Fischer, E. Perosino, M. Viera, and B. Dreno European Academy of Dermatology and Venereology, 24, 281- 292, 2010. 2. Lee SJ, Sung JH, Lee SJ, Moon CK and Lee BH Cancer Letters, 20, 39-43, 1999. 3. Choi K, Kim M, Ryu J and Choi C Neuroscience letters, 21, 37-41, 2007. 4. Lee HU, Bae EA, Han MJ, Kim NJ and Kim DH Liver International, 25, 1069-73, 2005. 한국생물공학회, 생물공학의 동향 : 2015.10
Metabolic Engineering Strategies towards Greener Processes B105 Seawater Based Industrial Biotechnology
George Guo-Qiang CHEN School of Life Sciences, Tsinghua University, Beijing 100084 China
Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the future challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium, practically, seawater, under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way in seawater. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production. At the same time, technology has been developed to change the shapes of Halomonas spp to allow convenient downstream processing. This lecture will focus on bioplastics polyhydroxyalkanoates (PHA) as an example to illustrate the seawater based industrial biotechnology.
Keywords : Halophiles, polyhydroxyalkanoates, Halomonas, industrial biotechnology, seawater
References 1. Yin Jin, Chen GQ, Halomonas spp, a Rising Star for Industrial Biotechnology. Biotechnology Advance 10.1016/j.biotechadv.2014.10.008 2. Li Teng, Chen Xiangbin, Chen JC, Wu Q and Chen GQ, Open and continuous fermentation: Products, conditions and bioprocess economy. Biotechnol J 9 (2014) 1503-1511 3. Yue HT, Chen XB, Ling C, Chen YL, Deng HT, Chen GQ, A Sea water based open and continuous process for polyhydroxyalkanoates production by Halomonas campaniensis LS21. Biotechnology for Biofuels 2014, 7:108 4. Fu XZ, Tan D, Aibaidula G, Wu Q, Chen JC and Chen GQ, Development of Halomonas TD01 as a host for open production of chemicals. Metabolic Engineering 23 (2014) 78-91
B106 Novel Metabolic Networks in Archaea
Haruyuki ATOMI Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510 and JST, CREST, 7, Gobancho, Chiyoda-ku, Tokyo 102-00
The Archaea represent the third domain of life and are phylogenetically distinct to the Bacteria and Eucarya. Archaea harbor many metabolic pathways that differ to previously recognized classical pathways in bacteria and eukaryotes. Based on the genome sequences of the Archaea, there are many cases in which a particular metabolic pathway seems to be absent or incomplete. The search for these “missing” pathways or enzymes has been an exciting field of research in the Archaea, and has led to the discovery of structurally novel enzymes or enzymes with novel activity. Until now, we have been focused on the metabolism of the hyperthermophilic archaeon Thermococcus kodakarensis. The organism is an obligate anaerobe and heterotroph, utilizing a wide range of organic compounds including peptides/amino acids, starch and maltooligosaccharides, and organic acids such as pyruvate. By searching for missing genes, we have identified a structurally novel fructose-1,6- bisphosphatase, a key enzyme in gluconeogenesis, and enzymes with novel activity, such as pantoate kinase and phosphopantothenate synthetase, both involved in coenzyme A biosynthesis in T. kodakarensis. Here we will present our results concerning pentose metabolism.
The pentose phosphate pathway is considered a ubiquitous pathway in bacteria and eukaryotes. It provides the reducing equivalents (NADPH) necessary for various biosynthetic pathways and generates erythrose 4-phosphate, a precursor for aromatic amino acid and folate biosynthesis. The pathway is also responsible for the interconversion of pentoses necessary for nucleic acid biosynthesis and the trioses/hexoses found in glycolysis and gluconeogenesis. It is known that many archaea do not harbor a complete pentose phosphate pathway. In terms of pentose synthesis from trioses/hexoses, the ribulose monophosphate pathway substitutes the pentose phosphate pathway in Archaea. However, a route through which the pentose moieties from nucleosides are directed to the trioses/hexoses in glycolysis and gluconeogenesis has not been demonstrated. Recently in T. kodakarensis, we identified a metabolic network that links the pentose moieties of nucleosides/nucleotides to central carbon metabolism. The network consists of three nucleoside phosphorylases, an ADP-dependent ribose-1-phosphate kinase and two enzymes of a previously identified NMP degradation pathway, ribose-1,5-bisphosphate isomerase and Type III ribulose-1,5-bisphosphate carboxylase/oxygenase (1-3). As ribose 1,5-bisphosphate and ribulose 1,5-bisphosphate are intermediates in this metabolism, we designated the pathway as the pentose bisphosphate pathway (4). Other related pathways in archaea will also be discussed.
Keywords : Archaea, metabolism, pentose, nucleoside
References 1. Ezaki S, Maeda N, Kishimoto T, Atomi H, Imanaka T. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. J. Biol. Chem. 274, 5078-5082, 1999. 2. Sato T, Atomi H, Imanaka T. Archaeal type III RuBisCOs function in a pathway for AMP metabolism. Science 315, 1003-1006, 2007. 3. Aono R, Sato T, Yano A, Yoshida S, Nishitani Y, Miki K, Imanaka T, Atomi H. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway. J. Bacteriol. 194, 6847-6855, 2012. 4. Aono R, Sato T, Imanaka T, Atomi H. A pentose bisphosphate pathway for nucleoside degradation in Archaea. Nat. Chem. Biol. 11, 355-360, 2015.
B107 Replenishing Acetyl-CoA in Synechococcus elongatus PCC 7942 Facilitates Photosynthetic Sole
Production of Acetone from CO2
Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, 2Green School, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea
Cyanobacteria have been gathering global interests regarding CO2 bio-mitigation due to the ability to direct utilization of
CO2. Engineered cyanobacteria have been enabled to produce industry-relevant chemicals from CO2. However, the final products have often been mixed with fermented metabolites during dark fermentation. Our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO2 to acetone as sole product via both ATP-driven malonyl-CoA synthesis pathway and heterologous phosphoketolase-phosphotransacetylase pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl-coA directly from CO2 in the engineered strain, led to sole production of acetone without changing nutritional constraints, and without an anaerobic shift. Subsequently, 5%
(v/v) CO2 gas-stripping-based recovery systems allowed collection of the sole production of acetone from CO2. Our engineered strains could be modified to create bio-solar cell factories for sustainable photosynthetic production of acetyl- CoA-derived biochemicals. This work was supported by Korea CCS R&D Center (KCRC) (no. 2014M1A8A1049277) grant-funded by the Korean Government (Ministry of Science, ICT & Future Planning).
Keywords : Metabolic Engieering, Cyanobacteria, Acetone
References 1. Chwa et al., 2015, Submitted.
B108 PHO13 Deletion-Induced Transcriptional Activation Prevents Sedoheptulose Accumulation during Xylose Metabolism in Engineered Saccharomyces cerevisiae
Kim SOO RIN1, Kim KYOUNG HEON2, Jin YONG-SU3 1Kyungpook National University, Daegu, Korea, 2Department of Biotechnology, Graduate School, Korea University, Seoul, Korea, 3Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
A phosphatase enzyme of unknown specificity in Saccharomyces cerevisiae, Pho13, plays a role in the transcriptional regulation of genes related to pentose phosphate pathway (PPP) and NADPH regeneration. It has been also reported that deletion of PHO13 (pho13Δ) in S. cerevisiae results in significantly improved xylose metabolism in engineered strains metabolizing xylose. However, it has not been elucidated if the transcriptional activation caused bypho13Δ is associated with the metabolic changes favoring xylose fermentation. We investigated the global metabolic changes in response to pho13Δ to understand underlying mechanisms of the enhanced xylose fermentation uponpho13Δ. Among the 134 metabolites identified in cells grown on xylose, the concentration of sedoheptulose decreased by 98% in the pho13Δ mutant as compared to that of a parental strain. Sedoheptulose is not a common metabolite but a dephosphorylated form of sedoheptulose-7-phosphate (S7P) was also significantly decreased by pho13Δ. As S7P is a substrate of transaldolase (TAL1) in PPP, we hypothesized that TAL1 upregulation induced by pho13Δ might be responsible for the reduced accumulation of S7P and sedoheptulose, leading to improved xylose fermentation. We constructed various mutants overexpressing TAL1, and monitored their metabolic and phenotypic changes. Increased TAL1 expression levels were correlated with reduced sedoheptulose levels, and increased xylose consumption rates. While other transcriptional and metabolic changes induced by pho13Δ were identified, we concluded that TAL1 upregulation that prevents sedoheptulose accumulation is the most important mechanism for improved xylose metabolism.
Keywords : GC-TOF/MS, NMR, metabolomics, Cas9-guided genome editing technique, RNA-seq
References 1. Kim, S. R., Xu, H., Lesmana, A., Kuzmanovic, U., Au, M., Florencia, C., Oh, E. J., Zhang, G., Kim, K. H., Jin, Y.-S., Deletion of PHO13 encoding HAD type IIA phosphatase results in upregulation of the pentose phosphate pathway in yeast. Appl Environ Microbiol (2014), Appl. Environ. Microbiol. 81 (5), 1601-1609
B109 Utilization of Renewable Resources for Production of Biochemicals
Hee Taek KIM Research Center for Biobased Chemistry, Korea Research Institute of Chemical Technology,
Recently, sustainable production of chemicals using renewable resources have been recognized as important issue due to depletion of fossil fuels, environment issue and creation of new growth engine. To develop platform chemicals and biomacromolecules from renewable resources, biochemical production platform including lactic acid and ethanol were developed and applied in industrial fields. However, only few cases such as lactic acid reached economic level of production due to cost of raw materials, titer and productivity. One of factors to hinder commercialization was composition complexity of biomass. During fermentation, certain component derived from either endogenous or exogenous factors gives negative effects on metabolic function and production. Therefore, for conversion of biomass or biowaste into fermentation suitable form, renewable sources such as red algae, corn steep liquor and biodiesel waste were pretreated and applied to biochemical production platform. Based on results, possible inhibitory factors were deduced and then suitable platform for fermentation was developed.
Keywords : Bioresource, biochemicals, pretreatment
References 1. H.T. Kim, E.J. Yun, D. Wang, J.H. Chung, I.-G. Choi, K.H. Kim, High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass (2013), Bioresour. Technol., 136, 582-587 한국생물공학회, 생물공학의 동향 : 2015.10
Recent Advances in Enzyme Technologies C106 Artificial Protein Complex via PCNA Scaffold for Self-Sufficient Biocatalysis
Nagamune NAGAMUNE, Hidehiko HIRAKAWA Department of Chemistry and Biotechnology, The University of Tokyo, Tokyo 113-8656, Japan
Most of bacterial cytochrome P450s (P450s) are cytosolic proteins and require intermolecular electron transfer from NAD(P)H through two redoxproteins, ferredoxinreductases and ferredoxins, to convert molecular oxygen into reactive species. However, high concentrations of redox proteins and expensive NAD(P)H are required to achieve sufficient reaction rate and high product yield.Furthermore, although multi-components of this cytochrome P450 system need to dynamically interact and form complex to transfer electron, it is very difficult to immobilize these components without loss of dynamic interaction among them.These are bottlenecks for practical application of cytochrome P450 systems to industry. We have created a functional nanoscaled self-sufficient P450 system (PUPPET system) by a bottom-up approach[1]. Putidaredoxinreductases (PdR), putidaredoxin (PdX) and P450cam from Pseudomonas putida are genetically fused to the C- termini of heterotrimeric Proliferating Cell Nuclear Antigen (PCNA) subunits (PCNA1, PCNA2 and PCNA3) from CrenarchaeotaSulfolobussolfataricus. PUPPET system was formed in self-assembly manner by PCNA-mediated heterotrimerization and showed high specific activity of P450cam (500 min-1 at 90 nMPUPPET system, 25oC) by efficient electron transfer within the complex. We further improved the activity and complex stability of PUPPET system by the following three strategies; (a) Stabilizing the interaction between PCNA subunits by introducing disulfide bonds at the interface of them [2], (b)Optimizing the length and the rigidity of the peptidyl linker between PdX and PCNA2 [3], and (c)Introducing two molecules of PdRand PdXinto the PUPPET system. As a result, higher specific activity of P450cam (1460 min-1 at 20 nMPUPPET system, 25 oC),which is 86% of the maximum specific activity of reconstituted P450cam system at the presence of high concentration redox proteins (1700 min-1 at 20 nM P450cam, 4 mM PdR and 50 mM PdX, 25oC), was achieved by combination of these strategies. We also applied this PUPPET system to enhance the activity of orphan cytochrome P450 mutant (CYP119D77R/ T214V)from Archaeaand succeeded in about 18-fold increase of lauric acid hydroxylation[4].Furthermore, a subunit of a homodimeric protein, phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri, was fused to the N-terminus of PCNA1 for NADH regeneration. As a result, the PUPPET with PTDH (PTDH- PUPPE) showed high catalytic activity and NADH turnover by effective electron transfer and cofactor regeneration within the complex[5]. For the immobilization of PTDH-PUPPET, this complexwas further cross-linked together by genetically fusing each PCNA subunit with a PTDH subunit, resulting in a gelation due to supramolecular network formation of PTDH-PUPPET[6]. However, the activity of water-insoluble large PTDH-PUPPET gel was suppressed by mass transfer limitation due to lower specific surface area of gel. To overcome this problem, PTDH-PUPPET was indirectly immobilizedon PCNA2-linked magnetic beads (NHS Mag Sepharose)by step-wise addition of each component of PTDH-PUPPET. Thin PTDH-PUPPET gel formed on magnetic beads consumed oxygen and hydroxylatedD-camphor in the presence of NAD+ and phosphite, indicating that PTDH played a dual function of regenerating cofactor NADH as well as cross-linking PUPPET. This indirect immobilization approach yielded a highly active cytochrome P450 and cofactor regeneration system complex as evident from the higher apparent specific activity that is comparable to free enzymes, suggesting the less mass transfer effect. This immobilization strategy via PCNA scaffold might be also applicable and useful for other multi-enzyme systems.
Keywords : Multi-enzyme system, Cytochrome P450 system, Cofactor regeneration system, PCNA, Immobilization
References 1. Hirakawa, H. and Nagamune, T.,ChemBioChem, 11, 1517-1520 (2010) 2. Hirakawa, H., Kakitani, A. and Nagamune, T., Biotechnol.Bioeng.,110, 1858-1864(2013) 3. Haga, T., Hirakawa, H. and Nagamune, T., PLOS ONE, 8(9):e75114(2013) 4. Suzuki, R., Hirakawa, H. and Nagamune, T.,Biotechnol. J., 9, 1573-1581(2014) 5. Watanabe, H., Hirakawa, H. and Nagamune, T., ChemCatChem,5, 3835-3840 (2013) 6. Tan, C. Y., Hirakawa, H. and Nagamune, T., Sci. Rep., 5, 8648 (2015)
C107 Bioelectrocatalytic Oxidation of Sucrose with an Artificial Metabolon Assembled on a DNA Scaffold
Shelley D. MINTEER, Fabien GIROUD, David P HICKEY, Khiem NGUYEN Departments of Chemistry and Materials Science & Engineering, 315 S 1400 E Rm 2020, Salt Lake City, UT 84112
Over the last decade, there has been substantial research focused on multi-enzyme cascades as anodic catalysts to improve the performance of enzymatic biofuel cells, as it allows for the conversion of more chemical energy stored in complex fuels, as well as the opportunity to increase the efficiency, current density, and power density. However, the performance of these enzyme systems is often limited by the mass transport of intermediate substrates between enzymes. Nature addresses this issue by organizing metabolic enzymes in a sequential and proximal manner to enhance the efficiency of metabolic pathways (referred to as a metabolon). In this work, we investigate the utilization of DNA as a structural scaffold for the assembly of the invertase/glucose oxidase enzymatic supercomplex to improve sucrose bioelectrocatalytic activity. It has been found that the DNA-assembled enzyme cascade has higher activity than the free cascade, both in solution and in the immobilized state on a bioanode surface. The organization of the enzyme cascade on the DNA scaffold leads to a 100% increase in the current density in amperometric measurements for a sucrose biosensor and a 75% increase in the power density of the biofuel cell. This is the first evidence of the advantages of utilizing DNA scaffords for improved bioelectrocatalysis in biofuel cells.
C108 Bio-inspired Carbonic Anhydrase-encapsulated Biocatalyst for CO2 Sequestration
Byung Hoon JO, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea
Here, we report on the development and characterization of a carbonic anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use in environmental CO2 sequestration. Encapsulation occurred simultaneously with autonomous silica synthesis by silica-condensing R5 peptide that was fused to recombinant CA. The encapsulation efficiency was greater than 95%, and the encapsulated CA was not leached from the silica matrix, demonstrating the highly efficient R5-mediated auto- encapsulation process. The catalytic efficiencies for both esterase and CO2 hydratase activities tended to increase with increasing pH; however, the catalytic efficiency for CO2 hydration was much more pH dependent, suggesting that proton transfer from silica to water is a rate limiting step, especially for CO2 hydration. In addition to good reusability, the encapsulated CA exhibited outstanding thermostability, even retaining 80% activity after 5 days at 50 °C. The thermoactivity was also remarkable, showing ~10-fold higher activity at 60 °C compared to that at 25 °C. The physical structure was observed to be highly compact with a low surface area, stressing the importance of the outermost surface for catalytic performance. We also demonstrated the applicability of the silica nanoparticle to the sequestration of CO2 in carbonate minerals. The rate of CaCO3 precipitation was remarkably accelerated by the encapsulated biocatalyst. The biosilica nanocomposite exhibited ~60% of the CO2 sequestrating power of the free enzyme, which is expected to be the maximal ability of the encapsulated CA. Thus, this silica-CA nanocomposite, efficiently synthesized via a biomimetic green route, can be successfully used as a robust biocatalyst for biomimetic sequestration of the greenhouse gas CO2.
References 1. Jo, B.H., Seo, J.H., Yang, Y.J., Baek, K., Choi, Y.S., Pack, S.P., Oh, S.H., and Cha, H.J., ACS Catalysis, 4, 4332– 4340 (2014) 2. Jo, B.H., Kim, I.G., Seo, J.H., Kang, D.G., and Cha, H.J., Applied and Environmental Microbiology, 79, 6697- 6705 (2013) 3. Kim, I.G., Jo, B.H., Kang, D.G., Kim, C.S., Choi, Y.S., and Cha, H.J., Chemosphere, 87, 1091-1096 (2012)
C109 Carbon Capture and Utilization System using Carbonic Anhydrase and Phosphoenolpyruvate Carboxylase from Microalgae
Hancheol JEON, Seungbum SEO, Kwang Suk CHANG, EonSeon JIN Department of Life Science, Hanyang University, Seoul, 133-791, Korea
The development and implementation of strategies for CO2 mitigation technology is necessary to counteract the greenhouse gas effect of carbon dioxide emissions. The only large-scale solution to the problem of CO2 emissions currently being considered is carbon capture and storage (CCS). There is also the possibility of the capture and utilization of CO2 (CCU) in which waste CO2 is recycled and converted into value added chemicals. In microalgae, carbonic anhydrase (CA) is an important biocatalyst that captures CO2 and produces bicarbonate. Another biocatalyst is phosphoenolpyruvate carboxylase (PEPCase) to produce C4 chemical, oxaloacetate, from bicarbonate. To introduce the possibility of CCU, we developed integrated systems using CA and PEPCase to mimic the in vivo process of carbon dioxide concentration mechanism. The recombinant CA of Dunaliella sp. and PEPCase of Phaeodactylum tricornutum successfully catalyzed a consecutive reaction to generate C4 di-carboxylic acid (OAA). Thus, combined CA-PEPCase specially derived from microalgae could open a possible platform technology for simultaneous CO2 capture and utilization. This integrated CA-PEPCase system applied to an in vivo conversion from carbon dioxide to value added organic compounds using microalgal transformation is now under investigation
Keywords : Marine microalgae, Carbonic anhydrase, Phosphoenolpyruvate carboxylase, bioconversion system
한국생물공학회, 생물공학의 동향 : 2015.10
Microbial Production of Secretary Proteins and Enzymes A201 Optimization of Secretion of Recombinant Proteins in Bacillus subtilis
Wolfgang SCHUMANN Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany
Industrial enzymes such as amylases, lipases, proteases, and many others are usually produced in Gram-positive bacteria and fungi. Both groups of organisms secrete proteins directly into the fermentation broth facilitating the downstream processing. B. subtilis and related species are able to secrete homologous proteins up to 25g/L concentrations. In contrast, secretion of various heterologous (recombinant) proteins, in particular proteins of eukaryotic origins, is frequently not efficient. I will compare the three major secretion pathways in E. coli and in B. subtilis and provide a detailed description of the stages of conversion of the secreted proteins from the unfolded preprotein chain into the correctly folded and fully active protein. Furthermore, bottlenecks in the production of recombinant proteins and ways to resolve them will be discussed.
Keywords : Bacillus subtilis, secretion, recombinant proteins
References 1. Sunghoon Park, Wolfgang Schumann, Optimization of the secretion pathway for heterologous proteins in Bacillus subtilis (2015) Biotechnology and Bioprocess Engineering, in press. 2. Kelly L. Mulder, Construction of an artificial secYEG operon allowing high level secretion of -amylase (2013), Protein Expression and Purification 89(1), 92-96 3. Marjo Simonen, Ilkka Palva, Protein secretion in Bacillus species (1993), Microbiological Revues 57(1), 109-137 4. Yafeng Song, Jonas M. Nikoloff, Dawei Zhang, Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis (2015), Journal of Microbiology and Biotechnology 25(7), 963-977
A202 Development of Secretion System for the Enhanced Production of Recombinant Proteins in Corynebacterium glutamicum
Ki Jun JEONG Dept. Chemical and Biomolecular Engineering, KAIST, Daejeon 305-701, Korea
Recently, beyond traditional amino acid production, Corynebacterium glutamicum also attracts attention as a platform for production of recombinant proteins, since it shows numerous ideal attributes for protein secretion. Signal peptide is a short peptide present at the N-terminus of the newly synthesized proteins which are destined to be secreted and plays a key role in directing the proteins to channels for secretion. Therefore, identifying efficient signal peptides is important to develop cost- effective secretory production system. Here, we isolated efficient signal peptides based on secretome analysis of C. glutamicum in high cell density cultivation conditions. First, secreted proteins by C. glutamicum in fed-batch cultivation condition were investigated by SDS-PAGE and two-dimensional gel electrophoresis, and one major protein spot could be detected. By extensive analysis of the protein spots, the one secreted protein could be identified. Novel secretory expression systems were constructed using the signal peptide of newly isolated protein, and the usefulness of the protein secretion systems were demonstrated by the secretory production of various recombinant proteins.
Keywords : Corynebacterium glutamicum, secretome, signal peptide, secretion
References 1. Becker J, Wittmann C. Bio-based production of chemicals, materials and fuels - Corynebacterium glutamicum as versatile cell factory. (2012) Curr Opin Biotechnol 23(4):631-640. 2. Pátek M, Nešvera J. Sigma factors and promoters in Corynebacterium glutamicum. (2011) J Biotechnol 154(2- 3):101-113. 3. Teramoto H, Watanabe K, Suzuki N, Inui M, Yukawa H. High yield secretion of heterologous proteins in Corynebacterium glutamicum using its own Tat-type signal sequence. (2011) Appl Microbiol Biotechnol 91(3):677-687. 4. Yim SS, An SJ, Kang M, Lee J, Jeong KJ. Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. (2013) Biotechnol Bioeng [Epub ahead of print]
A203 A Potent Expression System for Production of Recombinant Proteins in Bacillus subtilis
Hoang Duc NGUYEN VNUHCM-University of Science, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Vietnam
Bacillus subtilis is a model organism for Gram-positive bacteria, which is considered as a GRAS organism. This bacterium has been extensively used for extracellular enzyme production at industrial scale by biotechnology companies. This report focuses on the development of a strong expression system for production of recombinant protein in B. subtilis. We introduced a new synthetic promoter Pgrac01 derived from B. subtilis groESL promoter and E. coli lac operator. This promoter is 50 times stronger than Pspac, the most popular IPTG-inducible promoter used for B. subtilis. By changing the core promoter elements or the mRNA stabilizing elements, we showed that the second generation of Pgrac promoters could enhance production levels more than 30% of total cellular proteins in B. subtilis. Finally, we constructed potent plasmid- based expression vectors containing the second generation of promoter, Pgrac100 which could be useful for intracellular or extracellular production of recombinant proteins in B. subtilis.
Keywords : KSBB, biotechnology, Bacillus subtilis, Pgrac, Pgrac100, pHT vector
References 1. Phan TTP, Nguyen HD and Schumann W (2006). Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis, Protein Expr. Purif., 46, 189-195. 2. Phan TT, Nguyen HD, Schumann W (2012). Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements, J. Biotechnol. 157: 167-172. 3. Phan TTP, Nguyen HD, Wolfgang Schumann. (2013). Construction of a 5'-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis. J Biotechnol. 168(1): 32-39. 4. Phan TTP, Tran LT, Schumann W, Nguyen HD (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli, Microbial Cell Factories 14:72.
A204 Production and Biogenesis of Membrane Proteins in Saccharomyces cerevisiae
Hyun (Joy) KIM Department of Biological Sciences, Seoul National University, Seoul 08826, South Korea
Membrane proteins perform various crucial functions in cells such as signaling, ion and nutrient transport, and cell-cell interactions, and roughly comprise 1/3 of the genome. Due to their roles as communicator across the membrane, membrane proteins are the major drug targets. Although structural information is essential for structure-based drug design, less than 1 % of the protein structures deposited in protein database are of membrane proteins, thus structure determination of these proteins are much needed. The reasons that membrane protein research is under-represented are due to that membrane proteins are difficult to overexpress and purify for in vitro biochemical characterization, and also current understanding of membrane protein folding and synthesis is rather limited. Using Saccharomyces cerevisiae as a model organism, we have established an easy screening method for membrane protein overproduction and study biogenesis of membrane proteins.
Keywords : Membrane proteins, Saccharomyces cerevisiae, yeast, Sec61
A205 Environmental Application of Bacillus subtilis Spore Display System
Wooil KIM, Jonghwa PARK, Junehyung KIM* Department of Chemical Engineering, Dong-A University, Pusan, 604-714, South Korea
Surface display technology has been acknowledged as a useful tool in various fields of biotechnological application. Phage display and bacterial cell surface display are such well-developed technologies to screen biomolecules based upon affinity binding and catalytic activity. Similarly, ribosome display and mRNA display, which can easily increase the number of library pool without using the surface display techniques, were recently developed for screening of peptide ligand and antibody affinity maturation. Here, we report the environmental application of Bacillus subtilis spore based display system for synthetic industrial dyes decomposition using laccases having multi-copper oxidase activity. To express laccase protein on the surface of the spores, several anchoring motives such as CotE, CotG, CotY, and CotZ were used. His6 tag was added at the C-terminal of each laccase. The spore surface expression of target protein, CotA, was confirmed by flow cytometry using FITC labelled anti- His6 antibody. Synthetic dye decolorization was tried using constructed spore displayed laccase, CotA. We confirmed the decolorization of representative industrial dyes such as Indigo carmine, Acid red 18, and Acid green 25. And further possible applications will be discussed.
References 1. S. S. Sidhu, Curr. Opin. Biotechnol. 11, 610 (2000).
2. G. Georgiou, C. Stathopoulos, P. S. Daugherty, A. R. Nayak, B. L. Iverson, and R. Curtiss, Nature Biotechnol. 15, 29 (1997).
3. J. Kim and W. Schumann, Cell Cell. Mol. Life Sci. 66, 3127 (2009) 한국생물공학회, 생물공학의 동향 : 2015.10
Recent Developments in Industrial Biotechnology A206 Bacterial Diversity and Enzymes from Cold and Alkaline Ikaite Columns in Greenland.
Peter STOUGAARD Department of Plant and Environmental Sciences, University of Copenhagen, DK 1871 Frederiksberg C, Denmark.
Ikaite tufa columns in the Ikka Fjord, Southwest Greenland, constitute one of the few environments on Earth, which is alkaline (pH 10.5), low saline, and cold (2-6°C). The columns harbour a variety of Gram positive and Gram negative bacteria many of which represent new species and genera1). These extremophilic bacteria produce a number of enzymes many of which are active at low temperature and at high pH.
In this presentation, culture dependent and DNA-based screening for novel enzymes in ikaite-bacteria will be described. Both culture dependent and DNA-based methods results in great bias in the diversity that is subjected to screening3,4), but despite this bias, a number of novel enzymes have been isolated. For example, screening a culture collection revealed a novel cold active GH2 beta-galactosidase from a novel species (Alkalilactibacillus ikkensis)5). The purified enzyme showed high activity at low temperatures, e.g. more than 60% of its maximal activity at 0°C. Another example showed that a cold active alpha-amylase could be isolated by functional screening of a metagenome in E. coli2). Purified amylase displayed temperature optimum at 10-15°C and more than 70% of the relative activity was observed at 0°C. The pH optimum was at pH 8-9, and the amylase was active in two commercial laundry detergents. The enzyme was heat labile and was irreversible inactivated by incubating at 55°C for 5 minutes.
Keywords : Alkaliphile, psychrophile, functional metagenomics, cold-active, alkali-stable
References 1. Glaring MA, Vester JK, Lylloff JE, Abu Al-Soud W, Sørensen SJ, Stougaard P. PLoS One. Apr 27;10(4):e0124863. doi: 10.1371/journal.pone.0124863 (2015). 2. Glaring MA, Vester JK, Lylloff JE, Abu Al-Soud W, Sørensen SJ, Stougaard P. PLoS One. Apr 27;10(4):e0124863. doi: 10.1371/journal.pone.0124863 (2015). 3. Vester JK, Glaring MA, Stougaard P. Microb Cell Fact. May 20;13:72. doi: 10.1186/1475-2859-13-72. (2014). 4. Vester JK, Glaring MA, Stougaard P Can J Microbiol. Aug;59(8):581-6. doi: 10.1139/cjm-2013-0087. (2013). 5. Schmidt M, Stougaard P. Environ Technol 31, no10:1107-1114. (2010).
A207 Green Routes to Renewable Energy through Thermochemical and Biochemical Conversions
Seonah KIM National Bioenergy Center, National Renewable Energy Laboratory, Golden CO 80401, USA
Advanced biofuels from lignocellulosic biomass have the potential to produce valuable renewable liquid transportation fuels, biochemicals and biomaterials to be substitute for gasoline. The conversion of biomass to advanced biofuels can be achieved via biochemical and thermochemical processes. In NREL, we are actively developing and improving both conversion techniques. This presentation will describe our on-going efforts to address the green chemistry issues through both thermochemical and biochemical conversions. For the biochemical conversion process, we will discuss fatty acid decarboxylase for hydrocarbon biofuels and Lytic polysaccharide monooxygenases (LPMOs) for the enzymatic degradation of biomass. Fast pyrolysis of lignocellulosic biomass is a promising thermochemical technology for producing biofuels because it can produce oil yields of up to 75 wt% but poor quality. Here we will show new bi-functional catalysts in vapor phase pyrolysis upgrading process to improve the quality of the bio-oil.
Keywords : Biomass, biofuel, pyrolysis, LPMO, decarboxylase
References 1. S. Kim et al. “Quantum Mechanical Calculations suggest that Lytic Polysaccharide Monooxygenases employ a Copper-oxyl, Oxygen-rebound Mechanism” PNAS 111, 149-154 (2014). 2. S. Kim et al. “Ethanol Dehydration in HZSM-5 studied by Density Functional Theory: Evidence for Concerted Process” J. Phys. Chem A 119 (15), 3604-3614 (2015).
A208 Genetic Enzyme Screening System: A Method for High-Throughput Functional Screening of Novel Enzymes from Metagenomic Libraries
Haseong KIM1, Dae-Hee LEE1,2, Seung-Goo LEE1,2 1Synthetic Biology and Bioengineering Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon, 2University of Science and Technology, 217 Gajung-ro, Yuseong-gu, Daejeon
New enzymes and metabolic pathways are the primary requirements for sustainable biotechnology development, and many efforts have been made to obtain enzymes having new chemical reactivity, specificity, and stability. In the present study, we report a new platform for the screening of diverse enzyme genes from metagenome using a new genetic device, based on the recognition of catalytic products, which are created by enzymatic or metabolic reactions. The catalytic product activates a transcriptional factor to express a fluorescent reporter gene. In cells harboring this genetic system, various enzymes such as cellulase, lipase, phosphatase, tyrosine phenol-lyase, methyl parathion hydrolase, and isoprene synthase were successfully detected using a high throughput flow cytometry. Eventually, the high sensitivity, versatile applicability, and quantitative nature of this genetic circuit along with flow cytometry techniques can provide a widely applicable tool-kit for discovering and engineering novel enzymes and pathways in a single cell level.
Keywords : Enzyme screening, Fluorescence-assisted cell sorting, Genetic enzyme screening system, High-throughput screening
References 1. Kim H., Kwon KK, Rha E, Lee SG. Genetic Enzyme Screening System: A Method for High-Throughput Functional Screening of Novel Enzymes from Metagenomic Libraries (2015) Springer Protocols Handbooks. Date: 04 Apr 2. Lee DH, Choi SL, Rha E, Kim SJ, Yeom SJ, Moon JH, Lee SG. A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments. (2015) BMC Biotechnol. Jan 31;15:1. doi: 10.1186/s12896-015-0115-2 3. Choi SL et al., Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits (2014) ACS Synth Biol, 3(3), pp 163–171
A209 The Red Algal Sugar Platform Completed by the Novel Pathway of 3,6-Anhydro-L-Galactose Catabolism
Kyoung Heon KIM, Eun Ju YUN, In-Geol CHOI Department of Biotechnology, Graduate School, Korea University, Seoul 136-713, Korea
Although marine macroalgal biomass has received much attention as the resource for third-generation biofuels, macroalgae contain rare sugars which are not fermentable by terrestrial organisms. Therefore, it is important to understand the catabolic pathways of macroalgal sugars to fully utilize the carbohydrates of macroalgal biomass for the biofuel production and biorefinery. Especially, although 3,6-anhydro-L-galactose (AHG) is the main sugar component of red macroalgae, AHG has remained an untouched substrate for microbial fermentation. Based on our recent discovery of the catabolic pathway of AHG for the first time, we have established the enzymatic and microbial sugar platform for red macroalgae. Through our integrated and vigorous analyses of metabolite and transcriptome of a marine bacterium by using GC/TOF MS, 2-D NMR, and RNA Seq, two novel enzymes and one novel metabolite and one common metabolite were identified as the key components in the catabolic pathway of AHG. After two unique metabolic steps, the catabolism of AHG was found to merge into the central carbon metabolism. Our results have paved a new way to the full utilization of the red macroalgal carbohydrate as a renewable resource for microbial fermentation and bioconversion.
Keywords : Red macroalgae, Agar, Agarase, 3,6-anhydro-L-galactose, Biofuel, Biorefinery
References 1. Yun EJ et al. Red macroalgae as a sustainable resource for bio-based products. Trends in Biotechnology 33:247- 249 (2015) 2. Yun EJ et al. The novel catabolic pathway of 3,6-anhydro-L-galactose, the main component of red macroalgae, in a marine bacterium. Environmental Microbiology 17:1677-1688 (2015) 3. Yun EJ et al. Pretreatment and saccharification of red macroalgae to produce fermentable sugars. Bioresource Technology in press 4. Lee CH and Kim HT (co-first author) et al. A novel agarolytic β-galactosidase acts on agarooligosaccharides for complete hydrolysis of agarose into monomers. Applied and Environmental Microbiology 80:5965-5973 (2014) 5. Roh H and Yun EJ (co-first author) et al. Genome sequence of Vibrio sp. strain EJY3: an agarolytic marine bacterium metabolizing 3,6-anhydro-L-galactose as a sole carbon source. Journal of Bacteriology 194:2773 (2012) 6. Lee CH and Yun EJ (co-first author) et al. Saccharification of agar using hydrothermal pretreatment and enzymes supplemented with agarolytic -galactosidase. Process Biochemistry in press 7. Choi, IG et al. Novel 3,6-anhydro galactonic acid cycloisomerase and use thereof. Korea Patent 10-Filed 2013- 0051134 (2013) 8. Choi, IG et al. A noble 3,6-anhydro-L-galactose dehydrogenase to convert 3,6-anhydro-L-galactose into 3,6- anhydrogalactonic acid. Korea Patent Filed 10-2011-0006631 (2011), Registered 10-1316084 (2013) 9. In Choi, IG et al. Novel 3,6-anhydro-L-galactose dehydrogenase acting on 3,6-anhydro-L-galactose, and production of 3,6-anhydro-L-galctonic acid by using the enzyme. US Patent Filed 13/980211 (2013), Registered (2015)
A210 Integration of Metabolic Pathways for Enhanced Production of Biofuels and Chemicals from Renewable Biomass
Yong-Su JIN Department of Food Science and Human Nutrition, Carl R. Woese Institute for Genomic Biology, and Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
While there are anticipated benefits from using cellulosic biomass as a feedstock to produce biofuels and chemicals, commercial production of cellulosic biofuel and chemicals has been hampered by technical difficulties, such as inefficient fermentation of cellulosic sugars, and the toxicity of acetic acid which are abundant in plant cell wall hydrolysates. As an effort to overcome the problems, we constructed a highly engineered Saccharomyces cerevisiae strain capable of converting mixed carbon components (cellobiose, xylose, and acetate) into ethanol simultaneously. As S. cerevisiae cannot ferment xylose, we first constructed a rapid and efficient xylose fermenting S. cerevisiae (SR8) through a rational design that allows strong and balanced expression levels of the xylose metabolic genes (XYL1, XYL2, and XYL3), and evolution of the engineered strain for rapid xylose fermentation [1]. Through systems-level characterization of the evolved strain, we learned that deletion of PHO13 is a beneficial genetic perturbation eliciting up-regulation of the pentose phosphate pathway [2]. Second, we introduced a cellobiose metabolic pathway into the efficient xylose fermenting strain (SR8) for co-fermentation of cellobiose and xylose [3]. Again, a rational design for integrating genes (cdt-1 and gh1-1) coding for cellobiose transporter and intracellular β-glucosidase, and evolution of the engineered strain were combined to isolate an engineered strain (EJ4) co-fermenting cellobiose and xylose rapidly. After measuring the copy numbers of cdt-1 and gh1-1 in the evolved strain, we learned that cellobiose fermentation can be improved by amplification of cdt-1 and gh1-1. Finally, we introduced an acetate reduction pathway, which can enhance xylose fermentation through redox coupling [4], into the xylose and cellobiose co-fermenting strain (EJ4) for efficient co-utilization of cellobiose, xylose, and acetate. The resulting strain was able to produce ethanol from a mixture of cellobiose, xylose and acetate with a substantially higher yield and productivity than the control strains, demonstrating the synergistic effects from integration of multiple metabolic pathways [5]. The Design, Build, Test and Learn (DBTL) cycle has been formulated as a standard practice of synthetic biology to construct optimal microbial strains with desired traits. Our strain improvement efforts exemplifies the DBTL cycle.
Keywords : Cellulosic ethanol, Co-fermentation, Cellobiose, Xylose, Acetate, yeast
References 1. Kim, S. R., Skerker, J. M., Kang, W., Lesmana, A., Wei, N., Arkin, A. P., and Jin, Y. S. (2013) Rational and evolutionary engineering approaches uncover a small set of genetic changes efficient for rapid xylose fermentation in Saccharomyces cerevisiae. PloS ONE 8, e57048 2. Kim, S.R., Xu, H., Lesmana, A., Kuzmanovic, U., Au, M., Florencia, C., Oh, E.J., Zhang, G.C., Kim, K. H., Jin, Y.S. (2015) Deletion of PHO13 encoding HAD type IIA phosphatase results in upregulation of the pentose phosphate pathway in yeast. Applied and Enviromental Microbiology 81,1601-1609 3. Ha, S. J., Galazka, J. M., Kim, S. R., Choi, J. H., Yang, X., Seo, J. H., Glass, N. L., Cate, J. H., and Jin, Y. S. (2011) Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proceedings of the National Academy of Sciences of the United States of America 108, 504-509 4. Wei, N., Quarterman, J., Kim, S. R., Cate, J. H., and Jin, Y. S. (2013) Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast. Nature Communications 4: 2805 5. Wei, N., Oh, E.J., Million, G., Cate, J.H., Jin, Y.S (2015). Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform. ACS Synthetic Biology 19: 707-713
A211 Production, Conversion and Application of Bio-based Platform Chemicals and Polymer Materials
Sang-Hyun PYO Division of Biotechnology, Center for Chemistry and Chemical Engineering,Lund University, Box 124, SE-221 00 Lund, Sweden,Department of Food Bioscience and Technology, College of Life Sciences and Bio
Demands for the efficient, green, and sustainable production of chemicals, materials, and energy have led to the development of industrial biotechnology. We have developed biotechnological routes for the production of several bio-based chemicals from glycerol using Lactobacillus reuteri, including 3-hydroxypropionaldehyde, 3-hydroxypropionic acid, and 1,3- propanediol. The biotechnological process was integrated with chemical process in order to further valorize the platform chemicals. 3-Hydroxypropionic acid was converted into acrylic acid by catalytic dehydration, while methacrylic acid was produced quantitatively from 2-methyl-1,3-propanediol by an integration process consisted of biotransformation using Gluconobacter oxydans and catalytic dehydration. Bio-based polyols were functionalized to the corresponding hydroxy- carboxylic acids by microbial selective oxidation and to the corresponding methacrylated polyols by lipase-catalyzed transesterification. A facile, green process for producing various five- and six-membered cyclic carbonates from the corresponding polyols was developed. The resulting carbonates were polymerized to bisphenol-free polycarbonates (PC) and to isocyanate-free polyurethanes (PU) with (biogenic) polyamines by thermal ring-opening polymerization. Furthermore, the methacryl- and allyl-functional groups of these cyclic carbonates and various polymerization schemes were used on the three-dimensional printing and composition with nanoparticles. PC and PU scaffolds were fabricated by ultraviolet-induced digital projection printing and femtosecond laser-induced two-photon polymerization, and were evaluated as advanced biomaterials for biomedical applications by assessing their biocompatibility in cell proliferation tests. The utilization of bio-based (biogenic) chemicals and materials in biomedical applications would improve the safety and environmental profile of the products, and would contribute to the overall value of bioresources.
Keywords : Industrial biotechnology, Integration process, biobased chemicals and polymers, 3D-printing, biocompatibility
A212 Detoxification of Lignocellulosic Hydrolysate for Butyric acid Production by Clostridium tyrobutyricum
Kyung Min LEE1,2, Ki-Yeon KIM1, Han Min WOO1,3, Sung Ok HAN2, Youngsoon UM1,3 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul , 2School of Life Science and Biotechnology, Korea University, Seoul, 3Clean energy and Chemical engineering, Korea University of Science and Technology, Daejeon
Lignocellulosic biomass is preferred as a feedstock in biorefinery, but lignocellulosic hydrolysate usually contains microbial inhibitors. Among these inhibitors, phenolics are highly toxic to butyric acid-producing Clostridium even at a low concentration. Herein, two detoxification methods were developed to detoxify phenolic compounds in lignocellulosic hydrolysate for butyric acid production. First, a simple in situ detoxification method using a surfactant was developed for butyric acid production by Clostridium tyrobutyricum ATCC25755. Tween 80, a non-ionic surfactant, was chosen to sequester inhibitors by forming micelles, consequently preventing direct contact of inhibitors with cell membranes. Second, the electrochemical polymerization method was developed to detoxify phenolic compounds in lignocellulosic hydrolysate. Whereas the cell growth and metabolite production of C. tyrobutyricum ATCC25755 were completely inhibited in un- detoxified hydrolysate, those in detoxifying rice straw hydrolysate were recovered to 70~100% of the control cultures. The detoxification methods described herein provide an efficient strategy for producing butyric acid with lignocellulosic biomass.
Keywords : Lignocellulosic hydrolysate, detoxification, butyric acid, Clostridium tyrobutyricum
References 1. Lee, K. M.; Kim, K.-Y.; Choi, O.; Woo, H. M.; Kim, Y.; Han, S. O.; Sang, B.-I.; Um, Y., In situ detoxification of lignocellulosic hydrolysate using a surfactant for butyric acid production by Clostridium tyrobutyricum ATCC 25755. (2015) Process Biochem., 50 (4), 630-635. 2. Lee, K. M.; Min, K.; Choi, O.; Kim, K.-Y.; Woo, H. M.; Kim, Y.; Han, S. O.; Um, Y., Electrochemical detoxification of phenolic compounds in lignocellulosic hydrolysate for Clostridium fermentation. (2015) Bioresour. Technol., 187, 228-234.
A213 2,3-Butanediol Production at GS Caltex Hyohak SONG, Jong Myoung PARK, Duk Ki KIM, Sangjun JEON Research and Development Center, GS Caltex Corporation, Daejeon-city, 305-380 Since the establishment of GS Caltex Corporation in 1967 as the joint company of GS and Chevron, the company has continued to offer clean energy and chemicals. The R&D Center has launched a number of projects concerned with industrial biotechnology since 2006 with the goal of producing bio-based fuels and chemicals. GS Caltex successfully completed the laboratory and pilot scale studies of 2,3-butanediol(2,3-BD) production by microbial fermentation on the March of 2015, and its 300 ton/year demonstration plant is under construction with BioCombinat. 2,3-BD has been known as a skin-friendly solvent and humectant for cosmetic industry, and also it has functions to activate plants’ own defense systems against diseases, drought, and the elements for agricultural industry. Furthermore, 2,3-BD is easily converted to butadiene, monomer in the production of synthetic rubber, and methyl ethyl ketone, eco-friendly solvent. BioCombinat Process developed by GS Caltex, Changhae Ethanol, and Daesang can produce simultaneously more than three products, enabling us to improve the process efficiency. 2,3-BD is one of the target products to be produced from BioCombinat Process. Here, the strategies of Industrial Biotechnology of GS Caltex Corporation are to be introduced with 2,3-butanediol production technologies and BioCombint Process. This work was supported by the Industrial Strategic Technology Program (No. 10050407, MOTIE, Korea). Keywords : 2,3-Butanediol, BioCombinat, Fermentation, Industrial Biotechnology References 1. Jong Myoung Park, Hyohak Song, Hee Jong Lee, Doyoung Seung, In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production (2013) Journal of Industrial Microbiology, 40(9), 1057-1066 한국생물공학회, 생물공학의 동향 : 2015.10 Bio-inspired Biotechnology: Biomimetic Lessons from Biology and Material Interfaces B201 Continuous, Real-Time Detection of Biomolecules in Live Animals Tom H. SOH University of California at Santa Barbara, USA A biosensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients’ health and their response to therapeutics. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets (i.e. glucose, lactose, and oxygen) and existing platforms for continuous measurement are not generalizable for the monitoring of other analytes. In this presentation, we will present a universalreal- time biosensor technology capable of continuously tracking a wide range of circulating molecules in living animals. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC)requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system’s versatility, we will present real-time measurement of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats with sub-minute temporal resolution. Finally, we will present the first real-time, closed loop feedback control of drug concentration in live animals using the MEDIC system and discuss potential applications of our technology. B202 Highly Stabilized Organic-Inorganic-Enzyme Microbeads for Continuous Modular Cascade Enzymatic Reactions Man Bock GU, Bo Kuk SEO, Ji Eun PARK Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University Anam-dong, Seongbuk-Gu, Seoul, 136-701, South Korea The highly stabilized organic-inorganic-enzyme microbeads (OIEMs) composed of branched-polymer, magnetic bead, and silica shell were successfully developed with properties of mechanically robust, long-term stable, and recyclable (1). These OIEMs were successfully implemented with two of cascade enzymes, Pyruvate kinase (PK) and Lactic dehydrogenase (LDH), which is a modular in vitro cascade reaction system as a model of multi-enzymatic reactions. Phosphoenolpyruvate was converted to lactate through PK- and LDH modules, and the reaction rate firmly remained even after 3 restless cycles. In addition, Carbonic anhydrase (CA) and Phosphoenolpyruvate Carboxylase (PEPCase) were also immobilized and stabilized in these OIEMs. These two enzymes are crucial for CO2 mitigation and counteraction of greenhouse effect, because they capture CO2 and convert to organic acid. Both CA- and PEPCase-OIEMs were highly stable and retained up to 90% of its initial activity even after more than 30 times of reuse. For in vitro continuous production of oxaloacetate and CaCO3, both CA- and PEPCase-OIEMs were involved sequentially, and modular minibioreactors with a constant working volume were operated continuously. The reaction conditions for each module were optimized by controlling the number of OIEMs and dilution rate. This highly stabilized enzyme module may be developed to design an OIEM-based tool box, an on-table storage system of various OIEMs for the in vitro production of valuable products. Keywords : organic-inorganic-enzyme microbeads , Pyruvate kinase , Lactic dehydrogenase, Carbonic anhydrase , Phosphoenolpyruvate Carboxylase , CO2 mitigation , organic acid References 1. J. Chung, E.T. Hwang, J. H. Kim, B.C. Kim, and M.B. Gu (2014), Modular Multi-enzyme Cascade Process Using Highly Stabilized Enzyme Microbeads, Green Chemistry, 16 (3), 1163-1167 B203 Encapsulation of Single Cells with Bio-inspired Materials Sung Ho YANG Dept. of Chemistry Education, Korea National University of Education, Chung-buk 28173, South Korea Nature has found cryptobiotic ways to preserve genetic information and protect cellular components against external stresses, such as nutrient deprivation, desiccation, high temperatures, radiation, and caustic chemicals. For example, a bacterial endospore, usually formed in response to nutrient deficiency, is a non-dividing, dormant body, which possesses a thin but tough proteinaceous coat at its outmost layer. It has, however, been practically difficult to achieve biochemical compatibility of living cells with robust artificial shells, as the synthesis of robust materials generally requires harsh conditions that interfere with the viability of the cells. In this respect, the essential step for the formation of strong artificial shells is to develop cytocompatible materials and synthesis reactions. Several strategies have been suggested to provide robust artificial shells to encapsulate living cells, thereby forming artificial spores. The strategies are based on the chemical reactions occurring on the interface of living cells, which are inspired by biomineralization, adhesive mussel proteins, and tannic acid.1-7 Recent studies have sought to chemically control and tailor the metabolic behaviours of non-spore-forming cells, as well as enhancing their viability against adverse environmental conditions, by forming thin (<100 nm), tough artificial shells.8 Keywords : Cell encapsulation, Biomimetic, Bio-inspired, Cell protection, Cell interface References 1. S. H. Yang, D. Hong, J. Lee, E. H. Ko, I. S. Choi, Artificial spores: cytocompatible encapsulation of individual living cells within thin, tough artificial shells(2013), Small, 9, 178-186. 2. S. H. Yang, E. H. Ko, I. S. Choi, Bio-inspired functionalization of silica-encapsulated yeast cells(2011), Angew. Chem. Int. Ed., 50, 6115-6118. 3. S. H. Yang, K.-B. Lee, B. Kong, J.-H. Kim, H.-S. Kim, I. S. Choi, Biomimetic encapsulation of individual cells with silica(2009), Angew. Chem. Int. Ed., 48, 9160-6163. 4. S. H. Yang, E. H. Ko, I. S. Choi, Cytocompatible encapsulation of individual chlorella cells within titanium dioxide shells by a designed catalytic peptide(2012), Langmuir, 28, 2151-2155. 5. S. H. Yang, S. M. Kang, K.-B. Lee, T. D. Chung, H. Lee, I. S. Choi, Mussel-inspired encapsulation and functionalization of individual yeast cells(2011), J. Am. Chem. Soc., 133, 2795-2797. 6. J. H. Park, K. Kim, J. Lee, J. Y. Choi, D. Hong, S. H. Yang, F. Caruso, Y. Lee, I. S. Choi, A Cytoprotective and Degradable Metal-Polyphenol Nanoshell for Single-Cell Encapsulation(2014). Angew. Chem. Int. Ed., 53, 12420. 7. J. H. Park, I. S. Choi, S. H. Yang, Peptide-Catalyzed, Bioinspired Silicification for Single-Cell Encapsulation in the Imidazole-Buffered System(2015), Chem Commun., 51, 5523. 8. D. Hong, M. Park, S. H. Yang, J. Lee, Y.-G. Kim, I. S. Choi, Artificial spores: cytoprotective nanoencapsulation of living cells(2013), Trends Biotechnol., 31(8), 442-447. B204 Understanding of Calcium Carbonate Biomineralization of Pearl Oyster using Recombinant Acidic Shell Matrix Proteins Yoo Seong CHOI Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea Biominerals such as shells, bones and teeth are involved in various important biological functions including structural support, protection, cutting and grinding. Their superior mechanical properties are exceedingly attractive in a broad range of applications including bone implants, drug delivery, plastics and construction materials. In this aspect, calcium carbonate (CaCO3) has been intensively studied as one of the most abundant biominerals and a model compound for understanding biomineralization processes. Specifically, the CaCO3 biominerals in molluscan shells are relatively well studied, and it has been widely accepted that shell matrix proteins play important roles in crystal nucleation and growth inhibition. Here, we successfully produced some recombinant acidic shell matrix proteins, which are expected to be involved in the control of CaCO3 biomineralization in the pearl oyster Pinctada fucata. We tried to understand the participation of these matrix proteins in the calcification of mollusc shells including the association of the inorganic phase and polysaccharide template. The studies provide possible opportunities for understanding biomineralization mechanism of mollusk shells and designing novel marine-inspired organic-inorganic hybrid materials. Keywords : recombinant shell matrix protein, biomineralization, calcium carbonate B205 Bio-inspired silicafication: Silica-forming proteins and their uses Mi Ran KI, Seung Pil PACK Dept. of Biotechnlogy and Bioinformatics, Korea University, Sejong, 30019 Silica has a wide range of industrial uses. Thanks to nontoxic and highly biocompatible characteristics, silica has also been applied in biomaterials such as scaffolds for tissues, carriers for drug-delivery system, and matrixes for biosensors and imaging. Conventional silica synthesis methods typically require a combination of high temperatures and extreme pH and also have limitations in structure controlling. Interestingly, the principle biomolecules involved in biosilicification were found both in diatoms (silaffins and polyamines) and sponges (silicateins). These findings lead to the use of biosilicification processes as new preparation routes of biocompatible materials and have created a new paradigm for silica synthesis under ambient or mild conditions. The prerequisite for the utilization of biosilicification machinery is the availability of enzymatically active recombinant proteins involved in silica formation. In this presentations, several research approaches were introduced in temrs of the use and design of new recombinant silica-forming proteins (SFP) such as modified silicatein α, silicatein-like proteins, and silaffin-like peptides. In particular, SFP-mediated biosilication was employed for the preparation of biocatalyst-silica compoiste matrix. Keywords : Biosilicification, Silicatein, Silaffin, Biosilica References 1. M-R. Ki et al. “Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S- transferase (GST)”, Bioprocess and Biosystems Engineering, Vol. 36, pp 643-648, 2013. 2. M-R. Ki et al., “Hypothetical cathepsin-like protein from Nematostella vectensis and its mutant silicatein-like cathepsin for biosilica production”, Process Biochemistry, Vol. 49, pp 95–101, 2013. 한국생물공학회, 생물공학의 동향 : 2015.10 Animal Cell and Tissue Engineering C201 Obtaining Distinctive Microarchitecture Suitable for Chondrocyte Culture Using a Thermosensitive Peptide Hybrid Hydrogel Sydney PENG, I-Ming CHU Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 300, Taiwan In this study, we prepared a thermosensitive peptide hybrid copolymer that exhibits thermosensitivity and can form a hydrogel with distinctive stranded network-like microarchitecture that resembles the natural cartilage extracellular matrix (ECM). Chondrocytes encapsulated within the hydrogel retained their round phenotype and formed cell clusters after prolonged culture. Biochemical and gene expression analysis showed significant accumulation of the ECM glycosaminoglycans and sustained upregulation of chondrogenic genes, respectively. Scanning electron microscopy revealed the deposition of extracellular matrix fibrils within the hydrogel structure after cells were cultured within the scaffold for 42 days. These findings demonstrate the importance of hydrogel architecture in maintaining chondrocyte phenotype in a three dimensional hydrogel and that the thermosensitive peptide hydrogel provides a bio-mimetic architecture that is profoundly different from other fully synthetic hydrogel systems. Keywords : thermosensitive, hydrogel, chondrocyte, cartilage C202 Directed Evolution of New Adeno-Associated Viral Vectors for Gene Therapy David SCHAFFER Chemical and Biomolecular Engineering, Bioengineering, and Neuroscience, University of California, Berkeley, CA, USA Gene therapy – the delivery of genetic material to patients for therapeutic benefit – has been enjoying successes in an increasing number of human trials, including for hemophilia, two retinal degenerative diseases, b-thalassemia, and sickle cell anemia. These studies have firmly established that viral gene delivery vehicles or vectors – particularly ones based on adeno- associated virus (AAV) and lentivirus – are capable of efficient, safe, and therapeutic gene delivery to numerous human cells and tissues. However, a series of formidable gene delivery barriers now limit the extension of these clinical successes to the majority of human diseases. These problems include pre-existing immunity against the virus, limited biodistribution to target tissues, poor spread within those tissues, low infection of therapeutically relevant cells, and an inability for targeted delivery to specific cells. These challenges are not surprising, as nature did not evolve viruses for our convenience to use as human therapeutics. Over the past 15 years, we have developed directed vector evolution, a highly versatile approach that emulates the process of natural evolution, to greatly enhance the properties of numerous viral vectors for biomedical application.1 In particular, we have designed new AAV vectors with substantially improved gene delivery efficacy to the brain, retina, heart, lung, and other tissues, work that is increasingly translating towards the clinic. Keywords : gene therapy, adeno-associated virus, vector engineering, directed evolution References 1. Melissa Kotterman, David Schaffer, Engineering Adeno-Asssociated Viruses for Clinical Gene Therapy (2014), Nature Reviews Genetics, 15, 445-451. C203 Bovine Sourced Biomaterials – an old material doing astonishing new things Geoffrey BENNETT Collagen Solutions plc Collagen, pericardium, bone and other bovine-sourced biomaterials have a more than thirty year history of safe use in implantable medical devices. Changes in the regulatory environment of bovine derived material over the last 10 years have stimulated the exploration of alternative sources of biomaterials by R&D professionals. This presentation will show why bovine-sourced biomaterials remain pre-eminent for medical device development. The discussion will also demonstrate how regulatory obstacles can be avoided and the long history of safe use of bovine-sourced bio-materials can be used to your advantage. Join the many leading-edge product developers who are using this “old” material in many remarkable “new” applications such as stem cell seeding and remarkable ortho-biologics. C204 Extractables and leachables: An introductory overview Siow Leng SIM GE Healthcare Life Sciences, 1 Maritime Square, #13-01, Harbourfront Centre, Singapore 099253 An introductory overview on Extractables and Leachables (E&L) will be presented in the context of biopharm industry. Key definitions, global regulatory requirements, industry trends and guidelines will be discussed together with concepts underpinning the design and execution of E&L studies. Selected case studies of step-wise, risk-based E&L evaluations will be shown. C205 Upstream process development for monoclonal antibody production ByungPil LIM Celltrion, Inc. Incheon 406-840, Korea Upstream process for mammalian cell culture has advanced significantly over the last several decades and is now generally considered a reliable and robust technology in monoclonal antibody production. In order to achieve a robust manufacturing process from the bench to commercialization, a number of variables such as host cell-lines, culture media, bioreactor culture conditions, scale-up parameters and process control parameters should be optimized. This presentation will describe: 1. Requirements for successful upstream process development. 2. Cell-line development strategies for monoclonal antibody production. 3. Cell culture process development from the bench up to commercialization. 4. Consideration points for large-scale manufacturing. Keywords : Upstream process, Cell-line development, Cell culture process optimization, Large-scale manufacturing C206 Manufacturing a cell therapy: Past, present and future Brian S. NEWSOM ThermoFisher Scientific (Life Technologies; Invitrogen), 12506 High Forest Lane, 12506 High Forest Lane, USA Brian Newsom joined Life Technologies/ThermoFisher Scientific in business development after having worked in the development of cell therapies for over 15 years. His most recent prior post was as the Head of Research and Development at Opexa Therapeutics where he led the development of a T cell vaccination for Multiple Sclerosis into a phase IIb clinical trial. Prior to Opexa, Brian spent five years at Baylor College of Medicine, Center for Cell and Gene Therapy developing gene therapy applications for pediatric oncology. Brian began his career in cell therapy at Aastrom Biosciences, one of the pioneering companies in the industry, in 1992. Brian received his degree in Microbiology from Texas Tech University and an MBA from Texas A&M University. Brian’s current role is to support the commercial development of cell therapy client companies and growing the breadth and impact of Thermo Fisher Scientific’s portfolio of products in relation to cell therapy research, development and industrialization. Brian works across all divisions of Thermo Fisher Scientific to help find solutions to pain points in cell therapy workflows and to help create new solutions where current offerings are not available. 한국생물공학회, 생물공학의 동향 : 2015.10 AFOB : Recent Advances in Biochemical Engineering A301 Nanoparticle Engineering for Sensitive Bio-Detection and Bioimaging Tamotsu ZAKO Dept. Chemistry and Biology, Grad. Sch. Science and Engineering, Ehime Univ. Nanoparticles have become increasingly important in various fields, including industrial and biomedical application. Here I’d like to present our recent studies concerning i) sensitive detection of biomolecules (DNA, proteins) using aggregation of functionalized gold nanoparticles and ii) near infrared (NIR) imaging for cancer diagnostics and therapy using rare earth ion- doped ceramic nanoparticles. In the first topic, dark field imaging was employed to visualize and quantify complementary DNA-induced gold nanoparticle aggregation at the single-particle level, with a detection limit of 100 fM DNA. This method has been successfully applied in detection of protein amyloid aggregation. In the second topic, rare earth ion-doped ceramic nanoparticles were applied for bioimaging in near infrared (NIR) region between 800 and 2000 nm, called "biological window", which enables deep tissue penetration due to low scattering and autofluorescence. Here I would present our recent application in cancer detection and laparoscopic cancer surgery. References 1. Bu T, Zako T*, Fujita M and Maeda M* "Detection of DNA Induced Gold Nanoparticle Aggregation with Dark Field Imaging", Chem. Commun., 49, 7531-7533 (2013) 2. Zako T*, Yoshimoto M, Hyodo H, Kishimoto H, Ito M, Kaneko K, Soga K* and Maeda M* "Cancer-targeted Near Infrared imaging using rare earth ion-doped ceramic nanoparticles" Biomater. Sci. 3, 59-64 (2015) A302 A Novel Cell Penetrating Peptides for the Delivery of Trehalose into Mammalian Cells Yuping WEI, Jian WANG, Liang ZHANG, Xia XU State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, P.R. China, 100190 Stabilization of cells in desiccated state can significantly simplify the storage and transportation and save expenses for clinical applications. Introduction of impermeable disaccharide, trehalose, into cells is an important step to improve the desiccation tolerance of cells. In this study, a novel cell penetrating peptide, KRKRWHW (KW), was developed based on molecular simulations. The peptide exhibiting little cytotoxicity and high efficiency of penetrating into mammalian cells successfully delivered trehalose into the MEFs through hydrogen bond and π-π bond. The internalization of KW into living mammalian cells was principally via direct translocation below the concentration threshold (20 μM) and through both direct translocation and endocytosis above 20 μM. After depletion of GAGs on KW uptake, GAGs of cell outer surface participated in the direct translocation, whereas not involved in the endocytosis. The investigation of KW internalization mechanism provides a theoretical basis for design a cell penetrating peptide for delivering specific cargo through non- covalent bonding into mammalian cells. Keywords : cell penetrating peptide, trehalose, cellular internalization mechanism, mammalian cells A303 Development of In situ Crosslinkable Hydrogels for Anti-Adhesion Barriers, Hemostat, and Drug Carriers Taichi ITO Center for Disease Biology and Integrative Medicine, School of Medicine, The University of Tokyo In situ crosslinkable hydrogels are useful in biomedical engineering applications such as drug delivery, prevention of postoperative adhesions and hemostats, and tissue engineering as shown in Figure 1. They can be potentially utilized in vivo through various administration routes such as intraperitoneal, intrapleural, intracardiac, intraarticular, intrathecal, and so on. In addition, they can be utilized in vitro for the tools to encapsulate various cells to fabricate medical devices such as regenerated tissues or cell chips. Various in situ crosslinkable hydrogels at a constant temperature and pH are categorized into (i)covalently-crosslinkable hydrogels by mixing of two reactive precursor polymers or enzymatic reactions, (ii)ionically-crosslinkable hydrogels by mixing precursor and inorganic ions as shown in Figure 2. For example, fibrin is a natural covalently-crosslinkable hydrogels used therapeutically for hemostasis. We energetically research covalently-crosslinkable hyaluronic acid (HA) hydrogels using Shiff base formation or copper-free click reaction. Sodium alginate extracted from seaweed is one of ionically-crosslinkable hydrogels. We recently fabricate a new ionically- crosslinkable hydrogel composed of star-shaped block copolymer In this presentation, we report the application of new hydrogels and their application to an anti-adhesion barrier, hemostat, hyalurnic acid (HA), alginate (Arg), chitosan (Ch). A304 Microwell Array for the Integrative Analyses of Individual Immune Cells Responding to a Mixture of Pathogens Jonghoon CHOI Department of BioNano Engineering, Hanyang University ERICA, Ansan, Korea 426-791 In this study, we use a single-cell co-culture microwell assay to temporally resolve antigen-dependent cytokine responses exhibited by human immune cells ex vivo. Monocyte-derived human dendritic cells pulsed with peptides from common antigens (influenza, tetanus toxoid, cytomegalovirus, and Epstein-Barr virus) were co-incubated in arrays of microwells with individual autologous human CD4+ T cells, and the secretory responses were measured for three different analytes at two time points (6 h and 24 h). The results show that the reported single cell assay can probe the profile of functional responses from individual antigen-reactive immune cells persistently stimulated by antigen-presenting cells. A305 Design of Sensitive Peptide-Protein Interaction Detection System on Solid Surfaces Hiroyuki IMANAKA*, Runa MATSUSHITA, Naoyuki ISHIDA, Koreyoshi IMAMURA Division of Applied Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama Biomolecules such as proteins, peptides and nucleic acids interact with each other in the cells with strictly controlling their life activities. Therefore development of techniques that enables highly sensitive biomolecular interaction detection on solid substrate would be a good versatile way to elucidate the biological phenomena and to establish various applications such as biomolecular drug screening system, cell signaling characterization system, and so on. In this research, in order to clarify the factors affecting interaction detection sensitivity, we focused on the influence of ligand orientation and density in addition to spacer length of analyte on detection, applying cysteine synthase (CS) complex formation (between ligand-binding pocket of O-acetylserine sulfhydrylase (OASS) and C-terminal peptide of serine acetyltransferase (SAT)) as the model. In this model, as N- and C-terminus of OASS are located at the opposite side, the orientational control of OASS immobilization would be possible by the conjugation of surface affinity peptide tag at each terminus. After the addition of various mutants of SAT C-terminal peptide to OASS immobilized surface, detection sensitivities of interaction was shown to correlate with their respective affinities to OASS. However, the most striking differences of interaction detection emerged from the differences in orientations of OASS. This phenomenon might be occurred probably due to the steric hindrance as the ligand-binding pocket of OASS locates near the C-terminus. In addition, to overcome the steric hindrance, effectiveness of lengthening of the spacer of analyte SAT C-terminal peptide or lowering the density of immobilized ligand OASS on detection sensitivity was investigated. Although both strategies were, to some extent, efficient for increase in detection sensitivity, their impact were revealed to be moderate. As the results, from the various detailed investigations, useful information to design sensitive peptide-protein interaction detection system on solid substrate was successfully obtained. The most important factor was clarified to be the consideration of orientation of ligand molecules. And, in case the design of positive orientation is difficult, adjustment of ligand protein density and the degree of freedom of the analyte peptide would also improve the detection sensitivity. 한국생물공학회, 생물공학의 동향 : 2015.10 Delivery of Engineered Biomacromolecules and Cells D101 Modulation of HMGB1 Activity for Successful Outcome of Islet Transplantation Dong Yun LEE Department of Bioengineering, College of Engineering, and BK21 PLUS Future Biopharmaceutical Human Resource Training and Research Team, Hanyang University, Seoul 133-791, Republic of Korea Pancreatic islet transplantation has been emerged as the most potential therapy for the treatment of insulin-dependent diabetes mellitus (IDDM). However, transplanted islets are rejected due to host’s immune reactions. Among these immune reactions, recently, it is known that high-mobility group box 1 (HMGB1) protein can play crucial roles in host’s immune response after islet transplantation [1-4]. That is, HMGB1 proteins are secreted from transplanted islets during initial time of transplantation, which can strongly induce inflammatory responses through receptor molecules, such as TLR2, TLR4, and receptor for advanced glycation end product (RAGE), in the immune cells. Currently, we have developed a new regulator for inhibiting secretion of HMGB1 protein from transplanted islets. Firstly, we developed a cell-penetrating HMGB1A fragment that could bind with HMGB1 protein in the cells, thereby reducing the secretion of HMGB1A protein. Next, glycyrrhizin, a glycoconjugated triterpene produced by the licorice plant, could also bind with HMGB1 protein at the extracellular area. In this case, we chemically modified the islet surface with glycyrrhizin to modulate the release of HMGB1 protein from islets. Surface modification of islet successfully captured the secreted HMGB1 proteins and also attenuated the HMGB1 protein activity, thereby enhancing the islet graft survival. Collectively, this new remedy related to the inhibition of HMGB1 secretion would be effective therapy for successful outcome of islet transplantation in vivo. Keywords : Diabetes, pancreatic islet, HMGB1, transplantation, glcyrrhizin References 1. N. Matsuok, T. Itoh, H Watarai, et al., J. Clin. Invest. 120, 735 (2010) 2. J. Han, J. Zhong, W. Wei, et al., Diabetes 57, 2118 (2008) 3. T. Itoh, T. Nitta, H. Nishinakamuri, et al., Pancreas 44, 166 (2015) 4. D. Kuraya, M. Watanabe, Y. Koshizuka, et al., Transplantation 96, 445 (2013) D102 Engineering Therapeutic Proteins for the Prolonged Serum Half-Life and Reduced Aggregation Inchan KWON School of Materials Science and Engineering, GIST, 61005 Extending the therapeutic protein serum half-life will significantly enhance the utility of therapeutic proteins. The conjugation/fusion of a therapeutic protein to HSA becomes a very promising emerging technology to prolong the serum half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We showed that the genetic incorporation of a non-natural amino acid followed by chemoselective albumin conjugation to prolong the protein activity in vivo without significant loss of efficacy using urate oxidase as a model protein (1). Protein misfolding and aggregation are implicated in numerous human diseases. We demonstrated that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation using a model protein, human copper, zinc superoxide dismutase mutant associated with amyotrophic lateral sclerosis (2). Keywords : serum half-life, albumin, urate oxidase, aggregation, SOD1 References 1. Sung In Lim, Yukina Mizuta, Akinori Takasu, Young S. Hahn, Yong Hwan Kim, and Inchan Kwon: Site-Specific Fatty Acid-Conjugation to Prolong Protein Half-Life In Vivo (2013), Journal of Controlled Release, 170, 219-225 2. Simpson Gregoire, Shaojie Zhang, Joseph Costanzo, Kelly Wilson, Erik J. Fernandez, Inchan Kwon, Cis- Suppression to Arrest Protein Aggregation in Mammalian Cells (2014), Biotechnol. Bioeng., 111, 462-74 D103 Regenerative Medicine : Synergistic Effect of Stem Cells and Nanobiomaterials Byung-Soo KIM School of Chemical and Biological Engineering, Seoul National University Nanobiomaterials can direct stem-cell fate both in vitro and in vivo by displaying stem-cell-regulatory signals in a precise fashion. This presentation will show how new technologies of biomaterials can be used to regulate stem cell differentiation. Graphene and its derivatives can promote adsorption of cell-adhesion signals and soluble signals, which can be applied to enhancement of stem cell differentiation into cardiac (1,2) and chondrogenic lineages (3). Although these materials can promote the differentiation of stem cells, the difficulties associated with engineering graphene into 3D macrostructured scaffolds have hampered the application of graphene in tissue engineering and regenerative medicine. From a practical perspective, carbonized polyacrylonitrile (cPAN), a highly ordered carbon isomorph that resembles the graphitic structure of graphene, could be a promising alternative, as cPAN can be easily processed into 3D scaffolds. We demonstrate the fabrication of microporous 3D scaffolds of cPAN and excellent osteoinductivity of cPAN, suggesting utility of 3D cPAN scaffolds as synthetic bone graft materials (4). Iron oxide nanoparticles (IONPs) can be used to develop gap junctional crosstalk among cardiomyocytes and stem cells in co-culture and induce cardiomyogenic differentiation of stem cells, generating therapeutic cells that exceed the reparative potentials of conventional stem cells (5). Keywords : nanobiomaterial, stem cells , Regenerative medicine References 1. Park J, Kim YS, et. al., Advanced Functional Materials 25, 2590 (2015). 2. Park J, Kim B, et. al., ACS Nano 9(5), 4987 (2015). 3. Yoon HH, Bhang SH, et. al., Advanced Functional Materials 24, 6455 (2014). 4. Ryu S, Lee C, et. al., Angewandte Chemie-International Edition 53(35), 9213 (2014). 5. Han J, Kim B, et. al., ACS Nano 9(3), 2805 (2015). D104 Oral Nucleic Acid Delivery using Transport System Yong-kyu LEE Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungbuk 380-702 The precedence in oral delivery of biomolecules in targeting dreadful diseases like cancer is yet to be achieved since the GI barriers hamper their pharmacokinetics, stability and bioavailability. The surface functionalization of non-viral vectors with TCA resulted in shielding the biomolecules/nanocomplexes from GI degradations, facilitate preferential ASBT mediated active transport in ileum and aid in attaining effectual therapeutic indices with minimal doses. We devised GLP-1 pDNA and siRNA based oral gene delivery systems to treat T2DM and hepatic cancer respectively. The results were very promising where a singular dose of our engineered GLP-1 non-viral vectors could revamp the lost incretin effects in animal models and aid in regulating the insulin and blood glucose levels under homeostatic glycemic ranges for 21 days without inducing any systemic toxicity. We further aim to investigate the mechanisms involved in improving the therapeutic efficacies of our formulations over conventional oral gene delivery systems. The objective is to move towards developing a “gene pill” based platform technology and create a new hope in Translational medicine. Keywords : oral delivery, nucleic acid, transport References 1. Z. Khatun, M. Nurunnabi, G.R. Reeck, K.J. Cho, Y.K. Lee, J Control Release. 170, 74 (2013) 한국생물공학회, 생물공학의 동향 : 2015.10 Establishment of Infrastructure for Industrialization of the Korean Useful Microbes D105 Introduction on Establishment of Infrastructure for Industrialization of Korean Useful Microbes SungHo CHO Microbial Institute for Fermentation Industry, Sunchang 595-804, Korea Concept : The Korean useful microbe(KUM) is korean domestic microbes having industrial value. We will carry out on establishment of infrastructure for industrialization of Korean useful microbes. The aim of this research business is for securing the sovereignty of korean domestic microbe-industry. Through this research business, national advanced base of useful microbial industry will prepare and realize. Our research team are to conduct through the following activities: 1. Screening korean useful microbes(KUM) and building database 2. Evaluating afty and functionality of KUM - Establishing new evaluation technique; smart animal, intestine on a chip 3. Establishing guideline of value-evaluation of KUM 4. Supporting various companies using KUM - technology transfer and consulting 5. Providing information on KUM, fermentation industry, fermented food, fermentive microbes, etc - Infrastructure of the system for - microbe bank, primer bank, flavor library of yeast, big data Keywords : Korean useful microbe, Industrialization, Infrastructure References 1. DoYeon, Jeong. Business plan of foudation of technology infrastructure(2015), Minstry of Trade, Industry and Energy D106 Evaluation and Application of Funcional Yeasts Isolated from Traditional Feremented Foods Sang-Ho BAIK Department of Food Science and Human Nutrition, and Fermented Food Research Center, Chonbuk National University, Jeonju 561-756, Korea. In case of Korean soy sauce, the unique simultaneous fermentation process with meju also gives birth to a complex and discriminating yeast community. Moreover, relatively high numbers of yeasts from 2 to 6 log cfu/mL are usually found during the fermentation even if they are not added in methods of production. Nevertheless, no special attention has been given to studying, identifying or quantifying the yeast species present in Korean soy sauce fermentation so far although it is known that specific yeasts give rise to the desirable effects. But, the yeast community structure and dynamics as well as the relationship between yeast and fermentation parameters remain unclear. Moreover, yeast is one of the key microorganisms for production of high quality soy-sauce especially affecting various flavors production among the key factors such as used materials, production technology and specific microorganisms. In particular, salt-tolerant yeasts synthesize flavor compounds such as ethanol, higher alcohols and esters by consuming sugars and amino acids released during brine fermentation process as flavour formation and salt tolerance are complicated phenotypes controlled by multiple genes. Thereby, salt-tolerant yeasts are very important for production of high quality soy sauce but study on yeast strain in fermentation of soy product has been neglected and underestimated as contrasted with fungi and bacteria. The purpose of this study is. 1. To determine yeast microbial community succession and flavor change profiles during Korean soy sauce fermentation. 2. To screen high ethanol and isoamyl alcohol producing strains, as starter strains for development soy sauce fermentation technology at a low salt concentration without decreasing the quality of the prepared soy sauce References 1. Young-Ran Song, Do-Youn Jeong, Sang-Ho Baik, Monitoring of yeast communities and volatile flavor changes during traditional Korean soy sauce fermentation (2015) J. Food Sci. 2. Young-Ran Song, Do-Youn Jeong, Sang-Ho Baik, Effects of indigenous yeasts on physicochemical and microbial properties of Korean soy sauce prepared by low-salt fermentation (2015) Food Microbiology D107 New Strategy for Screening Probiotic Bacteria Isolated from Korean Traditional Foods using Caenorhabditis elegans Host Model Younghoon KIM BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science & Technology, Chonbuk National University, Jeonju 561-756, Korea In recent, a number of groups have used the nematode Caenorhabditis elegans to identify and study evolutionarily preserved traits associated with host-probiotic bacteria interactions. In these C. elegans pathogenesis models there is significant correlation between the microbial virulence traits during mammalian infection and the killing of nematodes. In addition, recent studies have described certain evolutionarily conserved defense mechanisms in the nematode such as the insulin/insulin-like growth factor (IGF-1) pathway, p38 mitogen-activated protein kinase (MAPK) pathway, the transforming growth factor (TGF)-b signaling pathway, and the b-catenin/HOX transcription factors signaling. Therefore, C. elegans has been accepted as an alternative infection model host for the study of microbial infection and a simple model with which to study evolutionarily preserved aspects of innate immunity [1]. Here we introduced the recent applications of C. elegans in vivo host model in the field of food microbiology; i) whole animal system for automated high-throughput screening (HTS) for probiotic bacteria [2], ii) simple in vivo animal model for host defense mechanisms to pathogen infections [3], and iii) effective in vivo host for host immunity and longevity in gut environment [2]. These insights derived from the present study can provide a novel strategy to identify the design of better approaches for screening probiotic bacteria from Korean traditional foods. Keywords : C. elegans, Probiotic bacteria, Korean traditional foods, Immunity, Longevity References 1. Irazoqui, J. E., Urbach, J. M., and Ausubel, F. M. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates (2010) Nature Reviews Immunology.10, 47-58. 2. Park, M., Yun, H., Son, S., Oh, S., and Kim, Y. Development of a direct in vivo screening model to identify potential probiotic bacteria using Caenorhabditis elegans (2014) Journal of Dairy Science. 97, 6828-6834. 3. Kim, Y., and Mylonakis, E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain NCFM enhances gram-positive immune responses (2012) Infection and immunity. 80, 2500-2508. D108 In Vivo Toxicological Assessment of Biologically Synthesized Silver Nanoparticles in Adult Zebrafish (Danio rerio) Soon-Il YUN Department of Food Science & Technology,College of Agriculture & Life Science, Chonbuk National University,Jeonju, 54896, Korea The keen use of zebrafish (Danio rereio) in the experiment is easy to handle, smaller size, controlled and visible embryological phase, genetics amenability and breeding potential, importantly similarity at the molecular and physiological levels with humans [1] renders these organisms as the most suited model organism for toxicological studies [2]. In the present study examines the deleterious effect of biologically synthesized silver nanoparticles (AgNPs) in adult zebrafish. AgNPs were synthesized by treating AgNO3with aqueous leaves extract of MalvacrispaLinn., a medicinal herb as source of reductants. For achieving the sub lethal concentrations (LC50), followed the OECD guidelines for testing chemicals (1992). Six different concentration of AgNPs (23.7, 47.4, 142.2, 237, 284.4, 331.8µg/l) were exposed and the LC50 at 96h was observed at142.2µg/l. In order to explore the underlying toxicity mechanisms of AgNPs, half of the LC50concentration(71.1µg/l) was exposed to adult zebrafish for 14 days. Cytological changes and intrahepatic localization of AgNPs were observed in gills and liver tissues respectively, and the results concluded a possible sign for oxidative stress. In addition to oxidative stress the genotoxic effect was observed in peripheral blood cells like presence of micronuclei, nuclear abnormalities and also loss in cell contact with irregular shape was observed in liver parenchyma cells. Hence to confirm the oxidative stress and genotoxic effects the mRNA expression of stress related (MTF-1, HSP70) and immune response related (TLR4, NFKB, IL1B, CEBP, TRF, TLR22) genes were analyzed in liver tissues and the results clearly concluded that the plant extract mediated synthesis of AgNPs leads to oxidative stress and immunotoxicity in adult zebrafish. Keywords : Zebrafish (Danio rerio), AgNPs, Intrahepatic localization, Gene expression References 1. M. Westerfield, “The Zebrafish Book”. In A Guide for the Laboratory Use of Zebra fish (Danio rerio). 4thed. Eugene: University of Oregon Press,2000. 2. O.B. Illan, R.M. Albrecht, V.E. Fako, D.Y. Furgeson, Toxicity Assesments of Multisized Gold and Silver Nanoparticles in zebrafish embryos. Small. 16 (2009) 1897-1910. D109 Microfluidic Gut-on-a-chip for Studying Intestinal Functions and Microbial Interaction Jong Hwan SUNG Chemical Engineering, Hongik University, Seoul, Korea Current in vitro cell culture models do not reflect human physiology, and various efforts have been taken to enhance existing models. Reconstitution of three-dimensional (3D) tissue structure has been one of the strategies, since 3D tissue structure provides essential cellular environmental cues for cell functions. Previously, we developed a novel hydrogel microfabrication technique for constructing an accurate 3D replica of human intestinal villi epithelium. In this study, genetic and physiological properties of the 3D villi model were examined to gain a better insight into the barrier function of gut epithelium and its interaction with microbes. Gene expression study of Caco-2 on the 3D villi scaffold revealed that expression of MUC17, which is one of transmembrane mucins, was highly enhanced in the 3D villi model, compared to a monolayer culture. Cells on the scaffold were almost immune to bacterial infection, while MUC17 knockdown in Caco-2 cells restored bacterial infectivity. The 3D villi model also exhibited changes in the barrier function compared to the 2D model, manifested by changes in transepithelial electrical resistance (TEER) and permeability of FITC-dextran. Knockdown of MUC17 resulted in reduction of tight junction protein expression and further increase in permeability, suggesting an important role of MUC17 in the barrier function against pathogens and xenobiotics. Our study suggests that mimicking the 3D tissue architecture of the small intestine induces physiological changes in human intestinal cells. Keywords : Microfluidic, gut-on-a-chip, organ-on-a-chip, microbes References 1. Meiying Chi, Banya Yi, Seunghan Oh, Dong-June Park, Jong Hwan Sung, and Sungsu Park, Microfluidic cell culture device (μFCCD) to culture epithelial cells with physiological and morphological properties that mimic those of the human intestine, Biomedical Microdevices, 2015, Jun, 17(3), 9966 2. Shi Hyn Kim, M Chi, B Yi, SH Kim, S Oh, S Park, JH Sung, Three-dimensional intestinal villi epithelium enhances protection of human intestinal cells from bacterial infection by inducing mucin expression, Integrative Biology, 2014, 6(12), 1122-31 3. Jong Hwan Sung, J. Yu, D. Luo, M.L. Shuler, and J.C. March, Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model. Lab Chip, 2011 Feb. 11(3): p. 389-92. 한국생물공학회, 생물공학의 동향 : 2015.10 Biobased C4~C6 Diacids and Diols as Alternative Precursors to Aliphatic and Aromatic Compounds D201 Current Status of Biorefinery Research towards Industrial Production of Biobased C4~C6 Molecules and Corresponding Polymers: Approaching via Convergence of Industrial Microbiology and Gree Sangyong KIM Green Materials and Process Group, KITECH, Cheonan, 31056 While biobased economy has been emerging as a sustainable solution for carbon neutral circular economy based on renewable feedstocks such as biomass replacing fossils we still lack in stable value chains of technology and materials. Biobased products as promising alternatives to petroleum derived chemicals and fuels require well-balanced feedstock supply and production units. Unlike optimized petroleum production lines which take refined C3-C6 hydrocarbons to convert them into conventional reactive functional small molecules and synthetic polymers, biomass derived chemicals are currently lacking in stable supply chains of optimized conversion pathways. The ultimate goal of biobased economy will be the most efficient integration of resources and technologies in terms of economical feedstock availability, byproduct-waste valorization, robust (bio)catalysts and processes. The development of bio-based materials has been extended from the synthesis of aliphatic molecules to aromatic molecules to balance the supply of renewable building blocks both in linear and ring structures to replace petroleum based drop-in compounds. While the bio-based platform chemicals are drawing more interests as potential monomer precursors for commodity bioplastics, a variety of synthesizing and processing pathways of sugar derived arene compounds has been investigated. As a corresponding research KITECH is aiming at sustainable production of C5~C6 cyclic and aromatic building blocks. The recent successful synthesis of diacids and diols such as itaconic acid, muconic acid, isosorbide and furanics from industrial sugar and crude polysaccharides may allow us to accept versatile non-food renewable feedstocks and replace oil based non-renewable drop-in aromatics such as terephthalate and bisphenol A preferentially. The noble catalysis and biocatalysis of bio-based small molecules along with inherent derivatization and copolymerization are in progress. Keywords : Biobased Economy, Value-chained Production, C3-C6 Chemicals, Industrial Fermentation, Green Synthesis References 1. BIO-TIC, A Roadmap to a Thriving Industrial Biotechnology Sector in Europe, Industrial Biotech for Europe, 2015 D202 Mass Production of Succinic acid and cis-cis Muconic acid: from Strain Improvement to Bioprocess Development Gie-Taek CHUN1, Sang Min PARK1, Seong-Ryeoul SEO2, Sun-Ok PARK2, Sang-Jong LEE2, Eung-Soo KIM3, Woo-Shik SHIN4, Do-Hoon LEE4, Sang-Young KIM4 1College of Biomedical Science, Kangwon National University, Chunchon, South Korea, 2STR Biotech. Ltd., Chunchon, South Korea, 3Inha University, Inchon, South Korea, 4Korea Institute of Industrial Technology, Chonan, south Korea Succinic acid(SA): SA, an anaerobic fermentation product biosynthesized by Actinobacillus succinogenes, has been reported as one of the top 10 valuable building block chemicals. For enhancing SA production, rapid and massive screening was performed through the use of high throughput system(HTS) developed in our laboratory, consisting of CO2- and humidity- controlled incubator and 24-well microplate-culture system for miniaturized fermentations inside the incubator. Significant enhancement in SA production was possible by employing cell-recycled continuous fermentation system(CRCFS). An efficient decantor system(i.e., cell separator) could separate high density of the suspended cells effectively from the outlet stream, thus overcoming the wash-out phenomenon encountered at relatively low dilution rate in the continuous process without cell-recycling. A strategy of feeding increased amount of MgCO3 through the inlet stream of the CRCFS turned out to be very efficient for enhanced production of SA, resulting in about 10 fold, and more than 6 fold higher volumetric productivity(DP) (i.e., g SA/L/hr) than the corresponding batch and continuous fermentation systems without cell-recycling, respectively. Cis-cis muconic acid(MA): MA, utilized as a raw material for new functional resins, pharmaceuticals and agrichemicals is an unsaturated dicarboxylic acid with six carbon atoms. MA was revealed to be easily converted to adipic acid, a valuable commodity chemical for bio-plastics including nylon 6-6 and polyurethane and polyethylene terephthalate(PET). For development of MA high-yielding recombinant Escherichia coli as well as C. glutamicum cells, additional genes from various sources were introduced into each of the aromatic amino acids auxotrophic mutants by use of plasmid expression vectors with various strong promoters. Intensive studies were conducted to develop an efficient fermentation process for overcoming catabolite repression phenomenon. Fermentation results from batch and fed-batch bioreactor operations performed with the recombinant cells will be presented in this paper. Keywords : KSBB, biotechnology, BT, symposium References 1. X. Tong, Y. Ma and Y. Li (2010) Biomass into chemicals: Conversion of sugars to furan derivatives by catalytic processes. Appl catal Gen. 385(1-2):1-13 2. Kathleen A. Curran, John M. Leavitt, Ashty S. Karim, Hal S. Alper, Metabolic engineering. 15, 55-66 (2013) 3. Moon C, Ahn JH, Kim SW, Sang BI, Um Y. Appl Biochem Biotechnol. 161:502-210(2010) D203 Itaconic Acid Production by Fungal Fermentation Dohoon LEE1, Sangyong KIM1, Woo-Shik SHIN1, Gie-Taek CHUN2 1Korea Institute of Industrial Technology(KITECH), Cheonan 31056, Korea, 2College of Biomedical Science, Kangwon National University, Chunchon 24341, Korea Itaconic acid (C5H6O4) is an essential platform chemical for the production of resins, biofuel components and its derivatives have been widely used in the chemical and pharmaceutical industries. Itaconic acid fermentation can be carried out by various microorganisms. Among them, a fungal strain, Aspergillus terreus, is known for the most prolific producer of itaconic acid. Fungi are natural producers of various valuable chemicals. Although most of the academic and industrial interests have been focused on their ability to produce useful secondary metabolites, they also have potential as remarkable producers of commodity chemicals like organic acids. Although strategies for the development of microbial and fungal processes share many technologies, careful consideration of the unique physiology of fungi is essential for the success, especially during the media selection, reactor design, and process monitoring and control. In this presentation, our recent research efforts to develop fungal bioprocesses converting renewable resources into itaconic acid will be introduced. Topics will cover improvement of fungal biocatalysts based on mutagenesis and expression of heterologous enzymes, control over catalyst physiology, and optimization of the fermentation process. Keywords : biorefinery, itaconic acid, fungi, fermentation References 1. Shin et al., Application of scale-up criterion of constant oxygen mass transfer coefficient (kLa) for production of itaconic acid in a 50 L pilot-scale fermentor by fungal cells of Aspergillus terreus (2013), J. Microbiol. Biotechnol., 23(10), 1445-1453 D204 Facile Route to Furanic Compounds from Lignocellulosic Biomass toward Bio-based Aromatics Jin Ku CHO Korea Institute of Industrial Technology Recently, much attention has been paid to conversion of lignocellulosic biomass into valuable chemicals to replace fossil- based resources with renewable and sustainable resources. One of hot topics is conversion of the carbohydrates contained in lignocellulosic biomass into a furanic precursor, such as 5-hydroxymethylfurfural (HMF), for bio-based aromatics. Despite the huge potential for versatile applications of HMF, an industrially adaptable and techno-economically viable pathway from lignocellulosic biomass to HMF is still not accomplished. Some major barriers toward the goal are its ineffective conversion, difficult isolation due to the hydrophilicity, and thermal/chemical instability. In this presentation, we propose 5-acetoxymethylfurfural (AMF) as an alternative precursor for furan-based chemicals and a facile pathway to produce it from lignocellulosic biomass. AMF can be produced in good to excellent yields under mild conditions from 5-chloromethylfurfural (CMF) and alkylammonium acetates. Both reactants could be obtained directly from readily available lignocellulosic biomass raw materials using a biphasic reaction and an anaerobic fermentation, respectively. AMF showed advantageous properties such as hydrophobicity and improved thermal stability, which made the practical isolation and purification of AMF possible. Various purification methods were examined to give AMF with high purity. Remarkably, the simple vacuum distillation of crude AMF could afford an extremely pure AMF (99.9%) without significant yield loss. AMF was successfully converted into some furan compounds, such as 2,5-furandicarboxylic acid (FDCA), 2,5- furandimethanol (FDM), and 5-hydroxymethyl-2-furanoic acid (HFA). Keywords : Lignocellulosic biomass, 5-acetoxymethylfurfural, 5-chloromethylfurfural, alkylammonium acetates, furan compounds References 1. E.-S. Kang et al., From Lignocellulosic Biomass to Furans via 5-Acetoxylmethylfurfural as an Alternative to 5- Hydroxymethylfurfural (2015), ChemSusChem, 8(7), 1179 D205 Isosorbide-based Bioplastics and Specialty Chemicals Seunghan SHIN Korea Institute of Industrial Technology Taking the issues on carbon footprint and depletion of petroleum reservoirs into account, isosorbide derived from glucose is highlighted as a potential substitute for petroleum-based aromatics. Isosorbide has chirality and low optical anisotropy and good thermal stability due to its wedge-shaped fused ring structure [1]. In this regard, recent application studies of isosorbide have been focused to utilize its unique and excellent properties as well as benefits of bio-based products. We have used isosorbide as a monomer for polycarbonates owing to its low optical anisotropy and tested various kinds of catalyst for melt polymerization to obtain high molecular weight and high glass transition temperature. As for dental adhesives, we have synthesized two different photo-polymerizable compounds with isosorbide backbone and 2-hydroxylproply methacrylate or methacrylate as bio-based alternatives of biphenyl-A glycidyl methacrylate and triethylene glycol dimethacrylate, a representative dental adhesive. In this presentation, we will show some recent results about above two cases. Keywords : isosorbide, bisphenol-A replacement, isosorbide-based polycarbonate, dental adhesive References 1. M. Rose and D.R. Palkovits, Isosorbide as a Renewable Platform chemical fro Versatile Applications (2012), ChemSusChem, 5, 167 한국생물공학회, 생물공학의 동향 : 2015.10 Vaccine Biotechnology and Current Issues B206 Bacterial Flagellin, the TLR5 Ligand, Can Serve a Versatile Adjuvant for Various Vaccines JoonHaeng RHEE Clinical Vaccine R&D Centerand Department of Microbiology,Chonnam National University Medical School, Gwangju, Republic of Korea Flagellinis the cognate ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation leads to activation of innate and subsequently modulates adaptive immune responses. TLR ligands are considered attractive adjuvantsfor vaccinesand immunotherapy. In this presentation, we show that flagellin has a uniqueimmunomodulating activity in the mucosal immune compartment. (1) Flagellin could be used as a mucosal adjuvant, which enables injectable vaccines to be used as a mucosal vaccine.Intranasal co-administration of aV. vulnificusflagellin(FlaB) with tetanus toxoid (TT) induced significantly enhanced TT-specific IgA responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plusFlaBwere completely protected from systemic challenge with supra-lethal dose of tetanus toxin. Intranasally administered FlaBcolocalized with CD11c as patches in DCs and caused an increase in the number of TLR5 expressing cells in cervical lymph nodes.Further, we tested whether FlaB could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans. Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high, influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. FlaB significantly potentiated influenza-specific cytokine production by immune splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high HIAb titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system.(2) Intranasal co-administration of flagellin with allergen could induce regulatory dendritic cells and could be used to treat allergic asthma.Balb/c mice sensitized with OVA were treated with OVA-FlaB mixture three times at 1-week intervals. The OVA-FlaB treatment significantly suppressed OVA-induced airway hyperresponsiveness (AHR), airway eosinophilic inflammation and OVA- specific Th2 cytokine productions in splenocytes. The adoptive transfer of in vitro FlaB-treated DCs inhibited the development of OVA-induced asthma. FlaB-treated splenic DCs from WT or TLR5-knock out (KO) mice were adoptively transferred 7 days before the OVA challenge. FlaB-treated DCs showed increased IL-10 and IL-12 productions compared to untreated DCs. AHR and eosinophils in bronchoalveolar lavages (BAL) were decreased in the group transferred by theFlaB- treated DCs from WT, but not KO mice. In BAL fluids, IL-4, IL-13 and IFN-g were reduced in the group received FlaB- treated spleen DCs from WT, but not those from KO mice. IL-10 and TGF-β were increased in the group received FlaB- treated DCs from WT, but not KO mice. Collectively, the adoptive transfer of FlaB-treated DCs from WT, but not KO mice, reduced AHR, eosinphilic airway inflammation and Th1 and Th2 responses, and enhanced Treg cell responses. These results indicate that flagellin can be considered as a component of allergen-specific immunotherapy and DC cell therapy for atopic diseases.(3) Flagellin effectively potentiates anti-peptide antigen immune responses.Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs.We investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specificantitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival.FlaB potentiated the CTL activity and Ag-specific IFN-g production of CD8+T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivodepletion of CD8+T cells and in TLR5 knockout (KO) or MyD88 KO mice. Keywords : Vaccine, Adjuvant, Flagellin, TLR5 References 1. Lee, S.E., Kim, S.Y., Jeong, B.C., Kim, Y.R., Bae, S.J., Ahn, O.S., Lee, J. J., Song, H.C., Kim, J. M., Choy, H. E., Chung, S. S., Kweon, M. N., Rhee, J. H.: A bacterial flagellin, Vibrio vulnificusFlaB, has a strong mucosal adjuvant activity to induce protective immunity. Infect. Immun. 74: 694-702. 2006. 2. Lee, S.E., Koh, Y.I., Kim, M.K., Kim, Y.R., Kim, S.Y., Nam, J.H., Choi, Y.D., Bae, S.J., Ko, Y.J., Ryu, H.J., Koh, J.T., Choy, H.E., Rhee, J.H. :Inhibition of airway allergic disease by co-administration of flagellin with allergen. J. Clin. Immunol. 28:157-65. 2008. 3. Nguyen CT, Kim SY, Kim MS, Lee SE, Rhee JH. Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice. Vaccine. 29:5731-9, 2011. 4. Hong SH, Byun YH, Nguyen CT, Kim SY, Seong BL, Park S, Woo GJ, Yoon Y, Koh JT, Fujihashi K, Rhee JH, Lee SE. Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection.Vaccine. 30:466-74, 2012. 5. Nguyen CT, Hong SH, Sin JI, Vu HV, Jeong K, Cho KO, Uematsu S, Akira S, Lee SE, Rhee JH. Flagellin enhances tumor-specific CD8+ T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model. Vaccine. 31:3879-87, 2013. 6. Lim JS, Nguyen KC, Nguyen CT, Jang IS, Han JM, Fabian C, Lee SE, Rhee JH, Cho KA. Flagellin-dependent TLR5/caveolin-1 as a promising immune activator in immunosenescence. Aging Cell. 2015 Jul 30.doi: 10.1111/acel.12383. [Epub ahead of print]. 7. Shim JU, Lee SE, Hwang W, Lee C, Park JW, Sohn JH, Nam JH, Kim Y, Rhee JH, Im SH, Koh YI. Flagellin suppresses experimental asthma by generating regulatory dendritic cells and T cells. J Allergy ClinImmunol. 2015 Aug 21.pii: S0091-6749(15)00952-5. doi: 10.1016/j.jaci.2015.07.010. [Epub ahead of print] B207 Engineered Immunomodulatory Biomaterials for Enhanced Immunity Yong Taik LIM SKKU Advanced Institute of Nanotechnology (SAINT), School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea The design and chemical synthesis of multifunctional nanomaterials have been providing potential applications in the control of the infectious disease and cancer therapy. In this talk, the recent research results on the development of engineered nanobiomaterials for enhanced immunity will be presented [1-3]. Nanobiomaterials were programmed for the combination of nanostructured materials and bioactive compounds to improve immune cells activity that is essential requirements in the control of infectious disease. Because most pathogens access the body through mucosal membranes, effective vaccines that protect these sites are highly needed. Recently, we focused on the development of a nanodelivery system for the effective mucus delivery of a viral antigen that can induce strong mucosal immunity without additional immunostimulatory adjuvant materials [2-3]. To accomplish this goal, we designed and synthesized a mucosal vaccine delivery system based on biosynthetic mucoadhesive polymer nanomicelles [2]. We also successfully generated a novel anti-influenza vaccine system combining conserved matrix protein 2 (sM2) and stalk domain of hemagglutinin (HA2) fusion protein (sM2HA2) and mucoadhesive polymer–based vaccine adjuvant systems [3]. Intranasal co-administration of sM2HA2 and the combination adjuvant was able to induce a high degree of protective mucosal, systemic, and cell-mediated immune responses. Keywords : Immunomodulation, nanobiomaterials, adjuvant, vaccine References 1. A. Seth, D.-B. Oh, Y.T. Lim, Nanomedicine, 10(6), 959-975 (2015) 2. Y.-W. Noh, et. al., Angew. Chem. Int. Ed., 52, 7684-89 (2013) 3. H. J. Noh, et.al., J. Immunol.,( in press,2015) B208 Pros and Cons of VP1-specific Maternal IgG for the Protection of Enterovirus 71 Infection Sun-Young CHANG College of Pharmacy, Ajou University, Suwon 443-749, Korea Enterovirus 71 (EV71) causes hand, foot, and mouth diseases and can result in severe neurological disorderswhen it infects the central nervous system. Thus, there is a need for the development of effective vaccines against EV71 infection. Here we report that viral capsid protein 1 (VP1), one of the main capsid proteins of EV71, efficiently elicited VP1-specific immunoglobulin G (IgG) in the serum of mice immunized with recombinant VP1. The VP1-specific IgG produced in female mice was efficiently transferred to their offspring, conferring protection against EV71 infection immediately after birth. VP1-specific antibody can neutralize EV71 infection and protect host cells. VP1-specific maternal IgG in offspring was maintained for over 6 months. However, the pre-existence of VP1-specific maternal IgG interfered with the production of VP1-specific IgG antibody by active immunization in offspring. Therefore, although our results showed the potential for VP1-specific maternal IgG protectionagainst EV71 in neonatal mice, other strategies must be developed to overcome the hindrance of maternal IgG in active immunization. In this study, we developed an effectiveand feasible animal model to evaluate the protective efficacy of humoral immunity against EV71 infection using a maternal immunity concept. Keywords : Enterovirus 71, VP1, maternal IgG, protective immunity, pre-existing immunity B209 Broadly protective Shigella vaccine development Min Jung Kim, Hee Joo Kim, Semi Rho, Seung Young Lee, Man Ki Song, Cecil Czerkinsky*, Dong Wook Kim*, Jae-Ouk Kim Lab Science/International Vaccine Institute, Seoul, Korea In developing countries Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis and we have named it pan-Shigella surface protein-1 (PSSP-1). PSSP-1- induced protection requires a mucosal administration route and co-administration of an adjuvant. Interestingly, blood samples from patients with recent onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. Further, we constructed the genetically modified mutant strain which enables better exposure of common protein antigens including PSSP-1 on the bacterial surface to the host immune system, which is potentially capable of eliciting stronger and broader cross-protective immunity than the strategy of targeting LPS alone. *Current affiliation: DW Kim- College of Pharmacy, Hanyang University, Ansan, Korea; C Czerkinsky-Institut de Pharmacologie Moléculaire et Cellulaire, CNRS,INSERM, Université de Nice-Sophia Antipolis, UMR 7275, Valbonne, Francec Keywords : Shigella, vaccine, conserved protein antigen References 1. Kim, J.O., et al. Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection. Clin Vaccine Immunol 22, 381-388 (2015). 한국생물공학회, 생물공학의 동향 : 2015.10 Bioenergy and Eco-Biotechnology for Creative Economics B210 Microalgal Cultivation and Algal Bloom Control: Flip-flop Yoon-E CHOI Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul, 136-713, Republic of Korea Microalgae have long been considered as promising microorganisms, due to their potential as sources of valuable pharmaceuticals, pigments, carbohydrates, biofuels, and the other fine chemicals. On the other hand, unwanted rapid increase in the population of algae in a water system causes harmful algal blooms, which are one of the major environmental problems. Therefore, it is evident that there are beneficial as well as detrimental aspects of microalgae. To address this issue further, we propose that microalgal cultivation and algal bloom control is analogous to flip and flop of our hands, respectively. In this study, we will first present our previous experience of various strategy of microalgal cultivation as a ‘flip’. Later, our eco-friendly approach against algal bloom will be provided as a ‘flop’. Particularly for a ‘flop’, in order to develop eco-friendly approach to suppress algal bloom, we isolated a novel bacterium, identified as Bacillus sp. (named T4) against Microcystis, the most common toxic microalgae. The bacterium could eliminate over 85% algal cells after 2 days, and the algicidal activity even reached 100% at day 5th. Interestingly, our study showed that microalgal cells were killed through indirect attack by compound(s) secreted from bacterial culture. Furthermore, the isolated bacteria strain holds genes such as mlrA, mlrB, mlrC, and mlrD, responsible for microalgal toxin degradation and its toxin removal ability was confirmed by ELISA. On the other hand, zooplankton plays a pivotal role in aquatic food webs as a food source for fish as well as a grazer on microalgae. To further enhance the removal effect of algal bloom, we attempted to simultaneously apply zooplankton and T4. Data suggested that the simultaneous applications of both zooplankton and T4 strain led to the best performance against algal bloom. Our approach based on eco-friendly application of either bacteria, zooplankton, or together will open a new way to control harmful algal bloom. Keywords : Microalgal cultivation, Algal control, Eco-friend approach References 1. Lionel Ho et al, Isolation and identification of a novel microcystin-degrading bacterium from a biological sand filter (2007), Water Research, 41, 4685-4695 B211 Combinatorial Engineering of Saccharomyces cerevisiae for Efficient Biofuels Production from Lignocellulosic Biomass Sun-Mi LEE Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, 136-791 Biofuels production from lignocellulosic biomass can be both sustainable and economical when all available carbon sources are completely utilized. Even with decades of research, the efficiency in pentose sugar utilization in Saccharomyces cerevisiae remains suboptimal requiring further improvement in pentose catabolism in this model organism for biofuels production. Here, we discuss the advantages of both combinatorial and evolutionary engineering approaches for the improvement of pentose sugar utilization. Specifically, we discuss the development of an efficient xylose fermenting strain harboring xylose isomerase-based pathway to achieve high yields during ethanol fermentation of lignocellulosic hydrolysate. We next discuss combinatorial and evolutionary engineering approaches to establish functional arabinose and xylan catabolic pathways. We show that alternative pentose sugar catabolic pathways in S. cerevisiae can perform better compared with previously reported pentose sugar pathways. Moreover, these studies suggest an effective combinatorial engineering strategy to develop efficient heterologous pathway in S. cerevisiae. Keywords : Saccharomyces cerevisiae, pentose sugars, combinatorial engineering References 1. S. Lee, T. Jellison, and H. Alper, Biotechnol. J. 10(4) 575 (2015) 2. S. Lee, T. Jellison, and H. Alper, Biotechnol. Biofuels 7(122) (2014) 3. S. Lee, T. Jellison, and H. Alper, Appl. Environ.Microbiol. 78(16), 5708 (2012) B212 Enhanced Biomethanation by Biostimulation with Conductive Iron Oxides Changsoo LEE School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST) Production of biogas, composed mainly of CH4 and CO2, from organic wastes by anaerobic digestion (AD) is regarded as a viable option to meet the increasing demand for renewable energy. Although having a great advantage of producing energy and reducing pollution load simultaneously, AD does have some limitations related to the slow reaction rate, i.e., longer retention time and lower organic removal as compared to aerobic treatment. As AD is a multi-step biological process mediated by diverse microbial groups, its performance basically relies on the activity of the microbial consortia involved. Electroactive bacteria (EABs) are a group of bacteria capable of transporting electrons over cell membranes to/from their extracellular environment [1]. The EABs in bioelectrochemical systems (BESs) are reported to be mostly MRBs, primarily Geobacter and Shewanella species, which can oxidize organic compounds using metal ions (e.g., Fe3+) as electron acceptors under anaerobic conditions. These bacteria directly or indirectly transfer electrons produced from the oxidation of organics to electrodes, generating an electric current. Although MRBs are not directly involved in the methanogenic pathway, some interesting observations related to their potential role in methanogenesis have recently been reported. A recent study observed significant increases in methanogenesis rates from acetate and ethanol by adding conductive iron oxides in batch AD tests using rice paddy soil as inoculum [2]. It was attributed to the stimulated growth of electroactive MRBs likely linked to the development of electric syntrophy between them and methanogens, indicating a potential role of EABs in promoting methanogenesis. Although a few studies have looked at this interesting potential, limited information is so far available on it. This talk will introduce recent research works and findings, including some from the author’s laboratory on the EAB biostimulation and bioaugmentation [3, 4], and discuss the potential of electric syntrophy as a route for improved biomethanation. Keywords : Biomethanation, Conductive iron oxides, Electric syntrophy, Electroactive bacteria, Methanogens References 1. A. Sydow, T. Krieg, F. Mayer, J. Schrader and D. Holtmann, Appl. Microbiol. Biotechnol. 98, 8481 (2014). 2. S. Kato, K. Hashimoto and K. Watanabe, Environ. Microbiol. 14, 1646 (2012). 3. G. Baek, J. Kim and C Lee, Bioresour. Technol. 166, 596 (2014). 4. G. Baek, J. Kim, K. Cho and C Lee, Appl. Microbiol. Biotechnol. In press (2015). B213 Heavy Metal Effects on the Fluorene Biodegradation by a Bacterium Isolated from PAH- contaminated Mine Soil In-Hyun NAM, Chul-Min CHON, Jae-Gon KIM Geologic Environment Division, Korea Institute of Geoscience and Mineral Resources (KIGAM), Daejeon, 305-350, Korea Polycyclic aromatic hydrocarbons (PAHs) are organic compounds with 2 or more benzene rings that are often recalcitrant to biodegradation due to their low aqueous solubilities and high sorption to soils. PAHs are mainly derived from coal processing and the incomplete combustion of petroleum fuels [1-3]. The present study examined Sphingobacterium sp. KM- 02-mediated degradation of fluorene, in the liquid or solid phase, in the presence of 5 common heavy metals, with use of mass spectrometry to identify all metabolic intermediates. A fluorene-degrading strain of Sphingobacterium was isolated from former mining soils that are contaminated with heavy metals and PAHs near a mine-impacted area in Korea. When Sphingobacterium sp. KM-02 was grown in a medium with fluorene as the sole carbon source, it removed 78.4% of this compound within 120 h. Identification of the metabolic intermediates (9-fluorenone, 4-hydroxy-9-fluorenone, and 8- hydroxy-3,4-benzocoumarin) were also performed. Composting experiments under laboratory conditions indicated that this microbe also removed fluorene from contaminated soil. In particular, treatment of microcosm soil with strain KM-02 for 20 days resulted in a 65.6% reduction of fluorene concentration. Experiments of the effects of heavy metals on fluorene removal by strain KM-02 showed that 10 mg/L cadmium, copper, zinc, lead, and reduced growth and fluorene degradation by this microbe. Cadmium and copper had strong effects at 10 mg/L, although zinc and lead had relatively slight inhibitory effect at concentrations of 10 mg/L and 100 mg/L. Arsenic had no effect on growth or fluorene degradation, even at 100 mg/L. This results suggest that strain KM-02, which is the first fluorene-degrading isolate identified from a copper mine- impacted soil, may be a potential candidate for the industrial elimination of PAHs from contaminated soils. Keywords : Heavy metal, Fluorene, Biodegradation , Bacterium References 1. B. Antizar-Ladislao, J. M. Lopez-Real, and A. J. Beck, J. Hazard. Mater. 137, 1583 (2006). 2. M. Banach-Szott, B. Debska, and E. Rosa, J. soils Sediments 14, 1169 (2014). 3. W. Wilcke, Geoderma 141, 157 (2007). B214 Laccase-catalyzed Biotransformation of Biomass-derived Phenolics Jong-Rok JEON College of Agriculture & Life Sciences, Gyeongsang National University Laccases are oxidases that contain several copper atoms, and catalyse single-electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low- molecular-weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into laccase applicability in green synthetic chemistry. Thus, I here review laccase-catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and I discuss how the discovered synthetic strategies mimic laccase-involved in vivo pathways, thus enhancing the green nature of such reactions. Keywords : Laccases, Natural phenols, Oxidative polymerization References 1. Jeon & Chang, Laccase-mediated oxidation of small organics: bifunctional roles for versatile applications, Trends in Biotechnology, 2013, 335-341. 한국생물공학회, 생물공학의 동향 : 2015.10 Recent Advances in Bio-nano/micro Hybridization C207 Use of Core-crosslinked Amphiphilic Polymer Nanoparticles as Nano-carriers for Target-drug Delivery and Oxygen Juyoung KIM1, Nahae KIM1, Nakwon CHOI2 1Dept of Advanced Materials Engineering, Kangwon National University, Gangwon-do 245-711, 2Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Korea Nanoparticles are emerging as a powerful platform for delivery of imaging [1] and therapeutic entities [2], particularly due to their potential applications in cancer therapy for early detection, enhanced therapeutic potency, and reduced side effects [3]. For example, microspheres, liposomes, nanoshells, dendrimers, and biodegradable polymers have been utilized as specific drug delivery systems [4]. Among them, self-assembled polymeric nanoparticles have been attracting a lot of attention because these particles have hydrophobic and hydrophilic segment at the same backbone. Their hydrophobic segments can encapsulate toxic hydrophobic dyes and drug, and hydrophilic segments can provide biocompatibility and water- dispersibility. So these amphiphilic polymer nanoparticles could carry out diagnosis, imaging and therapy simultaneously. However, most of amphiphilic polymer nanoparticles are prepared using amphiphilic polymers or dedrimers which are synthesized through very complicate process and lack in chemical diversity. In addition, their nanostructure formed by physical association (self-assembly) could be easily breakdown under high pressure and shear stress, which cause strong demand for develop physically and chemically robust nanoparticles. In this study, we presented core-crosslinked amphiphilic polymer (CCAP) nanoparticles as a new kind of robust nano-carrier for target drug delivery and oxygen sensing. CCAP nanoparticles could be synthesized using amphiphilic reactive polymer precursors which have hydrophilic polyethylene oxide segment and hydrophobic polypropylene oxide segment having reactive vinyl groups. Through simple suspension polymerization process, amphiphilic reactive polymer precursors could be converted to micelle-like CCAp nanoparticles where hydrophobic cores are chemically crosslinked via reaction between their vinyl groups, which makes CCAP nanoparticles very stable with very harsh conditions. In addition, free OH groups in the surface of CCAP nanoparticles could be conjugated with various chemicals which impart tumor targeting and oxygen sensing performance to CCAP nanoparticles. Lymphocyte function-associated antigen-1 (LFA-1) conjugated CCAP nanoparticles encapsulating hydrophobic cancer drugs could deliver cancer drugs to specific human cancer cells. Also, a ruthenium (Ru)-based phosphor (oxygen-sensitive phosphor) encapsulated within CCAP nanoparticles showed promising oxygen sensing performance in vivo. Keywords : nanoparticles, self-assembly, amphiphilic polymer, target drug delivery, oxygen sensing References 1. T. M. Allen and P. R. Cullis, Science, 303, 1818e22 (2004) 2. R. M. Mainardes, and L. P. Silva LP. Curr Drug Targets 5, 449e55 (2004). 3. M. E. Davis, Z. G. Chen, and D. M. Shin DM, Nat Rev Drug Discov 7, 771e82(2008). C208 Microfluidic Platform for Diagnosis and Others Kyoung G. LEE, Tae Jae LEE, Mon-Keun LEE, Su Jeong SHIN, Nam Ho BAE, Seok O YUN, Seok Jae LEE Department of Nano Bio Research, National Nanofab Center (NNFC), Daejeon 305-806, Republic of Korea Microfluidics can provide convenient and accurate diagnosis tools. In this talk, we present recent developments in microfluidics, with special emphasis on disposable plastic devices. In detail, we overview of the common methods used in the fabrication of polymer microfluidic systems, including replica and injection molding. Also described are the different methods by which on-chip operations—such as the pumping and valving of fluid flow, the mixing of different reagents, and the separation and detection of different biochemical species—have been implemented in a microfluidic format. Finally, a few select biotechnological applications of microfluidics are presented to illustrate both the utility of this technology and its potential for development in the near future. Keywords : Plastic Based Microfluidics, In Vitro Diagnosis, Injection Molding References 1. S. H. Seok et al, ACS Appl. Mater. Interfaces, 7, 4699 (2015) 2. K.G. Lee et al, Lab Chip, 15, 1412 (2015) 3. K.G. Lee et al, RSC Adv., 4, 32876 (2014) 4. K.G. Lee et al, Adv. Mater, 26, 6119 (2014) C209 Vapor-phase deposited functional polymeric thin films for the application to biomedical devices Sung Gap IM Department of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Korea Increased demand for high-end biomedical services prompted the need for development of new biomaterials with versatile material properties. Especially, the surface of the developed materials is of paramount importance, which greatly influence the performance of the biomedical devices. In this seminar, we introduce a new surface modification platform to control the surface of biomaterials using initiated chemical vapor deposition (iCVD) process. iCVD process utilizes very simple machinery with low power consumption. Since the vapor-phase method is performed at ambient temperature, the iCVD polymers are easily applicable to substrate materials vulnerable to liquid phase process and/or heat, such as paper, fabrics, and membranes, without damaging the substrates. Polymeric films can also be easily stacked with sharp interface, free of solvent-related problems. The functionality of polymeric coatings can be easily incorporated by introducing various kinds of monomers. Various chemical modification schemes allow production of new structures and wide tunability of the properties of nano-structures. A deeper insight of materials engineering also enables the development of a novel biomedical devices with various functionalities. With the developed technique, various kinds of biocompatible surfaces will be designed and controlled systemically, and their applications to various biomedical devices will also be reviewed. Keywords : Polymer thin films, surface chemistry, biocompatible surfaces, initiated chemical vapor deposition (iCVD), surface functionalization References 1. Hyun-Ji Park†, Seung Jung Yu†, Kisuk Yang, Yoonhee Jin, Ann-Na Cho, Jin Kim, Bora Lee, Hee Seok Yang, Sung Gap Im*, Seung-Woo Cho*, Biomaterials, 2014, 35 (37), 9811–9823 2. Kisuk Yang, Hyunjung Jung, Hak Rae Lee, Jong Seung Lee, Su Ran Kim, Ki Yeong Song, Eunji Cheong, Joona Bang*, Sung Gap Im*, and Seung-Woo Cho*, ACS Nano, 2014, 8(8), 7809-7822 3. Gu Min Jeong†, Hyejeong Seong†, Yong-Sung Kim, Sung-Gap Im*, and Ki Jun Jeong*, Polymer Chemistry, 2014, 5, 4459-4465. 4. Mun-Jung Kim†, Bora Lee†, Kisuk Yang, Junyong Park, Seokwoo Jeon, Soong Ho Um, Dong-Ik Kim, Sung Gap Im*, and Seung-Woo Cho*, Biomaterials, 2013, 34 (30), 7236-7246 5. Bora Lee, Alex Jiao, Seungjung Yu, Jae Bem You, Deok-Ho Kim*, and Sung Gap Im*, Acta Biomaterialia, 2013, 9 (8), 7691-7698 C210 Accelerated Induction of Drug Resistance in Cancer Galaxy Chip Sungsu PARK School of Mechanical Engineering, Sungkyunkwan University Suwon, Korea Glioblastoma multiforme (GBM) is the most common and aggressive primary tumor in humans. Emergence of drug resistance by tumor cell evolution is the most critical aspect of cancer treatment. Using microfluidic cell culture platform [1, 2], we have been developing ‘Cancer Galaxy Chip’ in order to understand how cancer cells evolve resistance to anticancer drugs. Cancer Galaxy Chip contains about five hundred interconnected hexagonal microchambers surrounded by perfusion microchannels. When GBM cells (U-87), a type of brain tumor, were grown under a gradient of doxorubicin and nutrient on Cancer Galaxy Chip for 3 days, cells survived only in the regions of low concentration of doxorubicin in the chip. At day 5, however, some cells gradually migrated into the regions of high concentrations of the drug, showing drug resistance to doxorubicin. Surprisingly, these resistant cells displayed about 30 times higher resistance to doxorubicin than its parental wild type U-87 cells. Exome sequencing of original and resistant cell lines identified 61 nonsynonymous mutations as candidate drivers for resistance development, while RNA sequencing reveals unexpected expression of certain genes related to drug detoxification. Mutations and differentially expressed genes were in excellent agreement with the known action mechanism of doxorubicin according to gene ontology and pathway analyses. Consequences of loss-of-function variants were verified by siRNA knockdown experiments. Our results suggest that Cancer Galaxy Chip [3] is an alternative to existing 2D culture models for cell biologists as well as medical researchers who are interested in understanding cancer resistance. Keywords : Evolution, cancer resistance, brain tumor, mutations References 1. S. M. Ong, C. Zhang, Y. -C. Toh, S. H. Kim, H. L. Foo, C. H. Tan, D. V. Noort, S. Park, H. Yu* Biomaterials 2008, 29(22), 3237-3244 2. J. Choi, S. Kim, J. Jung, Y. Lim, K. Kang, S. Park*, S. Kang* Biomaterials 2011, 32, 7013-7022. 3. Q. Zhang Q, G. Lambert, D. Liao, H. Kim, K. Robin, C. K. Tung, N. Pourmand, R. H. Austin . Science 2011, 333, 1764-1767 C211 Bioelectronic Nose based on Olfactory Receptor Proteins and Carbon Nanotubes Seunghun HONG Department of Physics, Institute of Applied Physics, Seoul National University, Seoul 151-747, Korea Extensive efforts have been given to develop artificial sensory devices which can imitate the responses of human noses and tongues using solid state electronics. However, such devices based on solid state electronics are usually inferior to human sensory systems in terms of its sensitivity and selectivity. In human olfactory systems, olfactory receptor molecules can selectively bind to specific odorant molecules, which allows humans to distinguish specific smells with a high sensitivity. In our work, we coated olfactory or taste receptors on carbon nanotube-based transistors to build bio-electronic noses or tongues which can imitate human sensory systems, respectively. In this device, when specific molecules bind selectively to the receptor molecules, charges are generated in the receptor molecules and alter the conductance of the underlyng carbon nanotube devices. Thus, one can selectively detect odorant or taste molecules simply by monitoring the currents in the underlying carbon nanotube devices. In this presentation, we will discuss the bioelectronic nose and tongues based on carbon nanotube devices and receptor proteins. Future prospect and possible applications of these devices also will be discussed. Keywords : bioelectronic nose, olfactory receptor, carbon nanotube, transistors References 1. T. H. Kim et.al., Single-Carbon-Atomic-Resolution Detection of Odorant Molecules using a Human Olfactory Receptor-based Bioelectronic Nose (2009), Advanced Materials, 21, 91-94. 한국생물공학회, 생물공학의 동향 : 2015.10 Biotecchnologyg Meets Cosmetics C212 A Synthetic C16 Omega-hydroxyceramide Restored Skin Barrier Function upon Perturbations of Epidermal Homeostasis Myoung Jin OH1, Jin Ju NAM1, Eun Ok LEE2, Jin Wook KIM2, Chang Seo PARK1 1Department of Chemical and Biochemical Engineering, Dongguk University, 3-26, Pil-dong, Chung-gu, Seoul, 100-715, Republic of Korea , 2LCS Biotech, Seodun-dong 103-2, Gwonseon-gu, Suwon-si, Gyeonggi-do, 441-857, Republic of Korea Omega-hydroxyceramides (w-OH-Cer) are the principal lipid components of the corneocyte lipid envelope placed on the extracellular surface of the cornified envelope and covalently attached to the outer surface of the CE linked to involucrin. w- OH-Cer role in epidermal barrier function and maintain the cohesion of corneocytes. In a previous study, we demonstrated that topical application of a synthetic C16 w-OH-Cer not only accelerated the rate of epidermal barrier restoration but also it could strengthen stratum corneum cohesion and integrity. Also, in a high calcium condition (0.6mM of calcium chloride), we identified a synthetic C16 w-OH-Cer treatment up-regulated expressions of keratinocyte differentiation marker proteins such as involucrin and transglutaminase-1 (TGase-1) by immuno blot assay. Ultraviolet-B (UVB) irradiation causes various adverse effects on skin. It is reported that decreased levels of w-OH-Cer is resulted from the UVB-exposed damage of skin barrier through reduction of covalently bound ceramides which bind to involucrin and decreased covalently bound ceramide levels affect the result of lowered value of transepidermal water loss (TEWL). Recently, it is discovered that UVB- irradiation affects down-regulated expressions of keratinocyte differentiation marker proteins and desmoglein-1 (DSG-1) which is a member of desmosomal cadherins and promotes keratinocyte differentiation. In epidermis, DSG-1 holds keratin intermediate filaments to the cell membrane and supports intercellular adhesion in desmosomes in order to form intact morphogenesis. In this study, epidermal barrier recovering effects of a synthetic C16 w-OH-Cer on UVB-induced perturbation of skin barrier were investigated. Followed by UVB-irradiation, skin barrier recovery aspects through increased expressions of keratinocyte differentiation and desmosomal component were assessed in cultured normal human keratinocytes. From the results, a synthesized C16 w-OH-Cer showed significant protective effect on epidermal barrier recovery against UVB-irradiation. C213 Production of high-functional Biooil and Biochemical Sun-Yeon HEO1, Jeong-Hyun JU1, Baek-rock OH1, Min-Su KWAK2, Min-Sung PARK2, Seung-Moon PARK3, Jeong-Woo SEO1, Won-Kyung HONG3 1Biorefinery Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup-si, Jeonbuk 580-185, Korea, 2Advanced Biomass R&D Center, Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea, 3Division of Biotechnology, College of Environmental & Bioresource Science, Chonbuk National Univ. Iksan Campus, Jeonbuk 570-752, Korea Oleaginous heterotrophic microalgae Thraustochytrids is a single cell oil producer which can accumulate high level of oil in the cells about 30 to 70% of dry cell weight and the oil contain high level of omega-3 polyunsaturated fatty acids such as DHA over 50% of total fatty acids. . Thraustochytrids are marine heterotrophic protists that have been extensively studied owing to their ability to accumulate health-beneficial lipids, especially omega-3 polyunsaturated fatty acids (PuFAs) such as docosahexaenoic acid (DHA, C22:6), which has the serological and cardiovascular benefits and the positive influence on retinal and brain development. Because microalgae contain high levels of fatty acids, their oil is valuable as a feedstock for the production of biofuels such as biodiesel through thermo-chemical or biological approaches. The major commercial source of DHA is fish oil, which faces challenges such as heavy metal (e.g., mercury) contamination, poor oxidative stability, and limited supply. In addition, fish oil is not suitable for vegetarians and its odor makes it unattractive. Microalgal oil have many advantages, it can be controlled by optimized process in defined quality. Also, it is not so mixture of pufa. So it saved the cost for purification. Finally it is clean. It is safe from environmental contamination like a dioxin, heavy metals. In most eukaryotic cells including thraustochytrid microalgae, the biosynthesis of unsaturated fatty acids is majorly driven by iterative reactions catalyzed by a set of fatty acyl desaturase and elongase enzymes. Additionally, a polyketide synthase- like protein complex (called PuFA synthase) was evolved to synthesize PuFAs by Thraustochytrid microglage. We are currently focusing on genetic and metabolic engineering of Thraustochytrids microalgae to improve the yield and composition of fatty acids. The results on genetic engineering based on the genome sequence are introduced. Also, Thraustochytrid Microalgae was cultured in pilot scale fermentor successfully. The 10.8 g/L day oil productivity were showed using glucose as carbon source, it is the highly competitive result in 6-ton air-lift type. And, additionally we succeed in cultivation of the microalgae in 34 ton bioreactor. In summary, production of hogh-functional biooil by heterologous thraustochytrud microalgae and and utilization of the oil component to synthesis of biochemicals. This would be have a potential as a model system of integrted biorefinery. Keywords : microalgae Thraustochytrids, polyunsaturated fatty acids (PuFAs), biochemical, integrted biorefinery References 1. W. K. Hong et al, Characterization of a Squalene Synthase from the Thraustochytrid Microalga Aurantiochytrium sp. KRS101, J Micribiol Biotechnol (2013), 23(6), 759 2. W. K. Hong et al, A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance, Bioprocess Biosyst Eng, (2013), 36(9), 1191 3. W. K. Hong et al, Large-Scale Production of Microalgal Lipids Containing High Levels of Docosahexaenoic Acid upon Fermentation of Aurantiochytrium sp. KRS101, Food and Nutrition Sciences 4, (2013), 9A, 1-5. 4. W. K. Hong et al, Production of lipids containing high levels of docosahexaenoic acid from empty palm fruit bunches by Aurantiochytrium sp. KRS101, Bioprocess Biosyst Eng, (2013), 36(7), 959 5. W. K. Hong et al, Growth of the oleaginous microalga Aurantiochytrium sp. KRS101 on cellulosic biomass and the production of lipids containing high levels of docosahexaenoic acid, Bioprocess Biosyst Eng, (2012), 35(1-2), 129 6. W. K. Hong et al, Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101, Appl Biochem Biotechnol, (2011), 164(8), 1468 C214 Cosmetics and Metabolic Engineering Meet Metabolomics Baek-Seok LEE Research Institute of Biotechnology, CJ CheilJedang, Seoul, 157-724 As our bodies age, many aspects of our metabolism also were changed1. Cellular damage and aging are associated with changes in amino acid, lipid and energy metabolites, antioxidant levels, detoxification reactions and nutrient status. Metabolomics has been introduced to explore this complex relationship and to investigate the role that bioactives used in consumer products may play in skin care, health, and disease2,3. Industrial fermentation processes are continuously being improved using a combination of strain improvement and bioprocess optimization4. Currently target selection, i.e. the identification of bottle-necks and bioprocess parameters that affect production yield and productivity, is the rate-limiting step in bioprocess optimization. With the recently introduced metabolomics technology it is now possible to compare wild-type's metabolic profile with overproduction strain’s and the metabolic profile of low production phase with high production phase. I have successfully applied this metabolomics platform for selecting targets for improvement of product yield and productivity based on different metabolic profile. As the biochemical level of the metabolome is closest to that of the phenotype (i.e. productivity, yield), metabolomics is the most relevant functional genomics tool for understanding biological production. By calculating the correlation between the large numbers of metabolites measured and the production outcome under different conditions, metabolomics enables the identification of bottlenecks in the production strain. After biological interpretation of the results, the genetic and bioprocess targets are identified. CJ has successfully applied this approach for identifying targets in metabolite production in bacteria. The targets have been validated and large improvements in yield and productivity of amino acids and nucleotides were achieved in these improved strains and fermentation optimization. Keywords : Metabolomics, Metabolic engineering, Cosmetics, Strain improvement, Bioprocess optimization References 1. Lawton KA, Berger A, Mitchell M, Milgram KE, Evans AM, Guo L, Hanson RW, Kalhan SC, Ryals JA, Milburn MV, Analysis of the adult human plasma metabolome (2008), Pharmacogenomics. 9, 383-397 2. Barnes VM, Teles R, Trivedi HM, Devizio W, Xu T, Lee DP, Mitchell MW, Wulff JE, Milburn MV, Guo L, Assessment of the effects of dentifrice on periodontal disease biomarkers in gingival crevicular fluid (2010), J. Periodontol. 81, 1273-1279 3. Barnes VM, Teles R, Trivedi HM, Devizio W, Xu T, Mitchell MW, Milburn MV, Guo L, Acceleration of purine degradation by periodontal diseases (2009), J. Dent. Res. 88, 851-855 4. Hollinshead W, He L, Tang YJ, Biofuel production: an odyssey from metabolic engineering to fermentation scale- up (2014), Front. Microbiol. 5, 1-8 C215 Characteristics of HPMC as a Potential Semi-synthetic Polymer in Cosmetic Fields Joo Yung JUNG1,2, Sung Hwan BANG1, Ju Hee SHIN1, Ji Seon JUNG1, Eun Kee KIM2 1Food & Pharm materials Development Team, Samsung Fine Chemical Co. Ltd., Incheon, Korea, 2Laboratory of Bioactive Materials, Dept. of Biological Engineering, Inha University, Incheon, Korea HPMC(Hydroxypropyl methylcellulose) can be soluble in mixed organic or aqueous solvent system and make the transparent film when solvent is removed. Generally, there is no chemical reaction with other active ingredients due to its non-ionic property and its hydrophilic matrix used along with HPMC hydrates to create a gel layer controlling the release profile of entrapped active ingredients. Therefore, it is widely used in parmaceutical industries for DDS(Drug Delivery System) to control the drug release pattern in wide range of pH. Once soluble in water and volatilized through solvent, HPMC can make transparent film with high tensile strength. It can also enhance binding properties and exponentially increase the solution viscosity in relation to the concentration. In csmetic fields, HPMC can be used as thickening agent, film forming agent, bio-adhesiveness enhancer and so on. It can also form the hydrogel matrix as it is combined with the certain biopolymers containing some of the gums. The hydrogel is widely used in cosmetic fields as a basic carrier for diverse active ingredients. In this study, we observed the thickening properties in aqueous solution systems, the chemical structure analyses through FTIR and XRD studies, the morphological properties using SEM analysis and the diverse applied properties such as film and gel strength and surficial adhesive force, etc. As HPMC is one of the crucial viscoelastic and bio-adhesive polymers used in various types of cosmetic binding applications and drug formulations, these studies open a new way in the area of novel cosmetic design including cosmeceutical fields. Keywords : HPMC, soluble cellulose, hydrogel, cosmeceutical References 1. J. Siepmann and N.A. Peppas, Advanced Drug Delivery Reviews 64, 163–174 (2012) 2. Eman S. El-Leithy, Dalia S. Shaker, Mohamed K. Ghorab and Rania S. Abdel-Rashid,AAPS PharmSciTech, Vol.11, No.4(2010) C216 Cosmetic Ingredient Research by Using a Production Method of Korean Traditional Rice Wine Chang Sung HAN Biotechnology R&D center, Bioland, Chungbuk, 363-951 In this research, a cosmetic ingredient was developed by using a production method of Korean traditional rice wine and its availability was proposed. After "Nuruk"(wheat) was produced by a traditional method, it was applied in a steamed rice for fermentation. Through a filtration and concentration process after fermentation, production metabolite(SENAS) was obtained. Aquaporin-3 gene expression[1] was tested to confirm an availability as a cosmetic ingredient of SENAS. The test result showed that Aquaporin-3 gene expression increased 2.5 times more than a control at SENAS 500ug/mL. Accordingly, SENAS was confirmed as an effective moisturizing ingredient. For the skin whitening effect, though SENAS itself showed less effective test result than a positive control(ARBUTIN), the skin whitening effect(melanogenesis inhibition effect) of ARBUIN increased 1.2-2 times by using SENAS together. The same test result was also detected in the test of oilsoluble licorice extract, natural origin skin whitening cosmetic ingredient. Accordingly, SENAS was confirmed to be available as a skin whitening effect booster. Based on those positive in-vitro test results, a clinical trial for skin brightness, even skin tone, skin translucency and skin gloss was conducted by a research center(Ellead Skin & Bio Research) on 20 women(age between 32-55). The test result showed statistically significant effect(p<0.05) so that SENAS was confirmed as an effective cosmetic ingredient for moisturizing and improving skin tone. Keywords : cosmetic ingredient , moisturizing , whitening , fermentation References 1. Tonghui Ma, Mariko Hara, Rachid Sougrat_, Jean-Marc Verbavatz_, and A. S. Verkman, Impaired Stratum Corneum Hydration in Mice Lacking Epidermal Water Channel Aquaporin-3(2002)J. Biol. Chem., Vol. 277, No. 19, pp. 17147–17153 C217 “Beauty” and Functional Food Young-Bum KIM, Sung Yong PARK, Gil-Yeon YOO, Mikyoung YOON IPON Co., Ltd., Daejeon 302-686, Korea Beauty is what most people are interestedregardless of the east and west, age, and sex, and country.A standard of external beauty varies depending on personal views. Therefore, various products and services and various functions have been demanded. The range of “beauty care” may cover cosmetics, diet, plastic surgery, etc. In recent years, the inner beauty market, i.e., the beauty food product market, has evolved and its related technology has been actively developed.Representatively, an individual recognition type functional ingredient of health/functional foods is beingdeveloped and utilized as various products, and also, used incosmetics for application to the skin to satisfy the consumers’ needs for beauty. However, the functionality of functional foods is tomaintain and improve a state of health bymaintaining normal functions or activating physiologic functionsin the human bodyrather than to directly treat or prevent diseases like medicines, and may include “nutrient function”, “reduction of disease risk”, and “physiologic activity”. The functional foods are limited in efficacy, but high in stability and continuous availability. Thus, it seems to be necessary to develop and commercialize the related technology. In the present study, we will discussa direction of raising a value of the inner beauty industry and commercializationthereof on the basis of the analysis ofdomesticand overseas markets, patents, technologies, and industry trends. Keywords : Beauty, Functional Food, Inner Beauty, 3P analysis References 1. Soo Chang Kim et al.,「Trend Reporton Overseas Health/Functional FoodIndustry」(2015) 한국생물공학회, 생물공학의 동향 : 2015.10 최신연구동향 심포지엄 (정책특강 & 신진연구자 발표회) D206 The Samsung Research Funding Program for Future Technology Hyun-Joo KIM Samsung Research Funding Center for Future Technology, Seoul 137-965, Korea Samsung has been maintaining various contribution programs with a belief in shared responsibility to our society. In August 2013, Samsung launched USD 1.5 billion 10-year grant programs to support creative and innovative R&Ds in the areas of Basic Science, Novel Materials and ICT Convergence. The programs aim to contribute to catalyzing creative and challenging R&Ds and building the infrastructure of technology innovation for the benefit of the broader society and economy, through supporting R&Ds at academia, public research institutes and R&D centers of small business in Korea. 1. The introduction of the research funding program and business philosophy 2. The proposal review process 3. The guideline to prepare the proposal Keywords : Research Funding, Basic Science, Novel Materials, ICT Convergence D207 Time Series Data Break-Detection Methods Adopted for Bio-Related Intellectual Property Policy Setup BeomYong KIM1, HyUP YANG1,2,3 1IP Promotion Programs, KNU, Samcheok-si Kangwondo KOREA , 2Dept. of Vehicle Engineering, KNU, Samcheok-si Kangwondo KOREA , 3Dept. of Mechanical Design Engineering, KNU, Samcheok-si KOREA On July 1987 Korea adopted the『composition of matter』(material) patent system, through which bio-technologies can get patents. The above system have caused both of patent-applied numbers and patent-registered numbers to be drastically increased since 1987. Domestic people have enjoyed the above adoption since 1987 but foreigners have after 1993. Foreigners applied earlier but took results later, and the domestic people did contrariwise. These event effects are proved thru statistics. The traditional CHOW test requires the iid condition. The structural change method of Torres-Reyna(2014) and Stock & Watson(2012) has been developed to be the iteratively moving QLR(Quandt Likelihood Ratio) test of Richard Quandt(1960). Three Steps for Proving Event Effects are as follows. 1st. Lag-Order Selection for Level Data or Log Data Using the stata command “varsoc”, we can select the lag-orders for the level data themselves or the natural log data of the level data. We may have to use the difference data from the above data. The selection standards are FPE, AIC, HQIC, SBIC. Balcombe, K. G. et al(2011) accept the lag order of up to 3. 2nd. Heteroskedasticity Tests for Models Using the stata command “cusum6”, we can select the appropriate & final model from the above candidate models. The selection standards are the cumulative sums(CUSUM) of the recursive residuals and their squares from the regression specified by the above models. 3rd. QLR Test for Finding Breaks Mitchell(2014) insists that in applying the general regression to the time series data we tend to exaggerate the both ends and so should adopt the centered 70% range. The dummy variable “di” indicates the point of the structural change(break). The multiplications of the dummy variable “di” and independent variables of the above final model are required for the above QLR test coding contents. Keywords : BIO(Material), Patent, Policy, Event References 1. Balcombe, Kelvin G., Fraser, Iain and Sharma, Abhijit , Bayesian model averaging and identification of structural breaks in time series, Applied Economics, 43, 3805–3818, 2011 2. Mitchell, Sara McLaughlin, Time Series Analysis: Method and Substance, Introductory Workshop on Time Series Analysis, Department of Political Science University of Iowa, p.16, 2014 3. Stock, James H & Mark M. Watson, Introduction to Econometrics, 3rd edition, Global Edition, Boston: Pearson Addison Wesley, pp. 598~602, 2012 4. Torres-Reyna, Oscar, Time Series(ver. 1.5), Princeton University, Data Consultant, 2014 D208 Cytoskeletal Forces in the Subcellular Machinery: Bridging between Bioengineering and Mechanobiology Dong-Hwee KIM KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Korea Accumulating evidence suggests that the three-dimensional organization of the nucleus regulates gene expression through lamina-chromosome interactions. The nuclear lamina is a thin filamentous meshwork that provides mechanical support to the nucleus and regulates essential cellular processes such as DNA replication, chromatin organization, cell division, and differentiation. Imaging using fluorescence and electron microscopy has long suggested that the nuclear lamina is composed of structurally different intermediate filamentous lamin proteins and nuclear lamin-associated membrane proteins that together form a thin shell largely confined to a narrow region underneath the nuclear envelope with a few filamentous structures extending to the intranuclear space. Moreover, chromosomes are known to be condensed differently along the radial direction from the periphery of the nucleus to the nuclear center; therefore, chromatin accessibility for gene expression is different along the nuclear radius. However, close comparison of confocal sections along the vertical axis of the nucleus indicates that the major lamin protein lamin A/C is dominantly localized at the apical and lateral surfaces of the nucleus, and largely absent from its basal section. Here we show that both A-type lamins and transcriptionally active chromatins are vertically polarized by the tension exercised by the perinuclear actin cap that is composed of highly contractile actomyosin fibers organized at the apical surface of the nucleus. Keywords : Cell mechanics, Bioengineering, Mechanosensation, Cytoskeleton, Nuclear mechanics References 1. Dong-Hwee Kim, et al., Volume regulation and shape bifurcation in the eukaryotic nucleus. J. Cell Sci., 2015, doi: 10.1242/jcs.166330. 2. Dong-Hwee Kim and Denis Wirtz., Cytoskeletal tension induces the polarized architecture of the nucleus. Biomaterials, 2015, 48, 161. 3. Dong-Hwee Kim, et al., Tight coupling between nucleus and cell migration through the perinuclear actin cap. J. Cell Sci., 2014, 127, 2528. 4. Dong-Hwee Kim, et al., The multi-faceted role of the actin cap in cellular mechanosensation and mechanotransduction. Soft Matter, 2013, 9, 5516. 5. Dong-Hwee Kim and Denis Wirtz, Focal adhesion size uniquely predicts cell migration. FASEB J., 2013, 27, 1351. D209 Graphene Oxide Combinatorial Patterns to Control Differentiation of Human Adipose-derived Mesenchymal Stem Cells Tae-Hyung KIM School of Integrative Technology, Chung Ang University, Seoul 156-756, Korea Stem cell engineering is a highly promising field that may provide possible solutions to cure a number of incurable diseases/disorders. Among many different stem cell-related researches, controlling stem cell fate into specific lineages is incredibly important for practical applications of stem cells, however, also has been a major barrier for the advent of stem cell-based therapies. Recently, the use of nanomaterials, including carbon-based materials such as graphene or graphene oxide, has emerged as an attractive approach that enables controlling stem cell behaviors including differentiation. Here, we report a novel method of generating combinatorial patterns of graphene oxide (GO) hybrid materials that is capable of guiding differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) into specific lineages (e.g. osteogenesis and neurogenesis) in highly efficient manner. GO micropatterns with different sizes and shapes were fabricated on any kind of biocompatible substrate including silicon-gold, tissue culture plate, polydimethylsiloxane (PDMS) and even poly(lactic-co-glycolic acid). Remarkably, the GO micropatterns combined with cell repulsive material were highly effective to modulate the morphologies of hADMSCs, which resulted in enhanced differentiation of hADMSCs into osteoblasts (GO line patterns) and neuron-like cells (GO-laminin hybrid grid patterns). Both conversion efficiencies of hADMSCs into osteoblasts and neurons were found to be 54.5% and 30% higher than control groups, respectively, which are hard to be achieved using conventional GO- or graphene-coated substrates. Since the GO patterns generated on different types of substrates were highly stable for long period of time, this approach can be utilized for the mass production of various kinds of patient-specific cells using GO-patterned tissue culture plates and biodegradable hydrogels. Additionally, since biophysical cues, such as elasticity, porosity and surface topography of the substrates are known to be key factors for steering the differentiation of stem cells into specific lineages via mechanotransductive pathways, the strategy developed here can be further applied to generate GO combinatorial patterns on elasticity-controlled biodegradable substrates, which could synergistically control stem cell differentiation in a more efficient manner. Hence, the platform introduced in this report can serve as a powerful tool for the development of cell type- and patient-specific stem cell-based regenerative therapies. Keywords : Adipose-derived stem cells, Differentiation, Graphene arrays, Cell morphology, Combinatorial pattern References 1. Paolo Bianco and Pamela G. Robey, Stem cells in tissue engineering (2001), Nature, 414, 118-121. 2. Matthew J. Dalby, Nikolaj Gadegaard and Richard O. C. Oreffo, Harnessing nanotopography and integrin–matrix interactions to influence stem cell fate (2011), Nature Materials, 13, 558-569. 3. Tapas R. Nayak, Henrik Andersen, Venkata S. Makam, Clement Khaw, Sukang Bae, Xiangfan Xu, Pui-Lai R. Ee, Jong-Hyun Ahn, Byung Hee Hong, Giorgia Pastorin and Barbaros Ozyilmaz, Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells (2011), ACS Nano, 5, 4670-4678. 4. Tae-Hyung Kim, Shreyas Shah, Letao Yang, Perry T. Yin, Md. K. Hossain, Brian Conley, Jeong-Woo Choi and Ki-Bum Lee, Controlling differentiation of adipose-derived stem cells using combinatorial graphene hybrid- pattern arrays (2015), 9, 3780-3790. 5. Adam J. Engler, Shamik Sen, H. L. Sweeney and Dennis E. Discher, Matrix elasticity directs stem cell lineage specification (2006), 126, 677-689. D210 A Novel Molecular Diagnostic Device for Genetic and Epigenetic Analysis in Human Diseases Yong SHIN Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympicro-43gil, Songpa-gu, Republic of Korea Medical device, especially molecular diagnostic, is defined to make immediate and informed decisions about patient for earlier diagnosis and disease management in primary care, which leads potentially to improved patient's management and outcome, cost-effectiveness, and reducing health inequalities. Diagnostic devices incorporate emerging techniques including silicon bio-photonic sensors, electrodes, and solid phase reagents that enable rapid assay reaction, reducing sample and reagent volumes, ease of use, and less technical skill. Recently, we have developed two novel techniques; a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure as a DNA extraction technique without centrifugation step and an isothermal solid-phase amplification/detection (ISAD) technique for the detection of biomarkers in human diseases. The DTS is useful for the extraction of DNA from a variety of sources, including cells, bacteria, blood, and urine in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis. In addition, the ISAD can be performed without labelling in real-time by utilizing both silicon microring-based solid-phase amplification and isothermal recombinase polymerase amplification (RPA) within 20 min. We demonstrated that the sensitivity of the ISAD technique was 100-times higher than that of RPA and conventional PCR methods. Therefore, an integrated device of two novel techniques will be useful for analysis of genetic and epigenetic alteration of human disease related biomarkers with simplicity, rapidity, and low-cost. Keywords : Molecular Diagnostic, Genetic, Epigenetic, Isothermal Solid-Phase Amplification/Detection References 1. Shin Y, Perera AP, Kim KW, Park MK. Lab on a Chip, 13, 2106-2114, 2013. 2. Shin Y, Perera AP, Wong CC, Park MK. Lab on a Chip, 14, 359-368, 2014. 3. Yoon JY, Park MK, Lee TY, Yoon YJ, Shin Y. Lab on a Chip. 15, 3530-3539, 2015 4. Liu Q, Lim SY, Park MK, Shin Y. Biosensors and Bioelectronics. 74, 865-871, 2015 D211 Direct Reprogramming of Primary Skin Fibroblasts into Functional Neuronal Cells using Polymer Nanoparticles Yoonhee JIN, Seung-Woo CHO Dept. of Biotechnology, Yonsei University, Soul, 120-749 Defined sets of transcription factors can directly reprogram differentiated cells into another linage without passing through a pluripotent stem cell-like state. Direct conversion of fibroblasts into functional induced neuronal cells can be achieved through genetic modification of three developmental transcriptional factors. Subsequent successful reports improvements on the conversion efficiency and production of specific neuronal subtypes have rapidly expanded this field, but so far most studies have replied on using viral vectors to deliver the transcriptional factor plasmids. Viral vectors are widely being used due to their high gene transfection efficiencies, but they are related to safety issues, and thus limits the scope of their potential clinical applications. Here, we report a novel method for reprogramming primary mouse fibroblasts directly into neuronal-like cells using non-viral vectors and applying exogenous stimulation and controlling microenvironment to provide more favorable environment for neuronal maturation. As a result, the efficiency of conversion showed comparable rate as the method delivered by viral vectors and the time required for generating induced neuronal cells was significantly reduced. These induced neuronal cells uniformly displayed morphological and molecular features of neuronal cells, and exhibited electrophysiological functions. Our non-viral direct conversion approach, with low cytotoxicity and high efficiency, may endorse the prospect of autologous cell therapy for neurodegenerative disorder. This work was supported by a grant (HI13C1479) from the Korea Health Technology R&D Project funded by the Ministry of Health and Welfare, Republic of Korea. Keywords : Direct reprogramming, nonviral gene delivery, induced neuronal cells, Regenerative medicine References 1. Vierbuchen et al., Direct conversion of fibroblasts to functional neurons by defined factors (2010), 142(3), 375- 386 2. Adler et al., Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells (2012), e32. doi: 10.1038/mtna.2012.25. D212 The Paradigm Shift of Research and Product Development in Cosmetics Industry Beom SHIM Skincare Research Institute, AMOREPACIFIC R&D Center, Yongin, Korea 화장품의 역사는 인류의 역사와 유사할 정도로 인간과 밀접한 관계를 가지며 발전해왔다. 현대적인 화장품의 발전은 20 세기 초부터 본격적으로 발전했다고 얘기할 수 있지만, 최근 20 여 년 동안 급격하게 발전을 이루어왔으며, 첨단 기술을 가장 적극적으로 활용하는 산업으로 부상하게 되었다. 화장품 연구는 피부의 다양한 문제에 대한 기초연구에서 출발하여 메커니즘을 규명하고, 이를 해결할 수 있는 활성 성분을 개발하고, 이를 제형 내에서 생리활성 안정성 및 인체에 대한 안전성을 확보하고, 다양한 임상을 통한 효능을 밝힘으로써 제품화를 가능하게 한다. 이런 전반적인 연구에 있어 전통적인 방식의 피부 기초 연구, 소재 개발 연구, 안정성 및 안전성 연구, 임상 연구가 Genetics 및 Proteomics 와 Bioinformatics 를 통한 메커니즘 규명, 제약산업에서 활용하는 신약개발 방식으로 신규 화장품 효능성분 개발, 동물실험을 대체하는 인공피부 실험 모델을 활용한 안전성 연구 등의 새로운 연구개발 방식으로 급격한 패러다임 전환이 이루어지고 있다. 최근 활발하게 이루어지고 있는 화장품 산업에서의 연구개발 패러다임 전환의 배경과 현황에 대해서 설명하고, 이를 통해 얻을 수 있는 연구개발 기회 및 인사이트에 대해서 의견을 제시하고자 한다. Keywords : Cosmetics, research paradigm shift, qqqqq D213 Developing Artificial Cell Membrane: Soft Nanoscience Minsub CHUNG Department of Chemical Engineering, Hongik University, Seoul 121-791, Korea Many cellular processes including cell-cell communications and regulated membrane transport are mediated by membrane proteins and depend upon the ability of lipid membranes to be a differentially permeable barrier. However, the roles and function of membrane proteins are often difficult to study due to the complexity of the native membranes and lack of reliable and flexible artificial model lipid membranes. We have developed new model membrane architecture, the DNA-tethered lipid bilayer. This separates the lipid membranes from surface interactions and provides a more favorable environment for integral membrane protein with large globular domains. The mobile DNA tethers between a tethered and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions. By careful choice of composition, improved stability was obtained and we can investigate the lateral segregation of DNA hybrids when different lengths are present. Based on a theoretical model, the effects of population, length and affinity of DNA complexes are simulated and described. This model system captures some of the essential physics of synapse formation and is a step toward understanding lipid membrane behavior in a cell-to-cell junction. To allow greater versatility and control, we sought a way to pattern tethered membrane patches. We present a method for creating spatially distinct tethered membrane patches on a glass slide using microarray printing. Surface-reactive DNA sequences are spotted onto the slide, incubated to covalently link the DNA to the surface, and DNA-GUVs patches are formed selectively on the printed DNA. By interfacing the bilayers with microfluidic flow cells, materials can be added on top of or fused into the membrane to change the composition of the bilayers. With further development, this approach would enable rapid screening of different patches in protein binding assays and would enable interfacing patches with electrical detectors. Keywords : lipid, cell membrane, DNA, pattern D214 Engineered Microfluidic Systems for High-throughput Analysis and Sensitive Detection of Biomolecules Hyewon LEE Korea Research Institute Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea High-throughput analysis of molecular information (DNA, RNA and proteins) from biological systems is increasingly important in clinical diagnostics, therapeutics and throughout bioengineering. However, traditional methods often limit applications due to the time-consuming and qualitative nature of phenotyping organisms. The recent development of microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Previously, I applied user-friendly microsystems and analytical tools to identify the regulatory mechanism in tryptophan hydroxylase (tph- 1), a key enzyme for serotonin synthesis in nematode Caenorhabditis elegans (C. elegans). I screened and identified 43 putative mutants using clonal imaging and statistical analysis in less than 25 hours, which would have taken substantially longer (many days or possibly months) using traditional methods. In addition, I demonstrated a versatile hydrogel-based microfluidic approach and novel amplification scheme for entirely on-chip, sensitive, and highly specific miRNA detection without risk of sequence bias. This system provides at least one order of magnitude better sensitivity than competing on-chip assays while allowing multiplexing, and quantification of miRNAs from low amounts (about 10.3 ng) of total RNA sample was demonstrated in a 2 hour and 15 minute assay. I anticipate that the results presented here will allow rapid and reliable clinical profiling, which can be further extended to cell analysis, drug screening, and the detection of any other important biomolecule. Keywords : diagnostics, hydrogels, microfluidics, high-throughput screening (HTS) technology References 1. Hyewon Lee, Rathi L. Srinivas, Ankur Gupta and Patrick S. Doyle, Sensitive and multiplexed on-chip microRNA profiling in oil-isolated hydrogel chambers (2015), Angewandte Chemie International Edition, 54(8), 2477–2481 2. Hyewon Lee, Rathi L. Srinivas, Ankur Gupta and Patrick S. Doyle, Sensitive and multiplexed on-chip microRNA profiling in oil-isolated hydrogel chambers (2015), Angewandte Chemie International Edition, 54(8), 2477–2481 3. Hyewon Lee, Shin Ae Kim, Sean Coakley, Paula Mugno, Marc Hammarlund, Massimo A. Hilliard and Hang Lu, A multi-channel device for high-density target-selective stimulation and long-term monitoring of cells and subcellular features in C. elegans (2014), Lab chip, 14(23), 4513-4522 D215 Fabrication of RNA-Quantum Dot Chimera Nanoparticle for Resistive Biomolecular Memory Application Taek LEE1, Jeong-Woo CHOI2, Peixuan GUO1 1Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA, 2Department of chemical and Biomolecular engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul, 121-742, Republic of Korea In the present study, authors suggested the pRNA-quantum dot chimera nanoparticle to the resistive biomemory application. The pRNA 3way junction (3WJ) and quantum dots (QD) were specifically conjugated using RNA apatmer-based conjugation method. The pRNA 3WJ-QD conjugates were confirmed by gel electrophoresis. The conjugated thiol-modified pRNA 3WJ-QD chimera nanoparticle was immobilized onto Au surface and it was confirmed by scanning tunneling microscopy (STM). To test the charge storage property, the scanning tunneling spectroscopy (STS) was carried out to verify the resistive memory function. As a result, the electrical properties of pRNA 3WJ-QD displayed stable electrical bistability and resistive memory function. Therefore, the proposed concept can be paved the way to develop the recent advances in bioelectronics field to industrial technologies with broad applications, including semiconductor industry and bionanotechnology. Authors envision the extension of this approach to bio-inspired electronics with adjustable storage performance through a molecular level manipulation. Keywords : RNA Nanotechnology, RNA-QD Chimera, Restitive Biomemory References 1. Taek Lee, Ajay Yagati Kumar, Fengmei Pi, Ashwani Sharma, Jeong-Woo Choi, Peixuan Guo, ACS NANO, Vol. 9, 6675-6682. 2. Taek Lee, Ajay Kumar Yagati, Junhong Min and Jeong-Woo Choi, Adv. Func. Mater., Vol. 24, 1781-1789. D216 Shape-Controlled Synthesis of Radio-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging Taejong PAIK School of Integrative Engineering, Chung-Ang University, Seoul 156-756, Korea Isotopically-labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal- based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we present size and shape controlled synthesis of rare earth fluoride nanocrystals doped with the beta- emitting radioisotope yttrium-90(90Y) is presented. Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts employed. We demonstrate the Cerenkov radioluminescence imaging, magnetic resonance imaging capabilities, and two-photon upconversion luminescence of synthesized nanocrystals, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy. Keywords : Nancrystal, Radiolabeling, lanthanide, upconversion, radioisotope References 1. Taejong Paik, Ann-Marie Chacko, John Mikitsh, Joseph S. Friedberg, Daniel A. Pryma, and Christopher B. Murray “Shape-Controlled Synthesis of Yttrium-90 Radio-labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging” ACS Nano, 2015, ASAP D217 Two-stage Pretreatment of Rice Straw using Aqueous Ammonia and Dilute Acid (and lactic acid production strategies) Jin-Woo KIM Department of Food Science, Sunmoon University, Asan-Si 336-708, Republic of Korea Lignocellulosic biomass is the most abundant renewable resources and thus, promising feedstocks for bio-based products. Liberation of fermentable sugars from recalcitrant lignocellulosic biomass is one of the key challenges in production of cellulosic ethanol. Here we developed a two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH3 was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Under the optimal pretreatment conditions, the enzymatic hydrolysis yields of the two-stage treated samples were 96.9% and 90.8% with enzyme loadings of 60 and 15 FPU/g of glucan, respectively. The overall sugar conversions of cellulose and hemicellulose into glucose and xylose by enzymatic and acid hydrolysis reached 89.0% and 71.7%, respectively. Keywords : Lignocellulose, Biomass, Pretreatment, Fermentable sugar, lactic acid References 1. Kim et al., Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid (2011) Bioresource Technology, 102: 8992-8999 2. Hsu et al., Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis (2010) Bioresource Technology, 101: 907–4913 3. Jung et al., Dilute acid pretreatment of lignocellulose for whole slurry ethanol fermentation (2013) Bioresource Technology, 132:109-114 한국생물공학회, 생물공학의 동향 : 2015.10 바이오산업 국제표준 (ISO/TC276) 설명회 및 전문가포럼 E202 Blue Ocean Opportunities in Standardization of Biological Resources Yeonhee LEE Department of Horticulture, Biotechnology & Landscape Architecture, Seoul Women's Univ., 621 Hwarang-ro, Nowon-gu, Seoul, 01797, Korea Upon the enactment of the Nagoya Protocol, countries around the world are aggressively preparing and implementing various strategies to secure biological resources to maximize their national interests. Since the publication of OECD guideline on biotechnology in 2007, International Organization for Standardization (ISO) initiated the efforts of standardizing every aspect concerning biological resource. On the 13th of December, 2013, Technical Committee (TC) 276 started with the first meeting at Berlin. With aims for conservation of resources and sustainable development of bioresource technology through information sharing, four Working Groups (WGs) – Terminology, Biobanks and Bioresources, Analytical Methods, and Bioprocessing – were initially created with later addition of a fifth WG, Data Processing and Integration. Participating members of TC 276 are 22 countries, including Germany, the secretariat, Korea, Japan, and the United States. TC 276 also has 13 observing members, including Israel and India, 16 internal liaisons and 5 Category A liaisons. Upcoming ISO/TC 276 meeting will be held at Tokyo on the 26th to 30th of October. This meeting is expected to become an arena of diverse opinions and proposals among countries with different agendas. Next general meeting of TC 276 will be held at USDA Training & Conference Center, USA on the 13th of May, 2016. E204 ISO/TC 276/WG 2: Biobanks and Bioresources Kyungsook AHN1, Paul Eunil JUNG1, Airan HAHN1, Yeonhee LEE2 1Korea National Research Resource Center (KNRRC), #324 Golden 50 Commemoration Hall, Seoul Women's Univ., 2Department of Horticulture, Biotechnology & Landscape Architecture, Seoul Women's Univ., 621 Hwarang-ro, Nowon-gu, Seoul, 01797, Korea The global bioindustry market is growing at an annual growth rate of 9.8% as of 2013 and expected to maintain such high rate until 2020. Korean bioindustry also has demonstrated the remarkable annual growth rate, reaching almost 14% (2013). International standards enacted by WG 2 of ISO/TC 276 will have crucial implication on the future of biobanks as a blueprint establishing overall landscape of biobanking activities. TC 276/WG 2 is currently working on creating standardized international guidelines of human, animal, plant, and microorganism biobanks*. The enactment of the standard will ensure the quality of bioresources and establish standardized operations for management of diverse types of bioresources. Prior to the Tokyo meeting on the 26th to 30th of October, TC 276/WG 2 has distributed the Working Draft (WD) to the member countries. The document includes not only general requirements of biobanks, but also competence assessment of staffs and traceability of samples and equipment. Members will indeed use this opportunity to submit their opinions which will maximize their national interests. Currently, there are 3 standard documents under development on technical requirements for operation and fulfillment of human biobanks, including on acquisition and processing of animal germplasms and human genetic resources by the proposal of China and Luxemburg. Also, WG 2 has assumed working on the New Work Item Proposal (NWIP) targeted at the stem cell market and other organisms. The Korea National Research Resource Center (KNRRC) is actively dealing with international standardization efforts by enacting the Best Practice Guidelines for Research Resource Centers in 2013. This standard is expected to have great influences as the only Korean guideline on biobanking and already has been utilized as a practical manual for operation of numerous biobanks. In comparison to other organizational standards, this standard is strongly unique and thorough in defining operation system and emphasizing satisfaction of stakeholders. Based on these strengths, we strive to express and add our opinions to ISO/TC 276. With the enactment of the biobanking guideline, every nation will share the same quality assurance processes which will ensure the credibility of the sample quality and satisfaction of all stakeholders, such as public institutions, industry officials, researchers, and customers. Furthermore, it will stimulate international collaboration for various bioindustrial activities, such as development of biomarkers and personalized medicines and contribute to the considerable growth of bioeconomy. * Resources for research and development (R&D) aspects, but excluding therapeutics and highly regulated agricultural productions E205 ISO TC276 Biotechnology, WG 3 Analytical Methods Jongwon KIM LabGenomics Co., Seongnam, 13488, Korea ISO TC276 was established in 2013 to work closely with related committees in order to identify standardization needs and gaps, and collaborate with other organisations to avoid duplications and overlapping standardization activities, similar to other TCs. There exists 5 WGs right now in TC276, and WG3, titled in “Analytical Methods”, aims to develop standards for accurate, reproducible and robust measurement and analysis in support of biotechnologies. The WG will develop a package of International Standards for biologically relevant molecules and entities, including nucleic acids, proteins, and cells. This WG will develop horizontal standards and, when applicable, vertical / particular standards for industry sectors. The WG will also coordinate with relevant technical committees and standardisation initiatives. The convenor of this WG 3 is right now Dr. Sheng Lin-Gibson (NIST, USA). There are several NWIPs which are discussing in WG 3 as follows; 1. Biotechnology – Cell Counting – Part 1: General guidance on cell counting methods (Dr . Sheng Lin- Gibson, NIST, USA) 2. Biotechnology – Cell Counting – Part 2: Experimental design and statistical analysis to quantify counting method performance (Dr . Sheng Lin- Gibson, NIST, USA) 3. Methods to evaluate the quality of the massive sequencing data, Chinese expert, Dr. Zhang 4. Oligonucleotide Quality Control, (Japanese Expert, Dr. Hiroki Nakae) 5. Quality considerations for targeted nucleic acid quantification methods, (English Expert, Dr. Helen Parkes ) 6. Methods to determine the concentration of total nucleic acids, (French Expert, Dr. Fay Betsou ) 7. Methods of quality evaluation and stability evaluation of nucleic acids as well as stability of nucleic acids during storage (France) Keywords : ISO TC 276, WG 3 Analytical Methods, biotechnology References 1. ISO TC 276 Biotechnology : Business Plan (www.iso.org) 한국생물공학회, 생물공학의 동향 : 2015.10 산업부 바이오 R&D사업 학회연계 성과발표회 G202 차세대 지속형 인성장호르몬의 임상 및 제품화기술 개발 김기완 CJ 헬스케어주식회사, 연구소 생물의약센터 차세대 인성장호르몬은 191 개의 아미노산으로 이루어진 인성장호르몬과 394 개의 아미노산으로 이루어진 A1AT 변이체를 유전공학적으로 융합(Fusion partner) 시킴으로써 인체내 신장에서의 제거 억제를 통하여 체내 반감기를 증가시키도록 고안된 단백질이다. 융합파트너로 사용된 A1AT (alpha 1-antitrypsin)는 인간의 혈중에 고농도로 존재하고 있으며, 인성장호르몬의 융합파트너로 사용된 A1AT 변이체는 point mutation 를 가하여 A1AT 의 고유 성질인 serine protease 저해능력을 제거함으로써 인체에 과량을 투여하여도 문제가 발생하지 않도록 안전성을 높였다. 차세대 인성장호르몬의 발현 세포주는 산업적으로 가장 많이 사용되고 있는 CHO 세포주를 사용하였으며, 안정화된 배양 발현은 1.0g/L 이상으로 일반적인 다른 유전자재조합 단백질 대비 높은 발현률을 얻었고, 고수율의 정제 기술이 적용되어 원가 경쟁력을 확보 하였다. Rat 과 monkey 에서 원천기술의 PoC 검증을 통하여 1 주 1 회 투여 제형으로써 개발 가능성을 확인하고 안전성과 안정성을 확보한 후, 사람에서의 유효성 및 안전성을 검증하기 위해 건강한 성인 남성 자원자를 대상으로 피하주사 시 안전성/내약성 및 약동학/약력학적 특성 평가를 위한 용량군별 무작위배정, 단일눈가림, 위약대조, 단회투여, 단계적 증량 방법으로 임상 1상 시험을 진행하였다. G203 The Development of novel diabetic retinopathy therapeutics EG-Mirotin Hyun Jong KIM Eyegene Inc., Seoul 157-200, Korea Eyegene Inc, has successfully developed human-originated recombinant protein EGT022 which composed of 58 amino acids, the part of human ADAM-15 disintegrin-like domain contains RGD motif. We have found that EGT022 induces normalization and stabilization of abnormal and/or immature blood vessels by recruiting pericyte through interacting platelet followed by angiogenic factor release thus can be utilized as therapeutic of ischemic disease. We have identified the structure, composition, immunochemical characteristics, and physicochemical characteristics, and set criteria and test methods for EGT022 to complete CMC. The nonclinical study for examining efficacy and safety of EGT022 is composed of pharmacology and efficacy evaluation and toxicity test. We have confirmed the efficacy of EGT022 on retinopathy using oxygen-induced retinopathy (OIR) animal model and streptozoticin induced diabetic retinopathy animal model, and repeated dose and other toxicity test have proved no toxicity of EGT022. Eyegene Inc, developed diabetic retinopathy therapeutics ‘EG-Mirotin’ using EGT022 and have successfully completed phase I study in Europe to healthy volunteers and type II diabetes mellitus patients. Eyegene Inc, now conducting phase IIa clinical trial for evaluating the efficacy of EG-Mirotin in France. Keywords : Eyegene, EG-Mirotin, EGT022, Diabetic retinopathy G204 Well-defined Scaffold for Regenerative Medicine H.B. LEE1, M.S. KIM1, J.H. LEE2, J.W. SHIN3, J.W. JANG4, H.P. YUN5 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Nature-Inspired Mechanical System, Korea Institute of Machinery and Materials, Daejeon, Korea, 3Department of Bioengineering, Inje University, Kimhae, Korea, 4Cellumed, Seoul, Korea, 5Anycasting, Seoul, Korea It has been well accepted that regenerative medicine is as an alternative approach to repair or replace damaged/diseased tissue and organs. Millions of patients suffer from end-stage organ failure or tissue loss annually. The development of regenerative medicine permits the transplantation of cells from a patient’s own tissue to regenerate damaged tissue or organ without causing immune response. For reconstruction of a neo-tissue by regenerative medicine, three major components have been issued those 1) cells dissociated from the donor tissue, 2) biomaterials as scaffold substrate for cell attachment and growing basement at the desire site, and 3) growth factors for promoting or preventing cell adhesion, proliferation, migration and differentiation. Three-dimensional biomaterial scaffolds are initially developed for the temporary substrate to grow cells in formed structure. It has been known that the three-dimensional organization of cell related with cellular adhesion affects the cellular development. Therefore, biodegradable and biocompatible polymers have used to fabricate three dimensional scaffold for regenerative medicine. Scaffolds require a highly open porous structure with good interconnectivity and sufficient mechanical strength for cellular in-growth. Drug or growth factors releasing scaffolds are ideal for modulating tissue regeneration. In order to harmony of above three components, it is necessary to fulfill requirement of biological scaffold for successful tissue regeneration. Recently, three-dimensional (3D) printing technology has been widely used in tissue regeneration applications because of its controllable feature and well-defined product. During last five years, we are developing 3D well-defined scaffold for bone and nerve regeneration as well as biodegradable polymers, 3D printer and bioreactor. In this presentation, the author will inform the results of progress achieved from a project “Development of well-defined scaffold for regenerative medicine” supported by the Ministry of Industry. Acknowlegement This study is supported partially by the Ministry of Industry Keywords : regenerative medicine, well-defined scaffold, biodegradable polymer, 3D printer G206 차세대 생명정보 활용을 위한 다양한 소프트웨어 개발 Je Hee LEE CHUNLAB Inc. #307, Bldg. 105-1, Seoul National University, Daehak-dong, Gwanak-gu, Seoul, Korea, 151-742 NGS 의 발달에 따라 유전체, 전사체, metagenome data 등은 기하 급수적으로 쌓이고 있고, 이를 분석하려는 수요도 많지만, 이를 분석할 수 있는 툴의 발전은 이에 못 미치고 있는 실정이다. 특히 기존의 생물정보 분석툴은 고사양의 서버와 리눅스 등의 운영체제에서 생물정보지식을 알아야 분석할 수 있어서 일반 연구자들이 쉽게 사용할 수 없는 경우가 많다. 대용량의 NGS data 를 쉽게 분석하고, 다른 data 와 비교 분석할 수 있는 소프트웨어 및 이를 처리할 수 있는 클라우드 시스템 개발이 필요하며, 또한 다른 데이터와 비교 할 수 있도록 2 차 데이터베이스 구축이 필요하다고 할 수 있다. 본 연구팀은 NGS data 를 이용하여, 유전체, 전사체, metagenome 분석 등을 쉽게 분석하는 분석 시스템 및 소프트웨어, 데이터베이스 등을 구축하여 이를 통한 사업화를 진행 중에 있다. Keywords : NGS, Bioinformatics, Cloud, Software, Genomics, Transcriptomics, Metagenomics G207 차세대 생명정보 통합분석을 이용한 다중 바이오마커 개발 오태정 ㈜지노믹트리, 305-510 대전시 유성구 테크노 10 로 44-6 본 과제는 2011 년 산업통상자원부가 지원하는 포스트게놈 다부처 유전체사업의 지원 (과제번호: 10040174)을 받아 수행되었다. 차세대 생명정보 분석 기술의 발달은 많은 생명현상을 이해하는데 획기적인 변화를 가져왔다. 본 과제에서는 대용량 차세대 생명정보를 활용하여 방광암/췌장암/담도암을 조기에 진단하거나, 모니터링 그리고 예후 예측을 위한 신규 바이오마커를 발굴하고 이를 이용한 비침습적 분자진단 기술을 개발하여 상용화하는 것을 목표하고 있다. 이러한 목표를 달성하기 위하여 방광암/췌장암/담도암을 대상으로 대량의 multi-omics 기반의 (유전적/후성유전적) 차세대 생명정보 (NGS/microarray)를 생산하였고, 생명정보 통합 분석을 위한 알고리즘 개발 그리고 이를 활용하여 암 분자진단용 신규 바이오마커들을 다수 발굴하였다. 발굴된 바이오마커들을 상용화하기 위하여 임상전문의료진과의 임상중개연구를 통하여 바이오마커들의 임상적 유용성을 비침습적인 소변과 혈액에서 검증하였다. 검증된 바이오마커들을 임상현장에 사용하기 위하여 암 분자진단용 시제품을 개발하고, 시제품의 안정성 및 유효성을 확인하여 한국식품의약품안전처의 품목허가를 위한 임상검증 연구를 진행하고 있다. Keywords : 차세대 생명정보, 암 분자진단, 바이오마커, Multiomics 한국생물공학회, 생물공학의 동향 : 2015.10 From Protein Expression to Microbial Engineering B301 Novel Synthetic Expression Regulators for Microbial Engineering Gyoo Yeol JUNG Department of Chemical Engineering/I-Bio Program, POSTECH, Pohang, 790-784 Pathway optimization of microbial metabolism is essential for the production of commercially valuable chemicals such as biofuels, platform chemicals and biologically active compounds. To achieve the successful design or redesign of microbial metabolism, robustness of naturally occurring biological systems has to be relieved so that cells can be easily redesigned. Although extremely huge efforts have been made to find genetic target to improve metabolic function of the microorganisms, there still exists the additional room for the non-rational approach. Currently, typical approach for metabolic engineering uses both rational approach as well as non-rational methods such as combinatorial and evolutionary methods. One of the most critical problems of metabolic engineering is especially robustness of the biological systems. Bacterial cells are generally evolved at the various levels from DNA to protein for maintaining their robustness against the changing circumstances. Therefore, general strategy to modify cellular physiology depending the robustness or flexibility of the biological systems should be required. In this study, we developed the general tools to modify the biological robustness at the various levels including translation and protein levels. For translation level control, a model to predict the translation efficiency based on the mRNA’s secondary structure and consequently expression level can be precisely controllable for any genes of interest. Additionally, intracellular metabolite sensor named “riboselector” to regulate metabolic distribution will be presented. The potentials of the platform technology developed in this study for the application to the production of biofuels and commodity chemicals. Keywords : Metabolic engineering, synthetic biology, riboselector, UTR Designer References 1. Yang, J., Seo, S.W., Jang, S., Shin, S.-I., Lim, C.H., Roh, T.-Y., and Jung, G.Y. (2013) “Synthetic RNA devices to expedite evolution of metabolite-producing microbes” Nature Commun., 4, 1413 2. Seo, S.W., Yang, J.-S., Kim, I., Yang, J., Min, B.E., Kim, S.*, and Jung, G.Y.* (2013) “Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency” Metab. Eng., 15, 67-74 3. Seo, S.W., Yang, J.-S., Cho, H.-S., Yang, J., Kim, S.C., Park, J.M., Kim, S.*, and Jung, G.Y.* (2014) “Predictive combinatorial design method for systematic optimization of gene expression”, Sci. Reports, 4, 4515 B302 New Logic and Expression Module for Functional Overexpression of Proteins Geun-Joong KIM Department of Biological Sciences, Chonnam National University, Gwangju 500-757, Korea The need for a systematic approach to provide microbial cells as whole cell enzyme, factory and sensor is continuously increasing. Thus, the related research is proliferating as an emerging field. In this context, the elucidation of underlined mechanism for the control of gene expression at the level of transcriptional and/or translational is prerequisite for systematic design of engineered cells. As generally well known, cell innately controls gene expression using a set of various cis- and trans-acting elements such as DNA, RNA, protein and DNA-modifying regulators. It was also recognized well that interactions between these elements were logically programed to modulate gene expression. A lot of researches, therefore, have attempted to understand the logic of the genetically installed program on genome for regulating gene expression. Any of these findings could have a deep impact on biotechnology, such as designing module to express the proteins of interest in a predictable manner, generating cells to sense and respond to signals from internal or external environment, and constructing cell factory that can overproduce a valuable protein and metabolite of interest. However, building synthetic module and genetic circuit, based on the innate principle and logic of cells for gene expression, remains one of the greatest challenges in the field, where even simple expression module is labor intensive to build and unable to perform suitable function comparable in-vivo to its natural counterpart. As described above, the research field in gene expression has experienced many challenges in discovering and developing new tools for functional overexpression of valuable proteins, especially for functional expression of difficult-to-express proteins with promising activity. In response, a lot of novel concepts and trials are reported to be working to pave a novel route for the development of practical application process. In line with this, we present here a novel concept and two expression modules all of which can function individually and will have a synergistic effect when integrated in a genetic circuit for gene expressions. The validation processes are currently being undertaken to evaluate the performance of transformed cells with these constructs for cancer therapy. Keywords : Expression module, genetic circuit, ramp function, translation rate, auto-induction, positive feedback References 1. KS Kim, JH Jeong et al., Molecular Therapy – Oncolytics, 2015, 2, 15007 2. H Hong, D Lim et al, Cell Cycle, 2014, 13(19), 3112-3120 3. WJ Park, SH You et al., 2015, Acta Biochimie et Biophysica Sinica, 47(7), 488-495 B303 Engineering of SRP Pathway for the Enhanced Production of Recombinant Proteins in Escherichia coli Ki Jun JEONG Dept. Chemical and Biomolecular Engineering, KAIST, Daejeon 305-701, Republic of Korea For a long time, recombinant protein production in E. coli has been extensively studied due to the various advantages such as rapid growth rate, cost effectiveness, ease of genetic manipulation and convenience of high-cell-density cultivation (HCDC). In many cases, therapeutically important proteins (e.g., antibodies, scaffold proteins, vaccines, enzymes, receptors) require the correct formation of disulfide bonds for the desired biological activity, and so the strategy of secretion into periplasm which can provide oxidizing condition and suitable for the disulfide bonds formation, has been primarily employed for the production of functional therapeutic proteins. In E. coli, the major pathway for protein secretion is Sec-dependent pathway and many recombinant protein has been produced by secretion through this pathway. But, many proteins (particularly big and complex proteins such as full-length IgG) were easily aggregated in the cytoplasm before translocation into periplasm, and the successful results could not be achieved. As an alternative pathway for protein secretion, SRP (Signal Recognition Particle) pathway can be considered, in which target protein can be secreted via co-translational translocation process. In this pathway, target proteins has much less chance to be aggregated in the cytoplasm and more protein can be secreted into periplasm. By simple fusion with SRP-dependent signal peptide (ex DsbA), the target protein can be translocated via SRP pathway, and we successfully demonstrated more efficient secretion of several therapeutic proteins including antibody fragments (scFv) than secretion by Sec-dependent pathway. In addition, by further engineering of SRP pathway, the secretion efficiency in E. coli could be improved significantly. In this talk, the general concept of SRP pathway and the strategy of SRP pathway engineering with several successful examples will be presented. Keywords : SRP pathway, Escherichia coli, secretion References 1. Makino T, Skretas G, Georgiou G (2011) Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 10:32. 2. Lee YJ, Kim HS, Ryu AJ, Jeong KJ (2013) Enhanced production of full-length immunoglobulin G via the signal recognition particle (SRP)-dependent pathway in Escherichia coli. J Biotechnol 165(2):102-108. 3. Driessen AJ, Nouwen N. (2008) Protein translocation across the bacterial cytoplasmic membrane. Annu Rev Biochem. 77:643-667. B304 Shift from Intractable Natural Producer to Tractable Microorganism for Production of Valuable Isoprenoids Chonglong WANG, Jung-Hun KIM, Ji-Bin PARK, Ju-Eon PARK, Seon-Won KIM Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea Isoprenoids, also called terpenoids, are a large and diverse class of naturally-occurring compounds. They are present in all living organisms and include many important drugs, valuable flavor and fragrance compounds, pigments, antioxidants, steroids and natural polymers. However, most of valuable isoprenoids are produced in a trace amount as secondary metabolites from intractable slow growing organisms such as plants, actinomycetes, archaea, etc. Isoprenoids are derived from five-carbon universal building blocks (IPP and DMAPP), and the increased building blocks synthesis is a way to enhance the production of isoprenoids. Using E. coli as a host, we successfully produced the valuable compounds of isoprene, geraniol, farnesol, farnesene, squalene, santalene, bisabolol and retinoids using the platform E. coli strain resulting from the carotenoids works. This work is supported by a grant (NRF-2013R1A1A2008289) and a grant (NRF- 2012M1A2A2671831) from the National Research Foundation, and a grant from the Next-Generation BioGreen21 Program (SSAC, grant#: PJ01106201), RDA, Korea. Keywords : Metabolic engineering, Isoprenoids, Carotenoids, Escherichia coli, MVA pathway References 1. J Zhou et al., Geranyl diphosphate synthase, an important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli, Enzyme and Microbial Technology, 68:50-55 (2015) 2. Chonglong Wang, Jung-Hun Kim and Seon-Won Kim, Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects, Mar. Drugs, 12(9):4810-4832 (2014) 3. HJ Jang et al., Comparison of extraction phases for two-phase culture of retinoids-producing recombinant E. coli, Biotechnology Letters, 36(3):497-505 (2014) 한국생물공학회, 생물공학의 동향 : 2015.10 부문위원회 Sessions Division Symposia C101Integration of Multiple Processes and Nutrient Cycling in a Bio-Energy System Ben J. STUART Department of Civil and Environmental Engineering, Old Dominion University, Norfolk, VA23508 Microalgae are hold great potential as feedstocks for bioproduct and biofuel production. Realizing this potential requires the economic integration of multiple processes, including but not limited to: cultivation, harvesting, dewatering, extraction and/or transformation. In addition, the economic viability also requires careful attention to the origin and management of necessary resources such as nutrients, water and process energy streams. It is necessary to employ a holistic approach to these systems and resource management if commercial algae-based systems are to become a part of the industrial landscape. The present study exploits the management of the organic fraction of domestic solid waste streams to produce energy through biogas production, recycles the digestate liquor from the anaerobic digestion process to cultivate algae in open and hybrid growth systems, harvests and dewaters the algal biomass, extracts the biomass for valuable products and performs a life cycle analysis of the integrated processes. This paper will present our progress to date, including such topics as: · The biochemical methane potential of waste organics via anaerobic digestion (AD) · AD liquor nutrient characterization and pretreatment · Algae cultivation utilizing AD liquor as a primary nutrient source · Algae harvesting and dewatering through enhanced coagulation and settling · Comparison of solvent and supercritical CO2 extraction of algal lipids · Establishing a framework for the life cycle analysis of the integrated processes This work was funded entirely by the National Science Foundation (NSF) through the Sustainable Energy Pathways (SEP) program (Award # 1230961). C102Enzymatic Degradation of Biodegradable Plastic Agricultural Mulch Films Hiroko KITAMOTO, Motoo KOITABASHI, Takashi WATANABE, Yukiko SHINOZAKI, Kimiko YAMAMOTO- TAMURA, Yuka KIMIKO, Ken SUZUKI, Hirokazu UEDA, Shigenobu YOSHIDA, Tohru YARIMIZU National Institute for Agro-Environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604 Japan Agricultural materials made from biodegradable polymers (BP) have been used to decrease the burden of plastic waste recovery and recycling. However, their degradations depend largely on environmental conditions and sometimes do not proceed as desired. NIAES studies to control the degradation speed of BP mulch films at agricultural field. 1. Investigated the key factor controlling the degradation speed of BP films in cultivated soils in Japan. 2. Isolation of BP degradable yeast and fungus from plant surfaces. 3. Characterization of BP degradable enzymes. 4. Enhancement of the productivity of BP degradable enzymes. Keywords : Biodegradable plastic degrading enzyme, plant, soil, yeast, fungus, mulch film References 1. Kitamoto, H.K., et al., (2011) Phyllosphere yeasts rapidly break down biodegradable plastics. AMB Express1, 44. 2. Koitabashi, M. , et al., (2012) Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants. AMB Express2, 40. 3. Shinozaki, Y. , et al., (2013) Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica: surface plasmon resonance and atomic force microscopy study. Appl Microbiol Biotechnol97, 8591-8598. 4. Watanabe, T. , et al., (2014) Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase- like enzyme which efficiently degrades biodegradable plastics. J Biosci Bioeng117, 325-329. 5. Yamamoto-Tamura, K., et al., (2015) Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils. AMB Express5, 10. C103Synthetic Biology-Supporting Future Space Missions Asif RAHMAN Bioengineering Branch, Space Biosciences Division, NASA-Ames Research Center, Moffett Field, CA The field of Synthetic Biology has a long term goal of supporting and improving life on Earth. Synthetic Biology also holds the promise of advancing long term Space Flight with the benefit of utilizing recombinant technologies in the production of medicine, food, materials, and energy. Currently, during Space missions we rely on the four R’s: resupply, repair by replacement, redundant hardware, and retreat to Earth. A “make it there, don’t take it there” approach is necessary for mission success and this is where Synthetic Biology can support missions. Maintaining and utilizing finite resources during long-term spaceflight will require cyclical biological systems to both reduce and recycle materials. The advantages of using microorganisms over traditional physio-chemical processes is of interest to NASA from the standpoint of being flexible, reduced payload mass, low power consumption, and the need for in-space manufacturing. In this presentation, Synthetic Biology with NASA related applications will be discussed. Keywords : Space Biology, Bioengineering, Synthetic Biology C104Evaluation of Different Pretreatments on the Enzymatic Saccharification of Rice Waste Biomass and their Utilization for Bioethanol Production Ganesh D. SARATALE, Min Kyu OH 1Department of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul, South Korea 136-713 In this study we have evaluated the comparative assessment of individual and multiple pretreatment processes for whole rice waste biomass (RWB). Individual alkaline pretreatment (2% NaOH) on autoclaving gave maximum yield of 505 mg of reducing sugar with 69.17% hydrolysis yield using enzyme dosage of 20 FPU/g of RWB. The sequential pretreatments of acidified sodium chlorite and sodium bicarbonate to alkaline pretreated RWB biomass gave maximum yield of 725 mg of reducing sugar with 90.65% hydrolysis yield using less enzyme dosage (10 FPU/g of RWB) enabling the process more feasible. The effects of different pretreatments on chemical composition, structure and complexity of RWB biomass and its relation with saccharification were investigated using various analytical techniques. Finally the enzymatic hydrolysates of RWB were used for ethanol production by developed strainSaccharomyces cerevisiae SR8. Under optimal conditions, RWB hydrolysates (20 g/L), the maximum sugar consumption, ethanol production and ethanol yield were 95%, 8.85 g/L and 0.465 g/g of sugar, respectively. The result increases the possibility in the preparation of biomass feedstock for economic biofuel production. Keywords : Rice waste biomass, acidified sodium chlorite pretreatment, Saccharomyces cerevisiae SR8 References 1. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M. J., Pretreatment for an efficient bioethanol production process based on enzymatic hydrolysis: A review. 2010. Bioresour. Technol. 101, 4851-4861. 2. Kim, S., Holtzapple M. T., Effect of structural features on enzyme digestibility of corn stover. 2006. Bioresour. Technol. 97, 583–591. 3. Saratale, G.D., Oh, M.K., Characterization of poly-3-hydroxybutyrate produced from Ralstonia eutropha using an alkali-pretreated biomass feedstock. 2015. Int. J. Biol. Macro. 80, 627-635. C105Development of Ionic Liquid tolerant Yeast for a Single-pot bioethanol production from lingo- cellulosic biomass Vivek MORYA, Siyeon YOON, Yoon-Mo KOO Department of Biological Engineering Inha University, Incheon 402-751, Korea Considerable research attention has focused on the potentials of Lignocellulosic agricultural waste materials as a source of fermentable sugar and successive bio-ethanol production. Major challenge with lignocellulosic biomass (LCB) is their recalcitrant nature; therefore, an efficient pretreatment process is being needed, which is an expensive and labor-intensive process, thus it become a big hurdle in commercial viability of bio-ethanol from LCB. In recent years, Ionic liquids (ILs) have gained immense attention for pretreatment of LCB. However, the toxicity due to ILs significantly affects the productivity. Therefore, an extensive washing step is involved in ILs based process [1]. This present study is focused to minimize the number of steps and process in bio-ethanol production from LCB. To meet this goal, an ILs tolerant microbe is being needed, and it was achieved by genetic modification, i.e. heterologous expression of artificially designed gene (a kind of channel protein). This protein helps in removal of ILs from the cytoplasm to against the gradient. A known bacterial gene responsible for ILs tolerance was selected and optimized for expression in yeast. Two genes for same protein were cloned in pYes2.1V5-His TOPO plasmid vector and transferred in yeast strain (INVSc1, S. cerevisiae) by LiOAc method. The transformed colony showed a better tolerance toward different imidazolium based ionic liquids and tolerant up to 100 mM concentration of ILs stress. Further investigation on ILs treated biomass and their hydrolysis by the halotolerant fungi, and the culture filtrates was carried to achieve saccharification of LCB. The hydrolysate was directly subjected to fermentation by yeast containing ILRG. A 74% of yield was achieved in 96 hours of fermentation condition. In this process, a 10-time less Ionic Liquid is required and most importantly the wasting step in not needed. Therefore, at the end of the process, the maximum recovery of ILs and product (Bioethanol) is possible. The optimization of the scale-up, product and ILs recovery is being under process. Based on the results, it is anticipated that this single pot cost effective process may be helpful in the development of a commercially compatible bio-ethanol production from LCB. Keywords : Ionic Liquid, Lignocellulosic Biomass, Bioethanol, genetic transformation, ILs tolerance References 1. Brandt, A., Gräsvik, J., Hallett, J. P., & Welton, T. Green chemistry, 15(3), 550-583 (2013) C301Screening of (R)-form Specific ω-aminotransferases by Exploring Evolutionary Sequence Space Joo-Hyun SEO, Eun-Mi KIM, Joon Ho PARK, Byung-Gee KIM School of Chemical and Biological Engineering, Seoul National University, Seoul 120-750 (R)-form amines are industrially important amines and covers about 70% of the chiral amines used as pharmaceutical building block. Although several (R)-form specific ω-aminotransferases (R-ωATs) have been reported, the definition of the R-ωAT family is not clear. On the other hand, it is generally supposed that R-ωATs are variants of aminotransferase group III proteins which consist of branched-chain amino acid aminotransferase (bcAT) and D-alanine aminotransferase (D-AT). Based on this assumption, we attempted to search the R-ωAT family which is supposed to be the neighbor subgroup of bcAT and D-AT. Using the family profiles of bcAT and D-AT, sequences in RefSeq database were scored and sequences having two profile analysis scores were plotted in 2-dimensional score space. In the score space, we took a region which shows low similarities both to bcAT and D-AT. Three sequences were selected and investigated whether they have R-ωAT activity or not. Experimental results revealed that three selected enzymes - amino acid aminotransferase from Saccharopolyspora erythraea NRRL 2338, branched-chain amino acid aminotransferase from Bacillus cellulosilyticus DSM 2522 and branched- chain amino acid aminotransferase from Bacillus thuringiensis serovar berliner ATCC 10792 - have R-ωAT activity. Keywords : R-form amine, omega-aminotransferase, sequence profile analysis C302A New Assay Methods for Sensitive Measurement of Glycerol Dehydratase activity and Glycerol Dehydratase Inactivation by Reactive Oxygen Species Eul-Soo PARK, Jong-Shik SHIN Dept. of Biotechnology, Yonsei University, Seoul, 120-749 3-Hydroxypropionic acid (3-HP) is an important platform chemical, which can be converted into many commercially valuable chemicals, such as acrylic acid, methyl acrylate, propiolactone, malonic acid, acrylamide and so on. Microbial production of 3-HP from glycerol has been studied by using recombinant bacterial host with two enzymes, glycerol dehydratase and aldehyde dehydrogenase. Glycerol dehydratase is the key enzyme that catalyzes conversion of glycerol to 3- hydroxypropanal, which involves radical intermediates. For the efficient optimization of biosynthetic preparation of 3-HP, accurate assay for the measurement of glycerol dehydratase activity is indispensable. Here we report a new spectrophotometric assay method for the sensitive detection of the glycerol dehydratase activity by using aldehyde dehydrogenase as a reporter enzyme. In this method, 3-hydroxypropanal is continuously converted to 3-HP by aldehyde dehydrogenase and the conversion of NAD+ to NADH is recorded as an increase in the UV absorbance at 340 nm. A new assay was used to determine kinetic parameters of glyceol dehydratase for glycerol and study glycerol dehydratase inactivation in the presence of reactive oxygen radicals. Keywords : 3-Hydroxypropionic acid, Glycerol dehydratase, Assay Method, reactive oxygen radicals References 1. Kumar V, Ashok S, Park S. Recent advances in biological production of 3-hydroxypropionic acid(2013), Biotechnology Advances. 31, 945-961 C303Development of Microbial Platform to Produce Fine Chemicals from Renewable Biomass Eun-Mi KIM1, Youngsoon UM1, Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea, 2Department of Chemistry, and Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea We present two kinds of microbial platforms: First, Corynebacterium glutamicum was engineered to produce succinate using levoglucosan as a sole carbon source. To utilize levoglucosan as sole carbon source, levoglucosan kinase (lgk) gene from Lipomyces starkeyi was introduced in C. glutamicum. The recombinant strain showed a higher succinate production (4.92 g/L) from 2 % levoglucosan than production of the BL-1 strain from 2% glucose (3.88 g/L), confirming that levoglucosan is an attractive carbon substrate for producer strains. Second, for production of myrcene, Escherichia coli was metabolically engineered by introducing a heterologous mevalonate pathway and overexpressing tailoring enzymes, such as geranyl diphosphate synthase (GPPS) and myrcene synthase (MS). Engineered E. colistrains harboring MS from Quercus ilex L. produced only myrcene (1.67 ± 0.029 mg/L). Subsequent engineering resulted in higher production of myrcene. The production level of myrcene (58.19 ± 12.13 mg/L) was enhanced by 34-fold using in situ two-phase extraction to eliminate cellular toxicity and the evaporation of myrcene. [1] Keywords : Levoglucosan , Levoglucosan kinase, Succinate, Myrcene, Myrcene Synthase References 1. Kim, E.-M., Eom, J.-H., Um, Y., Kim, Y., Woo, H. M (2015), Microbial synthesis of myrcene by metabolically- engineered Escherichia coli. Journal of Agricultural and Food Chemistry, 63(18), 4606-4612 C304Enzymatic Synthesis of Mandarin Oligosaccharide and Anti-plaque Juice Thi Thanh Hanh NGUYEN1, Jaeyoung CHO1, Sun LEE1,2, Shinhye YU3, Eunbae AN3, Jiyoun KIM3, Namhyeon PARK3, Jungmin HA3, Heejung LIM3, Songhee HAN3, Chunhyung KIM1, Doman KIM1,3 1Research Institute of Bio Food Industry, Institute of Green Bio Science & Technology (GBST), Seoul National University, Pyeongchang-gun, Gangwon-do, 232-916, Korea, 2School of Biological Sciences and Technology, Chonnam National University. 77 Yongbong-ro, Gwangju 500-757, Korea, 3Graduate School of International Agricultural Technology, 1447 Pyeongchang-daero, Gangwon-do, 232-916, Korea Sugar-sweetened beverages contribute to weight gain and increase type 2 diabetes and cardiovascular risks independently of obesity because of their added-sugar content, low satiety, and potential incomplete compensation for total energy, leading to increased energy intake. A simpler and cost-effective way is to consume foods with reduced energy while maintaining taste and nutrition to reduce the amount of energy consumed per day. In this study, over 99% of sucrose in mandarin juice (57.1 g·l−1 in original juice to 428.4 g·l−1 in concentrated juice) was enzymatically converted to glucooligosaccharides using 3 U·ml −1 glucansucrase prepared from Leuconostoc mesenteroides at 28°C. The oligosaccharide synthesis yields were 50.7% and 46.6% for the original and the concentrated mandarin juice, respectively. The degree of polymerization of oligosaccharides in the enzyme-modified juice was 2–7. Calories in the original and modified mandarin juice were 432.6 kcal·l−1 and 300.7 kcal·l−1 (30.5% reduction). Compared with the original juice, the enzyme-modified juice showed 81.6% inhibition of insoluble glucan formation by mutansucrase from Streptococcus mutans. A sensory evaluation of the juices revealed that the original and modified mandarin juices had sweetness values of 4.5 and 4.9 for and the same values for overall acceptability. Keywords : Leuconostoc mesenteroides, Low calorie, mandarin juice, oligosaccharides, Glucansucrase References 1. T.T.H. Nguyen, J.Y. Cho, Y.S. Seo, H.J. Woo, H.K. Kim, G.J. Kim, D.Y. Jhon, and D. Kim, Biotechnol. Lett. 37, 711 (2014). C305A Marine-derived Collagen-like Protein for Durable Hydrogel Component Yun Jung YANG, Dooyup JUNG, Hyung Joon CHA 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 Collagen has been considered as a promising component of protein-based scaffolds due to its abundance and wide distribution in our body. As the primary component of the ECM, collagen provides adequate mechanical supporting and space for facilitating cell growth and tissue regeneration. However, the commercialized collagens were usually extracted from animal’ tissues, and therefore, it is not economical and safe from graft rejection or disease transmission. In this study, the new collagen-like protein (named collane) derived from sea anemone’s skin was produced as a recombinant protein, and fabricated into durable hydrogel through photo-initiated dityrosine crosslinking method. The presented mechanical strength of collane hydrogel was comparable with wet collagen. In addition to superior mechanical resemblance with collagen, collane hydrogel was able to control a wide range of mechanical strength and stiffness by changing crosslinking density. The tuning mechanical property of hydrogel would be also advantageous not only for microenvironment construction and mechanical property diversity, but also for controlling biological properties. Keywords : Collagen-like protein, Hydrogel, Sea anemone, Photo-crosslink, Mechanical property References 1. Yang, Y.J.; Choi, Y.S.; Jung, D.; Park, B.R.; Hwang, W.B.; Kim, H.W.; Cha, H.J. Production of a novel silk-like protein from sea anemone and fabrication of wet-spun and electrospun marine-derived silk fibers. NPG Asia Materials. 2013, 5, e50. D301Environmental Application of Bacillus subtilis Spore Display System Wooil KIM, Jonghwa PARK, Junehyung KIM Department of Chemical Engineering, Dong-A University, Pusan, 604-714 Surface display technology has been acknowledged as a useful tool in various fields of biotechnological application. Phage display and bacterial cell surface display are such well-developed technologies to screen biomolecules based upon affinity binding and catalytic activity. Similarly, ribosome display and mRNA display, which can easily increase the number of library pool without using the surface display techniques, were recently developed for screening of peptide ligand and antibody affinity maturation. Here, we report the environmental application of Bacillus subtilis spore based display system for synthetic industrial dyes decomposition using laccases having multi-copper oxidase activity. To express laccase protein on the surface of the spores, several anchoring motives such as CotE, CotG, CotY, and CotZ were used. His6 tag was added at the C-terminal of each laccase. The spore surface expression of target protein, CotA, was confirmed by flow cytometry using FITC labelled anti- His6 antibody. Synthetic dye decolorization was tried using constructed spore displayed laccase, CotA. We confirmed the decolorization of representative industrial dyes such as Indigo carmine, Acid red 18, and Acid green 25. And further possible applications will be discussed. Keywords : Bacillus subtilis, Surface display, Spore, Laccase, Anchoring motif References 1. S. S. Sidhu, Curr. Opin. Biotechnol. 11, 610 (2000). 2. G. Georgiou, C. Stathopoulos, P. S. Daugherty, A. R. Nayak, B. L. Iverson, and R. Curtiss, Nature Biotechnol. 15, 29 (1997). 3. J. Kim and W. Schumann, Cell Cell. Mol. Life Sci. 66, 3127 (2009) D302Antioxidant activity of three Paeonia lactiflora species flower extracts LEE HA-JUNG1, LEE EUN-JEONG2, Choi YONG-BOCK3, Kim CHANG-HO1, LEE SEH-YOON1, Park SUK-KEUN1, KIM JUNE-HYUN4 1Dept. of Biotechnology and bioscience, Univesity of Suwon, Hwaseong, Kyunggi-do, 445-743, 2Dept. of Biotechnology and bioscience, Univesity of Suwon, Hwaseong, Kyunggi-do, 445-743, 3Dept. of Biotechnology and bioscience, Univesity of Suwon, Hwaseong, Kyunggi-do, 445-743, 4Dept. of Biotechnology and bioscience, Univesity of Suwon, Hwaseong, Kyunggi- do, 445-743, 5Dept. of Chemistry, Univresity of Suwon,Hwaseong, Kyunggi-do, 445-743, 6Iksu R&D Center,Iksu Pharmaceutical CO. ,LTD, 2-3 FL,315, Gucheonmyeon-ro,Gandong-gu, Seoul, 134-050, 7Korea Flower Tea Associations, Jayang3-dong,Gwangin-gu, Seoul, 227-131 This study was conducted to determine the antioxidant potentials of the three Paeonia lactiflora species flower. The antioxidant activity was evaluated by determining the antioxidant activity reducing powers and 2,2-diphenyl-1- picrylhydrazyl (DPPH) scavenging activities of water and 70% ethanol extracts from Paeonia lactiflora cv.'Coral Sunset', Paeonia lactiflora cv.'Kan Sas' and Paeonia lactiflora cv.'Red Charm'. The DPPH radical scavenging activity of the extracts was 87.9-90.4% at 0.1mg/ml. This result showed that these extracts have about approximately 10-fold more stronger antioxidant potential than a previous report with root extracts [1]. In addition, there were no significant differences in antioxidant activity among the three Paeonia lactiflora species tested in this study. Taken together, this suggests that the further study with these three Paeonia lactiflora species extracts might provide a basis to develop natural preservatives for cosmetics and foods in the near future. References Bang et al., Kor. Soc. Agric. Chem. Biotechnol. 42, 170 (1999). Keywords : Antioxidant, DPPH radical scavenging , flower extracts, Cosmetics. Paeonia lactiflora, cosmetics References 1. Bang et al., Kor. Soc. Agric. Chem. Biotechnol. 42, 170 (1999). D303Targeted Polymeric Nanoparticles for Antifungal Drug and Antitumor Gene Delivery MI-Kyeong JANG, Seong-Cheol PARK, Nam-Hong KIM Dept. of Polymer Science and Engineering, Sunchon National University, Suncheon, 540-950 The specific tissue targeting has been considered as an important factor for design of drug or gene carriers. Especially, polymeric nanoparticle based on peptide is emerging as a promising effective targeting inducing recptor-mediated endocytosis process. In particular, ligand molecules targeting receptors which are overexpressed in pathogen or tumor cells provides immense opportunities for the concentrative delivery of bioactive cargos into targeting cells and the minimum toxicity in normal cells. Among targeting ligands, cholecalciferol binds with vitamin D receptor which is over-expressed onto ovarian cancer cell surface (1). Therefore, the conjugation of a pathogenic cell-specific ligand molecules to polymeric nanoparticles is an important strategy in the design of drug delivery carrier. The present studies describe a notable use of antifungal peptide as targeting ligand toward fungal pathogen and the applications of new ligands in tumor gene therapy. This work (Grants No. C0276732) was supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2015. References C. Jean-Pierre, K. Guruswamy, and M. Yves, Biochimica et Biophysica Acta(BBA)-Biomembrane, 1617, (2003) Keywords : targeting ligand, peptide, polymeric nanoparticle, gene therapy References 1. C. Jean-Pierre, K. Guruswamy, and M. Yves, Biochimica et Biophysica Acta(BBA)-Biomembrane, 1617, (2003) D304Coacervate-Mediated Dual Growth Factor Delivery for Scar Reduction using in vivo Skin Flap Model Kyobum KIM5 1Minsuk Lee, Department of Nanobiomedical Science, Dankook University, Cheonan, Korea, 2Taufiq Ahmad, Department of Bioenginering, Hanyang University, Seoul, Korea, 3Heungsoo Shin, Department of Bioenginering, Hanyang University, Seoul, Korea, 4Heesok Yang, Department of Nanobiomedical Science, Dankook University, Cheonan, Korea, 5Kyobum Kim, Division of Bioengineering, Incheon National University, Incheon, Korea A series of previous in vivo studies demonstrated that self-assembled polymeric complex using polycations (poly(ethylene argininylaspartate diglyceride) (PEAD) synthesized using aspartic acid) effectively deliver the heparin-binding growth factors. Effective delivery of heparin-binding growth factors using this coacervate complex will be a promising protein therapeutic strategy. The tertiary structure of growth factor/heparin/polycation has several significant advantages: high encapsulation efficiency, simple fabrication process in aqueous condition, easy usage during medical operations, protection of cargo growth factors in in vivo physiological condition, and sustained release of incorporated growth factors. Therefore, a successful delivery of therapeutic growth factors facilitates skin tissue regeneration and suppresses scar reduction. Scar formation is generally resulted from incision and subsequent sutures during many cosmetic/plastic surgical procedures, C- section, and the treatment of cancer resection. A sequential delivery of transforming growth factor-beta3 and vascular endothelial growth factor using coacervates could be a therapeutic approach to reduce the deposition of excess collagen and subsequent scar formation. The in vivo result demonstrated that a delivery of dual growth factors using coacervate-coated nanofibrous PLGA scaffolds in a mice skin flap model facilitated a collagen remodeling, scar reduction, and early stage of would healing. Keywords : Polycation, Coacervate, Skin Tissue Engineering, Fibrous Scaffold, VEGF, TGFbeta3 D305Ultrasonic Delivery of Silica–gold Nanoshells for Photothermolysis of Sebaceous glands in Humans: Nanotechnology from the Bench to Clinic Byeong Hee HWANG1,2, Dilip PAITHANKAR3, Girish MUNAVALLI4, Arielle KAUVAR5, Jenifer LLOYD6, Richard BLOMGREN3, Linda FAUPEL3, Todd MEYER3, Samir MITRAGOTRI2 1Div. of Bioengineering, Incheon National University, Incheon, 406-772, South Korea, 2Dept. of Chem. Eng., University of California, Santa Barbara, CA, 93106, United States, 3Sebacia, Inc., Duluth, GA 30097, United States, 4Dermatology, Charlotte, NC 28207, United States, 5New York Later & Skin Care, New York, NY 10028, United States, 6Lloyd Dermatology & Laser Center, Youngstown, OH 44512, United States Recent advances in nanotechnology have provided numerous opportunities to transform medical therapies for the treatment of diseases including cancer, atherosclerosis, and thrombosis. Here, we report, through in vitro studies and in vivo human pilot clinical studies, the use of inert, inorganic silica–gold nanoshells for the treatment of a widely prevalent and researched, yet poorly treated disease of acne. We use ~ 150 nm silica–gold nanoshells, tuned to absorb near-IR light and near-IR laser irradiation to thermally disrupt overactive sebaceous glands in the skin which define the etiology of acne-related problems. Low-frequency ultrasound was used to facilitate deep glandular penetration of the nanoshells. Upon delivery of the nanoshells into the follicles and glands, followed by wiping of superficial nanoshells from skin surface and exposure of skin to near-infrared laser, nanoshells localized in the follicles absorb light, get heated, and induce focal thermolysis of sebaceous glands. Pilot human clinical studies confirmed the efficacy of ultrasonically-delivered silica–gold nanoshells in inducing photothermal disruption of sebaceous glands without damaging collateral skin. Keywords : Untrasonic Delivery, Silica-Gold nanoshells, Photothermolysis, Sebaceous glands, Acne, Clinic References 1. D. Paithanker, B. H. Hwang, G. Munavalli, A. Kauvar, J. Lloyd, R. Blomgren, L. Faupel, T. Meyer, and S. Mitragotri, J. Control. Release 206, 30 (2015). D306Mussel Adhesive Protein as a Functional Binder of Xenograft in Bone Regeneration Bong-Hyuk CHOI, Hyo Jeong KIM, Hogyun CHEONG, Hyung Joon CHA 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea Xenograft bone substitutes, such as deproteinized bovine bone mineral (DBBM), have been widely employed as osteoconductive structural materials for bone tissue engineering. However, xenograft bone substitute particles are not well retained at the defect site, their displacement reduces osteoconductivity before bone formation is properly generated. Diverse approaches have been attempted to solve these problems caused by using xenograft particles such as employing a physical barrier (non-resorbable or resorbable materials) or mixing with sticky biomaterials (collagen and hyaluronic acid). However, these strategies are hard to solve the complication of xenograft application. Mussel adhesive protein (MAP) has been paid great attention as biomaterials in tissue and biomedical engineering because of their superior properties, including biocompatibility, formability and surface-independent adhesiveness, even in a wet environment. In this presentation, we evaluated in vivo potential of MAP as a functional binder material for bone regeneration in bone tissue engineering. We observed that two forms of MAP (modified MAP and coacervated MAP) were able to bind DBBM particles easily to make an aggregate, and grafted DBBM particles were effectively retained in a defect in the rat calvaria during the healing period. Importantly, grafting of a MAP-bound DBBM aggregate resulted in remarkably accelerated in vivo bone regeneration and even bone remodeling. Collectively, MAP is a promising functional binder material for xenograft bone substitute-assisted bone regeneration with greatly enhanced osteoconductivity and acquired osteoinductivity. Keywords : Mussel adhesive protein, Xenograft , bone tissue engineering References 1. Choi et. al, Engineered mussel bioglue as a functional osteoinductive binder for grafting of bone substitute particles to accelerate in vivo bone regeneration (2015), Journal of Materials Chemistry B, 3(4), 546-555 한국생물공학회, 생물공학의 동향 : 2015.10 학생구두발표 Oral Presentation OP1001Bacterial Cellulose-Poly (3,4-ethylenedioxythiophene) Polystyrene Sulfonate (BC-PEDOT: PSS) Conducting Composites for Application in Optoelectronic Devices Shaukat KHAN1, Mazhar UL-ISLAM1,2, Muhammad Wajid ULLAH1, Yeji KIM1, Jae Hyun JANG1, Tae Yong NOH1, Joong Kon PARK1 1Department of Chemical Engineering, Kyungpook National University, Daegu 702-701, 2Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah, 211, Oman Highly conducting bacterial cellulose (BC) films were prepared by the ex-situ incorporation of Poly (3, 4- ethylenedioxythiophene) Polystyrene sulfonate (PEDOT:PSS) into the BC matrix. The BC pellicles were immersed into aqueous PEDOT:PSS and the resultant composites were vacuum dried. The structural characterization of the composites was done through X-ray Photoelectron Spectroscopy (XPS), Field Emission Scanning Electron Microscopy (FE-SEM), Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD). XPS confirmed the synthesis, SEM showed uniformity of the composites, FTIR spectra exhibited characteristic bands for both BC and PEDOT:PSS, and XRD indicated a slight decrease in crystallinity during composite preparation. The electrical conductivity of the composites with 31.24 wt% PEDOT:PSS in the BC matrix was 12.17 S/cm. These highly conducting BC-PEDOT:PSS composites are potential candidate for applications in optoelectronic devices. Keywords : Bacterial cellulose, Poly (3, 4-ethylenedioxythiophene) Polystyrene sulfonate, X-ray Photoelectron Spectroscopy, fourier transform infrared spectroscopy (FTIR), optoelectronic References 1. Khan S, Ul-Islam M, Khattak WA, Ullah MW, Park JK,Bacterial cellulose-poly(3,4-ethylenedioxythiophene)- poly(styrenesulfonate) composites for optoelectronic applications(2015), Carbohydrate Polymers, 127, 86-93 OP1002Nanomechanics of Poly(Catecholamine) Coating in Aqueous Solutions Chanoong LIM1, Jun HUANG2, Sunjin KIM3, Haeshin LEE3, Hongbo ZENG2, Dong Soo HWANG4 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784,2Department of Chemical and Materials Engineering, University of Alberta, Edmonton, T6G 2V4,3Department of Chemistry, Graduate School of Nanoscience and Technology (WCU) and KAIST institute NanoCentury, KAIST, Daejeon, 305-701, 4Integrative Biosciences and Biotechnology, POSTECH, Pohang, 790-784 Mussel inspired self-polymerized catecholamine coating has been widely utilized as a versatile coating strategy which works for a variety of substrates. Here, we report the nanomechanical measurement and contribution of primary amine group to poly(catecholamine) coating by using a surface forces apparatus, for the first time. The adhesive strength between poly(catecholamine) layers is 30 times higher than that of poly(catechol) coating. The origin of strong attraction between poly(catecholamine) layers is likely due to p-p stacking (the quadrupole-quardrupole interaction of indolic crosslinks), and cation-p interaction (the monopole-quadrupole interaction between positively charged amine groups and the indolic crosslinks). Keywords : dopamine, norepinephrine, pyrocatechol, catecholamine, SFA, cation-pi, adhesion References 1. H. Lee, S. M. Dellatore, W. M. Miller, P. B. Messersmith, Science, 318, 426-430 (2007). 2. S. M. Kang, J. Rho, I. S. Choi, P. B. Messersmith, H. Lee, J. Am. Chem. Soc. 131, 13224-13225 (2009). 3. Q. Lu, D. X. Oh, Y. Lee, Y. Jho, D. S. Hwang, H. Zeng, Angew. Chem. Int. Ed. 125, 4036-4040 (2013). OP1003Endoglin Depletion and Focal Adhesion Increment Enhance Cell Osteogenesis through Cell Surface Thiol Reduction Hyun Bum KIM1, Ji-Yong KIM1, Su-Hwan KIM1, Nathanial HWANG1,2,3 1School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, 2Institute for Chemical Processing, Seoul National University, Seoul, 151-742, 3NBio Institute, Seoul National University, Seoul, 151-742 Cell-based approaches for skeletal regeneration have been highlighted as a potential avenue to promote osteogenesis. In particular adipose derived mesenchymals stem cells have been shown to present a promising tool for regenerative medicine with its ability to enhance osteogenic differentiation.As with this phenomenon, in this study we have modified the outer cell plasma membrane with mild reducing agent TCEP, to reduce disulfide bonds on the membrane and resulted in depletion of endoglin protein and accrue cell focal adhesion. Reduction of endoglin is likely involved in osteogenic differentiation by lowering TGF-β1/Smad2 signaling, which were activated during bone resorption by osteoclasts. Also, Runx2, which is crucial for regulating the expression of bone-specific genes, is increased in early stage of osteogenesis by reducing TGFβ/HDAC signaling. In addition, greater numbers of focal adhesions are known to enhance osteogenic differentiation. Overall, we hypothesize our cell surface modification strategy may be applied to enhance cell osteogenesis. Keywords : Osteogenesis, Endoglin, Focal adhesion, Thiol reduction, Cell surface engineering References 1. Benjamin Levi, CD105 Protein Depletion Enhances Human Adipose-derived Stromal Cell Osteogenesis through Reduction of Transforming Growth Factor 1 (TGF-1) Signaling (2011), J. Bio. Chem, vol. 286, pp. 39497– 39509 2. Per Anderson, CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities (2013), PLos ONE, 8(10): e76979 3. Eun-Joo Jeon, Bone Morphogenetic Protein-2 Stimulates Runx2 Acetylation (2006), J. Bio. Chem, vol. 281, pp. 16502–16511 OP1004Bioinformatical Sequence Analysis of Spider Silk Proteins: Unearthing Their Relations with Evolution and Mechanical Properties Dooyup JUNG, Yun Jung YANG, Hyung Joon CHA Department of Chemical Engineering, POSTECH, Pohang, 790-784 Spider silks, which have outstanding mechanical strengths and extensibilities, are used for constructing webs for catching prey or protecting eggs in nature. Previous researches have investigated that mechanical properties of spider silks have been diversified while spiders have been diverged to various species. Because main components of spider silks are proteins and amino acid sequence is the most fundamental information of protein, it is valuable to find a correlation between spider silk protein sequences with mechanical properties and evolutionary steps. However, spider silk protein sequences were mostly regarded as a hard information to be analyzed due to its imperfectness and repetitiveness. Therefore, in the present work, bioinformatical tools, motif counting and graph-based method, were newly employed to systemically analyze group of silk protein sequences from different spider species. Filtered and aligned motifs resulted from this analysis were drawn interest because every time new types of spiders had been emerged, novel motifs with three amino acids were appeared. The result could be further used for identification of phylogenic relations among spiders and prediction of mechanical properties of different types of silks. These serial process can be a platform technology and applicable for analyzing other group of protein sequences which are thought to be evolutionarily-and-functionally-related. Keywords : Spider silk protein, Bioinformatics, Evolution, Mechanical property References 1. Blackledge, T.A. et al., Sequential origin in the high performance properties of orb spider dragline silk (2012), Scientific Reports, 2 (782), 1-5 OP1005Hyaluronic Acid-Based Electroconductive Hydrogel for Neural Stem Cell Engineering Jisoo SHIN1, Eun Jung CHOI2, Jung-Ho CHO1, Changsik SONG2, Seung-Woo CHO1 1Department of Biotechnology, Yonsei University, Seoul, South Korea, 2Department of Chemistry, Sungkyunkwan University, Suwon, Kyunggi-do 440-746, South Korea The electrically conductive hydrogel can provide simultaneously three-dimensional (3D) matrix and electrical stimulation for neural stem cell (NSC) differentiation. Enhanced electrical conductivity could promote neuronal differentiation of NSCs by generating electrical shortcuts. Thus, the electrical conductive hydrogel could provide more suitable microenvironment for NSCs to differentiate into neuronal lineage. In this study, we developed the electrically conductive hydrogel by incorporating single-walled carbon nanotube (SWNT) into catechol conjugated hyaluronic acid (HA-CA) hydrogel for neuronal differentiation of human NSCs. The resulting SWNT-HA-CA hydrogel showed more improved elastic modulus and enhanced electrical conductivity than bare HA-CA hydrogel. To increase the conductivity effect of the hydrogel, we additionally incorporated pyrrole, a conducting polymer, into SWNT-HA-CA hydrogel. Consequently, the improved electrical conductivity of SWNT-HA-CA hydrogel led to enhanced neuronal differentiation of human NSCs. In addition to these mechanical and electrical advantages, SWNT-HA-CA hydrogel provided biocompatibility suitable for human NSCs. Therefore, hyaluronic acid-based hydrogel incorporated with electroconductive materials could provide a therapeutic approach for neurodegenerative diseases. Keywords : Hydrogel, Carbon nanotube, neural stem cell References 1. Eunji Lee, Jiyoung Park, Sung Gap Im* and Changsik Song*, Synthesis of single-walled carbon nanotube- incorporated polymer hydrogels via click chemistry (2012), Polym. Chem. 3, 2451-2455 OP1006Where Stiff Meets Soft: Sweetness Mitigates Contact Damage Hee Young YOO1, Jun HUANG2, Lin LI2, Mathias FOO3, Hongbo ZENG2, Dong Soo HWANG1,2 1Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea, 2Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2V4, Canada, 3School of Engineering, University of Warwick, Coventry, CV4 7AL, United Kingdom, 4School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. The byssal threads of fan shell mussels (Atrina pectinata) once served as a surrogate fiber for fine fabrics in Western civilizations long before the introduction of silkworm silk by the Silk Road. Like byssal threads in other mussel species, Atrina threads provide a holdfast and are composed of proteins containing the catecholic amino acid 3,4- dihydroxyphenylalanine (DOPA). Atrina threads differ from other mussel threads in being deeply rooted to the byssal adductor muscle, thus passing through much of the body before emerging from the shell. Given that byssal threads are nonliving functional materials intimately associated with living tissue, Atrina byssus provides an intriguing paradigm of a bionic interface for a robust load bearing device. An interfacial load bearing protein with both DOPA-containing and mannose-binding domains (Atrina pectinata Foot protein-1, apfp-1) has been characterized from the fan shell foot. Apfp-1 was localized at the interface between the byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between byssus and the soft tissue, i.e., DOPA containing domain interacts with the byssus via Fe(III)-DOPA complexes and a mannose binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated binding is reversible and robust under wet conditions. The combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices. Keywords : atrina, apfp-1, byssus References 1. Sun C, Lucas JM, Waite JH (2002) Collagen-binding matrix proteins from elastomeric extraorganismic byssal fibers. Biomacromolecules 3(6):1240-1248. OP1007Fatty Acid Production from Various Marine Microalgae Using Light-Emitting Diodes (LEDs) as Light Source Jang Hyun JUNG, Chae Hun RA, Hyo Seon KIM, Yong Woo KIM, Sung-Koo KIM Department of Biotechnology, Pukyoung National University, Busan 608-737, Korea Omega-3 fatty acids have received growing attention due to their significant roles in human health. Currently the main source of these nutritionally and medically important fatty acids is marine fish, which could not meet increasing global demand. Marine microalgae are an important alternative source to produce those fatty acids. In this study, Dunaliella teritolecta, Phaeodatylum tricornutum, , Nannochloropsis oculata, Nannochloropsis salina, Isocrhysis galbana was cultured in F/2 media and blue LED wavelength (465 nm) and Fluorescent light, 12:12 LD cycle, 20℃ and 100 μmol/m2/s light intensity. Studies on the biomass production of various marine microalgae using nitrate concentrations as growth factors were carried out to evaluate optimal culture conditions in 1L batch bioreacter. The oil accumulation was also evaluated with two stage culture using salt stress and nitrate stress. The dry cell weight and oil accumulation were determined and fatty acid composition of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and various fatty acid of microalgae were evaluated. Keywords : Omega-3, Fatty acid, Marine microalgae, LED, F/2 media References 1. . Seok-Joo Lee, Sugeun Go, Gwi-Taek Jeong, and Sung-Koo Kim, Oil Production from Five Marine Microalgae for the Production of Biodiesel (2011). Biotechnology and Bioprocess Engineering, 16, 561-566 2. Sugeun Go, Seok-Joo Lee, Gwi-Taek Jeong and Sung-Koo Kim. Factor affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica (2012). Bioprocess Biosystems Engineering, 35, 145-150 3. Yangmin Gong, Xia Wan, Mulan Jiang, Chuanjiong Hu, Hanhua Hu, Fenghong Huang. Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids (2014). Progress in lipid research , v.56 , pp.19 - 35 OP1008Nanocomplexation of Curcumin with Proteins and Its Implications on Biological Activity Md. Abdur RAZZAK1, Shin Sik CHOI1,2 1Department of Energy Science and Technology,Myongji University, Yongin, Korea., 2Department of Food and Nutrition, Myongji University, Yongin, Korea. Curcumin (CU), a phenolic compound, originated from Curcuma longa (turmeric), has been widely used as spice or food colorant, nutritional supplement and herbal medicine in many Asian countries for thousands of years. In recent years, it has attracted extensive research attention, mainly due to its beneficial functional properties, such as antioxidant, anticancer, anti- inflammatory, antimicrobial, wound healing and potential prevention ability of neurodegenerative diseases. However, poor bioavailability caused by its low aqueous solubility and stability is considered as a major barrier for its fortification into food or pharmaceutical products. In this study, we fabricated CU-protein nanocomplexes to improve its stability and bioavailability. As a result, CU formed nanocomplexes with ovalbumin (OA); a predominant protein in egg white mainly through hydrophobic intermolecular interactions. Spectroscopic studies also revealed that about 90% of CU in CU-protein nanocomplexes remained stable after 10 hours of incubation, which would be sufficient time for its intestinal absorption and conferring its maximum medicinal functionalities. These stable CU-protein nanocomplexes have a great potential for their integration into functional food and pharmaceutical products. Keywords : Curcumin, Proteins, Stability References 1. Naksuriya, O., Okonogi, S., Schiffelers, R. M., & Hennink, W. E, Biomaterials, 35(10), 3365 (2014). 2. Li, M., Ma, Y., & Ngadi, M. O, Food Chemistry, 141(2), 1504 (2013). OP1009Dopamine-Mediated Sclerotization of Regenerated Chitin in Ionic Liquid Suyoung LEE, Dongsoo HWANG Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 790-784 Chitin is a promising structural material for biomedical applications, due to its many advantageous properties and abundance in nature. However, its usage and development in the biomedical field have been stagnant, because of chitin’s poor mechanical properties in wet conditions and the difficulties in transforming it into an applicable form. To overcome these challenges, we created a novel biomimetic chitin composite. This regenerated chitin, prepared with ionic liquid, showed improved mechanical properties in wet conditions by mimicking insect cuticle and squid beak sclerotization, i.e., catechol- meditated cross-linking. By ionic liquid-based heat treatment, dopamine oxidation produced melanin-like compounds and dopamine-meditated cross-links without any solvent evaporation and oxidant utilization. The dopamine-meditated sclerotization increased the ultimate tensile strength (UTS) of the regenerated chitin by 2.52-fold, measured after six weeks of phosphate-buffered saline (PBS) submersion. In addition, the linear swelling ratio (LSR) of the chitin film was reduced by about 22%. This strategy raises a possibility of using regenerated chitin as an artificial hard tissue in wet conditions. Keywords : dopamine , regenerated chitin, sclerotization, water-absorption, mechanical properties, ionic liquid References 1. European Chitin Society. Advances in Chitin Science; Universität Potsdam: Potsdam, Germany, 2000. 2. Nishino, T.; Matsui, R.; Nakamae, K. Elastic modulus of the crystalline regions of chitin and chitosan. J. Polym. Sci. B Polym. Phys. 1999, 37, 1191–1196 3. Pillai, C.K.S.; Paul, W.; Sharma, C.P. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog. Polym. Sci. 2009, 34, 641–678. 4. Qin, Y.; Lu, X.; Sun, N.; Rogers, R.D. Dissolution or extraction of crustacean shells using ionic liquids to obtain high molecular weight purified chitin and direct production of chitin films and fibers. Green Chem. 2010, 12, 968–971. 5. Prasad, K.; Murakami, M.A.; Kaneko, Y.; Takada, A.; Nakamura, Y.; Kadokawa, J. Weak gel of chitin with ionic liquid, 1-allyl-3-methylimidazolium bromide. Int. J. Biol. Macromol. 2009, 45, 221–225. OP1010Pyrogallol/Metal Ion Complex for the Instant and Efficient Dental Sensitivity Treatment by Tunicate-Inspired Mineralization Strategy Ekavianty PRAJATELISTIA1, Dong Soo HWANG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea., 2Integrative Bioscience and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. Commercially available dental desensitizers are generally effective only on a specific and localized area and their performance depends on a clinician's skill. Herein, tunicate-mimetic mineralization strategy has been employed to treat dental sensitivity by a simple rinse with an aqueous solution of the Tannic acid (TA) or gallic acid (GA) with metal ion to make them as a complex. As pyrogallol groups of tunicate make conjugation with the metal ion to heal the torn body armor, we mimicked the ingenuity mechanism by optimizing TA or GA as an alternative of pyrogallol groups of the tunicate. The human molars disc was treated with /metal ion solution for 4 min followed by 7 days remineralization in artificial saliva. Morphological studies confirmed the dentinal tubules remarkably occluded with densely packed remineralized hydroxyapatites (HA) with the high degree of regularity and homogeneity. We found that the GA/Fe3+ complex exhibited promising results to induced hydroxyapatite nucleation on dental slices and facilitated dentin remineralization resulting instant 57% blockage in tubules within 4 min and 89% was observed after 7days immersion in artificial saliva, which was nearly two-fold greater than previously reported desensitizing solution. We developed a patient-friendly, efficient and instant dental hypersensitivity treatment inspired by the biological system. Keywords : Dental hypersensitivity, Gallic acid, Tannic acid, TOPA References 1. Oh, D. X.; Prajatelistia, E.; Ju, S.-W.; Jeong Kim, H.; Baek, S.-J.; Joon Cha, H.; Ho Jun, S.; Ahn, J.-S.; Soo Hwang, D, Sci. Rep.5, (2015). 2. Zhou, Y.-Z.; Cao, Y.; Liu, W.; Chu, C. H.; Li, Q.-L, ACS Applied Materials & Interfaces, 4 (12), 6901-6910, (2012). 3. Ejima, H.; Richardson, J. J.; Liang, K.; Best, J. P.; van Koeverden, M. P.; Such, G. K.; Cui, J.; Caruso, F., Science (New York, N.Y.), 341 (6142), 154-157, (2013) OP1011Development of Auto-inducible Gene Expression Xystem inCorynebacterium glutamicum. Min Jeong KIM, Sung Sun YIM, Jae Woong CHOI, Ki Jun JEONG Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141 Corynebacterium glutamicum is a Gram-positive bacterium that has been used for industrial production of various products. To enhance its productivity, many techniques have been developed to control the expression of the target genes. One of the most effective techniques is engineering of the promoter. Among the various types of promoters, auto-inducible promoters have advantages in industrial-scale use because they allow controlled regulation of gene expression at desired point without adding inducers. However, no suitable auto-inducible promoters have been developed in C. glutamicum. In this study, we developed auto-inducible gene expression system that turn on at the transition phase between exponential phase and stationary phase in C. glutamicum. We created synthetic promoter libraries based on C. glutamicum native promoter and isolated auto-inducible promoters with different strengths and induction ratios using fluorescence-activated cell sorter. The expression of the isolated promoters were verified with the production of glutathione S-transferase. The isolated promoters exhibited up to 30-fold increased expression level compared to the original promoter. Keywords : Corynebacterium glutamicum, auto-inducible promoter, synthetic promoter, FACS OP1012Production and Structural Diversification of Select Secondary Metabolites from Actinomycetes sps. Using Metabolic Engineering and Synthetic Biological Tools Dipesh DHAKAL, Jae Kyung SOHNG Institute of Biomolecule Reconstruction, Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea. Actinomycetes produces various secondary metabolite (SM)[1].Nargenicin A1, an antibacterial polyketide produced by Nocardia sp.CS682, was enhanced by rational metabolic engineering and subsequently glycosylated by efficient one vessel reaction systems containing glycosyltransferase (GT) from Bacillus licheniformis. The novel products were structurally characterized[2]. In another study, using multi-monocistronic vector containing user defined parts and subsequent precursor refactoring strategy the titer of nargenicin A1 was significantly enhanced. Further the system was modified to generate hydroxylated nargenicin A1 using substrate flexible CYP 450, PikC. Herboxidiene, a herbicidal cum antitumor compound produced by Streptomyces chromofuscus was structurally modified by hydroxylation/epoxidation/glycosylation by using different substrate-flexible cytochrome P450s and glycosyltransferase[3]. Pradimicin A is an angucyclic antifugal antibiotic produced by Actinomadura hibisca P157-2(ATCC53557). By insertional inactivation of two glycosyltransferase, pdmQ and pdmS,the exact order of glycosylation was determined alongwith generation of novel analogues[4].Thus, by utilizing different metabolic engineering and synthetic biological tools, purposive enhancement or structural diversification were achieved from different Actinomycetes. Keywords : Secondary metabolites, Actinomycetes, Synthetic Biology, Metabolic Engineering, Enhancement, Modification References 1. Chaudhary, A. K., Dhakal, D., & Sohng, J. K. (2013). An insight into the “-omics” based engineering of streptomycetes for secondary metabolite overproduction. BioMed research international, 2013: 968518 2. Dhakal, D., Le, T. T., Pandey, R. P., Jha, A. K., Gurung, R., Parajuli, P., Pokhrel, A.R., Yoo, J.C. & Sohng, J. K. (2015). Enhanced Production of Nargenicin A1 and Generation of Novel Glycosylated Derivatives. Applied biochemistry and biotechnology, 175: 1-16. 3. Jha, A. K., Dhakal, D., Van, P. T. T., Pokhrel, A. R., Yamaguchi, T., Jung, H. J., Yoon, Y.J., & Sohng, J. K. (2015). Structural modification of herboxidiene by substrate-flexible cytochrome P450 and glycosyltransferase. Applied microbiology and biotechnology, 99:3421-3431. 4. Jha, A. K., Paudel, S., Dhakal, D., Van, P. T. T., Ghimire, G. P., & Sohng, J. K. (2015). Genetic evidence for the involvement of glycosyltransferase PdmQ and PdmS in biosynthesis of pradimicin from Actinomadura hibisca. Microbiological Research, 174: 9-16. OP1013Integrated Approach for Metabolic Engineering Using Rational and Evolutionary Synthetic Biology Tools in Escherichia coli for Production of L-Tryptophan SungHo JANG1, Sang Woo SEO1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, 2School of Interdisciplinary Bioscience and Bioengineering , Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, Korea Metabolic engineering of microorganisms has been traditionally relied on rational approach based on detailed knowledge of the producing host and the target pathway. However, combinatorial approach also has been proving its ability in metabolic engineering by utilizing nature’s genuine mechanism, diversification and selection. Current limitations of rational and combinatorial approach are fine and predictive expression of pathway enzymes and screening of superior and optimized variant out of vast library, respectively. Here, we present an integrated synthetic biology approach which settled the problems of rational and combinatorial approach to produce L-tryptophan from Escherichia coli. First, we removed known bottlenecks on L-tryptophan synthesis pathway by using a predictive model of protein expression level, UTR Designer, which can precisely predict expression level of enzymes based on folding energy of specific features in mRNA structure. UTR sequences of bottleneck enzymes were re-designed to maximize the metabolic flux. Then we optimized the producing strain by utilizing a riboswitch-based screening tool, riboselector, which modulates expression level of selective marker gene in response to the target molecule. Riboselector for L-tryptophan was able to select superior variants out of vast library which were constructed from rationally engineered strain. We claim here that efficient producing strains could be obtained through synthetic biology approach by resolving current problems of metabolic engineering. Keywords : Metabolic Engineering, Synthetic Biology, Escherichia coli, L-tryptophan References 1. Seo, S.W. and Yang, J.S. et al., Metab. Eng., 15, 67-74., (2013). 2. Yang, J. and Seo, S.W.et al., Nat. Commun., 4, 1413. (2013). OP1014Cell-Free System: A Highly Productive Approach for Bio-cellulose Synthesis Muhammad Wajid ULLAH1, Mazhar UL-ISLAM1,2, Shaukat KHAN1, Yeji KIM1, Jae Hyun JANG1, Tae Yong NOH1, Joong Kon PARK1 1Department of Chemical Engineering, Kyungpook National University, Daegu 702-701, 2Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah 211, Oman Microbial cellulose represents the most pure form of cellulose produced by a class of acetic acid bacteria. Its production by microbial cells encounters several limitations that lower the overall yield. Currently, cell-free technology has received immense interest in production of various biocommodities. In the current study, we have developed a Gluconacetobacter hansenii PJK based cell-free system via bead beating for bio-cellulose synthesis. The efficacy of beating process was analyzed via colony forming unit (CFU) and microscopic analysis. The presence of essential enzymes and cofactors in the cellular lysate was confirmed through liquid chromatography–mass spectrometry/mass spectrometry linear trap quadrupole (LC-MS/MS LTQ) orbitrap and assay kits, respectively. The system successfully produced bio-cellulose at a higher yield compared to microbial cells. Further, CFU analysis and microscopic observations confirmed the complete absence of any microbial cells indicating bio-cellulose production by cell-free enzymes only. The developed system could effectively address the limitations associated with cellulose production by microbial cells. Keywords : Cell-free system, Bead beating, Characterization, Bio-cellulose synthesis References 1. Muhammad Wajid Ullah, Waleed Ahmad Khattak, Mazhar Ul-Islam, Shaukat Khan, Joong Kon Park. Bio-ethanol production through simultaneous saccharification and fermentation using an encapsulated reconstituted cell-free enzyme system. (2014) Biochemical Engineering Journal , 91,110–119. 2. Muhammad Wajid Ullah, Waleed Ahmad Khattak, Mazhar Ul-Islam, Shaukat Khan, and Joong Kon Park. Encapsulated Yeast Cell-free System: A Strategy for Cost-effective and Sustainable Production of Bio-ethanol in Consecutive Batches.(2015) Biotechnology and Bioprocess Engineering, 20 , 561-575. OP1015Systems Level Metabolic Engineering of Escherichia coli for Enhanced Production of L- Phenylalanine Yoon Hyeok LEE1, Hyun Bae BANG1, Ki Jun JEONG1,2 1Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, Republic of Korea, 2Institute for the BioCentury, KAIST, Daejeon 305-701, Republic of Korea L-phenylalanine (L-Phe) is one of the important amino acid which has 5th largest market in world amino acid market. The market size of L-Phe has been gradually increased by considerable demand for the aspartame, food additives and pharmaceuticals. In this study, we successfully developed the E. coli strain can overproduce L-Phe by systems level metabolic engineering: (1) Moderating the rate of substrate uptake to reduce the metabolite overflows; (2) Gene deletion to alleviate the tight regulation of aromatic amino acid synthesis related regulon; (3) Overexpression of enzymes to improve the carbon flow through the shikimate pathway; (4) Gene deletion to prevent loss of carbon flow towards competing pathway; (5) Gene deletion to enrich the precursor and balance the flux between growth and L-Phe production. In the flask and fed- batch fermentation, the final recombinant E. coli mutant revealed the good performance in cell growth and much enhanced titer of L-Phe compared with parental strain. By applying the systems metabolic engineering strategies described in here, the wild type strain and the other hosts also can be from zero to hero strain in L-Phe production Keywords : Metabolic engineering, Escherichia coli, L-Phenylalanine References 1. Zhou et.al, Enhanced L-phenylalanine production by recombinant Escherichia coli BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach (2011), J Ind Microbial Biotechnol, 38:1219-1227 2. Liu et.al, A systems level engineered E. coli capable of efficiently producing L-phenylalanine(2014), Process Biochemistry 49:751–757 3. Rodriguez et.al, Engineering Escherichia coli to overproduce (2014), Microbial Cell Factories, 13:126 OP1016Improved Enzyme Digestibility of Miscanthus by a Two-Stage Process Using Gaseous Anhydrous Ammonia and Hot Water Roent Dune CAYETANO Department of Environmental Engineering, Kongju National University, Cheonan Miscanthus, lignocellulosic biomass, is currently considered to be a promising energy crop for cellulosic biofuel production because of its rapid growth, high carbohydrate content, and low ash content[1]. However, biomass conversion into biofuel is still challenging because it has various chemical and physical barriers. These barriers in biological conversion of biomass can be overcome by adoption of adequate pretreatment process. Therefore, effective pretreatment is necessary for achieving a high efficiency in the biomass conversion into fermentable sugars and subsequently biofuels and other value added products. A two-stage process for the pretreatment of Miscanthus was investigated with the aim of increasing sugar recovery and fractionation of each component. In the first stage, Miscanthus was treated with gaseous ammonia at moderate temperature. Then, the ammonia treated Miscanthus was subjected into the second stage which uses hot-water in a flow-through column reactor. After two-stage processing, remaining solid contains mostly glucan (cellulose), which becomes highly digestible by enzyme. In this paper, the effects of various treatment conditions are evaluated in terms of compositional changes. The enzymatic digestibility tests are also conducted for the treated solids under various treatment conditions. Other technical aspects pertinent to development of pretreatment and fractionation process are presented. Keywords : pretreatment, ammonia, hot water, enzyme digestibility, miscanthus References 1. H-Q.Li et al., Pretreatment on Miscanthus lutarioriparious by liquid hot water for efficient ethanol production (2013), Biotechnols Biofuels. 6:76 , 1-10 OP1017Utilization Seaweed Waste from Gwangalli, Busan as the Biomass for Ethanol Production InYung SUNWOO, ChaeHun RA, JungEun KWON, Sung-Koo KIM Dept. of Biotechnology, Pukyong National University, Busan, 608-737, Korea When the typhoon was hit in Korea, large amount of seaweeds are dumped on the beach Gwangalli, Busan, Korea. To dispose of seaweed waste efficiently, bioethanol production processes were carried out with thermal acid hydrolysis, enzyme treatment and fermentation of the mixture of seaweeds. Among the biomasses, Sargassum hulvellum comprised most of the biomass as 72%. Optimal pretreatment conditions were 8% slurry contents, 1.5% sulfuric acid concentration and 60 min of treatment time for the thermal acid hydrolysis of mixture of seaweeds obtained in the beach (10 species). To obtain high monosaccharide, enzyme treatment was carried out with 16 unit/ml of Viscozyme L for 48 hours. After pretreatment and enzyme treatment, 28 g/L of monosaccharide was obtained. The ethanol fermentation was performed with Pichia angophorae KCTC 1757, Pichia stipitis KCTC7228 and Saccharomyces cerevisiae KCCM 1129. The ethanol productions of 10.12 g/L (YEtOH=0.36), 12.49 g/L(YEtOH=0.45) and 11.39 g/L(YEtOH=0.41) were produced using P. angophorae, P. stipitis and S. cerevisiae, respectively. Keywords : Seaweed waste, Thermal acid hydtolysis, Enzyme treatment, Fermentation, Ethanol References 1. M. G. Borines, R. L. Leon, J. L. Cuello. Bioresour. Technol. 138, 22 (2013) 2. H. Rouhollah, N. Iraj, E. Giti, A. Sorah. Afr. J. Biotechnol.6, 1110(2007) OP1018Comparison of NaCl Adapted Yeast and Specific Sugar Adapted Yeast on Bioethanol Production from Gracilaria verrucosa by SHF Trung Hau NGUYEN, Chae Hun RA, Mi-Ra PARK, Sung-Koo KIM Dept. of Biotechnology, Pukyong National University, Busan, 608-737 Bioethanol was produced using the separate hydrolysis and fermentation (SHF) method with macroalgae polysaccharide from the seaweed, Gracilaria verrucosa as biomass. This study focused on the pretreatment, enzyme saccharification and fermentation of brown macroalgae, G. verrucosa. The pretreatment to obtain monosaccharide (mainly galactose) was carried out with 12% (w/v) seaweed slurry and 364 mM H2SO4 at 121℃ for 60 min. A monosaccharide (mainly glucose) was obtained from enzyme hydrolysis further was performed with 16 U/mL of Celluclast 1.5L at 45℃ for 48 h. Ethanol fermentation with 12% (w/v) seaweed hydrolysate was performed using non-adapted and adaptedSaccharomyces cerevisiae KCTC 1126 to high concentrations of galactose and NaCl. When non-adaptedS. cerevisiae was used, the ethanol production of 16.6 g/L with ethanol yield of 0.39 was produced. The ethanol production of 18.8 g/L and 21.2 g/L with ethanol yield of 0.43 and 0.48 were produced using adapted S. cerevisiae to high concentration of galactose and NaCl, respectively. Therefore, adapted S. cerevisiae to galactose or NaCl increased the ethanol yield comparing to that with non- adaptedS.cerevisiae. Keywords : Gracilaria verrucosa, Thermal acid hydrolysis, Enzymatic saccharification, Fermentation, Adaptation References 1. Cho HY, Ra CH, Kim SK. J. Microbiol. Biotechnol. 24, 264 (2014). 2. Ra CH, Jung JH, Sunwoo IY, Kang CH, Jeong GT, Kim SK. J. Microbiol. Biotechnol. 25, 856 (2015). OP1019Isolation of 1,3-Propanediol Local Producers from Palm Oil Mill Effluent (POME) and Biodiesel Wastewater Sludge ZHAO KANG TEE1, JAMALIAH MD JAHIM1, BYUNG HONG KIM2 1Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia., 2Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. The emerging of 1,3-propanediol (PDO) production shows its commodity needs at current market. Polytrimethylene terephthalate (PTT), a type of polyester from PDO and terephtalic acid, contributes its usage as textile, antifreeze and engineering thermoplastic applications. Since the first PDO bio-refinery was commissioned on year 2006 by DuPont Tate & Lyle, transformation of chemical synthesis of PDO has gone into fermentative process. Glycerol serves the feedstock for PDO production, which is excessively produced from bioethanol plant and biodiesel industry. Transesterification process of biodiesel produces one-tenth glycerol mass with every unit mass of biodiesel. Isolation work has been done to identify the PDO local producer obtained from mill effluent and wastewater sludge. Non-pathogenic and the ability to grow on cheaper media are chosen for the isolation criteria. In this study, wastewater from palm oil mill was heat-treated and cultured on glycerol-based media. Purification of isolates are done to obtain one species culture in one media plate. They are then screened for PDO production with various concentration of calcium carbonate. Hydrogen gas, acetic acid, butyric acid and butanol are co-produced throughout the fermentation. Clostridium butyricum JCM1391 and Clostridium pasteurianum DSM525 were determined by 16S rRNA gene sequence. Keywords : 1,3-propanediol, isolation, Clostridium, fermentation, glycerol References 1. Kaur G, Srivastava AK, Chand S, Biochem Eng J 64 106 (2012) OP2001Biosynthesis of Poly (3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Glucose by a Metabolically Engineering Approach Jung Eun YANG1, Seung Hwan LEE2, Bong Keun SONG3, Si Jae PARK4, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 373-1, 2Dept. of Biotechnology and Bioengineering, Chonnam National University, Gwangju 500-757, 3Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, Daejeon, 4Dept. of Environmental Engineering and Energy, Myongji University, Yongin-si 449-728 Due to its biodegradability and biocompatibility, polyhydroxyalkanoate has drawn many interest as a termoplastic. Among the different types of PHA co-polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has lower melting point and better flexibility compared to the 3-hydroxybutyrate. Until now, toxic second auxiliary carbon source is being added for the production of P(3HB-co-3HV). Therefore, metabolically engineered Escherichia coli that can synthesize 3HB-CoA and 3HV-CoA in a stable manner with the ratio being controlled was developed from glucose independent of the feeding of the carbon source.“This work was supported by the Technology Development Program to Solve Climate Changes from National Research Foundation of Korea (Development of systems metabolic engineering platform technologies for biorefineries; NRF-2012-C1AAA001-2012M1A2A2026556) and Intelligent Synthetic Biology Center (2011-0031963) of Korea through the Global Frontier Research Program of the Ministry of Education, Science and Technology (MEST). Further supports by the World Class University program (R32-2008-000-10142-0) of the MEST are appreciated.” Keywords : Metabolic engineering, Biopolymer, Polyhydroxyalkanoates, P(3HB-co-3HV) References 1. Yang, J.E., Choi, Y.J., Lee, S.J., Kang, K.-H., Lee, H., Oh, Y.H., Lee, S.H., Park, S.J., and Lee, S.Y OP2002Enzymatic Carbon Dioxide Conversion and Utilization for Expedited Microalgae Growth Sung-Gil HONG1, Hancheol JEON2, Hansol KIM1, Seung-Hyun JUN1, EonSeon JIN2, Jungbae KIM1,3 1Dept. of Chemical and Biological Engineering, Korea University, Seoul, 136-701, 2Dept. of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, 133-791, 3Green School, Korea University, Seoul, 136-701 Enzymes have gathered attention due to their promising applications [1]. In this study, we developed an enzymatic process for one-pot CO2 conversion and utilization. Carbonic anhydrase was immobilized on carboxylated polyaniline nanofibers (cPANFs) via the approach of magnetically-separable enzyme precipitate coatings (Mag-EPC), which consists of covalent enzyme attachment, enzyme precipitation and crosslinking with amine-functionalized magnetic nanoparticles. Under shaking (200 rpm), Mag-EPC showed a half-life of 236 days, even resistance to 70% ethanol sterilization, and recyclability via facile magnetic separation. For enzymatic CO2 conversion and utilization, Mag-EPC was used to accelerate the growth of microalga by supplying bicarbonate from CO2, and it showed 1.8-fold increase of cell concentration when compared to the control sample. After two repeated uses of Mag-EPC via magnetic separation, cell concentration with Mag-EPC was maintained as high as the first cycle of microalgae cultivation. This process, which demands no energy input, can be an alternative as well as complementary process to adsorption-based CO2 capture and storage as an environmentally-friendly approach. Keywords : Carboxylated polyaniline nanofibers, Magnetic nanoparticles, Enzyme precipitate coatings, Carbonic anhydrase, CO2 conversion and utilization References 1. J. Kim, J. W. Grate and P. Wang. Trends. Biotechnol. 26, 639 (2008). OP2003Inducible Expression of Efflux Pump for Antibiotics Overproduction Min Woo KIM, Byung-Gee KIM Interdisciplinary Program for Biochemical Engineering, Seoul National University, Seoul, Korea Streptomyces is a soil dwelling gram positive bacteria that has the ability to produce bioactive secondary metabolites such as antivirals, antifungals, and antibiotics. Especially, this strain is important in that it produces most natural antibiotics used in human and veterinary medicine. Therefore, there is much interest in enhancing the level of production of theses secondary metabolite Multidrug efflux pump is responsible for expel toxic compounds. It is well reported that efflux pump enhance not only the ability of resistance against toxic compounds but also the level of production of toxic compounds. In this study, we tried to overproduce target secondary metabolite by using multidrug efflux pump overexpression strategy. We overexpressed efflux pump with growth phase specific inducible promoter because, constitutive overexpression of efflux pump has negative effect on cell growth. With this strategy, we could enhance more than 10 fold in the level of production of target secondary metabolites Keywords : streptomyces, efflux pump, antibiotics References 1. Piddock, Laura JV(2006),Nature Review Microbiology,4, 629-636 2. Webber, M. and Piddock, L (2003). Journal of Antimicrobial Chemotherapy, 51, 9-11 3. Wagner, S., Baars, L., Ytterberg, A.J., Klussmeier, A., Wagner, C.S., Nord, O., Nygren, P.A., van Wijk, K.J. and de Gier, J.W.(2007), Molecular & cellular proteomics : MCP, 6, 1527-1550 OP2004Metabolic Engineering of Escherichia coli for 2-Hydroxyacids Containing Polyhydroxyalkanoates (PHAs) So Young CHOI1, Si Jae PARK2, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, 2Dept. of of Environmental Engineering and Energy, Myongji University, Gyeonggido, 449-728 Polyhydroxyalkanoates (PHAs) are bacterial polyesters having biodegradability and biocompatibility with general properties of plastics. To produce various PHAs for wide range of applications, many hydroxyacids are co-polymerized with various ratio. One of them, 2-hydroxyacids are non-natural substrate of PHA synthesis system. Here, we produced lactate (2- hydroxypropionate), 2-hydroxybutyrate and glycolate (2-hydroxyacetate) containing PHAs with evolved PHA synthase from Pseudomonas sp. MBEL 6-19 and propionyl-CoA transferase from Clostridium propionicum. For production from unrelated carbon sources such as glucose or xylose, rational metabolic engineering was done to redirect flux toward lactate and glycolate by introducing heterogeneous metabolic pathway. It resulted in the production of lactate and glycolate containing polymers from glucose and xylose that can substitute the current chemical process. [This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF- 2012M1A2A2026556).] Keywords : Metabolic engineering, Polyhydroxyalkanoates, Escherichia coli , Polylactic acid, Polyglycolic acid, PLGA, 2- hydroxybutyrate References 1. Y.K. Jung, S.Y. Lee, J.Biotechnol 151; 94-101. (2011) OP2005Designing Antibiotics Overproducers through Integrative Analysis of Transcriptomic Data into Metabolic Model Minsuk KIM, Jeong Sang YI, Byung-Gee KIM School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, 151-742 In recent years, genome-scale metabolic models have been widely applied for metabolic engineering. Dozens of in silico strain design algorithms have been developed to identify novel engineering targets from the metabolic models. However, none of the algorithms is able to use high-throughput data for making predictions. In this study, we developed a strain design algorithm which can utilize transcriptomic data, for the first time, to predict better engineering targets. Our new algorithm uses integrative metabolic analysis tool (iMAT) (Zur et al. (2010) Bioinformatics) to integrate transcriptomic data into metabolic model, and then searches for gene overexpression targets which can increase the product yields. We applied this algorithm for designing antibiotics overproducers of Streptomyces coelicolor, and experimentally validated the predictions. In short, by employing the transcriptomic data in integrative manner, we can widen the applicability of strain design algorithms for secondary metabolite overproducer design. Keywords : Metabolic model, Transcriptomics, Streptomyces OP2006Engineering of Omega-Transaminase to Improve Activity for Asymmetric Synthesis of Chiral Amines Sang-Woo HAN, Eul-Soo PARK, Joo-Young DONG, Jong-Shik SHIN Dept. of Biotechnology, Yonsei University, Seoul, 120-749 Preparation of enantiopure chiral amines is an interesting issue in pharmaceutical industry. Asymmetric synthesis has an advantage of 2-fold theoretical yield compared to kinetic resolution. However, an unfavorable reaction equilibrium (i.e., -4 Keq=8.8×10 between acetophenone and alanine) and low activities of omega-transaminases (omega-TAs) for ketones (i.e., <1% relative to pyruvate) have obstructed asymmetric synthesis of chiral amines using omega-TAs. Here, we engineered an (S)-selective omega-TA from Ochrobactrum anthropi to elevate the activity for ketones while retaining its stereoselectivity. With a single point mutation, the impressive activity improvement for ketones was achieved (i.e., 340-fold increase in kcat/KM for acetophenone) and several chiral amines had been synthesized from ketones with high enantiopurity (i.e., >99% ee) using the engineered omega-TA variant with isopropylamine as an amino donor. Keywords : chiral amine, omega-transaminase, asymmetric synthesis, protein engineering References 1. Christopher K. Savile et al, Biocatlytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufature (2010), Science, 329, 305-308 OP2007Improving ω-Hydroxylation Activity of CYP153 toward Hexadecanoic Acid by Rational Enzyme Design EunOk JUNG, Beom-gi PARK, Hee-Wang YOO, Byung-Gee KIM School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742 ω-Hydroxy fatty acids (ω-OHFAs) are valuable chemicals for adhesives, lubricants, cosmetic intermediates and potential anticancer agents. Especially, ω-OHFAs have a potential as precursors for pseudo-ceramides. Although there are several processes for ω-OHFAs production by chemical synthesis, those have low yield and harsh reaction condition with high temperature. On the other hand, CYP153 is well known as ω-hydroxylase to linear fatty acids as well as alkanes with high region-specificity. In this study, the structures of CYP153 were predicted by homology modelling and the major cavities were revealed by CAVER 3.0 and site-directed mutagenesis. Furthermore, several amino acids on entrance of active site were substituted by both random mutagenesis and site-directed mutagenesis rationally to improve hydroxylation activity toward hexadecanoic acid. Finally, molecular docking simulation were investigated by Autodock Vina to calculate docking energy with hexadicanoic acid and enzymes. Keywords : ω-OHFAs, P450, enzyme design References 1. Sumire Honda Malca, Bacterial CYP153A monooxygenases for the synthesis of omega-hydroxylated fatty acids (2012), ChemComm, 48, 5115-5117 OP2008Solar-to-Chemical Conversion Platform by Robust Cytochrome P450-P(3HB) Complex Lee JAE HYUNG1, Nam DONG HEON2, Lee SAHNG HA2, Park JONG HYUN1, Park CHAN BEUM2, Jeong KI JUN1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, 2Dept. of Materials Science and Engineering, KAIST, Daejeon, 305-701 Cytochrome P450 monooxygenases (CYPs or P450s) have been given steady attention in the fields of industrial chemical reaction, pharmaceutical and metabolite synthesis because of its powerful and attractive biocatalytic ability for the regio- and stereospecific oxidation of non-activated hydrocarbons [1, 2]. However, despite the high potential of P450s, their practical applications is hindered by costly nicotinamide cofactors (e.g., NAD(P)H), which serve as a redox equivalent in P450- catalyzed reaction. Here, we develop a solar-driven chemical conversion system that combines visible light-driven NADPH regeneration and in situ immobilization of P450-BM3 on biopolymer granules. Through fusion with phasin, P450-BM3 can be easily immobilized on poly(3-hydroxybutyrate) granules in Escherichia coli. The immobilized P450 exhibited higher stability and catalytic activity compared to free P450 against changes of pH, temperature, and concentrations of urea and ions [3]. Through photochemical NADPH regeneration using eosin Y as a photosensitizer, P450-P(3HB) complex successfully catalyzed a visible light-driven O-dealkylation reaction. Furthermore, large scale of P450 light catalytic reaction was performed with P450-P(3HB) complex in long term scale. Using the robust P450-P(3HB) complex and a solar-tracking module, we achieved a P450-catalyzed reaction under natural sunlight for four consecutive days on a preparative reactor scale (500 mL). Keywords : cytochrome P450 monooxygenases, solar energy conversion, enzyme catalysis, poly(3-hydroxybutyrate) granules, solar-tracking system References 1. S. Kille, F. E. Zilly, J. P. Acevedo, M. T. Reetz, Nat. Chem. 3 738 (2011). 2. P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science 339 307 (2013). 3. J. H. Lee, D. H. Nam, S. H. Lee, J. H. Park, S. J. Park, S. H. Lee, C. B. Park, K. J. Jeong, Bioconjug. Chem. 25 2101 (2014) OP2009Expression of Native-sized Spider Silk Protein in Escherichia colithrough Use of Metabolic Engineering and Synthetic Biology Approach Hannah CHUNG1, Xiao-Xia Xia XIA1,2, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering Laboratory, Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141, 2BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, Daejeon, 34141 Spider silk proteins are proteins found in nature, which attract much attention due to its extraordinary physical properties such as elasticity coming from the highly repeated sequence and large size. However, due to the expectionally large size and protein structure, expressions in heterologous hosts, such as bacteria and yeast, are hindered. Metabolic engineering approach as adopted to correctly express the native-sized spider silk protein in Escherichia coli for the first time. Cellular resource was increased, specifically, glycine tRNA and corresponding tRNA synthetase were elevated to increase the tRNA pool. Furthermore, synthetic small RNA system was applied for further increase in protein expression, which yielded up to 0.9 g/L of native-sized spider silk protein. The results provide insight into approaches to control expression of recombinant proteins containing high molecular weight and highly repetitive sequence. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF- 2012M1A2A2026556); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea] Keywords : Spider silk protein, Metabolic engineering, sRNA system, tRNA pool References 1. Xia, X.-X., Qian, Z.-G., Ki, C.S., Park, Y.H., Kaplan, D.L., and Lee, S.Y., "Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber (2010), PNAS, 107(32): 14059-14063 2. Na, D., Yoo, S.M., Chung, H., Park, H., Park, J.H., and Lee, S.Y., "Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs (2013), Nature, 31(2):170-174 OP2010Conversion of Glycerol to Dihydroxyacetone Using Stabilized Glycerol Dehydrogenase Youngho WEE1, Gudi Satheesh KUMAR1, Ho Jin SUN1, Xueyan ZHAO2, Shunxiang XIA2, Ping WANG2, Jungbae KIM1,3 1Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea, 2Bioproducts and Biosystems Engineering, and Biotechnology Institute, University of Minnesota, Saint Paul, Minnesota 55108, United States , 3Green School, Korea University, Seoul 136-701, Republic of Korea Biodiesel industry generates glycerol as a main by-product, and the conversion of glycerol to value-added products is an important issue [1]. An enzyme, called glycerol dehydrogenase (GDH), can catalyze the conversion of glycerol to dihydroxyacetone (DHA), which is used as a tanning agent in cosmetics and much more expensive than glycerol. In this study, GDH was adsorbed (ADS) into magnetically-separable mesoporous silica with 38 nm mesocellular pores connected via 18 nm window mesopores (ADS), and further crosslinked via a simple glutaraldehyde treatment to prepare ‘nanoscale enzyme reactors’ of GDH (NER) [2]. When the stabilities of free and immobilized GDHs were checked in aqueous buffer under shaking (200 rpm), the residual activities of free GDH and ADS could not be measured due to the inactivation of GDH after 8 days and 22 days, respectively, while NER maintained 64% of initial activity even after 24-day incubation. Magnetically-separable NER maintained 39% of initial activity after seven cycles of repeated uses while the residual activity of ADS dropped to 13% of initial activity only after two recycled uses. Keywords : Enzyme, immobilization, glycerol , dihydroxyacetone, nanoscale enzyme reactor, glycerol dehydrogenase , enzyme stabilization References 1. 1. M. Pagliaro, R. Ciriminna, H. Kimura, M. Rossi, C. Della Pina, Angew. Chem. Int. Ed. 46, 4434-4440 (2007). 2. 2. J. Kim, J. Lee, H.B. Na, B.C. Kim, J.K. Youn, J.H. Kwak, K. Moon, E. Lee, J. Kim, J. Park, A. Dohnalkova, H.G. Park, M.B. Gu, H.N. Chang, J.W. Grate, T. Hyeon, Small 1 1203-1207 (2005). OP2011Isobutanol Production from an Engineered Shewanella oneidensis MR-1 Jong-Min JEON1, Hyojung PARK2, Hyung-Min SEO1, Hyun-Joong KIM1, Jung-Ho KIM1, Kwon-Young CHOI3, Byoung- In SANG2, Yung-Hun YANG1,4 1Department of BiologicalEngineering, College of Engineering, Konkuk University, Seoul, 133-791, South Korea, 2Department of Chemical Engineering, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea, 3Department of Environmental Engineering, Ajou University, 206, World cup-ro, Yeongtong-gu, Suwon, Gyeonggi- do, 443-749, South Korea, 4Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701, South Korea Shewanella oneidensis MR-1 is one of the most well-known metal reducing bacteria and it has been extensively studied for microbial fuel cell and bioremediation aspects. In this study, we have examined S. oneidensis MR-1 as an isobutanol producing host by accessing three key factors such as isobutanol synthetic genes, carbon sources, and electron supply systems. Heterologous Ehrlich pathway genes, kivDencoding ketoisovalerate decarboxylase and adh encoding alcohol dehydrogenase, was constructed in S. oneidensis MR-1. Among the composition of carbon sources examined, 2% N- acetylglucosamine (NAG), 1.5% pyruvate and 2% lactate were found to be the most optimal nutrients and resulted in 10.3 mg/L of isobutanol production with 48 h of microaerobic incubation. Finally, the effects of metal ions (electron acceptor) and direct electron transfer systems on isobutanol production were investigated, and Fe2+ ions increased the isobutanol production up to 35%. Interestingly, the deletion of mtrA and mtrBgenes did not have any significant impact on the isobutanol production while those genes were responsible for the membrane transport systems. Finally, we applied engineered S. oneidensis MR-1 to a bioelectrical reactor system to investigate the effect of direct electron supply system on isobutanol production, and it resulted in an increase growth and isobutanol production (up to 19.3 mg/L). In conclusion the correlation between isobutanol production and significant factors were investigated and the optimal conditions found in this study will have the great implications in greener bioremediation and bio-production of value-added products using S. oneidensis MR-1. Keywords : Isobutanol, Shewanella oneidensis MR-1, Bio-electrical system References 1. Jong-Min Jeon, Isobutanol production from an engineered Shewanella oneidensis MR-1 (2015), Bioprocess Biosyst Eng, OP2012Metabolically Engineered Escherichia coli for Production of 1,3-Diaminopropane: A Three Carbon Diamine Tong Un CHAE1, Won Jun KIM1, Sol CHOI1, Si Jae PARK4, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), Center for Systems and Synthetic Biotechnology, Institute , 2Bioinformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701,3BioProcess Engineering Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701,4Department of Environmental Engineering and Energy, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin, Gyeonggido 449-728 Bio-based production of chemicals is important for sustainable chemical industry. Here, Escherichia coli is metabolically engineered to produce 1,3-diaminopropane (1,3-DAP), a monomer for polyamide. Comparison of heterologous C4 and C5 pathways for 1,3-DAP production by in silico flux analysis revealed that the C4 pathway employing Acinetobacter baumannii dat and ddc genes, encoding 2-ketoglutarate 4-aminotransferase and L-2,4-diaminobutanolate decarboxylase, respectively, was more efficient. In a strain having feedback resistant aspartokinases, the ppc and aspC genes were overexpressed to increase flux towards 1,3-DAP synthesis. Also, knocking out pfkA was found to increase 1,3-DAP production by applying 128 synthetic small RNAs. Overexpression of the ppc and aspC genes in the pfkAdeleted strain resulted in even higher production of 1,3-DAP. Fed-batch fermentation of the final engineered E. coli strain allowed production of 13 g/L of 1,3-DAP in a glucose minimal medium. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556).] Keywords : metabolic engineering, 1,3-diaminopropane, synthetic small RNA References 1. Chae, T. U. et al. Metabolic engineering of Escherichia coli for the production of 1,3-diaminopropane, a three carbon diamine. (2015) Sci. Rep. 5, 13040 OP2013A Highly Multiplex and Sensitive SNP Genotyping Method Using Three Color Fluorescence Labeled Ligase Detection Reaction and High Resolution CE-SSCP Woong CHOI1, Gyoo Yeol JUNG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology, Accuracy, simplicity, sensitivity and cost-effectiveness are the most important criteria for a genotyping method for single- nucleotide polymorphisms (SNP). One method developed for SNP genotyping, ligase-based coupled capillary electrophoresis (CE) is considered for clinical diagnosis. However, conventional CE system is the design process and multiplex assay procedure are complicated because of the DNA size-based separation principle. In this study, we developed a simple, accurate and sensitive multiplex genotyping method using ligase detection reaction and high-resolution CE-based single-strand conformation polymorphism (CE-SSCP). With this high-resolution CE-SSCP system, we were able to use similar-sized probes, simplifying the design step and assay process. We found that this method could accurately discriminate single base mismatches on SNPs of tp53 gene used as targets for multiplex detection using multi-color fluorescence labeled probes. This research was supported by the Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning and Technology (2012M3A2A1051681). Keywords : SNP genotyping, Single Nucleotide polymorphisms, Molecular diagnosis, CE-SSCP References 1. Manolio TA, Collins FS., The Hapmap and genome-wide association studies in diagnosis and therapy. (2009), Annu Rev Med (60), 443-456 2. Landegren U, Kaiser R, Sanders J, Hood L., A ligase-mediated gene detection technique. (1988), Science (241), 1077- 1080 3. Shin GW, Hwang HS, Seo SW, Oh M-H, Ryu CY, Salvo CJ, Feldman S, Doh J, Jung GY., A novel pathogen detection system based on high-resolution CE-SSCP using a triblock copolymer matrix. (2010), J Sep Sci (33), 1639-1643 OP2014Aptamer-based Sandwich Assay for detecting Lipocalin2 (LCN2) in Hepatocellular Carcinoma (HCC) Kyeong-Ah LEE1, Ji-Young AHN1, Sang-Hee LEE1, Simranjeet Singh SEKHON1, Jiho MIN2, Yang-Hoon KIM1 1Dept. of Microbiology, Chungbuk national University, Cheongju, 362-763, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk national University, Jeonju, 561-763 Lipocalin2 (LCN2) is a secretory glycoprotein overexpressed from various inflammation and cancers. Recently, the high expression of LCN2 has been observed by HCC-microarray analysis study, suggesting the potential of LCN2 as a quantitative biomarker. We demonstrate a single-stranded, DNA aptamer-based, diagnostic method capable of detecting LCN2 biomarker from clinically-relevant HCC patient serum in sandwich assay format. Nine aptamers for LCN2 were screened by SELEX processes and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen by SPR and dot blotting analysis. LCN2 was sensitively detected in the concentration range of 2.5 ~ 500 ng mL-1. The aptamer sandwich assay demonstrated an excellent dynamic range at clinically relevant serum levels. The results of the study have demonstrated the capability of the aptamer sandwich assay platform for HCC diagnosis. This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of Monitoring and Diagnostic Method for Environmental Animal Disease, Project No: PJ010530)" Rural Development Administration, Republic of Korea. Keywords : LCN2, Lipocalin2, Aptamer, Hepatocellular Carcinoma, HCC, Aptamer-based Sandwich Assay References 1. Wang, Y. P. et al. Lipocalin-2 negatively modulates the epithelial-to-mesenchymal transition in hepatocellular carcinoma through the epidermal growth factor (TGF-beta1)/Lcn2/Twist1 pathway. Hepatology 58, 1349-1361 (2013). 2. Kim, M. et al. Arsenic removal from Vietnamese groundwater using the arsenic-binding DNA aptamer. Environ Sci Technol. 43, 9335-9340 (2009). OP2015Novel carbohydrate microarray platform for both on-chip enzymatic synthesis of glycan and analysis of carbohydrate-protein interaction Hyung Joon CHA1, Chang Sup KIM1,2, Jeong Hyun SEO1,3, Hye Ryoung HEO1 1Dept. of Chemical Engineering, Pohang University of Science and Technology,790-784, 2School of Biotechnology and Graduate School of Biochemistry, Yeungnam University,712-749, 3School of Chemical Engineering, Yeungnam University,712-749 Synthesizing the specific glycan is important for analysis and understanding of interaction with glycan-binding proteins (GBP). Especially, on-chip enzymatic synthesis of glycan can be efficient and useful tool because this method can directly provide diverse glycans to use for analyzing carbohydrate-protein interaction on the chip. However, effective identification of yield of the synthesized glycan on the chip remains a major challenge. In this work, we prepared novel carbohydrate microarray platform in which glycan-conjugated single-stranded DNA was synthesized using DNA synthesizer, and the glycan-DNA conjugates were immobilized onto the complementary single-stranded DNA-modified glass slide. Also, the carbohydrate microarray was combined with microfluidic system to efficiently synthesize glycans on the chip using a small amount of samples and obtain the synthesized glycan without great loss. Due to the property of DNA to be possible for reversible hybridization and denaturation under certain condition, we effectively obtained the synthesized glycan on the chip, and optimized the synthesis condition for target glycans. We expect that this novel carbohydrate microarray combined with microfluidic system can be successfully used in a number of glycan-related studies, including analysis of diverse carbohydrate-protein interactions, characterization of carbohydrate-processing enzymes, and detection of pathogens for diagnosis. Keywords : biochip, carbohydrate microarray, glycan References 1. Kim, C.S., Seo, J.H., and Cha, H.J., Anal. Chem.(2012), 84, 6884 2. Juan C. Morales, Bioconjugate Chem.(2010), 21, 1280 OP2016The analysis of mating response of budding yeast in programmable static droplet array device Si Hyung JIN1, Sung Sik LEE2,3, Matthias PETER3, Chang-Soo LEE1 1Department of Chemical Engineering, Chungnam National University, Daejeon, Republic of Korea,2ScopeM, Optofluidics Unit, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland, 3Institute of Biochemistry, ETH Zurich, Zurich, Switzerland The yeast Saccharomyces cerevisiae is a promising model organism to understand an intracellular signal transduction pathway [1]. The mating response in yeast cell is one of the best understood signaling pathways in eukaryotes which is initiated by the secretion of peptide pheromones that are recognized by receptors in surface of opposite mating type. The mating pheromone, α-factor, can be artificially synthesized thus many of leading groups were studied with it [2]. However the real mating response between the opposite mating types of yeast has been hampered by technical difficulties. Here we present a programmable static droplet array for analyzing the mating responses of budding yeast. This system includes cell encapsulated droplet generation, array, storage, cell culture on a single device. By using this system, we can precisely culture and analysis the co-cultured yeast cells in confined droplet which enhance the contacting efficiency between opposite mating type cells. In addition we use three sets of genetically modified yeast cells to analysis the signal transduction pathway. When culturing the single mating type of yeast in droplet, each of yeast cells are well grown and generates budding yeast normally. On the other hands, when culturing the both of opposite mating type of yeast cells (wild type) in a single droplet, the cell cycles are arrested and some of cells tends to be polarized (shmooing) and fused to each other (mating). In case of genetically modified yeast cells, one set of yeast is well polarized (∆Bar1) but the other is not affected by other mating types (∆Ste12). According to those results, we successfully understood the characteristic of mating related proteins (Bar1, Ste12) which involves in mating signal transduction pathway. Thus, we expected that the proposed system will be a powerful tool to study single cell behavior or intercellular signal transduction pathway. Keywords : Yeast mating, Droplet array, Microfluidics References 1. F. Naider and J. M. Becker, Peptides, 25, 1441 (2004). 2. S. S. Lee, P. Horvath, S. Pelet, B. Hegemann, L. P. Lee and M. Peter, Integr. Biol., 4, 381 (2012) OP2017Human-Like Bioelectronic Nose and Tongue for the Assessment of Food Quality Manki SON1, Daesan KIM2, Kyung Mi LEE3, Ju Hee KANG4, Hyang Sook CHUN4, Shin Sik CHOI3, Seunghun HONG2,5, Tai Hyun PARK1,6,7 1Interdisciplinary Program for Bioengineering, Seoul National University, Korea, 2Department of Physics and Astronomy, Seoul National University, Korea, 3Department of Food and Nutrition, Myungji University, Korea, 4Department of Food Science and Technology, Chung-Ang University, Korea,5Institute of Applied Physics, Seoul National University, Korea, 6School of Chemical and Biological Engineering, Seoul National University, Korea, 7Advanced Institutes of Convergence Technology, Korea Multi-channel bioelectronic sensor for the detection of food contamination indicators was developed with human olfactory receptor and taster receptor-functionalized carbon nanotube field-effect transistor. Human OR2J2, OR2W1, TAAR5 and TAS2R38 were expressed as inclusion bodies in Escherichia coli, purified with his-tag resin and refolded into detergent micelles. The receptors were directly immobilized onto Ni-NTA-modified carbon nanotubes with uniform orientation. Electrical properties of the sensor were maintained after the immobilization of the receptors. The sensor was designed to contain 4x4 channels and simultaneously detect four odorants and tastants against four food samples. To evaluate the sensing ability of the bioelectronic nose, the data was compared with GC/MS and human sensory analysis. As results, the sensor showed a positive correlation with other methods and the signal change was two days faster. We believe that this simple and easy method could be used as an effective assessment tool for food freshness. Keywords : bioelectronic nose, bioelectronic tongue, human olfactory receptor, human taster receptor, carbon nanotube field-effect transistor, food quality assessment References 1. Lim, J. H., Park, J., Ahn, J. H., Jin, H. J., Hong, S., & Park, T. H. (2013). A peptide receptor-based bioelectronic nose for the real-time determination of seafood quality. Biosensors and Bioelectronics, 39(1), 244-249. 2. Song, H. S., Kwon, O. S., Lee, S. H., Park, S. J., Kim, U. K., Jang, J., & Park, T. H. (2012). Human taste receptor- functionalized field effect transistor as a human-like nanobioelectronic tongue. Nano letters, 13(1), 172-178. 3. Park, J., Lim, J. H., Jin, H. J., Namgung, S., Lee, S. H., Park, T. H., & Hong, S. (2012). A bioelectronic sensor based on canine olfactory nanovesicle–carbon nanotube hybrid structures for the fast assessment of food quality. Analyst, 137(14), 3249-3254. OP2018High Performance Phenol Sensors based on Chemically-Crosslinked Laccase over Polyaniline Nanofibers Jae Hyun KIM1, Sung-Gil HONG1, Su HA2, Jungbae KIM1,3 1Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea, 2Department of Chemical Engineering, Washington State University, Pullman, Washington 99164, USA, 3Green School, Korea University, Seoul 136-701, Republic of Korea Phenolic compounds have poor biodegradability and high toxicity on ecological system as well as human body [1]. Electrochemical enzyme biosensors can provide a simple and fast way for the determination of phenols. In this study, we fabricated the laccase (LAC) based amperometric biosensor for detection of phenolic compounds. LAC was immobilized into the porous matrix of polyaniline nanofibers (PANFs) in a three-step process, consisting of enzyme adsorption, precipitation, and crosslinking (EAPC). Immobilized LAC on PANF in the form of EAPC was highly active and stable when compared to control samples of enzyme adsorption (EA) and enzyme adsorption and crosslinking (EAC) samples. For example, the activity of EAPC was 19.7 and 15.1 times higher than those of EA and EAC per unit weight of PANF, respectively. The sensitivities of biosensors with EA, EAC, and EAPC were 20.3±5.9, 26.6±5.4 and 518±11 mAmM-1cm-2, respectively. At 50 ˚C for 5 hrs, EAPC electrode maintained 80% of its initial sensitivity, while EA and EAC electrode showed 0% and 19% of their initial sensitivities, respectively. Keywords : Biosensor, Phenolic compound, laccase, enzyme References 1. Ahmaruzzaman, M., Adv. Colloid Interface Sci. 143, 48 (2008). OP2019Cation–π Interaction in DOPA-deficient Mussel Adhesive Protein mfp-1 Sangsik KIM, Dong Soo HWANG School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang, 790-784 Here we report the possible contribution of cation–π interaction to underwater adhesion of mussels by using DOPA-deficient recombinant mussel adhesive proteins. Considering the instability of DOPA in an oxidative environment, the cation– π interaction in DOPA-deficient biopolymers provides a complementary cross-linking mechanism for the design of novel underwater adhesives. Keywords : cation–π interaction , underwater adhesives, mussel adhesive protein References 1. Dennis A.Dougherty, Cation-pi interactions in Chemistry and Biology: A New View of Benzene, Phe, Tyr, and Trp(1996), Science, 271,163 2. Hongbo Zeng at al, Strong reversible Fe3+ mediated bridging between dopa-containing protein films in water(2010), PNAS, 107(29), 12850 OP2020Dopa-Iron Complexation of Mussel Adhesive Proteins at Plaque-Substrate Interface Byeongseon YANG1, Chanoong LIM2, Bong-Hyuk CHOI1, Dong Soo HWANG2, Hyung Joon CHA1 1Dept. of Chemical Engineering, POSTECH, Pohang, 790-784, 2School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784 Metal complexation in biological system and biomaterials plays many important roles such as metal ion accumulation, cross- linking, assembly, and reinforcement of mechanical properties. 3,4-Dihydroxyphenylalanine (Dopa), key molecule of underwater mussel adhesion, strongly binds to iron ion and forms mono, bis, and tris Dopa-iron complex, contributing high hardness and high extensibility to mussel byssal cuticle. While Dopa-iron complexation of mussel adhesive proteins (MAPs) has mainly focused on cuticle of byssus and coating protein fp-1 based on its intra-/inter-molecular bridging and cohesive interaction, Dopa-iron complexations of fp-3 and fp-5 located at plaque-substrate interface have not been studied, overlooking the fact that fp-3 and fp-5 have high Dopa contents and raman resonance peaks of Dopa-iron complexation were detected at plaque-substrate interface. In the present work, we identified and characterized Dopa-iron complexation of MAPs at plaque-substrate interface with Dopa-incorporated recombinant fp-3 and fp-5 proteins, exploring their effects on physical properties. Keywords : Dopa, iron, mussel adhesive protein, metal complexation References 1. Hwang DS, Zeng H, Masic A, Harrington MJ, Israelachvili JN, Waite JH. 2010. Protein- and metal-dependent interactions of a prominent protein in mussel adhesive plaques. J Biol Chem. 285, 25850-25858 2. Yang B, Ayyadurai N, Yun H, Choi YS, Hwang BH, Huang J, Lu Q, Zeng H, Cha HJ. 2014. In Vivo Residue-Specific Dopa-Incorporated Engineered Mussel Bioglue with Enhanced Adhesion and Water Resistance. Angew Chem Int Ed. 53, 13360-13364 OP2021Quantitative Analysis of Multiple microRNAs Based on Isothermal Exponential Amplification with CE-SSCP Jeongkyeong NA1, Gi Won SHIN2, Hee Sung HWANG2, Gyoo Yeol JUNG1,3 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784, 2Institute of Environmental and Energy Technology, POSTECH, Pohang, 790-784 , 3Dept. of Chemical Engineering, POSTECH, Pohang, 790-784 MicroRNAs (miRNAs), a class of endogenous and noncoding small RNA, play an important role in a wide range of biological processes, such as proliferation, organismal aging, tumorigenesis. With the increased in the number of miRNAs shown alteration its expression during specific biological process, multiplex and quantitative analysis technology is desired to understand aging and associated pathogenesis. Despite of its importance, miRNAs are challenging molecules to amplify by PCR due to limitations in designing PCR primer for miRNAs. Cleavage-based signal amplification of RNA, recently developed method, is isothermal exponential amplification for the detection miRNAs with high specificity and sensitivity. Here we developed this isothermal amplification method that employ single-strand conformation polymorphism (CE-SSCP), which is suitable for multiplex analysis. We tested our new method by detecting the well-known miRNAs, like lin-4, miR-48, miR-84 and so on. Keywords : microRNA, CE-SSCP, Multiplex, Quantitative References 1. Sachi Inukai and Frank Slack et al., MicroRNAs and the Genetic Network in Aging (2013), J. Mol. Biol., 425, 3601-3608 2. Pengbo Zhang et al., Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction (2014), Anal. Chem., 86(2), 1076-1082 3. Thomas D. Schmittgen et al., Real-time PCR quantification of precursor and mature microRNA (2008), Methods., 44(1), 31-38 4. Yongyun Zhao et al., Cleavage-based signal amplification of RNA (2013), Nat. Commun., 4, doi:10.1038/ncomms2492 5. Ethan J. Kaufman et al., The microRNAs of Caenorhabditis elegans (2010), Semin. Cell Dev. Biol., 21, 728-737 OP2022Ribosome-dependent Growth Inhibition by YhaV in Escherichia coli Won Ho CHOI Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungcheongbuk-do, 363-883, South Korea. Escherichia coli contains at least 38 toxin genes and theses toxins induce the growth inhibition and eventual death. The activity of toxin is blocked by the formation of complex with their cognate antitoxins in operons called toxin-antitoxin system. PrlF-YhaV operon is one of toxin-antitoxin systems located in Escherichia coli genome. As other type II toxin- antitoxin system, YhaV also forms a stable complex with its cognate antitoxin, PrlF under normal growth conditions, however, under stress conditions, unstable PrlF is preferentially degraded to release free YhaV in the cells, which then cleaves cellular mRNAs to inhibit protein synthesis, leading to growth arrest. Here we identify that YhaV is ribosome-dependent toxin, not ribosome-independent as a previous report. To test this hypothesis, DNA replication, RNA transcription, and protein translation level were analyzed in Escherichia coli with or without ribosome. Also in vitro primer extension experiment is planned to decide the cleavage site in RNA. Up to date, our results demonstrate that YhaV is a ribosome-dependent mRNA interferase. Keywords : Toxin-antitoxin system, mRNA interferase, ribosome, endonuclease References 1. Park, J.H., Y. Yamaguchi & M. Inouye, (2011) Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase. FEBS letters 585: 2526-2532 OP2023Development of Feeder-free Human Pluripotent Stem Cell Culture Substrate by Mussel- inspired Peptide Immobilization Hyun-Ji PARK1, Kisuk YANG1, Jiho JANG2, Mihyun LEE3, Yoonhee JIN1, Dong-Wook KIM2, Haeshin LEE3, Seung-Woo CHO1 1Dept. of Biotechnology, Yonsei University, Seoul, 2Dept. of Physiology, Yonsei University College of Medicine, Seoul, 3Dept. of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon Human pluripotent stem cells (hPSCs) hold a great promise as useful resources for regenerative medicine. In the past decade, various methods for hPSC culture have been established with animal-derived feeder cells (e.g., mouse embryonic fibroblasts) or animal tissue-derived matrices (e.g., Matrigel) for clonal expansion. However, the use of these xenogenic sources raises safety issues such as pathogen transmission or immunogenicity, and are limited due to the difficulty in quality control originated from batch-to-batch inconsistency. Therefore, the development of chemically defined, xeno-free substrates for hPSC culture are urgently required. Here, we developed bio-inspired peptide-grafted substrates for feeder-free and xeno- free culture of hPSCs. The extracellular matrix-derived peptides were efficiently immobilized by mussel-inspired surface chemistry, which promoted specific integrin-mediated focal adhesion and cell-cell interactions. These enhanced focal adhesion and cellular interactions consequently led to the promotion of pluripotency and self-renewal of hPSCs. The biomimetic peptide-grafted surfaces are compatible with several passaging methods, various commercial media, and hPSC cell lines. Moreover, biomimetic peptide immobilization could support feeder-free growth of hPSCs in a surface material- independent manner. The bio-inspired peptide-grafted surfaces developed in this study provides novel culture platforms enabling feeder-free hPSC culture on a variety of biomedical systems. Acknowledgments This work was supported by a grant (NRF2013R1A1A2A10061422) from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea. This work was also supported in part by a grant (HI13C1479) from the Korea Health Technology R&D Project of the Ministry of Health & Welfare. Keywords : Human pluripotent stem cell, Mussel-inspired chemistry, peptide immobilization References 1. Hyun-Ji Park et al., Bio-inspired oligovitronection-grafted surface for enhanced self-renewal and long-term maintenance of human pluripotent stem cells under feeder-free conditions (2015), Biomaterials, 50, 127-139 OP2024Enhancement of Monoclonal Antibody Production through High Zinc Ion Addition in Recombinant Chinese Hamster Ovary Cell Suspension Culture Bong Gyun KIM, Hong Woo PARK Dept. of Chemical Engineering, Hanyang University, Seoul, 133-791 Development of chemical defined media (CDM) that support high cell densities and high production rates have generally been preferred in large-scale monoclonal antibody (mAb) production using recombinant Chinese hamster ovary (rCHO) cell because of economic reason and safety concerns. Addition of trace elements to cell culture media has been shown to significantly increase maximum viable cell density, mAb production, and cell longevity [1, 2]. Despite the importance of trace elements in rCHO cell suspension culture, it has been not report which component is effect for cell growth and mAb production. For clarifying effects of trace elements in rCHO cell suspension culture, further studies are needed in a quantitative manner. In this study, we selected six trace elements such as copper, selenium, vanadium, zinc, manganese, and molybdenum and used the combination of these six elements as a control. The trace element concentration that best enhances mAb production was identified in batch suspension cultures of rCHO cell line. Zinc ion was identified as an effective component for improving mAb production among tested trace elements. We determined the concentration of zinc ion that yield maximum mAb production in in-house CDM and identified the effects of zinc ion on mAb production in various commercial CDMs. Keywords : Chemically defined medium, Chinese hamster ovary cells, Monoclonal antibody production, Suspension culture, Zinc ion References 1. 1. L. Xie and D. I. C. Wang, High cell density and high mnoclonal antibody production through medium design and rational control in a bioreactor (1996), Biotechnol. Bioeng., 51, 725-729 2. 2. Y. M. Huang, W. Hu, E. Rustandi, K. Chang, H. Yusuf-Makagiansar, and T. Ryll, Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment (2010), Biotechnol. Prog., 26, 1400-1410 OP3001Biosynthesis of cis,cis-Muconic Acid in Corynebacterium glutamicumthrough Re-designing Metabolic Pathway Ji-yeon KIM1, Ji-soo SONG1, Si-sun CHOI1, Sun ok PARK2, Sang joung LEE2, Gie-Taek CHUN3, Sang yong KIM4, Eung- Soo KIM1 1Department of Biological Engineering, Inha University, 100, Inha-ro, Nam-gu, Incheon, Korea, 2STR Biotech Co.,Ltd., Bioplaza4-3, 56, Soyang gang-ro, Chun cheon City, Kangwondo.200-957,Korea,3Department of Molecular Bio-science, Kangwon National University,1 Kangwon daehak-gil, Chuncheon-si, Gangwon-do, 200-701, Korea, 4Korea Insititue of Industrial Technology, 89 Yangdaegiro-gil, Ipjang-myeon, Seobuk-gu, Cheonansi, Chungcheongnam-do, 331-822, Korea Muconic acid is one of the platform chemicals that can be used to synthesize useful materials such as nylon and plastic. It can be produced by petrochemical cause serious environmental issues and exhaustion of petroleum. To overcome such problem, there are growing interests in biotechnological processes using microbial fermentation. In this study, engineered Corynebacterium glutamicum were developed for biosynthesis of cis,cis-Muconic acid(CCM), a type of Muconic acid. C. glutamicum has been widely used to produce amino acids, and metabolic system is well known. To biosynthesize CCM in Escherichiacoli, Dehydroshikimate(DHS) dehydratase, Protocatechuate(PCA) decarboxylase and Catechol 1,2- dioxygenase are required. However, C. glutamicum already has DHS dehydratase and Catechol 1,2-dioxygenase, so only one foreign gene encoding PCA decarboxylase was inserted by genetic engineering. And we blocked competitive pathways to accumulate of DHS and PCA, and also blocked CCM degrading pathway to protect final product. Finally, engineered C. glutamicum strains were obtained and produced a considerable amount of CCM production by liquid culture. Keywords : cis,cis-Muconic acid, Corynebacterium glutamicum, Re-designing Pathway, Metabolic engineering OP3002Homologous Expression, Purification and Characterization of Formate Dehydrogenase in Methanol Utilizing Bacteria MinGee JANG, DaeHaeng CHO, Kyoungseon MIN, Yong Hwan KIM Department of Chemical Engineering, Kwangwoon University, Seoul 139-701 The conversion of CO2 by biocatalyst is the one of alternatives for green house gas CO2 mitigation and production of renewable fuels and chemicals. In recent study, we succeeded in the continuous production of formate from CO2 using Methylobacterium extorquens whole cell as a biocatalyst in an electrode system. Formate dehydrogenase from methanol utilizing bacteria was supposed the most probable biocatalyst that gives CO2-reducing activity to the whole cell biocatalyst. In order to prove the expression and CO2-reducing catalytic activity of formate dehydrogenase, a homologous over-expression system in methylotrophic bacteria first of all should be set up. In this study, we have tried to perform homologous expression of formate dehydrogenase in methyltrophic bacteria. Keywords : Formate dehydrogenase, Homologous expression, Purification , Characterization References 1. Hyojin Hwang, Electro-biocatalytic production of formate from carbon dioxide using an oxygen-stable whole cell biocatalyst(2015),Bioresour Technol, 185, 35-9. OP3003Production of Bio-isoprene from Recombinant Escherichia coliharboring codon optimizes isoprene synthase Seon-Won KIM1, Myeong-Seok CHA1, Jung-Hun KIM1, Chong-Long WANG1, Li-yang YANG1, Ju-Eon PARK1, Eui-Sung CHOI2 1Division of Applied Life Science (BK21), PMBBRC, Gyeongsang National University,, 2Korea Research Institute of Bioscience & Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon, Korea Isoprene (2-methyl-1,3-butadiene) is a C5 terpenoid with the formula CH2=C(CH3)CH=CH2. Isoprene is a highly volatile compound with no color and is soluble in ether and hydrocarbon solvents. Isoprene is the base compound for the production of synthetic rubber and at the same time, a major constituent of natural rubber. It is primarily used to make tires, medical supplies, adhesives, etc. It can be produced by isoprene synthase from dimethylallyl diphosphate (DMAPP) formed in mevalonate (MVA) pathway and methyl erythritol 1-phosphate (MEP) pathway. Microbial production of isoprene from renewable sources is an attractive route because of cost-effectiveness and sustainable development. In this study, we constructed plasmids containing isoprene synthase (ispS) from Populus trichocarpa, Pueraria montana (kudzu), and Populus alba and introduced them into E. coli. GC analysis was used to confirm isoprene production from recombinant E. coli. To enhance isoprene production, we introduced isoprene synthesis plasmid containing codon-optimized ispS for conversion of DMAPP to isoprene using the MVA pathway plasmid for DMAPP supply. This work was supported by a grant (NRF- 2012M1A2A2671831) from the National Research Foundation, MSIP, and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#:PJ01106201), RDA, Korea. Keywords : Isoprene, Isoprene synthase, E. coli, MVA pathway References 1. (1) Kuzuma, J., and R. Fall “Bacteria produce the volatile hydrocarbon isoprene, 7th International Symposium on the Genetics of Industrial Microorganisms” (1994) Jun. 26-Jul. 1. Quebec, Canada, abstract p. 248 2. (2) Barbara. M., Christa. O., and Wolfgang. Z. “First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli” (2001) Planta. 213: 483-487 OP3004Bio-conversion of Mixed Sugars and Acetic Acid in Lignocellulosic Hydrolysate to Butyric Acid by Clostridium sp. LMS Min Sun KIM1,2, Ki-Yeon KIM1, Kyung Min LEE1, Min-Kyu OH2, Han Min WOO1, Youngsoon UM1 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seongbuk-gu, Seoul 136-791, South Korea, 2Department of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul 136-713, South Korea Previously, Clostridium sp. LMS was found to consume mixed sugars simultaneously and produce butyric acid at high yields by consuming acetic acid. This unique characteristic is valuable in utilizing acetic acid and fermentable sugars (glucose, xylose and mannose) in lignocellulosic hydrolysate. In this study, we hypothesized that the strain LMS could generate NADH by NADH:Ferredoxin (Fd) oxidoreductase (NFOR)-like activity instead of producing H2 from Fd, which might cause the consumption of acetic acid as an electron acceptor. To validate our hypothesis, we compared H2/CO2 ratio of the strain LMS and butyrate-producing C. tyrobutyricum. The strain LMS showed a lower H2/CO2ratio than C. tyrobutyricum, indicating less H2 production by LMS. Using pyruvate, the strain LMS produced butyric acid, while C. tyrobutyricum did not. Based on the result, unlike C. tyrobutyricum, LMS was likely to generate NADH through NFOR-like activity in addition to glycolysis-derived NADH generation, resulting in the production of butyric acid from pyruvate. Finally, when real lignocellulosic hydrolysate was used, the strain LMS produced butyric acid successfully by consuming mixed sugars and acetic acid. Keywords : Bio-conversion, Acetic acid, Butyric acid, Lignocellulosic hydrolysate, Clostridium sp. LMS OP3005A MALDI-MS-based Quantitative Targeted Glycomics (MALDI-QTaG) for Total N-glycan Analysis Kyoung-Jin KIM, Cheol-Hwan HWANG, Han-Gyu PARK, Yun-Gon KIM Dept. of Chemical Engineering, Soongsil University, Seoul, 156-743 In this study, MALDI-MS based quantitative targeted glycomics (MALDI-QTaG) was developed for monitoring the abnormal glycosylation of clinical and biopharmaceutical products. MALDI-QTaG was proposed for sensitive and quantitatvie analysis of total N-glycans. The derivatization reactions (i.e., amidation of sialic acid and incorporation of a positive charge moiety into the reducing end) dramatically increased the linearity (R2>0.99) and sensitivity (limit of detection is 05 pmol/glycoprotein) relative to underivatized glycans. In addition, the analytical strategy was chromatographic purification-free and non-laborious process accessible to the high-throughput analyses. We used teh MALDI-QTaG method to cell line (Huh7 cells). The total percentage of core-fucosylated AFP N-glycans from Huh7 cells and normal cord blood was 98.0% and 17.8%, respectively. This MALDI-MS based glycomics technology has wide application in many clinical and bioengineering fields requiring sensitive, quantitative and fast N-glycosylation validation. Keywords : Chemical derivatization, MALDI-MS, N-glycans, Quantitative analysis, UPLC References 1. Jang KS, Kim YG, Gil GC, Park SH and Kim BG, Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines, Anal Biochem, 19, 3421- 3428 2. Gil GC, Iliff B, Cerny R, Velander WH, Van Cott KE, High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry, Anal Chem, 82, 6613- 6620 3. Wang C, Wu Z, Yuan J, Wang B, Zhang Y, Wang Z, Huang L, Simplified quantitative glycomics using the stable isotope label Girard's reagent p by electrospray ionization mass spectrometry, J Proteome Res, 13, 372-384 OP3006Shikimate Biosynthesis Pathway Redesign for Production of cis,cis-Muconic Acid Precursor in Escherichia coli. Bo-Ram LEE1, Si-sun CHOI1, Sun ok PARK2, Sang joung LEE2, Gie-Taek CHUN3, Sang yong KIM4, Eung-Soo KIM1 11Department of Biological Engineering, Inha University, Incheon, 402-751, Korea, 2STR Biotech Co., Ltd., Bioplaza 4-3, 56, Soyanggang-ro, Chuncheon City, Kangwon do. 200-957, Korea , 3Department of Molecular Bio-science, Kangwon National University,1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 200-701, Korea, 4Korea Insititue of Industrial Technology, 89 Yangdaegiro-gil, Ipjang-myeon, Seobuk-gu, Cheonan si, Chungcheongnam-do, 331-822, Korea cis, cis-muconic acid (CCM) is an important starting chemical for the synthesis of the biodegradable polymer. The structural isomers of CCM are precursors of important bulk chemicals including adipic acid and terephthalic acid, which can be applied to various products such as nylon and plastic. However, the synthesis of CCM is dependent on petroleum-based feedback and on highly concentrated heavy metal catalyst. Because of various environmental and economic issues including global warming, exhaustion of resources as well as rising oil prices of fossil fuel, it is necessary to study biosynthetic strategies using eco-friendly and renewable microorganisms. To solve these problems, the biosynthetic strategies utilizing environmentally renewable microorganisms have been actively studied. In this study, metabolic pathway of E.coli was re-designed for CCM biosynthesis. The shikimate biosynthetic pathway generates DHS as a CCM precursor. We emphasize a re-designed the shikimate biosynthetic pathway to accumulate. The ptsG involved in conversion of the PEP to pyruvate and tyrR in suppression of the migration of the PEP and E4P to DAHP were removed. Also, tktA was over-expressed to encrypt the transketolase for the synthesis of E4P. More detailed results will be discussed. Keywords : 3-dehydroshikimate, cis, cis-muconic acid, metabolic engineering OP3007Artificial Photosynthetic Escherichia coli having Calvin-Benson-Bassham Cycle Operon Isolated from Rhodobacter sphaeroides Young Su KIM1, Ju-Yong PARK1, Yang-Hoon KIM2, Jiho MIN1 1Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 664-14 Deokjin-dong, 1Ga Deokjin-Gu Jeonju 561-756, South Korea, 2Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763, South Korea In recent years, the concentration of CO2 that cause the global warming was increase. One way to solve this problem is biological conversion. Rhodobacter sphaeroides which is one of non-sulfur purple photosynthetic bacteria have calvin cycle. Five genes of Form I(cbbI) operon were inserted in pET-28b and six genes of Form II(cbbII) operon were inserted pET-21a. Recombinant Escherichia coli BL21 (DE3) strain were contained Form I::pET-28b and Form II::pET-21a as a dual plasmid system. In LB medium and M9 medium, relatively remained CO2 in overexpressed recombinant E. coli having Form I::pET- 28b and Form II::pET-21a is higher and CO2 production per cell is lower than others. In M9 medium, relatively remained CO2 containing ribulose 1,5-bisphosphate(RuBP) or fructose-6-bisphosphate(FBP) recombinant E. coli having Form I::pET- 28b and Form II::pET-21a is similar than did not add those and CO2 production per cell is lower than others. Intracellular ATP concentration was decrease adding IPTG, RuBP. Furthermore, NAD+/NADH ratio was increase adding IPTG, RuBP. However, intracellular ATP and NAD+/NADH ratio adding FBP was same with adding IPTG. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No: PJ01051502)” Rural Development Administration, Republic of Korea. The authors are grateful for their support. Keywords : Calvin-Benson-Bassham cycle, Artificial photosynthesis, Carbon dioxide reduction References 1. K. Kim and A. R. Portis Jr, Kinetic analysis of the slow inactivation of Rubisco during catalysis: effects of temperature, O2 and Mg++.(2006). Photosynthesis Research, 87(2), 195-204. 2. W. Bonacci, P. K. Teng, B. Afonso, H. Niederholtmeyer, P. Grob, P. A. Silver, and D. F. Savage, Modularity of a carbon- fixing protein organelle. (2012). Proceedings of the National Academy of Sciences, 109(2), 478-483. 3. Zhuang, Zong-Yu, and Si-Yu Li. Rubisco-based engineered Escherichia coli for in situ carbon dioxide recycling. (2013). Bioresource technology 150:79-88. OP3008Isolation and Characterization of Exosomes and Detection of Exosome miRNAs Using Molecular Beacons Ji Hye LEE, Min Hee KWON, Seonga JUNG, Jin Hee LEE, Won Jong RHEE Devision of bioengineering, University of Inchoen, Incheon, 119 Exosomes are extracellular vesicles with the size of 30-100 nm that are secreted by the most types of cells. They can be found in the most of body fluids including blood, urine, saliva, as well as cell culture medium. They are composed of proteins, DNAs, RNAs, and miRNAs derived from their originating cells, which indicates that exosomes contain useful information on the state of bodies. Among them, exosome miRNAs can be used for the early prediction of diseases in human. Therefore, exosome miRNAs are considered to be novel biomarkers in the diagnosis and prognosis of diseases including cancers. To analyze exosome miRNAs, we have isolated and characterized exosomes from cancer cells by different methods including precipitation and ultracentrifugation. In addition we provided a comparative evaluation of exosome proteins and RNAs among different isolation methods. Finally, we have demonstrated that miRNAs can be successfully detected in the isolated exosomes using oligonucleotide probe, molecular beacon. The method developed here is simple, easy, and sensitive, so it will offer great opportunities for diagnosis and prognosis of many diseases. Keywords : Exosomes, isolation, miRNA, molecular beacon, diagnosis References 1. J.H.LEE, J.A.Kim, M.H.Kwon, Ji Yoon Kang, W.J.Rhee, in situ single step detection of exosome microRNA using molecular beacon, 54, 116-125 OP3009Constructing the Vascular Tissue Using Microfluidic Devices Boa SONG1,2, Hongsoo JEONG1, Ping DU1, Min Hee PARK1, Hojeong JEON1, Kangwon LEE3, Sangheon KIM1,2 1Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Korea, 02792, 2Dept. of Biomedical Engineering, University of Science and Technology, Daejeon, Korea, 34113, 3Dept. of Convergence Science and Technology, Seoul National University, Suwon, Korea, 433-270 The actual living organisms are in three-dimensional (3D) environments. Therefore, bulk up the size of 3D vascularized tissues containing blood vessels is critical factor to be overcome in tissue engineering field. However, due to the lack of effective delivery of nutrient and oxygen to all tissue regiens constructing cells and tissues over 500 ㎛ in thickness is challenging. Herein, we designed perfusable channeled device to fulfill the vascularized 3D tissue structure. The microfluidic device contains three perfusable channels (500 ㎛ diameter), and collagen/fibrin mixed hydrogels were used as interstitial matrix. We seeded GFP-, RFP-transfected human umbilical vein endothelial cells (HUVECs) in the channel to visualize the vessel formed, and the cells were fully proliferated to form endothelial monolayer in channel wall (~ 500 cells/㎟) with ≥70 % of cell viability. We introduced the co-culture system using HUVECs and mesenchymal stem cells (MSCs) to investigate the maturation effect on stable vascularization by MSC in this system. Vascular channels formed by angiogenesis were further investigated to check channel interconnectivities through anastomosis. Our perfusable vascular bed can be applied to the drug screening, tissue regeneration studies or studing various ex-vivo disease models. Keywords : vascularization, microfluidic device, angiogenesis, vessel maturation References 1. S. Kim, H. Lee, M. Chung, and N.L. Jeon, Lap Chip. 13, 1489 (2013) 2. V.K. Lee, A.M. Lanzi, H. Ngo, S.S. Yoo, P.A. Vincent and G. Dai, Cellular and Molecular Bioengineering., Vol. 7, Issue 3 (2014) OP3010Anti-apoptotic and Anti-oxidative Effects of Silkworm Storage Protein 1 in HeLa Cell Ji hye LEE, Ji eun BAIK, Won jong RHEE Division of Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 406-772, Republic of Korea Silkworm contains several distinct proteins that have anti-apoptotic activities in mammalian and insect cells. Among them, 30K proteins have been well investigated for their characteristics while storage proteins (SPs) have not. SP1, a type of insect hemocyanin protein, is a female-specific and heat stable protein in silkworm and consists of high portion of methionine. In this article, an anti-apoptotic effect of SP1 was investigated in HeLa cell apoptosis. In addition, SP1 exhibited the resistance to oxidative stress. To investigate anti-apoptotic and anti-oxidative effects of SP1, the stable cell line expressing SP1 was constructed, and the cell line was selected by using trypan blue exclusion assay, microscopic analysis of nuclear fragmentation, and flow cytometry. SP1 expressing cell line showed strong anti-apoptotic activity against the treatment of staurosporine but not TNFa meaning that SP1 is a protein inhibitor of apoptosis caused by intrinsic mitochondria-mediated apoptosis pathway. Additionally, it was identified that oxidative stresses induced by UV B irradiation or hydrogen peroxide were reduced in cells expressing SP1 As SP1 is the novel anti-apoptotic and anti-oxidative protein, the regulation of apoptosis by SP1 will offer a new strategy to biopharmaceutical industry where the minimization of cell death and the maximization of productivity are required. Keywords : silkwrm, SP1, apoptosis, anti-apoptotic effect, anti-oxidant effect References 1. Rhee, W. J., E. H. Lee, and T. H. Park (2009) Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti- apoptotic effect in Sf9 cell. Biotechnol. Bioprocess Eng. 14: 645-650. 2. Wang, Z., J. H. Park, H. H. Park, W. Tan, and T. H. Park (2011) Enhancement of recombinant human EPO production and sialylation in Chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression. Biotechnol. Bioeng. 108: 1634-1642. 3. Wang, Z., X. Ma, L. Fan, W. J. Rhee, T. H. Park, L. Zhao, and W. S. Tan (2012) Understanding the mechanistic roles of 30Kc6 gene in apoptosis and specific productivity in antibody-producing Chinese hamster ovary cells. Appl. Microbiol. Biotechnol. 94: 1243-1253. 4. Yu, W., H. Ying, F. Tong, C. Zhang, Y, Quan, and Y. Zhang (2013) Protective effect of the silkworm protein 30Kc6 on human vascular endothelial cells damaged by oxidized low density lipoprotein (Ox-LDL). PLoS One 8: e68746. OP3011Vascularized Artificial Dermal Substitute by Three-Dimensionally Cultured Adipose-Derived Stem Cells for Wound Healing Ji Eun YEOM1,2, Se Won PARK3, Joon Ho SHIM3, Dong-Youn LEE3, Sang-Heon KIM1,2 1Center for Biomaterials, Biomedical Engineering Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea, 2Department of Biomedical Engineering, University of Science and Technology, Daejeon, Korea, 3Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Wound healing is composed of matrix remodeling, re-epithelization, vascularization, and contraction phases. Artificial dermal substitutes have been recommended for patients who need skin implantation. There is no ideal substitute for wound healing in the market because current artificial substitutes lack blood vessel networks, which can be implanted only temporarily and can cause immune rejection and infection problem. The aim of this study is to modify an artificial dermal equivalent (DE) by adding blood vessels for more effective skin regeneration. We used a three-dimensional cell mass, termed AngioclusterTM, previously reported, for blood vessels formation [1]. For the formation of AngioclusterTM, hASCs were cultured on a novel FGF2-coated surface, an artificial cell adhesion matrix [2], for 2 days in normal growth media and the AngioclusterTM was seeded on DE made of fibroblast cells. AngioclusterTM-DE complex was transplanted to the wounded dorsum of nude mouse and analyzed by examination with the naked eyes and immunohistochemical staining. The AngioclusterTM-DE group was more vascularization, thicker re-epithelialization, smaller scar formation and less wound contraction compared with other groups. These results indicate AngioclusterTM promotes denser vascularization and anticipate wound healing much faster with a proper matrix organization. Keywords : Wound healing, Three-dimensional cell mass, angiogenesis, artificial dermal substitute References 1. I. S. Park et al., Cytotherapy, 16, 508 (2014) 2. J.M. Kang et al., Acta Biomaterialia, 8, 1759 (2012) OP3012Physico-Chemical and Hydrogel Properties of Agar from Gracilaria verrucosa from the Coast of Jeonnam, South Korea Yong-Woon KIM1,1, Hyun-Jae SHIN1,2 1Dept. of Chemical Engineering, chosun university, Gwangju, 501-759 , 2Dept. of chemical, biochemical and polymer Engineering, chosun university, Gwangju, 501-759 Hydrogels are gaining importance in food, cosmetic and medical industries due to unique functionalities as well as texture. This study is mainly focused on the development of resources technique for the marine debris from domestic red algae, Gracilaria verrucosa. Most of the algae were neglected after harvest period of spring season. A novel agar extraction process was developed for G. verrucosa produced from the coast of Jeonnam (near Wan-do) by investigating the various extraction variables such as alkali soaking concentration and time on agar yield and physico-chemical properties. The optimized variables were extraction type, algae to water ratio, alkali concentration, and extraction time. The results showed optimal gelling and melting temperature, the gel strength, and the sulfate contents. The yield of agar was the maximum in 5% alkali concentration (34.8%) and minimum in 1% alkali concentration (17.0%). Agar gel strengths fluctuated from 462 to 662 g cm-2. The gel micro structures of agar showed porous network structures, and the pores were similar to those of bacto-agar and commercial-agar product. In summary, high-quality hydrogel was successfully extracted from G. verrucosa with novel alkaline extraction pretreatment and microwave treatment. Keywords : Gracilaria verrucosa, hydrogel, agar, Physico-chemical properties References 1. S. Yarnpakdee and S. Benjakul and P. Kingswascharapong, Food Hydrocolloids. 51, 217-226 (2015) 2. K. Xia and X. Liu and J. Zhao and X. Zhang, Carbohyd. Polym. 110, 32-37 (2014) OP3013Engineering of thermostable Neisseria gonorrhea-originated carbonic anhydrase through in silico design for efficient carbon sequestration Tae-yoon PARK1, Byung Hoon JO1, Young Joo YEON2, Hyun Jun PARK2, Young Je YOO2, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784, 2School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742 Various strategies for carbon capture and storage (CCS) are being suggested to overcome global warming issue. Solutions to reduce carbon dioxide (CO2) have been the indispensable part of future. One of the researches actively studied is carbonic anhydrase (CA)-based CCS. However, the use of CA in CCS is currently limited due to harsh operational environments, especially high temperature. Recently, several studies are progressed to significantly enhance thermostability while retaining catalytic activity. This work focused on developing thermostable Neisseria gonorrhea–originated CA (ngCA) with an additional disulfide bond generated by site-directed mutagenesis, based on in silico design. Several ngCA mutants were constructed and expressed in Escherichia coli system, and we found that their extra disulfide bonds were well-formed. The tolerance of all mutants at high temperature was higher than wild type counterpart and, especially, one of mutants maintained its activity ~16.2 fold higher than wild type at 70 °C. In addition, we confirmed that an additional disulfide bond formation could contribute to improvement on conformational stability by thermodynamic parameters analysis (Tm, ΔH, and ΔS). Thus, it was concluded that disulfide bond engineering is the promising means of enhancing CA thermostability. Keywords : disulfide engineered bacterial catalyst, CO2 sequestration, carbonic anhydrase, in silico design, thermostability References 1. S. Huang,Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide (1998), J. of Molecular Biology, 283(1), 301-310 OP3014Influence of Zeta Potential on Fractional Precipitation Behavior of (+)-Dihydromyricetin Geon-Soo HA, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, 330-717 This study evaluated the influence of the zeta potential of silica-alumina on the behavior in terms of purity, yield, and precipitate shape and size of fractional precipitation in the fractional precipitation process for the purification of (+)- dihydromyricetin. The optimal silica-alumina amount (surface area per working volume of reacting solution) for zeta potential control was 100 mm-1. As the zeta potential value of silica-alumina increased, (+)-dihydromyricetin yield and precipitate size were increased. The use of silica with the highest value of the zeta potential (+4.99 mV) as a zeta potential- controlling material increased the (+)-dihydromyricetin yield by 2-fold compared with that of the use of alumina with the lowest value of the zeta potential (-19.00 mV). In addition, the (+)-dihydromyricetin yield and precipitate size was inversely correlated with the absolute value of the zeta potential. On the other hand, the purity of (+)-dihydromyricetin had almost no effect on changes in the zeta potential of silica-alumina.Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : (+)-Dihydromyricetin, Purification, Fractional Precipitation, Silica-Alumina, Zeta Potential References 1. Lim MK, Kim JH, Improvement of the Fractional Precipitation Process for the Purification of (+)- Dihydromyricetin(2014), Korean J. Biotechnol. Bioeng., 42, 25-31. 2. Han MG, Kim JH, Evaluation of a High Surface Area Fractional Precipitation Process for the Purification of Paclitaxel from Taxus chinensis(2012), Biotechnol. Bioproc. Eng., 17, 1018-1024. 3. Ryu HK, Kim JH, Effect of Zeta Potential on Fractional Precipitation for the Purification of Paclitaxel from Plant Cell Cultures of Taxus chinensis(2014), Korean J. Biotechnol. Bioeng., 42, 114-120. 4. Cho EB, Cho WK, Cha KH, Park JS, Enhanced Dissolution of Megestrol Acetate Microcrystals Prepared by Antisolvent Precipitation Process Using Hydrophilic Additives(2010), Int J. Pharm., 396, 91-98. OP3015In vitro Antibacterial Effect of Sargassum serratifolium against Bacterial Skin Pathogens Yun-Hye KIM1, Song Hee KIM1, Gab jin KIM1, Young-Mog KIM1, Hyeung Rak KIM2 1Dept. of Food Science & Technology, Pukyong National University, Busan, 608-737, 2Dept. of Food Science & Nutrition, Pukyong National University, Busan, 608-737 Acne vulgaris, simply acne is a common human skin disease. It has been known that a commensal bacterium Propionibacterium acnes can cause inflammation related with acne, which may result in scarring or hyperpigmentation. Also, Staphylococcus epidermidis, S. aureus, and Pseudomonas aeruginosa are often involved in the development of abnormal follicular keratinization and inflammation. The currently available therapeutic option for these bacterial skin pathogens is usually antibiotic treatment to kill the bacteria. However, the application of antibiotic traetment is often associated with several side effects, such as the emergence of antibiotic resistance, organ damage, and immune hypersensitivity if these medicines are used for a long period. To overcome the problem of side effects, we tried to find an alternative way to treat bacterial skin pathogens using marine algae. Ongoing this study, it was observed that the ethanolic extract of edible brown algae Sargassum serratifolium exhibited potential antibacterial activity against bacterial skin pathogens including P. acnes S. epidermidis, S. aureus and P. aeruginosa. Among of solvent soluble fractions from the ethanolic extract, the hexane soluble (Hexane) fraction showed the strongest antibacterial activity against all tested bacteria in this study. Successively, three compounds [sargaquinoic acid (SQA), sargahydroquinoic acid (SHQA) and sargacromenol (SCM)] were isolated from the Hexane fraction flowed by chromatography. Three compounds also showed potential antibacterial activity against bacterial skin pathogens. Consequently, the results of this study suggest that the compounds derived from the edible brown algae S. serratifolium can be a potential source of natural antibacterial agents and a pharmaceutical component against bacterial skin pathogens. Keywords : Antibacterial, Sargassum serratifolium, Bacterial Skin Pathogens, Acne References 1. Clinical and Laboratory Standards Institute (2006) Methods for dilution antimicrobial 2. Lee et al. (2014) In vitro antibacterial and synergistic effect of phlorotannins isolated 3. Yoon et al. (2013) Epigallocatechin-3-Gallate Improves Acne in Humans by Modulating OP3016Antibacterial and Anti-inflammatory Effect of Chitosan-phytochemical Conjugates against Acne-related Bacteria Ji Hoon KIM, Seung Mok YEOM, Song Hee SONG HEE, Hong Yeop PARK Department of Food Science and T608-737echnology, Pukyung National University, Busan, Korea, Acne vulgaris is one of the most common skin diseases, affecting nearly 80% of young adults aged to 11 to 30 years. Propionibacterium acnes, Staphylococcus epidermidis, S. aureus, and Pseudomonas aeruginosa are often involved in the development of abnormal follicular keratinization and inflammation. The currently available therapeutic option for acne is usually used to treat acne vulgaris to inhibit inflammation or kill the bacteria using antibiotics. However, these antibiotics are often associated with several side effects such as the emergence of resistant bacteria, organ damage, and immune hypersensitivity if these medicines are used for a long period. To overcome the problem of side effects, medicinal plants and marine organisms have been investigated for the treatment of acne. Chitosan is a naturally occurring mucopolysaccharide. Several bioactivities of chitosan and chitosan-derived conjugates such as antioxidant, anticancer, antimicrobial, and enzyme inhibitory effects have also been reported. However, there is no further information on the antibacterial and anti- inflammatory effect of the conjugates against acne-related bacteria. In the present work, we investigated the antibacterial and anti-inflammatory activity of the chitosan-phytochemical conjugates against acne-related bacteria. In this study, we prepared three of chitosan-phytochemical conjugates; chitosan-caffeic acid (CCA), -ferulic acid (CFA) and -sinapic acid (CSA) conjugates. Among of them, CCA exhibited the highest antibacterial activity against all of acne-related bacteria tested in this study. Moreover, CCA and other chitosan conjugates showed strong inhibitory effects on nitric oxide (NO) production in P. acnes-induced inflammatory model using HaCaT cells. The results obtained in this study suggest that the compounds derived from chitosan can be a potential source to treat or prevent acne vulgaris since these compounds exhibited both strong antibacterial activity against acne-related bacteria causing acne vulgaris and anti-inflammatory activity against P. acnes- induced inflammation in HaCaT cells. Keywords : HaCaT cell, Antibacterial effect, Anti-inflammatory effect, Acne-related bacteria, Chitosan-phytochemical cojugates References 1. Lee JH, Eom SH, Kim YM., 2014. In vitro antibacterial and synergistic effect of phlorotannins isolated from edible brown seaweed Eisenia bicyclis against acne-related bacteria. 2. Lee DS, Eom SH, JE JY., 2014. Antibacterial and synergic effects of gallic acid-grafted-chitosan with ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA). OP3017Identification of Gaseous Food Spoilage Indicators for Non-destructive and Early-detective Food Freshness Monitoring Kyung Mi LEE1, Shin Sik CHOI1,2 1Department of Energy Science and Technology, Myongji University, Yongin 449-728, Republic of Korea, 2Department of Food and Nutrition, Myongji University, Yongin 449-728, Republic of Korea Oyster (Crassostrea gigas) is a shellfish which has been consumed for a while as a raw seafood in many countries. Since the freshness of oyster is required for the safety as well as sensory quality including flavour and taste, a precise and rapid monitoring system to assess freshness of oyster is necessary in food service chain. Conventional analysis methods such as gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) have been employed to monitor food spoilage or freshness. However, these classical methods require time- and labour-consuming destructive pre-treatment of samples along with a sensory evaluation by trained panels. In this study, we identified a non- destructive and early-detective headspace gaseous indicators accompanying with food storage or spoilage for oyster freshness monitoring. Keywords : oyster, freshness, food storage References 1. Z. Zhang et al, Study on the volatile profile characteristics of oyster Crassostrea gigas during storage by a combination sampling method coupled with GC/MS(2009), Food Chemistry, Vol (115), 1150-1157 OP3018Development of An Efficient Drying Method for Removal of Residual Solvents from Paclitaxel by Pre-treatment with Ethanol and Water Chung-Gi LEE, Jin-Hyun KIM* Dept. of Chemical Engineering, Kongju Natinoal University, Cheonan, 330-717 In this study, a drying method was developed for the effective removal of the residual solvents, methylene chloride and methanol, in purified paclitaxel. When the sample purified by silica-HPLC was concentrated using a rotary evaporator, the residual methanol level easily met the ICH-specified value (3000 ppm), but methylene chloride did not meet the ICH- specified value (600 ppm). However, residual methylene chloride was easily and conveniently removed below the ICH- specified value by rotary evaporation (~1 h) alone after pre-treatment of a sample (methylene chloride: 17,600 ppm) with 95% ethanol. In addition, residual ethanol (>14,300 ppm) met the ICH-Specified value (5000 ppm) after simple rotary evaporation (~1 h) alone with pre-treatment with water, and residual water also met the specified value(<4%) for active pharmaceutical ingredients. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Residual Solvent, Removal, Pre-Treatment, Drying References 1. Kim JH, Park HB, Gi US, Kang IS, Choi HK, Hong SS. Removal of residual solvents in paclitaxel by supercritical carbon dioxide(2001). Korean J Biotechnol Bioeng., 16, 233–6. 2. Pyo SH, Park HB, Song BK, Han BH, Kim JH. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis(2004), Process Biochem., 39, 1985-91. 3. Lee JY, Kim JH. Microwave-assisted drying of paclitaxel for removal of residual solvents(2013). Process Biochem., 48, 545-550. OP3019Production and Regulation of gamma-amino butyric acid (GABA)-enriched Sea Tangle Extract by Lactic Acid Bacterial Fermentation Deok Hoon KIM, Song Hee KIM, Gab Jin KIM, Young-Mog KIM Department of Food science and Technology, Pukyong National University, Busan, 608-737 This study was conducted to develop a value-added product using sea tangle Saccharina japonica. It has been known that glutamate converts into gamma-amino butyric acid (GABA) by lactic acid bacterial fermentation. Among of lactic acid bacterial strains tested in our previous studies, Lactobacillus brevis BJ-20 exhibited the highest efficacy in converting GABA in sea tangle water extract (SWE). In order to optimize the GABA production, the lactic acid bacteria was inoculated into and cultivated in the water extract. The GABA production was determined by HPLC analysis before following fermentation in addition of several growth factors such as carbon sources, nitrogen sources and amino acids. Among them, the addition of yeast extract (YE) in SWE was resulted in the most efficient GABA production. However, no GABA production in SWE was observed in the absence of YE, suggesting that YE will be an essential on GABA conversion by L. brevis BJ-20 strain in SWE. In order to address these phenomena, we conducted RT-PCR to monitor the expression of a gene(s) related GABA conversion in the presence or absence of YE. RT-PCR results indicated that glutamate decarboxylase transcriptional regulator gene was only expressed in the presence of YE. Considering above results, we concluded that YE is one of critical factor in the bioconversion of glutamate into GABA by L. brevis BJ-20. Keywords : Gamma amino butyric acid, Lactic acid Bacteria, Fermentation, Glutamate Decarboxylase gene References 1. Kang YM, Qian ZI, Lee BJ and Kim YM. 2011. Protective effect of GABA- enriched fermented sea tangle against ethanol-induced cytoxicity in HepG2 cell. Biotechnol Bioprocess Eng 16, 966-970 2. 3. Bae HN and Kim YM. 2010. Improvement of the functional qualities of sea tangle extract through fermentation by Aspergillus oryzae. Fish Aquat Sci 13, 12-17 OP3020Structural Elucidation of Aptamer-based Sandwich Assay of Cystatin B - DNA Aptamer for HCC Detection Woo-Ri SHIN1, Kyeong-Ah LEE1, Simranjeet Singh SEKHON1, Hyun-Ju UM1, Dae-Ghon KIM3, Jiho MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 362-763, South Korea, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 664-14, 1-Ga, Deokjin-Dong, Deokjin-Gu, Jeonju 561-156, South Korea, 3Research Institute of Clinical Medicine of Chonbuk National University, Biomedical Research Institute of Chonbuk National University Hospital When Hepatocellular carcinoma (HCC) is diagnosed in the later stage, there is very poor with a survival rate than 15%. In general, HCC detection is measured by identification of Alpha-fetoprotein (AFP) expression level in serum. Recently, it increased that associated Cystatin B can be useful biomarker for early diagnosis of HCC. The cystatin B (CSTB) protein is a member of the cysteine protease inhibitor superfamily and intracellular thiol protease inhibitor. CSTB mRNA is overexpressed in most HCC and is elevated in the serum. In this study, we carried out SELEX process to obtain CSTB specific binding DNA aptamers. Using the SPR and dot blot assay, 2 aptamers (apta_3 and apta_22) were selected the high affinity and specificity with CSTB. Aptamer-based sandwich assay were compared with antibody-based ELISA kit, its measurement of HCC patients serum. And analysis of the CSTB-aptamer complex structure, it provide insights into the mode of their interaction which diagnosis of HCC and recurrence cancer advance in early pertinent treatment while continuous surveillance. This study was supported by Fund of Biomedical Research Institute, Chonbuk National University Hospital. (20120801002) Keywords : Cystatin B, Aptamer-based sandwich assay, Protein-aptamer structure References 1. 1. Mi-Jin Lee, Gyung-RanYu, Seon-Hwa Park, Baik-Hwan Cho, Jong-Seong Ahn, Hae-Joon Park, Eun-Young Song, and Dae-Ghon Kim. Identification of Cystatin B as a Potential SerumMarker in Hepatocellular Carcinoma (2008). Clin Cancer Res. 14(4). 1080-1089 2. 2. Hau-Tsai Cheng, Ya-Hui Chang, Yang-Yuan Chen, Tzong-Hsien Lee, Dar-In Tai1, Deng-Yn Lin. AFPL3 in Chronic Liver Diseases with Persistent Elevation of Alpha-fetoprotein (2007). Chin Med Assoc. 70(8). 310-317 OP3021Microfluidic Animal-on-a-chip for Nanoparticle Assessment Jin Ho KIM1, Seung Hwan LEE3, Sung Jin HONG4, Sang Kug CHUNG4, Tai Hyun PARK3,5,6, Shin Sik CHOI1,2 1Dept. of Energy Science and Technology, Myongji University, Yongin, 17058, 2Dept. of Food and Nutrition, Myongji University, Yongin, 17058, 3Dept. of Chemical and Biological Engineering, Seoul National University, Seoul, 08826, 4Dept. of Mechanical Engineering, Myongji University, Yongin, 17058, 5Bio-MAX Institute, Seoul National University, Seoul, 08826, 6Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do, 16229 Numerous nanomaterials with various sizes, core molecules and surface functional groups are consumed in a wide range of industry. Among them, the use of silver nanoparticles (AgNPs) has been rapidly enlarged for human convenience and health such as consumer products and medical applications. However, environmental and biological toxicity of AgNPs have not been sufficiently understood. In this study, we fabricated microfluidic animal-on-a-chip using a soil nematode Caenorhabditis elegans by soft-lithography technique for the qualitative and quantitative nanoparticle toxicity assessment. Given that the body length of C. elegans was shorten by AgNPs, nanoparticles’ toxicity could be visualized on a microfluidic channels by entrapping the worms. Using a transgenic C. elegans mutants with fluorescence genetic markers (mtl-2::gfp), we observed the increase of fluorescence intensity in the animals with exposure to AgNPs. Results demonstrate that a genetic mechanisms of nanoparticle toxicity in C. elegansmight enable the identification and measurement of specific nanoparticles. Keywords : Microfluidic animal-on-a-chip, Caenorhabditis elegans, Nanoparticles, Silver References 1. J.Y. Roh et al, Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics (2009), Environ. Sci. Technol. 43, 3933–3940 2. S. H. Lee et al, Microfluidic bead-based sensing platform for monitoring kinase activity (2014), Biosensors and Bioelectronics 57, 1-9 OP3022Development of Anti-obesity Agents on a Micro Cell Pattern Chip Gi Yong KIM1,2, Chang-Soo LEE2, Changhyun ROH1 1Division of Biotechnology, Advanced Radiation Technology Institute (ARTI), Jeung-up, 56212 ,2Department of Chemical Engineering, Chungnam National University, Daejeon, 305-764 Obesity is of growing global problem. Obesity is the fifth leading risk for global deaths. Thus, anti-obesity agents are highly spotlighted. The research of lipid inhibitor for the biological effects of obesity is necessarily needed. The discovery of bioactive compounds from foods is one of possible ways to prevent or reduce the risks of various obesity-related diseases. Recently, various of microfluidic devices have also been developed to increase cell viability. However, these techniques have limitations, such as hard fabricating a microfluidic chip, difficult operating, oxygen is limited. This technique is often not compatible with existing liquid handling robots for performing high-throughput screening (HTS). Alternatively, PDMS based micro cell pattern chip are frequently applied to cell studies due to many advantages such as short-term reaction, high sensitivity and easy observation [1, 2]. In this study, we generated a simple method to make uniform micro cell pattern chip poly(dimethyl siloxane) (PDMS) as a new platform. Here, we demonstrated this platform can be used for the real-time analyzing of adipocytes differentiation and confirmed lipid inhibition. Thus, this simple approach provides a useful platform technology, which means a method to pattern cells with high efficiency and reliability by generating cell adhesion for adipocyte differentiation on array chip regions. Keywords : Micropatterning, Anti-obesity agents, Screening References 1. Jeong H-H, Kim Y-G, Jang S-C, Yi H and Lee C-S, Lab on a Chip 12: 3290-3295 (2012) 2. Hertzberg RP and Pope AJ, Current opinion in chemical biology 4: 445-451 (2000) OP3023Rapid identification of newly emerging influenza viruses by surface-enhanced Raman spectroscopy Se-eun YANG1, Jung-soo NAM2, Jae-young LIM1, Hyunku SHIN1, Yoon-ha JANG2, Gyu-Un BAE3, Taewook KANG4, Kwang-il LIM2, Yeonho CHOI1,5 1Department of Bio-convergence Engineering, Korea University, Seoul, 136-713, Korea, 2Department of Medical & Pharmaceutical Sciences, Sookmyung Women’s University, Seoul, 140-742, Korea,3Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul, 140-742, Korea, 4Department of Chemical &Biomolecular Engineering, Sogang University, Seoul, 121-742, Korea, 5School of Biomedical Engineering, Korea University, Seoul, 136- 713, Korea In this work, we demonstrate in-situ virus identification based onsurface-enhanced Raman scattering (SERS). We hypothesized that newly emerginginfluenza viruses possess surface proteins and lipids that can generate distinctive Raman signals. To test this hypothesis, SERS signals were measuredfrom the surface of a non-influenza virus, two different influenza viruses, and a geneticallyshuffled influenza virus. To ensure the safety for experimenters we constructed non- replicating pseudotyped viruses that display main influenza virus surface components. Pseudotype with influenza virus components produced enhanced Raman peaks, on gold nanoparticles, that are easily distinguishable from those of pseudotype with a non-influenza virus component, vesicular stomatitis virus G protein (VSVG). Furthermore, virus with the surface components of a newly emerging influenza strain, A/California/04/2009 (H1N1), generated Raman peaks different from those of viruses with components of the conventional laboratory-adapted influenza strain, A/WSN/33 (H1N1). Interestingly, the virus simultaneously displaying surface components of both influenza strains, a model mutant with genome reassortment, also produced a Raman signal pattern that is clearly distinguishable from those of each strain.This work highlights that SERS can provide a powerful label-free strategy to quickly identify newly emerging and potentially fatal influenza viruses. Keywords : Raman scattering, SERS, Influenza virus References 1. A, K. L.; Oster, L.; Cardinal, R.; de Champlain, J., Role of NO and angiotensin II in the early development of endothelial functions impairment and cardiac hypertrophy in deoxycorticosterone acetate-salt hypertension. Canadian journal of physiology and pharmacology 1998,76 (6), 665- 2. Lim, K. I.; Klimczak, R.; Yu, J. H.; Schaffer, D. V., Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties. P Natl Acad Sci USA 2010,107 (28), 12475-12480. 3. Ge, B.; Li, Q.; Liu, G.; Lu, M.; Li, S.; Wang, H., Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR. Archives of virology 2013,158 (6), 1181-7. 4. Dawood, F. S.; Iuliano, A. D.; Reed, C., Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study (vol 12, pg 687, 2012). Lancet Infect Dis 2012,12 (9), 655-655. 5. , L.; Seeger, W.; Ermert, L.; Hanze, J.; Stahl, U.; Grimminger, F.; Kummer, W.; Bohle, R. M., Real-time quantitative RT- PCR after laser-assisted cell picking. Nature medicine 1998,4 (11), 1329-33. OP3024Fabrication of Water-Splitting Photoelectrode by Layer-by-Layer Assembly for Photobiocatalysis Dasom JEON, Jungki RYU, Yujin HAN, Hyunjin CHO Dept. of Energy engineering, Ulsan National Institute of Science and Technology, Ulsan Industrial application of biocatalysts have long been a dream of biochemists and bioengineers because of many advantages of biocatalysts over man-made catalysts. In principle, it is possible to synthesize virtually any kinds of chemicals with a high yield and selectivity by using enzymes under mild reaction conditions (e.g., aqueous solution, neutral pH, ambient temperature and pressure). To date, however, industrial application of biocatalysts has been very limited especially due to dependence of many enzymes on expensive cofactors. To solve such a problem, many efforts have been made to develop efficient way to recycle cofactors. Among a number of different methods, photochemical regeneration of cofactor has attracted numerous attention from researchers due to its environmental friendliness. For example, it has been reported that chiral model compounds such as L-glutamic acid can be continuously produced by glutamate dehydrogenase upon in situ regeneration of NAD(P)H cofactors with photosensitizers and electron mediators, the so-called biocatalytic artificial photosynthesis. Despite its huge potential, biocatalytic artificial photosynthesis is still at its infancy and far from true artificial photosynthesis because it still requires sacrificial electron donors for regeneration of cofactors. To address such a problem, in the present study we have developed a novel method to fabricate photoelectrodes, which can split water and produce electrons, provide them to electron mediators, and eventually enable regeneration of cofactors. We found that multiple components for photocatalytic water-splitting can be readily assembled on substrate by a simple layer-by-layer assembly technique and the prepared electrode shows water-splitting activity under visible irradiation. Based on these findings, we are currently trying to further integrate biocatalytic assemblies on the photoelectrode to take a step closer to realization of biocatalytic artificial photosynthesis. Keywords : biocatalysis, cofactor regeneration, artificial photosynthesis, water-splitting References 1. J.Ryu et al., Chem. Eur. J. 20, 12020(2014) OP3025Screening Biomarkers for Triclosan Using Daphnia Magna and Potential Application of Transgenic Organism in Toxicity Detection HwaYoon SHIN1, Yang-Hoon KIM2, Jiho MIN3 1Graduate School of Semiconductor and Chemical Engineering,Chonbuk National University, 664-14 Duckjin-dong, Jeonju 561-756, 2Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju , 3Graduate School of Semiconductor and Chemical Engineering,Chonbuk National University, 664-14 Duckjin- dong, Jeonju 561-756 Triclosan (TCS) has been used as an additive in a variety of consuming products. In this study, acute toxicity tests were performed according to EPA protocol to assess the impacts of triclosan on Daphnia magna. Additionally, the proteomic profile of treated D.magna was also analysed using two dimensional electrophoresis (2DE) technique. The comparison in protein expression pattern between control and TCS-treated organisms was then carried out using Progenesis software to explore the differentially expressed proteins (DEPs). After that, MALDI-TOF analysis was also conducted to identify the proteins dots. From MALDI-TOF results, certain promoters that can express certain DEPs were inserted upstream to the GFP gene of plasmid vector. Then plasmid transfection was carried out on Hela cell to confirm the working ability of these promoters. The changed protein spots due to TCS treatment can be used as novel biomarker candidates to detect triclosan as being mentioned above. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No: PJ01051501)” Rural Development Administration, Republic of Korea. The authors are grateful for their support. Keywords : Daphnia magna, triclosan, proteomic References 1. Thai-Hoang Le, Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers, 2013, Chemosphere93, 2341-2348 OP3026Chitosan-Coated Magnetic Mesoporous Silica with Pre-adsorbed Carbonic Anhydrase for Carbon Dioxide Capture Kie Moon WOO1, Inseon LEE1, Sung-Gil HONG1, Sunhyung AN2, Jinwoo LEE2, Euichaul OH3, Jungbae KIM1,4 1Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea, 3College of Pharmacy, the Catholic University of Korea, Bucheon 420–743, Republic of Korea, 4Green School, Korea University, Seoul 136-701, Republic of Korea A new approach of enzyme immobilization was developed by coating enzyme-adsorbed magnetic mesoporous silica with cross-linked chitosan. An enzyme, called carbonic anhydrase (CA), can catalyze the conversion of CO2 to bicarbonate at a 6 high turnover rate (kcat) of 10 per second [1]. In this study, we immobilized the CA by using a protocol of cross-linked chitosan. CA was adsorbed into magnetically-separable spherical mesocellular siliceous foam (Mag-S-MCF) [ADS-CA]. Then, the chitosan coating was performed by adsorbing chitosan (CS) on the surface of Mag-S-MCF with pre-adsorbed enzymes [ADS-CA/CS] and cross-linking chitosan by using glutaraldehyde [ADS-CA/CS-GA samples]. Under shaking condition, the ADS-CA/CS-GA sample could effectively prevent leaching of enzymes, without decrease of enzyme activity for 85 days. In the recycle test, ADS-CA/CS-GA also showed no activity decrease, and could be successfully used for the biocatalytic CO2 conversion to bicarbonate. By that way, the approach of chitosan coating improved both loading and stability of CA, which can be successfully employed in applications of carbon dioxide conversion. Keywords : Crosslinked chitosan coating, Carbonic anhydrase, Enzyme immobilization and stabilization, Magnetic mesoporous silica, Carbon conversion and utilization, Calcium carbonate References 1. H. Steiner, B.H. Jonsson, S. Lindskog. Eur. J. Biochem. 59, 253-259 (1975) 한국생물공학회, 생물공학의 동향 : 2015.10 포스터 3분 스피치 PSP01 Development of Waterproof Protein Glue for Urinary Fistula Sealing Hyo Jeong KIM, Seonghye LIM, Byeong Hee HWANG, Bong-Hyuk CHOI, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea For several decades, tissue adhesives have been considered as alternatives for traumatic surgical repairs. However, it still remains a challenge due to difficulties in attaining a balance in strong adhesive strength and low cytotoxicity. The conventional adhesives have their own shortcomings, for example, synthetic adhesives (such as cyanoacrylate) are not suitable for internal organs due to their cytotoxicity and high stiffness. Whereas, biologically derived adhesives (such as fibrin glue) exhibit relatively low adhesive strength. Therefore, developmental focus of a novel tissue adhesive which has high bulk adhesion strength and biocompatibility has become more prevalent. In this study, we developed novel tissue adhesive based on complex coacervate formulation of mussel adhesive protein (MAP). The complex coacervate between recombinant MAP and hyaluronic acid (HA) (MAP/HA coacervate) exhibited strong waterproof ability and extraordinary underwater adhesive strength on both inorganic and organic surfaces. Additionally, ex vivo pressure test using rat bladders indicated that the gel-like cross linked MAP/HA caocervate has high durability and compliance. Collectively, our results provided evidence that MAP/HA coacervate could be successfully used as an advanced underwater tissue bioadhesive in our body. Keywords : Mussel adhesive protein, Complex coacervation, Underwater adhesion, Urinary fistula References 1. Lim S. et al. The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins (2010), Biomaterials, 31(13), 3715-3722 2. Hwang D.S. et al. Practical recombinant hybrid mussel bioadhesive fp-151 (2007), Biomaterials, 28(24), 3560- 3568 3. Ridder D. Vesicovaginal fistula: a major healthcare problem (2009), Curr. Opin. Urol. 19(4), 358-361 PSP02 Decrease in the Particle Size of Paclitaxel by Fractional Precipitation Process Using Ion Exchange Resin and Hydrophilic Polymer Yong Kyu LIM, Jin-Hyun KIM* Dept. Chemical Engineering, Kongju National University, Cheonan, 330-717 In this study, we have for the first time applied increased surface area fractional precipitation with hydrophilic polymer in order to decrease the particle size of the anticancer agent paclitaxel from plant cell cultures. When compared with the case where no hydrophilic polymer was employed, the addition of polymer in increased surface area fractional precipitation resulted in a considerable decrease in the size of the paclitaxel precipitate. The use of a polymer had an effect on inhibition of precipitate growth compared to prepared particles without polymer. Among the polymers used, PVP-K90 was the most effective for inhibition of precipitate growth. The size of the paclitaxel precipitate also depended on the type of polymer, a result considered to be due to differences in the affinity between the particular polymer used and the paclitaxel particles. In addition, a polymer concentration of 0.2 wt% yielded the smallest particle size. However, the particle size increased as the concentration of polymer increased to greater than 0.2 wt%. This result might be due to inhibiting an appropriate diffusion of the solvent toward the antisolvent (water) caused by the high viscosity of the solution containing polymer. Paclitaxel, with a reduced particle size due to the addition of a polymer during the increased surface area fractional precipitation process, is believed to be particularly useful for practical applications of the drug. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Increased Surface Area Fractional Precipitation , Particle size, Decrease, Polymer References 1. Han MG, Jeon KY, Mun SY, Kim JH, Development of a micelle-fractional precipitation hybrid process for the pre- purification of paclitaxel from plant cell celtures(2010), Process Biochem., 45, 1368-1374. 2. Cho EB, Cho WK, Cha KH, Park JS, Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additive(2010), Int. J. Pharm., 396, 91-98. 3. Lee JY, Kim JH, Decease in the particle size of paclitaxel by increased surface area fractional precipitation(2012), Korean J. Microbiol. Biotechnol., 40, 169-174. PSP03 Enhancement of Acetone-Water Fractional Precipitation Efficiency for Purification of Paclitaxel from Biomass Tae Wan KIM, Jin-Hyun KIM* Dept. of Chemical Engineering, Kongju National University, Cheonan, 330-717 This study investigated the efficiency of acetone/water fractional precipitation. When acetone/water ratio was 40/60, 30/70, 20/80, and 10/90 (v/v), the yield of paclitaxel was 54.3, 89.1, 95.5, and 97.6%, respectively. The yield of paclitaxel increased with increasing acetone/water ratio. Also, when adding distilled water until acetone/water ratio was 40/60 (v/v), follwed by mixing for 10 min was performed at low temperature (4℃), the high yield (~87.9%) of paclitaxel was obtained immediately. However, when the same method was performed at room temperature, the low yield (~54.3%) of paclitaxel was obtained compared to the addition of distilled water at low temperature (4℃) and after additional mixing at room temperature for 2 hr, the yield (~86%) of paclitaxel was achieved that was equivalent to the result (~87.9%) when method was conducted at low temperature (4℃). Thus, when the addition of distilled water was performed by acetone/water at low temperature (4℃), the high yield of high-purity paclitaxel was achieved during quite a short time. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Purification Efficiency, Acetone/water Fractional Precipitation, Improvement References 1. Lee CG, Kim JH, Improved fractional precipitation method for purification of paclitaxel(2014), Process Biochem., 49, 1370-1376. 2. Kim JH, Paclitaxel : recovery and purification in commercialization step(2006), Biotechnol. Bioeng., 21, 1-10. 3. Tertius L. Fonseca, Kaline Coutinho, Sylvio Canuto, Hydrogen bond interactions between acetone and supercritical water(2010), Phys. Chem. Chem. Phys., 12, 6660-6665. PSP04 Adsorption Behavior and Kinetic Characteristic of Paclitaxel by Diaion HP-20 Hong Sik SHIN, Jin-Hyun KIM* Dept. of Chemical Engineering, Kongju National University, Cheonan, 330-717 Batch experiment studies were carried out for adsorption of anticancer agent paclitaxel using Diaion HP-20 with various parameters such as initial paclitaxel concentration, contact time and solution temperature. The obtained adsorption data were fitted to the Langmuir, Freundlich, Temkin and Dubin-Radushkevich isotherm equations. The comparison results revealed that Langmuir model could account for the adsorption equilibrium data of paclitaxel with the highest accuracy among the four adsorption models considered. Base on Temkin constant and Dubinin-Radushkevich constant, this adsorption process is physical adsorption. Adsorption kinetics has been tested using pseudo-first-order, pseudo-second-order and intraparticle diffusion models. The experimental results agreed well with the second-order adsorption model. Thermodynamic parameters, such as standard enthalpy (), standard entropy () and standard gibbs free energy () change, were investigated. The results indicated that the adsorption process was endothermic, irreversible and spontaneous. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Diaion HP-20, Adsorption Isotherm, Kinetics, Thermodynamics References 1. I.A.W. Tan, A.L. Ahmad, B.H. Hameed, Adsorption of basic dye on high-surface-area activated carbon prepared from coconut husk: Equilibrium, kinetic and thermodynamic studies(2008), ScienceDirect, 154, 337-346. 2. Lee JJ, Adsorption behavior and kinetic characteristic of cibacron brilliant red 3B-A by granular activated carbon(2014), Korean Chem. Eng. Res., 52(4), 486-491. 3. *P.Sivakumar and P.N.Palanisamy, Adsorption studies of basic red 29 by a nonconventional activated carbon prepared from euphorbia antiquorum l(2009), International Journal of ChemTech Res., 1(3), 502-510. PSP05 An Efficient Removal Method of Residual Ionic Liquid from Paclitaxel Extract by Treatment with Water Seul Ki KIM, Jin-Hyun KIM* Dept. of Chemical Engineering, Kongju National University, Cheonan, 330-717 When using an ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate, [Bmim]BF4) as co-solvent, the extraction efficiency of anticancer agent paclitaxel from biomass was dramatically improved. However, the residual ionic liquid had a significant negative effect on convenient and feasibility of following concentration and drying steps. In this study, a novel method was developed for the effective removal of the residual ionic liquid in biomass extract. The residual ionic liquid was easily and conveniently removed by drying after treatment of a sample recovered by biomass extraction with water. The optimal crude extract/water ratio for water treatment was 1:100 (w/v). Furthermore, the operation methods (sonication- or microwave-assisted operation) and major parameters (amount of water, stirrer speed and treatment time) of water treatment process were also investigated. These results should prove useful for the removal of ionic liquid in the mass production of paclitaxel from biomass extraction using ionic liquid as co-solvent. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Extraction, Residual Ionic Liquid, Removal, Drying References 1. Ha SH, Applications and prospects of ionic liquids in microbiology and biochemical engineering(2013), Korean J. Microbiol. Biotechnol., 41, 1-7. 2. Kim GJ, Kim JH, Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures(2015), Process Biochem., 50, 279-284. 3. Kim GJ, Kim JH, Enhancement of extraction efficiency of paclitaxel from biomass using ionic liquid-methanol co-solvents under acidic conditions(2015), Process Biochem., 50, 989-996. PSP06 Controlling Particle Size of a Poorly Water-Soluble Paclitaxel Using Hydrophilic Polymer in Fractional Precipitation Min Jae KIM, Jin-Hyun KIM* Dept. of Chemical Engineering, Kongju National University, Cheonan, 330-717 In this study, we have for the first time applied fractional precipitation with hydrophilic polymer in order to decrease the particle size of the anticancer agent paclitaxel from plant cell cultures. When compared with the case where no hydrophilic polymer was employed, the addition of hydrophilic polymer in fractional precipitation resulted in a considerable decrease in the size of the paclitaxel precipitate. The use of a polymer had an effect on inhibition of precipitate growth compared to prepared particles without polymer. Among the polymers used, HPMC 2910 was the most effective for inhibition of precipitate growth. The size of the paclitaxel precipitate also depended on the type of polymer, a result considered to be due to differences in the affinity between the particular polymer used and the paclitaxel particles. In addition, a polymer concentration of 0.2 wt% yielded the smallest particle size. However, the particle size increased as the concentration of polymer increased to greater than 0.2 wt%. This result might be due to inhibiting an appropriate diffusion of the solvent toward the antisolvent (water) caused by the high viscosity of the solution containing polymer. Paclitaxel, with a reduced particle size due to the addition of a polymer during the increased surface area fractional precipitation process, is believed to be particularly useful for practical applications of the drug. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). Keywords : Paclitaxel, Fractional Precipitation, Polymer, Particle Size, Decrease References 1. Lee JY, Kim JH, Decrease in the particle size of paclitaxel by increased surface area fractional precipitation(2012), Korean J. Microbiol. Biotechnol., 40, 169-174. 2. Cho EB, Cho WK, Cha KH, Park HJ, Hwang SJ, Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additives(2010), Int. J. Pharm., 396, 91-98. 3. Han MG, Jeon KY, Mun SY Kim JH, Development of a micelle- fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures(2010), Process Biochem., 45, 1368-1374. PSP07 Light-Activated Instant Mussel Protein-Based Surgical Glue Eun Young JEON1, Byeong Hee HWANG2, Yun Jung YANG1, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, 2Division of Bioengineering, Incheon National University Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine photo-crosslinking chemistry[1]. Because dityrosine crosslinks are known to confer mechanical and conformational stability and elasticity to insect structural protein including resilins of dragonfly wings, fibroins of silk worms, and locust cuticles, their introduction into tyrosine-rich (approximately 20 mol%) MAP may significantly increase structural support and adhesive properties as a very stable bridge. As a result, LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs. Keywords : tissue adhesives, mussel adhesive proteins, light-activated hydrogel References 1. D. A. Fancy and T. Kodadek, Chemistry for the analysis of protein–protein interactions: rapid and efficient cross- linking triggered by long wavelength light (1999), Proc. Natl. Acad. Sci, Vol (96), 6020 PSP08 Dynamic Range Enhancement of SoxRS as a Genetic Circuit via High-throughput Screening Jong Hyun PARK, Yong Jae LEE, Min Ah CHOI, Ki Jun JEONG Dept. Chemical and Biomolecular Engineering, KAIST Recent studies about high-throughput screening of enzymes have focused on construction of genetic circuit with native or synthetic promoters. In cytoplasm of microbial host, those promoters can express reporter proteins such as fluorescent or antibiotic proteins induced by products, intermediates or something from enzymatic reaction. Among them, SoxRS was known as a promoter concerned in defensive mechanism of Escherichia coli which can be induced oxidative stress (i.e. reactive oxygen species, ROS). Consequently, SoxRS has been reported as a biosensor for screening of NADPH consuming enzymes producing ROS as an intermediate. By using this biosensor, specific expression of fluorescent protein could allow high-throughput screening with fluorescence activated cell sorting (FACS). However, native SoxRS has not only narrow dynamic range but also very sensitive response to nonspecific effects. For these reasons, more specific screening depending on enzymatic reaction requires widened dynamic range of promoter. Here, we have developed enhanced dynamic range of SoxRS for a powerful screening tool with FACS. In order to widen dynamic range, we have repeated FACS sorting with randomized SoxRS in presence of inducer or not, and then we could successfully sort suitable mutant for genetic circuit. Keywords : Biosensor, Genetic circuit, High-throughput screening, FACS References 1. J. Wu, B. Weiss, J Bacteriol 1992, 174, 3915-3920 2. P. Gaudu, N. Moon, B. Weiss, J Biol Chem 1997, 272, 5082-5086 3. S. Siedler, G. Schendzielorz, S. Binder, L. Eggeling, S. Bringer, M. Bott, Acs Synth Biol 2014, 3, 41-47 PSP09 Adhesive Protein Nanoparticle using Iron-DOPA Coordination for pH-Responsive Drug Delivery Yeonsu JEONG, Bum Jin KIM, Yun Kee JO, Bong-Hyuk CHOI, Hyung Joon CHA Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 A bio-inspired strategy for the synthesis of protein nanoparticles (NPs) was proposed for controlled release of an anticancer drug via pH-responsive transition in the coordinative stoichiometry of Fe(Ⅲ)-3,4-dihydroxyphenylalanine (DOPA) complexes. Doxorubicin-loaded polymeric NPs based on the DOPA-modified recombinant mussel adhesive protein that contain Fe(Ⅲ)-DOPA complexes were synthesized through a co-electrospraying process. The fabricated NPs could control the release of doxorubicin through the acidic-pH-induced transition in the structure of Fe(Ⅲ)-DOPA complexes. It was also exhibited effective cytotoxicity towards cancer cells through efficient cellular uptake and cytosolic release. This suggested that mussel-inspired protein NPs containing Fe(Ⅲ)-DOPA complexes can be used as a potential drug carrier for diverse pH- responsive drug delivery applications. Keywords : mussel adhesive protein, pH-responsive nanoparticle, iron complex, drug delivery References 1. Bun Jin Kim, Hogyun Cheong, Byeong Hee Hwang, and Hyung Joon Cha, Mussel-Inspired Protein Nanoparticle containing Iron(Ⅲ)-DOPA Complexes for pH-Responsive Drug delivery (2015), Angew. Chem., 54, 1-6 2. Khin Yin Win, Si-Shen Feng, Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticcancer drugs (2005), Biomaterials, 26, 2713-22 PSP10 Microcystis aeruginosa a Feasible and Carbohydrates Rich Source for Bioethanol Production Muhammad Imran KHAN1, Jong Deog KIM1,6, Moon Geon LEE1, Hyo Jin SEO1,6, Jin Hyuk SHIN1, Tai Sun SHIN2,6, Yang Ho YOON3, Min Yong KIM4,6, Jong Il CHOI5 1Dept. of Biotechnology, Chonnam National University, 2Dept. of Food Science and Nutrition, Chonnam University, 3Dept. of Environmental oceanography,Chonnam University, 4Dept. of Refrigeration Engineering, 5Dept. of Biotechnology and Bioengineering, Chonnam National University, 6Research center on Anti-Obesity and Health Care, Chonnam National University Bioalcohol is one of the most important renewable energy source and a competitive alternative to the petroleum based fossil fuels as comparatively cheap and environmentally safe technology. The rapid increase in the global demand of ethanol needs to explore new renewable biomass sources. Present study aimed to reveal the potential of using a sugar-riched microalga microcystis aeruginosa as feedstock for bioethanol production. Microcystis aeruginosa was cultured on MF Media (BG 11 modified media) under Red light with 16-8 hour’s alternate light and dark periods with continuous supply of CO2. Carbohydrates were obtained from M. aeruginosa by treating the biomass with UV, microwaves and acid hydrolysis. Final yield of glucose was fermented to bioethanole using combinly, three different microbial species i.e. klebsilla oxytoca, saccharomyces cerevisae and pichia stipitis which enhance the fermentation process efficiently. Production of bioethanole in the fermented media was conformed through dichromate analysis. A good yield of bioethonole conformed M. aeruginosa to be a feasible feedstock for bioethanle production. Keywords : Bioalcohol, BG 11 modified media, Microcystis aeruginosa, Microwaves, Saccharomyces cerevisae, Fermentation References 1. S. H. Ho, S. W. Huang, C. Y. Chen, T. Hasunuma, A. Kondo, and J. S. Chang, Bioresour Technol.(2013); 29:191- 198. 2. R.P. John , G.S. Anisha , K.M. Nampoothiri , A. Pandey, Bioresour Technol. (2011); 102: 186–193. 3. H.B. Seo, H.J. Kim, O.K. Lee, J.H. Ha, H.Y. Lee and K.H. Jung, J Ind Microbiol Biotechnol. (2009); 36: 285-92. PSP11 Aldehyde Treatment Using Aldehyde Dehydrogenase from Egg White Seyoung LEE1, Jihee YOON1, Seung Hyuck BANG1, Yang-Hoon KIM2, Jiho MIN1 1Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 664-14 Deokjin-dong, 1Ga Deokjin-Gu Jeonju 561-756, South Korea, 2Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763, South Korea Aldehyde dehydrogenase are a group of enzyme that catalyze the oxidation of a variety of aldehyde substrates to their corresponding carboxylic acids. In this study, we have designed novel method using aldehyde dehydrogenase to treat aldehyde. We successfully found the removal of formaldehyde and 2-nonenal using aldehyde dehydrogenase extracted from Hen’s egg white. First, luminescence of Vibrio fischeri was measured following formaldehyde concentration. Under the condition of formaldehyde which was treated the enzymes, it was increase to release of light. Trans-2-nonenal was decrease depending on the enzymes treatment time. Furthermore, in specific enzymes concentration, it was shown the result that the best treatment efficiency of trans-2-nonenal. Thus, based on this results, we expect that it would be applied to the field of deodorizing preparations and removal of hazard materials. This work was carried out with the support of “Cooperative Research Program for agriculture Science & Technology Development (Project title: Extraction and utilization technology development of functional materials in poultry egg white, Project No:PJ01164101)’ Rural Development Administration, Republic of Korea. The authors are grateful for their support. Keywords : aldehyde dehydrogenase, formaldehyde, 2-nonenal References 1. Haze S, Gozu Y, Nakashiyuki S, Kohno Y, Sawano K, Ohta H, Yamazaki K, 2-Nonenal Newly Found in Human Body Odor Tends to Increase with Aging, Journal of Investigative Dermatology(2001), 116, 520-524 2. William B. Rizzo, Fatty aldehyde and fatty alcohol metabolism: Review and importance for epidermal structure and function, Biochimica et Biophysica Acta 1841(2014), 377-389 3. Markus B, Jan G, Fenja S, Melanie P, Jens S, Oxidation of fatty aldehydes to fatty acids by Escherichia coli cells expressing the Vibrio harveyi fatty aldehyde dehydrogenase(FALDH), World J Microbiol Biotechnol(2013), 29:569-575 PSP12 Characterization of Isolate, Thermophile Clostridium sp., Isolated from Thermophilic Microbial Fuel Cell as Tentative Electrochemically Active Bacterium Mihwa LEE1, Serah CHOI1, Phuc THI HA2, In Seop CHANG1 1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST),261 Cheomdan- gwagiro, Buk-gu, Gwangju 500-712, South Korea , 2The voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman WA 99164-6515, USA A Gram-positive, moderate thermophile, anaerobic, rod-shaped, an indigo-reducing, strain GISTMFC-FeD9, was isolated from the anode electrode of distillery wastewater-fed thermophilic microbial fuel cell (MFC). The 16S rRNA analysis revealed that GISTMFC-FeD9 is phylogenetically close to known Clostridium isatidis strain Wv6T which is saccharolytic T with the similarity of 99%. The C. isatidis Wv6 has been known that gases (H2 and CO2), organic acids (acetic, lactic, and formic acid), and ethanol were produced on PYG medium. We determined that the isolate, GISTMFC-FeD9, could grow on glucose, sucrose, formate, and acetate as substrate, but not on galactose, fructose, glycerol, butyrate, fumarate, and lactate. The acetate, propionate, ethanol, H2, and CO2 were produced on glucose culture condition. We confirmed the electrochemical activity of GISTMFC-FeD9 on a graphite electrode during glucose fermentation from cyclic voltammetry (CV) test. The reduction peak was significantly showed at -0.35 V versus Ag/AgCl during CV measurement. Keywords : Thermophilic bacteria, Thermophlic microbial fuel cell, Glucose fermentation, Electrochemically Active Bacterium References 1. A. Nikki Padden , Vivian M. Dillon, John Edmonds, M. David Collin, Nerea Alvarez and Philip John, J. Systematic Bacteriology. 49, 1025-1031 (1999) PSP13 Colorimetric Detection of Glucose Based on the Enhanced Peroxidase Activity of PEG- Derivatized Ligand-Modified Magnetic Nanoparticles Ho Yun SHIN1, Bong-Geun KIM2, Hyon Bin NA2, Moon Il KIM1 1Dept. of BioNano Technology, Gachon University, Gyeonggi, 461-701, 2Dept. of Chemical Engineering, Myongji University, Gyeonggi, 449-728 Fe3O4 magnetic nanoparticles (MNPs), which were generally recognized to be chemically and biologically inert, have attracted significant recent attention as potent peroxidase mimetics [1]. Compared to natural peroxidase enzymes, they exhibited highly stable catalytic activity over a broad range of pH and temperature values and sustained an incubation or storage period. In this study, we report a colorimetric biosensor for the determination of H2O2 and glucose based on the peroxidase-mimicking activity of poly(ethylene glycol) (PEG)-derivatized ligand-modified MNPs (PEG-MNPs). Two kinds of PEG-ligands conjugated with phosphine oxide and phospholipid were simply synthesized and modified on the surface of MNPs, which leads to enhanced peroxidase activity by providing more monodispersed MNPs in aqueous buffer without self- aggregation. The PEG-MNPs were then used to catalyze the oxidation of peroxidase substrate into a colored end product, which provides a colorimetric detection of H2O2. Moreover, a highly sensitive and selective glucose biosensing strategy was developed based on the coupled catalytic action between glucose oxidase (GOx) and PEG-MNPs. Using this strategy, a highly linear absorbance enhancement was verified when the concentration of glucose was increased in a wide range from 5 μM to 1 mM with a lower detection limit of 3 μM, suggesting that PEG-MNPs can serve as potent peroxidase mimetics with a range of applications in diverse fields of biotechnology. Keywords : Colorimetric biosensor, Peroxidase mimetics, Magnetic nanoparticles References 1. H. Wei and E. Wang, Nanomaterials with enzyme-like characteristics (Nanozymes): Next-generation artificial enzymes (2013), Chem. Soc. Rev. 42 (14), 6060 PSP14 The Detection of Viroid by Using Loop-Mediated Isothermal Amplification Se Hee LEE1, Ga-Young PARK1, Gna AHN1, Eunji LEE1, Yae-Eun JEON1, Yang-Hoon KIM1, Hong-Soo CHOI2, Ji-Young AHN1 1Dept. of Microbiology, Chungbuk National University, Cheong-Ju, Korea, 2National Institute of Animal Science, RDA, Korea Viroid are about 250-400 base pair of short single strand RNA fragments and pathogenicity [1]. Plants are only hosts of viroid, which cause serious damage and disease. The detection methods of viroid are northern blotting, dot blotting by using specific probe and Real-time PCR based on reverse transcription. However the methods require much expensive equipment and complex procedure. It is now essential to develop simple detection methods which are applicable in field. We here designed a way for detection of viroid by grafting loop mediated isothermal amplification (LAMP) which can amplify target in isothermal without the need to change temperature [2]. At first, we extracted total RNA of apple and pear leaves infected with each apple scar skin viroid (ASSVd) and pear blister canker viroid (PBCVd), respectively. Extracted RNAs were converted to cDNA by using a cDNA synthesis kit and specific primer sets. For each viroid, identification process was performed by T-vector cloning and sequencing. LAMP primers were then specifically designed from the sequence information. LAMP experimental condition for amplifying ASSVd/PBCVd was critically optimized and amplification efficiency was compared with. This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Development of fast sensing platform for viroids in subtropical plants, PJ011642)" Rural Development Administration, Republic of Korea. Keywords : Viroid, LAMP, ASSVd, PBCVd, detecting methods References 1. T.O.Diener., The Viroids. Plenum press, New York. 1-344 (1987). 2. T. Notomi et al. Nucleic Acids Res., 28(12), e63 (2000). PSP16 Optimization of Agitation Conditions for High Xylitol Production by Kluyveromyces marxianus 36907-FMEL1 Jin-Seong KIM, Jae-Bum PARK, Seung-Won JANG, Deok-Ho KWON, Suk-Jin HA Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Republic of Korea Xylitol can be used as a building block for valuable chemicals as well as it is well known sugar substitute having low calorie and non-carcinogenicity. Kluyveromyces marxianus has been developed for the production of xylitol, because thermotolerant K. marxianus has several advantages over mesophilic yeasts in practical aspects. Through a directed evolution and random mutagenesis, the mutant Kluyveromyces marxianus 36907- FMEL1 was isolated for efficient xylitol production. When agitation conditions were varied, xylitol production yields and productivities were highly changed at 30oC or 40oC. At 30oC, K. marxianus 36907-FMEL1 produced 41.21 g/L of xylitol with 51% yield and 0.57 g/L∙h productivity under 400 rpm and 0.5 vvm for 72 h. Interestingly, K. marxianus 36907-FMEL1 produced similar amount of xylitol (43.42 g/L) with higher productivity (1.20 g/L∙h) at 40oC than that from 30oC under same agitation and aeration conditions because fermentation was completed within 36 h. According to these results, K. marxianus 36907-FMEL1 produced xylitol at 40oC the fold faster than that from 30oC. Keywords : Xylitol, mutant, agitation, fermentation References 1. Jin-Seong Kim, Jae-Bum Park, Seung-Won Jang, Suk-Jin Ha,Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity, Applied Biochemistry and Biotechnology, 176, 1975-1984 2. Jae-Bum Park, Jin-Seong Kim, Seung-Won Jang, Eunsoo Hong, Suk-Jin Ha, The Application of Thermotolerant Yeast Kluyveromyces marxianus as a Potential Industrial Workhorse for Biofuel Production, KSBB Journal, 2015, 30.3: 125-131 PSP17 Enhanced Xylitol Production by An Engineered Kluyveromyces marxianus 17555-JBP2 through Overexpressing KmXYL1 Gene. Jae-Bum PARK1, Jin-Seong KIM1, Jin-Woo KIM2, Dae-Hyuk KWEON3, Jin-Ho SEO2, Suk-Jin HA1 1Department of Bioengineering and Technology, College of engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea , 2Department of Agricultural Biotechnology, Seoul National University, Seoul 152-742, Republic of Korea, 3Department of Genetic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Xylitol is usually used in food industry because of its non-cariogenic nature and low calorie content. Moreover, xylitol is kwon as a valuable synthetic building block which is one of the top 12 value-added materials produced from cellulosic biomass. Kluyveromyces marxianus, known as thermotolerant yeast, is robust and able to grow at 45°C which is the major advantage of K. marxianus for applying SSF process. Recently, several recombinant K. marxianus strains were engineered for xylitol production through expression of heterologous XR genes from mesophiles. In this study, we engineered K. marxianus KCTC 17555 by the multi-copy integration of KmXYL1 gene from K. marxianus 36907-FMEL1. When fermentations were performed using xylose as a sole carbon source at 30°C, K. marxianus 17555-JBP2 exhibited 46.85 g L-1 xylitol production from 72.62 g L-1 of xylose with 0.65 g g-1 yield and 0.39 g L-1 h-1 productivity. Interestingly, K. marxianus 17555-JBP2 produced more xylitol at 40°C than that from 30°C. 53.17 g L-1 of xylitol was produced at 40°C with 0.73 g g-1 yield and 0.55 g L-1 h-1 productivity. Keywords : Xylitol, Xylose reductase, Multi-copy integration, K. marxianus References 1. Jae-Bum Park, Jin-Seong Kim, Seung-Won Jang, Eunsoo Hong, Suk-Jin Ha, The Application of Thermotolerant Yeast Kluyveromyces marxianus as a Potential Industrial Workhorse for Biofuel Production, KSBB Journal, 2015, 30.3: 125-131. 2. Jin-Seong Kim, Jae-Bum Park, Seung-Won Jang, Suk-Jin Ha,Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity, Applied Biochemistry and Biotechnology, 176, 1975-1984 PSP18 Selection of Enzyme and Molar Ratio for Enzymatic Production of Phenethyl Acetate Hyunah KIM1, Jinkwang JOO1, Ho Jin YANG1, Jinyoung LEE2, Sung Ok HAN3, Chulhwan PARK*1 1Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea, 2Department of Plant and Food Science, Sangmyung University, Cheonan 330-720, Republic of Korea, 3Department of Biotechnology, Korea University, Seoul 136-701, Republic of Korea Phenethyl acetate, one of aromatic volatile acetate esters with rose-flavor, is representative for cosmetics, perfumes, soaps and other odor products. The goal of this study was to obtain the optimum conditions for phenethyl acetate production. The reaction optimization was performed with immobilized lipase, enzyme amounts, acetate selection, and the molar ratio of substrate for the highest conversion of lipase reaction. Phenethyl acetate production with Novozym 435 from Candida antarctica lipase B was tested with two acetates (vinyl acetate and acetic anhydride). In the results, the enzyme reaction with 10-40 g/L of Novozym 435 and 1:2–1:6 molar ratio of phenethyl alcohol to acetates showed high conversions. We are going to study the reaction temperature and solvent selection. Our work is expected to support the process cost of phenethyl acetate production. Keywords : Phenethyl acetate, optimization, immobilized lipase, Novozym 435, acetates(vinyl acetate and acetic anhydride) References 1. A. Widjaja, T.-H. Yeh, Y.-H. Ju, Enzymatic synthesis of caffeic acid phenethyl ester (2008), J. Chin. Inst. Chem. Eng, 39, 413-418. 2. C.-H. Kuo,S.-H. Chiang, H.-Y. Ju, Y.-M. Chen, M.-Y. Liao, Y.-C. Liu and C.-J. Shieh, Enzymatic synthesis of rose aromatic ester (2-phenylethyl acetate) by lipase (2012), J. Sci. Food Agric, 92, 2141-2147. 3. S. Oh, C. Park, Enzymatic production of glycerol acetate from glycerol (2015), Enzyme Microb. Technol., 69, 19- 23. PSP19 Effects of Phenolic Compounds on 2,3-Butanediol Production by Enterobacter aerogenes Jinkwang JOO1, Hyunah KIM1, Hanyong KIM1, Min Sang YOO1, Sang Jun LEE2, Jaehoon CHO3, Seung Wook KIM2, Chulhwan PARK*1 1Department of Chemical Engineering, Kwangwoon University, Seoul, 139-701, Republic of Korea, 2Department of Chemical and Biological Engineering, Korea University, Seoul, 136-701, Republic of Korea, 3Green Process and Materials R&D Group, Korea Institute of Industrial Technology (KITECH), ChonAn, 331-822, Republic of Korea To solve the environmental problem, lignocellulosic biomass has been received great attention as a alternative of petroleums. Lignocellulosic biomass has several advantages such as variety of carbon sources, low cost of raw materials, possibility of using microbial production. Lignocellulosic biomass requires pretreatment process. The pretreatment process can dissolve cellulose and hemicellulose into carbon sources. However, organic acids, furans, and phenolic compounds are usually generated during pretreatment process. They make negative effects on microbial production of 2,3-butanediol which is widely known as one of the value-added chemicals. In this study, the effect of organic acid, furans and phenolic compounds was investigated on cell growth and production of 2,3-butanediol from xylose by Enterobacter aerogenes. Keywords : Lignocellulosic biomass, 2,3-Butanediol, Microbial production, Inhibitors, Enterobacter aerogenes References 1. J. Jarmander, B. M. Hallstrom, G. Larsson, Simultaneous uptake of lignocellulose-based monosaccharides by Escherichia coli (2014), Biotechnology and Bioengineering, 111, 1108-1115. 2. F. R. Frazer, T. A. McCaskey, Effect of components of acid hydrolysed hardwood on conversion of D-xylose to 2,3-butanediol by Klebsiella pneumoniae (1991), Enzyme and Microbial Technology, 13, 110-115. 3. M.-Y. Jung, C. Y. Ng, H. Song, J. Lee, M.-K. Oh, Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production (2012), Applied Microbiology and Biotechnology, 95, 461-469. PSP20 Transciptomic Analysis of Metabolic Changes by Temperature Stress in Megasphaera elsdenii SU1 for the Production of Hexanoic Acid from Jerusalem Artichoke Hyunjin KIM1, Byoung Seung JEON1, Tae Ho LEE2, Byoung-In SANG1 1Department of Chemical Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea, 2The Research Institute of Industrial Science, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea When Megasphaera elsdenii SU1 utilized Jerusalem artichoke (JA) hydrolysate as a substrate at 37℃, the growth rate was rapid but hexanoic acid production was relatively low. During the cultivation at 30℃ as sub-optimal temperature, Megasphaera elsdenii SU1 showed slower growth and the productivity of hexanoic acid was lower than that at 37℃. However the final titer of hexanoic acid and the ratio of hexanoic acid to butyric acid were higher than those results of cultivation at 37℃. Interestingly, when hexanoic acid production curves were fit using non-linear regression with a modified Gompertz equation, lag time and final concentration of hexanoic acid minus butyric acid have a linear relationship. In the case of Clostridial acetone-butanol-ethanol (ABE) fermentation, in rapid growth period, cells have a higher energy demand and more ATPs are required, resulting in the production of acetate. More production of butyrate and butanol are occasionally observed in slower growing cultures. Similarly, for hexanoic acid fermentation, the shift in the production pattern of produced acids was observed from one of primarily butyrate seen during the exponential growth phase to a high proportion of hexanoate production during the stationary phase. To investigate the effect of culture temperature on the metabolic changes during the hexanoic acid production, the transcriptomic analysis was conducted. Keywords : Transcriptome, Hexanoic acid, Temperature, Megasphaera elsdenii, Jerusalem artichoke References 1. Li, J. Z., et al. (2014). "Acetone-butanol-ethanol fermentation of corn stover by Clostridium species: present status and future perspectives." World Journal of Microbiology & Biotechnology 30(4): 1145-1157 한국생물공학회, 생물공학의 동향 : 2015.10 포스터 발표 한국생물공학회, 생물공학의 동향 : 2015.10 미생물공학 P0001 Adaptive Evolution for Efficient Production of D-Lactic Acid in Saccharomyces cerevisiae Eunice Y. KWON, Seung-Ho BAEK, Ji-Sook HAHN School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744 There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereo-complex PLA with improved properties. Although Saccharomyces cerevisiae does not synthesize LA naturally, it has emerged as a promising alternative to produce LA because of high acid tolerance. We developed D-LA-producing S. cerevisiae strains by using metabolic pathway engineering, but D-LA production was limited by the growth inhibitory effect of D-LA. To further increase D-LA production levels, we performed adaptive laboratory evolution using serial subcultures with a gradual increase in LA concentrations and selected the evolved strains based on glucose consumption ability and LA production level. Consequently, we generated an evolved strain showing 1.6 and 2.1 fold higher glucose consumption and LA production abilities respectively than those of unevolved strain. Keywords : Adaptive laboratory evolution, D-lactic acid, Saccharomyces cerevisiae P0002 Production of 5-Aminolevulinic Acid by Recombinant Streptomyces Cells Expressing HemA Gene of Rhodobacter spharoides Nu Thi TRAN, Chang Joon KIM, Sung Bae KIM Dept. of Chemical Engineering, Gyeongsang National University, Jinju 660-701, South Korea 5-aminolevulinate (ALA) has medical applications for photodynamic cancer theraphy and tumor diagnosis. It can be also utilized as a biodegradable herbicide or insecticide in agriculture. In previous study, recombinant Streptomyces cells expressing HemA gene were constructed and it was found that this strain produced 60 mg/L of ALA in YH medium supplemented with 5 g/L of glycine and 10 g/L of succinic acid. In this study, we aimed to further improve the ALA production of this recombinant Streptomyces cells. Several media (YEME, YMG, R2YE, M9, YH, LB, 2 xYT) supplemented with glycine and succinic acid were tested. The effect of nitrogen sources (yeast extract, peptone, casamino acid, corn steep liquor, malt extract, soybean flour and (NH4)2SO4) was examined at different concentrations. The effect of carbon sources (glucose, glycerol, starch, soybean oil) was also examined. It was observed that the highest amount of ALA was produced in YH than other complex media. In addition, the carbon sources were not effective for production of ALA. The glucose was chosen to be the carbon source for futher culture. The ALA concentration increased by supplementing 5 g/L of corn steep liquor, 10g/L of soybean flour, 5g/L of (NH4)2SO4, additionally. The highest amount of ALA, 170 mg/L, was produced when cells were cultured in YH supplemented with glycine, succinic acid, corn step liquor, (NH4)2SO4, soybean flour. Keywords : Recombinant Streptomyces cells, 5-aminolevulinic acid, Production, Medium effect References 1. Mariet J, Van der Werf, and J.Gregory Zeikus. MARIE, 5-Aminolevulinate Production byEscherichia coliContaining the Rhodobacter sphaeroides hemA Gene (1996),American Society for Microbiology .62, 3560 2. K. Sasaki, M. Watanabe, T. Tanaka and T. Tanaka, Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid (2002) Microbiol Biotechnol. 58, 23 3. T.Kieser, M.J.Bibb, M.J.Buttner, K.F.Chater and D.A.Hopwood, Partical streptomyces genetics (2000)International Microbiology.3, 260 P0003 Depolymerization of Agarose by an AgaA7-secreting Bacillus subtilis Kristine Rose RAMOS1,1, Kris Niño VALDEHUESA1, Llewelyn MORON1, Heejin YANG1, Rhudith CABULONG1, Grace NISOLA1, Wonkeun LEE2, Wookjin CHUNG1 1Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728, 2Division of Bioscience and Bioinformatics, Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728 The products of agar hydrolysis have been reported to possess various medical and pharmaceutical effects. In this study, an extracellular β-agarase, AgaA7, recently isolated from Pseudoalteromonas hodoensis sp. nov was cloned in Bacillus subtilis. B. subtilis was chosen as host because of its capability to overproduce and secrete functional enzymes. Phenotypic analysis showed that the engineered B. subtilis was capable of degrading agarose and that the maximum enzyme activity was observed during the late logarithmic phase. To further improve the secretion of AgaA7, an expression library consisting of 173 naturally occurring B. subtilis signal peptides (SP) fused to the amino terminal of AgaA7 was created. The amount of AgaA7 secreted by the clones were compared through activity assay, immuno-blot, and purification via affinity chromatography. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2009-0093816). Keywords : agarase, Bacillus subtilis, secretion, signal peptide References 1. W. J. Chi, J. S. Park, D. K. Kang and S. K. Hong, Production and characterization of a novel thermostable extracellular agarase from Pseudoalteromonas hodoensis newly isolated from the West Sea of South Korea (2014) Appl. Biochem. Biotechnol. 173 (7), 1703-1716 P0004 Immunogenicity of an Oral Vaccine against Dengue Virus Generated in Saccharomyces cerevisiae Jyotiranjan BAL1, Nguyen Ngoc LUONG2, Hee-Young JUNG1, Yong-Suk JANG3, Dae-Hyuk KIM1 1Department of Bioactive Material Sciences, Chonbuk National University,Jeonju, 561-756, South Korea, 2Department of Biology, College of Sciences, Hue University,Hue city, Vietnam, 3Research Center of Bioactive Materials, Chonbuk National University,Jeonju, 561-756, South Korea Dengue virus (DENV), consisting of four different serotypes (DENV1 to 4), is a potentially deadly mosquito borne virus [1] affecting millions of people annually. Lack of specific therapeutics has led its prevention to be limited to vector control measures only. Oral administration is an attractive alternative to the conventional parenteral route. The effect of M-Glucan, from Saccharomyces cerevisiae, as an adjuvant in vaccines [2] and having a highly evolved expression system, it was selected as the delivery vehicle. We tried to explore the efficacies of E.coli heat labile toxin protein (LTB) fused synthetic consensus envelop domain III (scEDIII) expressed whole yeast cells as well as cell free extracts as possible oral vaccines in Balb/c mice. Mice were orally gavaged with whole recombinant yeast cells as well as cell free extracts (CFE) 4 times with 2 weeks interval. Specific anti-Dengue serum IgG was detected in mice fed with CFE from 3-11 weeks from the start of immunization protocol, whereas in case of whole cell fed mice it was detected at 5-11 weeks. Specific anti-Dengue IgA in feces was detected from 7-10 weeks in both cases. Although the immune response due to recombinant whole cells was comparatively lower to that induced by CFE, but surprisingly surged and matched with that of CFE after intra peritoneal booster of purified scEDIII antigen, which confirms its efficacy as a potent oral vaccine. Keywords : Dengue, Saccharomyces cerevisiae, EDIII References 1. S. Bhatt et al., Nature. 496, 504 (2013). 2. Gunnar Rørstad, Per Martin Aasjord and Børre Robertsen, Fish Shellfish Immunol., 3, 179 (1993). P0005 Expression of Fusion Protein of Dengue Synthetic Consensus Envelope Domain III through Cell Surface Display on Saccharomyces cerevisiae for Use as Oral Vaccine. Hee-Young JUNG1, Ngoc Luong NGUYEN2, Jyotiranjan BAL1, Jung-Mi KIM3, Dae-Hyuk KIM1 1Department of Bioactive Material Science, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju 561-756, Korea, 2Dept. of Biology Colleae of Science, Hue University, Vietnam, 3Department of Bio-Environmental Chemistry, Wonkwang University, Iksan 570-749, Korea Dengue fever a mosquito borne disease is caused by one of the four serotypes of dengue virus. Dengue virus has four similar but nonidentical serotypes and each serotype induces different immune response. Recently there is a dramatic increase in the number of dengue infections reported by WHO annually.[1] Therefore, an effective vaccine is required against all four serotypes because of the unavailability of commercially available vaccine against this virus. Therefore in this study, we attempt to develop an oral vaccine against dengue virus by expressing fusion protein of consensus sequence of envelop domain III(scEDⅢ) through cell surface display in Saccharomyces cerevisiae. The fusion construct (Co1-scEDIII-AGA1) was cloned into yeast episomal pYEGPD vector and then transformed into S. cerevisiae. The transformants were confirmed through colony PCR, E.coli back transformation and expression of recombinant scEDⅢ was confirmed through western blot and northern blot analysis. Furthermore, surface display of scEDⅢ was confirmed through immunostaining with FITC conjugated antibody. Keywords : cell surface display, dengue virus, scEDIII References 1. Maria G. Guzman et al., Dengue: a continuing global threat. Nature Reviews Microbiology , S7-S16(2010) P0006 Screening of New Xylose Dehydrogenases for the Production of D-Xylonic Acid from D-Xylose in Escherichia coli Kris Niño G. VALDEHUESA1, Kristine Rose M. RAMOS1, Hee-Jin YANG1, Llewelyn S. MORON1, Rhudith B. CABULONG1, Grace M. NISOLA1, Won-Keun LEE2, Wook-Jin CHUNG1 1Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728, 2Division of Bioscience and Bioinformatics, Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728 Xylonic acid has several industrial applications as chelator, as concrete dispersion agent, and as precursor for synthesis of polymers and polyalcohols. This compound is derived from D-xylose though enzymatic catalysis of xylose dehydrogenases (Xdh), which are found in a few Bacteria (Caulobacter and Gluconobacter) and Archaea (Haloarcula and Haloferax). Screening for candidate Xdh from other microorganisms was performed by mining available databases for annotated ORFs with high homology to C. crescentus Xdh. Three candidate genes were selected from three different microorganisms, then cloned and expressed in Escherichia coli. The purified Xdh were characterized for substrate specificity, co-factor preference, and enzyme activity. The best performing Xdh was then tested for efficient conversion of D-xylose to D-xylonic acid in metabolically engineered E. coli. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2009-0093816). Keywords : xylose dehydrogenase, xylonic acid, Escherichia coli References 1. Valdehuesa et al. (2014) Process Biochemistry 49:25-32 2. Liu et al (2012) Bioresource Technology 115:224-248 P0007 Effect of Some Genetic Modifications of Recombinant Escherichia coli Host on the Biocatalytic Production of 2’-Deoxycytidine Byung Kyun KIM, Kang Hyun CHOI, Ji Soo SONG, Kap Soo NOH ST Pharm. Co. LTD.,231, Hyeomnyeok-ro, Siheung-si, Gyeonggi-do 429-848 2’-Deoxycytidine is commercially useful precursor in various nucleoside analogue drugs and intermediates of antisense oligonucleotides. 2’-Deoxycytidine can be produced using thymidine and cytosine as starting material by enzymatic transglycosylation process of nucleoside N-deoxyribosyltransferase. While using recombinant whole cell enzyme for production of 2’-deoxycytidine, uracil and 2’-deoxyuridine were formed probably due to cytosine deaminase and cytidine deaminase derived from Escherichia coli host. The side products formation results in lower production yield of 2’- deoxycytidine and makes following purification process more complex. Escherichia coli host JM109 was genetically modified by deleting cod(cytosine deaminase gene) and cdd(cytidine deaminase gene) to study the effect of the genes on the formation of uracil and 2’-deoxyuridine. Then, recombinant plasmid NDT-pFRPT encoding class II nucleoside N- deoxyribosyltransferase was transformed into E. coli JM109(Δcod, Δcdd). When the recombinant Escherichia coli whole cell was used as an enzyme for the production of 2’-deoxycytidine, there were no observable production of uracil and 2’- deoxyuridine implying that they were formed by host strain-derived cytosine deaminase and cytidine deaminase. Keywords : 2'-Deoxycytidine, N-deoxyribosyltransferase, cytosine deaminase, cytidine deaminase, transglycosylation References 1. Y. Miyatomo, T. Masaki, S. Chohnan, Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. Lactis (2007), Biochimica et Biophysica Acta, 1774, 1323-1330 2. R. Cardinaud, Nucleoside deoxyribosyltransferase from Lactobacillus helveticus (1978), Methods Enzymol. 51, 446-455 3. J. Fernandez-Lucas, L.A. Condezo, F. Martinez-Lagos, J.V. Sinisterra, Synthesis of 2’-deoxyribosylnucleosides using new 2’-deoxyribosyltransferase microorganism producers (2007), Enzyme Microb. Technol. 40, 1147-1155 4. K. Okuyama, S. Shibuya, T. Hamamoti, T. Noguchi, Enzymatic synthesis of 2’-deoxyguanosine with nucleoside deoxyribosyltransferase-II (2003), Biotehnol. Biochem. 67, 985-995 P0008 Mathematical Interpretation of Cell Lethality by Sequential Actions of Ionizing Radiation and Hyperthermia Jin Kyu KIM1,2, Vladislav G. PETIN3, Remigius A. KAWALA1,2, Jin-Hong KIM1,2 1Korea Atomic Energy Research Institute, Jeongeup 56212, Korea, 2University of Science and Technology, Daejeon 34113, Korea, 3Medical Radiological Research Center, Obninsk 249036, Russian Federation The combined action of hyperthermia with ionizing radiation is widely used in hyperthermic oncology. The combined effect of heat and radiation on cell lethality is dependent on both the sequence of application and temperature. The mechanism of synergistic interaction of hyperthermia and radiation may be brought about by an inhibition of the repair from sub-lethal and potentially lethal damage at the cellular level. The dependence of the thermal enhancement ratio after a sequential action of heat and radiation on the dose and dose rate was studied in yeast cells. The effectiveness of treatment with heat and radiation was greatly dependent on the duration of heat exposure. For an equal amount of cell killing from hyperthermia alone, long action of heat (50℃) was more effective for radiosensitization than a short acute action of higher heat (58℃). For heating at 50℃, heating after irradiation produced more radiosensitization than heating before irradiation. However, high heating at 58℃ before irradiation gave the same radiosensitization as heating after irradiation. The results were interpreted by means of a mathematical model for the sequential application of heat and radiation. The mathematical interpretation appears to be appropriate and the conclusions are valid. Keywords : ionizing radiation, hyperthermia, sequential action, mathematical model, yeast cells References 1. Streffer C, Vaupel P, Hahn G. Biological Basis of Oncologic Thermotherapy. Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong: Springer (1990). 2. Kim JK, Petin VG, Tkhabisimova MD. Survival and recovery of yeast cells after simultaneous treatment of UV light radiation and heat. Photochem. Photobiol. 79: 349-355 (2004). 3. Petin VG, Kim JK, Zhurakovskaya GP, Rassokhina AV. Mathematical description of synergistic interaction of UV light and hyperthermia for yeast cells. J. Photochem. Photobiol. B:Biol. 55: 74-79 (2000). P0009 Bioethanol Production from Hydrolysates of the Red Agal Gelidium amansii by Evolved Saccharomyces cerevisiae Hye-Jin LEE1, Soo-Jung KIM2, Hyun-Woo KIM1, Jeong-Jun YOON3, Yong-Cheol PARK1 1Dept. of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 136-702, Republic of Korea, 2Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea, 3IT Convergence Materials R&BD Group, Korea Institute of Industrial Technology, Cheonan, 331-822, Korea Marine biomass has been receiving attention as a renewable resource for sustainable bioethanol production. Among the marine biomass, red algae mainly composed of agar and cellulose are easily hydrolyzed to galactose and glucose. For efficient production of bioethanol by Saccharomyces cerevisiae, it is necessary to metabolize galactose fast. In this study, we constructed S. cerevisiae HJ7-14 capable of rapidly metabolizing galactose by applying evolutionary engineering. In order to evaluate fermentative performances of HJ7-14 strain, batch and repeated-batch fermentations using galactose and glucose were carried out. The HJ7-14 strain metabolized 5-fold more galactose and produced ethanol 2.1-fold faster compared with the parental strain. Transcription analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes, which might enable HJ7-14 to metabolize galactose rapidly. Finally, the HJ7-14 strain produced 7.4 g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12 h, which was 1.5-times faster than that observed with D452-2. Keywords : Saccharomyces cerevisiae, Galactose, Catabolite repression References 1. HJ Lee, Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol (2015), Bioresource Technology, 191:445-451 P0010 Selection and Characterization of ssDNA Aptamers Directed against the Glypican-3 Protein Targets HCC Quang-Thai NGUYEN1, Sang-Hee LEE1, Simranjeet Singh SEKHON1, Ji-Young AHN1, Jiho MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University, , 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, Glypicans are heparan sulfate proteoglycans linked to the cell surface and play an important role in regulation of cell growth, differentiation, and migration. While six members of the glypican family, Glypican-3 is an efficient diagnostic marker of human hepatocellular carcinomas. Aptamers are single-strand oligonucleotides able to bind specifically and sensibility to target molecules, offering great potential for applications in diagnosis and therapy. In addition, aptamers generally have higher specificity and stability for their targets than corresponding antibodies. ssDNA aptamers were selected using the Systematic Evolution of Ligands by EXponential enrichment methodology from an initial library containing a 40 bases long variable region. After in vitro selection, the structural stability as affinity ligands of these aptamers specifically were quantified by strict criteria of equilibrium (Kd) using surface plasmon resonance and compared with the corresponding monoclonal antibody. This study was carried out with the support of "Cooperative Research Program for Agricultural Science & Technology Development (PJ011661)", Rural Development Administration, Republic of Korea. Keywords : aptamer, Glypican3, SELEX, HCC References 1. Filmus J, Capurro M, Rast J, Glypicans(2008), Genome Biol, 9(5):224. 2. Graham JC, Zarbl H, Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells(2012), PLoS One, 7(4):e36103. 3. Song KM, Lee S, Ban C, Aptamers and their biological applications(2012), Sensors (Basel), 12(1):612-31. 4. Yan B, Wei JJ, Qian YM, Zhao XL, Zhang WW, Xu AM, Zhang SH, Expression and clinicopathologic significance of glypican 3 in hepatocellular carcinoma(2011), Ann Diagn Pathol, (3):162-9. P0011 Overexpression of a Mutated SPT15 Gene Contributes to Improvement of Ethanol Productivity of Saccharomyces cerevisiae Haeseong PARK, Jae-Hong MIN, Hyeun-Soo LEE, Bo-Mi KIM, Yong-Cheol PARK Dept. of Bio and Fermentation Convergence and Technology, Kookmin University, Seoul 136-702, Korea In yeast fermentation, osmotic stress caused by high sugar and ethanol concentrations result in reduction of bioethanol productivity. In order to overcome this problem, global transcription machinery engineering (gTME) was applied by introducing mutated SPT15 variants coding for TATA-binding proteins into S. cerevisiae and S. cerevisiae ETS3 showing improved ethanol producing ability under high osmotic pressure was selected. In this study, fermentative properties of the ETS3 strain were evaluated in batch and fed-batch cultures using high glucose concentration under different aeration conditions. In oxygen-limited condition, ETS3 strain existed about 13–16% increases in dry cell weight, ethanol concentration and ethanol productivity relative to those of the S. cerevisiae BY4741 host strain in batch fermentation with 300 g/L glucose. In aerobic condition, ETS3 strain showed elevated ethanol productivity to 2.49 g/L-h without significant changes of other fermentation parameters compared to that of host strain. In a fed-batch fermentation using 400 g/L glucose and oxygen-limited condition, ETS3 strain produced up to 151.7 g/L ethanol with a maximum ethanol yield of 0.402 g/g which is 16 % increase in ethanol concentration and productivity in comparison to the host strain. These results suggest that gTME is an effective tool to obtain a robust S. cerevisiae strain against high osmotic pressure. Keywords : global transcription machinery engineering (gTME), SPT15, fed-batch fermentation References 1. Yang, J., Bae, J.Y., Lee, Y.M., Kwon, H., Moon, H.Y., Kang, H.A., Yee, S.B., Kim, W., Choi, W. Construction of Saccharomyces cerevisiae Strains With Enhanced Ethanol Tolerance by Mutagenesis of the TATA-Binding Protein Gene and Identification of Novel Genes Associated With Ethanol Tolerance.(2011) Biotechnology and Bioengineering, 108(8), 1776-1787. P0012 Production of 2,3-Butanediol by Pdc-deficient Saccharomyces cerevisiae Expressing of Lactococcus lactis NADH Oxidase Jin-Woo KIM1, Seung-Oh SEO2, Guo-Chang ZHANG2, Yong-Su JIN2, Jin-Ho SEO1 1Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151- 921, Republic of Korea, 2Department of Food Science and Human Nutrition, and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA To minimize glycerol production during 2,3-BD fermentation by the engineered Saccharomyces cerevisiae, the Lactococcus lactis water-forming NADH oxidase gene (noxE) was expressed at five different levels. The expression of NADH oxidase substantially decreased the intracellular NADH/NAD+ ratio. The S. cerevisiae BD5_T2nox strain expressing noxE produced 2,3-BD with yield of 0.359 g 2,3-BD/g glucose and glycerol with 0.069 g glycerol/g glucose, which are 23.8% higher and 65.3% lower than those of the isogenic strain without noxE. These results demonstrate that the carbon flux could be redirected from glycerol to 2,3-BD through alteration of the NADH/NAD+ ratio by the expression of NADH oxidase. Keywords : 2,3-butanediel, Pdc-deficient Saccharomyces cerevisiae, NADH oxidase References 1. J.W. Kim et al., Expression of Lactococcus lactis NADH oxidase increases 2,3-butanediol production in Pdc- deficient Saccharomyces cerevisiae (2015), Bioresouce Technology, 191, 512-519 P0013 Enhanced Production of 2’-Fucosyllactose in Engineered Escherichia coli BL21 Star(DE3) by Modulation of Lactose Metabolism and Fucosyltransferase Young-Wook CHIN1, Ji-Yeong KIM1, Won-Heong LEE2, Jin-Ho SEO1 1Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151- 921, Republic of Korea, 2Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500- 757, Republic of Korea 2-Fucosyllactose (2-FL) is one of most abundant functional oligosaccharides in human milk, which is involved in many biological functions for human health. To date, most microbial systems for 2-FL production have been limited to use Escherichia coli JM strains since they cannot metabolize lactose. In this study, E. coli BL21 star (DE3) was engineered through deletion of the whole endogenous lactose operon and introduction of the modified lactose operon containing lacZ∆M15 from E. coli K-12. Expression of genes for guanosine 5 -diphosphate (GDP)-L-fucose biosynthetic enzymes andheterologous-1,2-fucosyltransferase (FucT2) from Helicobacter pylori allowed the engineered E. coli BL21 star (DE3) to produce 2-FL with 3-times enhanced yield than the non-engineered E. coli BL21 star (DE3). In addition, the titer and yield of 2-FL were further improved by adding the three aspartate molecules at the N-terminal of FucT2. Overall, 6.4 g/L 2-FL with the yield of 0.225 g 2-FL/g lactose was obtained in fed- batch fermentation of the engineered E. coli BL21 star (DE3) expressing GDP-L-fucose biosynthetic enzymes and three aspartate tagged FucT2. Keywords : 2'-fucosyllactose, Escherichia coli, fucosyltransferase References 1. Y.W. Jin et al., Enhanced production of 2’-fucosyllactose in engineered Escherichia coli BL21 star(DE3) by modulation of lactose metabolism and fucosyltransferase (2015), Journcal of Biotechnology, 201, 107-115 P0014 Deciphering and Characterization of Glucose Homeostasis of Escherichia coli, strain K-12 substr. W3110 Seong Gyeong KIM1, Jae Hyung LIM1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, POSTECH, Pohang, 790-784, 2School of Interdisciplinary Bioscience and Bioengineering (I-Bio), POSTECH, Pohang, 790-784 Glucose homeostasis is tightly controlled in all domains of life including bacteria, such as E. coli. A key regulatory point of glucose homeostasis resides in phosphosugar glucose-6-phosphate (G6P), generated during membrane translocation of glucose by the phosphotransferase system (PTS). Although the charged phosphate group is vital for retention of intracellular glucose, high levels of G6P are toxic to cells. Previous works suggested the mechanism for glucose homeostasis in E. coli with the small regulatory RNA SgrS and the haloacid dehalogenase (HAD)-like enzyme YigL. In this study, we want to decipher glucose homeostasis in E. coli, strain K-12 substr. W3110. This work will be expanded our knowledge for glucose homeostasis and understanding physiological implications depending on different genetic backgrounds. Keywords : phosphosugar, glucose homeostasis, small non-coding RNA References 1. Papenforb et al., Small RNA-Mediated Activation of Sugar Phosphatase mRNA Regulates Glucose Homeostasis. Cell. 2013. 153, 426-437 2. Vanderpool et al., The Novel Transcription Factor SgrR Coordinates the Response to Glucose-Phosphate Stress. J. Bacteriol. 2007. 189(6): 2238 3. Negrete et al., Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21). Microbial Cell Factories. 2010, 9:75 P0015 Production of Glucooligosaccharides by Leuconostoc mesenteroides Fermentation with Efficient pH Control Using Calcium Hydroxide-Sucrose Solution Sun LEE1,3, Namhyeon PARK2, JungMin HA2, Song-Hee HAN2, Heejung LIM2, Dong-Gu LEE2, Tae-Kyoung LEE2, Bon- Cheul GU2, Chun-Hyung KIM3, Do Man KIM2,3 1School of Biological Sciences and Technology, Chonnam National University. 77 Yongbong-ro, Gwangju 500-757, 2Graduate School of International Agricultural Technology, 1447 Pyeongchang-daero, Gangwon-do, 232-916, 3Institute of Food Industrialization, Institutes of Green Bio Sciences & Technology, Seoul National University, 1447 Pyeongchang-daero, Gangwon-do, 232-916 95.3% of the sucrose in a fed batch fermentation (300 g/L) was hydrolyzed and the glucose from sucrose together with 91.6 % of the maltose (200 g/L) produced by Leuconostoc mesenteroides subp. mesenteroides NRRL B23188 glucansucrase formed glucooligosaccharides (GOS) with DP over 2. The saccharate from lime (lime sucrate) was used to control pH during fermentation. The GOS obtained had 2 – 8 degrees of polymerization. When mutansucrase (0.1 U/mL reaction digest) from Streptococcus mutans was reacted with 0.1% sucrose, addition of 0.1 to 10% GOS to the mutansucrase reaction digest resulted in 56 to 90% reduction of mutan formation. GOS also reduced the growth of E. coli (74.4%) and Salmonella sp (over 40.0%) when 2.5% GOS was used as a single carbon source compared to growth on glucose. The calculated glycemic index and glycemic load of GOS were 8 and 1, respectively, based on a 10 g carbohydrate serving, and GOS was calculated to contain 2.43 kcal/g. In a glucose tolerance test using C57BL/6 mice, the increase of plasma glucose in mice treated with GOS was 59.4% lower than in those treated with maltose Keywords : Glucansucrase, Leuconostoc mesenteroides, Glucooligosaccharides, Calcium hydroxide References 1. Chung C, Day D (2002) Glucooligosaccharides from Leuconostoc mesenteroides B-742 (ATCC 13146): a potential prebiotic. J Ind Microbiol Biot 29(4):196-199 2. Moon YH, Madsen L, Chung C-H, Kim D, Day DF (2014) Lime application for the efficient production of nutraceutical glucooligosaccharides from Leuconostoc mesenteroides NRRL B-742 (ATCC13146). J Ind Microbiol Biot:1-7 3. Nguyen TTH, Cho J-Y, Seo Y-S, Woo H-J, Kim H-K, Kim GJ, Jhon D-Y, Kim D (2014) Production of a low calorie mandarin juice by enzymatic conversion of constituent sugars to oligosaccharides and prevention of insoluble glucan formation. Biotechnol Lett:1-6 4. Robyt JF, Walseth TF (1978) The mechanism of acceptor reactions of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydr Res 61:433-45 5. Seo ES, Kim D, Robyt JF, Day DF, Kim DW, Park HJ, Park HJ (2004) Modified oligosaccharides as potential dental plaque control materials. Biotechnol Prog 20(5):1550-4 P0016 Removal of Cu2+ and Ni2+ Using Surface Display of Poly Histidine on Bacillus subtilis Spore by Use of the cotE Anchor Protein June-Hyung KIM, Woo-Il KIM, Ju-ryeong KIM 1Department of Chemical Engineering, Dong-a University, Busan, 604-714, Korea We studied removing heavy metal using Bacillus subtilis spore surface display system. We used cot E protein as an anchoring motif because of its high abundance in coat layer and inserted double 6histidine tag at the C-terminal end of anchoring motif. We checked the surface expression of histidine tag using flow cytometry with FITC labelled anti-his6- antibody. Histogram of flow cytometry showed higher fluorescence intensity, possibly due to their outer location on Bacillus subtilis spore. We tried Cu2+ and Ni2+ adsorption with recombinant spore(CotE-His12) and DB104(wild type) used by atomic adsorption spectrometer. We confirmed that Cu2+ and Ni2+ adsorption capacity of CotE-His12 is higher than DB104(wild type). Keywords : Surface Display , Removal heavy Metal, Bacillus subtilis spore References 1. ADAM DRIKS, American Society for Microbiology, Vol.63, No 1. p. 1-20 (1999) 2. June-Hyung Kim, Jae-Gu Pan, Byung-Gee Kim (2011) Korean Society for Biotechnology and Bioengineering Journal 26: 199-205 (2011) 3. June-Hyung Kim, Soo-Keun Choi, Heung-Chae Jung, Jae-Gu Pan, Byung-Gee Kim Korean Society for Biotechnology and Bioengineering Journal 26: 243-247 (2011) 4. June-Hyung Kim, Chang-Soo Lee, Byung-Gee Kim Biochemical and biophysical Research Communications 331 210-214 (2005) 5. Bum-Yeol Hwang, Jae-Gu Pan, Byung-Gee Kim, June-Hyung Kim Jouranl of Nanoscience and Nanotechnology Vol. 12, 1-7 (2012) P0017 Aptamer Detection of TZS Protein in High Mass Transgenic Poplar Using Aptamer Kyung-Min JANG1,2, Yang-Hoon KIM2, Ji-Young PARK1, Jung-Ho PARK1 1Bio-evaluation center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), cheongju, 363-883, South Korea, 2Department of Microbiology, Chungbuk National University, Cheongju, 361-763, South Korea Bacterial trans-zeatin secretion gene (tzs gene) is inserted into the poplar using Nonpaline-type (pTiC58) Agrobacterium tumefaciens. The transgenic poplar shows the phenotype as follows: increased the amount of total biomass in shoots, leaf and delayed growth. We have developed an aptamer-based biosensor (aptasensor) for the detection of poplar with TZS protein in environment. Aptamers are small single-strand DNA or RNA sequences and have their ability to bind highly specific molecular targets. To develop the target specific aptamer, an in vitro process called Systematic Evolution of Ligands by Exponential enrichment (SELEX) was used from very large populations of random sequence oligomer for expressed TZS protein in E.coli (BL21). After SELEX, high specific aptamer pool was selected using Surface Plasmon Resonance (SPR), which detects Tzs protein adsorption onto aptamer microarray. Therefore, we will evaluate the contamination of GM poplar in environment using the aptasensor that detects these GMO-associated specific targets at a range of concentrations, even in milieu of complex environment. Keywords : Aptamer, Poplar, TZS References 1. Yan, Fangfang, Fang Wang, and Zilin Chen. P0018 Expression of Particulate Methane Monooxygenase Gene in Methylotrophic Bacteria Thi Ngoc Diep NGUYEN, Thi Thu NGUYEN, Eun Yeol LEE Dept. of Chemical engineering, Kyung Hee university,Yongin, 440-746 Methylosinus trichosporium OB3b is a methanotrophic bacterium utilizing methane as a sole source of carbon and energy [1]. Methane oxidation is catalyzed by methane monooxygenase (MMO) [2]. There are two types of MMO: a membrane- associated or particulate MMO (pMMO) [3] and a cytoplasmic or soluble MMO (sMMO) [4]. Compared to sMMO, cells expressing pMMO show higher affinity toward methane, so pMMO can be considered as the more efficient system for oxidation of methane. Especially, pMMO is located in specialized internal membrane structures, called intracytoplasmic membranes [5]. Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on methanol but not methane [6]. It is of considerable interest to understand the biochemistry of pMMO in a heterologous host in methylotrophs. In this study, three subunits pmoC, pmoA, and pmoB of pMMO gene from M. trichosporium OB3b were successfully amplified by PCR, and cloned into pBBR1MCS2 vector. The vector was introduced into Escherichia coli S17-1, and transferred into M. extorquens AM1 by conjugation. Keywords : methanotroph, methylotroph, methane monooxygenase References 1. R. S. Hanson and T. E. Hanson, Methanotrophic bacteria (1996) , Microbiol. Rev. 60, 439-471 2. L. C. Crossman, J. W. B. Moir, J. J. Enticknap, D.J. Richardson and S. Spiro, Heterologous expression of heterotrophic nitrification genes (1997) , Microbiol., 143, 3775-3783 3. J. A. Zahn and A. A. DiSpirito, Membrane associated methane monooxygenase from Methylococcus capsulatus (Bath) (1996), J. Bacteriol. 178, 1018-1029 4. H. Dalton, P. Wilkins and Y. Jiang, Structure and mechanism of action of the hydroxylase of soluble methane monooxygenase (1993), Microbial Growth on C1 Compounds, 65-80. 5. M. Kalyuzhnaya, A. W. Puri and M. E. Lidstrom, Metabolic engineering in methanotrophic bacteria (2015) , Metab. Eng. 29, 142-152 6. D. Peel and J. R. Quayle, Microbial growth on C1 compounds: isolation and characterization of Pseudomonas AM1 (1961). J. Biochem. 81, 465-469 P0019 Mass Production of the Human Growth Factor Using Recombinant E. coli jae-woo LEE1,2, Won-Ho CHOI1, Ji-young PARK1, Kyung-min JANG1, Jung-Ho PARK1, Hyeon-Young JEGAL1 1Bio-evaluation center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), cheongju, 363-883, South Korea, 2Chungnam National Univ., Gung-dong, Yuseong-gu, Daejeon, Korea 305-764 Epidermal growth factor (EGF) is a growth factor inducing cell growth and proliferation, cell differentiation by activating EFG receptor. Insulin-like growth factor-1 (IGF-1) has an important role in the growth and development of many tissues and control overall growth. And Thioredoxin (TRX) is working as an oxidation-reduction enzyme, a transcription factor involved in cell growth and control. From previous studies, the expression of EGF, IGF-1 and TRX are not convenient due to their insolubility from protein expression and isolation. In this study, we have tried to purify three proteins for increasing protein solubility using refolding methods and various partner tags. These proteins expressed in E. coli to be used effectively in functional products and can be used for human risk assessment to evaluate the toxicity and activity in human at a later time. Keywords : EGF, IGF, TRX References 1. Carpenter, Graham, and Stanley Cohen. "Epidermal growth factor." Annual review of biochemistry 48.1 (1979): 193-216. P0020 Comprehensive Genome Sequencing and Comparative Annotation of a Probiotic Bacterium, Bacillus polyfermenticus Isolated from Bispan, a Commercial Mixed Strain Product Jae Bong LEE1, Ji Eun JEONG2, Se Won KANG2, Hee Ju HWANG2, Sung-Hwa CHAE3, Young-Keun KWAK4, Inger KüHN4, Roland MöLLBY4, Bharat Bhusan PATNAIK2,6, Yeon Soo HAN5, Chang Hwa JEONG7, Yong Seok LEE2 1Institute of Agriculture and Life Sciences, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongsangnam-do, Republic of Korea, 2Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan, Chungchungnam-do, Republic of Korea, 3Research Institute, GnC BIO Co. LTD., 621-6 Banseok-dong, Yuseong-gu, Daejeon, Republic of Korea, 4Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden, 5College of Agriculture and Life Science, Chonnam National University, 300 Yongbong-Dong, Buk-gu, Gwangju, Republic of Korea, 6Trident School of Biotech Sciences, Trident Academy of Creative Technology (TACT), Bhubaneswar- 751024, Odisha, India, 7JNS Tech Inc. Floor 4, Samchang B/D, 1745-21, Daeyeon-5dong, Nam-gu, Busan, Republic of Korea Bacillus polyfermenticus (B.P.), a probiotic bacterium, secrete over 20 distinct enzymes, metabolize nutrients, and synthesize vitamins B1, B2, and K, suggesting potential demand of the species for both nutritional and industrial applications. In this study, we analyzed the complete genome sequence of B.P. isolated from the commercial mixed strain product, Bispan, using whole-genome shotgun/pyrosequencing approach. We compared the whole genome of the strain to the annotated genome sequence of Bacillus subtiltis 168 (NC_000964) and Bacillus amyloliquefaciens FZB42 (NC_009725.1). Pyrosequencing generated a total of 519,111 reads at an average read length of 257 bp, equivalent to 33X coverage of the B.P. genome. The total genome size of B.P. was estimated at 4,149,352 bp. A total of 4,048 coding sequence (CDS) regions were identified for B.P., which is larger than those of B. amyloliquefaciens FZB42 (3,813 CDSs) and smaller than those of B. subtilis 168 (4,422 CDSs). A Venn diagram plot show 3,526 overlapping ORFs between B. polyfermenticus and B. amyloliquefaciens FZB42, and only 1,306 ORFs common to all three species. The result suggests that B.P. genome is more similar to B. amyloliquefaciens FZB42 than to B. subtilis 168 despite biochemical fingerprinting analyses suggesting a closer relationship between B. polyfermenticus and B. subtilis. The findings provide new insights to explore the gene-based functional mechanisms of B.P. as an efficient probiotic and to improve its utility for the prevention of inflammation and infection in the gut. Keywords : Bacillus polyfermenticus, Pyrosequencing, Probiotics References 1. M.H. Floch, W.A. Walker, K. Madsen, M.E. Sanders, G.T. Macfarlane, H.J. Flint, L.A. Dieleman, Y. Ringel, S. Guandalini, C.P. Kelly, L.J. Brandt, Recommendations for probiotic use-2011 update, J. Clin. Gastroenterol. 45 (2011) S168-171. P0021 Development of Human Anti-aging Factor (IGF, EGF, TRX) Aptamers for Affinity Chromatography Heon-Young JEGAL1, Kyung-Min JANG1, Jung-Ho PARK1 1Bio-evaluation center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), cheongju, 363-883, South Korea, 2JUNGWON UNIVERSITY, Biomedical science The anti-aging needs are growing from people under the precautionary effective recovery of recognition. Therefore, anti- aging factors is required highly efficient method to purify the factors like antibody affinity purification methods. We developed human growth factor EGF, IGF, TRX binding aptamer using the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are small single-strand DNA or RNA sequences from very large populations of random sequence oligomer for expressed EGF, IGF, TRX protein in E.coli (BL21) and have their ability to bind highly specific molecular targets. SELEX process is a combinatorial chemistry method that determines the identification of specific oligonucleotide sequences. After SELEX, high specific aptamer pool was selected using Surface Plasmon Resonance (SPR), which detects EGF, IGF, TRX protein adsorption onto aptamer microarray. Finally three proteins will be purified using aptamer column that should be higher specificity than common antibody affinity purification column. Keywords : Aptamer, SELEX, EGF, IGF, TRX References 1. Hamaguchi, Nobuko, Andrew Ellington, and Martin Stanton. "Aptamer beacons for the direct detection of proteins." Analytical biochemistry 294.2 (2001): 126-131. P0022 Detection of Porcine Epidemic Diarrhea (PED) Viral Spike Protein Using Saccharomyces cerevisiae Cell Surface Expression by CWP2 Signaling Eunji LEE1, Ga-Young PARK1, Gna AHN1, Se Hee LEE1, Yang-Hoon KIM1, Hobaek YOON2, Ji-Young AHN1 1Dept. of Microbiology, Chungbuk National University, Cheong-Ju, Korea, 2National Institute of Animal Science, RDA, Korea Porcine epidemic Diarrhoea Virus (PEDv) is disease that occur diarrhea only in pigs. Our study was conducted to stable vector system for expressing PED virus (PEDv) spike proteins on yeast cell surface. The cell surface of the budding yeast, Saccharomyces cerevisiae, was engineered to anchor the PEDv spike proteins on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for immunogenic animal feed. In this study, the expression vector pYES2 includes a DNA sequences constructed from the signal sequence from the carboxyl-terminal of the yeast cell wall protein 2 (CWP2) gene, HA tag, the systemically designed PEDv spike genes, And to confirm for proper expression of recombinant yeast, we conduct dot blotting assay. Then 3 transformants of 40 yeast transformants were selected which were able to bind with anti-HA antibody, indicating successful expression of the viral spike fragment gene. Western blotting assay indicated that binding on HA primary antibody conjugated HRP of selected 3 transformants. Also It was indicated that binding on three species of PEDv blood serum with recombinant yeast using immunofluorescence assay. This study showed that the both expression and anchoring efficiency are much stable. This work was supported by "CRP for Agriculture Science & Technology Development (PJ010530)" Rural Development Administration, Republic of Korea Keywords : PED Virus, CWP2, Surface display, Saccharomyces cerevisiae, Yesast expression vector, Swine blood serum References 1. Wenshan Liu et al., Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein(2009), Biotechnol Lett, Vol(32), 255-260 2. Peter Lee et al., Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis(2006), Microbiology, Vol(152), 1981-1990 P0023 Surface Layer Protein of Bacillus cereus for Mosquito Control- A New Report Mani CHINNASAMY1, Subbiah POOPATHI2, Yong Hun JO1, Yong Seok LEE3, Yeon Soo HAN1 1Department of Applied Biology, Chonnam National University,, 2Vector Control Research Centre, Medical Complex, Indra Nagar, Puducherry, 605006, India, 3Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan, Chungchungnam-do, South Korea Mosquitoes are the furthermost medically significant insect among the phylum arthropods and communicate many life aggressive parasitic and viral diseases such as Japanese encephalitis, West Nile fever, malaria, filariasis, dengue fever, and yellow fever, and among humans or from animals to humans. Last few decades have been reported synthetic insecticide create many ecological problem and toxic to non-target organism, etc. Mosquitocidal bacteria are eco-friendly, alternatives to synthetic insecticides for switch off mosquito vector based diseases, and Bacillus sphaericus (Bs), Bacillus thuringiensis israelensis (Bti) are the spore forming bacteria previously described for mosquitocidal activity. Nevertheless, the recent progress of resistance to these bacterial strains (Bs and Bti) has blocked the development in application in the field. So, it is very urgent to find new bacterial strains for the control of mosquito vectors. Therefore, there have been extraordinarily world-wide efforts to discover new bacterial agents from marine environment. In our current study, marine fish such as, Lutjanus sanguineous (Red snapper) principally available in the East coastal zone (Bay of Bengal) of Union territory of Pondicherry (India) were screened for mosquitocidal bacteria. This isolate was characterized by 16S rRNA gene sequence and identified as Bacillus cereus VCRC B540 (JN377787). Biochemical study and protein synthesis exposed that the strain is reasonably useful for mosquito control. Toxicity assay showed that the vector of filariasis Culex quinquefasciatus is more susceptible and followed by Anopheles stephensi (malaria) and Aedes aegypti (dengue). No effect on non-target organisms and further, Characterize that the factor responsible for mosquitocidal effect was due to surface layer protein (90 kDa). Phylogenetic analyses of 16S rDNA gene sequence of B.cereus revealed the occurrence of homology with closely related strains. Moreover, several bacteria shows their pathogenicity due to S-layer especially Bacillus species groups like B. cereus, and B. anthracis this signal create platform for investigate new isolate which presence S-layer protein. The S-layer protein have offer many role in bacteria such as cell shape formation, protective coats, cell, adhesion, ion traps molecular filter, and also virulence factors in pathogenic bacteria which protect from bacterial parasite where as in archaea, its play major role in cell outline and cell division. Therefore, it is concluded that the S-layer protein from marine bacterium (B.cereus) is possibly be useful for the biological control of mosquito vectors. Keywords : Mosquito, control, bacteria References 1. Poopathi S1, Mani C, Thirugnanasambantham K, Praba VL, Ahangar NA, Balagangadharan K. Identification and characterization of a novel marine Bacillus cereus for mosquito control (2014). Parasitol Res. 2014 Jan;113(1):323-32. doi: 10.1007/s00436-013-3658 P0024 Discovery of the Novel Metabolic Pathway of the Rare Sugar from Red Macroalgae by the Integrative Metabolomic and Transcriptomic Analyses Eun Ju YUN, Saeyoung LEE, In-Geol CHOI, Kyoung Heon KIM Department of Biotechnology, Graduate School, Korea University, Seoul, 136-713, Republic of Korea The metabolic pathway of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as sustainable resource for producing bio-based products. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NAD(P)+-dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key steps for AHG metabolism. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of the two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into bio-based products. Keywords : 3,6-Anhydro-L-galactose, Red macroalgae, Vibrio sp. EJY3 References 1. Yun EJ et al. The novel catabolic pathway of 3,6-anhydro-L-galactose, the main component of red macroalgae, in a marine bacterium. Environmental Microbiology 2015, 17(5):1677-1688 (IF=6.240) 2. Yun EJ et al. Red macroalgae as a sustainable resource for bio-based products. Trends in Biotechnology 2015, 33(5):247-249 (IF=11.958) P0025 Identification of Ferrichrome Transporter from Aspergillus fumigatus Ju-Yeon KIM, Yong-Sung PARK, Cheol-Won YUN Department of Life Sciences, Korea University, Seoul, 136-701 Although TAFC (triacetyl fusarinine C) and reductive iron uptake systems have been identified from Aspergillus fumigatus which is an opportunistic fungal pathogen, FOB (ferrioxamine B) or FC (ferrichrome) transporters are not identified yet. Recently, we looked for yeast Sit1 homologues from A. fumigatus genome database and found 4 uncharacterized genes which have high homologues with yeast Sit1. Two of them were named as sit1 and sit2 and they complemented FOB and FC uptake activity and suppressed growth defect of SIT1/ARN1/ARN2 triple deletion mutant of S. cerevisiae functionally. However, the other two genes failed to complement yeast SIT1 functionally. Gene expressions of sit1 and sit2 were regulated by iron utilization and they were found from plasma membrane of S. cerevisiae when expressed heterologously. Single or double deletion mutants of Sit1 and Sit2 were constructed and uptake activities of FC and FOB were decreased dramatically when sit1 and sit2 were deleted simultaneously. Also, sit1 and sit2 were found from plasma membrane of A. fumigatus. Furthermore, deletion of sit1 and sit2 showed higher survival rate than wild type cells in conidial killing assay. Taken these results together, we suggested that sit1 and sit2 encode FC and FOB transporters and is a virulence factor potentially. Keywords : A.fumigatus, FOB, FC, Sit1, S.cerevisiae, sit1, sit2, iron , virulence factor References 1. Haas H et al., Siderophore in fungal physiology and virulence (2008), Annu Rev Phytopathol, 46, 149-187. P0026 Development of New Anchoring Motif for Bacillus subtilis Cell Surface Display Using YuaB Biofilm Component June-hyung KIM, Da-eun KIM, Ju-ryeong KIM Department. Chemical Engineering, Dong-A University, Busan, 604-714 Almost of Bacillus subtilis surface display system has been developed by using spore coat protein. To develop new Bacillus subtilis cell surface display system, we used YuaB as an anchoring motif. YuaB is the major component of Bacillus subtilis biofilm. YuaB is located in B. subtilis cytoplasmic membrane and plays roll of making repellency of B. subtilis biofilm after cell clusters are formed. We fused His6 tag at C-terminal YuaB to comfirm surface display and use this for heavy metal adsorption. As we expect, we confirmed that YuaB can be used as an anchoring motif by flow cytometry. AA analysis result shows that His6 tag has activities for heavy metal adsorption on the B. subtilis surface. Keywords : Biofilm, Cell Surface Display , Bacillus subtilis References 1. Steven S. Branda,A major protein component of the Bacillus subtilis biofilm matrix, Molecular Microbiology (2006) 59(4), 1229-1238 2. Laura Hobleya,BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm, PNAS vol. 110 no.33, 13600-13605 3. Kazuo Kobayashi, BslA(YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms, Molecular Microbiology (2012) 85(1), 51–66 4. Adam Ostrowski,YuaB Functions Synergistically with the Exopolysaccharide and TasA Amyloid Fibers To Allow Biofilm Formation by Bacillus subtilis, J. Bacteriol. 2011, 193(18):4821. DOI: 10.1128/JB.00223-11. P0027 Hydroxylation of the Phloretin Using Streptomyces avermitilis MA4680 Wooil KIM, June-hyung KIM Department of Chemical Engineering, Dong-A Uinversity, Busan, Korea. Twelve microorganisms were initially screened for their abilities to catalyze biotransformation of phloretin. Streptomyces avermitilis, two main products were identified in GC/MS analysis. They were interpreted as hydroxylated products of phloretin in A-ring at different position. (mass increase 179→267, 192→ 280) This result confirmed hydroxylation considering BSTFA derivatization of hydroxylated product. Maximum conversion was 6.7%, which was achieved for 1 hours of reaction, and the substrate (phloretin) and reaction product was completely metabolized after 3 hours of reaction. We confirmed P450 activity in the future research. Three kinds of Cytochrome P450 inhibitor, Coumarin, Erythromycin and Quinidine was added with 0.5mM final concentration. Keywords : Flavonoid, phloretin, biotransformation References 1. M. Sanchez-Gonzalez and J.P.N Rosazza ,Microbial Trasnformations of Chacones: Hydroxylation, O- Demethylation, and Cyclization to Flavanones(2004) J.nat Prod 67,553-558 2. Debbie Fonseca and Wendy E.Ward, Daidzein together with high calcium preserve bone mass and biomechanical strength at multiple site in overiectomized mice(2004),BONE 35, 489-497 3. Bishnu Prasad Pandy, Changhyun Roh, Kwon-Young Choi, Nahum Lee, Eun Jung Kim, Sungghi Ko, Taejin Kim, Hyundon Youn,Byung-Gee Kim, Rigioselective Hydroxylation of Daidzein Using P450(CYP105D7) From Streptomyces avermitilis MA4680(2010), Biotechnol Bioeng. 105(4):697-704 P0028 Synthetic Dyes Decolorization by CotA Laccase of Bacillus subtilis Spores Wooil KIM, Jong-Wha PARK, June-Hyung KIM Dept. of Chemical engineering, Dong-A University, Busan, 604-714 In this study, we tried to decompose synthetic industrial dyes using Bacillus subtilis spore, which has CotA spore coat protein known as having multi-copper oxidase (laccase) activity. We confirmed the decolorization rate of Indigo carmine by 30 %, Acid red 18 by 10 %, and Acid green 25 by 20 % in 1 hr using a wild-type Bacillus subtilis spore. And wild type Bacillus subtilis spore showed optimum decolorization activity on Indigo carmine dyes at 65 ℃. To express CotA protein on the surface of the spores, several anchoring motives such as CotE, CotG, and CotY were used. His6-tag was added at the C-terminal of target protein, CotA. The spore surface expression of target protein, CotA, was confirmed by flow cytometry using FITC labelled anti-His6 antibody. Synthetic dye decolorization was tried using constructed spore displayed laccase, CotA. Keywords : laccase, CotA, decolorization, spore display References 1. Zaharia, C., Suteu D., Muresan, A. Options and Solutions for Textile Effluent Decolorization Using Some Specific Physico-Chemical Treatment Steps.(2012) Enviromental Engineering and Management Journal, 11(2), 493-509 2. Cho, E.A., Seo, J., D.W., Pan, J.G Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores.(2011) Enzyme Microb Technol, 49(1) 100-4 3. Kim J.H., Lee, C.S., Kim B.G, Spore-displayed streptavidin: a live diagnostic tool in biotechnology (2005) Biochem Biophys Res Commun, 331(1), 210-4 P0029 Industrial Production of 2,3-Butanediol from the Engineered Corynebacterium glutamicum Jeongmo YANG Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, Republic of Korea The platform chemical 2,3-butanediol (2,3-BDO) is a valuable product that can be converted into several petroleum-based chemicals via simple chemical reactions. Here, we produced 2,3-BDO with the non-pathogenic and rapidly growing Corynebacterium glutamicum. To enhance the 2,3-BDO production capacity of C. glutamicum, we introduced budA encoding acetolactate decarboxylase from Klebsiella pneumoniae, a powerful 2,3-BDO producer. Additionally, budB (encoding α-acetolactate synthase) and budC (encoding acetoin reductase) were introduced from K. pneumoniae to reinforce the carbon flux in the 2,3-BDO production. Because budC had a negative effect on 2,3-BDO production in C. glutamicum, the budB and budA introduced strain, SGCG102, was selected for 2,3-BDO production, and batch culture was performed at 30 °C, 250 rpm and pH 6.86 with pure glucose, molasses, and cassava powder as carbon substrates. After batch culture, significant amount of 2,3-BDO (18.9 and 12.0 g/L, respectively) was produced from 80 g/L of pure glucose and cassava powder. Keywords : Corynebacterium glutamicum, 2,3-Butanediol, Cassava powder References 1. Kim, B. R., Lee, S. J., Jeong, D. U., Yang, J. M., Oh, M. K., & Lee, J. W. (2014). Redistribution of carbon flux toward 2,3-butanediol production in Klebsiella pneumoniae by metabolic engineering. PLoS One, 9(10), e105322. 2. Kim, B. R., Lee, S. J., Yang, J.M., Jeong, D.U., Shin, S. H.,Kook, J. H.,Yang, K. S.,& Lee, J.W. (2015). The influence of budA deletion on glucose metabolism related in 2,3-butanediol production by Klebsiella pneumoniae. Enzyme and Microbial Technol. doi:10.1016/j.enzmictec.2015.03.002. 3. Kim, B. R., Lee, S. J., Park, J. H., Lu, M. S., Oh, M. K., Kim, Y. R., & Lee, J. W. (2012). Enhanced 2,3- butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes. Journal of Microbiology and Biotechnology, 22, 1258–1263. P0030 Screening of Methanol Utilizing Strain Pseudoxanthomonas sp. from Sewage and Its Characterization Yuhyun KWON, Sangwoo KIM, Wangjun LEE, Jinwon LEE Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742 This study aimed to isolate methanol utilizing microorganism from sewage. Methanol has a potential for high polymer as a key component. To isolate a bacterium from sewage, 16S rRNA sequence and NCBI Genbank were used to detect a bacterium. In order to optimize growth conditions for the isolate, optimal culture condition including temperature, pH, rpm, and growth medium were determined. Utilizing a various carbon source as an energy source with ammonium nitrate mineral salts medium (ANMS) medium was tested. This study is the first report of a newly discovered strain, Pseudoxanthomonas sp. which is capable of utilization of methanol as a sole carbon source. Keywords : methanol, isolation, Pseudoxanthomonas, ANMS References 1. Benoit Van Aken, Caroline M. Peres, Sharon Lafferty Doty, Jong Moon Yoon, Jerald L. Schnoor, Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane- utilizing bacterium isolated from poplar trees (Populus deltoides6nigra DN34), International Journal of Systematic and Evolutionary Microbiology 54, 1191–1196 (2004) 2. Anand S. Nayak, M.H. Vijaykumar, T.B. Karegoudar, Characterization of biosurfactant produced by Pseudoxanthomonas sp. PNK-04 and its application in bioremediation, International Biodeterioration & Biodegradation 63, 73–79 (2009) P0031 Involvement of Antioxidant Defense System in Organic Solvent Tolerant Bacteria Exposed to Toluene Hyejung CHOI, Bora LIM, Woohong JOO Department of Biology, Changwon National University, Changwon 51140 Pseudomonas sp. BCNU 106 and BCNU 171, isolated on the basis of its ability to grow on high concentration toluene, was distinguishable from other Pseudomonas strains. Pseudomonas sp. BCNU 106 and BCNU 171 were investigated to elucidate the solvent tolerance under specific culture conditions with the presence of solvents and its adaptive mechanisms to those conditions with reference to the antioxidant system. Therefore, the investigation of their antioxidant properties (catalase, superoxide dismutase, glutathione S-transferase and total anti-oxidative capacities) will be useful for further study on toluene-tolerance of bacteria and the defense mechanism of antioxidant enzymes against toluene or other organic solvents. Compared to non toluene-tolerant P. putida, toluene-tolerant bacteria had relatively high tolerance to toluene stress, especially Pseudomonas sp. BCNU 106. The results demonstrated all toluene-tolerant bacteria possessed a toluene tolerance mechanism that may scavenge reactive oxygen species produced by toluene. Keywords : Pseudomonas sp. strains, Solvent-tolerant bacterium, Antioxidant defense, Toluene adaptation References 1. Y. Zhang, D. Meng, Z. wang, H. Guo, Y. Wang, X. Wang and X. Dong, J. Harzrd. Mater. 229-230, 434- 438(2012). P0032 Proteomic Analysis of Pseudomonas sp. BCNU 106 during Exposure to Toluene Hye Jung CHOI, Bora LIM, Woohong JOO Department of Biology, Changwon National University, Changwon 51140 Pseudomonas sp. BCNU 106 is tolerant to toluene and other toxic organic solvents through extrusion of the toxic compounds from the cell by means of efflux pumps. Our quantitative proteomic approach revealed the remarkable ability of Pseudomonas sp. BCNU 106 to severely change its protein expression profile upon toluene exposure. The average number of protein spots on 2-D gels detected for cultures at 1% toluene stress conditions for 1h and 2h were 876, 734 and 897, respectively. The pair wise comparisons of protein spots from the toluene stress gels and control gels showed 51 up- regulated, 88 down-regulated spots and 10 new spots. The increased and/or reduced 149 protein spots were selected and identified by MALTI-TOF mass spectrometry. Blast analysis revealed energy metabolism, protein synthesis, amino acid biosynthesis, energy production and conversion, and protein fate were found to display an adaptative activation to toluene. Thorough understanding of these mechanistic insights, the adaptative mechanisms of Pseudomonas sp. BCNU 106 to toxic organic solvents may be elucidated. Keywords : Pseudomonas sp. strains, Toluene-tolerant bacterium, Proteomic References 1. A. Segura, P. Godoy, P. Dillewijn, et al., J. Bacteriol. 187, 5937-5945 (2005). P0033 Enhancement of Solvent Tolerance in Pseudomonas sp. BCNU 106 by Addition of Trehalose to Culture Media. Bora LIM, Hyejung CHOI, Woohong JOO Department of Biology and Chemistry, Changwon National University, Changwon 641-240 Solvent hyper-resistant Pseudomonas sp. BCNU 106 experiences limited growth with solvents, a strategy is therefore needed to allow better growth to broaden its performance in biotechnological applications. Pseudomonas sp. BCNU 106 was cultivated in a medium supplemented with 50 mM trehalose, and cell survival was then observed during subsequent growth with 1% (v/v) toluene. Exogenously-added trehalose was transported into the cells and conferred protection against toluene stress. BCNU 106 grown in the presence of exogenous trehalose showed higher solvent tolerance, it can thus have more potential for biotransformation and biodegradation. Keywords : Exogenous trehalose, Pseudomonas sp., Stress tolerance, Toluene tolerance, Trehalose References 1. H. Jiang, Y. Ma, G. Qiu , F. Wu and S.Chen. 46, 703-708(2011) P0034 Enhancement of Various Solvent Tolerance in Pseudomonas sp. BCNU 106 by Addition of Trehalose to Culture Media. Bora LIM, Hyejung CHOI, Woohong JOO Department of Biology and Chemistry, Changwon National University, Changwon 641-240, Korea Solvent hyper-resistant Pseudomonas sp. BCNU 106 has some limits in growth with solvents. Pseudomonas sp. BCNU 106 was cultivated in a medium supplemented with 50 mM trehalose, and cell survival were observed during the time grown with various solvents such as hexane, cyclohexane, xylene, and propylbenzene. As a result, solvent tolerance of Pseudomonas sp. BCNU 106 can be improved by growing it in media supplemented with trehalose. Pretreatment also protected cells against solvent stress. Thus BCNU 106 can be more potentially available for biotransformation and biodegradation. Keywords : trehalose, Pseudomonas sp., solvent-stress, solvent-tolerance, biodegradation References 1. H. Jiang, Y. Ma, G. Qiu , F. Wu and S.Chen. 46, 703-708(2011) P0035 Mushroom Tyrosinase Inhibitory Activity and Antioxidant Activities of Yeast Strains Isolated from Fruit and Flower Sungmin HA, Hyejung CHOI, Woohong JOO Dept of Biology and Chemistry, Changwon National University, Changwon, 51140 Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o- diphenols and the oxidation of o-diphenols into o-quinones, and then forms brown or black pigments. In the present study, several yeasts were isolated from fruit and flower such as banana, apple, and cherry blossomes, camellia flower etc. all isolates were identified by 18S rRNA sequencing. Antioxidative activities of culture filtrates were examined by 2,2- diphenyl-1-picrylhy-drazyl radical scavenging method (DPPH), xanthine oxidant(XO) inhibition activity, reducing power, superoxide anion radical, and mushroom tyrosinase activity were also determined by following the ordinary methods. The mushroom tyrosinase inhibitor was also determined to be produced maximally when Sporobolomyces sp have been cultured at 26℃ for 24 h in MG broth medium. The culture filtrate of Sporobolomyces sp exhibited the highest mushroom tyrosinase inhibitory activities of 40.42%. The culture filtrate of Pichia sp exhibited the highest DPPH radical scavenging of 11.39%. The culture filtrate of Bullera sp exhibited the highest superoxide anion radical of 79.79% . The culture filtrate of Bullera sp exhibited the highest XO of 41.55%. Therefore, Bullera sp, Sporobolomyces sp, and Pichia sp. Isolates may be potential resources for the development of new cosmetics and for biomedical applications. Keywords : antioxidant, yeast, tyrosinase References 1. Ahotupa M, Saxelin M, & Korpela R. Antioxidative properties of Lactobacillus GG. Nutrition Today. 31, 51–52. (1996) 한국생물공학회, 생물공학의 동향 : 2015.10 동식물세포 및 조직공학 P0101 Immobilization of BMP-2 Using Mussel Bioglue for Guided Bone Regeneration Yun Kee JO1, Cong ZHOU2, Bong-Hyuk CHOI1, Jin-Soo AHN3, Sang Ho JUN4, Hyung Joon CHA1 1Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-794, 2Dept. of Medicine, Korea University Graduate School, Seoul, 136-705, 3Dental Research Institute and Dept. of Biomaterials Science, Seoul National University, Seoul, 110-749, 4Dept. of Dentistry, Anam Hospital, Korea University Medical Center, Seoul, 136-705 Guided bone regeneration (GBR) has been employed to enhance bone formation on the large bone defect, or to reconstruct alveolar ridge before dental implant insertion. As a GBR barrier membrane, titanium mesh (Ti-mesh) has been extensively used to provide space and prevent soft tissue invasion due to its excellent biocompatibility, mechanical properties, and clinical manageability. However, pure titanium does not have specific biofunctionality, and thus, the surface functionalization of titanium surface by appropriate signaling molecules is required to accelerate bone formation. Mussel adhesive proteins (MAPs) secreted from mussels have been suggested as promising bioadhesives due to strong adhesion, coating ability, and biocompatibility in tissue engineering and medicine. Here, we immobilized bone morphogenetic protein- 2 (BMP-2), an osteoinductive signaling protein, onto Ti-mesh surface using the recombinant MAP to improve bone regeneration, and then we investigated the osteoinductivity of the BMP-2-immobilized Ti-mesh surfaces in vivo and in vitro. We found that BMP-2-immobilized Ti-mesh surface displayed improved in vivo bone regeneration and maturation of new bone in a rat calvarial defect. In addition, in vitro cellular behaviors such as attachment, proliferation, and osteogenic differentiation of pre-osteoblast cells were significantly increased on BMP-2-immobilized Ti-mesh surface. Consequently, the mussel bioglue MAP can be successfully utilized as a promising biomaterial for impartment of osteoinductivity through BMP-2 immobilization onto titanium-based GBR memebrane. Keywords : Mussel adhesive protein, Bone morphogenetic protein-2, Guided bone regeneration, Titanium mesh, Osteoinductivity References 1. Kang J, Tada S, Kitajima T, Son TI, Aigaki T, Ito Y, Immobilization of BMP on DOPA- or Dopamine-Treated Titanium Surfaces to Enhance Osseointegration, BioMed Res. Int., 2013 , 6 P0102 Development of Stable CHO-S Cell Line for the Production of Human Monoclonal Antibody Ab417 Giyoung KWON1, Munsik JEONG1, Seulki CHO2, Haejung KIM3, Hyo Jeong HONG1,2 1Department of Systems Immunology, College of Biomedical Science, Kangwon National University, , 2Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, 3Department of Biology, College of National Science, Kangwon National University, Chuncheon 200-701 Monoclonal antibodies (mAbs) account for the largest segment of the therapeutic protein market. Mammalian cells are suitable for therapeutic protein production because of high capacity for post translational modification that is important for protein efficacy. Among mammalian cells, Chinese hamster ovary (CHO) cell is the most common host for stable cell line development. Particularly, CHO-S, adapted to serum-free suspension culture condition, is ideal for large scale culture. Ab417 is a human mAb that binds to human and mouse L1 cell adhesion molecule and has antitumor activity. In this study, the stable CHO-S cell line expressing the Ab417 was developed. CHO-S cells were transfected with expression plasmid (pdCMV-dhfr-Ab417) containing both heavy and light chain genes encoding Ab417. The transfected cells were selected with G418 and adapted to increasing concentration of methotrexate for gene amplification while high producer cells were sorted using flow cytometry. Finally, three clones with highest productivity were selected and their stability was evaluated. The stable CHO-S cell lines will be useful for large scale production of Ab417. Keywords : CHO-S, Stable cell line, Monoclonal antibody References 1. Okumura, Takeshi, Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry(2015), Journal of bioscience and bioengineering. 2. Chusainow, Janet, A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer?(2009), Biotechnology and bioengineering 102.4, 1182-1196. 3. Ghorbaniaghdam, Atefeh, Analyzing clonal variation of monoclonal antibody-producing CHO cell lines using an in silico metabolomic platform (2014), PloS one 9.3. P0103 Improved of the Method for Preparation of Calcium-Phosphate Nanoparticles as a Gene Delivery Tool Yeonsun YU, Duk Jae OH Dept. of Integrative Bioscience and Biotechnology, Sejong University, Seoul, 05006 Calcium phosphate nanoparticles(CPN) have been one of the most common non-viral transfection tool with little toxicity. Though CPN are known to have a high potential for transferring DNA molecules into cells efficiently, dynamic change of particel size and aggregation between particles were major issues in practical use of CPN. In this study, in order to develop the method to fabricate CPN with improved stability in particle size, key parametors that affect the characteristics of pre- formed CPN were investigated. Time dependency in size of particles that were pre-formed but crushed by sonication, use of filtered particles and particle stabilization using proteins and surfactants were evaluated for production of stable and size controlled CPN. This newly developed preparation method of CPN seemed to be a useful tool for gene delivery. Keywords : Calcium phosphate , Calcium phosphate nanoparticles, Gene delivery, Non-viral transfection tool References 1. J. Li, Y. C. Chen, Y. C. Tseng and L. Huang, J Control Release. 142(3), 6 (2010) P0104 Fed Batch Culture of Recombinant Chinese Hamster Ovary Cells for Enhanced Productivity of Recombinant Antibody using In-house Serum Free Medium, SJ-PFM3.0 Hyoung Sup YOON, Duk Jae OH Dept.of Integrative Bioscience & Biotechnology, Sejong University Many biopharmaceutical production platforms from animal cell cultures are based on fed-batch cell culture protocols, which can support high volumetric productivity. In this research, fed batch culture for rCHO cells was investigated to enhance the productivity of recombinant antibody using in-house serum free medium(SJ-PFM3.0). The recombinant Chinese hamster ovary(rCHO) cells previously grown in commercial medium(PowerCHOⓇ-2CD) were adapted to SJ-PFM3.0, then various commercial feeds were evaluated for fed batch culture to discover the most adequate feed for rCHO cell culture in SJ- PFM3.0. As a result, Cell Boost 6(HycloneTM) among various commercial feeds showed the result in the highest antibody yield and cell growth. When culture temperature was shifted from 37℃ to 30℃ in fed batch culture, it was shown that productivity increased ten times higher than that from batch cultures. As the fed batch culture showed high production of recombinant antibody using SJ-PFM3.0, it can be an attractive choice for production recombinant antibody from rCHO cells. Keywords : Fed batch culture, recombinant antibody, rCHO cells References 1. Lee, J. C., H. S. Kwon, D. J. Oh, and H. N. Chang Production of recombinant human growth hormone by rCHO cells in a depth filter perfusion system. Biotechnol. Bioproc Eng. 19: 1097-1104(2014) P0105 In vitro Study of The Cytotoxic Response of Mouse Embryo Fibroblast NIH3T3 Cells to Hydrogen Peroxide Compared to Acute and Chronic Stress Chae JI-HYE, Kim IK-HWAN Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea Cellular reactive oxygen species (ROS) are generated endogenously as in the process of mitochondrial oxidative phosphorylation, or they may arise from interactions with exogenous sources. Of the ROS, hydrogen peroxide (H2O2) and superoxide (O2-) are both produced in a number of cellular reactions. H2O2 has been widely used to study the oxidative stress response in several experimental models, including cell and tissue cultures. The objective of this study was to evaluate the effects of acute or chronic oxidative stresses on the cellular properties of NIH3T3 fibroblasts. The cells were exposed to exogenous H2O2 at doses of 200 μM and 500 μM for acute (24 hours) and chronic period (2 weeks). Their effects on cell growth/survival and the intracellular protein expression level related to oxidative stress were determined by cell viability assay and Western blot analyses. Our results showed that acute oxidative stress exhibited a cell growth inhibitory effect, while the chronic stress resulted in more change in intracellular protein levels. In summary, the cellular response against oxidative stress is time- and toxic level-dependent. Further studies are needed to determine the change of intracellular protein levels involved in acute and chronic stresses on NIH3T3 cells. Keywords : Oxidative stress, Fibroblast, Hydrogen peroxide, Reactive oxygen species References 1. Miguel F, Augusto AC, Gurgueira SA. Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities.(2009 Apr) Free Radic Res.;43(4):340-7. 2. Carvour M, Song C, Kaul S, Anantharam V, Kanthasamy A, Kanthasamy A. Chronic low-dose oxidative stress induces caspase-3-dependent PKCdelta proteolytic activation and apoptosis in a cell culture model of dopaminergic neurodegeneration.(2008 Oct) Ann N Y Acad Sci.;1139:197-205. 3. Mahalingaiah PK, Singh KP. Chronic oxidative stress increases growth and tumorigenic potential of MCF-7 breast cancer cells.(2014 Jan) PLoS One. 28;9(1):e87371 4. Cunha-Oliveira T1, Rego AC, Morgadinho MT, Macedo T, Oliveira CR. Differential cytotoxic responses of PC12 cells chronically exposed to psychostimulants or to hydrogen peroxide.(2006 Jan) Toxicology. 5;217(1):54-62. 5. Liu Y1, He M, Sun X, Peng K, Zhao L. Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H₂O₂.(2010 Oct) Cell Biochem Funct.;28(7):578-84. P0106 Development of Arthroscopic Injectable Scaffolds for Effective Cartilage Regeneration Z-Hun KIM1,2, Sun-Mi KIM1,2, Yoo-Song CHOI1 1Dept. of Biotechnology, CHA University, Seongnam, 463-400, 2Bundang CHA Hospital, Basic Medical Research Team, Seongnam, 463-400 We have developed an injectable hydrogel scaffolds which promote regeneration of damaged or degenerative cartilages through an arthroscopy. The hydrogel, composed of cross-linked hyaluronic acid and type I collagen isolated from human umbilical cord, was prepared with different ratios. Physical properties of the hydrogel (adhesive strength, swelling/shrinking and degradation rate) were evaluated and selected effective ratio. Furthermore, in order to identify expression pattern of cytokines in the absence or presence of hydrogel, the secretomes of UC-MSCs were analyzed. We confirmed that UC-MSCs with hydrogel could alter therapeutic cytokines secretion pattern, which promote migration of bone marrow-MSCs and demote catabolic and inflammatory mechanism of cartilage. For efficacy test, UC-MSCs with the hydrogel were smoothly implanted into the rabbit full-thickness model by syringe injection. After 10 weeks of transplantation, histological analysis showed significant regeneration of cartilage without inflammation on the damaged site compared with untreated group. In conclusion, our study shows that the hydrogel has promising effects for the treatment of degenerative osteoarthritis via arthroscopic procedures. Keywords : hydrogel scaffold, arthroscopy, hyaluronic acid, umbilical cord collagen References 1. A.R. Memon and J. F. Quinlan, Adv. Orthop. (2012). P0107 Umbilical Cord Mesenchymal Stem Cell-Conditioned Media Prevent Muscle Atrophy by Suppressing Muscle Atrophy-related Proteins and ROS Generation Park CHANMI Department of Biotechnology, CHA University, 335 Pankyo-ro The therapeutic potential of mesenchymal stem cell-conditioned media (MSC-CM) has been reported with various types of disease models. Here, we examine the therapeutic effect of umbilical cord MSC-CM (UCMSC-CM) on muscle-related disease, using a dexamethasone (Dex)-induced muscle atrophy in vitro model. The expression of muscle atrophy-related proteins (MuRF-1 and MAFbx) and muscle-specific proteins (desmin and myogenin) were evaluated by Western blot analysis. The level of production of reactive oxygen species (ROS) was determined using a 2', 7'-Dichlorofluorescein diacetate (DCFDA) dye assay. The expression of antioxidant enzymes (Cu/Zn-SOD, MnSOD, GPx-1 and CAT) was verified by RT-PCR. When L6 cells were exposed to Dex, the expression of muscle atrophy-related proteins was increased by 50- 70%, and the expression of muscle-specific proteins was in turn decreased by 23-40%. Conversely, when the L6 cells were co-treated with UCMSC-CM and Dex, the expression of muscle atrophy-related proteins was reduced in a UCMSC-CM dose-dependent manner and the expression of muscle-specific proteins were restored to nearly normal levels. Moreover, ROS generation was effectively suppressed and the expression of antioxidant enzymes was recovered to a normal degree. These data imply that UCMSC-CM clearly has the potential to prevent muscle atrophy. Thus, our present study offers fundamental data on the potential treatment of muscle-related disease using UCMSC-CM. Keywords : Umbilical cord conditioned medium, Muscle atrophy, Dexamethasone References 1. Kim MJ, Kim YM, Kim Z-H, Heo S-H, Kim S-M, Hwang J-W, Chang W-J, Baek MJ, Choi YS, J Stem Cell Res Ther. 5:2 (2015). P0108 The Melanogenesis Regulation through p-CREB and MITF by Electromagnetic Fields. Young-Kwon SEO1, Sang-Eun CHO1, Kye-hong KANG2, Jung-Keug PARK1 1Medical Biotechnology, Dongguk University, Goyang, Gyeonggi-do, Korea, 410-820, 2R&D center, NOWCOS, Seoul, Korea, 153-803 Melanocytes differentiated from melanoblasts are able to synthesize the pigment melanin in process called melanogenesis. There are several diseases such as the vitiligo when the melanocytes are not able to produce melanin because melanocytes are not present or not to functions. Also, a typical aging process of hair graying is caused by defects or frailty in melanocytes. For the increasing of melanin synthesis in melanocyte of vitiligo patient, many of the physical treatment methods were tried. However, previous studies did not perform the research applied the low-frequency electromagnetic fields (ELF-EMFs) to stimulating synthesis melanin in melanocytes. The purpose of this study was to evaluate the effect of ELF-EMFs according to various frequency for melanogenesis with melanocyte in vitro. Melanocytes were treated with a frequency of 30 ~ 75Hz at an intensity of 2 mT of EMF for 3 days and then the melanogenesis was analyzed. As results, 50 and 60Hz of ELF-EMFs increased CREB, it is activated and MITF is upregulated. MITF upregulation leads to enhancement of melanogenesis related genes, tyrosinase, TRP-1, TRP-2, which are stimulated by melanin synthesis. So, the present study showed that melanogenesis stimulation could be successfully induced by 50Hz of ELF-EMFs exposure and it can be applied anti-gray hair treatment system or therapeutic device to induce repigmentation in the skin of vitiligo Keywords : extremely low-frequency electromagnetic fields (ELF-EMFs), melanogenesis, p-CREB, MITF References 1. P. Wan, Y. Hu, and L. He, Mol Cell Biochem. 354, 241 (2011). 2. I. F. Videira, D. F. Moura, and S. Magina, An Bras Dermatol. 88, 76 (2013). 3. S. K. Nath, P. P. Majumder, and J. J. Nordlund, Am J Hum Genet. 55, 981 (1994). P0109 Enhanced Cell Migration Using the Plant-based Extracts Hoomin LEE1, Hongyeol CHOI2, Sangmin LIM3, Soonjo KWON4 1Integrated Tissue Engineering Lab., Department of Biological Engineering, Inha University, Incheon, 402-751, Korea, 2Cell Culture Engineering Lab., Department of Biological Engineering, Inha University, Incheon, 402-751, Korea, 3Marine Bioenergy Research Center, Department of Biological Engineering, Inha University, Incheon, 402-751, Korea, 4Integrated Tissue Engineering Lab., Department of Biological Engineering, Inha University, Incheon, 402-751, Korea Cell migration is an important marker for damaged skin repair. We investigated cell migration of skin cells in the presence of plant-based extracts in vitro. We used two different types of cells such as fibroblasts and keratinocytes to observe migration assay. And identified our cell lines about migration property by EGFR through the immunofluorescence. Plant-based extracts were made by the two different ways (hot-water and ethanol). Plant-based extracts were added to the culture media of cells. Cell migration and expression of EGFR were analyzed in the presence of plant-based extracts in the media. Plant- based extracts were analyzed by HPLC. We confirmed the presence of EGFR of keratinocytes by immunofluorescence. Plant-based extracts were effective in fibroblast migration. We are analyzing the composition of plant-based extracts to identify which components affect the cell migration and proliferation. Keywords : Migration, Extracts, Keratinocyte, Fibroblast References 1. Tokumaru, S., Higashiyama, S., Endo, T., Nakagawa, T., Miyagawa, J. -., Yamamoru, K., . . . Taniguchi, N. (2000). Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing. Journal of Cell Biology, 151(2), 209-219. 2. Peplow, P. V., & Chatterjee, M. P. (2013). A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration. Cytokine, 62(1), 1-21. P0110 Slanted Roller Bottle System for Suspension Cell Culture Se Young PARK, Ik-Hwan Kim KIM Department of Biotechnology, Korea University, Seoul, Korea Roller bottle has traditionally been used as a popular and reliable bioreactor with adherent cells for production of vaccines and biologicals. Because of the limitations of the bottle surface, however, the roller bottle showedlow cell yield and productivity, and requires a large physical workspace, especially in suspension cell culture.In this work, the performance of a new slanted-roller bottle system was tested using CHO cells. The culture volume in the slanted bottlescould be three-fold higher than horizontally placed bottles. In our new system, fed-batch culture could also be performed usingoptimized nutrient feeding. Our results suggestthatslanted-roller bottle system can be used as single- or multiple-use cell culture reactors for the commercial production of biologicals. Keywords : Roller bottle, Chinese hamster ovary cells, Suspension cell culture, Fed-batch, Cell productivity References 1. E. I. TSAO, Optimization of a Roller Bottle Process for the Production of Recombinant Erythropoietin(1992), Annals of the New York Academy of science, 13;665, p127-128 2. Jung Youn Baik, Optimization of bioprocess conditions improve production of a CHO cell-derived, bioengineered herapin(2015), Biotechnology Journal, 10(7), p2,12 3. Jee Yon Kim, CHO cells in biotechnology for production of recombinant proteins: current state and further potential(2012), Applied Microbiology and Biotechnology, 93(3), p917, 926 4. Ryoichi Kunitake, Fully-automated roller bottle handling system for large scale culture of mammalian cells(1997), Journal of Biotechnology, 52, p290 P0111 Evaluation of Wound Healing Activity of Plant-based Extracts Using in vitro Assays Jin-Ho PARK1, Z-Hun KIM1,2, Chan-Mi PARK1, Young-Soo CHOI1 1Department of Biotechnology, CHA University, 335 Pankyo-ro, Seongnam, Gyeonggi-do 463-400, Korea, 2Bundang CHA Hospital / Basic Medical Research Team, 335 Pankyo-ro, It is known that the proliferative activity of dermal fibroblast and keratinocyte plays an important role in wound healing. In this study, we evaluated the wound healing properties of plant-based extracts prepared by ethanolic and pressurized hot water using in vitro studies. Cytotoxicity and migratory abilities of the plant-based extracts on human dermal fibroblasts and keratinocytes were examined and compared, respectively. When fibroblasts and keratinocytes were treated with different concentrations of the plant-based extracts ranging from 25 to 100 ug/mL, the both extracts did not show any cytotoxicity on both cell lines. Also, from the result of cell migration assay, we found that treatment of plant-based extracts could significantly promote fibroblasts and keratinocytes migratory abilities compared to a negative control. These in vitro results demonstrate potential of plant-based extracts as wound healing natural materials. Keywords : Plant-based extract, Wound healing, keratinocyte, fibroblast References 1. L. Sanguigno. et al, Oligosaccharidic fractions derived from Triticum vulgare extract accelerate tissutal repairing processes in in vitro and in vivo models of skin lesions (2015) J. Ethnopharmacol. 159, 198-208 P0112 Aptamer Conjugated Live Immune Cell Biosensors for the Accurate Detection of C-reactive Protein and Their Migratory Behavior Jangsun HWANG1, Jonghoon CHOI1,2 1Dept of Bionano Technology, Graduate School, Hanyang University, Seoul 133-791, Korea, 2Dept of Bionano Engineering, Hanyang University ERICA, Ansan 426-791, Korea Detecting specific biomolecules are highly required for early diagnosis and prognosis of diseases. Among those biomolecules, C-reactive protein (C-RP) is well known as an inflammatory biomolecule. C-RP is a pentameric protein presents in a blood stream where inflammatory events occur: liver failure, leukemia and/or bacterial infection1.The level of C-RP indicates the progress and prognosis of diseases especially acute phase of inflammatory events. Normal concentration of C-RP in blood is 1-3mg/L. However, such inflammatory events increases the level of C-RP concentration up to 500 time so C-RP is a very useful biomarker that indicates inflammatory disease2.In our work, we demonstrate the preparation of ssDNA-aptamer conjugated peripheral blood mononuclear cells (Apta-PBMCs) specifically capturing human C-RP. Live PBMCs functionalized with aptamers could detect different levels of human C-RP in serum by producing immune- complexes visualized with labeled anti-CRP antibodies. The migratory behavior of Apta-PBMCs toward the higher concentrated C-RP site (40µg/mL) to lower concentrated C-RP site (0µg/mL) was also investigated. The immune responses of Apta-PBMCs were evaluated by measuring TNF-alpha secretion profiles after stimulating PBMCs with lipopolysaccharide (LPS) by ELISA. In sum, engineered Apta-PBMCs demonstrated their potentials in possible applications for live cell biosensing of biomolecules and in vivo tracing of the original sites involved with C-RP secretion. Keywords : C-RP, Aptamer, live cell sensor, PBMC References 1. J. E. Volanakis, Mol. Immunol., 38, 189-197 (2001). 2. W. K. Lagrand, C. A. Visser, W. T. Hermens, H. W. Niessen, F. W. Verheugt, G.-J. Wolbink and C. E. Hack, Circulation, 100, 96-102 (1999). P0113 Cytotoxic Activity of Solvent Extracts from Korean Plants against HeLa cells Bo A KIM, Seung Hun JEON, Je Jong LEE, In Taek PARK, Seok Bong YOO, Bong Sup SHIM, An Jin WI, Whoa Shig PARK Forest Resources Research Institute, 7 Dado-ro, Sanpo-myeon, Naju city, Jeonnam province 58213, Korea Tree ethanol and water extracts prepared from Korean plants were tested for their cytotoxic activity against HeLa cells, a human cervical cancer cell line by MTT assay system [1]. Camptotheca acuminata extracts showed cytotoxic rates of HeLa cells of less than 50% at 300μg/mL of the ethanol and water extracts. Then Cinnamomum camphora and cinnamomum yabunikkei were less than 15% [2]. Among them Ethanol extracts from leaves of Cinnamomum camphora completely inhibited the cytotoxicity of HeLa cells at the concentration of 30μg/mL. Also, Ethanol and water extracts from Seed of Zanthoxylum ailanthoides showed cytotoxic effect on HeLa cells. Keywords : Cytotoxic Activity, Cinnamomum camphora, HeLa cells References 1. J. K. Lee and S. J. Koo, Cytotoxic effects of Methanol Extract and Fractions from Echinacea angustigolia on Cancer Cells. Korean J. Food SCI. Technol. Vol. 34, No. 1, pp. 123-127 (2002). 2. N. M. Cuong, Cytotoxic Activity of Vietnamese Herbal Medicines against A549 cells. Kor. J. Pharmacogn. 33(1) : 64~68 (2002). P0114 Anti-inflammatory Effect of Yeast-based Lipid Extracts Myeongkwan SONG, Sekyung KIM, Hyunshik YUN, Soonjo KWON Dept. of Biological Engineering, Inha University, Incheon, 402-751, South Korea Prolonged inflammatory response accelerates cells’ aging to break down the tissues in our body. In this study, we extracted the lipids from Saccharomyces cerevisiae and investigated the anti-inflammatory effects of lipid extracts from Saccharomyces cerevisiae. To prove their anti-inflammatory effects, nitric oxide (NO) was measured using Griess reaction and gene expression for inducible nitric oxide synthase (iNOS) was analyzed using RT-qPCR on RAW 264.7 murine macrophage cell line. Yeast-based lipid extracts showed anti-inflammatory effect at the range of 1.5mg/ml – 50mg/ml, Yeast-based lipid extracts did not affect cell toxicity at these concentrations. LPS-induced NO decreases in dose-dependent manner. Additional gene expression for iNOS, COX-2, TNF-α is being analyzed to verify the anti-inflammatory effects and their mechanisms Keywords : anti-inflammation, yeast-based lipid, extract References 1. Jung, H.A., Jin, S.E., Ahn, B.R., Lee, C.M. & Choi, J.S. 2013, "Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPS-stimulated RAW264.7 macrophages", Food and Chemical Toxicology, vol. 59, pp. 199-206. 2. Marietta, M.A. 1993, "Minireview nitric oxide synthase structure and mechanism", Journal of Biological Chemistry, vol. 268, no. 17, pp. 12231-12234. 3. Park, S.-., Jung, S.-., Ha, K.-., Sin, H.-., Jang, S.-., Chae, H.-. & Chae, S.-. 2015, "Anti-inflammatory effects of Cordyceps mycelium (Paecilomyces hepiali, CBG-CS-2) in Raw264.7 murine macrophages", Oriental Pharmacy and Experimental Medicine, vol. 15, no. 1, pp. 7-12. P0115 Biological Activity of Essential Oil from Oriental Herbal Medicine* against Melanin Production in B16F10 Cells Seul Gi LIM, Hyang-Bok LEE, Birendra Kumar SINGH, Leonard G.A, Young-Ae GOO, Sung Pil SEO, Seo Hye RIM, Hyeon-Mi LEE, Eun Ki KIM* Department of Biological Engineering, Inha University, National Research Lab of Skinbioactive Materials, 253 Yonghyun- dong, Nam-gu, Incheon 402-751, Korea Skin pigmentation depends on the formation of melanin inside the melanosome through complex process of melanogenesis. This study was aimed to investigate the antimelanogenic and antioxidant properties of essential oil extracted from Oriental Herbal Medicine *. Essential oil extracted form Oriental Herbal Medicine * was subjected to mushroom tyrosinase assay and was observed that it inhibits mushroom tyrosinase activity in a dose dependent manner. Antioxidant property was assessed through DPPH assay (2, 2-diphenyl-1-picryl-hydrazyl) and it was found that it significantly scavenged DPPH radicals. Dose dependent inhibition of the melanin content was observed in B16F10 cells without sell toxicity, furthermore the volatile chemical composition of the essential oil was analyzed through gas chromatography-mass spectrometry (GC/MS). This result showed that essential oil extracted from Oriental Herbal Medicine * might be useful and safe as a new whitening agent in cosmetics and as a natural antioxidant in skin care products. * The name of Oriental Herbal Medicine * is being patented, so we are not able to reveal the name. Keywords : Distillation, Essential oil, Tyrosinase, Melanin, Antioxidant, B16F10 References 1. H. Huang, H. Wang, K. Yih, L. Chang , T. Chang, Int. J. Mol. Sci. 13, 14679-14697 (2012) 2. M. Miyazawa, T. Oshima, K. Koshio, Y. Itsuzaki, J. Anzai, J. Agric. Food Chem. 51, 6953−6956 (2003) 3. H. Huang, T. Chang, L. Chang, H. Wang, K. Yih, W. Hsieh, T. Chang, Molecules 17, 3902-3916 (2012) 4. S. Kim, S. Lee, C. Hong, K. Gwak, H. Yeo, J. Lee, I. Choi, J. Korean Wood Sci. & Tech. 39(4), 291∼302 (2011) P0116 EMF Induced Efficient Cell Reprogramming JONGPIL KIM Dept of Biomedical engineering, Dongguk University, Seoul, 100-710 Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency. Keywords : electromagnetic fields , iPS cells, reprogramming References 1. ACS Nano. 2014 Oct 28;8(10):10125-38. P0117 Characterization of Dynamic Culture Model for Vocal Fold Tissues Dongjoo KIM1, Joo-Hyung KIM2, Jae-Yol LIM3, Soonjo KWON1 1Dept. of Biological Engineering, Inha University, Incheon, 402-751, 2Dept. of Mechanical Engineering, Inha University, Incheon, 402-751, 3Dept. of Otorhinolaryngology, Inha University School of Medicine, Incheon, 400-712 As a part of treatment of scarred vocal folds, it is essential to understand how physiological characteristics of vocal folds tissue changes in response to external perturbation. In this study, we designed the simple vibrational culture model to mimic vibratory microenvironments observed in vivo. This model consists of a flexible culture plate, three linear actuators, a stereo splitter, and a function generator. The human vocal fold fibroblast (hVFF) monolayers were established on the flexible membrane, to which normal phonatory vibration is delivered from linear actuators and a function generator. The hVFF monolayers were exposed to 2, 6, and 10 hours vibration at frequency of 205 Hz, and maximum displacement of 47.1 μm followed by a 6 hours rest. We are observing the changes in cell morphology, cell viability, and gene expression related to extracellular matrix (ECM) components. Especially, RT-qPCR results shows that the expression of ECM related genes was changed in response to vibrational stresses. This work will contribute to provide the dynamic culture device platform and further, ECM multilayered structure for vocal fold treatment. Keywords : vocal fold tissues, phonatory vibration, ECM proteins, dynamic culture References 1. S. Gray, Cellular physiology of the vocal folds. (2000), Otolaryngologic clinics of North America, 33 (4), 679-698 2. I. Titze, On the relation between subglottal pressure and fundamental frequency in phonation. (1989), The Journal of the Acoustical Society of America, 85 (2), 901-906 3. A. Farran, S. Teller, F. Jia et al., Design and characterization of a dynamic vibrational culture system. (2013), Journal of Tissue Engineering and Regenerative Medicine, 7 (3), 213-225 4. J. Kutty, K. Webb, Engineered Micro-Environments and Vibrational Culture Systems for Vocal Fold Tissue Engineering. (2008), Gene Expression 5. I. Titze, R. Hitchcock, K. Broadhead et al., Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses. (2004), Journal of Biomechanics, 37 (10), 1521-1529 6. J. Wolchok, C. Brokopp, C. Underwood et al., The effect of bioreactor induced vibrational stimulation on extracellular matrix production from human derived fibroblasts. (2009), Biomaterials, 30 (3), 327-335 7. X. Chen, S. Thibeault, Novel isolation and biochemical characterization of immortalized fibroblasts for tissue engineering vocal fold lamina propria. (2009), Tissue engineering. Part C, Methods, 15 (2), 201-212 P0118 Inhibition of Melanogenesis by Phenylalanine hydroxylase siRNA in Melan-a Melanocytes, in a Quest of New Antimelanogenic Target. Birendra Kumar SINGH, Hyang-Bok LEE, Eun-Ki KIM* Dept. of Biological Engineering, Inha University, Incheon, South Korea, 402-751 Melanogenesis is a complex process in which several enzymes and metabolites are being involved. PAH, one of the decisive enzymes, is involved in the conversion of L-phenylalanine to L-tyrosine. The biosynthesis of melanin is affected by the cellular availability of L-tyrosine as its oxidation product produced melanin. Thus, PAH plays a major role in providing the L-tyrosine. The purpose of this study was aimed to establish the PAH as a target for its use in antimelanogenic therapy. The down regulation of PAH was mediated by PAH-siRNA transfection to melan-a melanocytes. Transfection was optimized and to ensure the specificity and safety, measurement of melanin contents and cell toxicity assay was also done, respectively. It was observed that melanin synthesis was significantly inhibited in response of siRNA mediated down regulation of PAH without showing any toxic effect on melan-a cells. The expression of tyrosinase enzyme was unaffected. However, tyrosine related protein-1 and 2 was down regulated. The known inhibitor of PAH is also down regulating the melanin via inhibition of PAH enzyme. Together these preliminary results suggest that a further exploration is being needed to establish this gene (or product) as antimelanogenic target. Keywords : Phenylalanine hydroxylase (PAH), Melanogenesis, siRNA transfection, Melan-a cell References 1. S. Fuchs, V. Behrends, J.G. Bundy, A. Crisanti, T. Nolan., Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito (2014), Plos One. 1: 9: e84865. doi:10.1371/journal.pone.0084865. 2. K. U. Schallreuter, S. Kothari , J. D. Chavan B Spencer., Regulation of melanogenesis – controversies and new concepts (2008), Exp. Dermatol. 17, 395–404. 3. K.U. Schallreuter, J.M. Wood.,The importance of L-phenylalanine transport and its autocrine turnover to L- tyrosine for melanogenesis in human epidermal melanocytes (1999), Biochem. Biophys. Res. Commun., 262, 423–428. P0119 Attachment-free Growth of 3T3 Cells Using Fibroin Microspheres Da Yeong RYU, Won HUR Dept. of Bioengineering and technology, Kangwon national university, Chuncheon, 200-701 Suspension culture has several advantages over two-dimensional monolayer culture of animal cells in terms of productivity and feasibility in scale up and engineering [1]. Anchorage dependent HEK293 cells were cultivated in suspension using biodegradable polymer microspheres [2]. Thus we investigated suspension growth 3T3 cells using fibroin microspheres in a non-adhesion culture plates. Fibroin microspheres were prepared and added to 3T3 culture. Microsphere supplementation enhanced 3T3 cell growth in a non-adhesion culture plate as well as in an ordinary cell culture plate. The cells were growing in the forms of multicellular aggregates. The average rate of glucose consumption of 3T3 cells supplemented with microspheres (0.5mg/mL) was 6.59 ±0.22 nmol/cell∙day. Mesenchymal stem cells also showed enhanced cell growth in the presence of microspheres in the culture medium. Dead and live assay and morphology of multicellular aggregates were also investigated. Keywords : Animal cell, Microspheres, Fibroin References 1. A. W. Nienow, Cytotech. 50, 9 (2006). 2. J. H. Ryu, D. J. Oh, C. Y. Choi, and B. S. Kim, Biotech. Lett. 25, 1363 (2003). P0120 Three Dimensional Co-Cultivation of Murine Fibroblast 3T3 Cells with Murine Monocyte Raw 264.7 Cells Based on Magnetic Levitation Jae Kwon SHIM, Won HUR Dept. of Bioengineering and technology, Kangwon national university, Chuncheon, 200-701 Murine fibroblast 3T3 cells were grown to a three dimensional (3D) millimeter-scale spheroid with an internal cavity using a magnetic levitation technique [1]. As a necrotic core develops and cellular debris accumulates in vivo, macrophages are supposed to engulf and digest the cell debris. Thus, we co-cultured murine monocyte Raw264.7 cells with a 3D culture of 3T3 cells. Macrophages are expected to repair the internal hollow of 3T3 spheroid as they do in wound healing process [2]. Cytokines are also produced by would macrophages to stimulate fibroblast growth [3]. The specific growth rates of 3T3 and Raw 264.7 cells were 0.05 h-1 and 0.07 h-1, respectively. Both cells taken up microspheres at a similar rate. Fe3O4-containing microspheres (0.1 mg/mL) were added to cultures of 3T3 cells and Raw 264.7 cells, followed by incubation under magnets. Magnetic levitation of 3T3 cells (1 ~ 3 × 106 cells) that taken up resulted in a multicellular spheres of 2.5 ~ 3.6 mm in diameter. However, Raw 264.7 cells (1 ~ 3 × 106 cells) formed a planar disk of 14.5 ~ 15.5 mm in diameter and 65 ~ 90 μm in thickness. Co-culture of 3T3 cells with Raw 264.7 formed a disk with a core, suggesting that Raw 264.7 was not activated by the cell debris of 3T3 cell spheroid and outgrowth over 3T3 cells. Co-culture of 3T3 cells with LPS-activated Raw 264.7 formed multicellular spheroids. The internal structure of the co-cultured spheroid was also investigated. Keywords : Co-culture, 3D culture, 3T3 cells, Raw 264.7 References 1. 1. J. H. Lee and W. Hur, Biotech. Bioengin. 111, 1038 (2014). 2. 2. S. K. Brancato, and J. E. Albina, Am. J. Path. 178(1), 19 (2011). 3. 3. M. P. Rodero, and K Khosrotehrani, Int. J. Exp. Pathol. 3(7), 643 (2010). P0121 Preparation of Mesenchymal Stem Cell Spheroids Using Magnetic Levitation Jin Sil LEE, Won HUR Dept. of Bioengineering and Technology, Kangwon National University, Chuncheon, 200-701 Mesenchymal stem cells (MSCs) are defined as pluripotent cells that isolated from colonies of adherent fibroblast-like cells on plastic tissue culture dishes [1]. MSCs secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities [2]. However, MSCs a two-dimensional polystyrene substrate may have different biological responses such as receptor expression, transcriptional expression, cellular migration, and apoptosis that arising from the original organ or tissue [3]. In answer to these problems, a number of three-dimensional (3D) methods have been suggested. In this report, MSCs were cultivated in 3D environment based on magnetic levitation. Fe3O4-containing microspheres were prepared and added to MSCs and incubated under magnetic force. Multicellular aggregates of several millimeters in diameter were routinely obtained by magnetic levitation. The 3D culture had a rough surface and spherical or planar shapes depending on the levitation conditions. MSCs aggregates were embedded, sectioned and stained with antibodies to investigate physiological changes in the multicellular spheroid. We also investigated tropic factors produced by 3D culture of MSCs. Keywords : MSC, 3D culture, Spheroid References 1. A. R. Williams, and J. M. Hare, Circ. Res. 109, 923 (2011). 2. A. I. Caplan, and J. E. Dennis, J. Cell. Biochem. 98, 1076 (2006). 3. A. Abbott, Nature 424, 870–872 (2003) P0122 Reduction of Protein Degradation by Using Protein-stabilizing Agents in Transgenic Rice Cell Cultures Yeri YOO, Hyung-Jin NAM, Hong-Yeol CHOI, Mi-Kyung GONG, Dong-Il KIM* Dept. of Biological Engineering, Inha University, Incheon, 22212, Korea Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was expressed in transgenic rice cell cultures using RAmy3D promoter which is induced by sugar starvation. RAmy3D promoter has been used as an effective tool to increase the expression, but the sugar starvation condition causes cell death and triggers the release of proteases by cell lysis. Consequently the target protein is degraded rapidly by protease. Also protein production in bioreactors is greatly affected by physical factors such as aeration and agitation [1]. High aeration rates in plant cell cultures may result in severe foaming which can inhibit cell growth and high agitation rates induce shear damage, reducing cell growth and production of recombinant proteins. In order to reduce the degradation of hCTLA4Ig by aeration, agitation and proteases, protection effects of protein-stabilizing agents such as polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) were evaluated [2]. It was found that these agents lowered the degradation of target protein by plant proteases and improved the production level in transgenic rice cell cultures. In conclusion, supplementation of 0.1 g/L PVA and 0.5 g/L PVP enhanced hCTLA4Ig production 1.5-fold and 2.8-fold, respectively. Keywords : transgenic rice cell cultures , RAmy3D, polyvinyl alcohol, polyvinyl pyrrolidone, hCTLA4Ig, degradation, protease References 1. A. Hohe, T. Winkelmann and H-G. Schwenkel, CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos(1999), Plant Cell Rep. 18, 863- 867 2. W. LaCount, G. An and J.M. Lee. The effect of polyvinylpyrrolidone (PVP) on the heavy chain monoclonal antibody production from plant suspension cultures(1997), Biotechnol. Lett. 19, 93-96 P0123 Substitution of Glutamine by Glutamate and Alanyl-glutamine to Reduce Ammonia Production in Transgenic Rice Cell Cultures Mi-Kyung GONG, Hyung-Jin NAM, Hong-Yeol CHOI, Yeri YOO, Dong-Il KIM* Dept. of Biological Engineering, Inha University, Incheon 22212, Korea Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced in transgenic rice cells. In this system, inducible RAmy3D promoter was used to produce the target protein. Cell death was occurred in production phase because RAmy3D promoter system should be induced by sugar starvation. Glutamine was added as a nitrogen source. Supplement of glutamine enhanced cell viability during production phase. However, ammonia formed from glutamine metabolism inhibited cell growth and production of target protein. When glutamine was added at different concentrations (0, 6, 12 mM) to the medium, it was found that ammonia level was increased and hCTLA4Ig production was decreased at 12 mM. To reduce ammonia, glutamate and alanyl-glutamine were used as the substitute for glutamine [1, 2]. As expected, ammonia level was decreased in medium supplemented with glutamate compared to that with glutamine. Alanyl-glutamine, alanine, glutamine as well as both alanine and glutamine were added at the same concentration. Cell viability was maintained at a high level in culture with glutamine, and maximum hCTLA4Ig production was observed in culture with alanine. Keywords : hCTLA4Ig, RAmy3D promoter , Glutamine, ammonia, glutamate, alanyl-glutamine, plant cell cultures References 1. J. K. Hong, S. M. Cho and S. K. Yoon, Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells (2010), Appl. Microbiol. Biotechnol., 88(4), 869-876 2. D. Y. Kim, M. A. Chaudhry, M. L. Kennard, M. A. Jardon, K. Braasch, B. Dionne M. Butler and J. M. Piret, Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production (2013), Biotechnol. Progr., 29(1), 165-175 P0124 Reduction of Cell Death by Using Storage-protein 2 from Silkworm Hemolymph in Chinese Hamster Ovary Cell Culture Hye-Eun SHIM, Jin-Hyuk LIM, Hyun-Myoung CHA, Joon-Serk SEO, Yeon-Kyeong SHIN, Soo-Ah SHIN, Beom-Jun CHO, Dong-Il KIM Department of Biological Engineering, Inha University, Incheon 402-751 Chinese hamster ovary (CHO) cells have been widely used in recombinant protein production. Cell death is one of the major problems in mammalian cell cultures. Many factors cause the cell death during culture processes. To increase cell viability and productivity, various anti-apoptotic agents have been used to extend the culture period. Storage-protein 2 (SP2) is a soluble protein derived from silkworm hemolymph. Molecular weight of SP2 is approximately 70 kDa and amino acid sequence is confirmed. In this study, we investigated the effects of SP2 addition in transfected CHO cells producing albumin-erythropoietin (Alb-EPO). When SP2 was added to culture media, viable cell density and viability were enhanced. Productivity was increased up to 1.48-fold than the control. It was found that cell aggregation rate was decreased by 20% with 2 mg/mL of SP2. To identify the effect of SP2 as an anti-apoptotic agent, annexin V-FITC/propidium iodide staining was performed to detect cell death. Compared with the control, the fraction of apoptotic cells was reduced by 80%. In conclusion, utilization of SP2 inhibited cell death, thereby enhanced the productivity. Keywords : CHO cells, Albumin-erythropoietin, Cell death, Storage-protein 2, Silkworm Hemolymph References 1. Choi SS, Rhee WJ, Park TH, Beneficial effect of silkworm hemolymph on a CHO cell system: inhibition of apoptosis and increase of EPO Production (2005), Biotechnol. Bioeng. 90, 793-800. 2. Arden N, Betenbaugh MJ, Life and death in mammalian cell culture: strategies for apoptosis inhibition (2004), Trends Biotechnol. 22, 174-180. P0125 The Effect of Inducing Differentiation and Migration of Melanoblasts Treated with Cryptanthus sp. Leaf Extract Seo-Hye RIM1, Leonard G.A1, Birendra Kumar SINGH1, Hyang-Bok LEE1, Jeong-Hyun SHIN2, Eun-Ki KIM1 1Department of Biological Engineering, Inha University, National Research Lab of Skinbioactive Materials, 253 Yonghyun- dong, Nam-gu, Incheon 402-751, Korea, 2Department of Dermatology, Inha University Hospital, 27, Inhang-ro, Jung-gu, Incheon 22332, Korea Skin pigmentation depends entirely on the presence of melanin, produced by melanocytes. Vitiligo is a progressive hypomelanotic skin disorder and causes loss of melanocytes. It is known that the migration of melanocyte precursors (melanoblasts) from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial to the repigmentation of vitiliginous skin. However, the pathogenesis of this condition is still unknown and continuous search for treatment is progressing. In the course to find a stimulating compound for melanin biosynthesis, we evaluated the effects of Cryptanthus sp. (Fam. Bromeliaceae) on the melb-a. Results show induction in melanin when treated with different concentrations (3.125-12.5 ppm). Dose dependent significant results were recorded and the extract is not toxic to the cells in the stated concentration. Migration assay was conducted to further elucidate the effect of the extract into the cell and found stimulation on migration of melanoblast cells. Dendrite formation is also visible in the migrated cells that confirm differentiation upon the treatment of Cryptanthus sp. (Fam Bromeliacee) leaf extract. This study showed that Cryptanthus sp. (Fam Bromeliacee) leaf extract is potential candidates that could be useful in the treatment of vitiligo condition. Keywords : Vitiligo, Migration, Crptanthus sp, Differentiation References 1. Babitha, S., , D.H. Nguyen, S.J. Park , J.H. Shin G.A. Reyes , A. Caburian and E.K. Kim (2010), Potential of Cassia alata leaf extract in inducing differentiation and migration of mouse melanoblast. 15:1071-1076 2. Cui, J., L.Y. Shen, G.C. Wang (1991), Role of hair follicles in the repigmentation of vitiligo. J. Invest Dermatol. 97:410-416 3. Njoo, M. D., P. I. Spuls, J. D. Bos, W. Westerhof, and P. M. M. Bossuyt (1998), Nonsurgical Repigmentation Therapies in Vitiligo. Arch Dermatol.134:1532-1540 P0126 Anti-aging Activities of Soymilk Fermented with Lactic Acid Bacteria Hyeon Mi LEE, Sung Pil SEO, Hyang Bok LEE, Birendra Kumar SINGH, Young Ae GOO, Eun Ki KIM Department of Biological Engineering, Inha University, National Research Lab of Skinbioactive Materials, 253 Yonghyun- dong, Nam-gu, Incheon 402-751, Korea Isoflavones mostly exists in glycosidic form and to a lesser extent in aglycones form in soybeans. During the process of fermentation with the help of β-glucosidase, glucoside conjugates of isoflavones are converted into aglycone which has the antioxidant property. The aim of the present study to investigate the comparative analysis of the anti-ageing effect of the isoflavone obtained from the fermented and non-fermented soymilk. Lactobacillus Plantarum was used for the fermentation process which has the β-glucosidase activity and converts isoflavone glycoside to aglycones. We observed antioxidant activity in the methanol extract of both fermented and non-fermented extract, which was assessed from DPPH assay. Inhibition in the expression profile of MMP-1 was also observed in human dermal fibroblast cell line. Both fermented and non-fermented soymilk has the anti-ageing effect but fermented soymilk has strong anti-aging activity than non-fermented soymilk. Keywords : Lactobacillus plantarum, Antiaging, Soymilk, Fermentation References 1. Yi-Chieh Wang, Roch-Chui Yu, Cheng-Chun Chou. Antioxidative activities of soymilk fermented with lactic acid bacteria and bifidobacteria. Food Microbiology; 23 (2006) 128–135 2. Yi Na Wang, Wei Wub, Hong Chao Chen, Hong Fang. Genistein protects against UVB-induced senescence-like characteristics in human dermal fibroblast by p66Shc down-regulation. Journal of Dermatological Science; 58 (2010) 19–27 P0127 Single Cell Adaptation of Vero Cell in Serum-Free Media Soo-Hyun KIM, Youn-Je PARK Dept. of Food Science & Technology, Kongju National University. Yesan, Chungnam, 340-702 In biopharmaceutical field, the suspension adaptation of single cell from adhesion mammalian cell line has considered to be very important for large-scale cell culture processes over the last few years. Many mammalian cells such as CHO, MDCK and PER.C6 are widely applied to suspension culture processes in serum-free media for producing recombinant protein including antibodies and vaccines. Vero cell line derived from the kidney epithelial cell of an adult African green monkey has been extensively used for producing viral vaccines. However Vero cell is difficult to convert to single cell in serum-free media because of strong cell-to-cell aggregation unlike other cells. In this study, we investigated the effective serum-free media and anti-adhesion materials for single cell suspension adaptation from Vero cell. In the addition of anti-adhesion materials, though the aggregation formation is delayed compared to control, the cell growth was inhibited and cell density was reduced, consequently. So, further studies are needed to increase growth rate of adapted single cell and to maintain as single cell in suspension culture condition. Keywords : Vero, Single cell, adaptation References 1. Yu Jing et al, Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells (2014), Cytotechnology. P0128 Development of Water-immiscible and Facile Binder for Effective Bone Grafting to Accelerate in vivo Bone Regeneration Hyo Jeong KIM1, Bong-Hyuk CHOI1, Sang Ho JUN2, Hyung Joon CHA1 1Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, 2Division of Oral and Maxillofactal Surgery, Dept. of Dentistry, Anam Hospital, Korea University Medical Center, Seoul 136-705 Bone is a mineralized connective tissue having a unique ability to regenerate itself and recover its original functions. However, when bone mass loss is significant due to traumatic injuries or diseases, orthopedic reconstructive surgeries are required. Bone grafts are commonly used in the reconstructive procedures to provide appropriate mechanical properties and stimulate bone regeneration. Especially, xenograft bone substitutes (originated from other species) such as deproteinized bovine bone mineral (DBBM) are widely used in the current orthopedic and dental operations because of their effective cost and good osteogenic performance. But, clinically, the bone substitute particles are used with some adjuvant materials such as putty, paste, gel, and formable strips because the bone graft particles are rarely agglomerated and retained at the defect site. To date, many attempts have been striving to overcome this displacement of the graft particles diminishing the osteoconductivity. Therefore, in this study, a novel water-immiscible binder material is developed to make bone grafting (preparing) process simple and effective in terms of labor and cost. Complex coacervateion (a liquid-liquid separation phenomenon caused by the electrostatic interactions of two oppositely charged polyelectrolytes) of recombinant mussel adhesive protein (rMAP) and hyaluronic acid (HA) exhibited certain level of adhesiveness, water-immiscibility, and low interfacial tension. There properties of rMAP/HA coacervate makes it a proper candidate for binding bone graft particles. The effective agglomerating of DBBM particles with rMAP/HA coacervate and its stability against water were confirmed in this study. We also evaluated improved mechanical properties, blood response, in vitro cell behaviors, and in vivo potential for bone regeneration. Keywords : Bone regeneration, musse adhesive protein, complex coacervation, bone graft materials References 1. B.-H. Choi et al. Engineered mussel bioglue as a functional osteoinductive binder for grafting of bone substitute particles to accelerate in vivo bone regeneration (2015), Journal of Materials Chemistry B, 3, 546-555 P0129 Decamer Repeats Derived from Protein of Xenopus tropicalis for Electrospun Nanofiber Fabrication Yunkyeoung KWON1, Yun Jung YANG1, Tae-yoon PARK1, Dooyup JUNG1, Byeong Hee HWANG2, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, 2Division of Bioengineering, Incheon National University, Incheon 406-772 Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiberbased sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein-based material. The codon-optimized recombinant frog repeat protein, referred to as ‘xetro’, was produced at a high rate in a bacterial system, and an acid extraction-based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet-spun xetro microfibers demonstrated about 2- and 1.5-fold higher tensile strength compared with synthetic polymer polylactic acid and cross-linked collagen, respectively. In addition, the wet-spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber- based material for biomedical engineering. Keywords : repeat motif, recombinant protein, mechanical property, fiber References 1. Kumar, M.; Sanford, K. J.; Cuevas, W. A.; Du, M.; Collier, K. D.; Chow, N., 2006, Biomacromolecules, 7, 2543– 2551. P0130 Bio-conjugation of Peptide to Mussel Adhesive Protein for Bone Tissue Engineering Bong-Hyuk CHOI, Yun Kee JO, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea The current paradigm of tissue engineering has not only focused on designing structurally and characteristically similar biomaterials to the extracellular matrix (ECM), but also considered for reproducing its molecular properties including bioactivity, biocompatibility, and ability to bind growth factors. In this aspect, the concept of multicomposite and multifunctional artificial ECM is important approach in tissue engineering fields to mimic the real ECM for tissue repair. However, it is required that development of facile and efficient technique for multicomponent coating on artificial ECM surface. Recently, we reported artificial ECM mimics based on fusion of mussel adhesive protein (MAP) with the biofunctional ECM peptides. Adhesive properties of MAP enabled efficient immobilization of ECM peptides without any protein and/or surface modifications, which significantly enhanced cellular behaviors on each ECM mimics. Here, we focused on the conjugated bioactive peptides related to bone-cell specific active peptides, such as RGD, basic fibroblast growth factor, and human bone morphogenetic protein-2 (hBMP-2). Diverse biological activities such as adhesion, proliferation, and differentiation on artificial ECM mimic mixture-coated surfaces were investigated for several bone cell lines. We found that multicomposite artificial ECM showed superior abilities on cells to single component ECM mimics. The osteogenic differentiation of MC3T3-E1 cells was also characterized by evaluating expression of several osteogenic differentiation markers including RUNX2, osterix, type 1 collagen, and bone morphogenetic protein-2. After selection of the most effective multicomposite artificial ECM in vitro, we conducted in vivo experiment using multicomposite artificial ECM-coated bone substitutes. Thus, multicomposite artificial ECM based on bone-cell specific active peptide-conjugated MAP can be successfully applied in bone tissue engineering. Keywords : multicomposite artificial ECM, mussel adhesive protein, human bone morphogenetic protein-2 References 1. B.-H. Choi, Y. S. Choi, D. G. Kang, B. J. Kim, Y. H. Song, H. J. Cha, Biomaterials, 2010, 31, 8980. P0131 Poly (Ethylene Glycol) Based Cryogel Scaffolds for Cartilage Tissue Engineering Min-Eui HAN1, Su-Hwan KIM1, Hwan D KIM2, Hyun-Gu YIM2, Tae-Il KIM3, Nathaniel S HWANG1,2 1Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, Republic of Korea, 2Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, Republic of Korea, 3Department of Periodontology, Seoul National University School of Dentistry, Seoul, Republic of Korea In this study, we fabricated cryogels composed of chondroitin sulfate (CS) or hyaluronic acid (HA) for cartilage tissue engineering. For fabricating highly porous structure, methacrylated CS (MeCS) or methacrylated HA (MeHA) was crosslinked with poly (ethylene glycol) diacrylate (PEGDA) via APS/TEMED followed by freezing and thawing cycles. This procedure allowed macroporous structure after thawing. The developed cryogels supported chondrocyte proliferation and ECM secretion. In particular, CS-based cryogels stimulated aggrecan gene expression, whereas HA-based cryogels stimulated type II collagen gene expression. These results indicated that as the biomimetic cryogel can support specific ECM secretion for cartilage tissue engineering. Keywords : Cryogel, Hyaluronic acid, Chondroitin sulfate, Cartilage Tissue engineering, poly (ethylene glycol) diacrylate References 1. Kim, H.D., et al., Extracellular-matrix-based and Arg-Gly-Asp-modified photopolymerizing hydrogels for cartilage tissue engineering. Tissue Eng Part A, 2015. 21(3-4): p. 757-66 P0132 Non-viral Minicircle Delivery Inducing Plasticity and Osteogenic Differentiation in Human Umbilical Vein Endothelial Cells Seung Hyun L. KIM1, Hwajin LEE3, Eunjee A. LEE2, Nathaniel S. HWANG1,2 1Dept. of Interdisciplinary program in Bioengineering, Seoul National University, Seoul, 151-742 , 2School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, 3School of Medicine, Johns Hopkins University, Maryland, 21218 In order to compensate for the limited cell sources in autologous tissue engineering, various advances in controlling cell fate has been seen in the past decade. Among these advances, the most notable and effective strategies are direct or indirect conversions and transdifferentiation. By changing fully differentiated cells into another differentiated state, using patient’s own cells for tissue engineering became possible. In addition, non-viral delivery is another attractive method in gene transfer for its low immune response and other potentially harmful consequences from using viruses. Here, an indirect conversion of Human umbilical vein endothelial cells (HUVEC) has been achieved to show osteogenic commitment. This was accomplished by non-virally delivering Thomson Factors1, Oct4, Sox2, Nanog, and LIN28, which are known to generate iPSCs. With the induction of these factors, HUVECs enter the plastic stage in which they exhibit stem cell-like phenotypes. Then, osteogenic differentiation was induced by expanding them in osteogenic medium. Keywords : Transdifferentiation, osteogenesis, HUVEC, non viral delivery References 1. Chao Zhang, Development of poly(β-amino ester)-based biodegradable nanoparticles for nonviral delivery of minicircle DNA, ACS nano, 7(8):7241-50. P0133 Preparation and Characterization of Highly Porous Poly (Vinyl Alcohol) (PVA) Gel Filled with Extracellular Matrix (ECM) Components for Cartilage Regeneration Yunsup LEE, Nathaniel S. HWANG Department of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea Articular cartilage has a very limited self-repair ability due to its avascular structure. Cartilage defect causes several dysfunctions of articular cartilage such as decreasing load resistance and lubrication in joint motion. In this study, we focused on the fabrication and characterization of porous poly (vinyl alcohol) (PVA) hydrogel filled with bioactive components for the repair of cartilage. PVA has been proposed as a non-degradable and biocompatible FDA-approved synthetic biomaterial, for a novel cartilage defect treatment. Highly porous PVA sponges provided from Merocel (Medtronic) was characterized prior to cell response examination. We further introduced photopolymerizing chondroitin sulfates (CS) or hyaluronic acid (HA) to fill the pores. Our study showed that both CS and HA provided a suitable microenvironment for functional cartilage regeneration by enhancing chondrocyte gene expression. Overall this study demonstrates the fabrication of porous and mechanically durable PVA hydrogel incorporated with bioactive components for functional cartilage tissue engineering. Keywords : Hydrogel, Poly (Vinyl Alcohol), Chondroitin Sulfate, Hyaluronic Acid References 1. H Bodugoz-Senturk, C. E. Macias, J. H. Kung, O. K. Muratoglu, Poly(vinyl alcohol)-acrylamide hydrogels as load-bearing cartilage substitue (2009), Biomaterials, 30(4), 589 2. D. A. Bichara, et al, Porous Poly(vinyl alcohol)-Alginate Gel Hybrid Construct for Neocartilage Formation Using Human Nasospetal Cells (2010), Journal of Surgical Research 163(2), 331 P0134 Optimization of the Buffer System Using HEPES and Sodium Bicarbonate in Animal Cell Culture Medium Kang HEO, Sin Kyu KANG, Joo Young YOON, Tae Gyeong LEE, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, 209 Neungdong-ro Gwangjin-gu, Seoul 05006, Korea Maintaining the pH level of cell culture medium is vital to cell viability. CHO (Chinese hamster ovary) cells were grown in an in-house serum free medium (SJ-PFM 2.0) buffered with a mixture of HEPES and sodium bicarbonate. Since, HEPES accounted for the highest cost in SJ-PFM 2.0, we wanted to reduce the usage of HEPES while buffering capacity is maintained similar or better. The concentrations of sodium bicarbonate and soy hydrolysate which consists mainly of amino acids as zwitterions were carefully adjusted to compensate the reduced usage of HEPES. Unlikely we expected, the reduced usage of HEPES did not change the buffering capacity much while the concentration of sodium bicarbonate was critical to keep the pH of the medium. We cultured CHO cells in selected media with optimized buffer system and compared cell culture characteristics to evaluate the new buffer conditions. Keywords : HEPES, Buffer system, CHO cell, Animal cell culture medium P0135 Gas-Permeable Membrane Enhances Cell Growth, Metabolic Activity and Production by Increased Gas Transport to the Cell in Cultures Seong Min KIM1, Duk Jae OH1, Dong Hoon KIM2 1Dept. of Integrative Bioscience and Biotechnology, Sejong University, 209 Neungdong-Ro, Gwangjin-Gu, Seoul, 143- 747(05006), 2SPL Lifesciences, 570 Eumhyeon-Ri, Naechone-Myeon, Pocheon-Si, Gyeonggi-Do 487-835(11192) Worldwide biopharmaceuticals is being produced by the animal cell culture. Under these circumstances, the usage of commonly used cell culturewares is rapidly increasing and development of single-use cell culture vessel have been required. When using a disposable cell culture products currently in use, the amount of culturable cells work limited and when culturing the cells at a certain time or more, low-oxygen environment may be composed. It can lead to unexpected adverse outcomes in biopharmaceuticals produced by these cells. To overcome these issues, we developed special containers utilizing gas-permeable membrane. It supports rapid equilibration between the partial pressures of gases in the atmosphere and the concentration of dissolved gases at the pericellular level of cells. The gas-permeable cell culture bag is proven to offer superior permeability to O2 and CO2 and helps maintaining cell viability for extended time and improving cell expansion without the need for changing culture vessel. Inparticular, metabolic activity of cultured cells was observed for further studies. As an example, lactate accumulation was dramatically reduced in some cases. Keywords : Gas permeable, cell culture, culture bag References 1. Lynn et al., 2011 2. de Souza N, 2007 한국생물공학회, 생물공학의 동향 : 2015.10 효소/단백질공학 P0201 Production and Characterization of Trace Amine-Associated Receptor 13c for the Selective Detection of Death-Associated Odor Cadaverine Heehong YANG1, Tai Hyun PARK1,2 1School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea, 2Advanced Institutes of Convergence Technology, Suwon 443-270, Republic of Korea Trace amine-associated receptors (TAARs), belonging to G protein-coupled receptors (GPCRs), are receptors related to biogenic amine receptors. Among the TAARs family, TAAR 13c, zebrafish TAAR, could be a general diamine sensing element with selective binding to death-associated odor cadaverine. A selective binding of receptor with its ligand can be useful for the application to biosensors. In addition, more selective interaction of receptor with cadaverine would substantially increase its potential for safety applications. In this study, we demonstrate the dose-dependent and selective binding of TAAR13c with cadaverine and various amines. TAAR13c was successfully expressed in Escherichia coli (E. coli), purified with column chromatography, and reconstituted using nanodisc. The size of nanodisc containing TAAR13c and its biological characteristics were confirmed by using dynamic light scattering and tryptophan fluorescence assay. Moreover, functional study of TAAR13c expressed in HEK-293 cell was successfully confirmed by using dual luminescence-based reporter gene assay. These results can be utilized as GPCR characterization, TAAR-ligand interaction and protein-based biosensor researches. Keywords : trace amine-associated receptor, G protein-coupled receptor, cadaverine, nanodisc References 1. Yang et al., Purification and functional reconstitution of human olfactory receptor expressed in Escherichia coli (2015), Biotechnology and Bioprocess Engineering 20, 423-430. 2. Hussain et al., High-affinity olfactory receptor for the death-associated odor cadaverine (2013), Proceedings of the National Academy of Sciences 110, 19579-19584. 3. Ritchie et al., Chapter eleven-reconstitution of membrane proteins in phospholipid bilayer nanodiscs (2009), Methods in Enzymology 464, 211-231. P0202 Highly Stabilized Enzyme Modules in Two-Enzyme Cascade Reactors Ji Eun PARK, Bo Kuk SEO, Man Bock GU College of Life Sciences and Biotechnology, Korea University Anam-dong, Seongbuk-Gu, Seoul, 136-701, South Korea Carbonic anhydrase (CA) catalyzes the conversion of carbon dioxide to bicarbonate, and phosphoenolpyruvate carboxylase (PEPCase) catalyzes the addition of bicarbonate to phosphoenolpyruvate to form the oxaloacetate (C4 chemical). These cascade reactions are used for carbon fixation in many organisms including CAM plants, C4 plants, and many bacteria. In this study, CA and PEPCase were stabilized and immobilized to silica coated branched polymer microbeads for stable and long-term usages in CO2 bioconversion and valuable chemical production. It was found that both enzymes were sustained its high activity and stability even in long-term continuous production system. For in vitro continuous production of oxaloacetate and calcium carbonate, modular minibioreactors (working volume 10 mL) with a constant volume were operated continuously. These immobilized enzymes were found to be maintained highly stable, enabling oxaloacetate and CaCO3 to be produced for more than 24 hours and 6 days, respectively. Keywords : Carbonic anhydrase, Phosphoenolpyruvate carboxylase, oxaloacetate, calcium carbonate, microbead, immobilization, CO2 bioconversion References 1. Jinyang Chung, Ee Taek Hwang, Ji Hoon Kim, Byoung Chan Kim and Man Bock Gu. Modular Multi-enzyme Cascade Process Using Highly Stabilized Enzyme Microbeads (2014), Green Chemistry, 16, 1163. 2. Kwang Suk Chang, Hancheol Jeon, Man Bock Gu, Seung Pil Pack, EonSeon Jin, Conversion of carbon dioxide to oxaloacetate using integrated carbonic anhydrase and phosphoenolpyruvate carboxylase (2013), Bioprocess Biosystems Engineering, 36, 1923 P0203 Development of Overexpression System of α-Agarase and β-Agarase by Artificial Chromosome Manipulation Lee JI-SU, Kim JAE-WOON, Ha JIN-HO, Seok JI-HWAN, Nam SOO-WAN, Kim YEON-HEE Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Korea, Agar, a well-characterized polysaccharide present in the cell walls of red algae, has a wide variety of uses due to its stabilizing and gelling characteristics and is used primarily in microbiological media and in the food industry. Moreover, oligosaccharides derived from agar also have industrially useful properties such as delayed starch degradation, bacterial growth inhibition, anticancer and antioxidation activities and a whitening effect. According to the mode of action on the agarose, agarases are classified into two groups α-agarase and β-agarase. In this study, we tried development of overexpression system of α-agarase and β-agarase genes by artificial chromosome manipulation. The α-agarase and β- agarase genes were subcloned into integrative plasmid for stable expression and pRSI-AgaA and pRSI-AgaB palsmids were constructed, respectively. These plasmids were integrated into target region (FUR4 gene and KAP104 gene region) of yeast chromosome II and each agarase was effectively expressed and secreted. Subsequently, the chromosomal region harboring the agarase expression cassette was manipulated as yeast artificial chromosome (YAC) by PCS (PCR-mediated chromosome splitting) technique and the agarase genes on the YAC will be overexpressed by chromosome copy number amplification. Keywords : α-agarase, β-agarase, yesst artificial chromosome, PCR-mediated chromosome splitting References 1. Seok, J.H., Kim, H.S., Hatada Y., Nam, S.W., and Kim, Y.H. Construction of an expression system for the secretory production of recombinant α-agarase in yeast (2012) Biotech. letters. 34(6) 1041-1049. P0204 An Enzyme-coupled Assay for Spectrophotometric Measurements of Omega-Transaminase Activities for Ketones Sang-Woo HAN, Yu-Nam LEE, Jong-Shik SHIN Dept. of Biotechnology, Yonsei University, Seoul, 120-749 Mapping substrate specificity of omega-transaminases (omega-TAs) is an essential procedure to develop biocatalytic process or engineer its properties. Especially elevating ketone reactivity of omega-TAs is an interesting issue for preparing enantiopure amines from ketones. However, profiling of enzyme activities for ketones is time-consuming when using a conventional analytical method such as HPLC. To expedite characterization of omega-TA activities for ketones, a novel assay method has been required. Here, we developed a general and rapid assay method using benzylamine, one of universal amino donors of omega-TAs, and with the assay method, (S)-selective omega-TA from Paracoccus denitrificans, (R)- selective omega-TA from Arthrobacter sp. and (S)-selective omega-TA variants from Ochrobactrum anthropi were characterized. Keywords : assay method, omega-transaminase, substrate profiling, ketones References 1. Sam Mathew et al, High Throughput Screening Methods for Omega-Transaminases (2013), Biotechnology and Bioprocess Engineering, 18, 1-7 P0205 Preparation of Enantiopure Chiral Amines from Racemic Mixtures Using Omega- Transaminases Jun-Hyun SONG, Jong-Shik SHIN Dept. of Biotechnology, Yonsei University, Seoul, 120-749 In this study, we designed a preparation process of enantiopure (S)-amines from racemic amines using (S)- and (R)-selective ω-transaminases (ω-TAs). (R)-amine present in a racemic mixture and pyruvate are converted to a ketone and D-alanine, respectively, by an (R)-selective w-TA. And then the produced ketone and isopropylamine are converted to (S)-amine and acetone by an (S)-selective w-TA. Because a wild-type (S)–selective w-transaminase also uses pyruvate as an amino acceptor, we engineered the (S)–selective w-transaminase to lose an activity for pyruvate. To overcome unfavorable equilibrium of the reaction between ketone and isopropylamine, reactions were carried out under reduced pressure. Using this method, various chiral amines were converted to enantiopure (S)-amines (ee > 98.5 %) with> 99 % yield. Keywords : Omega-transaminase, equilibrium shift, chiral amines, isopropylamine, protein engineering References 1. Eul-Soo Park, M. Shaheer Malik, Joo-Yong Dong, and Jong-Shik Shin, One-Pot Production of Enantiopure Alkylamines and Arylalkylamines of Opposite Chirality catalyzed by Omega-Transaminase (2013), ChemCatChem, 5, 1734-1738 P0206 w-Transaminase-catalysed Asymmetric Synthesis of Chiral Amines Using Benzylamine as an Amino Donor Hong-Gon KIM, Jong-Shik SHIN Dept. of Biotechnology, Yonsei University, Seoul, 03722 Chiral amines in enantiopure forms are important building blocks for pharmaceutical drugs. w-Transaminase (w-TA) receives a great attention as a tool to prepare chiral amines because of its unique catalytic capacity to carry out asymmetric amination of ketones. Benzylamine is an ideal amino donor for w-TA because of its reactivity higher than isopropylamine which is a typical substrate for w-TA. In addition, benzylamine is an inexpensive achiral amino donor. However, enzyme inhibition by benzaldehyde was a major hurdle in carrying out efficient w-TA reactions. To overcome product inhibition and unfavorable equilibrium, we shifted the unfavorable equilibrium to a product side by removing a co-product (i.e. benzaldehyde) using auxiliary enzymes. Keywords : w-transaminase, asymmetric synthesis, equlibrium shift, benzylamine, chiral amine References 1. Jong-Shik shin, Byung-Gee Kim, Asymmetric synthesis of chiral amines with w-transaminase (1998), Biotechnology and bioengineering, 65(2), 206-211. P0207 Double Clicking for Site-specific Coupling of Multiple Enzymes Jinhwan CHO1, Sung In LIM2, Inchan KWON1,2 1School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan Gwagi- ro, Buk-gu, Gwangju, 61005, Republic of Korea. , 2Department of Chemical Engineering, University of Virginia, 102 Engineers’ Way, Charlottesville, VA 22904, United States. We demonstrated a novel strategy to site-specifically couple multiple enzymes using two orthogonal click reactions (SPAAC and IEDDA) and site-specific incorporation of a non-natural amino acid. Conjugated multiple enzymes retained activities and showed the enhanced catalytic efficiency over free enzymes. Keywords : click chemistry, strain-promoted azide-alkyne cycloaddition, site-specifically couple multiple enzymes References 1. Lim, Sung In, Jinhwan Cho, and Inchan Kwon. P0208 Enzyme Engineering Using Tripartite Split GFP System Roo Jin LEE1, Yong Jae LEE1, Ae Jin RYU1, Ki Jun JEONG1,2 1Department of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea, 22 KI for the Biocentury, KAIST Green fluorescent protein (GFP) exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range, and for a long time, it has been widely used as a powerful reporter system in the field of bioimaging, protein interaction as well as protein engineering. Particularly, Due to the recent development of high-speed flow cytometer system, the usefulness of GFP in high throughput screening is growing more and more. Recently, the split form of GFP was developed in which the original GFP was divided into two fragments and, more recently, the tripartite split form of GFP was also developed, in which the original GFP was divided into three fragments and it can give high fluorescence only when they were correctly assembled into single molecules. Both split systems have been useful tools in various protein interaction studies. We developed the new protein engineering platform with tripartite split GFP system combined with high-speed FACS sorter, and the application of this platform toward enzyme engineering was successfully demonstrated. Keywords : Sortase A, Calcium ion, Tripartite split GFP, FACS, Random mutation References 1. Sarkis K. Mazmanian et al., Staphylococcus aureus Sortase, an Enzyme that Anchors Surface Proteins to the Cell Wall(1999), Science, 285, 760 2. Geoffrey S. Waldo et al., A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association(2013), Scientific Reports, 3: 2854., P0209 Identification UV-Damaged DNA Repair Effect from a Photolyase of the Nemopilema nomurai Using E.coli Protein Expression System Gna AHN, Ga-Young PARK, Se Hee LEE, Eunji LEE, Yae-Eun JEON, Yang-Hoon KIM, Ji-Young AHN Department of Microbiology, Chungbuk National University, Cheong-Ju 361-763, Korea One of DNA mutation is thymine-thymine dimer (T-T dimer) formation, it generated from solar UV radiation (especially UV-B) causing negatively effect to organism. Photolyase are light-activated enzymes that trigger an electron transfer from a reduced FADH- to the thymine dimer. So the photolyase enables using the visible-lay to cutting T-T dimer off. Generally, most of organisms have photolyase but mammals have photolyase-like enzyme not photolyase. Our group used photolyase of Nemopilema nomurai (N.nomurai) that exist as the oldest marine organism and preserve many genomes for a long time. In the current study, we evaluated the influence of codon bias on recombinant production of N.nomurai photolyase (NnP) in E.coli by expressing several variants of codon-modified open reading frames (ORFs) encoding NnP protein. The NnP protein expression in E.coli can be optimized by adjusting codon composition towards the most preferred codon adaptation. By utilizing these enzymes it will be able to take advantage of functional sun care treatments, beacon assay and cell repair test. Keywords : Photolyase, UV damaged repair enzyme, Protein expression, Nemopilema nomurai, Jellyfish References 1. J Li, Z Liu, C Tan, X Guo, L Wang, A Sancar and D Zhong, Dynamics and mechanism of repair of ultraviolet- induced (6–4) photoproduct by photolyase(2010), Nature, Vol(466), 887–890. 2. L.O.Essen and T.Klar, Light-driven DNA repair by photolyase,review(2006), cellular and Molecular Life Science, Vol(63), 1266-1277. 3. D.-P. Hader, H.D.Kumar,R.C.Smithc and R.C.Worrestd, Effects of solar UV radiation on aquatic ecosystems and interactions with climate change(2007), Photochemical & Photobiological Sciences, Vol(6), 267-285. P0210 Effect of Concentrations of Cell and Substrate on Cadaverine Production from L-Lysine Hanyong KIM, Hyunah KIM, Seongwook JEONG, Dae Haeng CHO, Yong Hwan KIM, Chulhwan PARK* Department of Chemical Engineering, Kwangwoon University, Seoul, Republic of Korea L-lysine, used as a color fixing agent or additives in animal feed, is gradually lowering price that combined with the over- production and the international corn-soybean decrease price. And these issues emerged as a problem in China, the major producer country of L-lysine. We considered as solve these problems, over-production and low price, that material was the cadaverine (1,5-diaminopentane). There are many applicants in the chemical industry for nylon production especially. Cadaverine can be obtained using from lysine decarboxylase (LDC). Through a preparatory experiment and the literature survey, Bactrium cadaveris (Hafnia alvei) was selected as a source of LDC as a wholecell catalyst. We investigated the conversion of cadaverine with reaction time, cell concentration and substrate concentration. Keywords : L-lyinse, lysine decarboxylase, cadaverine, wholecell catalyst References 1. J. Mandelstam, nduced biosynthesis of lysine decarboxylase in Bacterium cadaveris (1954), J. gen. Microbiol, 11, 426-437. 2. F. Kondo, Studies on the lysine decarboxylase formation of Bacterium cadaveris (1962), Tohoku Journal of Agricultural Research, 13. 3. Z. Qian, X. Xia, S.-Y. Lee, Metabolic engineering of Escherichai coli for the production of cadaverine : a five carbon diamine (2011), Biotechnology and Bioengineering, No. 1, 108. P0211 Molecular Cloning and Overexpression of a Novel Thermostable Lipase Gene from Thermophilic Geobacillus thermoleovorans KL3 in E.coli. Madhavaraj LAVANYA, Si Wouk KIM Department of Environmental Engineering, Chosun University, Gwangju, 61-452 South of Korea A novel thermostable lipase producing G. themeleovorans was isolated from chicken feather dumping site. Totally 32 different bacterial species were isolated and screened. Among all the isolates, G. themeleovorans KL3 was found to be the high potential thermostable lipase producing capacity at elevated temperature. Recently, many research groups mainly focused on stress tolerant lipases production such as thermostable, salt tolerant and pH stability. In this study, a novel thermostable lipase gene (Tslip) from G. themeleovorans KL3 was cloned into pET28a (+) vector and expressed in E.coli DH5α. The amino acid sequence of lipase enzyme revealed that higher amount of hydrophobic content and grand average of hydropathicity (GRAVY) were calculated to be 0.205. The instability index (II) was computed to be 31.20, this clarifies the purified lipase are stable. The purified recombinant lipase exhibited an optimal activity at pH and temperature was 8.5 and 65°C respectively. Further, the lipase showed activity over a wide range of pH 6.5-10.5. The maximum activity of the purified thermostable lipase was 1160U/ml. The molecular weight of this lipase was estimated to be 48kDa by SDS-PAGE analysis. Purified thermostable lipase enzyme was strongly inhibited by metal ions and chemical reagents such as, Zn2+, Ag+, Hg2+, Pb2+, Tween 20, Triton-X 100 and SDS. However, the enzyme activity was remarkably enhanced in the presence of Ca2+, Mn2+, Fe2+, Ni2+ and Mg2+. From the results, the expressed thermostable lipase has potential characteristics to perform well in industrial applications, especially in the fatty acid hydrolysis for enhanced biodiesel production at high temperature operational conditions. Keywords : Recombinant lipase, Tslip, G. themeleovorans References 1. M.H. Kim, H.K. Kim, J.K. Lee, S.Y. Park, T.K. Oh, Biosci. Biotechnol. Biochem. 64(2), 280 (2000). P0212 Enhanced Production of Lipophilic Molecules through the Formation of Caveola-like Structures inside Escherichia coli Jonghyeok SHIN1, Paul HEO1, Myungseo PARK1, Younghun JUNG1, Da-Hyeong CHO1, Byoung-jae KONG1, Junghoon IN1, Jun-Bum PARK1, Jin-Byung PARK2, Dae-Hyuk KWEON1 1Department of Biotechnology and Bioengineering, Sungkyunkwan University, 2Department of Food Science and Engineering, Ewha Womans University Caveolae are membrane-budding structures that exist in many vertebrate cells. One of the important functions of caveolae is to form membrane curvature (caveolae) and endocytic vesicles (endosome). Caveolae-like structures are formed in Escherichia coli through the expression of caveolin-1. We hypothesized that the caveolae-like sturcture might expand membrane surface area of E. coli, providing more storage space for lipophilic products and membrane bound-Proteins. Enlarged membrane surface area of E. coli provided tolerance for toxic fatty acid and augmented expression of membrane protein. The biotransformation of ricinoleic acid into ester by an alcohol dehydrogenase (ADH) and Baeyer-Villiger monooxygenase (BVMO) was utilized as the model system. The recombinant E.coli co-expressing ADH, BVMO and caveolin-1 showed durable bioconversion activity and ~80% improved bioconversion activity. Collection of lipophilic products (ester) in the caveolae-like structures is expected to improve the production of lipophilic products in E.coli. Keywords : Caveolae, Baeyer-Villiger monooxygenase, alcohol dehydrogenase, Fatty acid References 1. Piers J. Walser, Constitutive Formation of Caveolae in a Bacterium (2012), CELL, 150, 752–763 P0213 One-pot Enzymatic Conversion of Levulinic Acid to 4-Valerolactone Young Joo YEON1, Sung-heuck KANG2, Hoe-Suk LEE3, Young Je YOO2,3 1The Institute of Molecular Biology and Genetics, Seoul National University, Seoul, 151-742, 2School of chemical and biological engineering, Seoul National University, Seoul, 151-742, 3Graduate Program of Bioengineering, Seoul National University, Seoul, 151-742 The utilization of renewable biomass for the production of fuels and chemicals is one of the most interesting issues in industrial biotechnology. Among forms of biomass, cellulosic biomass is particularly suitable for industrial applications because of its large-scale availability, low cost and non-food character. A general approach to utilizing cellulosic biomass is the use of cellulase enzyme for the hydrolysis of cellulose to corresponding sugars, which then can be directly feed to industrial microbes to produce commercially valuable chemicals. However, the recalcitrant character of cellulose and the low efficiency with high cost of cellulase enzyme make the industrial use of cellulosic biomass a hard work. Use of levulinic acid (4-oxopentanoic acid) could be an alternative way for the utilization of cellulosic biomass. Levulinic acid can be obtained from cellulosic biomass through a simple chemical treatment with acid catalyst. 4-Valerolactone is a versatile compound which can be used as bio-fuels, solvents and precursors of carbon-based chemicals. Lots of reports have been published describing the conversion of levulinic acid to 4-valerolactone. However, most of the methods are based on chemical reactions which require harsh conditions with high temperature and pressure and/or use of expensive metal catalysts, and no enzymatic reaction schemes have been reported so far. In this study, two-step enzymatic conversion of levulinic acid to 4-valerolactone was designed by use of the engineered 3-hydroxybutyrated dehydrogenase and paraoxonase 1. The engineered 3-hydroxybutyrated dehydrogenase can convert levuinic acid to 4-valeric acid, and paraoxonase 1 can convert 4-valeric acid to 4-valerolactone. The designed reaction was performed in one-pot system, and approximately 33% conversion of levulinic acid to 4-valerolactone could be achieved in 24h. Keywords : Levulinic Acid, 4-Valerolactone, 3-Hydroxybutyrate dehydrogenase, Paraoxonase 1, One-pot enzymatic reaction References 1. Young Joo Yeon, Hyung-Yeon Park, Young Je Yoo, Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase(2013), Bioresource Technology, 134, 377-380 P0214 Cloning and Characterization of the KDPG Aldolase from Sphingomonas sp. MJ-3 Eun Jeong LEE1, Ok Kyung LEE1, Hee Sook KIM2, Eun Yeol LEE1 1Dept. of Chemical Engineering, KyungHee University, Gyeonggi-do, 446-701, 2Dept. of Food Science and biotechnology, Kyung Sung University, Busan, 608-736 The most abundant sugar in brown algae is alginate. However, conventional industrial microbes cannot be used alginate for biofuel and chemical production. In order to utilize alginate, it is required various enzymes related to Entner–Doudoroff (ED) pathway. Among the various enzymes, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase of the ED pathway, catalyzes the reversible cleavage of KDPG into pyruvate and glyceraldehyde-3-phosphate. We carried out the cloning, overexpression and characterization of a recombinant KDPG aldolase from Sphingomonas sp. MJ-3. In order to use KDPG aldolase as a cost-effective biocatalyst, the culture and expression conditions for the recombinant E. coli harboring a KDPG aldolase gene were optimized. The KDPG aldolase obtained under optimal culture condition was used to evaluate enzyme activity by kinetics study. Keywords : aldolase, KDPG, biocatalyst, ED pathway, Sphingomonas sp. MJ-3 References 1. M. Ryu, E. Y. Lee, Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3 (2011) , J. Ind. Eng. Chem. , 17(5), 853 2. S. S. Jagtap, J. H. Hehemann, M. F. Polz, J. K. Lee, H. Zhao, Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations (2014) , Appl. Environ. Microbiol. , 80 (14), 4207 P0215 Comparison of Serum Half-life Extension between PEGylated and Albuminated Proteins in Animal Studies Byungseop YANG, Inchan KWON School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea Albumin has an unusually long serum half-life of almost three weeks. Compared to the conventional poly(ethylene glycol) (PEG)-conjugation, conjugation of human serum albumin (HSA) is an attractive emerging strategy for the extension of the serum half-life of a therapeutic protein. First, conventional PEG-conjugation mainly relies on renal filtration evasion [1], but HSA-conjugation takes advantages of neonatal Fc receptor (FcRn)-mediated recycling as well as reduced renal filtration. Second, anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins [2]. However, the serum half-lives of PEGyalted and albuminated proteins have not been directly compared. In this study, we will compare the serum half-life of PEGlyated and albuminated protein in vivo. We first achieved that the site-specific PEG- or HSA-conjugation to superfolder green fluorescent protein (sfGFP), a model protein, using site-specific incorporation of a reactive non-natural amino acid followed by bioorthogonal click reactions. This study will clarify whether the site specific albumination is an effective strategy, alternative to PEGylation, to prolong the protein serum half-life in vivo. Keywords : Albumin, poly(ethylene glycol), Half-life References 1. Sung In Lim, Young S. Hahn, Inchan Kwon, Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo (2015), J. Control. Release, 207, 93-100 2. Merry R. Sherman, L. David Willams, Monika A. Sobczyk, Shawnya J. Michaels, and Mark G. P. Saifer, Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates (2012), Bioconjug. Chem, 23, 485-499 P0216 Safety Evaluation and Risk Assessment of the DsRed2 and EGFP Proteins Introduced into the Transgenic Silkworm. Ji-Young PARK1, Kyung-Min JANG1, Kwang-Ho CHOI2, Jung-Ho PARK1 1Bio-evaluation center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 363-883, Korea., 2Department of Agricultural Biology, National Academy of Agricultural Science, RDA, JeonJu 560-500, Korea. Silk has had a reputation as a luxurious and sensuous fabric but it is not popular due to the expensive price and poor durability. To develop the silk materials that apply the various industries, the artificially synthesized gene can be introduced into the silkworm and expressed in the silk gland. Transgenic silkworms for the mass production of green fluorescent silks are generated using a fibroin H-chain expression system. For commercial use, safety assessment of the transgenic silkworms is essential. Based on the regulations of safety assessment, we have evaluated physical and chemical stability and functional equivalence. First, we tried to develop the separation and large-scale purification of EGFP and DsRed2 proteins. To evaluate their toxicity, we showed that EGFP and DsRed2 could be degraded in intestine and stomach from the test using artificial intestinal and gastric juices, so that EGFP and DsRed2 may be easily digested in 5 seconds through stomach not intestine. Also, in vitro heat stability tests have shown that introduced proteins exposed to high temperatures lose biochemical function. From the studies, we concluded EGFP and DsRed2 expressed in transgenic silkworm dose not induce allergen when proteins made by transgenic silkworms was taken in. Keywords : Safety evaluation , eGFP, DsRed2 References 1. Construction of fluorescent red silk using fibroin H-chain expression system P0217 Electro-biocatalytic Conversion of Carbon Dioxide to Formate Using Oxygen Tolerant Formate Dehydrogenase Eun-Gyu CHOI, Young Joo YEON, DaeHaeng CHO, Yong Hwan KIM* Dept. of Chemical Engineering, Kwangwoon University, Seoul, 139-701 Recently reduction of CO2 has been drawing attention since chemicals and fuels can be produced through the reduction of + CO2. In this study, the electro-biocatalytic conversion of CO2 to formate was attempted with NAD -dependant formate dehydrogenase. This protein is known encoded by the fds operon comprising five genes - fdsGBACD. Oxygen-tolerant formate dehydrogenase consists of a (αβγ)2 heterodimer in which the α-subunit binds the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor and five iron-sulfur clusters. the β-subunit FdsB that binds one iron-sulfur cluster and the flavin mononucleotide (FMN) cofactor. The γ-subunit FdsG binds an iron-sulfur cluster. Even though both FdsC and FdsD are not subunits of formate dehydrogenase, however these are known essential for the maturation of active formate dehydrogenase. These proteins have a role in bis-MGD incorporation and modification by insertion of a terminal sulfide- ligand in bis-MGD. These subunits were heterogeneously expressed in Escherichia coli MC1061 without incorporating FdsG and FdsB subunits. We demonstrated that partial subunit has the capability to convert CO2 to formate by supplying electron with electrodes in aerobic condition. Keywords : Formate dehydrogenase, Molybdoenzyme, Electro-biocatalytic reduction of carbon dioxide References 1. T. Hartmann and S. Leimkühler, The oxygen-tolerant and NAD+-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate (2013), FEBS J, 280, 6083-6096 2. N. Böhmer, T. Hartmann, and S. Leimkühler, The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor (2014), FEBS LETT, 588, 531-537 P0218 Structural Studies on O-methyltransferases and Synthesis of Functional O-methylated Phytochemicals Jin-Kyung CHOI1, Sang-Hyuk LEE2, Ju-Eun LEE2, Byung-Gee KIM1,2 1School of Chemical and Biological Engineering, Seoul National University, 151-742, Seoul, South Korea, 2Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, 151-742, Seoul, South Korea Tea extracts and (iso)flavonoids are well known phytochemicals for human health. These have been studied for their positive effects on anti-cancer, anti-obesity, anti-bacterial activity, and anti-allergic action. The latter, (iso)flavones often exhibit anti- oxidant, estrogenic and anticancer activity. Recently, however, it was reported that O-methylated catechins are better effects on relieving inflammation than are not. The most O-methyltransferases(OMTs) are from plant. These are S-adenosyl-L- methionine (SAM)-dependent OMTs and their structures have well conserved SAM binding residues but the residues that binds substrates are relatively diverse. So, it is hypothesized that structural analysis along with mutagenesis of OMTs may allow us to understand various substrate specificity and then regio-specific O-methylation of phytochemicals could be achieved. Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) from Camellia sinensis, isoflavone O- methyltransferase(IOMT) from Alfalfa are currently studied since the crystal structures of enzymes are available and computational analysis, homology modeling and docking simulation, can be performed based on the published structures. In this study, we tried to heterologously express the various O-methyltransferase in E. coli and performed enzymatic reactions on several types of catechins as substrates. The identification of the products will be analyzed by HPLC, GC/MS, and NMR. Keywords : Tea extracts, isoflavonoids, O-methyltransferase, Crystal structure References 1. Yue ZHANG, Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity(2015), Biomed & Biotechnol, 16(2), 103-112 2. MASANOBU KIRITA, Cloning of a Novel O-Methyltransferase from Camellia sinensis and Synthesis of O- Methylated EGCG and Evaluation of their Bioactivity(2010), J. Agric. Food Chem, 58, 7196–7201 3. Xian-Zhi He, Genetic Manipulation of Isoflavone 7-O-Methyltransferase Enhances Biosynthesis of 4’-O- Methylated IsoflavonoidPhytoalexins and Disease Resistance in Alfalfa(2000), The Plant Cell, 12, 1689–1702 4. Bettina E. Deavours, Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula(2006), Plant Mol Biol, 62, 715–733 P0219 Contact Analysis as a Novel Rational Design Strategy to Control Enzyme Enantioselectivity Hoe-Suk LEE1, Young Je YOO1,2 1Interdisciplinary Program in Bioengineering, College of Engineering, Seoul National University, Seoul, 151-744, 2School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744 In asymmetric synthesis of chiral compounds, enzymes are drawing attention because of their innate chiral structures and mild reaction conditions. A novel enzyme rational design strategy to control enantioselectivity, utilizing the concept of “contact analysis”, is proposed and epoxide hydrolase is analyzed as a model enzyme. Epoxide hydrolase from Aspergillus niger hydrolyzes glycidyl phenyl ether, to form a diol in slight favor of (S)-product (E=4.6). Enantioselectively improved mutants were generated with up to E=115 via directed evolution (Reetz, et al. 2006). The epoxide hydrolase structure was used to model the mutants’ structures and they were docked with each enantiomer of the substrate. Contact analysis on the enzyme-substrate complex listed individual interatomic contacts between the enzyme and the substrate. It was found that the unfavorable contacts increased for the undesired enantiomer while the desired enantiomer remained similar in enantioselectively enhanced mutants. This could be utilized to predict and design mutations suitable for controlling enantioselectivity. Keywords : Enzyme enantioselectivity, Rational design, Contact analysis, Epoxide hydrolase References 1. M. T. Reetz, L. W. Wang and M. Bocola, Angew. Chem. 45(16), 2494 (2006) P0220 Discovery of a Core 1 O-linked Glycan-Specific Binding Lectin from Mushroom Seonghun KIM Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup 580-185 Mushroom lectins harboring carbohydrate binding specificity are powerful tools to detect glycoconjugates with N-linked or O-linked glycans. In this study, we purified a novel core 1 O-linked glycan-specific lectin from the fruiting body extract of a mushroom strain by ion-exchange chromatography, affinity chromatography on fetuin-agarose, gel filtration chromatography. The purified lectin was designed as HEL1. Tricine-PAGE, MALDI-TOF mass spectrometry, and N- terminal amino acid sequencing indicated that the native lectin has an identical form with a molecular weight of approximate 15 kDa and a lectin-like structure sequence. Isoelectric focusing of the lectin showed bands near pI 5.4. Agglutination assay displayed HEL1 was more effective toward porcine’s erythrocyte rather than other animals red blood cells. Glycan microarray analysis showed that HEL1 interacts with Galβ1-3GalNAc present in Thomsen-Friedenreich antigen or core 1 O- linked glycan structure such as Peanut agglutinain (PNA). Comparing the glycan binding affinities in glycan microarray, both HEL1 and PNA bound core 1 O-linked glycan. Interestingly, HEL1 can bind a broad range of glycan structures with an extension on the β1-3Gal linked to the GalNAc as well as a terminal α(2-3) linked sialic acid which is not bound by PNA. These HEL1 binding affinities could be useful to apply the cancer research and diagnostic assay involving core 1 and extended core 1 glycan structures. Acknowledgement: This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009783)” Rural Development Administration. Keywords : Lectin, Mushroom, O-Glycan, Sialic acid, Fetuin, Agglutination, Glycan microarray, Thomsen-Friedenreich antigen , Affinity, Glycoconjugate References 1. Gabius et al. The glycobiology of the CD system: a dictionary for translating marker designations into glycan/lectin structure and function (2015) in press P0221 Gene Cloning and Biochemical Characterization of Neopullulanase from Bacillus cereus ATCC 14579 Bo-Ram PARK2, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Dept. Life Sciences, Incheon National University, Incheon, 406-772 Bacillus cereus is a gram-positive rod-shaped, motile β-hemolytic soil bacterium. It is ubiquitous in nature and an opportunistic pathogen, often related with two forms of human food-borne illnesses characterized by either diarrhea or vomiting. An enzyme annotated as neopullulanase in the B. cereus genome shared 47.6% and 45.5% of identity with maltodextrin glucosidase of Aliivibrio fischeri and maltogenic amylase of B. subtilis, respectively. A 1.7 kb DNA fragment carrying the putative neopullulanase gene of B. cereus was amplified by PCR, cloned in pET-28a, and overexpressed in E. coli BL21 to investigate its role in glycogen metabolism, sporulation, biofilm formation, and pathogenicity. The gene encoded a protein of 586 amino acids with a predicted molecular mass of 68.25 kDa. The optimal pH and temperature for the enzyme activity was 6.5 and 37℃, respectively. The purified enzyme hydrolyzed various substrates mostly to maltose in the order of α-cyclomaltodextrin (CD)>β-CD>maltotriose>γ-CD>starch, maltodextrin>glycogen, but not maltose and pullulan. Therefore, the enzyme was not likely to be a neopullulanase, but a cyclomaltodextrinase. Furthermore, the enzyme exhibited transglycosylation activity on CDs and maltotriose by forming α-1,4-glycosidic linkage. The enzyme activity was not affected by metal ions, but inhibited significantly by Co2+ and Cu2+. Keywords : Bacillus cereus, Neopullulanase, Cyclomaltodextrinase, Transglycosylation References 1. Shim, J., J. Park, J. Hong, K. Kim, M. Kim, J. Auh, Y. Kim, C. Park, W. Boos, J. Kim, K. Park. 2009. Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168. J. Bacteriol. 191:4835-4844. 2. Lee, M., S. Yang, J. Kim, H. Lee, J. Kim, and K. Park. 2007. Characterization of a thermostable cyclodextrin glucanotransferase from Pyrococcus furiosus DSM3638. Extremophiles. 11: 537-541. 3. Cheong, K. A., T. J. Kim, J. W. Yoon, C. S. Park, T. S. Lee, Y. B. Kim, K. H. Park, and J. W. Kim. 2002. Catalytic activities of intracellular dimeric neopullulanse on cyclodextrin, acarbose and maltose. Biotechnol. Appl. Biochem. 35 : 27-34. P0222 Molecular Cloning and Characterization of Intracellular Amylolytic Enzymes from Vibrio vulnificus MO6-24/O Bo-Ram PARK2, Hae-Young KIM1, So-Rah PARK1, Hae-Won SHIN1, Jung-Wan KIM1 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Dept. Life Sciences, Incheon National University, Incheon, 406-772 Vibrio vulnificus is comma shaped rod, gram-negative halophilic marine bacterium, which can cause septicemia in human via intake of raw seafoods or wound infection. Therefore, it needs to response to the alternative environments and ensure energy sources to support the life style. V. vulnificus accumulates more glycogen than other bacteria and carries various genes involved in glycogen metabolism. V. vulnificus MO6-24/O has various amylolytic enzymes such as glycosidase, oligo- 1, 6-glucosidase (MalL), pullulanase, glycosyltransferase, cytoplasmic α-amylase and periplasmic α-amylase (MalS). In this study, V. vulnificus intracellular amylolytic enzymes (MalL, MalS, and cytoplasmic α-amylase) were cloned and overexpressed in E. coli BL21 to investigate their role in glycogen/maltodextrin metabolism. They were purified and estimated molecular mass of the enzymes were 63 kDa, 82 kDa, 68 kDa when examined by SDS-PAGE. Optimal activity of the enzymes was obtained at pH 6.5, 5.5, 7.0 and 37℃, 40℃, 37℃ for hydrolytic activity. MalL and cytoplasmic α-amylase hydrolyzed maltodextrin most efficiently, while MalS hydrolyzed soluble starch to maltose-maltohexose. However, their role in glycogen/maltodextrin metabolism is yet to be elucidated. Keywords : Vibrio vulnificus, Oligo-1,6-glucosidase, Cytoplasmic α-amylase, Periplasmic α-amylase, glycogen metabolism References 1. Park, J., J. Shim, P. Tran, I. Hong, H. Yong, E. Oktavina, H. Nguyen, J. Kim, T. Lee, S. Park, W. Boos, K. Park. 2011. Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli. J. Bacteriol. 193:2517-2526. 2. Schönert, S., Seitz, S., Krafft, H., Feuerbaum, E.A., Andernach, I., Witz, G., Dahl, M.K., 2006. Maltose and maltodextrin utilization by Bacillus subtilis. J. Bacteriol. 188: 3911-3922. 3. Raha, M., Kawagishi, I., Muller, V., Kihara, M., Macnab, R.M.J. 1992. Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. J. Bacteriol. 174(20):6644-52 P0223 An Auxiliary Activity 9 from Chaetomium globosum Expressed in a Bacterial Host Increases Cellulase Activity in Cellulose Hydrolysis In Jung KIM, Hak Jin YOUN, Kyoung Heon KIM Department of Biotechnology, Graduate School, Korea University, Seoul 136-713 Auxiliary activity family 9 (AA9), which is previously known as glycoside hydrolase family 61 or polysaccharide monooxygenase, is a recently identified class of synergistic proteins originating from cellulolytic fungi. By chemically modifying the recalcitrant cellulose through the oxidative mode of action, AA9s are capable to improve the efficiency of cellulase in the hydrolysis of cellulose. In this study, we show an expression of AA9 originating from the fungus Chaetomium globosum (CgAA9) in a bacterial host (Escherichia coli) for the first time, and its synergistic activity in the cellulose hydrolysis by cellulase. The optimal synergism (1.7 fold) was observed when 0.9 mg/g cellulose of CgAA9 was reacted with 0.9 filter paper units of Celluclast 1.5 L/g cellulose at 50°C and pH 5. In addition, the synergistic activity of CgAA9 was enhanced by adding increasing concentrations of sodium azide. Functional expression of the fungal AA9 in E. coli and its optimization for synergism conducted in this study will pave the way for further studies on AA9s and their industrial applications. Keywords : AA9, PMO, Synergism , Cellulose hydrolysis, Cellulase References 1. Kim IJ et al. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis, Appl Microbiol Biotechnol, in press P0224 The First Discovery of The Agarolytic β-galactosidase Releasing Galactose from Agarooligosaccharides Cho KYUNG MUN, Lee CHAN HYOUNG, Yun EUN JU, Kim KYOUNG HEON Department of Biotechnology, Graduate School, Korea University, Seoul, 136-713, Republic of Korea Marine macroalgae are considered as sustainable resource for producing the bio-based products such as biofuels and industrial chemicals owing to the abundance of carbohydrates. Agarose is the major cell wall component of red macroalgae and approximately comprises ~55 wt% of total carbohydrates of red macroalgae such as Gelidium amansii. Agarose is a heteropolysaccharide composed of equal molar amounts of D-galactose and 3,6-anhydro-L-galactose. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium Vibrio sp. strain EJY3; this enzyme was annotated as agarolytic β-galactosidase (ABG). Contrary to the lacZ-encoded β- galactosidase from Escherichia coli, ABG does not hydrolyze general substrates like lactose and it can act only on the galactose moiety at the nonreducing end of AOS. On an agarotriose substrate, the optimum pH and temperature of ABG were 7 and 35°C, respectively. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of ABG offers an enormous advantage in the saccharification of red macroalgal carbohydrates for its use as a biomass feedstock for fermentable sugar production. Keywords : Agarose, agarotriose, 3,6-anhydro-L-galactose, D-galactose, agarolytic β-galactosidase References 1. Lee CH et al. A novel agarolytic β-galactosidase acts on agarooligosaccharides for complete hydrolysis of agarose into monomers. Applied and Environmental Microbiology 2014, 80(19):5965-5973 P0225 Pretreatment-specific Optimization of Cellulase Mixtures for Saccharification of Lignocellulose Hak Jin YOUN, In Jung KIM, Kyoung Heon KIM Department of Biotechnology, Graduate school, Korea University, Seoul 136-713 Lignocellulose is such a heterogeneous and complex biomass that its saccharification process prior to the conversion into biofuels or biochemicals is costly in biorefinery. The variability of carbohydrate composition and physical structure of lignocellulosic biomass depends on the biomass source and the pretreatment method. For this reason, it is necessary to utilize a customized cellulase mixture specific to pretreated lignocelluloses to maximize the efficiency of enzymatic saccharification of lignocellulose. In this study, we optimized the ratio of enzyme mixture consisting of three recombinant enzymes such as endoglucanase (Cel5H), cellobiohydrolase (E3), and endoxylanase (Xyn10C) for the saccharification of two differently pretreated rice straw (acid-pretreated; ACID and alkaline pretreated; ALKALI). Based on a statistical mixture design, the optimal ratios of Cel5H: E3: Xyn10C were 6.969:23.031:0 and 9.012:7.962:13.029 for ACID and ALKALI, respectively. This result indicates the optimal formulations of cellulase mixture are highly dependent on the type of pretreated rice straw. The results obtained from this study will be helpful for the development of customized enzyme mixture for utilizing variable pretreated lignocelluloses in industry. Keywords : Lignocellulose, Pretreatment, Cellulase mixture, Cellulose hydrolysis, Mixture design References 1. Agbor, V.B., Cicek, N., Sparling, R., Berlin, A., Levin, D.B. 2011. Biomass pretreatment: Fundamentals toward application. Biotechnology Advances, 29(6), 675-685. 2. Akin, D.E. 2007. Grass lignocellulose - Strategies to overcome recalcitrance. Applied Biochemistry and Biotechnology, 137, 3-15. 3. Anarjan, N., Tan, C.P. 2013. Developing a three component stabilizer system for producing astaxanthin nanodispersions. Food Hydrocolloids, 30(1), 437-447. 4. Bailey, M.J., Biely, P., Poutanen, K. 1992. Interlaboratory testing of methods for assay of xylanase activity. Journal of Biotechnology, 23(3), 257-270. 5. Bak, J.S., Ko, J.K., Han, Y.H., Lee, B.C., Choi, I.G., Kim, K.H. 2009. Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment. Bioresource Technology, 100(3), 1285-1290. P0226 Monomeric Sugar Production from Agar by Acid-base Buffer Pretreatment and Enzymatic Saccharification Supplemented with Agarolytic β-galactosidase Jung Hyun KIM, Chan Hyoung LEE, Eun Ju YUN, Kyoung Heon KIM Department of Biotechnology, Seoul, 136-713 Red macroalgae (Rhodophyta) are currently considered as sustainable resources owing to their high carbohydrate and low lignin and hemicellulose contents. However, utilization of red macroalgae has been hampered due to the lack of established methods for pretreatment and an effective saccharification system. The major carbohydrate in red macroalgae, specifically in an agarophyte, is agarose that is composed of D-galactose and 3,6-anhydro-L-galactose (AHG). So far, agarose saccharification was most efficiently achieved by prehydrolysis using a weak acid and subsequent enzymatic hydrolysis using β-agarase and neoagarobiose hydrolase (NABH) for producing the monomeric sugars. However, this process requires rigorous neutralization after acid prehydrolysis, resulting in the formation of salts. Furthermore, residual agarotriose is not further hydrolyzed by currently available β-agarases and NABH. In this study, we aimed at eliminating or minimizing the neutralization step by using a low-concentration acid-base buffer such as 20 mM (0.3%, w/v) Tris-HCl for hydrothermal pretreatment of agar at 170°C for 10 min. In order to hydrolyze agarotriose, a novel agarolytic β-galactosidase, acting on odd-numbered agarooligosaccharides, was introduced into the enzymatic saccharification process. Using these chemical and enzymatic processes, the monomeric sugar yields, 44.9% for D-galactose and 44.8% for AHG, were obtained from agar, which were based on the theoretical maximum from the initial agar before pretreatment. Keywords : agarolytic β-galactosidase, acid-base buffer pretreatment , L-AHG References 1. Lee CH et al. Saccharification of agar using hydrothermal pretreatment and enzymes supplemented with agarolytic β-galactosidase. Process Biochemistry in press. P0227 Expression, Purification and Refolding of Ligand-Binding Domain of Human Umami Taste Receptor Il Ha JANG, Sae Ryun AHN, Tai Hyun PARK School of Chemical and Biological Engineering, Seoul National University, 599 Gwanangro, Gwanak-gu, Seoul 151-742, Republic of Korea Taste is composed of five primary taste sensations: saltiness, sourness, bitterness, sweetness, and umami [1]. Bitter, sweet and umami taste receptors are G-protein coupled receptors (GPCR). Among these, umami taste is known to harmonize and enhance the flavors of ingredients in food. Umami has been actively investigated in food industries due to their usefulness as food additives. Human umami receptor is a heterodimer consisting of two class C GPCRs, T1R1 and T1R3. Many scientists have attempted to produce T1R1 and T1R3 proteins in E.coli; however, it has been difficult to express these proteins because of their big sizes around 90 kD membrane targeting expression properties, strong hydrophobicity and complicated structures. So we propose to express only N-terminal of T1R1 and T1R3, respectively, because the N-terminal domain was known to play a key role in binding to the ligand [2, 3]. Each N-terminal domain of T1R1 and T1R3 was cloned into pET-DEST42 vector, and overexpressed in E.coli and purified using FPLC. Their yield was 8.8 g/l and 7.3 g/l, respectively. The purified protein was successfully refolded and it was confirmed by CD spectrum. In this study, we suggest a new method to produce each of T1R1 and T1R3 N-terminal domains. Keywords : Umami, C class GPCR, Taste receptor, T1R1, T1R3, Expression, Purification, Refolding References 1. Sami Damak, Sience, vol. 301, no.5634 (2003) 2. Feng Zhang, PNAS, vol. 105, 20930 (2008) 3. Hong Xu, PNAS, vol. 101, 14258 (2004) P0228 Engineered Mussel Bioglue with Enhanced Adhesion and Water Resistance via Residue- Specific Dopa Incorporation Byeongseon YANG1, Niraikulam AYYADURAI2, Hyungdon YUN3, Yoo Seong CHOI4, Jun HUANG5, Qingye LU5, Hongbo ZENG5, Byeong Hee HWANG1, Hyung Joon CHA1 1Dept. of Chemical Engineering, POSTECH, Pohang 790-784, Korea, 2Dept. of Biotechnology, CLRI, Chennai 600-020, India, 3Dept. of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea, 4Dept. of Chemical Engineering, Chungnam National University, Daejon 350-764, Korea, 5Dept. of Chemical and Materials Engineering, University of Alberta, Edmonton T6G 2V4, Canada 3,4-Dihydroxyphenylalanine (Dopa) has been suggested to be the key factor for rapid and strong underwater adhesion. Mussel adhesive protein (MAP) is one example where Dopa chemistry is the core of their underwater adhesion with extremely high Dopa contents of ~10-25 mol%. The biosynthesis of recombinant MAPs in an Escherichia coli system was a good approach but lack of Dopa in recombinant MAPs critically limited the underwater adhesion. Here, we explore the in vivo residue-specific Dopa incorporation into recombinant MAPs allowing the quantitative replacement of tyrosine residues with a yield of over ~90 %, to create, for the first time, engineered MAPs in E. coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated engineered MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa- incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications. Keywords : Mussel adhesive protein, Dopa, bioglue References 1. Yang B, Ayyadurai N, Yun H, Choi YS, Hwang BH, Huang J, Lu Q, Zeng H, Cha HJ. 2014. In Vivo Residue- Specific Dopa-Incorporated Engineered Mussel Bioglue with Enhanced Adhesion and Water Resistance. Angew Chem Int Ed. 53, 13360-13364 P0229 The Unique Role of Novel Cellulosomal Subunit in Clostridium cellulovorans Explored by Cohesin Marker Related to Binding on Insoluble Cellulosic Substrate SoHyeong KIM, SangDuck SANG, SuJung KIM, JeongEun HYEON, BomYi PARK, SungOk HAN Dept. of Biotechnology, Korea University, Seoul, 02841, Republic of Korea Clostridium cellulovorans efficiently degrades components of lignocellulosic biomass as producing of an extracellular enzyme complex called the cellulosome. Cellulosome assembly depends on the interaction of a dockerin module in the cellulosomal subunit with one of several cohesin modules in the scaffolding protein. By using a cohesin biomarker, we identified a novel cellulosomal subunit (Clocel3193) which don’t belong to any family. The purified Clocel3193 didn’t have the enzymatic activity, but we found that it has just the insoluble substrate-binding ability. To measure the binding affinity of the dockerin in Clocel3193 for the cohesin such as Coh2, a competitive enzyme-linked interaction assay was performed. As competitors, Clocel3193 reduced the binding ability of Endoglucanase E (EngE) depending on concentration of competitors. Also, we made the complex with mCbpA, Clocel3193 and EngE. When we measured activities of the complex and the mixture of mCbpA, Clocel3193 and EngE, we found the highest activity of the complex in Lignin. These results demonstrate that a novel cellulosomal subunit has unique ability of binding on lignin to active EngE than other insoluble celluloses. Therefore, the Clocel3193 will assist cellulase by interacting with cohesins and enhance degradation by binding on insoluble cellulosic Substrate. Keywords : Clostridium cellulovorans, Cellulosome, Insoluble cellulosic substrate, Cellulosomal subunit, novel function References 1. CHO, W. J. et al., Cellulosomic profiling produced by Clostridium cellulovorans during growth on different carbon sources explored by the cohesin marker (2010) Journal of biotechnology 145.3: 233-239 2. JEON, S. D. et al., Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans, Applied and environmental microbiology 79.19: 5942-5948 (2013) 3. ARAI, T. et al., Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis, Journal of bacteriology 185.2: 504-512 (2003) P0230 Photo-controlled Site-specific Fluorescent Labeling of Proteins in Live Cells Hyunjin JEON, Youngeun KWON Dept. of medical engineering, Dongguk University, Seoul, Korea There have been many trials to introduce controllable molecular switches on the proteins of interest in order to change or modify their functions. Regulating the endogenous function of proteins is important on studying the location, interaction and roles of proteins in vivo. Our molecular switch are made an effective tools by investigation of cytotoxic protein with proper temporal and spatial resolution. Here, we introduce a system where the function of the proteins could be modulated by external stimulus. We utilize the trans-splicing reaction between engineering split Npu DnaE Inteins as a model system. In our approach, a bulky protecting group is introduced to the synthetic C-terminal fragment of Intein (IC) in order to disturb its proper binding to N-terminal fragment of Intein (IN). This steric modulation successfully inhibited the trans-splicing activity of the split intein. We then experimented whether the trans-splicing activity of intein complex could be reacquired. The photolysis of the photocages trigger off irradiating the trans-splicing reaction mixture with 365 nm wavelength light in living cells. The function of the protein was successfully restored upon the removal of the photo-cages. Keywords : split intein, fluorescent labeling, live cell, biosensing P0231 Efficient Labeling of Transmembrane Proteins Based on Engineered Split-Intein Mediated Protein Trans-splicing Euiyeon LEE, Youngeun KWON Dept. of Medical engineering, Dongguk University, Korea Proteomics has become one of the fastest growing fields in life science research, and the demand for tools to analyze proteins has increased significantly. Engineering and modifying proteins in vivo or in vitro can aid the understanding of the protein-protein interactions and cellular dynamics. For this purpose, live cell imaging using fluorescent proteins (FPs) has been widely utilized to visualize target proteins under a microscopy by building fusion proteins. However, methods of FP- based imaging have a few limitations, such as slow maturation kinetics or issues with photo-stability under laser illumination. In order to address these drawbacks, we employed a pair of engineered Npu DnaE split-intein for labeling of recombinant target membrane proteins. By utilizing short 14 amino-acids synthetic C-intein instead of 36 amino-acids native sequence, this method offered novel opportunity to build more practical and sufficiently efficient labeling system. The labeling reaction occurred in two different modes, namely either via specific binding of two split-intein fragments or via covalent bond formation through split-intein mediated protein trans-splicing reaction. No external energy was required for the labeling reaction. Labeling reaction was carried out efficiently on HeLa cell-line at the concentration of reagent as low as 4 mM within 1~30 min with minimal background staining. This approach will lead to new methodologies for investigating protein localization, transportation, and cell signaling. Keywords : split intein, trans-splicing, trans membrane splicing P0232 Identification and Characterization of A Novel SGNH-Hydrolase(Ef21) from Enterococcus faecalis V583. Deu Rae AN1,2, Bum Han RYU1,2, Kyeong Kyu KIM1, T. Doohun KIM2 1Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea, 2Dept. of chemistry, sookmyung woman university, Seoul 140-742, Republic of Korea Microbial hydrolases can be used to synthesize and produce of fine chemicals, food-ingredients, or drug intermediates. Here, a novel SGNH-hydrolase (Ef21) from Enterococcus faecalis V583 was identified, characterized, and immobilized for industrial applications. Primary sequence analysis of Ef21 identified a putative catalytic triad (Ser10-Asp166-His169) and a molecular weight of 21KDa. Ef21 was active towards short chain esters such as p-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. Biochemical methods including dynamic light scattering (DLS), time of flight (TOF) mass spectrometry, SDS-PAGE, and native PAGE were employed for its characterization. Furthermore, cross-linked enzyme aggregates (CLEA) of Ef21 exhibited high reusability, highlighting its applicability as an industrial biocatalyst. Keywords : SGNH hydrolase, Cross-linked enzyme aggregates, Ef21, Industrial applications References 1. Song Yi Bae et al,Characterization and immobilization of a novel SGNH(2013), Appl Microbiol Biotechnol, 97,1637–1647 2. Seulgi Kim et al, Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262(2012),International Journal of Biological Macromolecules, 50, 103–111 3. Tri Duc Ngo et al, Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/β- lactamase homologue from a metagenomic library(2014), Acta Cryst. D70, 2455–2466 4. Heejin Hwang et al, Characterization of a novel oligomeric SGNH-arylesterase from Sinorhizobium meliloti 1021(2010), International Journal of Biological Macromolecules, International Journal of Biological Macromolecules, 46, 145–152 5. Kyungmin Kim et al, Structural and biochemical characterization of a carbohydrate acetylesterase from Sinorhizobium meliloti 1021(2015), FEBS letters, 589, 117–122 P0233 Expression and Characterization of Hydrophobin in Pichia pastoris Yuanzheng WU1, Ho-Dong LIM2, Geun-Joong KIM2, Hyun-Jae SHIN1 1Department of Chemical and Biochemical Engineering, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759, 2Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Yong-Bong Dong, Buk-gu, Gwangju 500-757 Hydrophobins are extracellular surface active proteins produced by filamentous fungi with a molecular weight between 7 and 15 kDa. Hydrophobins show very little sequence conservation in general, apart from the presence of 8 conserved cysteine residues implicated in the formation of 4 disulfide bridges. Hydrophobins can assemble spontaneously into amphipathic monolayers at hydrophobic-hydrophilic interfaces. In this work 9 different hydrophobin genes were synthesized and inserted into shuttle vector pPICZα A. The corresponding pPICZα A-HFB recombinants were then transformed into Pichia pastoris GS115 for heterologous expression. The secreted hydrophobins were purified by ultrafiltration and affinity chromatography. Water contact angle (WCA) and soy oil emulsion demonstrated that purified hydrophobins could self- assemble on both hydrophobic siliconized glass and hydrophilic mica surfaces. The successful production of hydrophobins in P. pastoris will reduce the cost and open up several new applications in future P0234 The Novel Function of Deinococcal PprM : Enhanced Tolerance against Various Stresses Yongjun CHOI School of Environmental Engineering, University of Seoul, Seoul, 02504 Deinococcus radiodurans, an extremophilic bacterium, possess a unique characteristic that tolerant against various extreme stresses. PprM, a cold shock domain protein (CSP) from D. radiodurans, is known as a regulator protein of DNA damage repair system. Here, we demonstrated the novel role of PprM conferring oxidative stress resistance in Escherichia coli. The E. coli strain expressing PprM showed significantly improved tolerance to hydrogen peroxide (H2O2)-induced reactive oxygen species (ROS) stress. The transcription level of several genes involved in oxidative stress response such as mntH, hemH, and katE was increased by the overexpression of pprM. Moreover, upregulated catalase activity, the intracellular level of Mn/Fe ratio (2.8 fold), and enhanced ROS scavenging ability were observed in E. coli expressing PprM protein. These results suggested that PprM functions as a global regulator to protect cells against oxidative stress by regulating the expression of oxidative stress response-related genes, controlling manganese uptake, and stimulating catalase activity, which collectively makes E. coli more tolerant against oxidative stress. Keywords : protein engineering, oxidative stress, tolerance 한국생물공학회, 생물공학의 동향 : 2015.10 바이오에너지 및 환경생물공학 P0301 Model-based Prediction of Potential Distribution of Lycorma delicatula (Hemiptera: Fulgoridae) Using CLIMEX Jae-Min JUNG1, Sung-Hoon JUNG2, Wang-Hee LEE1 1Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Yuseung-gu, 305-764, South Korea, 2Department of Applied Biology, Chungnam National University, Daejeon, Yuseung-gu, 305-764, South Korea After the first report of Lycorma delicatula brought from China [1, 2], its population has been increased all over the places in Korea since 2006. Because it usually survives where grows fruit trees and its excreta lead to sooty mold, significant damages in fruit farming have been issued 2008 and 2009 [3]. This study aims to predict the potential geographical distribution of L. delicatula using CLIMEX software and to propose its possible usage in pest control. CLIMEX software, a modeling tool for predicting distribution and dispersion of species based on information regarding habitat of target species and climate, is the main tool for this study. CLIMEX determines suitability of a specific area for a species based on Ecoclimatic Index (EI) calculated by multiplying Growth Index (GI), Stress Index (SI), and Interaction Stress Index (SX). EI larger than 25 means that the area is fit for inhabitation of the target species, while an area with EI<10 is uninhabitable. We selected 73 representative areas in Korea and calculated the average climates from the data collected from Korea Meteorological Administration (KMA) to plug them into CLIMEX. Physiological data of L. delicatula is obtained from literatures studying on hatching time and survival rate of egg in winter [4, 5]. Among 73 representative areas, 11 areas were uninhabitable (EI<10), while 28 areas showed EI>25. Thirty four areas showed EI between 10 and 25. Thus, it is conclusive that Korea is generally adequate as a habitat of L. delicatula. In detail, either Jeolla or Gyeongsang province, the south part of Korea, showed higher EI. In contrast, the northern part of Gyeonggi including Seoul and the inland Gangwon may not be suitable because of high cold stress (Taebaek and Daegwallyeong resulted in EI=0). Current distribution of L. delicatula has been covered the south Gyeonggi including Seoul, Chungcheong, the north Jeolla and Gyeongsang provinces, which mostly agrees with CLIMEX simulation. For Jeonlla and Seoul, however, CLIMEX prediction was consistent with the current distribution. In Jeolla-do except Jeongeup, L. delicatula has not been reported, but CLIMEX simulated the high EI. This disagreement may be due to less grape farming areas (7%) compared to Gyeongsangbuk-do (45.5%) and Chungcheong-do (23.2%). On the other hand, Seoul showing low possibility of habitation of L. delicatula by CLIMEX is actual habitat. For Seoul, minimum temperature in January(-9.4C) is too low to inhabit. However, climatic change starting from the late 2000s could lead to inaccurate prediction because we used climatic data for past 30 years. This study demonstrated that the potential geographical distribution of L. delicatula simulated by CLIMEX was quite similar to the actual habitat, suggesting its possible usage in predicting expansion and distribution of a species under new environments. Because prediction of future distribution of pests helps reduce agricultural damages in advance, application of CLIMEX is expected in the field of agriculture. Moreover, it can be used to predict spread of a specific species carrying pathogens and viruses or destroying domestic ecosystem. Keywords : Lycoma delicatula, climate, CLIMEX, modeling, potential distribution References 1. J. M. Han, H. J. Kim, E. J. Lim, S. H. Lee, Y. J. Kwon and S. W. Cho, Entomological Research, 38, 281 (2008). 2. H. Doi, Journal of Chosen Natural History Society, 13, 30 (1932). 3. J. D. Park, M. Y. Kim, S. G. Lee, S. C. Shin, J. H. Kim and I. K. Park, Korean Journal of Applied Entomology, 48, 53 (2009). 4. D. S. Choi, D. I. Kim, S. J. Ko, B. R. Kang, J. D. park, S. G. kim and K. J. Choi, Korean Journal of Applied Entomology, 51, 371 (2012). 5. Y. S. Lee, M. J. Jang, J. Y. Kim and J. R. Kim, Korean Journal of Applied Entomology, 53, 311 (2014). P0302 Fermentation of Korean Food Waste with Diluted Acid Hydrolysis by Engineered Saccharomyces cerevisiae Yong Seon KIM1, Se Jin KIM1, Ji Yeon JANG2, Byung Hwan UM1 1Dept. of Chemical Engineering and Research, Hankyong National University, Anseong, Gyeonggi-do, 456-749, 2IT Convergence Materials R&BD Group, Korea Institute of Industrial Technology, Cheonan, Chungcheongnam-do, 331-822 Korean food waste consists of 23.1% of glucan on dry basis and has the potential to serve as an effective feedstock for production of ethanol and lactic acid through fermentation. Dilute acid pretreatment and hydrolysis at varied temperature, sulfuric acid concentration and reaction time were evaluated for conversion of glucan of Korean food waste to fermentable sugar. The maximum concentration of fermentable sugar from Korean food waste (24.1g/L) by pretreatment (0.4% w/v H2SO4, 64.5min, 160℃) and acid hydrolysis (pH 0.55, 1hr, 121℃).[1] The acid pretreatment was performed using a 1L and 7L stainless steel reactor. A reactor was loaded with 100g of air dried food waste and 400mL liquid (a final ratio of solid to liquid =1:4) at under this condition.[2] After pretreatment, liquid was separated from solid and acid hydrolysis was performed at the same condition in an autoclave. Autoclaved liquid was concentrated 4 times and extracted organic acid by Liquid-Liquid extraction using TAPO (Trialkylphosphine oxide). The concentration of glucose in the extracted liquid was about 84.2g/L. The extracted liquid was neutralized to pH 3 and 5.5 by calcium carbonate. In addition, a recombinant S. cerevisiae harboring a plasmid containing a codon-optimized lactate dehydrogeases (LDH) gene derived from Lactobacillus casei was constructed. We have also constructed another lactic acid producing S. cerevisiae in which pyruvate decarboxylases (PDC1 & PDC5) were replaced with lactate dehydrogease (LDH) on the chromosome. These strains were used to produce lactic acid from the glucose derived from food waste. This system with food waste as a biomass can be applied to produce other organic acids such as 3-hydroxypropionic acid, adipic acid, succinic acid, and other biochemicals such as isobutanol and isobutylene which will result in producing cost-effective biochemicals against petroleum-based chemicals. Keywords : Korean food waste, Pretreatment, Acid hydrolysis, Fermentation, Ethanol, Lactic acid, Liquid-liquid extraction, TAPO, Glucose References 1. B. C. Saha, L. B. Iten, M. A. Cotta, Y. V. Wu. Process Biochem. 40(12), 3693-3700 (2005) 2. S. J. Kim and B. H. Um, Appl. Biochem. Biotechnol. 175(5), 2501-2515 (2015) P0303 Green Fluorescent Protein-based Environmental Monitoring Young Hoon SONG, Jeong Hyun SEO School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749 Recombinant green fluorescent protein (GFP) was expressed in E. coli to look at the stress effect for the cell against diverse toxic conditions such as ethanol, caffeine, and phenol. GFP expressing cell shows fluorescence in vivo without cell lysis or purification of expressed GFP, thus we were able to easily investigate the stress level in diverse chemical agents or materials by measuring fluorescence intensity at 395nm(excitation)/509nm(emission). From this, we might successfully use GFP- expressing E. coil as a environmental biosensor for toxic conditions. Keywords : GFP, cell biosensor, monitoring, whole cell P0304 Characterization of Isolate, Thermophile Clostridium sp., Isolated from Thermophilic Microbial Fuel Cell as Tentative Electrochemically Active Bacterium Mihwa LEE1, Serah CHOI1, Phuc THI HA2, In Seop CHANG1 1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST),261 Cheomdan- gwagiro, Buk-gu, Gwangju 500-712, South Korea , 2The voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman WA 99164-6515, USA A Gram-positive, moderate thermophile, anaerobic, rod-shaped, an indigo-reducing, strain GISTMFC-FeD9, was isolated from the anode electrode of distillery wastewater-fed thermophilic microbial fuel cell (MFC). The 16S rRNA analysis revealed that GISTMFC-FeD9 is phylogenetically close to known Clostridium isatidis strain Wv6T which is saccharolytic T with the similarity of 99%. The C. isatidis Wv6 has been known that gases (H2 and CO2), organic acids (acetic, lactic, and formic acid), and ethanol were produced on PYG medium. We determined that the isolate, GISTMFC-FeD9, could grow on glucose, sucrose, formate, and acetate as substrate, but not on galactose, fructose, glycerol, butyrate, fumarate, and lactate. The acetate, propionate, ethanol, H2, and CO2 were produced on glucose culture condition. We confirmed the electrochemical activity of GISTMFC-FeD9 on a graphite electrode during glucose fermentation from cyclic voltammetry (CV) test. The reduction peak was significantly showed at -0.35 V versus Ag/AgCl during CV measurement. Keywords : Thermophilic bacteria, Thermophlic microbial fuel cell, Glucose fermentation, Electrochemically Active Bacterium References 1. A. Nikki Padden , Vivian M. Dillon, John Edmonds, M. David Collin, Nerea Alvarez and Philip John, An Indigo- reducing moderate thermophile from a woad vat, Clstridium isatidis sp. nov. (1999), J. Systematic Bacteriology, 49, 1025-1031 P0305 Biotransformation of Actinides and Fission Products Changhyun ROH Division of Biotechnology,Korea Atomic Energy Research Institute (KAERI), 1266 Shinjeong-dong, Jeongeup-si, Jeolabuk- do 580-185, Korea The successful bioremediation of radionuclides relies on a complex interplay of biological, chemical, and physical processes. Underpinning mechanisms include oxidation, reduction, dissolution, precipitation, sorption and leach, and these processes can influence the toxicity and transport of radionuclides in biogeochemical systems. When these mechanisms are performed by organisms, bioprocesses present opportunities for bioremediation of radionuclides in the environment ? either to immobilize them in place to accelerate their removal [1]. The goal of this article is to give an overview of the range of radioactive wastes remediated using such microorganisms, the mechanisms involved, and the possible applications for these processes. In this review, particular emphasis will be placed, on the bioremediation of actinides and fission products. Keywords : Biotransformation, Actinides, Fission Products, Biotransformation References 1. IAEA. Vienna, International Atomic energy Agency (2004) P0306 Application of By-products of Marine Alga, Tetraselmis sp. from Biodiesel Production Associated with High-Pressure Steaming Process Woon Yong CHOI1, Nam Young KIM1, Do Hyung KANG2, Hyeon Yong LEE3 1Dept. of Medical Biomaterials Engineering, Kangwon National University, Chuncheon 200-701, 2Korea Institute of Ocean Science and Technology (KIOST), Ansan P.O. Box 29, Seoul 426-744, 3Dept. Food Science and Engineering, Seowon University, Cheongju, 361-742 This study was to investigate the possibility of utilizing the by-products from marine alga, Tetraselmis sp. after extracting the lipids from Tetraselmis sp. The lipid extraction was combined with high pressure steaming pre-treatment process at 2450MHz and 1700W input power for 10min, to extract the total lipids under less harsh conditions, which could result in maintaining higher lipid extraction yield of 11.31 (%, w/v) than 8.11 (%, w/v) from conventional folch extraction process. It was also found that the by-products from this process can be well utilized in many areas of foods, feeds and other biomaterials because it has favorable amounts of total protein as 13.7 (%, w/w) with high quality amino acids profiles such as 7.1 (% w/w) of glutamate, 7.3 (% w/w) of leucine and 4.8 (% w/w) of arginin, respectively. In addition, this by-products showed relatively strong antioxidant activity as 37.01% of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging ability in adding 1.0 mg/mL of the by-products. Therefore, it is believed that the by-products of marine alga, Tetraselmis sp. applied with combined lipid extraction process can be widely used for animal or fish feeds as well as a new resource of biomaterials. Keywords : By-products , Marine Alga, High-Pressure Steaming Process References 1. I. Laing and C. G. Verdugo, Nutritional value of spray-dried Tetraselmis suecica for juvenile bivalves (1991), Aquaculture, 92, 207-218 2. M. R. Brown, The amino-acid and sugar composition of 16 species of microalgae used in mariculture (1991), Journal of experimental marine biology and ecology, 145, 79-99 P0307 Activation of Formate Hydrogen-lyase Pathway via Expression of Uptake [NiFe]-Hydrogenase in Escherichia coli BL21(DE3) Byung Hoon JO, Hyung Joon CHA Dept. of Chemical Engineering, POSTECH, Pohang, 790-784 Several recent studies have reported successful hydrogen (H2) production achieved via expression of recombinant uptake [NiFe]-hydrogenases in Escherichia coli BL21(DE3), a strain that lacks H2-evolving activity [1-4]. Herein, we demonstrate that the production of H2 was indeed induced via native formate hydrogen-lyase (FHL) activated by the expression of recombinant hydrogenases. Unlike wild-type BL21(DE3), the recombinant BL21(DE3) strains possessed FHL activities. Through experiments using fdhF (formate dehydrogenase-H) or hycE (hydrogenase-3) mutants, it was shown that H2 production was almost exclusively dependent on FHL. The two subunits of uptake [NiFe]-hydrogenase could activate FHL independently of each other, implying the presence of more than two different mechanisms for FHL activation in BL21(DE3). It was also revealed that the signal peptide was essential for activation of FHL via the small subunit. The recombinant strains with [NiFe]-hydrogenase appear to be unsuitable for practical in vivo H2 production due to their relatively low H2 yields and productivities. We suggest that an improved H2 production system could be designed by constructing a well characterized and overproduced synthetic H2 pathway and fully activating the native FHL in BL21(DE3). Keywords : hydrogenase, formate hydrogen lyase, Hydrogenovibrio marinus, Escherichia coli BL21(DE3), biohydrogen References 1. JYH Kim, BH Jo, HJ Cha, Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli (2010), Microbial Cell Factories, 9(54) 2. SY Lee,HJ Lee, JM Park, JH Lee, JS Park, HS Shin, YH Kim, J Min, Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture (2012), International Journal of Hydrogen Energy, 35(3), 1112-1116 3. JYH Kim, BH Jo, HJ Cha, Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli (2011), Journal of Biotechnology, 155, 312-319 4. P Zhou, Y Wang, R Gao, J Tong, Z Yang, Transferring [NiFe] hydrogenase gene from Rhodopeseudomonas palustris into E. coli BL21(DE3) for improving hydrogen production (2015), International Journal of Hydrogen Energy, 40(12), 4329-4336 P0308 Isolation of Streptomyces sp. strain 1154 Capable of Degrading Alkali Lignin Gyeongtaek GONG1,2, Han Min WOO1, Tai Hyun PARK2, Youngsoon UM1 1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, 02792, 2Interdisciplinary program of Bioengineering, Seoul National University, Seoul, 08826 Lignin is one of the main components of lignocellulose and is the most plentiful natural aromatic hetero-biopolymer. However, due to the recalcitrant characteristics of lignin, microbial degradation of lignin is difficult and complex. In this study, we report a lignin-degrading bacterial strain isolated form wood decaying soil. In order to screen lignin-degrading bacterial strains, we selected candidate isolates based on the bacterial growth in lignin-added agar plates. Among the twenty candidate bacterial isolates, the strain, designated Streptomyces sp. strain 1154 exhibited the gradual decrease of lignin contents in liquid lignin-YPG (yeast extract, peptone and glucose) medium. The degradation of lignin was evaluated by monitoring the decrease of absorbance at 280 nm, and the maximum degradation and decolorization rate of lignin was 66% and 81% respectively in five days at the modified ISP (International Streptomyces Project) medium 4. These results provide that the microbial lignin degradation using Streptomyces sp. strain 1154 is one of the promising clues for utilization of lignocellulosic bio-resources. Keywords : microbial lignin degradation, alkali lignin, Streptomyces P0309 Butanol-Isopropanol (BI) Fermentation by a Newly Isolated Clostridium sp. A1424 Sung Hun YOUN1, Jae-Hyung AHN1, Han Min WOO1,2, Youngsoon UM1,2 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, South Korea, 2Clean Energy and Chemical Engineering, Korea University of Science and Technology, Daejeon 34113, South Korea An alcohol-producing bacterium, designated as Clostridium sp. A1424, was isolated from soil. The 16S rRNA analysis exhibited 99.9% identity with both C. beijerinckii NCIMB 8052T and C. diolis DSM 5431T. However, the fermentation products were distinct from those microorganisms. Using glucose, Clostridium sp. A1424 produced butanol (BuOH) and isopropanol (PrOH), while C. beijerinckii produced BuOH and acetone. With glycerol, BuOH was mainly produced by Clostridium sp. A1424, while C. diolis produced 1,3-propanediol. Notably, BuOH and PrOH production by Clostridium sp. A1424 was sugar-specific: 4.4-5.0 g BuOH/L and 1.6-2.7 g PrOH/L were produced using each of glucose, mannose, fructose, cellobiose, and sucrose (18.2-19.7 g/L), while only acid (2.0-4.7 g butyric acid/L) was produced from xylose, arabinose, and galactose. From glycerol (17.1 g/L), 3.9 g BuOH/L and 3.4 g 1,3-propanediol/L were produced. Alcohol production was increased (8.6 g BuOH/L & 3.7 g PrOH/L) after utilizing 37 g glucose/L for 28 h. In conclusion, an isolated Clostridium sp. A1424 is capable to produce BuOH and PrOH as the main products from various sugars, providing great potential in BuOH- PrOH fermentation. Keywords : Clostridium sp. A1424, Butanol, Isopropanol References 1. Biebl, H. and C. Spröer, Taxonomy of the Glycerol Fermenting Clostridia and Description of Clostridium diolis sp. nov. (2002) Systematic and Applied Microbiology 25(4): 491-497 2. Qureshi, N., et al., Butanol production by Clostridium beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber (2008) Bioresource Technology 99(13): 5915-5922 3. Survase, S., et al., Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol (2013) Journal of Industrial Microbiology & Biotechnology 40(2): 209-215 P0310 Enhancement of Butyric Acid Production from Lignocellulosic Biomass with Supplementary Sugar Alcohol from Biodiesel-Waste Kyung Min LEE1,2, Ki-Yeon KIM1, Okkyoung CHOI1, Sung Hun YOUN1, Min Sun KIM1, Han Min WOO1,3, Sung Ok HAN2, Youngsoon UM1,3 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea, 2School of Life Science and Biotechnology, Korea University, Seoul, Republic of Korea, 3Clean energy and Chemical engineering, Korea University of Science and Technology, Daejeon, Republic of Korea Butyric acid is widely used in chemical, food and pharmaceutical industries. Recently, biological production of butyric acid has been investigated using renewable biomass because of fossil fuel depletion. Since substrate cost significantly influences biochemical price, we have focused on our research in the development of high yield fermentation process. Because glycerol (reductance degree, 4.66) which is a by-product from biodiesel production is a more reduced form than glucose (reductance degree, 4), the production of reduced compound such as butyric acid is likely to be favorable using glycerol. In this study, we investigated the optimum ratio of glycerol to glucose associated with growth and butyric acid production of Clostridium tyrobutyricum ATCC 25755. Interestingly, to solve redox imbalance, C. tyrobutyricum converted acetic acid to butyric acid when glycerol was added. When crude glycerol was added to lignocellulosic hydrolysate, C. tyrobutyricum utilized not only fermentable sugars (glucose and xylose) but also a nonfermentable by-product in hydrolysate (acetic acid). Therefore, the yield of butyric acid was increased from 0.37 g/g sugar to 0.45 g/g sugar. Keywords : lignocellulosic hydrolysate, crude glycerol, butyric acid, Clostridium P0311 Isolation and Characterization of Thermophile for Understanding Role of Anode Electrode in the Distillery Wastewater-fed Thermophilic Microbial Fuel Cell Serah CHOI1, Mihwa LEE1, Phuc THI HA1,2, Taeyoung KIM1, In Seop CHANG1 1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 500- 712, South Korea, 2The voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman WA, 99164-6515, USA Strain GISTMFC-FeM16 is one of strains that were previously isolated from anode electrode of distillery wastewater-fed thermophilic Microbial Fuel Cell (tMFC) operated on 55℃ using anaerobic solid culture technique. The 16S rRNA analysis revealed that GISTMFC-FeM16 is phylogenetically close to Thermosinus carboxydivorans type strain DSM 14886T Nor1T with the similarity of 97%. The glycerol and sugars (glucose, fructose, galactose, glycerol and sucrose) as substrate were investigated for cell growth and utilization. We discovered that GISTMFC_FeM16 produce ethanol, acetate, and propionate utilizing glucose, galactose, glycerol and sucrose. The isolate was determined electrochemical tests such as microbial fuel cell and cyclic voltammetry, and it gave us to see whether this strain has a role of electrochemical activity for electricity generation in the tMFC using distillery wastewater which contains carbohydrates. Keywords : thermophilic Microbial Fuel Cell, Glucose fermentation, thermophilic bacteria References 1. Okkyoung Choi; Taeyeon Kim; Han Min Woo; Youngsoon Um, Electricity-driven metabolic shift through direct electron uptake by electroactive heterotroph Clostridium pasteurianum (2014) , Scientific Reports , 4(6961), 1-10 P0312 Efficient Ethanol Production from Lignocellulosic Hydrolysate by Engineered Saccharomyces cerevisiae Harboring Xylose Isomerase-based Pathway Ja Kyong KO, Youngsoon UM, Sun-Mi LEE Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarang-ro 14 gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea Simultaneous fermentation of both glucose and xylose present in lignocellulosic biomass is necessary for the economically viable ethanol production from renewable resources. However, the efficiency in lignocellulosic hydrolysate fermentation remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the performance of SXA-R2P-E, an efficient xylose fermenting Saccharomyces cerevisiae strain with a xylose isomerase-based pathway [1], in anaerobic fermentation of lignocellulosic hydrolysates. In a mixed sugars fermentation with 70 g/L glucose and 40 g/L xylose, this strain produced ethanol with a high yield and titer of 0.43 g ethanol/g sugars and 50 g/L, respectively. Moreover, SXA-R2P-E resulted in the highest ethanol yield of 0.46 g/g among those of other strains (0.30-0.46 g/g) during the fermentation of lignocellulosic hydrolysates processed by acidic pretreatments [2, 3]. This study shows a xylose isomerase- expressing strain can efficiently ferment lignocellulosic hydrolysates to ethanol without intensive pathway engineering. Keywords : xylose isomerase, Saccharomyces cerevisiae, cofermentation, ethanol References 1. Lee S-M, Jellison T and Alper HS (2014) Biotechnol Biofuels 7:122. 2. Lu Y, Warner R, Sedlak M, Ho N and Mosier NS (2009) Biotechnol Prog 25:349-356. 3. Novy V, Krahulec S, Wegletier M, Muller G, Longus K, Klimacek M and Nidetzky (2014) Biotechnol Biofuels 7:49. P0313 Construction of a High Efficiency Copper Adsorption Bacterial System by Peptide Display and Its Application on Copper Dye Polluted Wastewater Murali kannan MARUTHAMUTHU, Jaewook KIM, Sang Woo JUNG, Soon Ho HONG Department of Chemical Engineering, University of Ulsan, Ulsan 680-749 For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display (543.27 µmol/g DCW Dry Cell Weight (DCW)) showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW). This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ01111601) by RDA, and Basic Science Research Program by the Ministry of Education (NRF-2014R1A1A2054726). Keywords : copper , peptide display , Escherichia coli References 1. T.S. Moon, J.E. Dueber, E. Shiue, P. Jones, and L. Kristala, Metabolic Engineering 12, 298 (2010). P0314 Characterization of the Efficient Cellulase in an Isolated Cellulolytic Bacterium Paenibacillus sp. CAA11 Sukhyeong CHO, Eun Sook KIM, Han Min WOO, Youngsoon UM Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul 136-791 Cellulosic biomass is the most abundant and attractive resource for biorefinery that aims to produce valuable bio-chemicals. In this study, we investigated an isolated cellulolytic bacterium, designated Paenibacillus sp. CAA11 from soil and its cellulase. The isolate was able to grow using carboxymethyl cellulose (CMC) and secreted cellulase by own secretion system. To determine an effective cellulase in Paenibacillus sp. CAA11, putative genes encoding cellulase in CAA11 were cloned and overexpressed in Bacillus subtilis RIK1285 which is deficient in two kinds of proteases. After Congo-red staining, the recombinant B. subtilis RIK1285 overexpressed CAA11_02340 showed 2.1-fold bigger halo zone on CMC containing plate than RIK1285 wild type, indicating that putative cellulase CAA11_02340 was functional to degrade cellulose. Furthermore, B. subtilis RIK1285 overexpressed CAA11_02340 was able to degrade insoluble regenerated amorphous cellulose. These results suggest that the recombinant B. subtilis RIK1285 containing CAA11_02340 could be used as a consolidated bioprocessing platform after metabolic pathway engineering toward a cost-effective biorefinery using cellulosic biomass. Keywords : Cellulosic biomass, cellulase, Paenibacillus References 1. Zhang X-Z, Sathitsuksanoh N, Zhu Z, Zhang Y-HP. One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis (2011) Metabolic engineering. 13:364-72. 2. Grange DC, den Haan R, van Zyl WH. Engineering cellulolytic ability into bioprocessing organisms (2010) Applied microbiology and biotechnology. 87:1195-208. P0315 Analysis of Extreme Furfural Tolerance of a Soil Bacterium Enterobacter cloacae GGT036 Seungjung CHOO1,3, Sun Young CHOI1,2, Gyeongtaek GONG1, Hong-Sil PARK1,2, Sun-Mi LEE1, Youngsoon UM1, Sung Ok HAN3, Sang Jun SIM2, Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, 02792, 2Green School, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea, 3Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea Detoxification process of furfural is essential for production of bio-based chemicals from lignocellulosic biomass. We isolated an extreme furfural-tolerant bacterium Enterobacter cloacae GGT036 from soil sample collected in Mt. Gwanak, Republic of Korea. Compared to the maximal half inhibitory concentration (IC50) of well-known industrial strains Escherichia coli (24.9 mM furfural) and Corynebacterium glutamicum (10 mM furfural) based on the cell density, IC50 of E. cloacae GGT036 (47.7 mM) was significantly higher after 24 h than the others. The conversion rates of furfural to furfuryl alcohol were 62.8 % for 20 mM furfural and 64.3% for 40 mM furfural after 12 h of inoculation. However, conversion rate of 60 mM furfural was shown as 32.3 % due to several growth defects. Since bacterial cell growth was exponentially inhibited depending on linearly increased furfural concentrations in the medium. In conclusion, E. cloacae GGT036 is an extreme furfural-tolerant bacterium. Recently, the genome sequence of E. cloacae GGT036 was completed and this could provide an insight for engineering of E. cloacae GGT036 itself or other industrially relevant bacteria. Keywords : Enterobacter cloacae, Furfural tolerance, Detoxification References 1. Glebes et al., Genome-wide mapping of furfural tolerance genes in Escherichia coli (2014), PLoS ONE, 9 (2014), e87540 P0316 Evolutionary Engineering of Saccharomyces cerevisiae for Efficient Xylose Consumption and Growth Inhibitor Tolerance Yeong-Je SEONG1, Yong-Han CHO1, Soo-Min LEE2, Yong-Cheol PARK1 1Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 136-702, Korea, 2Division of Wood Chemistry and Microbiology, Korea Forest Research Institute, Seoul 130-712, Korea For sustainable bioethanol production from cellulosic biomass, efficient consumption of xylose and tolerance against toxic chemicals derived from pretreatment of cellulosic biomass are required. In our previous work, we constructed recombinant Saccharomyces cerevisiae DXS strain capable of consuming xylose and producing ethanol by introducing xylose metabolic enzymes. Furthermore, we constructed S. cerevisiae DXSP strain with high xylose consuming ability by applying evolutionary engineering to the DXS strain in this study. In batch cultures with 40 g/L xylose as a sole carbon source or a mixture of 50 g/L xylose and 20 g/L glucose, the DXSP exhibited about 24 % and 13 % faster xylose consumption rate and ethanol production, respectively than the DXS strain did. In the presence of growth inhibitors such as acetate and furfural, the DXSP strain showed a range of 11 % to 92 % fold increases in xylose consumption rate and ethanol productivity, relative to the DXS strain. Considering the improvement of xylose metabolic ability and growth inhibitor tolerance, it is obvious that evolutionary engineering is an effective tool to obtain increased metabolic capacities. Keywords : Saccharomyces cerevisiae, bioethanol, xylose References 1. Lee S-H, Kodaki T, Park Y-C, Seo J-H. Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.(2012) J Biotechnol ;158:184-191. P0317 Production of Reducing Sugar from Defatted Coffee Waste by Hydrothermal Reaction and Enzymatic Hydrolysis Ji Wook KIM1, A-Ram KIM1, Don-Hee PARK2, Gwi-Taek JEONG1 1Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea, 2Department of Biotechnology and Bioengineering, Interdisciplinary Program of Graduate School for Bioenergy and Biomaterials, Chonnam National University, Gwangju 500-757, South Korea In this study, the production of reducing sugar from defatted coffee waste by hydrothermal reaction and subsequent enzymatic hydrolysis was investigated. In hydrothermal reaction, the highest yield of 21% was obtained at the condition of 200°C and 30 min. By subsequent enzymatic hydrolysis, the highest yield of 34% was achieved from hydrolysate obtained from hydrothermal reaction at 140°C and 45 min. Keywords : defatted coffee waste, reducing sugar, hydrothermal reaction, enzymatic hydrolysis References 1. Kim et al., Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis (2014), Bioresource Technology, 161, 348–353 P0318 Production of Reducing Sugar from Two Brown Macro-algaes for Growth of Yarrowia lipolytica Jae-Hyung PARK, Sang-Yeong YOON, Jae-Kun KIM, Hye-Min CHO, Sujin BAE, A-Ram KIM, Gwi-Taek JEONG Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea Marine macro-algaes as biomass feedstocks are focused on the production of biofuels. In this work, we investigate the cell growth of Yarrowia lipolytica for producing lipids using hydrolysate of Undaria pinnatifida and Saccharina japonica. Firstly, the optimization of saccharification condition of U. pinnatifida and S. japonica using hydrothermal reaction and subsequent enzymatic hydrolysis was investigated. As a results of hydrolysis, in the hydrothermal reaction condition of 100 mM H2SO4, 121°C for 90 min and subsequent enzymatic hydrolysis using enzyme mixture, 18.77g/L (U. pinnatifida) and 20.90g/L (S. japonica) total reducing sugars was obtained. Also, marine algae hydrolysate can be used to cell growth of Y. lipolytica. Keywords : macro-algaes , Yarrowia lipolytica , hydrolysate, cell growth References 1. Kim et al., Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis (2014), Bioresource Technology, 161, 348–353 P0319 Hydrolysis of Corn Husk by Microwave Irradiation Pretreatment Sang-Yeong YOON, A-Ram KIM, Sung-Koo KIM, Gwi-Taek JEONG Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea In this study, corn husk is used for producing total reducing sugar. To obtain TRS, microwave irradiation and co-catalyst were applied to pretreat corn husk. The optimal pretreatment condition for production of reducing sugar is 1% H2SO4, 0.05M (NH4)2SO4, 1:15 S/L ratio, during microwave irradiation of microwave strength 500W and 5 min. By subsequent enzymatic hydrolysis for 48 hr, 56.35% TRS yield was obtained. Keywords : corn husk, reducing sugar, pretreatment, microwave References 1. Kim et al., Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis (2014), Bioresource Technology, 161, 348–353 P0320 Enzymatic Conversion of ɑ-Cellulose into Fructose Hye-Min CHO, Ji Wook KIM, Sang-yeong YOON, Jae-Hyung PARK, A-Ram KIM, Gwi-Taek JEONG Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea In this study, we investigate the enzymatic conversion of ɑ-cellulose into fructose by two subsequent enzymatic reaction. Firstly, the hydrolysis of ɑ-cellulose into glucose was performed by cellulase and β-glucosidase. Secondly, the conversion of glucose into fructose was conducted by glucose isomerase. In the condition of hydrolysis of ɑ-cellulose (0.05M citrate buffer, pH4.8, 50°C), 67.1 g/L glucose was obtained. By subsequent enzymatic hydrolysis with glucose isomerase, 26.9 g/L fructose was obtained. Keywords : cellulose, enzymatic conversion, fructose References 1. Kim et al., Optimum Reaction Condition of Enzymatic Hydrolysis for Production of Reducing Sugar from Enteromorpha intestinalis (2015), KSBB Journal, 30(2), 53-57 P0321 Isolation of CO Utilizing Bacteria by Suppressing Heterotrophic Rumen Microorganisms Using Antibiotics Yeubin LEE, Shinyoung PARK, Nulee JANG, Muhammad YASIN, In Seop CHANG School of Environmental Science of Engineering, School of Environmental Science of Engineering, Gwangju Institute of Science and Technology, Gwangju, 500-712 The bioethanol produced from CO fermentation is drawing attention as alternative energy source. The CO is converted to bioethanol using acetogen as a biocatalyst. The acetogen that produce C2 and C4 compounds through Wood-Ljungdahl pathway promise biocatalyst for bioethanol production. Based on current research, animal feces are well-known habitat of CO utilizing bacteria. However, coexistence of heterotrophic rumen bacteria in animal feces has become a big hurdle for isolation of CO utilizer. In this study, we applied antibiotic for inhibit growth of rumen bacteria (especially, Gram-negative bacteria). In addition, CO was provided as a sole carbon source. And the Hungate roll tube was used to isolate CO utilizing bacteria. The result showed that CO utilizing consortia were isolated from inoculum source. These consortia produced less amount of propionate than control. Then, forty-five isolates were screened from these consortia. Among these isolates, four isolates that are capable of utilizing CO were isolated. Hence, the antibiotics can be used as an artificial selective pressure for suppressing coexisting rumen bacteria for isolation of CO utilizing bacteria from animal feces. Keywords : Bioethanol, CO utilizing bacteria, Isolation References 1. Bryan P Tracy, Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications (2012), Current Opinion in Biotechnology, 23, 364–381 2. Pamela P. Peralta-Yahya, Microbial engineering for the production of advanced biofuels (2012), Nature, 488, 320- 328 P0322 Improved Ethanol Production Due to High Xylitol Dehydrogenase Activity by Mutant Kluyveromyces marxianus 17694-DH1 Deok-Ho KWON, Jin-Seong KIM, Jae-Bum PARK, Seung-Won JANG, Suk-Jin HA Department of Bioengineering and Technology, college of engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea Kluyveormyces marxianus is well knwon thermeltolerent yeast which is capable of growing at 45oC. However ethanol production from xylose as a carbon source is not efficient. K. marxianus KCTC17694 was developed though a directed evolution and random mutagenesis to isolate efficient xylose fermenting mutantant K. marxianus strain. Xylose consumption rate and ethanol production rate by the mutant K. marxianus 17694-DH1 were 58% and 178% improved at 30 oC, respectively as compared to those from the parental strain. In addition, ethanol yield was highly increased from 0.17 g/g to 0.28 g/g by K. marxianus 17694-DH1. When xylose reductase (XR) activities were compared with the parental strain and K. marxianus 17694-DH1, they were not significantly changed. However, enzymatic activities of xylitol dehydrogenase (XDH) were highly improved by K. marxianus 17694-DH1. The activity of XDH from K. marxianus 17694-DH1 was 64.8% increased as compared to that from the parental strain. In order to confirm the reason of enhancement, nucleotide sequences of the parental strain and K. marxianus 17694-DH1 were analyzed. Keywords : Xylose, Xylitol dehydrogenase, XDH, Ethanol References 1. Jin-Seong Kim, Jae-Bum Park, Seung-Won Jang, Suk-Jin Ha,Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity, Applied Biochemistry and Biotechnology, 176, 1975-1984 2. Biao Zhang • Lulu Li • Jia Zhang • Xiaolian Gao •Dongmei Wang • Jiong Hong, Improving ethanol and xylitol fermentation at elevated temperature through substitution of xylose reductase in Kluyveromyces marxianus, Society for Industrial Microbiology and Biotechnology 2013, 40, 305-316 3. Jae-Bum Park, Jin-Seong Kim, Seung-Won Jang, Eunsoo Hong, Suk-Jin Ha, The Application of Thermotolerant Yeast Kluyveromyces marxianus as a Potential Industrial Workhorse for Biofuel Production, KSBB Journal, 2015, 30.3: 125-131 P0323 Effects of CNTs and Linker on Biosensor for Detection of Bacillus subtilis Min-Sang YOO1, Sung Bong KIM2, Dong Sup KIM2, Seung Wook KIM2, Jinyoung LEE*3, Chulhwan PARK*1 1Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea, 2Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea, 3Department of Plant and Food Science, Sangmyung University, Cheonan 330-720, Republic of Korea Biosensors using nanotechnolgy (NT) have been attractive for many research and industrial applications, such as biomedicine, environmental monitoring and food science. Carbon nanotubes (CNTs) have the structures with formations of rolling hexagonal arrangements of carbon atoms and result in superior characteristics with good electric conductivity. CNTs can be used in various devices of nanoscale for electrical equipment. In this study, we developed CNT-based nanobiosensor for detection of living cells (Bacillus subtilis KCCM 11316) which is available to be present in environmental dust. Anti- Bacillus subtilis antibody was used as a receptor to detect the bacteria. The antibody was immobilized using non-covalent bonding using pyrenebutanoic acid, succinimidyl ester (PBSE) linker. The electrical response (resistance) was obtained at the different conditions of CNTs, linker, and antibody. B. subtilis was detected by electrochemical method with resistance response and scanning electron microscope (SEM) images. This study is expected to provide a blueprint for future research on biosensors with immobilization. Keywords : Carbon nanotubes, Bacillus subtilis, Biosensor, Biological dust References 1. S. Lee, B. Choi, S.-M. Yi, G.P. Ko, Characterization of microbial community during Asian dust events in Korea (2009), Sci. Total Environ, 407, 5308-5314. 2. D. Ivnitski, I. Abdel-Hamid, P. Atanasov, E. Wilkins, Biosensors for detection of pathogenic bacteria (1999), Biosens. Bioelectron., 14, 599-624. 3. G. A. Campbell, R. Mutharasan, Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300 spores/ml (2006), Biosens. Bioelectron, 21, 1684-1692. P0324 Verification of Xylose Reductase Activities from 4 Kluyveromyces marxianus Strains Jong-Seok KIM, Jae-Bum PARK, Jin-Seong KIM, Suk-Jin HA Department of Bioengineering and Technology, Kangwon National University, Chuncheon 200-701, Republic of Korea Xylitol has non-calciogenic nature and low calorie content. Kluyveromyces marxianus has the capability of producing small amount of xylitol from xylose. In this study, xylitol producing capabilities by four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555) were verified. As results, xylitol producing capabilities were highly varied depending on strains. K. marxianus KCTC 7001 showed the highest xylitol production (13.18 g/L) with 47% of yield and 0.14 g/L·h of productivity. In order to determine the xylose reductase (XR) activity for xylitol production, NADH or NADPH-dependent XR activity was verified through enzymatic assay. K. marxianus KCTC 7001 showed high affinity to NADH and K. marxianus KCTC 7155 strain showed similar NADH and NADPH-dependent XR activity, whereas K. marxianus KCTC 17212 showed the lowest NADH or NADPH-dependent XR activity. K. marxianus KCTC 17555 showed high NADPH-dependent XR activity. Total XR activity of K. marxianus KCTC 7001 was higher than the other strains Keywords : Xylitol, Kluyveormyces marxianus, Xylose redutase, XR References 1. Jin-Seong Kim, Jae-Bum Park, Seung-Won JAng, Suk-Jin Ha, Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity, Applied Biochemistry and Biotechnology, 176, 1975-7984 2. Jae-Bum Park, Jin-Seong Kim, Seung-Won Jang, Eunsoo Hong, Suk-Jin Ha, The Application of Thermotolerant Yeast Kluyveromyces marxianus as a Potential Industrial Workhorse for Biofuel Production, KSBB journal, 2015, 30.3:125-131 P0325 Screening of 1-Ethyl-3-Methylimidazolium Acetate [EMIM][Ac] Resistance Yeast through a Directed Evolution. Yoo-Jin LEE, Jin-Seong KIM, Jae-Bum PARK, Suk-Jin HA Department of Bioengineering and Technology, Kangwon National University, Chuncheon 200-701, Republic of Korea Cellulosic biomass is a renewable resource for biofuel and high value-added biochemical production. In order to utilize cellulosic biomass efficiently, an optimum pretreatment process is required. Among various pretreatment processes, ionic liquid has been used recently. However, the toxicity of ionic liquid for microorganisms is a major huddle for applying on pretreatment. The purpose of this study was to achieve a strain raving resistance to ionic liquid through a directed evolution. In this study, 1-ethyl-3methylimidazolium acetate [EMIM][Ac] was used as a ionic liquid. At below 2% of [EMIM][Ac], two Saccharomyces cerevisiae (D452-2 and JAY289) and two Kluyveromyces marxianus (KCTC7155 and ACTT36907) were able to grow. When the concentration of [EMIM][Ac] was higher than 3%, four yeast strains were not able to grow at all. After a directed evolution, an evolved S. cerevisiae D452-2 consumed 71.86 g/L of glucose and produced 20.20 g/L of ethanol with 5% of [EMIM][Ac]. The evolved S. cerevisiae D452-2 might be used for biofuel production from [EMIM][Ac] pretreated cellulosic biomass. Keywords : Cellulosic biomass , pretreatment , ionic liquid , [EMIM][Ac], a directed evolution References 1. Irnayuli R. Sitepu1,2, Shuang Shi1, Blake A. Simmons3,4, Steven W. Singer3,5, Kyria Boundy-Mills1 &Christopher W. Simmons1,3, Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate, FEMS Yeast Research,Volume (14), 1286–1294 P0326 Screening of Microorganisms Able to Degrade Low-Rank Coal Isolated from Soil: Potential Coal Biosolubilization Mediators from Coal to Biochemicals Mary Grace BAYLON1, Yokimiko DAVID1, Sudheer DVN PAMIDIMARRI2, Kei-Anne BARITUGO1, Haejung KIM1, Cheolgi CHAE1, You Jin KIM1, Jeong Geol NA2, Park SI JAE1 1Department of Environmental Engineering and Energy, Myongji University, Yongin, Gyeonggido 449-728, 2Clean Fuel Department, Korea Institute of Energy Research, Yuseong-gu, Daejoen 305-343 Coal is a carbon-rich fossil fuel like petroleum. The discovery of bacteria capable of solubilizing coal and using it as a substrate would expand its application as a feedstock for chemical production. In this study, 32 bacterial strains isolated from soil enriched in MR medium containing coal were screened based on their growth in a medium with coal and ability to form colorless zones in RBBR plates. Shake flask cultivation were made for the selected isolates and the filtered supernatant from the cultures were analyzed by measuring its pH, absorbance (OD450) and COD; and detecting the presence of organic acids using HPLC. The filtered supernatants were also used to test if it can support the growth of E. coli without additional carbon source. Based on the results, the selected isolates were able to utilize and degrade coal. The filtered culture supernatants from the selected isolates were also found to be capable of supporting the growth of E. coli WL3110. This work was supported by the Energy Efficiency& Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea (No. 20132020000170). Keywords : Coal, coal biosolubilization, coal degradation, Biological refining References 1. Fakoussa, R. M., & Hofrichter, M., Biotechnology and microbiology of coal degradation, (1999), Applied Microbiology and Biotechnology, 52(1), 25–40. P0327 Screening of Microorganisms Able to Degrade Low-rank Coal in Aerobic Condition: Potential Coal Biosolubilization Mediators from Coal to Biochemicals Yokimiko DAVID1, Mary Grace BAYLON1, Sudheer DVN PAMIDIMARRI2, Kei-Anne BARITUGO1, Cheolgi CHAE1, You Jin KIM1, Haejung KIM1, Jeong Geol NA2, Si Jae PARK1 1Department of Environmental Engineering and Energy, Myongji University, Yongin, Gyeonggido, 449-728, 2Clean Fuel Department, Korea Institute of Energy Research, Daejoen, 305-343 The recalcitrant property of coal limits the use of such high carbon-containing raw material for the synthesis of various compounds. Chemical combustion, liquefaction and gasification are among the chemical treatments of coal which often leads to environmental hazards. An alternative As, biological degradation of coal using microorganisms becomes an ideal process for coal beneficiation. In this study, 46 bacteria were isolated from a coal-enriched sludge sample obtained from a full-scale anaerobic plant treating brewery wastewater. Of the pathogenicity of some isolated strains Because, bacteria coming from Genus: Cupriavidus, Pseudomonas and Arthrobacter were further examined for coal degradation. Coal solubilization products were analyzed in UV spectroscopy, HPLC, and GC-MS. Laccase-like activity were also found in some of the isolated strains. Microbial strains isolated from this study were found to degrade and convert coal into other useful compounds. This work was supported by the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea (No. 20132020000170). Keywords : Coal, Coal Biosolubilization, Coal Degradation, Biological refining References 1. Fakoussa, R. M., & Hofrichter, M., Biotechnology and microbiology of coal degradation (1999), Applied Microbiology and Biotechnology, 52(1), 25–40. P0328 Isolation of Cellulolytic and Lignolytic Bacteria from Soil and Their Characterization for Lignocellulosic Biomass Degradation Oh HYUN JU1, Gong GYEONGTAEK1, Cho SUKHYEONG1, Woo HAN MIN1, Oh MIN KYU2, Um YOUNGSOON1 1Korea Institute of Science and Technology , 2Department of Chemical & Biological Engineering, KOREA University, Seoul 136-713 Lignocellulosic biomass is an abundant resource for the sustainable production of high value chemicals. In this study, two potential cellulolytic and lignolytic bacteria, designated Enterobacter sp. 561 and Enterobacter sp. 641, were isolated from soil. The 16S rDNA sequences of Enterobacter sp. 561 and Enterobacter sp. 641 showed the highest similarity to that of E. mori R18-2 (98.65%) and E. asburiae JCM 6051(T) (98.97%), respectively. To investigate the ability of the isolates in degrading cellulose, two isolates were cultured in M9 plate containing 10 g/L caboxymethylcellulose, followed by staining with Congo red. After destaining, the plates showed clearing halo zones, indicating that two isolates were capable to degrade cellulose. Because bacterial dye-decolorizing peroxidase (Dyp) has attracted attention due to its potential in lignin degradation, the gene of DyP in two isolates were amplified based on the genome information of [u1] E. aerogenes KCTC 2190. When the recombinant DyP of two isolates (EDyP) was purified and used, EDyp showed a high ABTS oxidation activity (120 Umg-1). This result shows that the two isolates have a potential to degrade lignocellulsic biomass. Keywords : Enterobacter , dye-decolorizing peroxidase , lignocellulsic biomass References 1. Campos, E., et al. Microbiological research 169(2): 213-220. (2014). 2. Zhang, K., et al. Applied and environmental microbiology 80(8): 2592-2601. (2014). 3. Ruegg, T. L., et al. Nature communications 5. (2014). 4. Scully, E. D., et al. PLoS ONE 8(9): e73827. (2013). P0329 Isolation and Lipid Analysis of Chlamydomonas sp. KNM0029C from Arctic Sea Ice Eun Jae KIM1,2, Woongsic JUNG1, Suyoun LIM1, Sanghee KIM1, Han-Gu CHOI1, Se Jong HAN1,2 1Division of Life Sciences, Korea Polar Research Institute, KIOST, Incheon 406-840, Korea, 2Department of Polar Sciences, University of Science and Technology, Incheon 406-840, Korea Microalgae can efficiently utilize sunlight and carbon dioxide and store organic compounds which can be exploited for food and biofuels. The Arctic microalgae (KNM0029C) isolated from sea ice near Dasan Station in Ny-Ålesund was analyzed to evaluate the optimal growth condition and lipid contents. Arctic Chlamydomonas sp. KNM0029C has an optimal growth temperature of 4°C, reaching densities up to 1.4 × 107 cells/mL. Lipid body formation was visualized by BODIPY 505/515 staining and fluorescence microscopy. The fatty acid methyl ester (FAME) production level of KNM0029C was 178.6 mg/L culture and 2.3-fold higher than that of C. reinhardtii CC-125 at 4°C. Analysis of the FAME content showed predominance of polyunsaturated fatty acids such as C16:3, C18:2, C18:3 and C20:2. C18:3 fatty acids comprised the maximum portion (20.7%), and the polyunsaturated fatty acids content (39.6%) was higher than that of saturated fatty acids (6.8%) at 4°C. These results suggest that KNM0029C is promising feedstock for biodiesel and functional food production at low temperature. [The research was supported by a grant from iPET (PN14070) and KOPRI (PE15140)] Keywords : Arctic microalgae, biodiesel, fatty acids References 1. Y. Chisti, Biodiesel from microalgae. Biotechnol Adv 25 (3):294-306 (2007) P0330 Soil Extract Enhanced Mass Production of Biomass and Total Lipid of Marine Microalgal Species Geun Ho GIM1, Moon Jong KIM2, Sung Min PARK2, Si Wouk KIM1,2 1Department of Environmental Engineering, Chosun University, Gwangju 61-452, South Korea, 2Department of Energy Convergence, Chosun University, Gwangju 61-452, South Korea Effect of soil extract on the biomass and total lipid production of three marine microalgal strains, such as Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1 and Phaeodactylum tricornutum B128, were investigated. To make a soil extract, 100 g of specific soil sample was treated with 200 mL of distilled water and stand for 24 h, and then filtered and sterilized. Microalgal cells were cultured at 25℃ with a light intensity of 100 μmol/m2/s for 7 days. When cultured in a 1 L Erlenmeyer flask containing f/2 medium under photoautotrophic condition, the obtained biomass and total lipid concentration of three microalgal strains were as follows: 1.16 and 0.34 g/L for I. galbana LB987; 1.26 and 0.33 g/L for N. oculata CCAP849/1; and 1.19 and 0.54 g/L for P. tricornutum B128, respectively. However, the addition of soil extract to f/2 medium at a ratio of 4:6 increased the biomass and total lipid production as follows: 1.73 and 0.58 g/L for I. galbana LB987; 2.52 and 0.90 g/L for N. oculata CCAP849/1; and 2.08 and 1.12 g/L for P. tricornutum B128, respectively. Surprisingly, when the three microalgal cells were grown in a 10 L tubular photobioreactor under the same condition, the biomass and total lipid production increased 6-10 folds as follows: 11.58 and 4.06 g/L for I. galbana LB987; 9.66 and 2.92 g/L for N. oculata CCAP849/1; and 20.42 and 10.64 g/L for P. tricornutum B128, respectively. These results indicated that P. tricornutum B128 can be used as an alternative for biodiesel production when cultured in a large size photobioreactor containing some specific soil extract. Keywords : Marine Microalgae, Biomass, Total lipid, Soil extract References 1. A. Prakash, and M. A. Rashid, Influence of humic substances on the growth of marine phytoplankton: Dinoflagellates, Limnology Oceanography. 13, 598-606 (1968) P0331 Production and Identification of the Fusaricidin from Paenibacillus kribbensis CU01 Isolated from Yellow Loesses Jaewon RYU1, Moonjong KIM1, Si Wouk KIM1,2 1Department of Energy Convergence, Chosun University, Gwangju, 61-452 South of Korea, 2Department of Environmental Engineering, Chosun University, Gwangju, 61-452 South of Korea A novel bacterial strain was isolated from the yellow loesses in Haenam, Jellonam-do province, South Korea. On the basis of 16S rDNA sequence and biochemical tests, the isolate was named as Paenibacillus kribbensis CU01. Cells were cultured in a modified M9 medium with the addition of MnCl2. Culture broth showed strong antifungal activity against several phytopathogenic fungi such as Sclerotinia sclerotiorum KACC40457, Colletotrichum gloeosporidoides KACC40003, Phytophthora capsici KACC40157, Fusarium oxysporum KACC40031, and Botrytis cinerea KACC40574. This broth was centrifuged and the supernatant was treated with 100% butanol. Butanol extracted fraction was evaporated and then dissolved with 100% methanol. The solution was applied to HPLC and HPLC purified fractions having antifungal activity were analyzed by LC/MS. Major m/z values of 883 and 897 were observed from mass spectrum. Based on the results of MALDI-TOF/TOF MS, the antifungal substances was found to be LI-F type antibiotics (fusaricidin A and B) . Keywords : P. kribbensis, fusaricidin, phytopathogenic References 1. J. Vater, B. Niu, K. Dietel, R. Borriss, J. Am. Soc. Mass Spectrom. 26(9), 1548 (2015). P0332 Production of Lignols and Polyurethanes from Crude Glycerol Based Liquefaction of Empty Fruit Bunches Residues Ju-Hee LEE, Jae Yeong JUNG, Eun Yeol LEE Dept. of Chemical Engineering, Kyung Hee University, Gyeonggi-do, 446-701 The conversion of lignocellulosic biomass to biopolyols can be achieved by a liquefaction process, during which petrochemical polyhydric alcohols are generally used as liquefaction solvents. Lignin based polyols (lignols) can be used for production of polyester and polyurethane. Crude glycerol, a major by-product from the biodiesel production, can be used as a cost-effective solvothermal liquefaction solvent for the production of lignols and biopolyurethanes. The feasibility of using crude glycerol to liquefy empty fruit bunches residues for the production of lignols and biopolyurethanes was investigated in this study. The effects of various liquefaction parameters such as biomass loading, catalyst loading, liquefaction temperature, and liquefaction time on the lignols synthesis were investigated and optimized. The lignols obtained under optimal condition were used to synthesis the biopolyurethanes. Keywords : Liquefaction, Lignin, Polyol, Polyurethane References 1. Jo YJ, Ly HV, Kim J, Kim SS, Lee E, Preparation of biopolyol by liquefaction of palm kernel cake using PEG# 400 blended glycerol (2015), J IND ENG CHEM doi:10.1016/j.jiec.2015.04.010 2. Wang SK, Sung CSP, Fluorescence and IR characterization of cure in polyurea, polyurethane, and polyurethane- urea (2002), MACROMOLECULES, 35, 883-888 P0333 Bio-Conversion of C1-C3 Substrates to Value-added Products Using Methanotrophs Dong Hoon HUR, Eun Yeol LEE Dept. of Chemical engineering, Kyung Hee University, Gyeonggi-do, 446-701 C1-C3 substrates can be easily obtained from shale gas, natural gas and various byproducts of chemical processes. Methanotrophs can utilize various C1-C3 substrates as sole carbon source such as alkanes, alcohols and organic acids. In this work, we cultured methanotrophs supplying various C1-C3 substrates to produce value-added products such as lactate and fatty acids. Cell growth kinetics, substrate consumption, product yield and composition were investigated at different concentration of substrates and supplementation periods to increase productivity. As a result, C1-C3 substrates could be successfully converted into value-added products with high productivity. Our results show the potential of the utilization of C1-C3 substrates as feedstocks for bioprocess using methanotrophs as biocatalyst. Keywords : methanotroph, C1-C3 substrate, value-added product, lactate, fatty acid References 1. Hwang, In Yeub, et al, Batch Conversion of Methane to Methanol Using Methylosinus trichosporium OB3b as Biocatalyst (2015), Journal of microbiology and biotechnology, 25(3), 375-380 2. Crombie, Andrew T., and J. Colin Murrell, Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris (2014), Nature, 510(7503), 148-151 P0334 Cultivation of Parachlorella kessleri JD-076 Using Flat-panel Photobioreactor Eun Kyung KIM1, Gang-Guk CHOI2, Dae-Hyun CHO3, Ji-Won YANG2, Yong Keun CHANG1,2 1Advanced Biomass R&D Center, KAIST, Daejeon 305-701, 2Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon 305-701, 3Environmental Biotechnology Research Center, KRIBB, Daejeon 305-806 The microalgae are a very diverse group of photosynthetic organisms that have attracted global attention as feedstocks for biofuel production. In order to produce efficient algae based biofuels the optimization of culture condition is necessary. It has been recently proposed that mass and concentrated cultivation can be achieved through photobioreactor. In particular, flat panel PBR (fp-PBR) has been employed for enhancing the photosynthetic efficiency due to the fact that it allows algal cells to increase surface area for light penetration (Martin Gross, 2013). In addition, air bubbles delivered from bottom to top can lead sufficient mixing and gas exchange efficiencies. In this study, we investigated the optimal culture condition of cultivation of Parachlorella kessleri JD-076, a green unicellular alga in Chlorellaceae family and candidate of powerful biofuel producer (Přibyl et al., 2012). P. kessleri JD-076 was isolated from influent of sewage treatment plant in Daejeon, Korea. We carried out operation of 10-L scaled fp-PBR to find optimal culture condition against this strain. The maximum DCW was 17 g/L after 19 days of cultivation under 2 optimized conditions (800 uE/m /s light, 32 ℃ and 0.6 vvm with 3% of CO2). Futhermore, our cultivation results under continuous culture condition revealed that approximately 2~3 g/L of DCW was continuously maintained during 7 days after onset of continuos cultivation. These results suggest that the optimization of our results could be useful for mass cultivation of P. kessleri JD-076 in both Indoor/Outdoor culture systems. Keywords : biofuels , flat panel PBR, Parachlorella , mass cultivation References 1. Martin Gross, Development and optimization of algal cultivation systems, Iowa State University (2013) 2. Přibyl P, Cepák V, Zachleder V. Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris. Appl Microbiol Biotechnol 94:549–.561. (2012) P0335 Optimization of Biodiesel Production from Soybean and Rapeseed Oil under Supercritical Condition Hyoung-Min LEE1, Siti Fauziyah RAHMAN1, Seok-Hwan PARK1, Jae-Hee PARK2, Don-Hee PARK1,2 1Interdisciplinary Program of Graduate School for Bioenergy and Biomaterials,Chonnam National University, Gwangju, 500-757, Republic of Korea, 2Department of Biotechnology & Bioengineering, Chonnam National University,Gwangju 500- 757, Republic of Korea The objective of this study was to find the optimum composition of soybean and rapeseed oil using methanol supercritical fluid. In last study, the FAME content was obtained 95.8wt% at temperature of 340℃, reaction time 10min, and oil to methanol molar ratio of 1:50 from palm oil. In same condition, FAME content was obtained 84.1wt% and 87.1wt% from soybean oil and rapeseed oil. In this study, biodiesel was obtained from soybean oil and rapeseed oil using process for non- catalytic production biodiesel under supercritical condition methanol fluid. The reaction was carried on the 300mL batch supercritical reactor system under the condition of temperature 260~340℃, pressure 300 bars, reaction time 10~60 min. The contents of fatty acid methyl ester were analyzed by gas chromatography. The FAME content was obtained 96.64wt% at temperature of 300℃, reaction time 40min, and oil to methanol molar ratio of 1:45 by soybean oil and 94.81wt% at temperature of 300℃, reaction time 30min, and oil to methanol molar ratio of 1:45 by rapeseed oil. In contrast, FAME yield was increased when the temperature of the reaction increased, but it was decreased in the temperature more than 300℃. In addition, FAME contents was increased when the reaction time increased, but reaction time was not significantly influenced the FAME contents. The temperature condition was affected the FAME contents more than the reaction time. Keywords : Supercritical fluid, Biodisel, FAME, Vegetable oil References 1. S-H Park, Development of biodiesel production and optimum process under supercritical fluid conditions (2013), Chonnam National University 2. Gobikrishnan S, J-H Park, S-H Park, N. Indrawan, S.F. Rahman, D-H Park, Sonication-assisted production of biodiesel using soybean oil and supercritical methanol (2013), Bioprocess Biosyst. Eng, 36, 705-712 P0336 Lipase-Catalyzed In Situ Transesterification for Biodiesel Production from Heterotrophic Microalgae, Aurantiochytrium sp. KRS101 Keon Hee KIM, Ok Kyung LEE, Eun Yeol LEE Dept. of Chemical Engineering, Kyung Hee University, Gyeonggi-do, 446-701 Heterotrophic microalgae, Aurantiochytrium sp. have much attention to as a potential feedstock for biodiesel production and omega-3 PUFA (polyunsaturated fatty acid). The lipid content of Aurantiochytrium sp. KRS101 was increased up to 40-50% (w/w) by controlling the culture conditions. The microalgal lipid from Aurantiochytrium sp. KRS101 contains a high level of palmitate, which can be used for biodiesel production. In this study, we developed a dimethyl carbonate (DMC)-based in situ transesterification for production of FAMEs from Aurantiochytrium sp. KRS101 in the presence of Novozyme 435. DMC was used as a lipid extraction solvent, an acyl acceptor in transesterification for FAMEs synthesis. The effects of the microalgae biomass to DMC ratio, water content, microalgae biomass to enzyme ratio, and reaction temperature on the yields of FAMEs were analyzed and optimized. Keywords : heterotrophic microalgae, Aurantiochytrium sp. KRS101, in-situ transesterification, dimethyl carbonate, biodiesel References 1. Y. J. Jo, O. K. Lee, E. Y. Lee, Dimethyl carbonate-mediated lipid extraction and lipase-catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters and glycerol carbonate from Chlorella sp. KR-1 biomass (2014), Biores. Technol. 158, 105-110. 2. K. Kim, E. J. Kim, B. G. Ryu, S. Park, Y. E. Choi, J. W. Yang, A novel fed-batch process based on the biology of Aurantiochytrium sp. KRS101 for the production of biodiesel and docosahexaenoic acid (2013), Biores. Technol. 135, 269-274. P0337 Solvothermal Liquefaction of Red Pine Biomass by PEG Blended Crude Glycerol for Production of Polyols and Biopolyurethane Yumi LEE, Eun Yeol LEE Dept. of Chemical Engineering, Kyung Hee University, Gyeonggi-do, 446-701 Red pine is one of the most abundant softwood biomass in the world. Lignocellulosic biomass can be used as polyol for polyurethane preparation since they contain natural polymers with more than two hydroxyl groups per molecule. In this study, red pine was solvothermally liquefied using PEG-400 blended crude glycerol as a liquefaction solvent since it is economical and environment friendly polyol and has also high liquefaction rate. The effect of different liquefaction conditions such as solvent blending rate, biomass loading, sulfuric acid loading, temperature and time on polyol production was optimized. The biopolyol produced at the optimal condition was used to sythesize biopolyurethane. Biopolyurethane were synthesized by polyol and toluene diisocyanate (TDI). The polyols and biopolyurethane were analyzed for their structural characteristics using Fourier transform infrared (FT-IR). Keywords : red pine, PEG blend crude glycerol, polyol, biopolyurethane References 1. Y. J. Jo, H. V. Ly, J. Kim, S. S. Kim, and E. Y. Lee, Preparation of biopolyol by liquefaction of palm kernel cake using PEG#400 blended glycerol (2015), J. Ind. Eng. Chem. 29, 304-313. 2. S. Hu, X. Luo, and Y. Li, Polyols and polyurethanes from the liquefaction of lignocellulosic biomass (2014), ChemSusChem, 7, 66-72. P0338 Increasing Biomass and Lipid Productivity of Nannochloropsis salina by Overexpression of a bHLH Transcription Factor Nam Kyu KANG1, Seungjib JEON1, Sohee KWON1, Hyun Gi KOH1, Sung-Eun SHIN1, Bongsoo LEE1, Gang-Guk CHOI1, Ji-Won YANG1,2, Yong Keun CHANG1,2, Byeong-ryool JEONG1 1Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea, 2Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Microalgae have been considered a promising feedstock for biofuels. Nannochloropsis, marine microalga, has many advantages such as fast growth rate, high lipid content, and availability of genomic information. It is important to make economically feasible microalgae showing high lipid productivity by genetic manipulation [1]. There has been growing interest in engineering of transcription factors (TFs) which can regulate multiple metabolic enzymes [2]. We characterized two basic helix-loop-helix (bHLH) TFs in Nannochloropsis salina which are known as a stress regulator in plant. Among them, we overexpressed NsbHLH2 in N. salina and cultivated NsbHLH2 overexpressing transformants under normal and stress conditions (N limitation and osmotic stress). The qRT-PCR results indicated transformants highly expressed NsbHLH2 mRNA, and expression of NsbHLH2 was reduced under stress conditions. Overexpression of NsbHLH2 caused increase of growth rate, especially at early exponential phase, resulting in high biomass and fatty acid methyl ester (FAME) at day 8 under normal and stress conditions. Taken together, overexpression of NsbHLH2 can be used for production of microalgal based biodiesel. Keywords : Microalgae, Nannochloropsis salina, biodiesel, basic helix-loop-helix transcription factor, fatty acid methyl ester References 1. Radakovits, R., R.E. Jinkerson, A. Darzins, and M.C. Posewitz, Eukaryot. Cell 9, 486-501. (2010) 2. Courchesne, N.M.D., A. Parisien, B. Wang, and C.Q. Lan, J. Biotechnol. 141, 31-41 (2009). P0339 Effect of Maximum Power Point Tracking (MPPT) and Fixed Load Resistance Operation with Flat-plate Microbial Fuel Cell (FPMFC) Young Eun SONG1, Byong-Hun JEON2, Jung Rae KIM1 1School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea, 609-735, 2Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul, 133-791 Microbial fuel cells (MFCs) employ exoelectrogenic bacteria to generate electricity from wastewater and various organic contaminant. However, the low power and voltage have been drawback for scale-up and field application. In this study, we developed a logic based control algorithm, maximum power point tracking (MPPT) using LabVIEW™ and a circuit box for flat-plate MFC (FPM). The effects of operating parameters such as organic load rate (OLR), hydraulic retention time (HRT) and sampling-load shifting time have been extensively investigated. The results show that MPPT operation clearly improve power production and organic removal of FPMFC. These results will provide a strategy of MFC field scale application and consider the critical information for high power generation and sustainable bioenergy recovery with simultaneous wastewater treatment. Keywords : Microbial fuel cell, maximum power point tracking, OLR, HRT, FPMFC References 1. H. C. Boghani, J. R. Kim, R. M. Dinsdale, A. J. Guwy, and G. C. Premier, "Control of power sourced from a microbial fuel cell reduces its start-up time and increases bioelectrochemical activity," Bioresource Technology, vol. 140, pp. 277-285, 7 (2013) 2. G. C. Premier, J. R. Kim, I. Michie, R. M. Dinsdale, and A. J. Guwy, "Automatic control of load increases power and efficiency in a microbial fuel cell," Journal of Power Sources, vol. 196, pp. 2013-2019 (2011) 3. G. S. Jadhav and M. M. Ghangrekar, "Performance of microbial fuel cell subjected to variation in pH, temperature, external load and substrate concentration," Bioresource Technology, vol. 100, pp. 717-723, 1 (2009.) P0340 Metabolic Flux Analysis of K.pneumoniae L17 during Electricity Production from Glucose in Microbial Fuel Cell Changman KIM1, Satish Kumar AINALA1, Byong-Hun JEON2, Sunghoon PARK1, Jung Rae KIM1 1School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea, 609-735, 2Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul, 133-791 Microbial fuel cells (MFCs) are electricity generation systems which are using electron transfer mechanisms from microbe to electrode. However little is reported about intracellular metabolism of exoelectrogen during electron excretion in MFCs. In this study, we investigate the change of metabolites production of Klebsiella pneumoniae L17 by the activated electron transfer mechanism in MFC derived fermentation. Also stoichiometric analysis was performed for estimation of intracellular metabolic fluxes. Metabolites production results suggested significantly less production of reductive metabolites in MFC fermentation compared to that in non-MFC fermentation. Metabolic flux analysis also showed the intracellular metabolic flux changes in between MFC fermentation and non-MFC fermentation. The results indicated that MFC could be applied to anaerobic fermentation as a tool of disposing electron produced by respiration. Keywords : Microbial fuel cell, metabolic flux analysis, Klebsiella pneumoniae References 1. Zhang et al., Microbial fuel cell based on Klebsiella pneumoniae biofilm (2008), Electrochemistry Communications, 10, 1641–1643 2. LiFang et al., A study of electron-shuttle mechanism in Klebsiella pneumoniae based-microbial fuel cells (2010), Chinese Science Bulletin, 55, 99–104 3. Xia et al., Electricity generation from glucose by a Klebsiella sp. in microbial fuel cells (2010), Appl Microbiol Biotechnol 87, 383–390 P0341 Feasibility Test on Using of Bulking Agents for Sawdust Substitution in Composting of Dairy Cow Manure Jung Kon KIM, Kwang-Hwa JEONG, Won-Mo CHO, Dong Jun LEE, Jung-Hoon KWAG, Dong-Yoon CHOI Animal Environment Division, National Institute of Animal Science, Rural Development Administration, Wanju-Gun, Jeollabuk-Do, 565-851, South Korea The feasibility on using of bulking agents for sawdust substitution in composting of dairy cow manure (DCM) was investigated. Two bulking agents for sawdust (S) substitution such as decomposed manure (DM) and miscanthus (M) were used in this study. The composting effects in accordance with the volumetric mixing ratio of DCM and bulking agents (DCM only, DCM:DM=1:1, DCM:DM=1.5:0.5, DCM:DM=0.5:1.5, DCM:DM:S=1:0.5:0.5, DCM:S=1:1, DCM:M=1:1, DCM:M=1.5:0.5, DCM:M=0.5:1.5, DCM:M:S=1:0.5:0.5, DCM:S=1:1 and DCM:S=1.5:0.5) were investigated. Among the several volumetric mixing ratio conditions, DCM:DM:S=1:0.5:0.5 was shown the highest temperature of the compost as a 66℃ during composting period. When the miscanthus was used as bulking agent, DCM:M=1.5:0.5 was shown the highest temperature of the compost as a 58℃ during composting period. After 5 weeks composting, moisture contents of most experimental composting reactors using DM as bulking agent were less than 50%. On the other hand, in case of using of miscanthus as bulking agent, moisture content of compost was higher than one of decomposed manure. However, both of bulking agents can be used as bulking agents for sawdust substitution when it is used in mixture with sawdust. The optimum volumetric mixing ratio of dairy cow manure and bulking agent was 50% of livestock manure, 25% of decomposed manure or miscanthus and 25% of sawdust. Keywords : Bulking agents, composting, decomposted manure, miscanthus References 1. Z. H. Hu, Y. L. Liu, G. W. Chen, X. Y. Gui, T. H. Chen, X. M. Zhan, Characterization of organic matter degradation during composting of manure–straw mixtures spiked with tetracyclines (2011), Bioresource Technol. 102, 7329–7334. 2. X. Qian, G. Shen, Z. Wang, C. Guo, Y. Liu, Z. Lei, Z. Zhang, Co-composting of livestock manure with rice straw: Characterization and establishment of maturity evaluation system (2014), Waste Manage. 34, 530-535. P0342 Characterization of Methane Oxidizing Bacteria Isolated from Landfill Young Chan JEON, Eun Yeol LEE Dept. of Chemical engineering, Kyung Hee university, Gyeonggi-do, 446-701 Methane oxidizing bacteria (MOB) have been found in various habitats where methane and oxygen are readily available. We isolated methane oxidizing bacteria from landfill and characterized the strain using various techniques such as phylogenetic, morphologic and chemotaxonomic analysis. The culture conditions such as the gas supplement, temperature and concentration of copper ion in the medium were optimized to enhance the growth rate. Based on these results, cell growth kinetics and methane consumption at optimal culture condition were investigated. Furthermore, this isolate was cultured with the addition of various organic substances for the high cell density cultivation. In addition, this isolated MOB was feasible to produce methanol from methane in our experimental condition. Keywords : methane oxidizing bacteria, methane, high cell density culivation, methanol References 1. Kim, Hee Gon, et al., Isolation and taxonomic characterization of a novel type I methanotrophic bacterium (2008), The Journal of Microbiology, 46(1), 45-50 P0343 Pretreatment of Corn Stover Using Direct Applied Ammonia-Mist (DAAM) Method VI NGUYEN PHUONG TRUONG Dept. of Environmental Engineering, Kongju National University, Cheonan, 331-717 Pretreatment is one of the most important steps for effective bioconversion of lignocellulosic biomass into biofuels. Previous study showed that plant cell walls have a natural resistance to chemical, physical and enzymatic degradations [1]. Therefore, an efficient pretreatment process to open up rigid structure of lignocellulosic biomass is necessary. Pretreatment method using ammonia mist was investigated to enhance the enzymatic digestibility of corn stover. In this process, aqueous ammonia was applied to corn stover in a form of mist. For effective pretreatment, ammonia-mist is applied to corn stover and then it was mixed in the drum mixer for uniform reaction. In the first step, ammonium hydroxide was directly sprayed to the corn stover based on a target solid content and ammonia loading. The biomass with aqueous ammonia was treated at elevated temperature from 60oC to 150oC for an extended period of time. Effects on biomass composition and enzymatic digestibility were tested in this paper. It is speculated that pretreatment method using aqueous ammonia-mist reduces chemical and water inputs, which can produce fermentable sugars at low cost. Keywords : pretreatment, corn stover, ammonia, enzyme hydrolysis References 1. C. E. Wyman (Ed.), Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals. Ch. 3. 24-36. (2013) P0344 Development of Potential Control Strategy of Cathodic Biofilm for Intermediate Chemical Production Chae Ho IM1, Ganapathiraman MUNUSSAMI1, Young Eun SONG1, Byong-Hun JEON2, Jung Rae KIM1 1School of Polymer science and Chemical Engineering, Pusan National University University,, 2Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul, 133-791 Microbial electrosynthesis is alternative way for intermediate chemicals and biofuels production with electroactive microorganism using bioelectrochemical system. Reductive reaction on the cathode electrode is catalyzed by electroactive autotrophic bacteria and biofilm. CO2 serves as sole carbon source for microbial growth and chemical production in bioelectrochemical system. However, biofilm formation on cathode for electrosynthesis is rate limiting process which delays enrichment and decrease performance. In this study, we demonstrate that the regulate potential to electrode can promote the biofilm establishment on biocathode. Acetate production by electrosynthesis was shown with using continuous CO2 supply. This study will contribute to develop novel platform chemical production pathway from greenhouse gas, CO2 by using bioelectrosynthesis Keywords : CO2, electrosynthesis, bioproducts References 1. K.P. Nevin, T.L. Woodard, A.E. Franks, Z.M. Summers and D.R. Lovely, Mbio. 1, 1 (2010) 2. K.P. Nevin, S.A. Hensley, A.E. Franks, Z.M. Summers, J. Ou, T.L. Woodard, O.L Snoeyenbos-West and D.R. Lovely, Appl Environ Microbiol, 77, 2882 (2011) 3. C.W. Marshall, D.E. Ross, E.B. Fichot, R.S. Norman and H.D. May, Appl Environ Microbiol, 78, 8412 (2012) P0345 Metabolic Engineering of Shewanella oneidensis MR-1: A Novel Method of Platform Chemical Production from Glycerol in an Electroactive Bacterium Ganapathiraman MUNUSSAMI1, Somasundar ASHOK1, Young Eun SONG1, Byong-Hun JEON2, Sun-Gu LEE1, Sunghoon PARK1, Jung Rae KIM1 1School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 609-735 , 22Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul, 133-791 Biological production of platform chemicals from sustainable feedstock has been the central interest for alternative conventional chemical refinery process. Yet the biological production process is limited by various factors, among them is the internal redox imbalance, which is caused due to undisposed electrons, leading to high byproduct production, less conversion yield and eventually increase the final purification cost of process. These factors motivated us to create a novel biorefinery system, where we demonstrate the controlling of intracellular NADH/NAD+ redox balance using chemical mediators and Terminal electron acceptors (TEA). Recently, Shewanella oneidensis MR-1, an electrochemically active bacterium showed promising results to regulate redox imbalance complications without either using expensive and non- sustainable electron acceptors or by producing undesirable byproducts. S. oneidensis MR-1 can intrinsically transfer electrons directly to electrodes through a process called extracellular electron transfer (EET) there by providing a promising approach to control internal redox status of the cells. In this study, we demonstrate the biological production of 3-HP by a recombinant S. oneidensis MR-1dhaB gdrAB KGSADH strain grown in a conical flask as a batch culture with chemical mediators and TEA. The production of 3-HP proved a promising aspect to proceed further with Electrochemical Bioreactor Array (EBA). Here, we showed the recombinant S. oneidensis MR-1 dhaB gdrAB KGSADH as a potential electrode interfaced biorefinery system to produce 3-hydroxypropionic acid (3-HP) from glycerol. Keywords : bioelectrochemcial system, biorefinery, platform chemicals References 1. Flynn, J. M., D. E. Ross, K. A. Hunt, D. R. Bond, and J. A. Gralnick. Enabling unbalanced fermentations by using engineered electrode-interfaced bacteria. 2010. mBio. 2. S, Ashok, Subramanian M. Raj, C, Rathnasingh, S, Park. Development of recombinant Klebsiella pneumoniae ∆dhaT strain for the co-production of 3-hydroxypropionic acid and 1, 3-propanediol from glycerol. Applied Microbiology and Biotechnology 2011 P0346 The Yield of Simultaneous Saccharification and Fermentation (SSF) Using Pretreated Miscanthus with Recycled Black Liquor Sun-Il SEO, Young-Lok CHA, Hyo-Yeon CHOI, Gyeong-Dan YU, Ji-Eun LEE, Youn-Ho MOON, Gi Hong AN, Jong- Woong AHN, Kyeong-Bo LEE Bioenergy Crop Research Institute, National Institute of Crop Science, RDA, Jeonnam, 534-833 The goal of effective pretreatment is to increase the enzyme digestibility of lignocellulosic biomass. In this study, the continuous pretreatment using an alkaline solvent was conducted to extract sugar from Miscanthus biomass. One of the important conditions in this pretreatment was 1:6 of solid (Miscanthus biomass) and liquid (NaOH solvent) ratio. After the pretreatment, biomass was separated into solid phase and liquid phase (black liquor) by a screw type separator. The objective of this study was to analyze the yield of simultaneous saccharification and fermentation (SSF) of pretreated Miscanthus with recycled black liquor. The condition of solvent for pretreatment was 0.6 M NaOH. For the recycling of solvent, the primary black liquor was reused in second pretreatment. The pretreated solid with 8% of cellulose was used for SSF, which was adjusted to pH 5.0 with 1 M H2SO4. SSF was conducted with 30 FPU/g-cellulose by jar-fermenter (5L) at 33℃ for 72h. As a result, the ethanol concentration by the first pretreated biomass was 33.45 g/L, showing that the yield of fermentation was 90.73%. Ethanol concentrations by second and third pretreated biomass were 29.10 g/L and 24.01 g/L, respectively. And their yields of fermentation were 83.93% and 84.26%, respectively. Keywords : Miscanthus, pretreatment, black liquor, SSF References 1. Ko, J. K., Bak, J. S., Jung, M. W., Lee, H. J., Choi, I. G., Kim, T. H., & Kim, K. H. Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes. Bioresource Technology 100.19 (2009): 4374-4380. P0347 Detoxification of Lignocellulosic Derivatives Using Laccase Immobilized on Bacterial Cellulosic Nanofibers Thiyagarajan SARAVANAKUMAR1, Han-Sung PARK1, Ae-Young MO1, Myoung-Suk CHOI2, Dae-Hyuk KIM2, Seung- Moon PARK1 11Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570– 752, Korea, 2Institute of Molecular Biology and Genetics, College of Natural Sciences, Chonbuk National University, Jeonju, Jeonbuk 561-756, Republic of Korea Biotransformation of lignocellulose by microbial fermentation is usually preceded by thermo-chemical pretreatments followed by enzymatic hydrolysis of cellulose. Derivatives formed during the pretreatment of the lignocellulosic biomass inhibit enzymatic hydrolysis as well as microbial fermentation. Most derivatives are either furan or phenolic. In the present study, laccase was used to detoxify three different types of lignocellulosic derivatives that are highly toxic to microbial fermentation due to their low hydrophilic nature, namely furfural, acetosyringone, and coniferyl aldehyde. A minimal inhibitory concentration (MIC) test was carried out with Saccharomyces cerevisiae. The MIC of furfural, acetosyringone, and coniferyl aldehyde was 12 mM, 24 mM, and 1.5 mM, respectively. Laccase was immobilized on cellulose nanofiber produced by bacteria. Field emission scanning electron microscopy (FESEM), fourier transform infrared spectroscopy (FTIR) analysis, enzyme activity assay, and protein estimation clearly demonstrate the binding efficiency of laccase on the nanofiber. Immobilized laccase showed a better pH and thermal stability than free laccase. The three derivatives were used at a concentration of 25 mM for detoxification studies. Immobilized laccase completely degraded the derivatives after 36 h of incubation at 40 ºC. Finally, the degradation was confirmed by ultraviolet-visible spectroscopy (UV–VIS) spectra and high-performance liquid chromatography. It was found that the effect of degradation depends on the structural variation of the lignocellulosic derivatives as laccase alone detoxified the furfural and coniferyl aldehyde, whereas a redox mediator HOBt was needed for the detoxification of ketone based lignin derivative acetosyringone. Keywords : Lignocellulosic inhibitory derivatives, bacterial cellulose, laccase, immobilization, detoxification References 1. Olsson L, Hahn-Hagerdal B. 1996. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 18:312–331. 2. Sun Y, Cheng J. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83: 1–11 P0348 In Situ Biphasic Extractive Fermentation for Hexanoic Acid Production using Jerusalem Artichoke Tubers by Megasphaera elsdenii SU1 Hyunjin KIM1, Byoung Seung JEON1, Tae Ho LEE2, Byoung-In SANG1 1Department of Chemical Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea, 2The Research Institute of Industrial Science, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea Hexanoic acid is an attractive biorenewable chemical, due in part to its potential to be catalytically converted to a variety of industrial chemicals and fuels. Though several investigations on the biological production of hexanoic acid under anaerobic condition have been attempted, there is no report on commercial hexanoic acid production with the utilization of carbon sources with reasonable costs. Jerusalem artichoke (JA) has recently been recognized as a promising biomass for bioeconomy development, with a number of advantages over conventional crops. JA tuber is a rich source of carbohydrates, where 70-90% of them are inulin which could be hydrolyzed by enzymes or acids to generate fructose and glucose. These JA-derived monosaccharides are excellent carbon sources for the microbial production of various chemicals, such as ethanol, 2,3-butandiol, and lactic acid. In this study, JA hydrolysate obtained by dilute acid treatment was used as a carbon source for the production of hexanoic acid by Megasphaera elsdenii SU1, an obligately anaerobic bacterium. To avoid the toxicity of the produced carboxylic acids to the microbial biocatalyst, in situ biphasic extractive fermentation was conducted. High hexanoic acid production of 13 g l-1 and productivity of 0.5 g l-1 h-1 was achieved from the biphasic extractive fermentation with JA hydrolysate used as a carbon source for hexanoic acid production by M. elsdenii SU1. Keywords : Hexanoic acid, Jerusalem artichoke, Megasphaera elsdenii, Extractive fermentation, Biochemical References 1. Ge, X.-Y., et al. (2009). "Improvement of l-lactic acid production from Jerusalem artichoke tubers by mixed culture of Aspergillus niger and Lactobacillus sp." Bioresour Technol 100(5): 1872-1874. P0349 Peracetic Acid Mediated Rapid Degradation of Thiazine Dye Azure B and Azo Dye Reactive Orange 16 through Free Radical Reaction Mechanism Thiyagarajan SARAVANAKUMAR1, Han-Sung PARK1, Ae-Young MO1, Myoung-Suk CHOI2, Dae-Hyuk KIM2, Seung- Moon PARK1 1Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570– 752, Korea, 2Institute of Molecular Biology and Genetics, College of Natural Sciences, Chonbuk National University, Jeonju, Jeonbuk 561-756, Republic of Korea Peracetic acid (PAA) is a powerful oxidant for the degradation of lignin and aromatic ring compounds. In the present study, it was used for the degradation of thiazine based dye azure B (AZB) which is also primarily used as a substrate for lignin peroxidase, and the azo dye reactive orange 16 (RO16). It was found that 5 mM PAA was able to decolorize about 76% of 100 mg/L AZB and about 66 % of RO16, which could not be effectively decolorized by fungal phenol oxidases laccase or manganese peroxidase. PAA mediated degradation was catalyzed using simple salt MnSO4. Both the dyes were completely decolorized when MnSO4 was used with 5 mM PAA and the optimum concentration of MnSO4 was 2 mM. High performance liquid chromatography (HPLC) and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) analyses revealed the degradation of AZB and RO16. Free radicals formed by PAA with MnSO4 demethylate the AZB and finally cleave the aromatic ring by desulfonation, whereas in RO16, they cleave the azo bond first, followed by the desulfonation and demethylation, which finally leads to degradation. Keywords : Peracetic acid, Azure B, Reactive orange 16, Manganese (II) sulfate References 1. Aguiar, A., & Ferraz, A. (2007). Fe3+- and Cu2+- reduction by phenol derivatives associated with Azure B degradation in Fenton-like reactions. Chemosphere, 66, 947-954. 2. Houas, A., Lachheb, H., Ksibi, M., Elaloui, E., Guillard, C., & Herrmann, J.M. (2001). Photocatalytic degradation pathway of methylene blue in water. Applied Catalysis B: Environmental, 31, 145-157. P0350 Development of Microalgal Cultivation Process for Simultaneous Induction of Biomass and Lipid Accumulation Min Gyu SUNG1, Kyung Hwan RYU1, Kibok NAM1, Jay H. LEE1, Yong Keun CHANG1,2 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, 2Advanced Biomass R&D Center, Daejeon, 305-701 Microalgae are having attention for source of biofuel. They are introduced as 3rd generation biofuel, which uses sunlight and water to generate oil. Microalgae-derived oil is highly related to stress condition. At stress conditions such as nutrient limitation, microalgae accumulate massive amount of lipid to save energy. This energy, however, is originated from one for growth. [1] This inherent characteristics result in low lipid productivity, which is directly related to biodiesel production. Wide range of researches try to solve the problem. In cultivation stage, two-step cultivation approach shows impressive advances [2]. It, however, requires additional step so that overall cost of production increases. In this study, a novel cultivation strategy motivated from two-stage culture concept was developed. Four flat-panel photobioreactors are connected in series. Fresh medium is supplied to first reactor, and the system is operated chemostat. Natural gradient of cell concentration occurs along reactors due to different retention time. It was verified that concentrated Nannochloropsis gaditana biomass accumulates lipid at final reactors due to nitrogen starvation. Overall environment at each reactor is described by a simple mathematical model which describes growth and lipid accumulation. Keywords : Microalgae, Cultivation, Photobioreactor, Modeling References 1. Borowitzka, Algal biotechnology products and processes - matching science and economics (1992), Journal of Applied Phycology, 4(3), 267-279 2. Su et al., Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process (2011), Journal of Applied Phycology, 23 (5), 903-908 P0351 Transcriptomic Analysis of Endangered Freshwater Mussel Cristaria plicata Provides Valuable Resource for Species Conservation Bharat Bhusan PATNAIK1,2, Tae Hun WANG1, Se Won KANG1, Hee Ju HWANG1, So Young PARK1, Eun Bi PARK1, Jong Min CHUNG1, Dae Kwon SONG1, Changmu KIM3, Soonok KIM3, Jun Sang LEE4, Yeon Soo HAN5, Hong Seog PARK6, Yong Seok LEE1 1Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan, Chungchungnam-do, Republic of Korea, 2Trident School of Biotech Sciences, Trident Academy of Creative Technology (TACT), Bhubaneswar- 751024, Odisha, India, 3National Institute of Biological Resources, Incheon 404-170, Korea, 4Institute of Environmental Research, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do 200-701, Korea, 5College of Agriculture and Life Science, Chonnam National University, 300 Yongbong-Dong, Buk-gu, Gwangju, Republic of Korea, 6Research Institute, GnC BIO Co., LTD., 621-6 Banseok-dong, Yuseong-gu, Daejeon, 305-150, Republic of Korea The freshwater mussel Cristaria plicata (Bivalvia: Eulamellibranchia: Unionidae) has been assessed as endangered by the Korean Red List of Threatened Species and Data deficient by International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened species. The number of individuals been dwindling in recent times, due to indiscriminate collection of specimens and loss of natural habitats. In order to understand the strategic indices for the conservation of the species, we conducted de novo transcriptome sequencing, assembly, and annotation analysis using Illumina HiSeq2000 next-generation sequencing (NGS) technology, Trinity assembler, and BLAST2GO analysis, respectively. We obtained 98.31% of high-quality reads from a total of 286,152,584 raw read sequences. The assembly generated a total of 453,931 contigs having a mean length of 731.2 and N50 length of 1,254. The contig sequences were clustered to 374,794 unigenes with a mean length of 737.1 and N50 length of 1,262. A 100% coverage of C. plicata mitochondrial genes within two unigenes validated the quality of the assembler. The BLAST top-hit distribution of unigenes against SCHDB (79,960 hits) showed maximum homology to other molluscs. The NCBI-KOG annotation showed the maximum preference of the transcriptome towards Cellular Processes and Signaling mechanisms with a total of 4,916 belonging to the signal transduction mechanism category. BLAST2GO analysis was helpful to decipher the putative genes related to immunity and reproduction that would be beneficial for protection of the population. Keywords : Cristaria plicata, Transcriptome, Endangered species References 1. Dong ZG, Li L.J. Biodiversity and conservation of freshwater mollusks. Acta Hydrobiologica Sinica. 2004; 4: 440-444. P0352 Expressed Sequence Tags from the Threatened Freshwater Sphaeriid Clam Species, Pisidium (Neopisidium) coreanum: Discovery of Functional Genomic Resources Ji Eun JEONG2, Se Won KANG1, Hee Ju HWANG1, So Young PARK1, Bharat Bhusan PATNAIK1,3, Changmu KIM4, Soonok KIM4, Jae Bong LEE5, Tae Hun WANG1, Eun Bi PARK1, Sun Shin YI6, Yeon Soo HAN7, Jun Sang LEE8, Hong Seog PARK9, Yong Seok LEE1 1Department of Life Science and Biotechnology, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan city, Chungcheongnam-do 336-745, Korea., 2Department of Applied Biology, College of Natural Resources and Life Science, Dong-A University, 550-37 Nakdongdaero, Sahagu, Busan 604-714, Korea, 3School of Biotech Sciences, Trident Academy of Creative Technology, Bhubaneswar 751024, Odisha, India, 4National Institute of Biological Resources, Incheon 404-170, Korea, 5Korea Zoonosis Research Institute, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 561-756, Korea, 6Department of Biomedical Laboratory Science, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan city, Chungcheongnam-do 336-745, Korea., 7College of Agriculture and Life Science, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, Korea, 8Institute of Environmental Research, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do 200-701, Korea, 9Research Institute, GnC BIO Co., LTD., 621-6 Banseok-dong, Yuseong-gu, Daejeon, 305-150, Republic of Korea The freshwater Sphaeriid clam, Pisidium (Neopisidium) coreanum is endemic to Korea and there exist no conservation measures. The species has found relevance to biomedical applications due to its use in bone fracture treatment research and therapeutics development. Having said that, there has been no studies to understand the expressed sequence profiles from the species that would be helpful for cataloging the genetic information in the rich interest of ecologists and medical scientists alike. A study was undertaken to study the expressed sequence tags (ESTs) from the clam species by construction of cDNA library and traditional DNA sequencing. After quality assessment of the sequence reads the assembled EST clusters formed 791 contigs and 521 singletons. Out of a total of 5,656 ESTs, 3,265 and 3,314 EST sequences showed homology (E- value of <1e-5) to matches in mollusks sequence database and the NCBI non-redundant database, respectively. About 1,674 sequences show homology to sequences in the NCBI-Eukaryotic Clusters of Orthologous Genes (NCBI-KOG) database. About 18.61% of ESTs matching the NCBI-KOG belonged to the translation, ribosome structure, and biogenesis related genes followed by energy production and conversion (13.96%), and post-translational modification genes (13.29%) category. Most significantly, we were able to identify 16 EST transcripts showing high homology with bone morphogenetic protein 2- B that could be a valuable resource for treatment of bone-fractures and have considerable biomedical interest. The information generated here can also be used by researchers in genetic improvement programme of this species apart from its conservation in its natural habitat. Keywords : Pisidium (Neopisidium) coreanum, expressed sequence tags, bone morphogenetic proteins References 1. Altschul AA, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410 P0353 Functionally-relevant Expressed Sequence Tags from the Biomarker Organism, Physa acuta Exposed to Cadmium Ji Eun JEONG2, Bharat Bhusan PATNAIK1,3, Se Won KANG1, Hee-Ju HWANG1, So Young PARK1, Tae Hun WANG1, Eun Bi PARK1, Je Bong LEE4, Myung-Mo NAM5, Yong Hun JO6, Yeon Soo HAN6, Jun-Sang LEE7, Hong Seog PARK8, Yong Seok LEE1 1Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan city, Chungchungnam-do, 336-745, Republic of Korea, 2Department of Applied Biology, College of Natural Resources and Life Science, Dong-A University, 550-37 Nakdongdaero, Sahagu, Busan 604-714, Republic of Korea, 3Trident School of Biotech Sciences, Trident Academy of Creative Technology (TACT), Bhubaneswar- 751024, Odisha, India, 4Korea Zoonosis Research Institute, Chonbuk National University, 567 Baekje-daero, deokjin-gu, Jeonju-si, Jeollabuk-do 561-756, Republic of Korea, 5East Sea Fisheries Research Institute, NFRDI, Gangneung 210-861, Republic of Korea, 6College of Agriculture and Life Science, Chonnam National University, 300 Yongbong-Dong, Buk-gu, Gwangju 500-757, Republic of Korea, 7Institute of Environmental Research, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do 200-701, Korea, 8Research Institute, GnC BIO Co., LTD., 621-6 Banseok- dong, Yuseong-gu, Daejeon, 305-150, Republic of Korea Biomarker organisms have been routinely exploited to assess the environmental contamination load due to toxic chemicals, pesticides, and heavy metals. For conventional biomonitoring of polluted water-bodies and an environmental impact assessment of the contaminants, the use of molluscs have found a wider acceptance due to the sedentary life style and filter- feeding habit of the specimens. Physa acuta is a freshwater pulmonate and a widely established bioindicator species that can dwell on anthropogenically harsh conditions with reports on environmental impact assessment in relation to the contamination of herbicides, heavy metal exposure, and immunity against microbial stressors. We generated an Expressed Sequence Tag (EST) analysis of P. acuta under an environmental exposure of Cadmium at 500ppb for a duration of 8 hrs. We identified a total of 1,108 high-quality reads under Cadmium exposure that were clustered to 730 unique sequences representing 114 contigs and 616 singletons. Annotation of the ESTs showed 722, 696, and 398 significant hits against NCBI nr, Mollusks, and KOG databases, respectively. Among the upregulated transcripts included the serpin B3, ink toxin 3, alcohol dehydrogenase-like isoform, and ubiquitin c isoform, whereas the downregulated transcripts included the superoxide dismutase, cathepsin, metallothionein, G-type lysozyme, and calcium binding protein-1. The results can be utilized for the development of a scientific resource technology for environmental impact assessment of heavy metals using P. acuta as a bioindicator organism in freshwater environment. Keywords : Physa acuta, Cadmium, Expressed Sequence Tag References 1. Amiard-Triquet C (2009) Behavioral disturbances: the missing link between sub-organismal and supra-organismal responses to stress? Prospects based on aquatic research. Hum Ecol Risk Assess 15: 87-110. P0354 Production of Biopolymer from an Organic Acid Mixture by Azotobacter vinelandii UWD in Fed-batch Culture Chang-Ho PARK1, Harald DANHAMER2 1Department of Chemical Engineering, Kyung Hee University, Yongin-si, 446-701, South Korea, 2Technische Universitat- Braunschweig, Germany (Department of Agricultural Engineering, University of Minnesota, U.S.A.) Poly(hydroxybutyrate) (PHB) was produced by fed-batch culture of Azotobacter vinelandii UWD (ATCC 53799) in a computer-controlled 7 liter fermentor using an organic acid mixture (3.75 g/L acetic acid, 1.0 g/L propionic acid, and 1.0 g/L butyric acid) as substrates. During fermentation process concentration of acetic acid consistently decreased from its initial value but for the other two acids concentration decreased after reaching maximum values. The maximum specific acetic acid consumption rates occurred at 2.6-2.8 g/L for acetic acid, 2.6-3.3 g/L for propionic acid, and 3.7-4.2 g/L for butyric acid. After acetic acid concentration reached a low level (2.0-2.5 g/L), propionic acid was preferentially consumed to butyric acid. Upon exhaustion of acetic acid PHB production rate increased significantly from near zero value. Specific growth rate of residual biomass decreased significantly while propionic and butyric acids were utilized with concurrent decrease of dissolved oxygen concentration. A compound which was incorporated into the PHB was identified as poly-β-hydroxy-3- penten-1-acid by mass spectrometry. Keywords : PHB, Azotobacter vinelandii UWD, fed-batch culture, organic acids References 1. Ryu, H.-W., K.-S. Cho, P. R. Goodrich, and C.-H. Park (2008), Production of Polyhydroxyalkanoates by Azotobacter vinelandii UWD Using Swine Wastewater: Effect of Supplementing Glucose, Yeast Extract, and Inorganic Salts, Biotech. and Bioprocess Eng., 13, 651-658. 2. Cho, K.-S., H. W., Ryu, C.-H. Park, and P. R. Goodrich (2001), Utilization of Swine Wastewater as a Feedstock for the Production of Polyhydroxylakanoates by Azotobacter vinelandii UWD, J. Bioscience and Bioeng., 91, 129-133. 3. Page, W. J. (1989), Production of polyhydroxyalkanoates by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources, Appl. Microbial. Biotechnol., 31, 329-333. P0355 Application of Bacteria for Effective Microalgae Production System Seungjib JEON1, Nam Kyu KANG1, Ji-Won YANG1,2, Yong Keun CHANG1,2, Bongsoo LEE1 1Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea, 2Advanced Biomass R&D Center, #2502 Building W1-3, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Biodiesel production from microalgae has been recently considered a new energy source to replace fossil fuel. However, several technical barriers, such as the method development for cultivation and genetic tool box for production of oleaginous mutant still need to overcome through various methods. In attempt to improve these challenges, bio-stress experiment was conducted by adding bacterial supernatant in Nannochloropsis culture. The results showed remarkable changes in microalgal population and size when comparing with normal condition. Particularly, culture containing supernatant of KYC2844 strain induced continuous growth of N. salina. Dry cell weight of cells grown in culture containing 20% supernatant was approximately 20% higher than that in normal condition. In case of N. salina cells grown in media containing supernatant of KYC DZ2 and KYC1262 strain, they were three times bigger than cells grown in control media. We performed electroporation based genetic transformation with swollen N. salina cells. As a result, transformation using swollen cells was more efficient than transformation using normal cells. These results indicate that bacterial effects can be helped for effective microalgae production system . Keywords : Biodiesel, N. salina, Bacteria, Bio-stress References 1. Wang, H., Hill, R.T., Zheng, T., Hu, X., Wang, B. 2014. Effects of bacterial communities on biofuel-producing microalgae: stimulation, inhibition and harvesting. Crit Rev Biotechnol, 1-12 2. Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz, M.C. 2012. Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana. Nature Communications, 3. P0356 Degradation of Bisphenol A by Solution Plasma Process Davoodbasha MUBARAKALI1,2, Seong-Cheol KIM2,3, Sang-Yul LEE2,3, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Centre for Surface Technology and Applications, Korea Aerospace University, Goyang, 412-791, 3Dept. Materials Engineering, Korea Aerospace University, 412-791 Bisphenol A (BPA) is a phenolic polymer widely used as material for phenoxy or polyester resin and polycarbonate plastics. BPA causes serious problems in the cells due to its structural similarity to estrogens. In this study, a solution plasma process (SPP) was employed to degrade BPA more effectively. BPA solutions (100 mg/L) of pH 3-8 were subjected to plasma discharge at 800 V, 30 kHz for 0-20 min using an unipolar power supply or at 1,870 V and 30 kHz for 0-45 min using a bipolar power supply. Unipolar plasma discharge for 18 min removed BPA by 86% at pH 4.0, and 30 min of bipolar discharge for 92 % BPA removal at pH 4.0. GC-MS analysis of the intermediates formed during plasma discharge revealed characteristic ions of some hydroxyl bisphenols, carboxylic intermediates, phenolic intermediates, and other intermediates that were formed by the cleavage of benzene rings. Major intermediates were dimethyl esters, benzene, imidazole, pthalic acid, etc. The BPA solution treated with SPP showed almost no toxicity on the aquatic fern, Lemna minor. Based on the results, SPP has potential for application to waste water treatment and eco-friendly way to reduce the levels of various endocrine disrupting chemicals. Keywords : Solution plasma porcess, Bisphenol A, Toxicity, Degradation, intermediates References 1. Zhang, W., K. Yin, L. Chen. 2013. Bacteria-mediated bisphenol A degradation. Appl. Microbiol. Biotechnol. 97:5681-5689. 2. Kolvenbach, B., N. Schlaich, Z. Raoui1, J. Prell, S. Zühlke, A. Schäffer, F. P. Guengerich, and P. F. X. Corvini. 2007. Degradation Pathway of Bisphenol A: Does ipso Substitution Apply to Phenols Containing a Quaternary α- Carbon Structure in the para Position? Appl. Environ. Microbiol. 73:4776-4784. 3. Alonso-Magdalena, P., S. Morimoto, C. Ripoll, E. Fuentes, and A. Nadal. 2006. The Estrogenic Effect of Bisphenol A Disrupts Pancreatic β-Cell Function In Vivo and Induces Insulin Resistance. Envir. Health Pers. 114:106-112. P0357 Creation of High Reutilization Value from High-Salinity Anchovy Fishmeal Wastewater by the Microbial Degradation Hyun Yi JUNG, Joong Kyun KIM Dept. of biotechnology, Pukyong National University, Busan, 608-737 Fish consumption and wastes are increased steadily. So, eco-friendly treatment of fish wastes has been suggested. To provide an option for the reutilization of high-salinity anchovy fishmeal wastewater (FMW), generated during anchovy fishmeal manufacturing processes, its potential for biodegradation was assessed in 1-L 5-neck flasks using a halotolerant and proteolytic microbial consortium. During the first 41 h of biodegradation, the pH, DO, ORP, and dry-sludge weight decreased as the total cell number of the microbial consortium increased steadily. The removal percentages of CODCr and TN were 59.0% and 54.4%, respectively, and the dry-sludge weight decreased from 115.5 to 68.0 g, with a degradation rate of 0.59 g h−1, during the 80 h experiment. The supernatant from the culture of the anchovy FMW at 70 h (culture supernatant) was phytotoxin-free. In hydroponic cultures, the culture supernatant demonstrated a good fertilizing ability. The culture supernatant also exhibited a high degree of antioxidant activity, with a 52.3% hydroxyl radical scavenging activity and 0.16 reducing power. Moreover, the culture supernatant inhibited DNA damage from hydroxyl radicals, enhancing the reutilization value of anchovy FMW. Keywords : Reutilization value, Anchovy fishmeal wastewater, Liquid fertilizer, Antioxidant activity, DNA-protective activity References 1. Albrecht-Ruiz M, Salas-Maldonado A, Chemical composition of light and dark muscle of Peruvian anchovy (Engraulis ringens) and its seasonal variation (2015), J Aquat Food Prod Technol, 24 (2), 191–196 2. Azam MS, Kim EJ, Yang HS, Kim JK, High antioxidant and DNA protection activities of N-acetylglucosamine (GlcNAc) and chitobiose produced by exolytic chitinase from Bacillus cereus EW5 (2014), SpringerPlus, 3, 354– 364 3. Bellaaj OG, Jridi M, Khaled HB, Jellouli K, Nasri M, Bioconversion of shrimp shell waste for the production of antioxidant and chitosan used as fruit juice clarifier (2012), Int J Food Sci Technol, 47 (9), 1835–1841 P0358 Characteristics of a Novel Multiple Enzymes-Possessing NaCl-Tolerance Bacillus licheniformis TK3-Y Strain for Treatment of Green Seaweed Containing Fisheries Waste Hyun Yi JUNG, Kyeong Hwan KANG, Joong Kyun KIM Dept. of biotechnology, Pukyong National University, Busan, 608-737 Total amount of fishery output of Korea is approximately 3 million ton per year. Among the output, production of fishes and seaweeds account for 75%. Every year, 28 percents (0.7 million ton) of wastes occur during fishery process. Due to London dumping convection, ocean disposal of fishery wastes has been strictly prohibited since 2012, treatment and recycle of fishery wastes has been issued. For reutilization of fishery waste, a strain was isolated from coastal area in Busan, identified as Bacillus licheniformis TK3-Y. Plate assay revealed that the isolate clearly produced cellulase, protease, lipase and tolerant in high NaCl concentration as well. The optimal pH, temperature and NaCl concentration for the degradation of the polymers by TK3-Y strain were 6, 50℃ and 17.5% respectively. With 10% (v/v) inoculums size, degradation characteristics of TK3-Y carried out in the carboxymethylcellulose, skim milk and olive oil medium and TK3-Y produced cellulase, protease and lipase. The simultaneous multi-substrate degradation ability of TK3-Y was carried out in mixture of carboxymethylcellulose, skim milk and olive oil, it indicated possibility of TK3-Y strain as a good candidate to degrade non- separate collected fishery wastes. Keywords : Biodegradation, Bacillus licheniformis TK3-Y, NaCl-tolerance References 1. Sharma, C.K., Sharma, P.K., Kanwar, S.S., Optimization of production conditions of lipase from B. licheniformis MTCC-10498 (2012), Res. J. Recent. Sci. 1(7), 25-32 2. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods (2011), Mol. Biol. Evol, 28(10), 2731–2739 3. Toyokawa, Y., Takahara, H., Reungsang, A., Fukuta, M., Hachimine, Y., Tachibana, S., Yasuda, M., Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce (2010), Appl. Microbiol. Biotechnol. 86(6), 1867-1875 P0359 Saccharification and Ethanol Fermentation of The Whole Slurry of Rice Straw Pretreated with Maleic Acid Dong Hyun KIM, Young Hoon JUNG, Kyoung Heon KIM Dept. of Biotechnology, Graduate School, Korea University, Seoul, 136-713 To increase the yield of ethanol from lignocellulose, we evaluated the feasibility of whole slurry saccharification and fermentation of maleic acid-pretreated rice straw for ethanol production. The optimized conditions for pretreatment were to expose rice straw to a high temperature (190°C) with 1% (w/v) maleic acid for a short time duration (3 min ramping to the set temperature with 3 min holding) in a microwave digester. An enzymatic digestibility of 91.5 % (based on theoretical glucose yield) was exhibited using pretreated rice straw. Simultaneous saccharification and fermentation (SSF) of the whole slurry of pretreated rice straw for 60 h resulted in 83.2% final yield of ethanol based on the initial amount of glucan in untreated rice straw. These results indicate that maleic acid is a suitable catalyst not requiring detoxification steps for whole slurry fermentation of rice straw for ethanol production, thus improving the process economics. Also, the whole slurry saccharification and fermentation can significantly increase the biomass utilization by converting sugar from both solid and liquid phases of the pretreated slurry. Keywords : Pretreatment, Maleic acid, Saccharification, Fermentation, Rice straw References 1. Jung YH et al., Whole slurry saccharification and fermentation of maleic acid-pretreated rice straw for ethanol production (2015), Bioprocess and Biosystems Engineering, 38(9):1639-1644 P0360 Isolation of Acinetobacter sp. from Soil and Characterization of Its Methyl Acetate Biodegrading Capability Sangwoo KIM, Wangjun LEE, Yuhyun KWON, Jinwon LEE Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, Republic of Korea This study aimed to isolate methyl acetate biodegrading bacteria from a soil source and investigate a potential of substitute for methane gas and growth characterization of the strain. The origin of the isolated strain was revealed by its 16S rRNA sequence using NCBI GenBank. In order to characterize the isolated strain further, the optimal growth conditions including temperature, rpm, pH, and gas composition were determined and the microbiological characterization was conducted by scanning electron microscopy. The degradation abilities of the strain were analyzed by comparing cell-free and cell- inoculated cultures. The methyl acetate is easily vaporized in the cell free condition but it can be confirmed through the degradation performance which was 247 % greater in the inoculated culture compared with the cell-free culture. This study is the first report of a newly discovered strain, Acinetobacter sp., which is capable of methyl acetate uptake as its sole carbon source under aerobic condition. Keywords : Acinetobacter sp., biodegradation, methyl acetate References 1. Wang Ying, Tian Ye, Han Bin, Zhao Hua-bing, Bi Jian-nan, Cai Bao-li, Journal of Environmental Sciences 19, 222–225 (2007) 2. Mukherjee P, Roy P., Adv Microbiol 2, 284-294 (2012) P0361 Pretreatment of Lignocellulose by Mimicking the Fenton Reaction in the Fungal Wood Decay Ji Eun KIM, Young Hoon JUNG, Hyun Kyung KIM, Kyoung Heon KIM Department of Biotechnology, Graduate school, Korea University, Seoul 136-713 Lignocellulose is one of the most plentiful renewable resource in the world. The fungal decay of lignocellulose is caused not only by enzymes such as cellulase, hemicellulase, and ligninase, but also by chemical reagents such as the Fenton’s reagent. In the fungal degradation of lignocellulose, the Fenton’s reagent promotes chemical degradation by Fenton reaction. The Fenton reaction does not require extreme conditions such as high temperature, high pressure, or high concentration of chemicals. In this study, using the Fenton reaction which is naturally used by fungi for wood decay, pretreatment of rice straw was performed to increase the enzymatic digestibility for saccharification of lignocellulose. 93.2% of the theoretical glucose yield was acquired by employing an optimized Fenton’s reagent (FeCl3 and H2O2) for pretreatment. This is the first report of the application of the Fenton reaction to lignocellulose pretreatment at a moderate temperature (25 °C) and with a relatively high solids loading of biomass (10% (w/v)). These results suggest that using a Fenton reaction could achieve considerable improvement in the process economics of lignocellulosic biofuel and chemical building block production. Keywords : Fenton reaction, Lignocellulose, Enzymatic saccharification, Fungal wood decay References 1. Jung YH et al., Mimicking the Fenton reaction-induced wood decay by fungi for pretreatment of lignocellulose (2015), Bioresource Technology, Vol (179), 467-472 P0362 Bioinspired Silica Nanoparticle with Auto-Encapsulated Carbonic Anhydrase for Biomimetic CO2 Sequestration Byung Hoon JO1, Jeong Hyun SEO1,2, Yun Jung YANG1, Kyungjoon BAEK3, Yoo Seong CHOI4, Seung Pil PACK5, Sang Ho OH3, Hyung Joon CHA1 1Department of Chemical Engineering, POSTECH, Pohang 790-784, 2School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, 3Department of Materials Science and Engineering, POSTECH, Pohang 790-784, 4Department of Chemical Engineering, Chungnam National University, Daejeon 305-764, 5Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700 Here, we report on the development and characterization of a carbonic anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use in environmental CO2 sequestration. Encapsulation occurred simultaneously with autonomous silica synthesis by silica-condensing R5 peptide that was fused to recombinant CA. The encapsulation efficiency was greater than 95%, and the encapsulated CA was not leached from the silica matrix, demonstrating the highly efficient R5-mediated auto- encapsulation process. The catalytic efficiencies for both esterase and CO2 hydratase activities tended to increase with increasing pH; however, the catalytic efficiency for CO2 hydration was much more pH dependent, suggesting that proton transfer from silica to water is a rate limiting step, especially for CO2 hydration. In addition to good reusability, the encapsulated CA exhibited outstanding thermostability, even retaining 80% activity after 5 days at 50˚C. The thermoactivity was also remarkable, showing ~10-fold higher activity at 60˚C compared to that at 25˚C. The physical structure was observed to be highly compact with a low surface area, stressing the importance of the outermost surface for catalytic performance. We also demonstrated the applicability of the silica nanoparticle to the sequestration of CO2 in carbonate minerals. The rate of CaCO3 precipitation was remarkably accelerated by the encapsulated biocatalyst. The biosilica nanocomposite exhibited ~60% of the CO2 sequestrating power of the free enzyme, which is expected to be the maximal ability of the encapsulated CA. Thus, this silica-CA nanocomposite, efficiently synthesized via a biomimetic green route, can be successfully used as a robust biocatalyst for biomimetic sequestration of the greenhouse gas CO2. Keywords : carbonic anhydrase, immobilization, biosilica, CO2 sequestraion, silaffin References 1. BH Jo, JH Seo, YJ Yang, K Baek, YS Choi, SP Pack, SH Oh, HJ Cha, Bioinspired silica nanocomposite with autoencapsulated carbonic anhydrase as a robust biocatalyst for CO2 sequestration (2014), ACS catalysis, 4(12), 4332–4340 P0363 Construction of Global Cohesin Marker for Cellulosome from Clostridia spp with Cross- Species Cohesin-Dockerin Interaction Bom Yi PARK, Sang Duck JEON, Su Jung KIM, Jeong Eun HYEON, So heyong KIM, Sung Ok HAN Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea Anaerobic microorganism such as Clostridia produces the extracellular multi-enzyme complex called cellulosome for efficiently degradation of lignocelluose. Interaction of cohesin in the saffoldin and dockerin in the cellulosomal subuit is significant role for assembling cellulosome. Usually same strain-derived cohesin and dockerin has strolngly interaction like that of antigen and antibody. In previous study, researcher demonstrated that celluosomal subunits from Clostridium cellulovarans were detected by using fluorescence-labeled cohesin as biomarker. These result indicated that cellulosome can be detected by high affinity of cohesin-dockerin interaction. By using this method, we tried detecting of cellulosomal subunits within the same strain as well as different Clostridium spp. such as C. cellulovorans, C. thermocellum and C. cellulolyticum. Enzyme-linked interaction assay suggest that EngE from C. cellulovorans and CelE from C. thermocellum bound to cohesin from C. cellulovorans. This result demonstrate the possibilities of cohesin-biomarker can be used as a common marker to Clostridium spp. Consequently, developing the global marker will be able to explore to profile cellulolytic enzyme with flurescene-labeled cohesin as a biomarker. Keywords : Global cohesin marker, Anaerobic bacteria, Cohesin-dockerin interaction, Cellulosome References 1. Jeon, S. D. et al, Analysis of selecrive. High protein-protein binding interaction of cohesion-dockerin comples using biosensing methods(2012), 35, 382– 389 2. Hyeon, J. E et al, Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and application(2013), Biotechnology Advances ,31, 936–944 3. Cho, W. J. et al, Cellulosomic profiling produced by Clostridium cellulovorans during growth on different carbon sources explored by the cohesin marker(2009), Journal of Biotechnology , 145, 233–239 한국생물공학회, 생물공학의 동향 : 2015.10 대사공학 및 합성생물공학 P0401 Production of 3-Aminopropionic Acid in Metabolically Engineered Escherichia coli Tong Un CHAE1,3, Chan Woo SONG1,3, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 In this study, a novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP) in Escherichia coli. Using a fumaric-acid producing E. coli strain as a host, the C. glutamicum panD gene (encoding L-aspartate-α- decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase (AspA)-catalyzed reaction. Additional overexpression of the aspA and phosphoenolpyruvate carboxylase (ppc) genes, and the supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. This was further increased to 3.94 g/L by optimizing the expression level of PPC, which was achieved by evaluating 12 different combinations of synthetic promoters and RBS sequences. Fed-batch culture of the final strain yielded 17.9 g/L 3-AP in 89 h, with an overall yield and productivity of 0.186 g 3-AP/g glucose and 0.200 g/L/h, respectively. (Development of systems metabolic engineering platform technologies for biorefineries; NRF-2012- C1AAA001-2012M1A2A2026556) funded by the Ministry of Education, Science and Technology) Keywords : 3-aminopropionic acid, metabolic engineering, aspartase References 1. Song, C.W., Lee, J., Ko, Y.-S., and Lee, S.Y, Metabolic engineering of Escherichia coli for the production of 3- aminopropionic acid (2015), Metab. Eng., 30:121-129 P0402 Metabolically Engineered Escherichia coli for C5 Platform Chemicals: Aminovalerate and Glutarate Tong Un CHAE2, Si Jae PARK4, Eun Young KIM1, Won NOH1, Hye Min PARK2, Young Hoon OH1, Seung Hwan LEE1, Bong Keun SONG1, Jonggeon JEGAL1, Sang Yup LEE2,3 1Industrial Biochemicals Research Group, Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseon, 2Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, and Instit, 3Department of Bio and Brain Engineering, Department of Biological Sciences, BioProcess Engineering Research Center, and Bioinformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-70, 4Department of Environmental Engineering and Energy, Myongji University, San 38-2, Nam- dong, Cheoin-gu, Yongin-si, Gyeonggido 449-728, Republic of Korea 5-Aminovalerate (5AVA) is an important C5 platform chemical. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured and produced 3.6 g/L of 5AVA by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strain XQ56 allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Education, Science, and Technology (MEST) through the National Research Foundation of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556). Keywords : metabolic engineering, 5-aminovalerate, glutarate References 1. Park, S.J., Kim, E.Y., Noh, W., Park, H.M., Oh, Y.H., Lee, S.H., Song, B.K., Jegal, J., and Lee, S.Y, Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals (2013), Metab. Eng., 16: 42-47 P0403 Production of Gamma-Butyrolactone Using Metabolically Engineered Mannheimia succiniciproducens Tong Un CHAE1, Sol CHOI1,3, Hyun Uk KIM1,2, Won Jun KIM1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), Center for Systems and Synthetic Biotechnology, Institute for t, 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which has a wide range of industrial applications. GBL can be produced by acid treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic acid. Heterologous metabolic pathways were designed and established in succinic acid overproducing M. succiniciproducens LPK7 by the introduction of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) resulted in the production of 6.37 and 6.34 g/L of 4-HB, respectively. Finally, GBL was produced by acid treatment of the 4-HB obtained from the fermentation broth. This study demonstrates that 4-HB, and potentially other four carbon platform chemicals, can be produced by the engineered rumen bacterium M. succiniciproducens. [“This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556).] Keywords : γ-butyrolactone , 4-hydroxybutyric acid, metabolic engineering References 1. Choi, S., Kim, H.U., Kim, T.Y., Kim, W.J., Lee, M.H., and Lee, S.Y, Production of 4-hydroxybutyric acid by metabolically engineered Mannheimia succiniciproducens and its conversion to gamma-butyrolactone by acid treatment (2013), Metab. Eng., 20: 73-83 P0404 Synthesis and Production of Native Sized Spider Silk Protein in Escherichia coli Hannah CHUNG1, Xiao-Xia XIA1, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering Research Center, and Center for Systems and Synthetic Biot, 2Department of Bio and Brain and Bioinformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Naturally found spider silk and elastin protein attract attention due to its outstanding physical properties coming from the highly repeated structure and size. However, the exceptional structure and size limits expression in heterologous hosts, where the repetitive sequences in mRNA create extensive secondary structures. And these structures decrease ribosome processivity and assist mRNA degradation. Using the naturally found protein, spider dragline silk protein, we present techniques to solve biological problems that occurred: using metabolic engineering and increasing the cellular resource, more specifically, particular amino acid tRNA pool. Newly synthesized native-size spider dragline silk protein produced increased titer than those reported previously, therefore proving that the strategies used were efficient. The results provide insight into approaches to control expression of recombinant proteins containing high molecular weight and highly repetitive sequence. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea] Keywords : Spider silk protein, Protein engineering, Metabolic engineering References 1. Xiao-Xia, Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber (2010), PNAS, 107(32): 14059-14063 P0405 Biological Production of Phenol from Glucose in An Engineered E. coli Strain Hannah CHUNG1, Byoungjin KIM1, Hyegwon PARK1, Dokyun NA3, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems a, 2Department of Bio and Brain Engineering, Department of Biological Sciences and Bioinformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea, 3School of integrative engineering, ChungAng University, Seoul, Microbial phenol production has been limited due to weak metabolic flux through the complicated biosynthetic pathway and toxicity of phenol to microorganisms. To address these issues, we utilized the synthetic small RNA (sRNA)-based gene knockdown technology to rapidly engineer the biosynthetic pathway of aromatic amino acids in a number of Escherichia coli strains and developed a biphasic fermentation strategy which could minimize the toxicity of phenol by in situ extraction of phenol produced in aqueous culture medium. Overexpression of Pasteurella multocida tyrosine phenol-lyase and down- regulation of the two transcriptional regulators, tyrR and csrA generated E. coli strains efficiently producing phenol from glucose. Among the 18 E. coli strains, the engineered BL21(DE3) strain showed a phenol titer of 419 mg/L in flask culture and 1.69 g/L in fed-batch fermentation. The titer was increased up to 3.79g/L in biphasic fed-batch fermentation. [This work was supported by the Intelligent Synthetic Biology Center through the Global Frontier Project (2011- 0031963) of the Ministry of Science, ICT & Future Planning through the National Research Foundation of Korea.] Keywords : Phenol, Metabolic engineering, syntethic small RNA References 1. Kim, B., Park, H., Na, D., and Lee, S.Y., "Metabolic engineering of Escherichia coli for the production of phenol from glucose" (2014) Biotechnol. J., 9(5): 621-629 P0406 Biodiesel Production via Metabolic Engineering in the Oleaginous Microorganism Hannah CHUNG1, Hye-Mi KIM1, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems a, 2Department of Bio and Brain Engineering, Department of Biological Sciences and Bioinformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea The dramatic increase in demand for transportation fuels, related with depleting crude oil reserves, and the increase in environmental concerns have increased the emphasis on renewable energy. Biodiesel, refers to fatty acid methyl esters (FAMEs) derived from vegetable oils or animal fats, is one of promising alternative fuels. A triacylglycerol (TAG) consists of one glycerol and three fatty acid molecules, and fatty acids in TAG are used to produce FAMEs and FAEEs which can be used for a diesel alternative via transesterification process with methanol and ethanol, respectively. In this study, we engineered the oleaginous microorganism which can accumulate triacylglycerols in their cell bodies to produce fatty acid ethyl esters (FAEEs) as diesel derivatives. The genes encoding TAG lipases from other organisms were overexpressed in wild strain to enhance the production of fatty acids from TAGs. The engineered strain produced a large amount of free fatty acids from glucose in fed-batch fermentation. To produce FAEEs in vivo from fatty acids, we constructed an ethanol biosynthetic pathway and overexpressed wax ester synthase in fadE mutant strain. Finally, biosynthetic pathways for the production of FAEEs were successfully developed in the engineered oleaginous microorganism. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.] Keywords : Biodiesel, fatty acid ethyl esters, metabolic engineering References 1. Yasser Elbahloul and Alexander Steinbuchel, Pilot-scale production of fatty acid ethyl esters by an engineered Escherichia coli strain harboring the p(Microdiesel) plasmid (2010), Applied and environmental microbiology, 76, 13 P0407 Rapid One-step Genome Engineering in Escherichia coli Using a Newly Developed Integration- helper Plasmid Dongsoo YANG1, Chan Woo SONG1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioProcess Engineering Research Center, KAIST, Daejeon, Republic of Korea, 3BioInformatics Research Center, KAIST, Daejeon, Republic of Korea To facilitate genome manipulation in E. coli, the integration helper plasmid pCW611 was developed which expresses two recombinases (Red and Cre) by using two independent (IPTG and Arabinose) inducible systems [1]. Since iterative transformation of the conventional helper plasmids and repetitive curing of plasmids are not required, the time and effort it takes for genome engineering can be significantly reduced when compared to the conventional method of using several integration helper plasmids. Thus, by using this novel plasmid, pCW611, we could delete one target gene in just 3 days. To verify the effectiveness of this novel system, gene deletion experiments were performed by knocking out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). In addition, fumaric acid producing E. coil strain was developed by deleting four target genes (fumB, iclR, fumA, and fumC) in 10 days as a proof-of-concept study. [Development of systems metabolic engineering platform technologies for biorefineries; (NRF-2012-C1AAA001-2012M1A2A2026556) funded by the Ministry of Education, Science and Technology] Keywords : Cre recombinase, Escherichia coli, Gene manipulation, Metabolic engineering, Red recombinase References 1. C.W.Song and S.Y.Lee, Rapid one-step inactivation of single or multiple genes in Escherichia coli (2013), Biotechnol. J. 8, 776-784. P0408 Biosynthesis of 1-Propanol by Systems Metabolic Engineering Jung Eun YANG1, Yong Jun CHOI2, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 373-1 , 2School of Environmental Engineering, University of Seoul, Seoul The 1-propanol is a next generation biofuel used not only as a gasoline substitute, but also as various industrial products such as paint and cosmetics. In terms of energy density and combustion efficiency, it is advantageous than the traditional bio-fuel, ethanol. Previously, Atsumi et al. reported that a metabolically engineered Escherichia coli harboring an engineered citramalate synthase (CimA), which converts pyruvate directly into 2-ketobutyrate, from Methanococcus jannaschii produced the highest reported concentration of 1-propanol, 3.5 g L-1. We have previously reported an L-threonine overproducing E. coli TH20 strain, which was genetically modified to concentrate carbon fluxes towards L-threonine by systems metabolic engineering. The TH20 strain was further engineered for 1-propanol production. Toward this goal, novel synthetic pathway for 1-propanol production, deleting competing pathway and carbon source optimization based on the in silico flux response analysis was established. Additional metabolic engineering strategy of the resulting strain further improved the titer. [This work was supported by the Advanced Biomass R&D Center of Korea (ABC-2011-0028386) through the Global Frontier Research Program of the Ministry of Education, Science and Technology (MEST)]. Keywords : metabolic engineering, 1-propanol, biofuel References 1. Choi, Y. J., Park, J. H., Kim, T. Y., & Lee, S. Y. Metab. Eng., 14(5), 477-486. P0409 Metabolic Engineering of Escherichia coli for the Biosynthesis of Poly (3-Hydroxybutyrate-Co- 3-Hydroxyvalerate) Jung Eun YANG1, Yong Jun CHOI2, Seung Hwan LEE3, Bong Keun SONG4, Si Jae PARK5, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 373-1 , 2School of Environmental Engineering, University of Seoul, Seoul , 3Dept. of Biotechnology and Bioengineering, Chonnam National University, Gwangju 500-757, 4Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, Daejeon , 5Dept. of Environmental Engineering and Energy, Myongji University, Yongin-si 449-728 Polyhydroxyalkanoates(PHAs) are polyesters accumulated in many bacteria. Among lots of PHA copolymers, poly(3- hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is one of the important copolymers because of lower melting point and much better flexible compared to current used in many application. So far, for the production of P(3HB-co-3HV), adding of second auxiliary carbon source was needed. However, due to the toxicity of auxiliary carbon source, it is very hard to maintain the balance between cell growth and P(3HB-co-3HV) production. Thus, we developed the E. coli can stably synthesize 3HB-CoA and 3HV-CoA in controlled ratio from glucose without feeding of exogenous auxiliary carbon source by metabolic engineering. This engineered strain can efficiently synthesize P(3HB-co-3HV) independent with exogenous auxiliary carbon source. “This work was supported by the Technology Development Program to Solve Climate Changes from National Research Foundation of Korea (Development of systems metabolic engineering platform technologies for biorefineries; NRF-2012-C1AAA001-2012M1A2A2026556) and Intelligent Synthetic Biology Center (2011-0031963) of Korea through the Global Frontier Research Program of the Ministry of Education, Science and Technology (MEST). Further supports by the World Class University program (R32-2008-000-10142-0) of the MEST are appreciated.” Keywords : Biopolymer, Polyhydroxyalkanoates, Metabolic engineering References 1. Yang, J.E., Choi, Y.J., Lee, S.J., Kang, K.-H., Lee, H., Oh, Y.H., Lee, S.H., Park, S.J., and Lee, S.Y., P0410 Enhanced Biosynthesis of Poly(3-Hydroxybutyrate) Based on Proteome Analysis Jung Eun LEE1, Seung Hwan LEE2, Bong Keun SONG3, Si Jae PARK4, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 373-1, 2Dept. of Biotechnology and Bioengineering, Chonnam National University, Gwangju 500-757, 3Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, Daejeon, 4Dept. of Environmental Engineering and Energy, Myongji University, Yongin-si 449-728 We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose- bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.[This work was supported by the Technology Development Program to Solve Climate Changes (Systems metabolic engineering for biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the NRF (NRF-2012-C1AAA001-2012M1A2A2026556) is appreciated.] Keywords : poly(3-hydroxybutyrate), Proteome analysis, Metabolic engineering References 1. Lee, S.H., Kang, K.-H., Kim, E.Y., Chae, T.U., Oh, Y.H., Hong, S.H., Song, B.K., Jegal, J., Park, S.J., and Lee, S.Y., Biotechnol. Lett., 35(10): 1631-1637 P0411 Metabolic Engineering of Corynebacterium glutamicum as a Microbial Cell Factory to Produce L-Ornithine Jae Sung CHO1, Seo Yun KIM1, Joungmin LEE1, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 291 D, 2BioInformatics Research Center, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 3Institute for the BioCentury, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 4BioProcess Engineering Research Center, KAIST, 291 Daehak- Ro, Yuseong-Gu, Daejeon 305-701, South Korea L-Ornithine is a non-essential amino acid for various applications in food industry. We sought high-titer production of L- ornithine by Corynebacterium glutamicum ATCC 13032 through metabolic engineering. The proB and argF genes were first deleted to optimize metabolic pathway. The argR gene encoding the regulatory repressor of the L-arginine operon was also deleted to enhance the ornithine flux. After initial flask cultivation, this base strain was further engineered by plasmid-based overexpression of argCJBD genes. Changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter enriched the NADPH pool. Fed-batch cultivation of the final strain YW06 (pSY223) showed a titer of 51.5 g/L of L-ornithine in 40 h with productivity of 1.29 g/L/h. The results demonstrate efficient L-ornithine production with engineered C. glutamicum. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556).] Keywords : ornithine, metabolic engineering, Corynebacterium glutamicum References 1. Seo Yun Kim, Joungmin Lee, Sang Yup Lee, Metabolic Engineering of Corynebacterium glutamicum for the Production of L-Ornithine (2014), Biotechnology and Bioengineering, 112 (2), 416-421 P0412 Metabolic Engineering of Corynebacterium glutamicum to Produce 1,5-Diaminopentane Jae Sung CHO1, Seok Hyun PARK1, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 291 D, 2BioInformatics Research Center, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 3Institute for the BioCentury, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 4BioProcess Engineering Research Center, KAIST, 291 Daehak- Ro, Yuseong-Gu, Daejeon 305-701, South Korea Bio-based production of 1,5-diaminopentane from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a metabolically engineered Corynebacterium glutamicum to produce 1,5-diaminopentane. L-lysine decarboxylase, which converts L-lysine directly to 1,5-diaminopentane, was amplified by plasmid-based overexpression of the cadA gene under the tac promoter in an industrial L-lysine producer C. glutamicum (U2 strain). The recombinant C. glutamicum (U2/pCEcadA) did not produce 1,5-diaminopentane however, and L-lysine was detected in the culture medium. The cadA gene was thus modified using a codon adaptation program. With the redesigned cadA gene, we achieved a production titer of 31.94 g/L of 1,5-diaminopentane without exogenous feeding of L-lysine by fed-batch fermentation of C. glutamicum (U2/pCEcadA). [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556).] Keywords : cadaverine, 1,5-diaminopentane, metabolic engineering, corynebacterium glutamicum References 1. Zhi-Gang Qian, Xiao-Xia Xia, and Sang Yup Lee, Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine (2011), Biotechnology and Bioengineering, 108 (1), 93-103 P0413 Metabolic Engineering of Escherichia coli as a Microbial Cell Factory to Produce Cadaverine Jae Sung CHO1, Zhi-Gang QIAN1, Hye Min PARK1, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 291 D, 2BioInformatics Research Center, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 3Institute for the BioCentury, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 4BioProcess Engineering Research Center, KAIST, 291 Daehak- Ro, Yuseong-Gu, Daejeon 305-701, South Korea Cadaverine (1,5-diaminopentane) is a five-carbon diamine that can be used as a building block for sustainable polymer production. Bio-based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the conventional petroleum-based chemical synthesis. Here, we report a metabolically engineered Escherichia coli strain that produces cadaverine from glucose in defined medium. The pathways that lead to cadaverine degradation were blocked by genetic engineering. The final engineered strain produced 9.61 g/L of cadaverine with a productivity of 0.32 g/L/h by fed- batch cultivation. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] Keywords : cadaverine, 1,5-diaminopentane, escherichia coli, metabolic enigneering References 1. Zhi-Gang Qian, Xiao-Xia Xia, and Sang Yup Lee, Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine (2011), Biotechnology and Bioengineering, 108 (1), 93-103 P0414 Metabolic Engineering of Corynebacterium glutamicum as a Microbial Cell Factory to Produce L-arginine Jae Sung CHO1, Seok Hyun PARK1, Hyun Uk KIM1,2, Tae Yong KIM1,2, Jun Seok PARK5, Suok-su KIM5, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus rogram), Center for Systems and Synthetic Biotechnology KAIST, 291 D, 2BioInformatics Research Center, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 3Institute for the BioCentury, KAIST, 291 Daehak-Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 4BioProcess Engineering Research Center, KAIST, 291 Daehak- Ro, Yuseong-Gu, Daejeon 305-701, South Korea, 5Daesang corporation research center, Incheon-city, Gyeonggi-do 467- 813, South Korea Corynebacterium glutamicum was metabolically engineered for L-arginine production. Random mutagenesis was first performed on C. glutamicum to increase tolerance to L-arginine. Arginine operon repressor proteins were inactivated. The PPP flux was strengthened by downregulating the pgi gene and overexpressing the opcA, pgl, tal, tkt, and zwf genes. Next, the Ncgl1221 gene encoding L-glutamate exporter was inactivated to channel L-glutamate to L-arginine formation. Also, the expression levels of the argF and carAB genes were optimized for converting L-ornithine to L-citrulline effectively. Finally, the argGH operon was overexpressed. Fed-batch fermentation of the final strain was performed in a 1,500 L bioreactor resulting 81 g/L of L-arginine production. The approaches described here will be useful in developing strains of Corynebacteria regarding the production of arginine and its derivatives. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012- C1AAA001-2012M1A2A2026556).] Keywords : arginine, corynebacterium glutamicum, metabolic engineering References 1. Seok Hyun Park, Hyun Uk Kim, Tae Yong Kim, Jun Seok Park, Suok-Su Kim, and Sang Yup Lee, Metabolic engineering of Corynebacterium glutamicum for L-arginine production (2014), Nature Communications, 5 (4618) P0415 Visualization of Reconstructed Metabolic Network Using FBA-simVis Do Kyun KIM1, Sung-Won SEO2, Un-soo KIM2, Hye-Sun PARK2, Wang-Hee LEE1 1Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Yuseung-gu, 305-764, South Korea, 2Department of Animal Biosystem Science, Chungnam National University, Daejeon, Yuseung-gu, 305-764, South Korea Demands on advanced tools in analyzing complex metabolic system lead to devise metabolic reconstruction which comprehensively takes into account a large number of metabolic and genetic data. One of the core techniques for utilizing metabolic reconstruction is visualization of metabolic pathways for further analyses. Visualization is a drawing of a metabolic network map, allowing an insight of how reactions and metabolites are connected to construct metabolisms. There are a few available tools for metabolic visualization such as COBRA and FBA-simVis [1, 2]. Also, a public database named BiGG provides online tools for drawing large-scale metabolisms [3]. Among them, FBA-smVis developed based on VANTED program is specialized software for visualizing metabolic network and for carrying out flux balance analysis (FBA) or flux variability analysis (FVA). In this study, we are to use FBA-simVis software in order to visualize glycolysis pathway in bovine mammary gland epithelial cell. To use FBA-simVis, we used previously developed a tissue-specific model using COBRA that combined experimentally obtained gene expression data of cattle into glycolysis pathway so that we could identify which reactions and metabolites should be included. As a result, glycolysis pathway was visually constructed on computer with verification of expressed and unexpressed reactions. In the computationally visualized metabolic map, reactions and metabolites were represented by triangle and quadrangle blocks, respectively, and connected by arrows. It was easy to manipulate arrangement of system component and to add extra reactions and metabolites for expanding the metabolism. However, we currently focus on small-scale pathway that can be manually drawn. Hence, the next step has to consider large-scale mapping that requires retrieving big database. Also, in the future, we are plan to perform FBA based on the developed map and compare the result with other FBA results obtained by different tools (COBRA and direct mathematical formulation). Keywords : FBA-simVis, metabolic network map, visualization, glycolysis References 1. J. Schellenberger, I. Thiele, and J. D. Orth, Nature Protocol, 6, 1290 (2012). 2. E. Grafahrend-Belau, C. Klukas, B. H. Junker, F. Schreiber, Bioinformatics, 25, 2755 (2009). 3. J. Schellenberger, J. O. Park, T. M. Conrad, and B. Ø. Palsson, BMC Bioinformatics, 11, 213 (2010). P0416 Production of Short-Chain Alkanes in Escherichia coli by Systems Metabolic Engineering Dongsoo YANG1, Yong Jun CHOI1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioProcess Engineering Research Center, KAIST, Daejeon, Republic of Korea, 3BioInformatics Research Center, KAIST, Daejeon, Republic of Korea We have successfully developed the platform Escherichia coli strains which are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols. First, β-oxidation pathway was blocked by deleting the fadE gene. Also, to enhance the formation of short-chain fatty acids, fadR gene was deleted. Short-chain fatty acyl-ACPs were then converted to the corresponding FFAs by a modified thioesterase, which were further converted to SCAs by E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. From the final engineered strain, up to 580.8mg/L of SCAs were produced. The modified versions of this strain could produce short-chain FFAs and short-chain fatty esters. [Funded by the Advanced Biomass Research and Development Center of Korea (ABC-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF), and by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012- C1AAA001- 2012M1A2A2026556) by MSIP through NRF.] Keywords : Metabolic engineering, Escherichia coli, short-chain alkanes, gasoline References 1. Y.J.Choi and S.Y.Lee, Microbial production of short-chain alkanes (2013), Nature, 502, 571-574. P0417 Metabolic Engineering of Escherichia coli for The Production of 5-Aminolevulinic Acid Yoojin CHOI1, Yoosung KO1, Sol Choi CHOI1, Jae Won JANG1, Dong In KIM1, Hyun Uk KIM1,3, Si Jae PARK4, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea, 3BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea, 4Department of Environmental Engineering and Energy, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin, Gyeonggido 449-728, Republic of Korea Escherichia coli (E. coli) W3110 strain was metabolically engineered to produce 5-aminolevulinic acid (ALA), a non- standard five-carbon amino acid that is widely used in agricultural and medical industries. First, the plasmid pKE112hemA was constructed by cloning into a high copy number plasmid the codon-optimized Rhodobacter sphaeroides hemA gene encoding the ALA synthase (ALAS) catalyzing the condensation of succinyl-CoA and glycine to make ALA. Second, plasmid pKE112hemA was introduced into the lacI-deleted WL3110 strain; the WL3110 (pKE112hemA) strain produced 0.249 ± 0.027 g/L of ALA. Third, in silico knock-out simulation was carried out to identify additional gene knock-out targets to further improve ALA production. The gcvTHP genes, which are responsible for the glycine cleavage, were predicted as knockout targets. The JW01 strain (WL3110 ΔgcvTHP) harboring pKE112hemA produced 1.17 ± 0.035 g/L of ALA, which was 4.7 times higher than that obtained with the base strain. Finally, in order to increase the succinyl-CoA 0, the glyoxylate shunt flux was enhanced by the deletion of the iclR and sdhAB genes, while the TCA cycle flux was reinforced by the deletion of ptsG gene. The JW03 strain (JW01 ΔiclR ΔsdhAB ΔptsG) harboring pKE112hemA was able to produce 1.72 ± 0.001 g/L of ALA. Fed-batch culture of the JW03 (pKE112hemA) strain resulted in the production of 5.77 g/L (44 mM) of ALA in 41 h. This metabolically engineered strain can serve as a good base strain for developing further improved strains capable of efficiently producing ALA and related chemicals. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556).] Keywords : Systems metabolic engineering, Escherichia coli, 5-aminolevulinic synthase References 1. H. R. Jones and M. K. Wiles, (1999), J. Phys. Chem. 78, 8356 P0418 Production of Tyrosine and Cadaverine Using Synthetic Small Regulatory RNAs-based Metabolic Engineering Approach Yoojin CHOI1, Minho ROH1, Dokyun NA2, Seung Min YOO1, Sang Yup LEE1,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology (KAIST, 2School of Integrative Engineering, Chung-Ang University, Seoul 156-756, Republic of Korea, 3Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea, 4BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea Small regulatory RNAs (sRNAs) are the gene expression regulatory system which acts after transcription in bacteria. We developed synthetic small regulatory RNAs system to control the gene expression. The target mRNA binding region of MicC, one of the sRNAs in Escherichia coli, was replaced to translation initiation sequence of our target genes. We found that MicC scaffold based synthetic sRNA is properly repressed expression of DsRed2, a fluorescence protein. Synthetic sRNAs was used for metabolic engineering to enhance the production of tyrosine and cadaverine. Through screening of 14 different strains which harboring one or combination of the synthetic sRNAs targeting ppc, tyrR, csrA, and pgi, we isolated a tyrosine producer producing 2 g per liter of tyrosine. Using a library of 130 synthetic sRNAs, we screened knockdown targets that increase cadaverine productivity substantially. Repression of murE led to a 55% increase in cadaverine production compared to the base strain. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea] Keywords : Synthetic small regulatory RNAs, Metabolic engineering, Tyrosine, Cadaverine References 1. D. Na, S. M. Yoo, H. Chung, H. Park, J. H. Park and S. Y. Lee, Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs (2013),Nat. Biotechnol. 31(2), 170-4 P0419 Fine-tuned Knockdown Using Small Regulatory RNA and Application in Metabolic Engineering: Case Production of Putrescine Yoojin CHOI1, Minho ROH1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology (KAIST, 2Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea Putrescine, 1,4-diaminobutane, is an important chemical which has a high potential of application in chemical industry. In particular, putrescine is commercially used to synthesize nylon-4,6 by combined polymerization with adipic acid. Biosynthesis of putrescine from renewable biomass is a promising substitute of chemical synthesis from non-renewable petroleum. There was a trial of development of putrescine producing Escherichia coli strain through construction of biosynthesis pathway using knockout and overexpression. We expected that productivity of this strain is able to be increased by recently developed knockdown method, small regulatory RNA system. Using this system we repressed competitive branch pathway and increased flux to putrescine. Additionally, culture condition was modified to support the increased putrescine productivity. Finally the production titer, yield, and productivity were enhanced dramatically. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.] Keywords : Small regulatory RNA, Metabolic engineering, Putrescine References 1. Na, D., Yoo, S.M., Chung, H., Park, H., Park, J.H., and Lee, S.Y., Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs (2013), Nat. Biotechnol., 31(2), 170-174 P0420 Acetone-Butanol-Ethanol Production with High Productivity by Continuous Fermentation in the Membrane Cell-Recycle Bioreactor Won Jun KIM1, Yu-Sin JANG2, Sang Yup LEE1,2 11Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 22BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST Acetone-Butanol-Ethanol (ABE) fermentation is one of the famous solvent production processes by microorganism. Clostridium genus is generally known as producers of solvents by ABE fermentation and to improve the productivity of solvents, continuous fermentation can be a solution. However, continuous ABE fermentation has some limitations as cell wash-out at high dilution rates. Limitations of continuous fermentation were solved by using applying high cell density continuous fermentation in the membrane cell-recycle bioreactor. Developed C. acetobutylicum BKM19 mutant strain and C. pasteurianum MBEL_GLY2 mutant strain showed 21.1 g/l/h and 8.3 g/l/h of ABE productivity, respectively. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556); and the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2010-0029799)] Keywords : Continuous ABE fermentation, Membrane cell-recycle bioreactor, Clostridium, High cell density fermentation References 1. Jang et al., Acetone-Butanol-Ethanol Production With High Productivity Using Clostridium acetobutylicum BKM19(2013), Biotechnology and Bioengineering, 110(6), 1646-1653 2. Malaviya et al., Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum(2012), Appl Microbiol Biotechnol, 93,1485-1494 P0421 Development of Hyper-ABE P producing Clostridium acetobutylicum BKM19 and Genome Analysis Using Next-Generation Sequencing Won Jun KIM1, Changhee CHO1, Yu-Sin JANG2, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 2BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST Acetone-butanol-ethanol (ABE) are naturally produced by a Clostridium genus, but the biological production of solvents is not sufficient to cope with the petro-based production yet. Thus a hyper ABE producer is needed combined with the development of efficient bioprocess. To develop a hyper ABE producer, Mutagenesis of the Clostridium acetobutylicum PJC4BK strain was carried out by using N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and screened on fluoroacetate plates to isolate a mutant strain. A hyper ABE producing BKM19 strain was isolated. The BKM19 strain produced 32.5 g/L of ABE (17.6 g/L of butanol, 10.5 g/L of ethanol, and 4.4 g/L of acetone) from 85.2 g/L of glucose in batch fermentation exhibiting the total solvent production capability 30.5% and 90.5% higher than the PJC4BK and ATCC824 strains, respectively. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556); and the Advanced Biomass R&D Center of Korea (NRF-2010-0029799) through the Global Frontier Research Program of the MSIP.] Keywords : ABE-fermentation, Random mutagenesis, Butanol production References 1. Yu-Sin Jang, Alok Malaviya and Sang Yup Lee, Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19(2013), Biotechnology and Bioengineering, 110: 1646–1653 P0422 Highly Selective Butyric Acid Production Obtained by Disruption of the Butyrate Kinase Gene in Clostridium acetobutylicum Won Jun KIM1, Yu-Sin JANG2, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 2BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST Butyric acid is widely used in various field. Clostridium acetobutylicum produce butyric acid with acetic acid during acidogenic phase and to improve butyric acid selectivity metabolically engineered C. acetobutylicum strain was generated. The second butyrate kinase of C. acetobutylicum encoded by the bukII gene was employed instead of butyrate kinase I encoded by the buk gene. In addition additional metabolic pathways were engineered to enhance the NADH-driving force. The engineered C. acetobutylicum strain showed 32.5 g/l of butyric acid with a butyric acid-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/l of glucose by batch fermentation at pH 6.0. These results indicated that disruption of the buk gene was essential to get high butyric acid selectivity. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556) Keywords : Butyric acid, Clostridium acetobutylicum, Butyrate Kinase References 1. Jang et al., Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity(2014), Metabolic engineering, 23, 165-174 P0423 Isopropanol-Butanol-Ethanol Mixture Production as a Fuel in the Recombinant Clostridium acetobutylicum Won Jun KIM1, Joungmin LEE1, Yu-Sin JANG2, Sang Yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 2BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST 1-Butanol is an attractive biofuel that can replace gasoline, but its low octane rate might produce knocking effect. In the present study, we developed engineered Clostridium acetobutylicum strains that can produce high amount of isopropanol- butanol-ethanol (IBE) mixture. In addition to introduction of the adhB-593 gene encoding NADPH-dependent primary/secondary alcohol dehydrogenase, co-overexpression of the hydGB-593gene, encoding putative ferredoxin:NADP+ reductase, improved IBE titer further. Interestingly, C. acetobutylicum does not harbor any genes orthologous to hydGB-593 unlike other clostridial species, suggesting that C. acetobutylicum might use a different mechanism for NADPH regeneration. The final strain was employed as a host for a pilot-scale fermentation to examine its potential for industrial IBE production. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556) and by the Advanced Biomass R&D Center of Korea (ABC- 2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP).] Keywords : Clostridium acetobutylicum, Isopropanol, Mixed-alcohol fuel References 1. Lee et al., Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation(2012), 78(5), 1416-1423 P0424 Design and Extension of Novel Biochemical Synthetic Pathways for Production of Industrially Valuable Chemicals Jung Ho AHN1, Dong In KIM1, Ayoun CHO1, Hongseok YUN1,2,3, Jin Hwan PARK1,3, Sang Yup LEE1,2,3, Sunwon PARK1 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 Several systematic frameworks have been developed in the past years to predict the metabolic pathways for efficient production of desired chemicals. In this trend, we developed the framework to predict pathways and enzyme candidates through screening process to evaluate feasibility of designed pathways. The systematic framework consists of two parts, route generation and prioritization process. Route generation process designs pathways by the reaction rule sets which were constructed based on the logics acquired from analysis of reaction transformation pattern of existing biochemical reactions. After, five screening factors, binding site covalence, chemical similarity, thermodynamic favorability, pathway distance and organism specificity are evaluated to assess the feasibility of predicted pathways. For validation, novel metabolic pathways for isobutanol, 3-hydroxypropionate (3HP) and butyryl-CoA, were designed using this platform. This systematic framework should be a valuable resource useful for the practice of synthetic biology and metabolic engineering. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] Keywords : Systems metabolic engineering, Isobutanol, 3-Hydroxypropionate References 1. Shin, J.H., Kim, H.U., Kim, D.I., and Lee, S.Y., Biotechnol. Adv., 31(6): 925-935 (2013) P0425 Systemic Analysis of Mannheimia succiniciproducens for Efficient Succinic Acid Production Jung Ho AHN1, Won Jun KIM1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 Mannheimia succiniciproducens has been studied due to its native capabilities to produce a substantial amount of succinic acid. In order to gain further insights, we used elementary mode (EM) analysis, one of the well-established metabolic pathway analysis approaches, and conducted comparative analysis of M. succiniciproducens. Among the thousands of EMs, we used an efficient strategy, pathway clustering analysis, by clustering optimal EMs that have the higher succinic acid production without loss of growth rates to identify metabolism. Pathway clustering analysis could show linear relationships with biomass or succinic acid. In this proof-of-concept study, the metabolism of M. succiniciproducens was designed to enhance succinate production by overexpressing zwf gene and mdh gene in LPK7 strain. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF- 2012-C1AAA001-2012M1A2A2026556).] Keywords : Mannheimia succinciproducens, Succinic acid, Systems metabolic engineering References 1. Lee, S.J., Song, H. and Lee, S.Y., Appl. Environ. Microbiol. 72(3): 1939-1948 (2006) P0426 Biosynthesis of L-Malic Acid Using Metabolically Engineered Mannheimia succiniciproducens Strain Jung Ho AHN1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 L-malic acid is one of the commonly used chemicals in industry. Mannheimia succiniciproducens, a gram-negative facultative capnophilic rumen bacterium, produces succinic acid as its major metabolite using anaplerotic pathway under anaerobic condition. Wild type M. succiniciproducens strain does not naturally produce malate. Using M. succiniciproducens for the production of fumaric acid and malic acid, is advantageous due to its strong anaplerotic pathway under CO2 conditions. Recently, our group reported on the development of high succinic acid production strain by genome engineering. The malate production strain was constructed based on this strain. Especially, when the fumC gene encoding fumarase, which converts malate to fumaric acid, was deleted, this strain produced mostly malic acid instead of succinic acid. These results will provide useful information for the rational metabolic engineering by optimizing metabolic fluxes to improve malic acid production in strains having a strong anaplerotic pathway. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012- C1AAA001-2012M1A2A2026556).] Keywords : L-malic acid, Mannheimia succiniciproducens, Metabolic engineering References 1. Lee, S.J., Song, H. and Lee, S.Y., Appl. Environ. Microbiol. 72(3): 1939-1948 (2006) P0427 Succinic Acid Production Using Mannehimia succiniciproducens under Acidic Condition by Introduction of Acid Resistance System Jung Ho AHN1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701 Bio-based production of succinic acid (SA) from renewable feedstock have potential to replace petroleum-based production. Although great progress has been made, much needs to be developed further to minimize the production cost of the biotechnological process in order to increase its competitiveness with that of petrochemical process. During recovery and purification of SA from fermentation broth, conversion of succinate salt obtained in the course fermentation is converted into free acid. This stage is the most expensive stage influencing the final cost of SA and takes 60-70 % of the product cost. The most promising process implies fermentation without or minimal use of buffering agent where free form of SA is the main product. However, the SA producing bacterial strains up to date are unable to grow effectively at low pH condition. Escherichia coli has developed sophisticated acid resistance (AR) systems to survive in acidic environment and this system has been well-characterized. In this study, two AR systems, glutamine- and glutamate-mediate system, from E. coli were introduced into Mannheimia succiniciproducens, a well-known natural SA producer, for efficient SA production under acid condition. This result will provide useful information to improve strain growth and metabolic capability under acidic condition. Furthermore, reduction in buffering agent and simpler downstream process will lead to more economical SA production. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] Keywords : Acid resistance system, Mannheimia succiniciproducens, Succinic acid References 1. Lee, S.J., Song, H. and Lee, S.Y., Appl. Environ. Microbiol. 72(3): 1939-1948 (2006) P0428 Metabolic Engineering of a buk-Deficient Clostridium acetobutylicum PJC4BK and Its Derivative for Isopropanol-Butanol-Ethanol Production Dongsoo YANG1, Joungmin LEE1,2, Yu-Sin JANG1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioProcess Engineering Research Center, KAIST, Daejeon, Republic of Korea, 3BioInformatics Research Center, KAIST, Daejeon, Republic of Korea 1-Butanol is an attractive biofuel that can replace gasoline, but its low octane rate might lead to engine knocking. To overcome this problem, addition of isopropanol, which has a much higher octane rate than that of gasoline, can be blended. In order to obtaine a high amount of isopropanol-butanol-ethanol mixture, we engineered Clostridium acetobutylicum PJC4BK, which lacks the butyrate kinase gene (buk), and one of its mutant derived by random mutagenesis. In addition to introduction of the adhB-593 gene encoding NADPH-dependent primary/secondary alcohol dehydrogenase, co-overexpression of the hydGB-593 gene, encoding putative ferredoxin:NADP+ reductase, further improved IBE titer. The final strain was employed as a host for a pilot-scale fermentation to examine its potential for industrial IBE production. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556) and by the Advanced Biomass R&D Center of Korea (ABC-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP).] Keywords : Metabolic engineering, Clostridium acetobutylicum, biofuel, Isopropanol-butanol-ethanol References 1. V.G.Rassadin, O.Yu.Shlygin, N.M.Likhterova, V.N.Slavin, and A.V.Zharov, Problems in production of high- octane, unleaded automotive gasolines (2006), Chem. Tech. Fuels Oil, 42, 235-242. P0429 Malonyl-CoA-mediated Acetone Production from CO2 Using Engineered Cyanobacteria Mieun YI1,2, Jun-Won CHWA1,2, Sun-Mi LEE1, Youngsoon UM1, Sang Jun SIM2,3, Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, 2Green School, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea, 3Department of Chemical and Biological Engineering, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea Cyanobacteria have been gathering global interests regarding CO2 bio-mitigation, because of their ability to direct utilization of CO2. Applying metabolic engineering and synthetic biology technics, we metabolically engineered Synechococcus elongatus PCC7942 in order to produce acetone from CO2. Heterologous acetone-producing pathway was introduced into S. elongatus, showing no acetone production under the light and aerobic condition. Thus, we constructed acetone-producing strain harboring heterologous metabolic pathways via ATP-driven malonyl-CoA synthesis pathway. Our experiments of engineering Synechococcus elongatus enabled continuous conversion of CO2 to volatile acetone under the aerobic and lighted culture condition. The synthesized acetone in the medium was analyzed by GC-FID. The engineered strain produced small amount of acetone, compared to engineered E. coli strain. However, further metabolic engineering could be applied to enhance the titer of acetone in S. elongatus. This work was supported by Korea CCS R&D Center (KCRC) (no. 2014M1A8A1049277) grant-funded by the Korean Government (Ministry of Science, ICT & Future Planning). Keywords : Cyanobacteria, Malonyl-CoA, CO2 References 1. Lan E.I. et al., ATP drives direct photosynthetic production of 1-butanol in cyanobacteria (2012), PNAS, Vol(109), 6018–6023 P0430 Versatile SyneBrick Vectors for Gene Expression in Synechococcus elongatus PCC7942 Wook-Jin KIM1,2, Jun-Won CHWA1,2, Sun-Mi LEE1, Youngsoon UM1, Sang Jun SIM2,3, Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, 2Green School, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea , 3Department of Chemical and Biological Engineering, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul, Republic of Korea Due to uprising global warming and understanding of positive carbon cycle, a photosynthetic cyanobacterium has gather attentions to capture solar energy and convert CO2 to various metabolites. However, cyanobacteria generally lack genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for valuable biochemical from CO2. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC7942 as a model cyanobacterium. A standard SyneBrick vector with pTrc promoter showed strong and controllable expressions of the gfp gene as a reporter gene. In addition, a constitutive SyneBrick vector with CbbL Promoter was shown as the half strength of the pTrc promoter. This SyneBrick vectors will be useful to accelerate metabolic engineering of cyanobacterium by expressing and controlling multiple genes of interests. This work was supported by Korea CCS R&D Center (KCRC) (no. 2014M1A8A1049277) grant-funded by the Korean Government (Ministry of Science, ICT & Future Planning). Keywords : Cyanobacteria, Vector, Co2 References 1. Taek Soon Lee et al., BglBrick vectors and datasheets: A synthetic biology platform for gene expression(2011), Journal of Biological Engineering, 5(12) P0431 Automatic Generation of Secondary Metabolite Biosynthetic Reactions to Update Host Strain's Network Kyu-Sang KYU-SANG1,2, Hyun Uk KIM1,2,4, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea, 4The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark Exploration of novel natural products from various microorganisms to plants has explosively been increased by the evolving tool of synthetic biology at high speed combined with bioassay-based screening tool. Furthermore, rapid and more cost effective genome sequencing tools in combination with fast growing computational power have converged to transform previous experimental-based discovery toward a more rational and predictive pursuit. We have developed a rapid method of generation of metabolic reactions and concomitant chemical structures for multiple modular polyketide, nonribosomal peptide, and hybrid combination of polyketides and nonribosomal peptides with output in a text format that can be readily converted to sbml file using other software pipeline. Our program can assemble various metabolic reactions composed of twenty amino acids and eighteen other chain elongation intermediates used in nonribosomal peptide systems, ten acyl-CoA starter and extender units used in polyketide systems. This system also includes cofactors along the incorporation process for the assembly of their natural products. Using this approach we have established the system for rational prediction of gene manipulation target for target natural products from a diverse range of bacterial genomes. In accelerating genome mining data to analysis of metabolic engineering, this method bridges the interface between putative novel metabolite and experimental-based validation and enables rapid scanning for their compounds. Keywords : automatic modeling, genome-scale metabolic model, secondary metabolites, polyketide, non-ribosomal peptide, flux balance analysis References 1. Blin, K., et al. Nucleic acids research 2013;41(Web Server issue):W204-212. 2. Boddy, C.N. Journal of industrial microbiology & biotechnology 2014;41(2):443-450 3. Ebrahim, A., et al. BMC systems biology 2013;7:74 P0432 Enhanced Transcription in Clostridial Species by Using Synthetic Untranslated Regions Dongsoo YANG1, Joungmin LEE1,2, Yu-Sin JANG1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and Synthetic Biotechnology, Institute , 2BioProcess Engineering Research Center, KAIST, Daejeon, Republic of Korea, 3BioInformatics Research Center, KAIST, Daejeon, Republic of Korea Optimization of a gene expression system for clostridia has been relatively less focused on due to their difficult nature of gene knockout and chromosomal manipulation. In this study, we were able to enhance not only gene expression strengths but also gene knock-down efficiencies by antisense RNAs through re-investigating previously elucidated features of the native promoters, considering RNA decay, and employing a strong, synthetic terminator. After achieving strong gene expression in Clostridium acetobutylicum, controlled gene expression via fine-tuning of ribosomal binding sites was employed to the metabolic engineering of C. acetobutylicum strains. [This work was supported by the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) and by the Advanced Biomass R&D Center of Korea (ABC-2011-0028386) through the Global Frontier Project of the Ministry of Science, ICT and Future Planning (MSIP).] Keywords : Metabolic engineering, Clostridium, Gene expression, Synthetic untranslated regions References 1. Lutke-Eversloh, T. Application of new metabolic engineering tools for Clostridium acetobutylicum (2014), Appl Microbiol Biotechnol, 98, 5823-5837. P0433 Enhanced Production of D-psicose from Corynebacterium glutamicum by Engineering of Sugar Transportation. Dae-Yun LEE1, Seong-Hee JEONG1, Min-Jin CHOI1, Rachelle CANETE1, Sang-Hwal YOON2, Hyeon-Seo LEE2, Seon- Won KIM1 1Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea, 264 Dongburo 1259, Jinsung, Jinju, South korea Amicogen,Inc. Rare sugars, referred to as monosaccharides and their derivatives that rarely exist in nature, are known to have various biological functions and applications ranging from foodstuffs to pharmaceutical and cosmetic industry. Likewise, D-psicose, a C-3 epimer of D-fructose, is a rare sugar which is known to be hypolipidemic, neuroprotective and also has antioxidant activities and an insulin resistance effect. It is an ideal sucrose substitute, having 70% of its relative sweetness. In addition, it has only 0.3% of the calories of sucrose and has high water solubility of 4% (w/w) at 25 °C. However, production of psicose by natural and chemical means has been difficult. Few studies on the biological production of D-psicose from D-fructose have been reported. Conversion of D-fructose to D-psicose by whole cell bioconversion reaction with recombinant cells of Corynebacterium glutamicum requires the great influx of substrates of D-Psicose 3-epimerase such as D-fuctose. However, D-psicose formation is limited by the uptake of D-fructose in its free form. So, transporters which uptake D-fructose inside the cell need to be over-expressed. This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant# : PJ011062), RDA, Korea Keywords : Psicose, Fructose, Sugar transport, Corynebacterium glutamicum, D-Psicose 3-epimerase References 1. Tokuda, M.; Hossain, M. A.; Kitagaki, S. Rare sugar D-psicose ameliorates glucose intolerance through glucokinase activation in the liver and suppression of pancreatic islet injury in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. J. Gastroenterol. Hepatol. 2010, 25, A124. 2. Carsten Baumchen, Eva Krings, Stephanie Bringer, Lothar Eggeling & Hermann Sahm.(2009). Myo -inositol facilitators IolT1and IolT2 enhance D-mannitol formation from D-fructose in Corynebacterium glutamicum. FEMS Microbiol Lett 290:227–235. 3. Min-Kyoung Kang ; Jungseok Lee ; Youngsoon Um ; Taek Soon Lee ; Michael Bott ; Si Jae Park & HanMin Woo. Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization. Appl Microbiol Biotechnol (2014) 98:5991–6002 P0434 Optimization of Medium Components for 1,3-Propanediol Production by Enterobacter gergoviae Jinyeong KIM, Eunsoo HONG, Yeonwoo RYU Department of Molecular Science and Technology, Ajou University, Suwon, South Korea The production of glycerol as a byproduct of biodiesel industries is increasing, and price of glycerol has decreased. Therefore, glycerol is good feedstock for industrial fermentation and converted to various chemicals such as 1,3-propanediol. 1,3-propanediol as a bifunctional organic compound could potentially be used for many synthesis reactions, in particular as a monomer for polycondensations to produce polyesters, polyethers and polyurethanes. Various microorganisms were cultured in LB medium containing glycerol at aerobic condition for 1,3-propanediol production, and 1,3-propanediol production was compared by high performance liquid chromatography (HPLC) system. As a result, Enterobacter gergoviae showed the highest 1,3-propanediol production among various microorganisms. And then, medium components were optimized for increasing of 1,3-propanediol production from Enterobacter gergoviae. The effects of various medium components on 1,3- propanediol production were determined by one factor at a time method, and we selected the three factors (glycerol, Na2HPO4, and NH4Cl) which have great effects on 1,3-propanediol production. The optimum concentration of three factors was determined by response surface methodology (RSM) for maximum 1,3-propanediol production. Keywords : 1,3-propanediol, Enterobacter gergoviae, response surface methodology, Optimization References 1. F. Barbirato, Glycerol fermentation by a new 1,3-propanediol-producing microorganism Enterobacter agglomerans(1995), Applied Microbiology and Biotechnology, 43:786-793 2. Eunsoo Hong, Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4(2013), Bioprocess and Biosystems Engineering, 36:835–843 P0435 Production of 1,3-Propanediol from Glycerol by Engineered K. pneumoniae AJ4-ES01 by Inactivating Lipopolysaccharide Biosynthesis Eunsoo HONG, Jinyeong KIM, Yeonwoo RYU Department of Molecular Science and Technology, Ajou University, Suwon, 443-749, Korea The potential of a new isolate, K. pneumoniae AJ4, for the production of 1,3-PDO was investigated by optimization of medium constituents and culture conditions [1]. By adopting these optimization strategies, a titer of 52.59 g/L 1,3-PDO, productivity of 2.02 g L-1h-1, and yield of 0.63 mol/mol were obtained during batch fermentation. However, the pathogenic characteristics of K. pneumoniae could pose hurdles in the industrial production of 1,3-PDO. To eliminate the pathogenicity determinants from 1,3-PDO producing K. pneumoniae AJ4, wabG involved in the LPS biosynthesis was disrupted using homologous recombination technique. This gene encodes glucosyltransferase, which is involved in the transfer of the first outer core LPS residue (α-L-glycero-D-manno-heptopyranose II) at the O-3 position of the last inner core LPS residue (α-D- galactopyranosyluronic acid) in K. pneumoniae AJ4 [2]. In this study, wabG-inactivated strain, K. pneumoniae AJ4-ES01, was constructed and compared the titer, yield, and productivity of 1,3-PDO with the parental strain. In addition, the effect of initial glycerol concentration and initial calcium carbonate (CaCO3) concentration on cell growth and pH control were examined, respectively. K. pneumoniae AJ4-ES01 was found that it has immense potential for 1,3-PDO production, subject to elimination of the pathogenic natured of the strain. Keywords : Klebsiella pneumoniae, 1,3-Propanediol, Lipopolysaccharides References 1. Hong E, Yoon S, Kim J, Kim E, Kim D, Rhie S, and Ryu YW. Isolation of microorganisms able to produce 1,3- propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4 (2013), Bioprocess and biosystems engineering, 36, 835-843 2. Izquierdo L, Coderch N, Pique N, Bedini E, Corsaro MM, Merino S, Fresno S, Tomas JM, and Regue M. The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence (2003), Journal of bacteriology 185, 7213-7221. P0436 Selection of RNA Aptamer for Caprolactam by Systematic Evolution of Ligands by Exponential Enrichment Sungyeon JANG1, SungHo JANG1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam- Gu, Pohang, Gyeongbuk, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, Korea Caprolactam is a monomer of Nylon 6 used for fiber and plastic, and the global caprolactam market is estimated to $US 10.6 billion per year. RNA aptamer is applicable for diverse fields such as therapeutic agent, research reagent, and diagnosis. Because RNA aptamer that specifically binds to caprolactam has not been discovered or developed, we performed systematic evolution of ligands by exponential enrichment (SELEX) that consists of repeated series of processes in order to obtain synthetic RNA aptamer for caprolactam. We carried out several rounds of SELEX and obtained a pool of RNA aptamer and the enriched aptamer pool can be applied in various areas of biotechnology where in vitro or in vivo detection of caprolactam is needed. Keywords : Caprolactam, RNA aptamer, SELEX References 1. R. Stoltenburg, C. Reinemann, and B. Strehlitz, SELEX-A (r)evolutionary method to generate high-affinity nucleic acid ligands(2007), Biomolecular Engineering, 24(4), 381-403. P0437 Engineering Escherichia coli to Produce Coenzyme B12 for Biosynthetic 3-HP Production MyungHyun NOH1, HyunGyu LIM1, SungHoon PARK3, GyooYeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam- Gu, Pohang, Gyeongbuk, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, Korea, 3Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735, Republic of Korea As a solution of the environmental problem and energy crisis due to abuse of fossil fuel, carbon source needs to be renewable and sustainable. 3-Hydroxypropionic acid(3-HP) is one of the valuable platform chemicals and widely used in many industrial applications. Its production through microorganism was developed, however, this process requires supplement of an expensive coenzyme B12 for activation of 3-HP producing enzymes. In this research, B12 production pathway was introduced from Pseudomonas denitrificans and reconstructed in Escherichia coli for B12-independent 3-HP production. By manipulating 5'-UTR sequence using UTR designer which predicts the accurate expression level according to its sequence, enzyme balancing in metabolic node was achieved for maximum or optimum expression coenzyme B12. Also, using riboselector which is specific to B12, high producer strain will be selected and produce B12. Keywords : 3-HP, B12, coenzymeB12 References 1. Ko, Yeounjoo, et al. P0438 Multi-objective Optimization to Minimize Byproduct Secretions from an Engineered Strain Kyu-Sang HWANG1,2, Hyun Uk KIM1,2, Jong Myoung PARK1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea., 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea Separation and purification of a target chemical is a cost-intensive process in a microbial production of industrial chemicals. Therefore, minimization of byproducts secreted from engineered strains helps reducing the cost required for downstream process. In this study, we conduct multi-objective optimization of a microbial host strain’s metabolic model in order to minimize byproducts in addition to conventionally considered objectives of maximal target chemicals production and sufficiently high cellular growth rate [1]. This systematic approach was demonstrated by experimentally engineering E. coli strains producing acetic, lactic and succinic acids. E. coli strains producing homo-acetic and homo-lactic acids were successfully constructed through a minimal number of knockouts of computationally predicted gene targets. On the contrary, effective gene knockout targets for the homo-succinic acid production in E. coli failed to be predicted, suggesting that E. coli is not a suitable host strain for the production of a homo-succinic acid. This systems metabolic engineering approach can be considered for the production of other industrially valuable chemicals.[This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF- 2012M1A2A2026556). Further support from Samsung Advanced Institute of Technology is appreciated.] Keywords : Multi objective optimijation, genome-scale metabolic model, gene knockout target, succinic acid References 1. T. Y. Kim1, J. M. Park1, H. U. Kim1, K. M. Cho and S. Y. Lee, Metab. Eng. 28, 63-73 (2015) P0439 Metabolic Engineering of Escherichia coli for (-)-α-Bisabolol Production Ju-Eon PARK, Chonglong WANG, Seon-Won KIM Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju, 660-701 α-Bisabolol is a natural-occuring sesquiterpene with applications in cosmetics as whitening and soothing agent. It is synthesized from the universal precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are generated either through the mevalonate (MVA) pathway or the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway. Farnesyl pyrophosphate (FPP) synthase then catalyzes the condensation of IPP and DMAPP to the linear FPP, which is rearranged and cyclized by bisabolol synthases into (-)-α-bisabolol. Here, we compared various α-bisabolol synthases from different organisms including Lippia dulcis, Streptomyces citricolor, Santalum spicatum, Matricaria recutita, and Artemisia annua. MVA pathway and FPP synthase were also overexpressed to supply sufficient FPP for bisabolol synthesis in the recombinant E. coli. Bisabolol synthase from M. recutita shows the highest activity of bisabolol synthesis, by which α-bisabolol of 1.5g/L was produced in a test-tube culture. The production was further increased to 3.2g/L by a fed- batch culture. This work was supported by a grant (NRF-2013-R1A1A2-008289) from the National Research Foundation, and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009522003), RDA, Korea Keywords : bisabolol, sesquiterpene, E. coli, isoprenoid, MVA References 1. C.Wang, Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli (2013), Metabolic Enegineering, 18,53-59 2. J. Lee, Whitening effect of a-bisabolol in Asian women (2010), Int Journal of Cosmetic Science. 32, 200-303 3. YJ.Son, Enantioselective microbial synthesis of the indigenous natural product (−)-α-bisabolol by a sesquiterpene synthase from chamomile (2014) Biochem. J.463, 239-248 P0440 Metabolic Engineering for Production of 2-Methyl-1-propanol via Improving Mitochondrial Pyruvate Pool in Saccharomyces cerevisiae. Seong-Hee PARK, Ji-Sook HAHN School of Chemical and Biological Engineering Higher alcohols including isobutanol (2-methyl-1-propanol) are considered as next generation transport fuels because of their higher energy density and lower moisture absorption compared with ethanol. Saccharomyces cerevisiae, which produces small amounts of isobutanol via Ehrlich pathway from the catabolism of valine, is a promising host for the production of isobutanol because of its high tolerance to alcohols and other harsh industrial conditions. In this study, isobutanol was produced in mitochondria by targeting two cytosolic enzymes of the Ehrlich pathway (Aro10 and Adh2) to the mitochondria and increasing mitochondrial pyruvate pool. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. In addition, genes involved in isobutanol production (ILV2, ILV3, and ILV5), LEU3Δ601 which encodes a constitutively active form of Leu3 transcriptional activator for leucine biosynthesis, as well as ARO10 and ADH2 containing mitochondrial targeting sequences were overexpressed. To increase pyruvate flux to isobutanol synthesis in mitochondria, LPD1, one of component of pyruvate dehydrogenase complex, was deleted. Furthermore, mitochondrial pyruvate carrier (MPC) proteins were overexpressed to enhance the mitochondrial pyruvate pool. Among the combinatorial overexpression of three MPC proteins, Mpc1, Mpc2, and Mpc3, forming a hetero-oligomeric complex in the inner mitochondrial membrane, overexpression of MPC1 and MPC3 was most effective in increasing isobutanol production. The final engineered strain produced 330.9 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared with wild type. Keywords : Saccharomyces cerevisiae, Isobutanol, 2-Methyl-1-propanol, Yeast, Mitochondrial pyruvate carrier P0441 Identification of Endogenous Aldehyde Reductases in E. coli for the Improvement of Ethylene Glycol Production from D-Xylose Rhudith CABULONG1, Kris Niño VALDEHUESA1, Kristine Rose RAMOS1, Llewelyn MORON1, Hee-jin YANG1, Grace NISOLA1, Won–Keun LEE2, Wook-Jin CHUNG1 1Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), 2Division of Bioscience and Bioinformatics, Myongji University The chemical precursor ethylene glycol (EG) is produced commercially by hydration of ethylene, or through OMEGA process by Shell Company. Recently, a biosynthetic pathway for conversion of xylose to EG was developed in E. coli. This pathway consists of four steps: xylose → xylonate → 2-dehydro-3-deoxy-pentonate → glycoaldehyde → EG. The objective of the present study is to identify and over-express endogenous aldehyde reductase(s) (ALRs) involved in the final step of EG pathway, and to improve product titer. Four native ALR genes from E. coli (adhP, yahK, yjgB, and yqhD) were selected for further investigations. Interestingly, EG concentration was highest in the yjgB expression strain, especially when there is no addition of IPTG. Furthermore, the crude cell lysate of the strain harbouring yjgB overexpression showed the highest enzymatic activity towards glycoaldehyde reduction. These results show that overexpression of yjgB is beneficial for the improvement of ethylene glycol production. Keywords : yjgB, E. coli, ethylene glycol References 1. Liu H, Ramos KRM, Valdehuesa KNG, Nisola GM, Lee WK, Chung WJ (2013) Biosynthesis of ethylene glycol in Escherichia coli. Appl Microbiol Biotechnol 97: 3409-3417. P0442 Production of Polyhydroxyalkanoates by Ralstonia eutropha Using Sucrose So Young CHOI1, Si Jae PARK2, Young-Ah JANG3, Won NOH3, Young Hoon OH3, Hyuk LEE4, Yokimiko DAVID2, Mary Grace BAYLON2, Jihoon SHIN3, Jung Eun YANG1, Seung Hwan LEE3, Sang Yup LEE1 1Dept. of Chemical and Biomolecular engineering, KAIST, Daejeon, 305-701 , 2Dept. of Environmental Engineering and Energy, Myongji University, Gyeonggido, 449-728 , 3Industrial Biochemicals Research Group, Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon, 305- 600, 4Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon, 305-600 To decrease the microbial polyhydroxyalkanoates (PHAs) production cost, Ralstonia eutropha was engineered to utilize sucrose which is abundant and relatively inexpensive carbon sources from sugarcane and sugar beet. Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 expressed Mannheimia succiniciproducens MBEL55E sacC gene that encodes β- fructofuranosidase. The β-fructofuranosidase excreted and hydrolyzed sucrose to glucose and fructose, then these were used by R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in defined medium containing sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co- 21.5mol%lactate) by the expression of the sacC gene and the Escherichia coli ldhA gene. [This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF- 2012-C1AAA001-2012M1A2A2026556)] Keywords : Polyhydroxyalkanoates, PHA, Biopolymer, Sucrose, Metabolic engineering, Ralstonia eutropha References 1. S. J. Park. et al., Metab. Eng. 20:20-28 (2013) P0443 Enhanced Production of Poly(lactate-co-3-hydroxybutyrate) by Introducing of Phasin from Aeromonas hydrophila So Young CHOI1, Min Kyung KIM1, Si Jae PARK2, Sang Yup LEE1 1Dep. of Chemical and Biomolecular Engineering, Daejeon, 305-701 , 2Dep. of Environmental Engineering and Energy, Myongji University, Gyeonggido, 449-728 Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are accumulated in form of granules within cytoplasm of many bacteria as carbon storage materials. Phasins (PhaP) are granule associated proteins which accumulate during PHA synthesis. It is also known to stabilize the PHA formation, and to regulate monomer composition of their copolymer. Here, the gene phaPAh from Aeromonas hydrophila 4AK4 was cloned and characterized for production of poly(3-hydroxybutyrate- co-lactate). Recombinant strains harboring phaP gene and polyhydroxyalkanoate (PHA) synthase gene (phaC) accumulated poly(3-hydroxybutyrate-co-lactate) consisting of upto 47.2 mol% of LA and 41.52 wt% polymer contents. The introduction of phasin resulted in increase of the lactate fraction and polymer contents and different granule morphology. [This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556).] Keywords : Polyhydroxyalkanoates, Biopolymer, Polylactic acid, Metabolic engineering, Phasin References 1. Y.K. Jung, S.Y. Lee, J.Biotechnol 151; 94-101. (2011) 2. G. M. York, J. Stubbe, A. J. Sinskey, J.Biotechnol 184.1: 59-66. (2002) P0444 Metabolic Engineering for the Biosynthesis of 2-Hydroxycarboxylic Acid Containing Polymers in Ralstonia eutropha So Young CHOI1, Si Jae PARK2, Seung Hwan LEE3, Young Hoon OH3, Jung Eun YANG1, Sang Yup LEE1 1Dep. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, 2Dep. of Environmental Engineering and Energy, Myongji University, Gyeonggido, 449-728, 3Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon, 305-600 Polyhydroxyalkanoates (PHAs) are microbially synthesized polyesters in many microorganisms. Previously, we have metabolically engineered Escherichia coli for the production of 2-hydroxyacids (lactate and 2-hydroxybutyrate) containing PHAs from structurally unrelated carbon sources by one-step fermentative process. Here, we report the metabolic engineering strategies for the development of recombinant Ralstonia eutropha to synthesize PHAs containing 2- hydroxyacids as monomers. This could be achieved by replacing the R. eutropha phaC gene with either the R. eutropha phaC S506G A510K gene or the Pseudomonas sp. MBEL 6–19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. Expression of the E. coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) from glucose. [This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556) ] Keywords : Polyhydroxyalkanoates, Metabolic engineering, Ralstonia eutropha, Biopolymer, Polylactic acid, Poly(3- hydroxybutyrate) References 1. Y.K. Jung, S.Y. Lee, J.Biotechnol 151; 94-101. (2011) P0445 Biosynthesis of Polylactic Acid and Its Copolymers by Metabolically Engineered Escherichia coli So Young CHOI1, Si Jae PARK2, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering, KAIST, , Daejeon, 305-701, 2Dept. of Environmental Engineering and Energy, Myongji University, Gyeonggido, 449-728 Polylactic acid (PLA) has been considered as a good alternative to petroleum-based plastic as it possesses several desirable properties such as biocompatibility, biodegradability. For biosynthesis of PLA, we introduced the heterologous metabolic pathways including evolved propionyl-CoA transferase which converts lactate into lactyl-CoA and PHA synthase which converts lactyl-CoA into PLA. For efficient PLA production, E. coli was engineered to strengthen lactate flux based on in silico genome-scale metabolic flux analysis. Several target genes were inactivated and replaced the native ldhA promoter into strong trc promoter. It resulted in enhanced synthesis of PLA and P(3-hydroxybutyrate-co-LA) in E. coli. In this study, the strategy of combined systems-level metabolic engineering and enzyme engineering allowed one-step production of PLA and its copolymers in E. coli. [This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] Keywords : Polylactic acid, Polyhydroxyalkanoates, Metabolic engineering, Escherichia coli, Biopolymer References 1. Y.K. Jung, S.Y. Lee, J.Biotechnol 151; 94-101. (2011) P0446 Metabolic Engineering for Engineered E. coli Utilizing Alginate Polysaccharide in Brown Seaweed Donghun KWAK1, Hyun Gyu LIM2, Gyoo Yeol JUNG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 2Department of Chemical Engineering, Pohang University of Science and Technology Brown macroalgae, well-known seaweed in Asia, consists of abundant sugar components such as alginate, mannitol and glucan. However, it is difficult for industrial microorganisms to utilize alginate polysaccharide due to the lack of utilization pathway. Thus, to convert brown macroalgae into valuable chemicals, its utilization pathway should be introduced and optimized for maximum yield and productivity. In this research, two types of strategies were used to solve these problems. First, In order to degrade alginate polysaccharide, MJ3 alginate lyase which has exo- and endotype activity was overexpressed. And then, alginate media was digested by MJ3 enzyme. As a result, 5.5 g/L alginate monosaccharide called DEHU was analyzed through TLC method by using 1% alginate media. Second, alginate metabolic pathway was integrated into Escherichia coli chromosome to utilize alginate through the synthetic biology approach. The genes encoding alginate transporter and reductase were expression under synthetic promoter and optimized 5’-UTR level for maximum utilization. It is expected that the engineered strain will show capability to directly utilize non-fermentable sugars in brown macroalgae as carbon sources and this microbial platform will be further applied to produce fuels and chemicals. Keywords : alginate, metabolic engineering, synthetic biology, brown seaweed References 1. A. Wargacki et. al., Science, 2012: 335 (6066), 308-313. P0447 Leak-free Production of n-Butanol through Fine-tuning of Glycolytic Flux in E. coli Jae Hyung LIM1, Gyoo Yeol JUNG1,2 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Gyeongbuk, Korea., 2Department of Chemical Engineering, POSTECH, Pohang, Gyeongbuk, Korea. Maximizing the cell factory in terms of yield and productivity is important for high-volume (and low-value) biofuels, especially n-butanol. Although many engineering success improved n-butanol yield and productivity, considerable pyruvate- formation as by-product resulted in lowering the both yield and productivity of microbial factories. To address this, we speculated tuning the glycolytic flux through phosphotransferase system (PTS) that optimizes the efficiency of product- forming pathway, which we termed leak-free pathway engineering, is much beneficial to the cell factory so as not to waste carbon as the by-products, while it can retain maximal productivity of n-butanol. We demonstrated yield and productivity of n-butanol controlled depending on glycolytic flux (65% to 93% and 0.168 to 0.215 g/L/h, respectively). In result, the best trade-off strain improved 20% in yield, but the productivity showed nearly identical to the parental strain (0.214 g/L/h). Our approach would pave the way to explore the optimal glycolytic flux for the cost-effective production of various chemicals and fuels in the field of metabolic engineering and synthetic biology. Keywords : butanol, glycolysis, UTR engineering, Escherichia coli, PTS, synthetic biology, metabolic engineering References 1. Lim, J. H., Seo, S. W., Kim, S. Y., Jung, G. Y., 2013b. Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli. Metab. Eng. 20, 56-62. P0448 Comparative Proteome of Scenedesmus dimorphus Wild Type and Mutant with Higher Lipid Content Jong-il CHOI Department of Biotechnology and Bioengineering, Interdisciplinary program for Bioenergy and Biomaterials, Chonnam National University, Gwangju 500-757, South Korea This study was conducted to investigate the proteomic changes in Scenedesmus dimotphus mutant with enhanced lipid content. S. dimorphus was gamma-irradiated, and then a mutant (Sd-Pm210) with 25% increased lipid content was selected using Nile-red staining thodology. Several proteins related to lipid synthesis and energy metabolisms were verexpressed in the mutant strain. Keywords : Scenedesmus dimotphus , proteome, lipid References 1. Choi, J., Yoon, M., Joe M., Park H., Lee, S. G., Han, S. J., Lee, P. C. (2014) Bioprocess and Biosystems Engineering, 37: 2437-2444. P0449 Synthetic Scaffolds Introduction for the Efficient Production of Gamma-Aminobutyric Acid from Glucose in Recombinant Escherichia coli Van Dung PHAM, Jaewook KIM, Sang Woo JUNG, Soon Ho HONG Department of Chemical Engineering, University of Ulsan, Ulsan, 680-749 Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92 g/L of GABA was produced from 10 g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30oC, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3 g/L was obtained from 10 g/L glucose. The recombinant E.coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold. This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ01111601) by RDA, and Basic Science Research Program by the Ministry of Education (NRF-2014R1A1A2054726). Keywords : gamma-aminobutyric acid , synthetic protein scaffold, Escherichia coli References 1. D. Ma, P. Lu, C. Yan, C. Fan, P. Yin, J. Wang, and Y. Shi, Nature 483, 632 (2012). P0450 Designed Biosynthesis of FK506 Derivatives Containing Altered Side Chain by Genetic Engineering Ji Yoon BEOM, Eun Ji KIM, Jae-yeon HWANG, He Qing CUI, Xu ZHAO, Yeo Joon YOON Department of Chemistry and Nano Science, Ewha Womans University, 11-1, Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea FK506 is a 23-membered macrocyclic polyketide exhibiting various biological activities that was first discovered from the soil bacterium Streptomyces tsukubaensis in 1984. And it has become a clinically important drug used to prevent the rejection of organ transplants. FK506 is biosynthesized by a hybrid polyketide synthase (PKS) / nonribosomal peptide synthetase (NRPS) system. Polyketide backbone can be engineered by building block substitutions. In the assembly line of type I PKS, modular acyltransferase (AT) domain act as gatekeeper of extender unit. Although AT domain generally possess a strict substrate specificity, the AT domain of the fourth module in FK506 PKS has a quite flexible specificity. Recently a three gene operon encoding the biosynthesis of the PKS building block isobutyrylmalonyl-CoA (ibMCoA), associated with ansalactam A from the marine bacterium Streptomyces sp. CNH189, was identified. In this study, exogenous pathways of ibMCoA extender unit are replaced with the endogenous allylmalonyl-CoA pathway to produce the unnatural polyketide engineered biosynthesis of 36-methyl-FK506. The relaxed specificity of the native dehydrogenase tcsD and the AT4 domain in FkbB contribute to the successful engineering of 36-methyl-FK506 in Streptomyces sp. KCTC11604BP. Introducing biosynthetic gene cluster encoding new extender unit has the possibility to produce further new FK506 analogues, which are not produced in nature and have therapeutic potential. Keywords : FK506 derivatives, isobutyrylmalonyl-CoA, 36-methyl-FK506, Genetic engineering References 1. YH Ban, JH Lee, GR Gu, BLee, SJ Mo, HJ Kwon and YJ Yoon. Mutational biosynthesis of a FK506 analogue containing a non-natural starter unit (2013), Molecular BioSystems. 9, 944-947 2. SJ Mo, DH Kim, JH Lee, JW Park, DB. Basnet, YH Ban, YJ Yoo, SR Park, Ej Kim, SK Lee, JY Park, MOk Lee, KS Lee, SJ Kim, D Kim, BC Park, S Lee, HJ Kwon, JW Suh, BS. Moore, SK Lim, and Yeo Joon Yoon. Biosynthesis of the Allylmalonyl-CoA Extender Unit for the FK506 Polyketide Synthase Proceeds through a Dedicated Polyketide Synthase and Facilitates the Mutasynthesis of Analogues (2011), JACS 133 (4), 976-985 P0451 Metabolic Engineering of Klebsiella oxytoca for 2,3-Butanediol Production Based on Genome- Scale Simulation Jong Myoung PARK, Hyohak SONG, Doyoung SEUNG Research and Development Center, GS Caltex Corporation, 104-4 Munji-dong, Yuseong-gu, Daejeon 305-380, Republic of Korea 2,3-butanedol (2,3-BD), synthesized as one of the fermentation products, is a promising chemical owing to its wide industrial applications. Klebsiella oxytoca naturally produces a large amount of 2,3-BD along with various byproducts. In this study, the in silico gene knockout simulation was carried out by using the genome-scale metabolic model of K. oxytoca, KoxGSC1457 for the enhanced production of 2,3-BD. The genes of ldhA and pflB were given as knockout candidates of top priority based on the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed high 2,3-BD production yield of 0.44 g/g glucose (about 90% of theoretical maximum yield). Based on in silico investigation, the effect of agitation speed on 2,3-BD production was investigated. The condition of aeration during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis. Then, we optimized the condition of fed-batch fermentation and produced 113 g/L of 2,3-BD, with a yield of 0.45 g/g and a productivity of 2.1 g/L/h of 2,3-BD. [This work was supported by the Industrial Strategic Technology Development Program (No. 10050407) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).] Keywords : 2,3-butanediol, Klebsiella oxytoca, in silico, genome-scale metabolic model References 1. J. M. Park, H. Song, H. J. Lee, and D. Seung, In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production (2013), J. Ind. Microbiol. Biotechnol. 40, 1057- 1066 2. J. M. Park, H. Song, H. J. Lee, and D. Seung, Genome-scale reconstruction and in silico analysis of Klebsiella oxytoca for 2,3-butanediol production (2013), Microb. Cell Fact. 12, 20 P0452 Metabolic Engineering of Klebsiella oxytoca for the Enhanced Production of (R,R)-2,3- Butanediol Jong Myoung PARK, Chelladurai RATHNASINGH, Hyohak SONG, Doyoung SEUNG Research and Development Center, GS Caltex Corporation, 104-4 Munji-dong, Yuseong-gu, Daejeon 305-380, Republic of Korea A racemic mixture of 2,3-butanediol ((R,R)-BD, (S,S)-BD, and meso-BD) is generally produced by the fermentation of natural microorganisms. The meso-2,3-BD can make difficulties for its transportation and storage because of its higher freezing point than (R,R) and (S,S) forms. Thus, we engineered a Klebsiella oxytoca strain metabolically to overproduce (R,R)-2,3-BD. Klebsiella oxytoca ΔldhA ΔpflB strain, which shows an outstanding performance for the production of 2,3-BD with more than 90% of the meso form, was used as a host strain. A budC gene encoding 2,3-BD dehydrogenase in the K. oxytoca ΔldhA ΔpflB strain was replaced with an exogenous gene encoding (R,R)-2,3-BD dehydrogenase from Paenibacillus polymyxa. Then, the expression level of (R,R)-2,3-BD dehydrogenase was further amplified. The K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH (pBBR-PBDH) strain produced 106.7 g/L of (R,R)-2,3-BD [meso-2,3-BD, 9.3 g/L] by fed-batch fermentation, with a yield of 0.40 g/g and a productivity of 3.1 g/L/h. [This work was supported by the Industrial Strategic Technology Development Program (No. 10050407) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).] Keywords : (R,R)-2,3-butanediol, Klebsiella oxytoca, metabolic engineering References 1. J. M. Park, H. Song, H. J. Lee, and D. Seung, In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production (2013), J. Ind. Microbiol. Biotechnol. 40, 1057- 1066 2. J. M. Park, C. Rathnasingh, and H. Song, Enhanced production of (R,R)-2,3-butanediol by metabolically engineered Klebsiella oxytoca (2015), J. Ind. Microbiol. Biotechnol. In press P0453 Development of Microbial Cell Factory for High-Level Production of Bio-nematicide Hyun Bae BANG1, Yoon Hyeok LEE1, Ki Jun JEONG1,2 1Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141, 2Institute for the BioCentury, KAIST, Daejeon, 34141 Nematodes, plant-parasitic organisms, are harmful to agricultural crops or many plants, causing severe yield losses and death. To eliminate these organisms, chemical pesticides called nematicides have been developed and widely used. However, the use of chemically-synthesized pesticides has been dramatically restricted. In this reason, novel strategies for the production of pesticides have been required. Cinnamaldehyde, which is a volatile and yellow liquid, has been considered as an alternative, because of several reasons including: (i) it is nature-driven compound, and (ii) it shows high nematicidal activity. In this study, synthetic pathway for the production of cinnamaldehyde in Escherichia coli was developed. At first, three kinds of enzymes were used for the construction of basic production system, and then the biosynthesis of cinnamaldehyde was demonstrated. In addition, to increase the production yield of cinnamaldehyde, other enzymes from different organisms were examined. With the production system developed, strain engineering was also performed by systems metabolic engineering strategy for high-level production of phenylalanine which is used as a precursor. Using engineered strain, we could obtain the increased cinnamaldehyde titer compared to previous result which was mentioned above. Keywords : Cinnamaldehyde, Nematodes, Biopesticides, Phenylalanine, Escherichia coli, Systems metabolic engineering References 1. Ooi, L.S., Li, Y., Kam, S.L., Wang, H., Wong, E.Y., Ooi, V.E., Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume (2006), Am J Chin Med, 34(3), 511-522 2. Schneider, K., Hovel, K., Witzel, K., Hamberger, B., Schomburg, D., Kombrink, E., Stuible, H.P., The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase (2003), Proc Natl Acad Sci USA, 100(14), 8601-8606 3. Liu, S.P., Liu, R.X., Xiao, M.R., Zhang, L., Ding, Z.Y., Gu, Z.H., Shi, G.Y., A systems level engineered E. coli capable of efficiently producing L-phenylalanine (2014), Process Biochemistry, 49(5), 751-757 P0454 Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli Eun-Mi KIM1, Jin-Hee EOM1,2, Youngsoon UM1, Yunje KIM1, Han Min WOO1,2 1Clean Energy Research Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea, 2Department of Chemistry, and Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea Monoterpenes are an important and large class of natural products that are widely used in the cosmetic and pharmaceutical industries. [1,2] Among the monoterpenes, myrcene has been considered as a starting material for high-value compounds, such as geraniol/linalool and (−)-menthol. [1] In this study, we metabolically engineered Escherichia coli for production of myrcene by introducing a heterologous mevalonate pathway and overexpressing tailoring enzymes, such as geranyl diphosphate synthase (GPPS) and myrcene synthase (MS). Although MSs have broad ranges of functionality for producing various monoterpenes, our engineered E. coli strains harboring MS from Quercus ilex L. produced only myrcene (1.67 ± 0.029 mg/L). Subsequent engineering resulted in higher production of myrcene by optimizing the levels of GPPS in amino- acid-enriched (EZ-rich) defined medium, where glycerol as a carbon source was used. The production level of myrcene (58.19 ± 12.13 mg/L) was enhanced by 34-fold using in situ two-phase extraction to eliminate cellular toxicity and the evaporation of myrcene. Keywords : Monoterpene, Myrcene, Myrcene synthase References 1. Behr, A.; Johnen, L. Myrcene as a natural base chemical in sustainable chemistry: A critical review. ChemSusChem 2009, 2, 1072−1095 P0455 Expression of Biosynthetic Genes in Streptomyces Venezuelae for the Production of Flavones and Flavonols Shiying JIN, Hea Luyng SHIN, Myoun Su KIM, Jin A JUNG, Ji Young LEE, Yeo Joon YOON* Dept. of Chemistry and Nano Science and Institute of Nano-Biotechnology, Ewha Womans University, Seoul 120-750 Phenylpropanoids, including flavonoids and stilbenes, are plant secondary metabolites with potential pharmacological and nutraceutical properties. In this study, the codon-optimized flavone synthase I gene from Petroselium crispum under the control of the ermE* promoter was cloned using pSE34 as pYJ858 and introduced into the recombinant S. venezuelae DHS2001 bearing a deletion of native pikromycin polyketide synthase gene. As a result, the strain generated flavones from exogenously fed flavanones. LC-ESI-MS/MS analysis showed that 1.4mg/l of apigenin and 2.9 mg/l of chrysin were produced by S. venezuelae DHS2001/pYJ858 supplemented with 0.5 mM naringenin and pinocembrin, respectively. Quantification was conducted in multiple reactions monitoring (MRM) mode in MS/MS. In addition, the genes required for flavonol biosynthesis, synthetic codon-optimized flavanone 3β-hydroxylase gene and flavonol synthase gene from Citrus siensis and Citrus unshius were cloned into pSE34 to construct pYJ861. When 0.5 mM naringenin and pinocembrin were used as precursors, the recombinant strain of S. venezuelae expressing the plasmid (DHS2001/ pYJ861) produced 0.2 mg/l of kaempferol and 1.0 mg/l of galangin, respectively. Here, the flavonoid biosynthetic genes were expressed in an engineered strain of S. venezuelae. This result expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides. Keywords : Flavones, flavonol, heterologous production, Streptomyces venezuelae References 1. Park SR, Yoon JA, Paik JH, Park JW, Jung WS, Ban YH, Kim EJ, Yoo YJ, Han AR, Yoon YJ. Engineering of plant-specific phenylpropanoids biosynthesis in Streptomyces venezuelae (2009). Journal of Biotechnology. 141,181–188 2. Park SR, Park JW, Jung WS, Han AR, Ban YH, Kim EJ, Sohng JK, Sim SJ and Yoon YJ. Heterologous production of epothilones B and D in Streptomyces venezuelae (2008). Applied Microbiology and Biotechnology. 81, 109-117 P0456 Cell-free Synthesis of Cell Penetrating scFv with Enhanced Solubility and Disulfide Bond Formation. Seung Eui MIN1, Christy CATHERINE1, Devi KASI1, Dong Myung KIM1, Dong Ki CHOI2, Young Sung KIM2, Tae Hyeon YOO2 1Department of Applied Chemistry and Biological Engineering, Chungnam National University, 22Department of Applied chemistry and Biological Engineering, Ajou University, Conventional cell-based gene expression methods often fail to produce sufficient amount of functional scFv. In contrast, cell-free protein synthesis system provides a high accessibility to the translational mechanism, thereby providing optimal environments to increase the productivity and solubility of scFv. In this study we attempted to express in a cell-free protein synthesis system scFv antibodies that had been engineered to penetrate into the cells. To increase the solubility, we used DsbA, DsbC, FkpA, SurA enriched extract. We also used a cleavable ubiquitin signal sequence in front of the target scFvs. To assist the formation of correct disulfide bonds, we have changed redox conditions of the cell extract and reaction mixture using GSSG/GSH redox buffer. Taken together, these modifications of the cell-free synthesis system allowed substantial increase in the productivity and solubility of the expressed scFvs. We expect that the established conditions would be applied as a general strategy for producing active scFvs Keywords : Cell-free protein synthesis, scFv, Split GFP complementation, In situ clevage References 1. Oh, I.S., Kim, T.W., Ahn, J.H., Keum, J.W., Choi, C.Y., and Kim, D.M, Biotechnology and Bioprocess Engineering. 12 : 574-578(2007). 2. Oh, I.S., Lee, J.C., Lee, M.S., Chung, J.H., and Kim, D. M, Biotechnology and Bioprocess Engineering. 33 : 127- 132(2010). P0457 Improved L-Lactic Acid Production by Kluyveromyces marxianus Co-expressing Lactate Dehydrogenase Genes with Different pH-Optimum Junghoon IN, Jaewon LEE, Jun-Bum PARK, Jonghyeok SHIN, Da-hyeong CHO, Paul HEO, Myungseo PARK, Young hun JUNG, Byoung-jae KONG, Dae-hyuk KWEON Dept. of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon Plant- and crop-based renewable plastics, including polylactic acid (PLA), are polymeric materials, of which monomers can be produced by fermentation process. Compared to petroleum-based plastics, PLA is biodegradable, biocompatible and bioresorbable. Yeasts have been explored as a candidate for producing lactic acid because they are robust and tolerant to low pH. However, the decline in intracellular pH (pHi) inhibited the activity of lactate dehydrogenase (LDH) enzyme in yeast during fermentation, leading to production of low levels of lactic acid. In this study, we constructed Kluyveromyces marxianus (KM) co-expressing LDHs with different pH-optimum. There combinant KMs showed synergistic effects even at the decline in pHi. The result strains (KMAC, KMBC) produced 14.7 and 15.8 g/L lactic acid from 50 g/L glucose without a neutralizing agent, respectively, which were 1.8 to 3.7-folding higher than lactic acid production of the K. marxianus expressing single LDH gene. The result suggest that lactic acid production can be improved by the complementation between LDHs with different pH-optimum in yeast Keywords : Kluyveromyces marxianus, Lactic acid, Lactate dehydrogenase, Synergistic effect References 1. Timothy L. Turner, Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion (2015), Appl Mircrobial Biotechnol P0458 Inhibition of Methanol Dehydrogenase for Accumulation of Methanol Using Noncoding RNAs in Methanotroph In Yeub HWANG, Eun Yeol LEE Dept. of Chemical engineering, Kyung Hee university,Yongin, 440-746 Production of methanol from methane is possible to derive the production of alternative energy and the solution of global warming. Methanol can be used as a substrate for the production of various value-added products and liquid fuel itself. Methanotrophs can convert methane to methanol using methane monooxygenase (MMO) and generated methanol is converted to formaldehyde immediately by methanol dehydrogenase. Therefore, it is essential the inhibition of methanol dehydrogenase for the accumulation of methanol. Inhibition of methanol dehydrogenase using chemicals to accumulate methanol have been reported. However, addition of chemical compounds is not suitable for accumulation of methanol because of inhibition of other enzymes by chemicals [1]. In this study, we designed noncoding RNAs containing complementary sequences with genes encoding methanol dehydrogenase, which control the expression level of methanol dehydrogenase for the accumulation of methanol [2,3]. We developed a vector system that uses the cumate as inducer for transcription of RNAs. As a result, designed noncoding RNAs inhibit the expression of methanol dehydrogenase and methanol was successfully accumulated. Keywords : Methanotroph, Methanol dehydrogenase, noncoding RNA, methane, methanol References 1. I. Y. Hwang, D. H. Hur, J. H. Lee, C. Park, I. S. Chang, J. W. Lee and E. Y. Lee, Batch Conversion of Methane to Methanol Using Methylosinus trichosporium OB3b as Biocatalyst (2015) , J. Microbiol. Biotechnol., 25(3), 375- 380 2. S. M. Yoo, D. Na and S. Y. Lee, Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli (2013) , Nature, 8(9), 1694-1707 3. A. K. Chaudhary, D. Na, and E. Y. Lee, Rapid and high-throughput construction of microbial cell-factories with (2015), Biotechnol. Adv., 33(6), 914-930 P0459 Redirecting Carbon Flux for Enhancing Fatty Acids Production in Microalgae Tetraselmis sp. by Inhibition of Starch Synthesis Pathway Mi-Ae HAN1, Seong-Joo HONG2, Choul-Gyun LEE3 1National Marine Bioenergy Research center & Dept. of Biotechnology, Inha University, Incheon, Korea, 402-751, 2National Marine Bioenergy Research center & Dept. of Biotechnology, Inha University, Incheon, Korea, 402-751, 3National Marine Bioenergy Research center & Dept. of Biotechnology, Inha University, Incheon, Korea, 402-751 Microalgae are able to accumulate storage metabolites such as starch and lipids under stressful environmental conditions. The lipids are more energy-rich compounds than starch and considered as better feedstock for biofuel production [1,2]. Recently, numerous researches have been reported to enhance the lipid content in microalgae. This study focused on enhancement of the fatty acid production in microalga, Tetraselmis sp. KCTC12432BP, using combination of two chemical inhibitors for blocking of carbohydrate synthesis pathway under nitrogen sufficient (NS) or nitrogen deficient (ND) conditions. Tetraselmis sp. was isolated in Youngheung Island, Incheon, Korea by Marine Bioenergy Research Center (MBE), Inha University. The cells were grown under continuous illumination from fluorescent lamps at 80 µE∙m-2∙s-1 in modified f/2 medium, with aeration of 2% CO2 (balanced with air) at 20℃. Cells in the stationary phase were applied to NS or ND media for 3.5 days and two chemical inhibitors were added to the experimental group for blocking of starch synthesis pathway at the time of medium exchange. The results showed that carbohydrate productivity of the combination of two chemical inhibitiors decreased by 6.6% and 29.3%, while the FAMEs productivity was increased by 15.9% and 32% as compared without two chemical inhibitors under NS or ND conditions, respectively. This study demonstrated that redirection of carbohydrate synthesis to lipid synthesis without affecting growth could be the possible solution for enhancement of biofuel precursor’s productivity. Keywords : Microalgae, Fatty acids , Carbohydrate, Redirection References 1. P. Selvakumar and K. Umadevi, Enhanced lipid and fatty acid content under photoheterotrophic condition in the mass cultures of Tetraselmis gracilis and Platymonas convolutae (2014), Algal Reserach, 6, 180-185 2. R. Miller et al., Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism (2010), Plant Physiology, 154, 1737-1752 P0460 A Genetic Switch for Chemical High-Producing Escherichia coli using Flp-recombinase Jin Yi SONG1, Byung Eun MIN1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, Korea, 37673, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, Korea, 37673 Chemicals production using engineered microorganisms receives attention for the past few years. However, chemical production pathway conflicts with cell growth in a considerable proportion of cases. Thus, it is required to keep balance cell growth and chemical production or to separate growth phase and production phase. Genetic switch which converts promoter strength of cell growth pathway and chemical production pathway in metabolic node is constructed using Flp-recombinase and FRT sequence in Escherichia coli. To confirm the Flp-recombinase is operated, sGFP with strong promoter and mCherry with weak promoter were used to identify fluorescence change after Flp-recombinase is induced. Keywords : Metabolic Engineering, Flp recombinase, Synthetic metabolic pathway References 1. Friedland and Lu, Synthetic Gene Networks That Count (2009), Science, 324(5931), 1199-1202 P0461 Regulation of TCA Cycle for Increase Butanol Production Min-Ji HEO, Sang-Woo LEE, Dae-kyun IM, Seung-Jin KIM, Min-Kyu OH Department of Chemical & Biological Engineering Korea University, Seoul, 136-713, Korea Biobutanol is considered as a potential biofuel, because it is hydrophobic and its energy content is similar to gasoline. Many researchers conducted butanol synthesis experiments from Escherichia coli, an organism easily manipulated. To increase butanol production from Escherichia coli, we constructed butanol synthesis pathway using the genes from Candida boidinii, Salmonella enterica and Lactococcus lactis[1]. To increase butanol production, regulation of TCA cycle and increase of acetyl-CoA pool are important. The enzyme citrate synthase catalyzes the conversion of acetyl-CoA and oxaloacetate to CoA and citrate, which is an intermediate of the TCA cycle. In E.coli, citrate synthase is encoded in the gene gltA. Using CRISPR/CAS9, we modified gltA’s 5’-UTR sequence to decrease citrate synthase expression and regulate TCA cycle. So we obtained several E. coli strains with gradual decreased citrate synthase expression. This modification resulted in changes of growth rate and butanol production. Keywords : Butanol, CRISPR/CAS9, Escherichia coli, Citrate synthase References 1. E. I. Lan, Soo Y. Ro and J. C. Liao, Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria(2013), Energy Environ. Sci., 6, 2672-2681 P0462 An Improved Cell-free Protein Synthesis with Simplified and Cost-effective Continuous- exchange Cell-free System Jin-Ho KWON, Dong-Myung KIM Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea Cell-free protein synthesis (CFPS) is a powerful tool for rapid production of recombinant proteins as well as for functional analysis of genes. The unique flexibility and throughput of cell-free protein synthesis have made this technology a promising alternative to conventional cell-based protein production methods. Despite the versatility of CFPS, however, it requires further improvements in protein productivity. One of the measures for improving yield is a continuous-exchange cell-free (CECF) system. The CECF system supports extended syntheses of target proteins through continuous supply and removal of substrate materials and by-products, respectively. However, previous CECF platforms have suffered from the complexity of reaction devices and unacceptably high reagent costs. In this study, we tried to simplify CECF system with cost-effective substrates. First, we designed a simplified CECF device using common lab materials. In addition, we prepared a concentrated master mix solution to simplify the reaction schemes. Finally, nucleoside triphosphates (NTP) were replaced with nucleoside monophosphates (NMP) that are far more cost-effective. These simplified CECF platform with cost- effective substrates led to a significant increase yield of protein synthesis with a fraction of the cost required for conventional CECF reactions. Keywords : Cell-free Protein Synthesis (CFPS), Contimuous-exchange Cell-free System (CECF), nucleoside onophosphates (NMP) References 1. Kara A. Calhoun and James R. Swartz, An Economical Method for Cell-Free Protein Synthesis using Glucose and Nucleoside Monophosphates (2005), Biothechnology Progress, 21 (4), 1146-1153 2. Dong-Myung Kim and James R. Swartz, Prolonging Cell-Free Protein Synthesis by Selective Reagent Additions (2000), Biothechnology Progress, 16 (3), 385-390 3. Tae-Wan Kim, Dong-Myung Kim and Cha-Yong Choi, Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system (2005), Journal of Biotechnology, 124 (2), 373-380 4. Dong-Myung Kim and James R. Swartz, Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli (2004), Biotechnology and Bioengineering, 85 (2), 122-129 P0463 High-throughput Screening of Effective Antigenic Candidates for the Development of Vaccines or Antibodies against Cloronchis sinensis and Plasmodium vivax Catherine CHRISTY, Sung-Won LEE, Devi KASI, Dong-Myung KIM Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea Screening of suitable antigen is an important way to develop diagnosis and eradication of a particular disease in the medical field. This study describes the high-throughput screening of antigens in the in-situ condition. The well-established cell-free protein synthesis system is the coupling process of transcription and translation by using cellular components in the absence of live cell. This system favored to replace laborious cloning and screening process in the biotechnology for the gene expression. Here in this study, the library of Plasmodium vivax and Clonorchis sinensis genes were PCR amplified and expressed. To validate the antigenicity of the library, cell-free system is highly desirable for high throughput system. The gene library was amplified with biotinylated T7 promoter and terminator primers and immobilized on streptavidin agarose beads for the in situ expression with cell-free reaction mixture containing Escherichia coli cell extract. The expressed proteins were treated with patient’s sera for the screening of antigenic genes and was integrated with FACS to discriminate highly effective genes. This study will be a versatile platform to screen library of genes in various fields Keywords : Cell-free Protein Synthesis (CFPS), High-throughput screening (HTS), Antigenic protein References 1. T.S Kim, S.H Cho, S H, Y K, W.M Sohn, S.S Hwang, J.Y Chai, S.H Lee, Y.K Park, D.K Oh, J.K Lee, A Nationwide Survey on the Prevalence of Intestinal Parasitic Infections in the Republic of Korea (2004), Korean J. Parasitol, 47 (1), 37-47 2. K.H Lee, K.Y Lee, J.Y Byun, B.G Kim and D.M Kim, On-bead expression of recombinant proteins in an agarose gel matrix coated on a glass slide (2012), Lab on Chip, 12, 1605-1610 P0464 Microbial Production of Acetoin in Klebsiella pneumoniae Using Metabolic Engineering Ji-Woong JANG, Moo-Young JUNG, Hwi-Min JUNG, Min-kyu OH Dept. of Chemical & Biological Engineering, Korea University, Seoul, 136-713 Acetoin, naturally in some fruits or dairy products, is mainly used for flavor enhancer of foods and ingredients. It is also employed for precursor of chemical materials in chemical and cosmetic industries. Unlike acetoin production of chemical synthesis using chemical catalysts until now, microbial fermentation is relatively a environmentally friendly way. Klebsiella pneumoniae strain which is usually exploited for 2,3-butanediol was utilized for acetoin production in this study. In the former research of K. pneumoniae, wabG was removed for inactivation of LPS that is endotoxin. Besides, ldhA and pflB genes were deleted for minimizing byproducts. [1] Starting over this former research, budC which is acetoin reductase was additionally eliminated for acetoin production. Then, optimum culture condition was studied for high yield and productivity in flask level. It was found that aeration is important factor in K. pneumoniae for acetoin production. As a results, 47.1g/L (at 32h) was obtained in Fed-batch culture at 600rpm agitation speed and 3vvm aeration volume. In the future work, further deletion of unknown acetoin reductase and additional gene works will be studied. The best condition will be also found out for much higher production of acetoin. Keywords : acetoin, Klebsiella peumoniae, budC, ldhA, pflB, wabG, 2,3-butanediol, fed-batch References 1. Moo-Young Jung, Min-Kyu Oh et. al., Appl. Environ. Microbiol. 80, 6195 (2014). P0465 Cell-free Expression of DNA Immobilized on a Gold-coated Surface Yu Jin PARK1, Seok Jae LEE2, Kyung G. LEE2, Dong Myung KIM1 1Dept. of Applied Chemistry and Biological Engineering, Chungnam National University, Daejeon, 305-764, 2National Nanofab Center, Daejeon, 305-806 The unique flexibility of cell-free protein synthesis allows gene expression in various unconventional reactional configurations. In this work, to develop a rapid method for translational analysis of genes, we attempted cell-free protein expression on a chip surface coated with gold. Genes of target proteins were amplified with thiol-modified primers for their binding on the gold surface through the Au-thiol reaction. Upon the incubation in the reaction mixture for cell-free protein synthesis in the scales of microliters, immobilized DNAs were successfully translated into corresponding proteins. It was also shown that the DNAs immobilized on the chip directed protein synthesis over repeated wash steps. We are presently working to integrate PCR techniques with the chip-based cell-free protein synthesis for in situ amplification, expression and analysis of genes on the same chips. Keywords : Cell-free protein synthesis, Au-thiol reaction, Immobilized DNA References 1. JY Byun, KH Lee, KY Lee, MG Kim and DM Kim, In-gel expression and in situ immobilization of proteins for generation of three dimensional protein arrays in a hydrogel matrix(2013), Lab On a Chip. 13(5), 886-91 P0466 Expression of Phospholipase A1 from Serratia sp. MK1 in a Cell Free Protein Synthesis System Derived from Escherichia coli Hye Jin LIM1,2, Yu Jin PARK2, Yeon Jae JANG2, Jae Kwang SONG1, Dong Myung KIM2 1Hye Jin Lim, Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea, 2Department of Applied Chemistry and Biological Engineering, Chungnam National University, Daejeon 305-764, South Korea Phospholipase A1 (EC 3.1.1.32) is an enzyme that hydrolyzes phospholipids into fatty acids and other lipophilic substances. Phospholipase has potential industrial applications in such fields as oil degumming, food processing, and synthesis of pharmaceutical intermediates. The lipolytic activity of phospholipase, however, causes inhibition of cell growth and hinders its production in the conventional cell-based expression methods. Because it does not require membrane integrity for protein synthesis, cell-free protein synthesis system using cell lysates offers an alternative route to the production of this cell lytic enzyme. In this study, we attempted to express the phospholipase A1 gene cloned from the chromosomal DNA of Serratia sp. MK1 either in E.coli cells or a cell-free synthesis system derived from the lysate of the E.coli cells. It was found that the phospholipase can be efficiently produced in the cell-free protein synthesis system with substantially higher volumetric productivity compared to the recombinant E.coli cells (370.2 ug/mL vs 115.8 ug/mL, respectively). We expect that the cell- free approach can provide a solution for production of many other enzymes that interfere with the growth and viability of host cells. Keywords : Phospholipase A1, Cell-free protein synthesis, protein expression References 1. JK Kim, MK Kim, GH Chung, CS Choi and JS Rhee, J Microbiol Biotechnol 7 (4), 258-261 (1997) 2. JK Song, MK Kim and JS Rhee, J Biotechnol 72 (1-2), 103-114 (1999) 3. C Catherine, KH Lee, SJ Oh and DM Kim, Biotechnol Adv. 31 (6), 797-803 (2013) P0467 Microbial Production of Cadaverine Using Engineered Corynebacterium glutamicum Expressing a Lysine Decarboxylase from Hafnia alvei Young Hoon OH1, Tae Uk KANG2, Jae Woo CHOI1,3, Kyoung-Hee KANG1, Il-Kwon KIM4, Bong Keun SONG1, Si Jae PARK5, Jeong Chan JOO1 1Center for Bio-based Chemistry, KRICT, Daejeon, 305-600, 2Dept. of Biological Sciences, Chonbuk National University, Jeonju, 54896, 3Dept. of Biological and Chemical Engineering, Hongik University, Chungnam 339-701, 4Bio R&D Center, Paik Kwang Industry Co., 57 Oehang-4 Gil, Gunsan-si, Jeollabukdo, 5Dept. of Environmental Engineering and Energy, Myongji University, Yongin, 449-728 Synthesis of biomass-derived polymers has been extensively investigated due to increasing concerns on environmental issues and depletion of petroleum resources. Among several promising bio-polymers, polyamides recently attract scientific and industrial attention since these bio-polymers can be used as engineering plastics of which material properties are comparable with those of petroleum-derived plastics. Cadaverine, a monomeric precursor of bio-polyamides, can be synthesized from lysine by a one-step enzymatic reaction catalyzed by lysine decarboxylases (LDCs). Among several investigated LDCs, a LDC from Hafnia alvei has a distinguished feature that it can function as a monomer. Therefore, in this study, a recombinant Corynebacterium glutamicum expressing a H. alvei LDC has been constructed. The cadaverine producing ability of the recombinant C. glutamicum has been compared with those of recombinant C. glutamicum expressing Escherichia coli LdcC or CadA which function as decamers and are the most well studied LDCs in C. glutamicum for the production of cadaverine. In addition, culture conditions of recombinant C. glutamicum expressing H. alvei LDC have been optimized for the high titer production of cadaverine. Keywords : cadaverine, C. glutamicum, LDC, H. alvei References 1. Y. H. Oh, J. W. Choi, E. Y. Kim, B. K. Song, K. J. Jeong, K. Park, I. K. Kim, H. M. Woo, S. H. Lee, and S. J. Park, Appl. Biochem. Biotech., 176, 2065 (2015). P0468 Intracellular Metabolite Profiles of Escherichia coli and Saccharomyces cerevisiae Depending on Complex vs. Minimal Media Jungyeon KIM, Kyoung Heon KIM Dept of Biotechnology, Graduate school, Korea University, Seoul 136-713 Metabolomics is one of the omics studies which reflects global changes of the entire metabolites in various organisms. It directly shows the abundance of metabolic intermediates in response to genetic or environmental changes. In metabolomics studies, as an experimental factor, medium is one of the most important factor because ingestion of different nutrient directly alters the flux of biological pathways. However, no studies have evaluated the effect of media on metabolic activities of microorganism for metabolomics studies or metabolic engineering. For this reason, we systematically evaluated how metabolic activities are differently expressed on different medium using gas chromatography/time-of-flight mass spectrometry (GC/TOF MS). We identified 119 metabolites including various classes of metabolites, and conducted statistical analyses including principal component analysis(PCA) and hierarchical clustering analysis(HCA). The profiles of metabolic pattern were clearly distinguished between the two groups, which consisted of metabolites of Escherichia coli and Saccharomyces cerevisiae grown on rich and minimal media, respectively. These results suggest that the medium can affect the overall metabolism and metabolic status of the cells. Keywords : Metabolomics, Media, GC/TOF MS, Escherichia coli, Saccharomyces cerevisiae P0469 Biosynthesis of (S)-3-Hydroxybutyrate by Engineered Saccharomyces cerevisiae Sora YU, Eun Ju YUN, Kyoung Heon KIM Department of Biotechnology, Graduate School, Korea University, Seoul, 136-713 (S)-3-Hydroxybutyrate (S-3HB), an example of bio-based platform chemicals, can be used as a starting material for the production of the biodegradable polymers such as polyhydroxyalkanoate and for the production of the optically fine chemicals such as antibiotics, vitamins, perfumes, and pheromones. To produce S-3HB in yeast, the S-3HB biosynthetic pathway was introduced into Saccharomyces cerevisiae. The pathway consists of three enzymes from the different microbial sources. The three enzymes were acetyl-CoA C-acetyltransferase (ACCT), acetoacetyl-CoA reductase (ACR), and 3- hydroxybutyryl-CoA thioesterase (HBT). The engineered S. cerevisiae strain had the ability producing S-3HB from ethanol as well as enzyme activities of ACCT, ACR, and HBT. To obtain a higher titer of S-3HB, a fed-batch fermentation based on pulse feeding of ethanol as a C2 carbon source was conducted. The biologically synthesized S-3HB from the engineered S. cerevisiae was identified by gas chromatography-mass spectrometry and the concentration of S-3HB was quantified by high performance liquid chromatography. By the fed-batch fermentation, the final titer of S-3HB was 12.0 g/L. To our knowledge, this is the first report on the production of 3HB by S. cerevisiae and the use of ethanol as a carbon source for 3HB production. Keywords : (S)-3-hydroxybutyrate, Saccharomyces cerevisiae, Acetyl-CoA C-acetyltransferase, Acetoacetyl-CoA reductase, 3-Hydroxybutyryl-CoA thioesterase References 1. Yun EJ et al. Production of (S)-3-hydroxyburate by metabolically engineered Saccharomyces cerevisiae(2015). Journal of Biotechnology 209(10):23-30 P0470 Biological Conversion of Formate to Poly-β-Hydroxybutyric Acid Using Isolated Methylobacterium sp. as Biocatalyst Wangjun LEE, Sangwoo KIM, Yuhyun KWON, Jinwon LEE Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, South Korea This study is about screening formate utilizing bacteria and biological conversion of formate to poly-β-hydroxybutyric acid (PHB). The isolate was revealed by 16S rRNA gene sequences as Methylobacterium genus. The modified nitrate mineral salts (NMS) media were used as growth and production medium. Removing potassium nitrate in the production medium was most effective for PHB accumulation Keywords : Formate, Poly-β-hydroxybutyric acid, Methylobacterium sp. References 1. Karr, D.B., Waters, J.K., Emerich, D.W., Appl. Environ. Microbiol. 46,1339-1344 (1983). 2. Madison, Lara L., Gjalt W., Huisman, Microbiology and molecular biology reviews. 63(1),21-53 (1999). P0471 Alginate Saccharification by Combining Endo- and Exo-Type Alginate Lyases for the Optimal Production of 4-Deoxy-L-Erythro-5-Hexoseulose Uronic Acid Damao WANG, Kyoung Heon KIM Department of Biotechnology, Korea University, Seoul, 136-713 Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass. Keywords : Alginate lyases, Saccharification, yield, monosaccharide References 1. Wang, D. M et al. Optimal production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases. 2014(10), 2105-2111. Bioprocess and Biosystems Engineering 2. Kim, H. T. et al. Characterization of a recombinant endo-type alginate lyase (Alg7D) originating from Saccharophagus degradans 2-40.2012(34),1087-1092. Biotechnology Letters 3. Kim, H. T. et al. Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40. 2012(93), 2233-2239. Applied Microbiology and Biotechnology P0472 Functional Characterization of the Reversed β-Oxidation Pathway in Escherichia coli for the Production of Adipic Acid THIRUMALAISAMY BABU, KIM KYOUNG HEON Department of Biotechnology, Major in Systems Food Biotechnology, Korea University Graduate School, Seoul, Republic of Korea. Adipic acid is the most important straight-chain C6 dicarboxylic acid which is linked almost 90% to nylon production with hexamethylenediamine. Combinatorial assemble of the homologous and heterogeneous genes into the synthetic pathway in Escherichia coli to the complete metabolic pathway for converting glucose into acetyl-CoA and succinyl-CoA via glycolysis and TCA cycle, and then convert them into adipic acid. We have identified novel synthetic pathway genes and assembled them into adipic acid via five metabolic reactions. The identified C6 dicarboxylic acid pathway starts with the TCA intermediates, acetyl-CoA and succinyl-CoA, which subsequently condense into 3-oxoadipyl-CoA by 3-oxoadipyl-CoA thiolase, a reduction reaction forms 3-hydroxyladipyl-CoA by 3-hydroxyladipyl-CoA dehydrogenase, a dehydration reaction further converts them into 5-carboxypentanoyl-CoA. The final dehydrogenation of adipyl-CoA shows our pathway intermediates can form reverse β-oxidation end products such as dicarboxyl-CoAs and the ω-oxidation forms adipic acid. This work provides the progress towards bio-based production of adipic acid. Keywords : Adipic acid, Dicarboxylic acid, Acetyl-CoA, Succinyl-CoA, β-oxidation cycle, Acot8 References 1. James MC, Mathew DB, Jacob EV, Alexander C, Seo HK, Ramon Gonzalez. Integrated engineering of beta- oxidation reversal and omega-oxidation pathways for the synthesis of medium chain omega-functionalized carboxylic acids. Metab Eng 2015;28:202-212. P0473 Production, Purification, and Skin-Whitening and Anti-Inflammatory Activities of 3,6- Anhydro-L-Galactose from Agar Ah Reum LEE, Eun Ju YUN, Kyoung Heon KIM Department of Biotechnology, Korea University, Seoul, 136-713 Red macroalgae are being actively investigated as a renewable biomass source because of their advantageous characteristics such as abundant carbohydrate contents, low lignin contents, and the absence of conflicts with food production. The main carbohydrate of red macroalgae, specifically in an agarophyte, is agarose that is composed of D-galactose and 3,6-anhydro- L-galactose (L-AHG). So far, there is no information on the mass production, metabolic fate, or physiological activities of L- AHG. In this study, L-AHG was produced and purified from agarose through the following chemical and enzymatic steps: agarose was converted to agarooligosaccharides by chemical prehydrolysis using acetic acid, agarooligosaccharides were enzymatically hydrolyzed into L-AHG and D-galactose by an exo-type β-agarase and a neoagarobiose hydrolase, and lastly, L-AHG was purified to over 95% purity by adsorption and gel permeation chromatographies. The chemical structure of the purified L-AHG was confirmed by gas chromatography-mass spectrometry, nuclear magnetic resonance spectrometry, and polarimeter analyses. In a skin-whitening assay, 100 μg/mL of L-AHG showed significantly higher activity on melanin inhibition than arbutin. L-AHG at 100 and 200 μg/mL showed anti-inflammatory activity by suppressing nitrite production. These results are expected to initiate the industrial production and application of L-AHG from red macroalgae as an important health-benefiting agent. Keywords : L-AHG, skin-whitening , anti-inflammatory activities References 1. Yun et al, Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin- whitening and anti-inflammatory activities(2013). Applied Microbiology and Biotechnology, 97(7):2961-2970 P0474 Overexpression of cis-Aconitate Dcarboxylase and Upregulation of the TCA Cycle to Increase Itaconic Acid Production in Corynebacterium glutamicum Sang A SIM, Jeong Eun HYEON, Young-Chul JOO, Seung Kyou YOU, Sung Ok HAN Dept. of Biotechnology, (College of Life Sciences and Biotechnology), Korea University, Seoul, 02841 Itaconic acid, a C5-dicarboxylic acid, is used in industry as building block chemical for manufacture of plastics, fuels and paints. Corynebacterium glutamicum is well used host for industrial acid production but do not metabolize itaconic acid by wild-type strain. Thus, we engineered C. glutamicum to biosynthesize itaconic acid through overexpression of two gene related itaconic acid metabolic pathway. First, we introduced cadA gene encoding cis-aconitate decarboxylase (CadA) from Aspergillus terreus. This gene was codon optimized after removing an intron for expression in C. glutamicum. cis-Aconitic acid is intermediate of citric acid cycle and is converted into itaconic acid by CadA. As a result, engineered strain by overexpression of codon-optimized cadA gene was able to produce itaconic acid in the stationary growth phase. Second, the overexpression of global transcriptional regulator RamA (Cg2831) enhanced cis-aconitic acid production flux by activating citrate synthase (GltA) and aconitase (Acn). Simultaneous overexpression of CadA and RamA recombinant was achieved higher itaconic acid concentration than only CadA overexpressed in C. glutamicum. This study develops the useful strain for industrial biotechnology. Keywords : Itaconic acid, cis-Aconitate decarboxylase, transcriptional regulator, Corynebacterium glutamicum References 1. Vuoristo, K. S. et al, Metabolic engineering of itaconate production in Escherichia coli(2015), Applied Microbiology and Biotechnology, 99, 221-228 2. Blazeck, J. et al, Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production(2014), Applied Microbiology and Biotechnology, 98, 8155-8164 P0475 In vitro Single-Vessel Efficient Enzymatic Synthesis of Novel Resvera-A Glucosides Ju Yong SHIN, Ramesh PRASAD PANDEY, Ha Young JUNG, Song E KIM, Ju Hyun JANG, Jae Kyung SOHNG Institute of Biomolecule Reconstruction, Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea. An in vitro enzymatic glycosylation system is developed for the efficient synthesis of glucosides of 3,5-dihydroxy-N-(4- hydroxyphenyl) benzamide (resvera-A), a chemically synthesized molecule resembling resveratrol in structure. Resvera-A is a pharamacophore-based designed molecule that exhibits anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In this study, an alternative cost-effective uridine diphosphate (UDP) recycling system was established to produce UDP-a-D-glucose through two steps enzyme catalyzed reactions using easily available cheap sources. This UDP-a-D- glucose biosynthesis system was combined with a glycosyltransferase (YjiC, Bacillus licheniformis)-catalyzed reaction for the synthesis of glucoside derivatives of resvera-A. The side product of the glycosylation reaction, UDP, was used as a precursor for the biosynthesis of UDP-a-D-glucose, which is used by YjiC for glycosylation, thus recycling UDP. As a result, two novel molecules, resvera-A 3-O-•·-D-glucoside (42.33 mg/ml) and resvera-A 4'-O-•·-D-glucoside (99.38 mg/ml), were synthesized in practical quantities. Structures of both products were elucidated using liquid chromatography, mass spectroscopy, and nuclear magnetic resonance analysis. Keywords : Glycosylation, Biostnthesis, Resvera-A, Nucleotide diphosphate sugars, One-pot reaction References 1. Abril M, Negueruela AI, Perez C, Juan T, Estopanan G, Preliminary study of resveratrol content in Aragon red and rose wines. Food Chemistry 92 (2005) 729-736. 2. Roldán A, Palacios V, Caro I, Pérez L, Resveratrol content of Palomino fino grapes: influence of vintage and fungal infection. J. Agric. Food Chem. 51 (2003) 1464-1468. 3. Jerkovic V, Collin S, Occurrence of resveratrol and piceid in American and European hop cones. J. Agric. Food Chem. 55 (2007) 8754-8758. 4. Anisimova NY, Kiselevsky MV, Sosnov AV, Sadovnikov SV, Stankov IN, Gakh AA, Trans-, cis-, and dihydro- resveratrol: a comparative study. Chem. Cent. J. 5 (2011) 88. 5. Orallo F, Comparative studies of the antioxidant effects of Cis and Trans resveratrol. Curr. Med. Chem. 13 (2006) 87-98. 6. Szkudelski T, Resveratrol inhibits insulin secretion from rat pancreatic islets. Eur. J. Pharmacol. 552 (2006) 176- 181. 7. Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X, Zhai Q, SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B. Cell Meta b 6 (2007) 307-319. P0476 In-vivo Genetic Circuit Performance Prediction via In-vitro Modeling Bo-Young JEONG1, Haseong KIM1, Seung-Goo LEE1,2 1Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea, 2Biosystems and Bioengineering Program, University of Science and Technology, Daejeon, South Korea The design of synthetic circuits for controlling processes at the molecular scale is an important goal of synthetic biology (Yordanov et al., 2014). This shift toward an engineering model of synthetic devices has been designed and implemented in vivo using protein expression and gene regulation mechanisms (Galloway et al., 2013). However, the complexity of circuits is limited by the low availability of well-understood genetic components and interactions with the host strains that are hard to predict. Therefore, complementary to the design of in vivo synthetic circuits, in vitro systems resonate with the bottom-up approaches of synthetic biology and have indeed been exploited to create complex circuitry in cell-free systems (Kim et al., 2006; Kim and Winfree, 2011; Padirac et al., 2012). Here, we use implementations to design capable of regulating the output signal of a system according to repressor module, CelR and its inducible cellobiose to be designed and analyzed within a modeling framework. We compare to differences between in vivo and in vitro models of biochemical networks with stochastic tools that could be applied to the construction of cell-free expression system. References 1. Yordanov, B., Kim, J., Petersen, R. L., Shudy, A., Kulkarni, V. V., and Phillips, A. (2014) Computational design of nucleic acid feedback control circuits. ACS Synth. Biol. 3. 600-616. 2. Galloway, K. E., Franco, E., and Smolke, C. D. (2013) Dynamically reshaping signaling networks to program cell fate via genetic controllers. Science 341, 1235005. 3. Kim, J., White, K. S., and Winfree, E. (2006) Construction of an in vitro bistable circuit from synthetic transcriptional switches. Mol. Syst. Biol. 2, 68. 4. Kim, J., and Winfree, E. (2011) Synthetic in vitro transcriptional oscillators. Mol. Syst. Biol. 7, 465. 5. Padirac, A., Fujii, T., and Rondelez, Y. (2012) Bottom-up construction of in vitro switch able memories. Proc. Natl. Acad. Sci. U.S.A. 109, E3212–E3220. P0477 Cellulose Binding Domain Based Multi-Enzyme Complex for Enhancing Wonjae SEONG1,2, GuiHwan HAN1, Soo-Jin YEOM1, Paul Kyung-Seok YOON1, Seong Keun KIM1,2, Seung-Goo LEE1,2 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon 305-600, Korea , 2Biosystems and Bioengineering Program, University of Science and Technology (UST) Multi-enzyme complex engineering has become popularized for an efficient target production in a bacterial metabolic pathway due to its ability to reduce intermediate evasion through substrate channeling. Aware of cellulose binding domain (CBD) having the tendency to form aggregation with protein activities, we constructed a CBD based multi-enzyme complex in vivo, to optimize n-butanol production in Escherichia coli. To build a CBD based multi-enzyme complex, non-covalent associating antiparallel linkers were genetically fused on the N-terminals of the CBD and n-butanol pathway enzymes (AtoB, Hbd, Crt, Ter, and AdhE2). Through glucose batch fermentation, strain containing the multi-enzyme complex produced n- butanol at a titer of 10 % in TB medium containing 2% (w/v) glucose which is significantly higher than strains without the multi-enzyme complex. In conclusion, our engineered multi-enzyme complex can be applied as a matrix to gather targeted cytosolic proteins in cells while maintaining their original activities, which provides a unique platform for an efficient biochemical product production. 한국생물공학회, 생물공학의 동향 : 2015.10 해양생물공학 P0501 Antioxidant and Anti-aging Activities of Solvent Extracts from Eisenia bicyclis Hae-Joo KIM, Nyun-Ho PARK, Jung-Hee WOO Gyeongbuk Institute for Marine Bio-Industry (GIMB), Uljin 767-813, Gyeongbuk, Korea This study was carries out to analyzed antioxidant and anti-aging activities of methanol extract and its five different solvent sub fractions by using the solvent partition method (hexane[HE], chloroform[CE], butanol[BE], ethyl acetate[EE], and water[WE]) from marine brown algae, Eisenia bicyclis., The highest extraction yield was observed in EE(22.7%) and phenolic content was the highest in EE(94.24 mg/g). The antioxidant activity by DPPH/ABTS radical scavenging activities and reducing power ability were relatively higher in the EE. The IC50 (50% inhibition concentration) of EE on the DPPH and ABTS radical scavenging activities were 37.90 and 18.18 μg/mL. The values of absorbance at 700 nm of EE on the reducing power ability were 1.62 at 1 mg/mL. The collagenase inhibition activities of HE and CE (1 mg/mL) were observed to 44.2% and 35.1%, respectively. The elastase inhibition activities were showed CE and EE (1 mg/mL) to 47.1% and 46.7%, respectively. These results indicate that Eisenia bicyclis extracts could be used as an effective natural antioxidant and anti- aging agent in functional materials. This work was supported by Start-Up, Growth R&D Program funded Korea Small and Medium Business Administration in 2015. Keywords : Eisenia bicyclis, Antioxidant, Anti-aging References 1. V. L. Singleton, A. Joseph and J. Rossi, Am. J. Clin. Nutr. 68, 1474 (1958). 2. E. S. Saleh and A. Hameed, Food Chem. 114, 1271 (2008). P0502 A Framework Protein, Pif80, for Controlling Calcium Carbonate Biomineralization of Nacre So Yeong BAHN1, Byung Hoon JO2, Yoo Seong CHOI3, Hyung Joon CHA1,2 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784, 2Department of Chemical Engineering, POSTECH, Pohang, 790-784, 3Department of Chemical Engineering, Chungnam National University, Daejeon, 305-764 Molluscan shells are widespread natural calcium carbonate biominerals which are constructed for protection of internal soft bodies. The inner layer of these mineralized tissues, called nacre, provides exceptional toughness via its highly organized organic-inorganic composite structure. In spite of the small proportion, organic matrix composed of proteins and polysaccharides is regarded as a regulator for calcium carbonate aragonite crystal mineralization of nacre. Among the organic matrix, a recently known acidic aragonite-binding protein Pif80 from nacreous layer of pearl oyster Pinctada fucata, have been regarded as a significant component for the nacre formation. In this study, the recombinant Pif80 protein was designed and mass-produced in Escherichia coli system for its intensive characterization. The successively purified recombinant protein was used to investigate the interaction aspects with calcium ions and calcium carbonate mineral phases. From the effects of recombinant Pif80 on in vitro calcium carbonate mineralization in comparison with organic complex extracted from nacre of P. fucata, we expect that Pif80 protein plays a role in nacre-mimicking calcium carbonate mineralization. Keywords : Pif80, biomineralization, nacre, calcium carbonate, recombinant References 1. Suzuki, M.; Saruwatari, K.; Kogure, T.; Yamamoto, Y.; Nishimura, T.; Kato, T.; Nagasawa, H. Science 2009, 325, 1388−1390. P0503 Antiproliferative Effect of Artemisia scoparia against Growth of Human Cancer Cells Heejeong JEONG1, Hojun KIM2, Eunshin JU2, Youngwan SEO1,2 1Department of Convergence Study on the Ocean Science and technology School, Busan 606-791, Korea, 2Division of Marine Bioscience, Korea Maritime & Ocean University, Busan 606-791, Korea The Halophyte Artemisia scoparia was collected in Ganghwado (Korea). The collected samples were briefly dried under shade. The air dried sample of A. scoparia (480 g) was chopped into small pieces and soaked for 2 days with MeOH and CH2Cl2, sequentially. The combined crude extracts were evaporated under reduced pressure and partitioned between CH2Cl2 and water. The organic layer was further partitioned between 85% aq. MeOH and n-hexane, and then the aqueous layer was fractionated with n-BuOH and H2O, successively, to afford the n-hexane, 85% aq. MeOH, n-BuOH and water fractions. Inhibitory effect of crude extract and its solvent-partitioned fractions on growth of HT-1080 (fibrosarcoma cells), HT-29 (colon cancer cells), AGS (gastric cancer cells), and MCF-7 (breast cancer cells) human cancer cells was evaluated by MTT assay. Keywords : halophyte, Artemisia scoparia , human cancer cells References 1. Choi E, Park H, Lee J, Kim. Anticancer, antiobesity, and anti-inflammatory activity of Artemisa species in vitro (2013), Journal of traditional Chinese medicine, 33(1), 92-97 2. Habib M, Waheed I, Evaluation of anti-nociceptive, anti-inflammatory and antipyretic activities of Artemisia scoparia hydromethanolic extract (2013), Journal of ethnopharmacology, 145(1), 18-24 3. Neghban M, Moharramipour S, sefidkon F, Chemical Composition and Insecticidal Activity of Artemisia scoparia Essential Oil against Three Coleopteran Stored-Product Insects (2006), Journal od Asia-Pacific Entomology, 9(4), 381-388 P0504 Cytotoxic Activity of Tetragonia tetragonoides in Human Cancer Cells Hojun KIM1, Heejeong JEONG2, Eunshin JU1, Youngwan SEO1,2 1Division of Marine Bioscience, Korea Maritime & Ocean University, Busan 606-791, Korea, 2Department of Convergence Study on the Ocean Science and technology School, Busan 606-791, Korea Dried samples of the halophyte Tetragonia tetragonides were extracted four times: twice with CH2Cl2 and twice with methanol (MeOH), yielding two crude extracts (CH2Cl2 and MeOH), The two crude extracts were combined and then the combined crude extracts were partitioned between CH2Cl2 and H2O. The aqueous layer was re-partitioned between n-butanol (n-BuOH) and H2O and then the organic layer was re-partitioned between n-hexane and 85% aq.MeOH, affording four solvent-partioned fractions. Two crude extracts of Tetragonia tetragonides and four solvent fractions were tested for their cytotoxic activity using MTT assay in HT-1080 (fibrosarcoma cells), HT-29 (colon cancer cells), AGS (gastric cancer cells), and MCF-7 (breast cancer cells), respectively. Keywords : halophyte, Tetragonia tetragonides, cytotoxic activity References 1. Wilson C, Lesch S M., Grieve C M., Growth Stage Modulates Salinity Tolerance of New Zealand Spinach (Tetragonia tetragonoides, Pall.) and Red Orach (Atriplex hortensis L.) (2000), Annals of Botany, 85(4), 501-509 2. Aoki T, Takagi K, Hirata T, Suga T, Two naturally accurring acyclic diterpene and norditerpene aldehydes from Tetragonia tetragonoides (1982), Phytochemistry, 21(6), 1361-1363 3. Kong CS, Yeom YR, Lee JI, Kim YA, Lee JS, Seo Y, Inhibition effects of extracts and its solvent fractions from Limonium tetragonum on growth of human cancer cells (2008), Korea J. Biotechnol. Bioeng., 23(2), 177-182 P0505 Factors Stimulating Growth of M.aeruginosa a Novel Source for Bioethanol Production Jong Deog KIM, Muhammad imran KHAN, Moon Geon LEE, Jin Hyuk SHIN Department of Biotechnology, Chonnam National University The micro algae Microcystis aeruginosa contain sufficient amount of carbohydrates which can be utilized for bioethanol production. Growing M. aeruginosa with increased sugar contents is the key and basic step towards the higher yield of bioethanol. We investigated some growth promoting reagents and bacteria for Microcystis aeruginosa. Rapid and effective growth of Microcystis aeruginosa was observed by Addition of 2mM Aminolevulinic acid to the culture of Microcystis aeruginosa. Similarly Microcystis aeruginosa growth was increased by addition of 2.28 mM Lysine to the culture. 1mM alanine also assists growth of Microcystis aeruginosa but less than Lysine. Culture of Microcystis aeruginosa containing 1mM Napthylamine acetic acid as cytokine showed enhanced and effective growth as compared to the culture without cytokine. Salmonella and Pseudomonas assisted growth of Microcystis aeruginosa in a co-culture. Keywords : Microcystis aeruginosa, Aminolevulinic acid, Salmonella, Lysine, Bioethanol References 1. Khan, S. A., Rashmi., Hussain M. Z., Prasad, S., and Banerje, U. C. , “Prospects of biodiesel production from microalgae in India,” (2009), Renew. Sust. Energy Rev. 13, 2361- 2372 2. Hunt, R., Chinnasamy, S., Bhatnagar, A., & Das, K. C., Effect of Biochemical Stimulants on Biomass Productivity and Metabolite Content of the Microalga, Chlorella sorokiniana, (2010), Applied Biochemistry and Biotechnology, 162(8), 2400-2414. doi: 10.1007/s12010-010-9012-2 3. Ho, Y. K., and J. L ele, 8-Aminolevulinic acid dehydratase of Spirilum itersonii and the regulation of tetrapyrrole synthesis, (1971), Arch. Biochem. Biophys. 144: 734-740. P0506 Isolation and Identification of Agar-Degrading Marine Bacterium from Red Algae Palmaria sp. Llewelyn MORON1, Kris Niño VALDEHUESA1, Hee-jin YANG1, Kristine Rose RAMOS1, Rhudith CABULONG1, Grace NISOLA1, Won–Keun LEE2, Wook-Jin CHUNG1 1Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728, 2Division of Bioscience and Bioinformatics, Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728 Marine microorganisms are reported to produce agarases which catalyze the hydrolysis of agar present in macroalgae. In this study, agar-degrading bacterium designated as W5C was isolated from red macroalgae, Palmaria sp., collected from the seashore of Yeosu, South Korea. Samples were cultivated using marine agar 2216 (Difco) for 3-7 days at 28 ℃. Agar- degrading ability was screened by flooding the culture plate with iodine solution. Initial characterization identified the bacterium as Gram-negative rod-shaped, positive for catalase and gelatinase, negative for oxidase and amylase. Based on 16sRNA sequencing and phylogenetic analysis, W5C was identified as belonging to the genus Cellulophaga sp. being highly related to Cellulophaga lytica ATCC 23178T (99% sequence similarity). This isolate will be further studied on its application on marine biomass utilization. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2009-0093816). Keywords : agarase, red macroalgae, agar-degrading References 1. Chi et al., Isolation and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gen, nov, sp. no., from the Southern Sea, Korea, 23(11), 1509-1518 P0507 The Development of Microbe Carrier for Bioremediation of Heavy Metal by Using Desulfovibrio desulfuricans and Recycled Aggregate in Artificial Sea Water In-Hwa KIM1, Won-Jin LEE1, Eun-Jung LEE1, Byung-Keun OH1,2 1Dept. of Chemical & Biomolecular Engineering, Sogang University, Seoul, 121-742, 2Dept. Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 121-742 Marine is the final assembly area of the various contaminasts, including heavy metals. So, we have developed a microbe- carrier that is effective in removing heavy metals. Micrbe-carrier are made by using Desulfovibrio desulfuricans one of the sulfate reducing bacteria and recycled aggregates. Microbe-carrier showed a very high heavy metal removal efficiency compared to natural carriers. The result showed that microbe-carrier strengthened the removal efficiency of heavy metals (Cd, Cu, Zn, Ni, Cr). In addition, we conducted a research while changing the temperature and heavy metal concentration. The results showed that microbe-carriers is possible to remove the heavy metals at high concentrations above 50ppm. Also, heavy metals removal efficiency except Ni showed up more than 60% at 5℃. Acknowledgements: This research was a part of the project titled "Development of Sustainable Remediation Technology for Marine Contaminated Sediments" funded by the Ministry of Land, Transport and Maritime Affairs, Korea, This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (No. 2015R1A2A2A01007843). Keywords : heavy metal removal, microbe-carrier, Desulfovibrio desulfuricans References 1. Qin Zhou, Bioresource Technology. 136, 413-417 (2013). P0508 The Change of Heavy Metal Concentration in Marine Sediment by Bioremediation Using Desulfovibrio desulfuricans carrier Won-Jin LEE1, In-Hwa KIM1, Eun-jung LEE1, Byung-Keun OH1,2 1Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, 121-742, 2Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 121-742 In this study, Using zeolite carrier which absorbed Desulfovibrio desulfuricans as In-situ coating material, we examined the concentration change of heavy metals in precipitates such as Cu, Zn, Ni, Cr at 5℃, 25℃. In order to access the distribution after 30days, The type of concentration is devided each heavy metal into 5 parts in order of stability in seawater and precipitates system; exchageable, bound to carbonates, bound to Fe-Mn oxides, bound to organics, residual. The concentration is measured by sequential extraction suggested by Tessier. The results show that Cu, Zn are transformed into relatively stable phase and Ni, Cr are transformed into the most stable phase at 5℃, 25℃. The efficiency of stabilization is improved at higher temperature. Acknowledgements: This research was a part of the project titled "Development of Sustainable Remediation Technology for Marine Contaminated Sediments" funded by the Ministry of Land, Transport and Maritime Affairs, Korea, This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (No. 2015R1A2A2A01007843) Keywords : In-situ coating, sequential extraction, heavy metal References 1. A. Tessier, Analytical chemistry, 51(7), 844-854 (1979) P0509 Inhibition on Prorocentrum micans by Biodegraded Products of Brown Seaweed Wastes Hyun Yi JUNG, Ja Young CHO, Joong Kyun KIM Dept. of biotechnology, Pukyong National University, Busan, 608-737 Every year, seafood consumption has increased due to its bioactivity and health benefits. As increasing the seafood consumption, wastes have also increased. Especially, fishery wastes from industry fields, can affect negative damage to the marine environment such as Harmful Algal Blooms (HABs). This phenomenon causes mass mortalities of fish, marine mammal, shellfish, and other oceanic organisms due to the toxins and depletion of oxygen [1]. For this reason, the disposal and reutilization of fishery wastes have been essential for preservation of the marine environment and recycling of organic substances. In this study, we selected bacteria with optimum culture of degradation of carbohydrates, proteins and lipids and investigated for reutilization of brown-seaweed wastes. The culture supernatants collected during the biodegradation exhibited algal inhibition activity. [2, 3]. We will investigate the inhibition mechanism by brown seaweed wastes. Keywords : brown seaweed wastes, biodegradation, Prorocentrum micans References 1. A. J. Bourdelais, C. R. Tomas, J. Naar, J. Kubanek, and D. G. Baden, New Fish-Killing Alga in Coastal Delaware Produces Neurotoxins (2002), ENVIRON. HEALTH. PERSP. 110, 465. 2. J. H. Jeong, H. J. Jin, C. H. Sohn, K. H Suh and Y. K Hong, Algicidal activity of the seaweed Corallina pilulifera against red tide microalgae (2000), J. APPL. PHYCOL. 12, 37. 3. D. E. Terlizzi1, M. D. Ferrier, E. A. Armbrester and K. A. Anlauf, Inhibition of dinoflagellate growth by extracts of barley straw (Hordeum vulgare) (2002), J. APPL. PHYCOL. 14, 275. P0510 Optimization of Direct-Transesterification for Biodiesel Production from Marine Microalgal Biomass Choi CHANGYONG, Ryu YOUNG-JIN, Lim SANG-MIN, Lee CHOUL-GYUN National Marine Bioenergy Research Center & Department of Biological Engineering, As the price of conventional fossil fuels continues to escalate, so-called alternative fuels have become more attractive. Microalgae have been considered as better feedstock for bioenergy production than conventional energy crops for their superior productivity and other characteristics. However, the present production process of biodiesel from microalgal biomass requires is more complex than the processes for energy crops, increasing the price of algal biodiesel. Various unit operation steps involved in biodiesel production processes include drying, lipid extraction, transesterification, and biodiesel refining. The direct-transesterification can simplify the biodiesel production process by combining lipid extraction and transesterification in one step. In this study, the overall conversion efficiency was analyzed as functions of reaction temperature, reaction time, concentration of catalyst (sulfuric acid) and the volume fraction of methanol for optimization of direct-transesterification process for two marine microalgae, Dunaliella tertiolecta and Tetraselmis sp. The optimal conditions was found to be reaction temperature of 64℃, and sulfuric acid concentration of 3% for both microalgae. Optimal transesterification conditions for D. tertiolecta were 10 vol. (v/w) of volumetric fraction of methanol and 3 h of reaction time. For Tetraselmis sp., the optimal conditions were 7.5 vol. (v/w) and 5 h. The results of this study will help to design mass production of biodiesel from Dunaliella tertiolecta and Tetraselmis sp. Keywords : microalgae, biodiesel, Tetraselmis, Dunaliella, biofuel References 1. H. Tang, Potential of microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel (2011), Appl. Energy, 88, 3324–3330 P0511 Investigation of Effects of L/D Cycle on Fatty Acid Productivity in Tetraselmis sp. and Nannochloropsis oculata Youngsook SHIN, Hanwool PARK, Choul-Gyun LEE* National Marine Bioenergy Research Center & Department of Biological Engineering Microalgae are known as one of excellent sources for biofuels production. For sustainable production of biofuels, microalgae need to be cultivated using sunlight. During day, microalgae synthesize various energy storage compounds, such as lipids and carbohydrates, but during night, they consume the storage compounds, resulting in decreased biomass known as night biomass loss (NBL). In this study, two marine microalgae, Tetraselmis sp. MBEgh01L (KCTC12236BP) and Nannochloropsis oculata K-1281 (SCCAP) were cultivated in 12:12h L/D cycle for comparison of the effects of L/D cycle on their fatty acid productivities. The microalgae were cultivated in bubble columns with 2 L of modified f/2 medium in natural seawater and 2% CO2 gas aeration at 0.1 vvm. The light was supplied with 2 fluorescent light bulbs at 100 ± 10 µE/m2/sec. The L/D cycle decreased both biomass productivity and lipid contents, but the decreases in biomass productivities were greater than the reductions in lipid contents in both microalgae. The fatty acid contents were different in N. oculata and Tetraselmis sp., up to 20.6% and 8.4%, respectively, but the effect of L/D cycle on the fatty acid productivities in the two microalgae were similar as the productivities decreased by 65% and 72% in Tetraselmis sp. and N. oculata, respectively. In the present study, the presence of L/D cycle caused significant reduction in fatty acid productivities in both Tetraselmis sp. and N. oculata. For improving fatty acid productivity in large-scale, outdoor cultivation, development of economic methods to reduce the NBL will be needed. Keywords : L/D cycle, fatty acid productivity , night biomass loss References 1. Q. Hu, N. Kurano, M. Kawachi, I. Iwasaki, and S. Miyachi, Appl. Microbiol. Biotechnol. 49, 6 (1998). 2. L. L. Price, K. Yin, and P. J. Harrison, J. Exp. Mar. Biol. Ecol. 232, 2 (1998). P0512 Marine Hydroid Perisarc: A Chitin/Melanin Composite Reinforced with DOPA-iron (III) Complexes Ekavianty PRAJATELISTIA1, Dong Soo HWANG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea., 2Integrative Bioscience and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. Many marine invertebrates utilize chitin and melanin as a building block to form their hard tissues. Their hard tissues made of chitin and melanin containing composites are appealing prototype building blocks due to their extraordinary mechanical properties and light weight compare to the inorganic materials, correlated with energy efficiency. Here, a marine hydroid perisarc of Aglaophenia latirostris Nutting, 1900, was investigated to understand how nature design the hard tissues made of chitin and melanin containing components. Chitin and melanin were constituted 10wt %, 53 wt% of the perisarc, respectively. Interestingly, a DOPA (3, 4-dihydroxyphenylalanine) containing protein and iron was detected from the perisarc, as same as adhesive and coating of marine mussels. A resonance Raman spectrum due to DOPA-iron (III) complex formation was also detected in the chitin containing perisarc, implying the existence of DOPA-iron (III) interaction around chitin structure for the first time. Keywords : Hydroid, Perisarc, DOPA, Chitin References 1. Lichtenegger HC, Schoberl T, Ruokolainen JT, Cross JO, Heald SM, Birkedal H, et al. Proceedings of the National Academy of Sciences of the United States of America.100:9144-9, (2003). 2. Miserez A, Schneberk T, Sun C, Zok FW, Waite JH. Science. 319:1816-9, (2008). 3. Broomell CC, Khan RK, Moses DN, Miserez A, Pontin MG, Stucky GD, et al. Journal of the Royal Society, Interface / the Royal Society.4:19-31, (2007). P0513 Tunicate-Mimetic Nanofibrous Hydrogel Adhesive with Improved Wet Adhesion Suyoung LEE1,1, Dongyeop OH2,2, Dong Soo HWANG1,2 1Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH),Pohang 790-784, 2POSTECH Ocean Science and Technology Institute, Pohang University of Science and Technology (POSTECH),Pohang 790-784 The main impediment to medical application of biomaterial-based adhesives is their poor wet adhesion strength due to hydration-induced softening and dissolution. To solve this problem, we mimicked the wound healing process found in tunicates, which use a nanofiber structure and pyrogallol group to heal any damage on its tunic under sea water. We fabricated a tunicate-mimetic hydrogel adhesive based on a chitin nanofiber/gallic acid (a pyrogallol acid) composite. The pyrogallol group-mediated cross-linking and the nanofibrous structures improved the dissolution resistance and cohesion strength of the hydrogel compared to the amorphous polymeric hydrogels in wet condition. The tunicate-mimetic adhesives showed higher adhesion strength between fully hydrated skin tissues than did fibrin glue and musselmimetic adhesives. The tunicate mimetic hydrogels were produced at low cost from recyclable and abundant raw materials. This tunicate-mimetic adhesive system is an example of how natural materials can be engineered for biomedical applications Keywords : Tunicates, Underwater adhesives, Chitin nanofibers, Pyrogallol, Nanofibrous hydrogels References 1. Cha HJ, Hwang DS, Lim S, White JD, Matos-Perez CR, Wilker JJ. Bulk adhesive 2. Pasanphan W, Chirachanchai S. Conjugation of gallic acid onto chitosan: an 3. Ejima H, Richardson JJ, Liang K, Best JP, Van Koeverden MP, Such GK, et al. Onestep P0514 Effect of Fractionated Seawater on Biomass Productivity of Tetraselmis sp. KCTC12429BP Daewoo JUNG, Philhan KIM, Hanwool PARK, Choulgyun LEE National Marine Bioenergy Research Center & Department of Biological Engineering Inha University, Incheon 402-751, Korea Production of biofuels from microalgae has been receiving enormous attentions for their advantages over conventional energy crops. However, huge amount of fertilizers and water requirements are one of the constraints in commercialization of microalgal biofuels. Seawater contains nutrients for microalgal growth such as nitrogen, phosphorous, sulfur, etc. Therefore use of seawater for cultivation is preferred for sustainability and economic feasibility of microalgal biofuels production than using freshwater. The effects of various seawater pretreatment methods on the biomass productivity of marine microalgae were investigated. Algal biomass productivity was lower in untreated seawater than in sterilized seawater. In order to overcome the cause of the negative effect of untreated natural seawater, the seawater was filtered with membrane filters with different pore sizes: 0.2 μm, 4 μm, 8 μm and 25 μm. The fractionated seawater was used for preparation of modified f/2-Si media for cultivation of Tetraselmis sp. KCTC12429BP, isolated from Youngheung Island, Incheon. Both biomass productivity and total fatty acids productivity were highest in the 4 μm filter group. The overall biomass productivity of 4 μm filtered group was 12.5% and 31.8% higher than 0.2 μm filtered group and untreated seawater group, respectively. Total fatty acids productivity of 4 μm filtered group was 31% and 24% higher than those of 0.2 μm filtered group and untreated seawater group. Although the biomass productivity was higher in 0.2 μm filtered group than in untreated seawater group, total fatty acids productivity was higher than that in untreated seawater group due to higher fatty acid contents. The results indicate that natural seawater contains factors that affect not only algal growth but also fatty acids accumulation. Further research will investigate the factors contained in seawater which affect the microalgal growth and fatty acids contents. Keywords : microalgae, cultivation, pretreatment, filtration, natural seawater References 1. A. M. Lakaniemi, C. J. Hulatt, K. D. Wakeman, D. N. Thomas, and J. A. Puhakka, Bioresource Technol. 121 (2012) 2. V. Vasudevan, R. W. Stratton, and M. N. Pearlson, Environ. Sci. Technol., 46, 4 (2012). P0515 Seasonal Cultivation of Different Microalgal Strains for Improved Biomass Productivity Jong-Chan LEE, Hanwool PARK, Choul-Gyun LEE National Marine Bioenergy Research Center & Dept. of Biological Engineering, Inha University, Incheon, 402-751 Microalgae are capable of assimilating CO2 into energy storage compounds such as fatty acids that can be used for production of biodiesel. Cultivation of microalgae requires less area compared with terrestrial energy crops. However, national-scale production of biofuels from microalgae still requires large area, and culturing microalgae in the ocean could reduce the burden of area requirement. One of the environmental factors affecting microalgal biomass productivity is temperature. Seawater temperature is rather stable on daily basis, but it changes over a course of months. Since it is difficult to control seawater temperature, cultivation of microalgae with different optimal temperatures could be a method to ensure productivities in different seasons. Five different microalgal strains were isolated from Youngheung Island, Incheon, Korea: Tetraselmis sp. KCTC12429BP, KCTC12430BP, KCTC12431BP, KCTC12432BP, and KCTC12433BP. They were cultivated at 3 different temperatures, 10°C, 20°C, and 30°C, representing the seawater temperature in spring, fall and summer, respectively. Among five Tetraselmis sp., Tetraselmis sp. KCT12433BP showed highest productivities at all three temperatures. The highest biomass and fatty acid productivities were obtained at 20°C. Cultivation of the microalgae in marine photobioreactors deployed in the coastal seawater of Youngheung Island will be performed as the further study. Keywords : Microalgae, Biodiesel, Ocean cultivation, Photobioreactor References 1. S. M. Renaud, L. V. Thinh, G. Lambrinidis, and D. L. Parry, Aquaculture 211, 1 (2002) 2. L. Zhu, X. Zhang, L. Ji, X. Song, and C. Kuang, Process Biochem. 42, 2 (2007) P0516 Effects of Total Suspended Solids in the Coastal Seawater of Youngheung Island on Microalgal Growth Philhan KIM, Daewoo JUNG, Hanwool PARK, Choul-Gyun LEE National Marine Bioenergy Research Center & Dept. of Biological Engineering, Inha University, Incheon 402-751, Korea Seawater contains total suspended solids (TSSs) with various properties, and these TSSs may affect microalgal cell growth. From the previous experiments, we have found that TSSs generally inhibit the growth of algal cells. In the present study, TSSs were fractionated by sizes, and each fraction was investigated for its effect on the growth of microalgae. Seawater was collected from Youngheung Island, Incheon, Korea. TSSs in the seawater were fractionated using glass microfiber filters with different pore sizes: 0.2 μm (A), 0.7 μm (B), 1.2 μm (C), and 3.0 μm (D). A marine microalga, Tetraselmis sp. KCTC12433BP, was cultivated using f/2-Si medium prepared with the seawater with each fraction. The cultures with D fraction showed the highest specific biomass productivity of 0.27 g/L/day, followed by B, C, and A fractions. The biomass productivity of the cultures containing all TSSs showed only 43% of that of the cultures with fraction D. The results indicate that suspended solids with the size ranges from 1.2 μm to 3.0 μm may promote the growth of Tetraselmis sp. In addition, TSSs, with larger than 3.0 μm, showed inhibitory effect on algal growth. Further research will aim to identify the factors that promote or inhibit algal growth in TSSs. Keywords : Microalgae, Biomass productivity, Suspended solids References 1. V. Vasudevan, R. W. Stratton, and M. N. Pearlson, Environ. Sci. Technol., 46, 4 (2012) 2. S. G. Go, S. J. Lee, G. T. Jeong, and S. K. Kim, Bioproc. Biosyst. Eng., 35, 1-2, (2012) P0517 Isolation of Agarolytic Marine Bacterium from Marine Macroalgae Llewelyn MORON1, Hee Jin YANG1, Kris Niño VALDEHUESA1, Kristine Rose RAMOS1, Rhudith CABULONG1, Grace NISOLA1, Won–Keun LEE2, Wook-Jin CHUNG1 1Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728, 2Division of Bioscience and Bioinformatics, Myongji University Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 449-728 Marine macroalgae are considered as third generation biomass resources. Red macroalgae, in particular, contain high levels of carbohydrates that can be used as feedstock for the synthesis of biofuels. Thus, in recent years, research regarding isolation of agarolytic microorganisms has steadily increased. In this study, agarolytic microbes were isolated from seaweeds obtained from seashore of Wando and Manjaedo, South Korea. Samples were grown in marine agar and agarase-positive colonies were visualized by staining with lugol’s iodine solution. Morphological, biochemical, and 16 sRNA analyses were done to confirm the identification of the isolates. The isolated microbes were further evaluated for their application in marine biomass hydrolysis. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2009-0093816). Keywords : Agarolytic bacteria, isolation, red algae References 1. Chi et al. Isolation and characterization of a novel-agar degrading marine bacterium, Gayadomonas joobiniege gen, nov. sp. nov., from the southern sea, Korean (2013), Journal of Microbiology and Biotechnology 23(11), 1509–1518 P0518 Mussel Adhesive Protein-Blended Silk Fibroin Nanofiber for Advanced Cell and Molecular Adhesion Yun Jung YANG, Bong-Hyuk CHOI, Dooyup JUNG, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 Superior mechanical properties allow silkworm silk fibroin to be applied in diverse fields. However, silkworm silk’s lack of biological functions and relatively poor biodegradation has hindered its wide use in tissue engineering. In this study, we improved bulk-scale adhesive ability as well as micro-scale adhesiveness to cells and various biomolecules by blending silk fibroin (SF) with RGD peptide-fused recombinant mussel adhesive protein (MAP-RGD). Based on high protein productivity and simple addition of MAP-RGD, silkworm silk was able to acquire much enhanced adhesion, proliferation, and spreading of mammalian cells. Adhesive silk fiber also showed better attachment of biomolecules, including carbohydrate and protein compared to SF without MAP-RGD. MAP-RGD-blended SF material also demonstrated improved hydrophilicity, swelling, and biodegradability without notable hampering of the original mechanical properties of SF. Simple blending strategy can expand silk’s application as an adhesive substitute composite material with improved biological functions. Keywords : Adhesive silk, Silkworm silk fibroin, Mussel adhesive protein, Blending, Biological function, Physical property References 1. Y.J. Yang, Y. Kwon, B.H. Choi, D. Jung, J.H. Seo, K.H. Lee, and H.J. Cha, "Multifunctional adhesive silk fibroin with blending of RGD-bioconjugated mussel adhesive protein", Biomacromolecules 2014, 15, 1390 한국생물공학회, 생물공학의 동향 : 2015.10 의약생물공학 P0601 Curcumin Nature-Mimetics as Potent Vasodilator Targeted L-Type Calcium Channel and Endothelin A/B2 Receptors Ye Sol UM1, Yuan CUI1, Daeho KWON2, Woon-Seob SHIN2, Seokjoon LEE3, Byong-Gon PARK1 1Department of Physiology, Institute for Clinical and Translational Research, College of Medicine, Catholic Kwandong University, Gangneung, Korea, 210-701, 2Department of Microbiology, Institute for Clinical and Translational Research, College of Medicine, Catholic Kwandong University, Gangneung, Korea, 210-701, 3Department of Pharmacology, Institute for Clinical and Translational Research, College of Medicine, Catholic Kwandong University, Gangneung, Korea, 210-701 Curcumin isolated from the root of Curcuma longa L. has versatile and useful biological properties. It shows anti- inflammatory, antioxidant, antiviral, chemopreventive, anti-infective, and wound-healing properties [1, 2]. We synthesized a library of curcumin mimics with diverse alkylsulfonyl and substituted benzenesulfonyl modifications through a simple addition reaction of important intermediate, 1-(3-Amino-phenyl)-3-(4-hydroxy-3-methoxy-phenyl)-propenone, with various sulfonyl chloride reactants and then tested their vasodilatation effect on L-type Ca2+ (CaL) channel activation- and endothelin-1 (ET-1)-induced-basilar artery contraction. Generally, curcumin mimics with aromatic sulfonyl groups showed stronger vasodilation effect than alkyl sulfonylated curcumin mimics. Among the tested compounds, six curcumin mimics in a CaL channel activation-induced vasoconstriction and seven compounds in an ET-1-induced vasoconstriction showed strong vasodilation effect. When the oral administration of curcumin mimics, induced blood pressure down-regulation in spontaneous hypertension rats (SHR+/+) and these effects are maintained at least 24 hrs. Keywords : Curcumin, Vasodilatation, Calcium channel, Endothelin receptor, Cerebral artery References 1. Zhou H., Beevers C. S., and Huang S. Targets of curcumin (2011), Curr. Drug Targets. 12, 332 2. Ghosh S., Banerjee S., and Sil P. C. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update (2015), Food Chem. Toxicol. 83, 111 P0602 Preparation of Polymeric Hydrogels Incorporating Cyclic Dinucleotides via Emulsion-Solvent- Evaporation Method as Vaccine Adjuvants Eunjoo LEE, Heejung JUNG, Hyejung MOK Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea Cyclic dinucleotides have been well known as a natural cytosolic molecules synthesized by cGAMP synthase in the presence of viral DNA or bacterial DNA in mammalian cells. In particular, cyclic guanosine mono phosphate-adenosine monophosphate can activate stimulator of interferon genes pathway, which promote release of Type 1 interferon like IFN-α and IFN-β [1]. Thus, cyclic dinucleotides can be harnessed as vaccine adjuvants due to their excellent induction of Type 1 interferon in mammalian cells. However, intracellular uptake of cyclic dinucleotides themselves and their location in cytoplasm seem to be difficult. To enhance intracellular uptake of gene-based vaccines and vaccine adjuvants, diverse formulation techniques have been investigated. Previously, we designed thiolated linear polyethyleneimine to produce nano- sized hydrogels for efficient delivery to cancer cells [2]. In addition, hydrogels with a size of ~0.5~2 µm showed improved intracellular uptake of gene-based vaccines and vaccine adjuvants via phagocytosis to antigen presenting cells like macrophages. In this study, we formulated cationic hydrogels with a size around 500 nm using LPEI-SH and hyaluronic acids. As therapeutic oligonucleotides, cyclic dinucleotides were incorporated within hydrogels. Hydrodynamic size of LPEI-HA hydrogels was measured by dynamic light scattering. Encapsulation efficiency of cyclic dinucleotides was quantitatively determined. Cell viability of formulated hydrogels was evaluated using macrophage cells (RAW264.7 cells) and fibroblast cells (L929 cells), respectively. Keywords : hydrogel, Cyclic dinucleotides , vaccine adjuvant References 1. Wu J, Sun L, Chen X, Du F, Shi H, Chen C, Chen ZJ. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.(2013) Science. 339(6121):826 2. Hong CA, Kim JS, Lee SH, Kong WH, Park TG, Mok H, YS Nam. Reductively Dissociable siRNA-Polymer Hybrid Nanogels for Efficient Targeted Gene Silencing. (2013) Advanced Functional Materials. 23, 316 . P0603 Production of 30Kc19α Protein Nanoparticle for Drug Delivery Yeon Hwa WOO, Hee Ho PARK, Jina RYU, Tai Hyun PARK Interdisciplinary Program for Bioengineering, Seoul National University, Seoul 151-744, Republic of Korea Nanoparticles are an emerging strategy for drug delivery. Previously, we demonstrated that application of a recombinant 30Kc19 protein to human serum albumin (HSA) nanoparticles enhanced cellular uptake and stability of a nanoparticle cargo enzyme. Here, similar drug delivery potential could be achieved with nanoparticles prepared only with α-helix domain of the 30Kc19 protein (30Kc19α) without the addition of HSA. 30Kc19α nanoparticles were prepared using the desolvation method. Several parameters of the preparation process were evaluated; the pH of the 30Kc19α solution and the protein concentration were shown to be the crucial factors determining the nanoparticle size. Protein concentration of 1 mg/ml and pH 9-10 produced the optimal particle size between 180-230 nm. It was notable that the 30Kc19α protein particles were smaller in size than the 30Kc19-HSA protein nanoparticles. SEM images showed that 30Kc19α nanoparticles were uniformly spherical in shape. The 30Kc19α nanoparticles had negligible toxicity to cells. These results suggest that 30Kc19α nanoparticles could be employed as a versatile tool for drug delivery to various cells. Keywords : Nanoparticle, Drug delivery , 30Kc19α protein References 1. Lee HJ, Park HH, Kim JA, Park TH. Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles. Biomaterials 2014; 35(5):1696-1704 2. Von Storp B, Engel A, Boeker A, Ploeger M, Langer K. Albumin nanoparticles with predictable size by desolvation procedure. J Microencapsul 2012; 29(2):138-46 3. Park JH, Park HH, Choi SS, Park TH. Stabilization of enzymes by the recombinant 30Kc19 protein. Process Biochem 2012; 47(1):164-9 P0604 Human Genes with a Greater Number of Transcript Variants Tend to Show Biological Features of Housekeeping and Essential Genes Kyu-Sang HWANG1,2, Jae Yong RYU1,2, Hyun Uk KIM1,2,4, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea, 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea, 4The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark Alternative splicing is a process observed in gene expression that results in a multi-exon gene to produce multiple mRNA variants which might have different functions and activities. Although physiologically important, many aspects of genes with different number of transcript variants (or splice variants) still remain to be characterized. In this study, we provide bioinformatic evidences that genes with a greater number of transcript variants are more likely to play functionally important roles in cells, compared with those having fewer transcript variants. Among 21,983 human genes, 3,728 genes were found to have a single transcript, and the remaining genes had 2 to 77 transcript variants. The genes with more transcript variants exhibited greater frequencies of acting as housekeeping and essential genes rather than tissue-selective and non-essential genes. They were found to be more conserved among 64 vertebrate species as orthologs, subjected to regulations by transcription factors and microRNAs, and showed hub node-like properties in the human protein-protein interaction network. These findings were also confirmed by metabolic simulations of 60 cancer metabolic models. All these results indicate that genes with a greater number of transcript variants play biologically more fundamental roles. Keywords : transcript variants, multi-exon gene, alternative splicing, cancer metabolic models References 1. A. J. Butte, V. J. Dzau and S. B. Glueck, Physiol. Genomics, 2001, 7, 95-96. 2. C. W. Chang, W. C. Cheng, C. R. Chen, W. Y. Shu, M. L. Tsai, C. L. Huang and I. C. Hsu, PLoS One, 2011, 6, e22859. 3. B. Georgi, B. F. Voight and M. Bucan, PLoS Genet., 2013, 9, e1003484 P0605 Effects of Size and Surface Property of Gold Nanoparticles on Epithelial Cells : Characterization, Cellular Uptake, and Inflammatory Response Yoonjae SEO, Soonjo KWON Department of Biological Engineering, Inha University, Incheon, 402-751, Korea The evidence for applications of gold nanoparticles (AuNPs) to disease treatment has been increasing. However, effects of size and surface property of AuNPs on their translocation and toxicity have not been characterized well. In this study, we used the different size and surface property of AuNPs to measure the endo-/exocytosis. We also measured claudin-1 (tight junction forming protein) to check the perturbations in the barrier function of the epithelial cells. To correlate the endo- /exocytosis of AuNPs and inflammatory responses, we also monitored the nitric oxide concentration following exposure of different size and surface property of AuNPs to two different types of epithelial cells. Keywords : gold nanoparticle, barrier function, endo-/exocytosis, inflammatory response, epithelial cell References 1. Oh, N., & Park, J. -. (2014). Surface chemistry of gold nanoparticles mediates their exocytosis in macrophages. ACS Nano, 8(6), 6232-6241. 2. Furuse, M., Furuse, K., Sasaki, H., & Tsukita, S. (2001). Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into madin-darby canine kidney I cells. Journal of Cell Biology, 153(2), 263- 272. P0606 Generating Aptamers Capable of Binding to Antimicrobial Peptides: PG1, PR26 and PMAP36 Phat-Loc NGUYEN1, Kyeong-Ah LEE1, Simranjeet Singh SEKHON1, Jiho MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, PG1 (Protegrin-1, an antimicrobial agent in the treatment of local or systemic infections); PR26 (Proline-arginine-rich Neutrophil Antibacterial peptide, against enteric gram-negative bacteria) and PMAP36 (Porcine Myeloid Antibacterial peptide, a highly cationic and amphipathic α-helical peptide) are identified as important components of host defense mechanism [1-3]. Aptamers are oligonucleotide ligands capable of binding to detect or chemically modified these peptides. After 10 rounds of selection, ssDNA aptamers targeting these peptides with high affinity were generated by SELEX (Systematic Evolution of Ligands by Exponential enrichment) process from an initial library containing 76 or 100 bases long variable region. The secondary structure and affinity of aptamers binding peptide were analyzed using mfold-DNA folding form and surface plasmon resonance (SPR) assay. This research was supported by Bio-industry Technology Development Program for, Ministry for food, Agriculture, Forestry and Fisheries, Republic of Korea (311007-5). Keywords : Aptamer, SELEX, AMPs (Antimicrobial peptides), PG1, PR26, PMAP36 References 1. Scocchi, M.; Zelezetsky, I.; Benincasa, M.; Gennaro, R.; Mazzoli, A.; Tossi, A. Structural aspects and biological properties of the cathelicidin pmap-36. FEBS J 2005, 272, 4398-4406. 2. Korthuis, R.J.; Gute, D.C.; Blecha, F.; Ross, C.R. Pr-39, a proline/arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. Am J Physiol 1999, 277, H1007-1013. 3. Steinberg, D.A.; Hurst, M.A.; Fujii, C.A.; Kung, A.H.; Ho, J.F.; Cheng, F.C.; Loury, D.J.; Fiddes, J.C. Protegrin- 1: A broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrobial Agents and Chemotherapy 1997, 41, 1738-1742. P0607 Chemotherapy of Breast Cancer Cells during the Metastatic Phase by CEACAM6-Targeting Albumin Nanoparticles Hohyeon LEE1, Minji LEE1, Hyuncheol KIM1,2 1Department of Chemical & Biomolecular Engineering, Sogang University, #1 Shinsu-dong, Mapo-gu, Seoul, 121-742, Republic of Korea, 2Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Korea As an emerging cancer treatment strategy, drug delivery systems have great potentials to maximize an effect of chemotherapeutic drugs. However, in the case of metastatic tumor cells, basic systems have limited delivery efficiency due to the turbulent blood flow and absence of binding ability. Consequently, surface modification is highly required for enhancing the entrance of drug-encapsulating nanoparticles into the metastatic tumor cells. Here, we developed an albumin- based drug delivery system to target CEACAM6-overexpressing metastatic breast cancer cells by an ethanol desolvation method. As a molecular target of metastatic cancer cells, we selected CEACAM6 which are reported that highly expressed in various metastatic cancers. The results showed that the nanoparticles are quickly attached and entered to floating metastatic cancer cells and effectively inhibited the growth despite the short-time treatments in vitro. In summary, this system can be easily applied to many kinds of metastatic tumors, including breast, pancreatic and also gastric tumors, without the aid of other medical devices, such as ultrasound or MRI to treatment of floating metastatic cancer cells. Keywords : Anoikis-resistance, Breast cancer, Metastasis, CEACAM6, Antibody, Nanoparticles References 1. Duxbury, M. S., Ito, H., Zinner, M. J., Ashley, S. W., & Whang, E. E., CEACAM6 gene silencing impairs anoikis resistance and in vivo metastatic ability of pancreatic adenocarcinoma cells (2004), Oncogene, 23(2), 465-473 2. Lee, H., Park, S., Kim, J. B., Kim, J., & Kim, H., Entrapped doxorubicin nanoparticles for the treatment of metastatic anoikis-resistant cancer cells (2013), Cancer letters, 332(1), 110-119 P0608 Synthesis of Nerve Growth Factor Vector for the Treatment of Glaucoma by Regeneration of Retinal Ganglion Cell Layer Lee JIHWANG1,1, Lee SUNHO1,1, Kim JAMIN1,1, Han DAE JONG2,2, Kim, HYUNCHEOL1,1 1Department of Chemical and Biomolecular Engineering, Sogang University, Mapo-gu,Seoul 121-742, South Korea, 2Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea, 110-799 To develop a plasmid DNA-based therapeutic agent delivery system to the retina ganglion cell layer for the treatment of glaucoma, which means optic nerve degeneration. we established the strategy that regenerate impaired neuron by delivering nerve growth factor (NGF). NGF mRNAs were extracted from human lung epithelial A549 cells. Then the prepared NGF mRNAs were inserted into plasmid DNA. In order to confirm the NGF expression by the pcDNA-NGF vector, in vitro experiments were performed. RGC-5 cell which is NGF non-expressed cell line was transfected by lipofectamin and pcDNA-NGF vector complexes. The results showed over-expression of NGF protein about 20-folds at 1 day and 4-folds at 6 day by ELISA analysis. To evaluate the expression and activity of NGF, we transfected pcDNA-NGF vector to PC-12 cells by lipofectamine and observed the neurite outgrowth. After 7 days of post-transfection, neurite was observed between the cells and they were gradually increased in course of time by 27 day. The pcDNA-NGF show great potential at the treatment of glaucoma. neurite outgrowth will contribute to the treatment of glaucoma by showing the regeneration of nerve cells that can be hoped. Keywords : nerve growth factor, NGF, glaucoma, pcDNA-NGF vector References 1. H. Wang et al., BioMed research international 2014 (2014) 2. G. Roberti et al., j. Cell. Physiol. 229, 1130-1133 (2014) P0609 HPLC Fractionation and Pharmacological Assessment of Green Tea Seed Saponins for Anti- angiogenic and Hemolytic Activities Jong Deog KIM1, Muhammad Imran KHAN1, Tai Sun SHIN2 1 Department of Biotechnology, Chonnam National University, 2Department of Food Science and Nutrition, Chonnam National University Herbal medicinal products have proven to be safe, economical, and effective alternatives to synthetic chemical pharmaceuticals. The green tea plant (Camellia sinensis) is of profound medicinal value because it contains potent bioactive constituents. The purpose of the present work was to investigate saponins from green tea seeds for potential use as anti- angiogenic, and hemolytic agents. Green tea seed saponins were separated into six fractions by reverse phase HPLC. The presence of three aglycone chains in the saponins of each fraction was confirmed by acid hydrolysis. Anti-angiogenic activity was evaluated using saponin fractions at concentrations in the range 2.5-25 mg/ml. Our results confirm that green tea saponins have anti-angiogenic, and hemolytic activities, indicating their potential for development as natural pharmaceutical products. Keywords : Camellia sinensis, saponins, anti-angiogenic, aglycone chains, acid hydrolysis References 1. Woldemichael, G.M., and M. Wink (2001) Identification and biological activities of Triterpenoid saponins from Chenopodium quinoa. J Agr Food Chem. 49: 2327– 2330. 2. Gestetner B, Birk Y, Tencer, Y (1968) Soybean saponins. Fate of ingested saponins and the physiological aspect of their hemolytic activity. J. Sci. Food Agr. 16:1031– 1035. 3. Kim, J.D., N. Chaudhary, H.J. Seo, M.Y. Kim, and T.S. Shin (2014) Tea saponin E1 as effective ingredients for anti-angiogenesis and anti-obesity effects. Biosci Biotechnol Biochem.78(2):279-87 P0610 Isolation and Fractionation of Green Tea Seed Saponins and Investigation of Anti-Microbial Activities Jong Deog KIM1, Muhammad Imran KHAN1, Min Yong KIM2 1Dept. of Biotechnology, Chonnam National University , 2Dept. of Refrigeration Engineering, Chonnam National University Bacteria and Fungi are the major cause of sever plants and animal disease. The present synthetic antibiotics are failed to achieve their goal of broad spectrum due to high cost and adverse side effects. Bioactive constituents of medicinal plants are the safe and cheap therapeutic agents for a verity of medicinal uses. Green tea seed saponin was separated into six fractions by reverse phase HPLC. Presence of three aglycone chain in each saponin fractions was confirmed by acid hydrolysis. Antimicrobial activity of saponin fractions was performed against E.coli, S. mutans, Salmonella strains and A.niger. Tea saponins showed potent anti-angiogenesis effect. All saponins fraction exhibits hemolytic activity. The saponin fractions were more potent against zoonose bacteria of Salmonella and gram negative bacteria E.coli than gram positive bacteria S. mutans. Our results confirmed the green tea saponin to be the active agent against bacteria. Keywords : Bioactive substances, Saponins, Reverse phase HPLC, Zoonose bacteria, Aspergillus niger References 1. J. Chang (2000) Medicinal herbs drugs or dietary supplements. Biochem.Pharmacol. 59: 211–219. 2. A.H. Khalil, and T.A. Adawy (1994) Food Chem.50:197–201. 3. Sen, S., H.P. Makkar, S. Muetzel, and K. Becker (1998) Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli. Lett Appl Microbiol. 27: 35–38. P0611 Structural Similarity Analysis to Identify Human Pathways Potentially Targeted by Compounds in Traditional Oriental Medicine Kyu-Sang HWANG1,2, Hyun Uk KIM1,2, Jae Yong RYU1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute , 2BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea., 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea Traditional oriental medicine (TOM) is gaining increasing popularity around the world. Despite its well-known efficacies in various symptoms, full exploration of the TOM as a new source of drug development has been bottlenecked by lack of precise knowledge on its mechanisms of action. To this end, structural similarities between compounds found in the TOM and human metabolites were systematically analyzed as an initial attempt to map candidate human pathways potentially targeted by TOM compounds at a global scale. The objective of this strategy was to narrow down candidate target pathways that can potentially be affected by the TOM compounds.Multicomponent/multitargeting principle of TOM is also discussed wherein multiple components show synergistic effects to treat symptoms. These systems efforts will ultimately facilitate our better understanding of TOM and its integration with modern drug discovery pipeline.[This work was supported by the Bio- Synergy Research Project (2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation.] Keywords : Multicomponent/multitargeting, Traditional oriental medicine, drug discovery References 1. H. U. Kim, J. Y. Ryu, J. O. Lee and SY Lee, Nat. Biotechnol. 33 (3), 264-268 (2015) P0612 Islet Nano-Surface Modification by Multilayer PEGs to Improve Survival Outcome in Xenotransplantation Model Muhammad Rezwanul HAQUE1, Youngro BYUN1,2 1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 151-742, South Korea, 2Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 151-742, South Korea Islet transplantation is as much effective as pancreas transplantation for the treatment of Type-1 diabetes while being much less immunogenic. Nevertheless, islet transplantation has a great obstacle, immune rejection. In this study, Non-Human Primate (NHP) islets were used as a pre-clinical model for transplantation since they have close phylogenetic and immunological relationship with humans. The immune rejection process was tried to be delayed by the nano-surface modification of islets using nano thin layer of PEGs along with development of a combination of immunosuppressive drugs. In vitro viability and functionality were confirmed for the nano-surface modified islets before in vivo transplantation into a xeno-recipient, C57BL6 mice. The transplanted graft activity was monitored for a certain period of time while administering a combination of immunosuppressive drugs. Animals transplanted with nano-surface modified islets showed higher survival rate than that of the unmodified islet group. Survival rate was further increased and steady blood glucose level was achieved when a combination of immunosuppressive drugs was administered as well. Therefore, this study demonstrates that islet coverage with nano thin layer of PEGs barely hamper its viability and functionality, which in combination with an immunosuppressive drug protocol improves survival time of the transplanted islets in vivo. Keywords : Islets, Transplantation, immunosuppressive drug, PEGylation, Surface modification References 1. A. M. James Shapiro et al, The New England Journal of Medicine 343, 4 (2000). P0613 β-secretase Inhibitory Effects of Metanolic Extracts from Euphorbiaceae and Leguminosae against Alzheimer's Disease Eun-Young NOH, Hyun Jung LEE, Soonok KIM, Changmu KIM, Joo-Hong YEO, Woo Young BANG Environmental Research Complex, National Institute of Biological Resources (NIBR), Incheon, 404-708 β-Secretase activates production of β-amyloid peptides (Aβ), the major components of the toxic amyloid plaques found in the brains of patients with Alzheimer’s disease (AD). Thus, the β-secretase is an important target for development of drugs against the AD. For a screening of β-secretase inhibitors, we performed β-Secretase assay with 80% methanolic extracts from four Euphorbiaceae and four Leguminosae plants. Among them, the extracts from Euphorbia maculata L., Securinega suffruticosa (Pall.) Rehder and Melilotus alba Medicus significantly inhibited β-Secretase activity in 0.5 % DMSO, which shows that they may include secondary metabolites having suppressive effect on the AD. Furthermore, the all eight extracts showed significantly inhibitory effects on β-Secretase activity in 1 % DMSO. Altogether, our results support that methanolic extracts from Euphorbiaceae and Leguminosae plants include metabolites corresponding to β-secretase inhibitors, which may be useful resources for development of drugs against AD. Keywords : β-Secretase, Alzheimer’s disease, secondary metabolites, Euphorbiaceae, Leguminosae References 1. V.D. Ashwlayan and R. Singh (2011) J. Appl. Pharm. 3(2), 10–15 P0614 Development of Orally Available Nanoparticle for Transporter-Mediated Absorption in the GI Tract Using Deoxycholic Acid Jooho PARK1, Youngro BYUN1,2 1College of Pharmacy, Seoul National University, Seoul 151-742, 2Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 151-742 Oral delivery is the most acceptable drug delivery method to patients. Many drugs are already orally available, however, several hydrophilic drugs, most peptides and genes still have some troubles to be absorbed in the GI tract. Based on new nanotechnology and advanced delivery system, many research groups have tried to increase the oral bioavailability of injectable drug and peptides[1]. Here, we newly designed a deoxycholic acid conjugate and nanocomplex using FDA- approved heparin and protamine for oral delivery. Among functional polysaccharides and peptides, heparin is a famous anticoagulant, and protamine is the antidote peptide to heparin. Deoxycholic acid (DOCA), the one of bile acids, conjugated to heparin and protamine to promote the oral absorption of heparin-protamine nanocomplex. This newly designed hepaarin- DOCA and protamine nanocomplex binds to apical sodium-dependent bile acid transporter (ASBT) on caco-2 and MDCK cell. In animal experiment using cy5-conjugated nanocomplex, the orally available bile acid based nanoparticle presented potential to target the ASBT in the ileum. We measured the retention period of the nanoparticles and the binding sites in the GI tract. These results represent the recent advanced drug delivery technique about orally available nanoparticle as a drug and gene carrier. Keywords : nanoparticle, oral delivery, heparin, bile acid, ASBT References 1. T. A. Al-Hilal, F. Alam, Y. Byun, Adv. Drug Delivery Rev. 65, 845-864 (2013) P0615 A MALDI-MS-Based Quantitative Targeted Glycomics (MALDI-QTaG) for Total N-glycan Analysis Kyoung-Jin KIM, Cheol-Hwan HWANG, Han-Gyu PARK, Yun-Gon KIM Dept. of Chemical Engineering, Soongsil University, Seoul, 156-743 In this study, MALDI-MS based quantitative targeted glycomics (MALDI-QTaG) was developed for monitoring the abnormal glycosylation of clinical and biopharmaceutical products. MALDI-QTaG was proposed for sensitive and quantitatvie analysis of total N-glycans. The derivatization reactions (i.e., amidation of sialic acid and incorporation of a positive charge moiety into the reducing end) dramatically increased the linearity (R2>0.99) and sensitivity (limit of detection is 05 pmol/glycoprotein) relative to underivatized glycans. In addition, the analytical strategy was chromatographic purification-free and non-laborious process accessible to the high-throughput analyses. We used teh MALDI-QTaG method to cell line (Huh7 cells). The total percentage of core-fucosylated AFP N-glycans from Huh7 cells and normal cord blood was 98.0% and 17.8%, respectively. This MALDI-MS based glycomics technology has wide application in many clinical and bioengineering fields requiring sensitive, quantitative and fast N-glycosylation validation. Keywords : Chemical derivatization, MALDI-MS, N-glycans, Quantitative analysis, UPLC References 1. Jang KS, Kim YG, Gil GC, Park SH and Kim BG, Mass spectrometric quantification of neutral and sialylated N- glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines, Anal Biochem, 19, 3421-3428 2. Gil GC, Iliff B, Cerny R, Velander WH, Van Cott KE, High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry, Anal Chem, 82, 6613-6620 3. Wang C, Wu Z, Yuan J, Wang B, Zhang Y, Wang Z, Huang L, Simplified quantitative glycomics using the stable isotope label Girard's reagent p by electrospray ionization mass spectrometry, J Proteome Res, 13, 372-384 P0616 A Novel Albumin Nano-Complex with both siRNA and Au Nanorods for a Synergetic Therapeutic Dual Effect on Solid Cancer Jin-Ha CHOI1, Eun-Jung LEE1, In-Hwa KIM1, Byung-Keun OH1,2 1Department of Chemical & Biomolecular Engineering, Sogang. University, 35 Baekbeom-Ro, Mapo-gu, Seoul 121-742, 2Interdisciplinary Program of Integrated Biotechnology Sogang. University, 35 Baekbeom-Ro, Mapo-gu, Seoul 121-742 In recent years, advances in cancer treatment are made by diverse novel therapeutic methods and combined techniques. In this study, we developed two component encapsulated nano-complex based on bovine serum albumin (BSA) for effective breast cancer treatment through targeted photothermal treatment of encapsulated AuNR and RNAi effect of siBcl-2, which is targeted on Bcl-2 protein, critical factor for apoptosis pathway, simultaneously. RNA interference (RNAi) is a powerful tool for the treatment of various cancers on behalf of chemotherapeutic agents and photothermal therapy as therapeutic tool is an attractive method for treating cancer cells with a minimally side effect and maximally therapeutic effect. The siRNA and AuNRs encapsulated BSA (SREB) complexes is a function as effective delivering of siRNA, photothermal therapeutic agent simultaneously, able to cause synergetic effect on cancer treatment. Most of all, the optimized SREB complexes resulted in a very impressive therapeutic effect in breast cancer, which is ease to be confirmed photothermal effect, an apparently complete apoptosis of the cancer cell was achieved. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2015R1A2A2A01007843) Keywords : Bovine serum albumin (BSA), siRNA, Au nanorod, photothermal therapy, cancer treatment References 1. JH Choi et al., Nanoscale 7(20), 9229-2937 (2015) P0617 Design of Doxorubicin Containing Polymeric Microbeads for the Treatment of Hepatic Cancer Ratchapol JENJOB, Phruedsaporn TARANAMAI, Min-Kyung KIM, Su-Geun YANG Department of New Drug Development, School of Medicine, Inha University, Incheon 400-712, South Korea Transcatheter arterial chemoembolization (TACE) using microbeads has been an effective clinical practice in hepatocellular carcinoma (HCC) therapy, and it avoids the toxic effect of chemotherapy. The injected microbeads occlude the tumor’s blood supply, resulting in necrosis of the tumor cells. In this study, the doxorubicin (DOXO)-containing albumin microbeads (DOXO-MBs) cross-linked with glutaraldehyde as new TACE modality was prepared using a capillary microfluidic device. The DOXO-MBs showed narrow size distribution with 183.2±2.2 μm and the size was easily controlled by changing the flow of fluidic solutions. SEM imaging and DOXO release were performed. DOXO released in controlled manner for a month and the maximum release rate was around 15 and 46% in pH 7.2 and 5.4 buffer, respectively. The localization and distribution of DOXO beads in hepatic vessel was confirmed after intra-portal injection of DOXO beads on rats which proved DOXO-MBs accumulated in the hepatic vessels. Hepatic tissues, recovered at 24 days after portal injection, showed that the DOXO released from DOXO-MBs penetrated surrounding hepatic tissues at a depth of 200 μm and induced a change of cellular morphology. And in vivo therapeutic efficacy was estimated on xenograft hepatic cancer models which revealed that DOXO-MBs more efficiently inhibited tumor growth than I.V. injection (p<0.01). Acknowledgement; This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT and future Planning (2014R1A2A2A04006562) Keywords : Doxorubicin, Albumin, Microbead References 1. R. Xu, M. Fisher, and R. L. Juliano, Bioconjug. Chem. 22, 870 (2011). 2. D. Lombardi and P. S. Dittrich, Anal. Bioanal. Chem. 399,347(2011). P0618 Enhanced Conjugation Stability and Blood Circulation Time of New Macromolecular Gadolinium-DTPA Contrast Agent Phruedsaporn TARANAMAI, Ratchapol JENJOB, Lim Hyo SANG, Su-Geun YANG Department of New Drug Development, School of Medicine, Inha University, Incheon 400-712, South Korea In this study, we prepared macromolecular MR T1 contrast agent: pullulan-conjugated Gd diethylene triamine pentaacetate (Gd-DTPA-Pullulan) and estimated residual free Gd3+, chelation stability in competition with metal ions, plasma and tissue pharmacokinetics, and abdominal MR contrast on rats. Residual free Gd3+in Gd-DTPA-Pullulan was measured using colorimetric spectroscopy. The transmetalation of Gd3+ incubated with Ca2+ was performed by using a dialysis membrane (MWCO 100-500 Da) and investigated by ICP-OES. The plasma concentration profiles of Gd-DTPA-Pullulan were estimated after intravenous injection at a dose 0.1 mmol/kg of Gd. The coronal-plane abdominal images of normal rats were observed by MR imaging. The content of free Gd3+, the toxic residual form, was less than 0.01%. Chelation stability of Gd- DTPA-Pullulan was estimated, and only 0.2% of Gd3+ was released from Gd-DTPA-Pullulan after 2 h incubation with Ca2+. Gd-DTPA-Pullulan displayed the extended plasma half-life (t1/2,α = 0.43 h, t1/2,β = 2.32 h), much longer than 0.11 h and 0.79 h of Gd-EOB-DTPA. Abdominal MR imaging showed Gd-DTPA-Pullulan maintained initial MR contrast for 30 min. The extended plasma half-life of Gd-DTPA-Pullulan probably allows the prolonged MR acquisition time in clinic with enhanced MR contrast. Acknowledgement; This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2014K2A2A2000720) Keywords : Gadolinium, Contrast agent, Diethylene triamine pentaacetate References 1. H. Yim, S.-G. Yang, Y. S. Jeon, I. S. Park, M. Kim, D. H. Lee, Y. H. Bae, K. Na, Biomaterials. 32, 5187 (2011). 2. M. Moon, R. G. Thomas, S-u Heo, M-S Park, W K.Bae, S H.Heo, N.Y.Yim, Y.Y.Jeong, Mol. Imaging Biol. 17, 497 (2015). P0619 Nanofibrous Matrix Using Poly(e-caprolactone)/Poly(N-vinylpyrrolidone) for Tissue Adhesives Ye-Eun YOON, Jae-Hyung JANG* Dept. of Chemical & Biomolecular Engineering, Yonsei University, Seoul Nowadays there are many necessities for surgical bio-adhesives that can adhere to surfaces of organs and stop bleeding from suture cavities. Although many alternative products are already in the market, these present some drawback, such as toxicity and poor adhesion in wet environment. Here, we introduced a novel approach to fabricate nanofibrous matrix for tissue adhesives. These nanofibrous matrix was fabricated by electrospinning technique using poly(e-caprolactone) and poly(N- vinylpyrrolidone). The addition of poly(N-vinylpyrrolidone) resulted in a dramatic increase in adhesive property, which has a direct correlation with their hemostatic potential. Therefore, this study will be promising for drug delivery, cell therapy, and other biomedical application in further study. Keywords : electropspinning, adhesives, tissue engineering, scaffold, nanofiber References 1. Biomater. Sci., 2013, 1 , 239 P0620 Adeno-Associated Virus (AAV) Transferring Pro-Apoptotic Genes for Cancer Gene Therapy Yoojin KIM, Eunmi KIM, Ye-Eun YOON, Jae-Hyung JANG Department of chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea Viral gene delivery vehicles are one of the most effective methods of transferring therapeutic levels of the transgenes in gene therapy applications [1]. Adeno-associated viruses are leading candidates for the clinical cancer gene therapy vehicles based on their characteristics of non-pathogenic, non-toxic, and replication deficient [2]. Using these safe vehicles, we tried to deliver well-known pro-apoptotic genes, BIM, to induce the apoptosis in cancer cells. BIM, belongs to the BH3-only proteins, is considered as a pro-apoptotic protein because it induces Bax / Bak oligomerization on mitochondria and forms channels on the mitochondrial membrane [3]. And then this permeabilization leads to cytochrome c release and apoptosis in the pro-apoptotic gene transferred cancer cells [3]. We applied this therapeutic system to breast cancer cell and melanoma cancer cell, and confirmed the BIM protein expression and its simultaneous apoptotic activities in cancer cells [4]. Keywords : Adeno-associated virus, Viral gene delivery, pro-apoptotic genes References 1. Raghu Gogada · Neelu Yadav · Junwei Liu · Shaohua Tang · Dianmu Zhang · Andrea Schneider · Athul Seshadri · Leimin Sun · C Marcelo Aldaz · Dean G. Tang · Dhyan Chandra, Bim, a Proapoptotic Protein, Up-regulated via Transcription Factor E2F1-dependent Mechanism, Functions as a Prosurvival Molecule in Cancer (2012), Journal of Biological Chemistry, 288(1), 368-381 2. Susan M Faust, Peter Bell, Yanqing Zhu, Julio Sanmiguel and James M Wilson, The Role of Apoptosis in Immune Hyporesponsiveness Following AAV8 Liver Gene Transfer (2013) , Molecular Therapy, 21(12), 2227- 2235 3. O'Connor L, Strasser A, O'Reilly LA, Hausmann G, Adams JM, Cory S, Huang DC., Bim: a novel member of the Bcl-2 family that promotes apoptosis. (1998) , EMBO J. 15;17 (2):384-95 4. Georg Häcker., Kathrin Suttner, Hisashi Harada and Susanne Kirschnek, TLR-dependent Bim phosphorylation in macrophages is mediated by ERK and is connected to proteasomal degradation of the protein (2006) , Int. Immunol. 18 (12): 1749-1757 P0621 Adhesion Force of Chitosan: Effects of Acetylation, Contact Time, and Molecular Weight Chanoong LIM1, Dong Woog LEE2, Jacob ISRAELACHVILI2, Dong Soo HWANG3 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784, 2Dept. of Chemical Engineering, University of California at Santa Barbara, Santa Barbara, CA 93106, 3Integrative Biosciences and Biotechnology, POSTECH, Pohang, 790-784 Chitosan, a load-bearing biopolymer found in the exoskeletons of crustaceans and insects, is a promising biopolymer for the replacement of synthetic plastic compounds. Especially, low molecular weight chitosan (LMW chitosan, ~5 kDa) are widely used in biomedical applications such as anti-microbial, anti-tumor, and anti-diabetes. Here, surface interactions mediated by chitosan in aqueous solutions, including the effects of acetylation, contact time and molecular weight were investigated using a surface forces apparatus (SFA). Previously, we measured molecular interactions of chitosan focused on molecular weight and pH ranges. High molecular weight chitosan (HMW chitosan, ~110 kDa) and LMW chitosan were prepared in this experiment. Each of chitosan was reacetylated using acetic anhydride. Degree of acetylation (DA) of LMW chitosan was 11%, 61%, 98% and HMW chitosan was 19%, 28%, 50%. Molecular interactions of chitosan were measured by SFA, chitosan-mica surface (asym), chitosan-chitosan (sym). In LMW chitosan, highly acetylated chitosan (DA 98%) showed strongest adhesion at asym but lowest at sym mode. In HMW chitosan, chitosan of DA 50% showed both lowest adhesion at asym and sym mode. These differences are caused by molecular conformation during acetylation, converting into amine group to acetyl group changed inter- and intramolecular hydrogen bonding network. Keywords : chitosan, SFA, degree of acetylation, reacetylation, biopolymer, molecular interactions References 1. D. W. Lee, C. Lim, J. N. Israelachvili, D. S. Hwang, Langmuir, 29, 14222-14229 (2013) 2. C. Lim, D. W. Lee, J. N. Israelachvili, D. S. Hwang, Carb. Pol, 117, 887-894 (2014) 3. S. Hirano, R. Yamaguchi, Biopolymers, 15, 1685-1691 (1976) P0622 Development of LIMLET as an Anti-Tumor Agent Sujin JEONG, Sangah LEE, E.K. LEE Dept. of Bionano Engineering, Hanyang University, Ansan, 15588 HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cell) is known to have a selective cytotoxic activity against certain types of tumors [1,2]. It consists of lactalbumin, an abundant protein in mother’s milk, and oleic acid, a naturally occurring fatty acid. The cytotoxicity is known to stem from water-insoluble oleic acid, which may indicate lactalbumin may simply act as a natural carrier for the fatty acid. Considering this, we manufactured an alternative drug complex using liposome as a fatty acid delivery component. We named it LIMLET (LIposome Made LEthal to Tumor cell), in which oleic acid is inserted into the lipophilic phospholipid bilayer during a vesicle formation step [3]. Using LIMLET and employing WST-1 assay, we tested its cytotoxicity against two cancer cell lines; MDA-MB-231 (human breast cancer) and A549 (human lung cancer), and compared the results with that against a normal cell line; VERO (from monkey kidney). As a result, we found that: (1) LIMLET possesses selective, concentration-dependent toxicity against the cancer cells (several- fold difference in ED50 values), and (2) LIMLET shows distinctive cytotoxicity against A549 whereas plain liposomes (containing no oleic acid) has no toxicity at all. Our result clears shows the LIMLET can emulate with HAMLET as an interesting nanolipocomplex for tumor treatment. Keywords : LIMLET, Oleic acid, Liposome, Anti-tumor, cytotoxicity References 1. C. Svanborg et al, HAMLET (human a-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death (2009), International Journal of Cancer, 124(5), 1008-1019. 2. C. Svanborg et al, Apoptosis induced by a human milk protein(1995), PNAS , 92(17), 8064-68 3. E. K. Lee et al, Effects of operating parameters on the efficiency of liposomal encapsulation of enzymes(2012) , Colloids and Surfaces B: Biointerfaces , 94, 296–303 P0623 Functional Self-Expander Hydrogel for Sustained Release of Growth factor Ji-eun KIM1, Dongmyong KIM3,4, Hochang KANG4, Jonghoon CHOI1,2 1Dept. of Bionanotechnology, hanyang university, Seoul, 133-794, 2Dept. of BionanoEngineering, hanyang university, ansan, 426-791, 3Colloge of natural science, seoul university, seoul, 4Probiomimetic Res. Center, seongnam We synthesized copolymer of hydrogel composed of methyl-methacrylate and N-vinyl-2-pyrrolidone with tannic acid and in vitro release of growth factor from the self-expanded hydrogel in a physiological condition. The degree of expansion of self- expander copolymer was measured in various solutions. We induced absorption of drug through self-expansion caused by osmotic pressure into hydrogel composed of methyl methacrylate and N-vinyl-2-pyrrolidone. Cell proliferation effects of released growth factor were evaluated with NIH3T3 cell line. Growth factor contained self-expander hydrogel may have a potential in biomedical applications, for example, implantation at the tissue area that requires physical support as well as wound healing Keywords : hydrogel, MMA, NVP, cell proliferation References 1. Guoqin Liu. et al, J. Polym. Res. 18, 2109-2117 (2011). P0624 Poly(dopamine) Coating of Microsphere for Stem Cell Carrier and Gene Delivery. Jung-suk KIM, Minhee KIM, Eunmi KIM, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea The goals of tissue engineering are to restore, repair, or replace shortages in tissues and organs for transplantation therapy such as cells, proteins, or genes by developing tools for biological substitute. In order to create clinically usable volume of tissue, it is essential to keep the cells alive until graft survival, which is one of the important issues in the field of tissue engineering. Herein, we have developed the cell and viral gene delivery platform using microparticles which was modified with either norepinephrine or dopamine to enhance cell attachment and cell viability. Cells were immobilized onto the modified particles and the efficiency of attachment was enhanced in modified with poly (dopamine) (pDA). In addition, to nutritionally support cells after delivered to target sits, AAV was chosen as a carrier, releasing growth factor. AAV is a nonpathogenic and does not elicit immune responses in animal models. Cells were successfully immobilized on the modified particles and the improved cell viability on the modified with pDA was characterized. Through experiments, pDA coated three dimensional scaffolds can provide was proven to be an effective strategy and exhibited great potential in tissue engineering application. Keywords : Polycaprolactone, poly(dopamine), Surface modification, Cell adhesion, AAV, biointerface References 1. Jgamadze D, Bergen J, Stone D, Jang J-H, Schaffer DV, Isacoff EY, et al. Colloids as Mobile Substrates for the Implantation and Integration of Differentiated Neurons into the Mammalian Brain(2012), PLoS ONE, 7,e30293. P0625 PARIS Reprograms Glucose Metabolism through Transketolase Suppression Hyein KIM1, Joo-ho SHIN1 1Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University, School of Medicine, Suwon, 2Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University, School of Medicine, Suwon Parkinson’s disease (PD) is a progressive neurodegenerative disorder and is caused by selective loss of dopaminergic neurons (DA) in the substantia nigra pars compacta (SNpc). Several PD-associated genes, including parkin, α-synuclein, leucine rich repeat kinase 2 (LRRK2), DJ-1, PINK1, and ATP13A2 have been identified, and an investigation of their biology has been providing clues to study the pathogenesis of PD. Recently, PARIS is found to play a deleterious role in DA death by suppressing the expression level of PGC-1α. PARIS (a zinc-finger protein ZNF746) is a Parkin-interacting substrate. Accumulation of PARIS may lead to dopaminergic neuronal death via suppression of mitochondrial biogenesis, ROS defense, and oxidative phosphorylation in pathogenic condition (Shin et al., 2011). However, how the selective DA death caused by PARIS overexpression is still questionable due to the lack of knowledge on PARIS functionality. In order to understand how the accumulation of PARIS causes brain region-specific toxicity, we generated tTA-responsive PARIS transgenic mice using tet-off system (TetP-Flag-PARIS), which further is crossed with CamKII-tTA mice to express PARIS in neuronal cells (Tg-PARIS). Subsequently, we performed the LC-MS/MS-based quantitative proteomic analysis to profile the global proteomic alteration in the frontal cortex (FC), striatum (STR), ventral midbrain (VM), and cerebellum(CBM) of Tg-PARIS as compared with wild-type mice (WT). Pattern analysis reveals that seven proteins (neurofilament medium polypeptide, neural cell adhesion molecule 1, transketolase, alpha-internexin, glutamate decarboxylase 2, dihydropyrimidinase-related protein 3, and microtubule-associated protein) are exclusively down-regulated in VM but not in other regions. Since previous studies have demonstrated that reduced transketolase (TKT) activity was shown to be associated with neurodegenerative disorder including Alzheimer’s disease and PD (Sheu et al., 1988; Jimenez-Jimenez et al., 1999), we investigate the relationship between TKT and PARIS in PD pathogenesis. As a result, we found that PARIS transcriptionally suppressed the level of TKT in brain region and cell-specific manner. These findings suggest that TKT might be a target molecule in PARIS-induced selective DA death. Keywords : Parkinson’s disease , PARIS, Pattern analysis, Transketolase P0626 Dissection of Structure and Mode of Action Using N-terminal Penetrating Sequence of Frog Antimicrobial Peptide Eun-Ji KIM, Jae-Eun JEONG, Yung-Hoon PARK, Mi-Kyeong JANG, Seong-Cheol PARK* Dep. of Polymer Science and Engineering, College of Engineering, Sunchon National University, Jeonnam 540-950, Korea Abuse and the increasing use of antibiotic in biomedical fields has led to the emergence of antibiotic-resistant microbial pathogens. In recent, antimicrobial peptides (AMPs) are recognized to promising new antibiotic molecules. The aims of present study are to understand the mode of action of frog AMP in bacterial cells and to gain information for structural factors that influence to penetration and membranolytic action. Based on results, we found that IFGAIL sequence was a new-type motif penetrating into bacterial cytosol. Therefore, these results could aid in the design of penetrating-peptide agents with enhanced antimicrobial activity and reduced cytotoxicity. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded the Ministry of Education, Science and Technology (2013R1A1A4A01010701). Keywords : antimicrobial peptide, drug-resistance, antibiotic P0627 Development of VEGF-Specific Repebody for the Suppression of Choroidal Neovascularization Da-Eun HWANG1, Jeong-Hyun RYOU1,2, Jong Rok OH3, Jung Woo HAN3, Tae Kwann PARK3, Hak-Sung KIM1 1Dept. of Biological sciences, KAIST, Daejeon, 305-701, 2Graduate School of Medical Science & Engineering, KAIST, Daejeon, 305-701, 3Dept. of Ophthalmology, Soonchunhyang University, College of Medicine, Bucheon Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over age 60. Vascular endothelial growth factor (VEGF) plays major roles in pathological angiogenesis in AMD. Here, we present the development of VEGF-specific repebody which is a non-antibody scaffold consisting of leucine-rich repeat (LRR) modules. The newly generated repebody showed high binding affinity and specificity against VEGF. We demonstrated the resulting repebody effectively inhibited in vitro angiogenic cellular processes, such as proliferation and migration, by blocking VEGF- induced signaling pathways. We also revealed this repebody suppresses choroidal neovascularization (CNV) and vascular leakage significantly in vivo. Our results showed the repebody may have therapeutic potentials to neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers. Keywords : repebody, VEGF, choroidal neovascularization, age-related macular degeneration References 1. Sang-Chul Lee, et al. (2012), Proc Natl Acad Sci USA, 109(9), 3299 2. Joong-jae Lee, et al. (2014), Mol Ther, 22(7), 1254 3. Woosung Heu, et al. (2014), Anal Chem, 86(12), 6019 P0628 Modulation of ERK2 Signaling Pathway by Design and High-efficiency Delivery of Proteins Yoo-Kyoung SOHN1, Jeong-Hyun RYOU1,2, Hak-Sung KIM1 1Dept. of Biological sciences, KAIST, Daejeon, 305-701, 2Graduate School of Medical Science & Engineering, KAIST, Daejeon, 305-701 Biological phenomena occur through complicated processes which are maintained and directly regulated by the interactive functions of biomolecules, mainly involved in signaling pathways. Thus, dysregulation of intracellular signaling pathways can be immediate cause of diseases, and signaling-related proteins have been targeted over past decades for therapeutic effects, accordingly. We previously developed a highly efficient intracellular delivery system based on bacterial toxins. Here, we design and deliver a potent signaling modulator that can effectively modulate ERK2 signaling pathway. We first delivered a negative regulator of ERK2, PEA-15, which directly interacts with ERK2, and confirmed that the direct delivery of un-phosphorylated PEA-15 (PEA-15AA) can inhibit ERK2 signaling. Then, for the effective modulation, we further designed a potent signaling modulator by engineering PEA-15AA through phage peptide display. The successfully designed PEA-15 exhibited 800-fold higher affinity to ERK2 and showed much effective modulation of ERK2 signaling than PEA- 15AA when intracellularly delivered. Consequently, based on design and high-efficiency delivery of proteins, we believe that intracellular signaling modulation can be the most powerful therapeutic approach in diseases caused by dysregulation of signaling pathways, enabling biological research. Keywords : Intracellular delivery, Protein delivery, Bacterial toxin, Signaling pathway, ERK2, PEA-15, Peptide engineering, Phage display, Directed Evolution, Protein-protein interaction References 1. JH. Ryou and HS. Kim. Shiga-like toxin-based high-efficiency and receptor-specific intracellular delivery system for a protein (2015), Biochem. Biophys. Res. Commun. 464(4), 1282-1289 2. SC. Lee, K. Park, J. Han, JJ. Lee, HS. Kim. Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering (2012), PNAS, 109(9), 3299-3304 한국생물공학회, 생물공학의 동향 : 2015.10 식품생물공학 P0701 In Vitro Selection of Botulinum Neurotoxin Type C Specific Binding DNA Aptamers for Diagnosis of Botulism Ji-Man CHA1, Ji-Young AHN1, Jiho MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University, Cheongju, Chungbuk, 362-763 , 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, Jeonju 561-756 Botulinum neurotoxin Type C (BoNT/C) represents one of seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians through blocking the transmission of acetylcholine at the neuromuscular junction leading to flaccid paralysis. Also because of extreme toxicity, the speed of symptom onset and lack of treatment to reverse paralysis, a sensitive and rapid BoNT/C detection method is needed to diagnose botulism in suspected cases before paralysis occurs. Here we report the development of BoNT/C specific binding DNA aptamers for detection of BoNT/C in livestock using SELEX (Systematic Evolution of Ligands by EXoonential enrichment) that is able to rapidly and sensitively detect BoNT/C. We conducted optimization through SELEX and SPR test. The development of aptasensor can be applied to sensitive detection of BoNT/C from diseased livestock. This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of Monitoring and Diagnostic Method for Environmental Animal Disease, Project No: PJ010530)" Rural Development Administration, Republic of Korea. Keywords : Botulinum, Botulinum neurotoxin Type C, Aptamer, SELEX, aptasensor, Diagnosis References 1. Dongxia Wang, Joan Krilich, Jakub Baudys, John R. Barr, Suzanne R. Kalb, 1. Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates (2015), Bioorganic & Medicinal Chemistry, Vol 23 p 3667-73 2. Jones RG, Liu Y, Sesardic D, New highly specific botulinum type CI endopeptidase immunoassays utilizing SNAP25 or Syntaxin substrates (2009), Journal of immunological methods, Vol. 343 21-27 P0702 Combination of Emodin with Antibiotics against Methicillin-Resistant Staphylococcus aureus Isolated from Clinical Specimens Jeong-Dan CHA, Seung-Mi HWANG, Eun-Sil KO, Kyung-Min CHOI Department of Efficacy Research, Institute of Jinan Red Ginseng, Jinan, 567-801 South Korea Emodin (3-methyl-1,6,8-trihydroxyanthraquinone), a natural anthraquinone compound, is an active compound derivative isolated from the rhizome of Rheum undulatum L, an herb widely used as a laxative in traditional Korean medicine. Emodin has been reported to have a variety of biological activities, such as anti-cancer, vasorelaxation, immunosuppressive, anti- inflammatory, and wound healing properties [1,2]. In this study, emodin was evaluated against 20 clinical isolates of MRSA, either alone or in combination with antibiotics. The emodin exhibited strong antibacterial activity against isolates MRSA with MICs/MBCs ranged between 80/20-160 μg/mL, for ampicillin 64-1024/128-2048 μg/mL, and for oxacillin 8-32/16-64 μg/mL. The combination of emodin plus oxacillin or ampicillin was reduced by ≥4-fold against isolates MRSA tested, evidencing a synergistic effect as defined by a FICI of ≤ 0.5. Furthermore, a time-kill study evaluating the growth of the tested bacteria was completely attenuated after 2-5 h of treatment with the 1/2 MIC of emodin, regardless of whether it was administered alone or with oxacillin (1/2 MIC) or ampicillin (1/2 MIC). In conclusion, emodin exerted synergistic effects when administered with oxacillin or ampicillin and the antibacterial activity and resistant regulation of emodin against clinical isolates of MRSA might be useful in controlling MRSA infections. R0002929 Keywords : emodin, methicillin-resistant Staphylococcus aureus, minimum inhibitory concentrations, minimum bactericidal concentrations, time-kill curves, fractional inhibitory concentration References 1. Choi SZ, Lee SO, Jang KU, Chung SH, Park SH, Kang HC, Yang EY, Cho HJ, Lee KR. Antidiabetic stilbene and anthraquinone derivatives from Rheum undulatum. Arch Pharm Res. 2005;28(9):1027-30. 2. Kim JE, Kim HJ, Pandit S, Chang KW, Jeon JG. Inhibitory effect of a bioactivity-guided fraction from Rheum undulatum on the acid production of Streptococcus mutans biofilms at sub-MIC levels. Fitoterapia. 2011;82(3):352-6. P0703 Production of the Novel Lipase from Pichia petersonii NRRL YB-3808. Eun-Chong WON1, Jea-Han BAE2, Hak-Ryul KIM3 1School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, 2School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, 3School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701 Lipase (triacylglycerol acylhydrolases, E.C. 3.1.1.3.) are class of hydrolases which catalyze the hydrolysis of triglycerides to free fatty acids, diacylaglycerols, monoacylglycerols and glycerol over an oil-water interface. Lipases have the potentials in industrial applications. Because lipase can catalyze numerous different reactions, they have been widely used as biocatalysts for biotechnological applications such as a detergent, food, flavor industry, biocatalytic resolution of pharmaceuticals etc. Lipases are produced from varied microbial sources with carbon sources, such as oils, glycerol, fatty acids in the presence of an organic nitrogen source. However, there are still remained in development of new microbial strains for economical lipase production. In this study, we found out a novel lipase produced from a yeast strain, Pichia petersonii NRRL YB-3808 and we conducted optimization study for efficient production of a novel lipase from YB-3803. The strain produced maximum extracellular lipase at initial pH of 7.0 and at 25℃. Optimal substrate types for lipase production were the mustard oil. Keywords : Lipase, Production, Microbial lipase, Optimization, hydrolase, novel lipase, yeast, biotechnology, microbial engineering, Food chemistry References 1. Kim, H. R., Kim, I. H., Hou, C. T., Kwon, K. I., Shin, B. S. Production of a novel cold-active lipase from Pichia lynferdii Y-7723. Agric. Food Chem. 2010, 58, 1322-1326 Alquati, C., Gioia, L. D., Santarossa, G., Alberghina, L., Fantucci, P., Lotti, M. The cold-active lipase of Pseudomonas fragi. Eur. J. Biochem. 2002, 269, 3321-3328. P0704 Fed-batch Fermentation for Production of 7,10-Dihydroxy-8(E)-Octadecenoic Acid(DOD) from Pseudomonas aeruginosa KNU-2B Se-Rin KIM, In-Hae CHOI, Hyung-Geun LEE, Ji-Hye JUNG, Hak-Ryul KIM School of Food Science and Biotechnology, Kyungpook National University, Daegu, KOREA Hydroxy fatty acid(HFA) are known to have special properties such as high viscosity and reactivity. These special properties augmented high industrial potentials in a wide range of applications. Among those HFAs, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from Pseudomonas aeruginosa PR3 was well studied in terms of production and antimicrobial activities. However, for industrial applications of HFA, there are still problems remained to be solved, such as economical production cost and enhancement of biological properties. In this research, we used fed-batch fermentation for efficient production of DOD from KNU-2B, which is a newly developed Pseudomonas aeruginosa strain. As a carbon source, whey powder was used instead of glucose through the fed-batch fermentation in order to reduce production cost. DOD was efficiently produced from KNN-2B by fed-batch fermentation. The maximum DOD production from batch fermentation and fed-batch fermentation was 137.5mg/50mL, 326.7mg/50mL respectively, representing that fed-batch fermentation produced 2.3-fold more DOD than the batch fermentation. Results from this study demonstrated that whey powder could be used as efficient carbon source for DOD production from strain KNU-2B and fed-batch fermentation could be used to increase the maximum DOD production in this system. Keywords : Hydroxy fatty acid, Pseudomonas aeruginosa, fermentation, Batch fermentation, Fed-batch fermentation References 1. Hou CT, Bagby MO (1991) Production of a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid by Pseudomonas sp. PR3. J Ind Microbiol 7:123–130. 2. Min-Jung Suh ,et al., (2010) Production of 7,10-dihydroxy-8(E )-octadecenoic acid from olive oil by Pseudomonas aeruginosa PR3. Applied Microbiology and Biotechnology Volume 89, Issue 6, pp 1721-1727. P0705 Apoptotic Effect of Korean Wildlife Plant Extracts on A549, AGS and B16/F10 Cells. Jeong Eun SONG1, Ye Chan SIM1, Jong Seok LEE2, Sarah LEE2, Young Kyoung SON2, Hye Jin LEE2, Eock Kee HONG1 1Department of Bioengineering and Technology, Kangwon National University, 192-1, Hyoja 2-dong, Chuncheon, Kangwon-do 200-701, Korea, 2National Institute of Biological Resources, Incheon 404-708, Republic of Korea Apoptosis (programmed cell death), is an important physiological process of cell death and occurs during tissue remodeling, immune regulation and tumor regression. Most of the chemotherapeutic drugs kill cancer cells by inducing apoptotic death pathways. Cells undergoing apoptosis show a sequence of cardinal morphological features, including membrane blebbing, cellular shrinkage and condensation of chromatin. Although there are many therapeutic strategies to treat cancer, including chemotherapy, drug resistance have limited the successful outcomes in most cases. Therefore, natural product extract to required for anti-cancer effect. The sample used in this report is korean wildlife plants extracted from 70% ethanol, and three cell lines (A549, AGS and B16/F10) were used. Some of the reviewed sample, which confirmed by using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were selected. After that, apoptotic cells were comfirmed by using flow cytometry. In this study, it was investigated that anti-cancer effect of korean wildlife plants extracts (KWPE) on A549, AGS and B16/F10 cells via MTT assay and flow cytometry. Keywords : Apoptosis, Cancer, Flow cytometry References 1. Yoshihisa, I. and K. Masanori, Anti-apoptotic effects of quercetin: Intervention in the JNK- and ERK-mediated apoptotic pathway(2000), Kidney International, 58, 1078-1087. 2. Solaroglu, I., T. Tsubokawa, J. Cahill and J. H. Zhang, Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat(2006), Neuroscience, 148, 965-974. P0706 Korean Wildlife Plant Extracts Protect Hydrogen Peroxide-Induced Damage in MIN6N Pancreatic β-Cells Ye Chan SIM1, Jeong Eun SONG1, Jong Seok LEE2, Sarah LEE2, Young Kyoung SON2, Eock Kee HONG1 1Department of Bioengineering and Technology, Kangwon National University, 192-1, Hyoja 2-dong, Chuncheon, Kangwon-do 200-701, Korea, 2National Institute of Biological Resources, Incheon 404-708, Republic of Korea - Reactive oxygen species (ROS) such as superoxide anions (O2 ), hydroxyl radicals (-OH), and hydrogen peroxide (H2O2) were caused increasing diabetes pathogenesis. Diabetes mellitus is a common metabolic disease that was associated with chronic inflammation, hyperglycemia, obesity, hyperlipidemia, hyperinsulinemia and insulin resistance. It was mediated beta-cells apoptosis and other complications by constant oxidative stress in pancreas. Living organisms have been faced with the necessity to inactivate these free radicals, and they have developed several ways to protect themselves from oxidative attacks. these defense mechanisms include a variety of antioxidant enzymes like superoxide dismutase (SOD), catalase and glutathione peroxidase. This reaction is essential for the maintenance in oxidant-reduction processes, and also serves in detoxification. But, excessive production of ROS lead to imbalance its levels. Therefore, this study investigated that natural antioxidant effects of extract from korean wildlife plants, which previous was studied by antioxidant screening using 2, 2- diphenyl-1-picrylhydrazyl (DPPH), Oxygen radical antioxidant capacity (ORAC) assay can prevent hydrogen peroxide- induced oxidative damage in MIN6N pancreatic β-cells. Keywords : Antioxidant, Diabetes, Insulin, ROS (Reactive oxygen species) References 1. Lee, J. H., J. S. Lee, Y. R. Kim, W. C. Jung, K. E. Lee, S. Y. Lee and E. K. Hong, Hispidin isolated from Phellinus linteus protects against hydrogen peroxide–induced oxidative stress in pancreatic MIN6N β-cells, Journal of medicinal food, 14, 1431-1438(2011) 2. Kimoto, K., K. Suzuki, T. Kizaki, Y. Hitomi, H. Ishida, H. Katsuta, E. Itoh, T. Ookawara, K. Suzuki, K. Honke and H. Ohno, Gliclazide protects pancreatic β-cells from damage by hydrogen peroxide, Biochemical and biophysical research communications, 303, 112-119(2003) P0707 Anti-Inflammatory Effect of Sargachromanol G Isolated from Myagropsis myagroides Nan-Young BAE1, Ji-Hye PARK1, Sun-Hee PARK1, Koth-Bong-Woo-Ri KIM2, Min-Ji KIM2, Dong-Hyun AHN1 1Department of Food Science and Technology, Pukyong National University, Busan, 608-737, 2Institute of Fisheries Sciences, Pukyong National University, Busan, 619-911 Numerous studies have concentrated on the contribution of marine organisms, including seaweeds and marine microorganisms, in the search for new drugs from natural products. Currently, a strong interest in developing new anti- inflammatory drugs from various natural products has been growing. It may inhibit or prevent a variety of inflammatory diseases by suppressing the secretion of inflammatory mediators such as IL-6 and TNF-α in activated macrophages. This study is focused on searching for anti-inflammatory compounds from natural sources, so we investigated the effects of Myagropsis myagroides methanol extract (MMME) on inflammation induced by lipopolysaccharide (LPS) in Raw 264.7 cells. The n-hexane fraction exhibited the most potent inhibitory activity against IL-6 and TNF-α production. The n-hexane fraction was separated using silica gel column chromatography and the fraction eluted with chloroform/methanol (50:1) showed the highest anti-inflammatory activity. Subsequently, the active compound was separated using Sephadex LH-20 column chromatography, an octadecyl silica (ODS) Sepak cartridge, and silica gel TLC. The isolated compound, sargachromanol G, showed high anti-inflammatory effect on the IL-6 and TNF-α production. These results suggest that sargachromanol G may be a useful therapeutic agent on inflammatory diseases. Keywords : anti-inflammatory, sargachromanol g, myagropsis myagroides References 1. Lawrence T, Willoughby DA, Gilroy DW, Anti-inflammatory lipid mediators and insights into the resolution of inflammation(2002), Nat Rev Immunol, 2(10), 787-795 P0708 Anti-inflammatory Effects of Grifola frondosa Water Extract in LPS-induced Raw 264.7 Cells and Mouse Models Min-Ji KIM1, Ji-Hye PARK2, Nan-Young BAE2, Sun-Hee PARK2, Koth-Bong-Woo-Ri KIM1, Dong-Hyun AHN2 1Institute of Fisheries Sciences, Pukyong National University, Busan, 619-911, 2Department of Food Science and Technology, Pukyong National University, Busan, 608-737 A wide range of active compounds from herbal medicines and plants is used as sources for drug development in clinical applications. In this study, the anti-inflammatory effect of Grifola frondosa water extract (GFWE) was investigated using LPS-induced RAW 264.7 cells and mouse models. As a result, NO and pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) were inhibited up to over 50% with increasing concentration of GFWE without any cytotoxicity. In addition, GFWE suppressed the expression of inducible nitric oxide (iNOS), cyclooxygenase 2 (COX-2) and nuclear factor kappa B (NF-κB) in a dose-dependent manner. Furthermore, GFWE inhibited mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) signaling. In mice ear edema test, the formation of ear edema was reduced at the highest dose tested compared to that in the control and reduction of ear thickness and the number of mast cells was observed in histological analysis. In acute toxicity test, no moralities occurred in mice administered 5000 mg/kg body weight of GFWE over two week observation period. These results indicate that GFWE exhibits anti-inflammatory activity and can be used as a material to treat inflammatory diseases. Keywords : anti-inflammation, nuclear factor kappa B, mitogen-activated protein kinase References 1. A. Majdalawieh and H. S. Ro, Regulation of IκBα function and NF-κB signaling: AEBP1 is a novel pro- inflammatory mediator in macrophages (2010), Mediators Inflamm, 2010, 821-823 P0709 Anti-inflammatory Effects of Dieckol Isolated from Ecklonia cava Ju Hyung UM1, Han EUI JEONG1, Kim NA HYUNG1, Park SOO YEON1, Ahn GINNAE1,2 1Department of Food Technology and Nutrition, Chonnam National University, 2Department of Marine Bio-Food, Chonnam National University Ecklonia cava (E. cava), one of brown seaweed species (Laminariaceae), is widely distributed along the southern coasts of South Korea and Japan. E. cava is mainly composed of polysaccharide and phlorotannin components such as dieckol and phloroglucinol. Recent studies have suggested that E. cava strongly inhibits lipopolysaccharide (LPS)-induced macrophage activation. However, dieckol’s effects on inflammatory responses in in vivo model systems are still unclear. This study evaluated the anti-inflammatory effect of dieckol in in vivo models and its biological mechanism. In TPA-caused mouse ear edema model, dieckol decreased the thickness and hyperplasia of mouse ear. In further experiments, LPS treatment significantly increased the ROS and NO levels in zebrafish embryos without toxicity, whereas it was inhibited by the application of dieckol. Moreover, dieckol has a protective effect by decreasing the cell death and the yolk sac edema size increased by LPS exposure in zebrafish embryos. On the basis of these results, we suggest that dieckol’s anti-inflammatory effect might contribute to therapy of macrophage- related to inflammation diseases. Keywords : Ecklonia cava, Dieckol, Macrophages, Anti-inflammatory effect, Zebrafish embryos, Mouse ear edema model References 1. Guiry MD, Bulunden G, (1991), Seaweed Resource in Europe: Uses and potential, John Wiley & Sons Ltd, Chichester West Sussex, England. P0710 Identification of Antioxidant Properties of Various Extracts from Mealworm, Tenebrio molitor, for Development of Functional Food Il-Suk KIM1, Mi Ra YANG1, Gun Ryoung NO1, Dong Hyun KIM1, Woo Young BANG2, Sam Woong KIM1 1Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Gyeongnam, 660-758, 2National Institute of Biological Resources, Environmental Research Complex, Incheon 404-708 Insects have been highly evaluated for availability as new food materials in recent years owing to a high protein source and functional potential. Here we identified the antioxidant activity of extracts from Tenebrio molitor, to find a new material for functional foods. After extraction from the freeze-dried T. molitor larvae with various solvents on time course, the ones, extracted with water for 5 h and 10 h, showed highest DPPH activities per total protein and solid, respectively. When the water extract was fractionated depending on methanol concentration, high concentration of methanol led to the reduced level of the high molecular weight proteins and the increased level of the DPPH activity in supernatants, suggesting that the low molecular weight peptides may mediate the antioxidant activity in the supernatant. The most of organic solvents excluding butanol also showed similar activity. Thermal stability of the organic solvent partition fraction exhibited a 28~44% decrease in the activity, implying that the unstable components to heat is present in the fraction. The treatment of proteinase K to water extract increased 10~20% of DPPH activity, suggesting that peptides, released from total proteins, partially increased the antioxidant activity. Altogether, we suggest that the insect extracts having the anti-oxidative property are very useful resources to develop a functional food. Keywords : mealworm, antioxidant, functional food, peptide References 1. Juan A. Morales-Ramos, M. Guadalupe Rojas, Effect of Larval Density on Food Utilization Efficiency of Tenebrio molitor (2015) Journal of Economic Entomology, DOI: http://dx.doi.org/10.1093/jee/tov208 P0711 Structural Analysis of Fucoidan Degraded by Gamma Irradiation Jong-il CHOI Department of Biotechnology and Bioengineering, Chonnam National University,Gwangju 500-757, South Korea The change of molecular structure of fucoidan after gamma irradiation was studied. After irradiation, the polydispersity decreased because of further degradation of higher weight molecules. Ultraviolet absorption and Fourier-transform infrared spectroscopy analyses were carried out.Carboxylgroups and carbon double bonds increased by gamma irradiation; however, sulfate content remained unchanged. Keywords : fucoidan, structure, gamma irradiation References 1. J. CHOI, Radiation Physics and Chemistry 100 (2014) 54–58 P0712 Hydrolysis of Chitosan Using Organic Acid and Commercial Enzymes Jae-Kun KIM, Sujin BAE, A-Ram KIM, Gwi-Taek JEONG Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea Chitosan is natural polysaccharides, which are non-toxic, tasteless, and odorless. They can be recovered from crustaceans’ shells and exoskeletons of insects. Chitosan and its oligomers are applied to healthy food, agriculture, water and waste treatment, cosmetics, and pharmaceutical and medical resources. Moreover, the use of chitosan in the field of biofuels production is recently increasing [1]. In this work, the hydrolysis of chitosan using organic acid and commercial enzymes was investigated. The organic acids such as oxalic acid, maleic acid, succinic acid and citric acid were introduced. Also, commercial enzymes such as cellulase, lipase, pectinase were applied to enzymatic hydrolysis. Keywords : chitosan, hydrolysis, organic acid, enzymatic hydrolysis References 1. Lee and Jeong, Catalytic Conversion of Chitosan to 5-Hydroxymethylfurfural Under Low Temperature Hydrothermal Process (2015), Appl Biochem Biotechnol, 176(4), 1151-61 P0713 Isolation of Soil Bacteria Having High Cyclodextrin Glucanotransferase Activity by Random Mutagenesis Sun-Jin HONG, Jin-Sung KIM, Jae-Bum PARK, Suk-Jin HA Dept. of Bioengineering and Technology, Kangwon National University, Chuncheon 200-701 Cyclodextrin glucanotransferase (CGTase) is a commercially important enzyme which catalyses the formation of cyclodextrins (CD) from starch. In order to isolate soil bacteria having CGTase activity, we collected several soil sample and verified CGTase activities using phenolphthalein. After isolation of soil bacteria having CGTase activity, random mutagenesis was performed to improve CGTase activity. As results, mutant strain (5-52) exhibited 27% improved CGTased activity. Open reading frame of CGTase genes from the parental strain and mutant strain were sequenced and compared. The mutant strain having high CGTase activity might be used for food industry. Keywords : Cyclodextrin glucanotransferase, soil bacteria, Random Mutagenesis References 1. Graciette Matioli. Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil. Applied Biochemistry and Biotechnology. Volume 70-72. Issue 1. pp 267-275 2. Hajime Aga, Masaru Yoneyama, Shuzo Sakai & Itaru Yamamoto. Synthesis of 2-O-α-d-Glucopyranosyl l- Ascorbic Acid by Cyclomaltodextrin Glucanotransferase from Bacillus stearothermophilus. Volume 55. Issue 7. 1991 3. Zichen Zhang, Jianghua Li, Long Liu,Jun Sun, Zhaozhe Hua, Guocheng Du, Jian Chen. Enzymatic transformation of 2-O-α-D-glucopyranosyl-L-ascorbic acid by α-cyclodextrin glucanotransferase from recombinant Escherichia coli. Biotechnology and Bioprocess Engineering. February 2011. Volume 16. Issue 1. pp 107-113 P0714 Enhancement of Antioxidant Activities from Spinach or Blackberry Juices through Acetic Acid Bacteria Fermentation Joong-Hee PARK1, Jae-Bum PARK1, Hyun-Su SIM2, Hun-Joo KWON2, Myoung-Dong KIM2, Suk-Jin HA1 1Department of Bioengineering and Technology, Kangwon National University, Chuncheon, 200-701, Korea, 2Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, 200-701, Korea Vegetable and berry are known to have high antioxidant activities and they were very helpful for the prevention of various diseases caused by oxidative stress. In this study, spinach or blackberry juices were fermented by four acetic acid bacteria (Acetobacter pomorum KCTC 22319, Acetobacter pasteurianus subsp. Pasteurianus KCTC 22322, Acetobacter estunensis KCCM 32831, Acetobacter pasteurianus KCCM 40011) for enhancement of antioxidant activities. After fermentation, antioxidant activity of spinach juice was increased from 168.90 μmol TE/g to 248.08 μmol TE/g by Acetobacter pomorum KCTC 22319 using the oxygen radical absorbance capacity (ORAC) assay. For blackberry juice, antioxidant activity was increased from 177.85 μmol TE/g to 231.05 μmol TE/g by Acetobacter estunensis KCCM 32831. In order to confirm the reason of antioxidant activity improvements, total polyphenol and total flavonoid contents were assayed. Keywords : Antioxidant activities, Polyphenol, Flavonoid, Spinach, Blackberry, Acetic acid bacteria References 1. Kim DB, Shin GH, Cho JH, Baik SO, and Lee OH. 2013. Antioxidant activities of beverage concentrates and purees. J. Korean Soc. Food Sci. Nutr. 42: 997-1002. 2. Lee BH, Kim SY, Cho CH, Chung DG, Chun OK, and Kim DO. 2011. Estimation of daily per capita intake of total phenolics, total flavonoids, and antioxidant capacities from fruit and vegetable juices in the korean diet based on the Korea national health and nutrition examination survey 2008. Korea J. Food Sci. 43: 475-482. P0715 Overexpression of Recombinant Glucoamylase from Deinococcus geothermalis by E. coli and Its High Acido-stable Capability Seung-Won JANG1, Deok-Ho KWON1, Suk-Jin HA1, Jong-Hyun JUNG2, Sang-Yong LIM2 1Dept. of Bioengineering and Technology, Kangwon National University, Chuncheon, 200-701, 2Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 Glucoamylase gene was cloned from Deinococcus geothermalis and overexpressed by pET-21a vector using E. coli BL21 (DE3). In order to characterize starch degrading activity of recombinant glucoamylase, enzyme was purified using HisPur Ni-NTA column. The recombinant glucoamylase from D. geothermalis exhibited the optimum temperature as 45°C for starch degradation activity. And highly acido-stable starch degrading activity was shown at pH 2. For further optimization of starch degrading activity with metal ion, various metal ions (AgCl2, HgCl2, MnSO4•4H2O, CoCl2•6H2O, MgSO4, ZnSO4•7H2O, K2SO4, FeCl2•4H2O, NaCl, or CaCl2•2H2O) were added for enzyme reaction. As results, It was found that FeCl2•4H2O and MnSO4•4H2O addition resulted in 17% and 9% improved starch degrading activity, respectively. The recombinant glucoamylase from D. geothermalis might be used for SSF process at high acidic conditions. Keywords : Recombinant glucoamylase, Overexpression, Deinococcus, Starch degrading, Acido-stable, Metal ion References 1. Characterization of glucoamylase immobilized on magnetic nanoparticles 2. Molecular Cloning and Characterization of Maltogenic Amylase from, KOREAN J. FOOD SCI. TECHNOL. Vol. 43, No. 3, pp. 369~374 (2011) 3. alomo M, Kralj S, van der Maarel MJ, Dijkhuizen L. Theunique branching patterns of Deinococcus glycogen branching enzymes are determined by their N-terminal domains. Appl. Environ. Microb. 75: 1355-1362 (2009) 4. Jung JH, Seo DH, Ha SJ, Song MC, Cha J, Yoo SH, Kim TJ, Baek NI, Baik MY, Park CS. Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea. Carbohyd. Res. 344: 1612-1619 (2009) P0716 Microbial Succession of Culturable Bacteria in Nuruks Made with Different Water and Raw Materials Kang NAM2, Nam Keun LEE1, Dae Hyuk KIM3, Soo Hwan YEO4, Yong Seob JEONG2 1Research Center for Industrial Development of Biofood Material, Chonbuk National University, Jeonju, 54896, 2Dept. of Food Science and Technology, Chonbuk National University, Jeonju, 54896, 3Dept. of Molecular Biology, Chonbuk National University, Jeonju, 54896, 4Fermentation and Food Processing Division, Dept. of Agrofood Resources, Rural Development Administration, Jeonju, 54875 In this study, culturable bacterial succession in Nuruks produced according to water and raw materials contents was investigated. All culturable bacteria were then isolated at various times point(0, 3, 6, 10, 20 and 30 days) and identified by 16S rDNA sequence analysis. The following major bacterial strains were identified from various Nuruks: (1) from type 1-1 (barley:mung bean = 6:1) added water 20%, Pantoea sp. at 0, 30 days, Pediococcus sp. at fermentation 3-10 days and Staphylococcus sp. at 3-30 days; from type 2-1 (barley:mung bean:whole wheat = 6:1:6) added water 20%, Pediococcus sp. at all times, Staphylococcus sp. at 3-30 days; (2) from type 1-2 added water 26%, Enterococcus sp. at 0 and 10 days, Pediococcus sp. at fermentation 3-30 days and Enterobacter sp. at 10-30 days; from type 2-2 added water 26%, Pseudomonas and Leclercia sp. at 3 days, Enterobacter sp. at 3-6 days and Pantoea sp. at 10-30 days; (3) from type 1-3 added water 30%, Enterococcus, Weissella and Pediococcus sp. at 0-6, 20-30 days, Pantoea sp. at fermentation 6-30 days; from type 2-3 added water 30%, Enterococcus sp. at 0 days, Pediococcus sp. at 6-30 days. Keywords : Nuruk, Korean fermentation starter, Microbial succession References 1. Ahn BH, et al., Characterization of Yakju Brewed from Glutinous Rice and Wild-Type Yeast Strains Isolated from Nuruks(2010), J. Microbiol. Biotechnol., 20(12), 1702–1710 P0717 Screening and Evaluation of Edible Plant Extracts for Anti-Obesity effect Jong Hoon JEONG1, Nam Keun LEE1,2, Hyeji LEE1, Yong-Seob JEONG1 1Department of Food Science and Technology, Chonbuk National University, Jeonju, Jeollabuk-do 561-756, Republic of Korea, 2Research Center for Industrial Development of Biofood Materials, Chonbuk National University, Jeonju, Jeollabuk- do 561-756, Republic of Korea The goal of this study was to evaluate the edible plant extracts (EPEs) and EPEs after passing through the three-dimentional intestinal villi model (EPEs-3D villi) for anti-obesity effect by in vitro examining possible biological influence of EPEs and EPEs-3D villi treatment on 3T3-L1 preadipocytes. Eighty eight types of EPEs were selected by evaluation of cell viability (>80% at 0.25 mg/mL) using the cell counting kit-8, and then evaluated an inhibition of differentiation for 3T3-L1 preadipocytes. Our results demonstrate that some EPEs inhibited 3T3-L1 preadipocytes differentiation at 0.25 mg/mL. Accumulation of fat in adipocytes according to treatment of EPEs - Hovenia dulcis thunb. leaves, Aster spathulifolius Maxim. leaves, Acanthopanax koreanum leaves, Kalopanax pictus leaves, and Chrysanthemum indicum - were 35.08±3.39, 37.70±3.90, 37.70±4.24, 39.11±2.51, and 39.11±2.75%, respectively, compared to the control (100%). The positive control, epigallocatechin gallate, was 46.67±3.77%. These results suggest that some EPEs with inhibitory effects of preadipocytes differentiation could be beneficial to prevention of diabetic-related metabolic diseases. Keywords : edible plant extracts, 3T3-L1 preadipocytes, anti-obesity References 1. K. Zebisch et. al., Analytical Biochemistry. 425, 88-90 (2012) 2. T. S. Oh et. al., Biotechnoloy and Bioprocess Engineering, 16, 978-986 (2011) P0718 Schizandrin Prevents Cytoplasmic TDP-43 Accumulation in Neuronal Cells Hyeji LEE1, Jisun OH2, Nam Keun LEE1, Yong-Seob JEONG1 1Department of Food Science and Technology, Chonbuk National University, Jeonju 561-756, 2School of Food Science and Biotechnology (BK21 plus), Kyungpook National University, Daegu 702-701 Transactive response DNA binding protein 43 kDa (TDP-43, encoded by the TARDBP gene) is involved in transcriptional regulation and alternative splicing process. TDP-43 proteins are located in the nucleus and shuttles transcripts to the cytoplasm in normal neurons; however, they are observed in the forms of cytoplasmic inclusions in the degenerating cells. The abnormal accumulation of TDP-43 proteins is a pathologic feature of neurodegenerative diseases, such as Lou Gehrig's disease and dementia including frontotemporal lobar degeneration and Alzheimer's disease. In this study, we examined whether schizandrin, a main effective compound of Schisandra chinensis (Turcz.), so called Omija, reduces TDP-43 accumulation in the hippocampal neurons in vitro. An immortalized hippocampus cell line, HT-22 and hippocampal neural stem cell-derived neuronal cells were treated with a proteasome inhibitor, MG132 in the absence or presence of schizandrin. We found that schizandrin treatment reduced MG-132-induced cytoplasmic accumulation of TDP-43. Schizandrin may be a promising compound for development of functional food materials beneficial to the neuronal protection against TDP-43 proteinopathies. Keywords : Schizandrin, TDP-43, Neuronal Cell References 1. Y. C. Liu, P. M. Chiang and K. J. Tsai, Int. J. Mol. Sci. 14, 20079 (2013) P0719 Enhancement of Functional Compounds of Platycodon gradiflorum (Doraji) Using Two Mushroom Mycelia, Inonotus obliquus and Lentinula edodes Hae-mi KIM1, Nam Keun LEE1,2, Seok hwan YANG3, Yong-Seob JEONG1 1Department of Food Science and Technology, Chonbuk National University, Jeonju, Jeollabuk-do 561-756, Republic of Korea, 2Research Center for Industrial Development of Biofood Materials, Chonbuk National University, Jeonju, Jeollabuk- do 561-756, Republic of Korea, 3Sanmaeul, Mujugun, Jeollabuk-do 55500, Republic of Korea The purpose of this study was to investigate the potential possibility of enhancing functional food compounds in Platycodon grandiflorum (Doraji) with two mushroom mycelia, Inonotus obliquus and Lentinula edodes. Doraji was autoclaved twice at 121℃ for 20 min and fermented with 2% (v/w) mushroom mycelia at 80% humidity, 25℃ for 3 days. The fermented Doraji was then freeze-dried and its compounds were extracted using methanol. Total phenolics and platycodin D contents in the extract were analyzed and the total phenol content of fermented Doraji was approximately twice as high as that of unfermented Doraji. The platycodin D content, in Doraji fermented with I. obliquus and L. edodes was increased 28%, 23% respectively compared to the control. These results suggest that the level of functional components in Doraji can be augmented by mushroom mycelia. Thus, the fermented products have the potential to be used as functional food materials. Keywords : Mushroom mycelia, Platycodon grandiflorum, Solid-fermentation, Platycodin References 1. Cho JH, et al., Deveolpment of functional Food Materials from Acanthopanax senticosus-fermented Mushroom Mycelia (2014) , J Korea Soc Food Sci Nurt 43: 411-418 P0720 A Study on Effect of Cudrania tricuspidata Leaf Hot Water Extract on Fermentation of Yogurt Starter Kim SUJIN1, Lee NAM KEUN1,2, Kim YOUNG HOON3, Ha YOUNG SIK4, Jeong YONG-SEOB1 1Department of Food Science and Technology, Chonbuk National University, Jeonju, Jeollabuk-do 561-756, South Korea, 2Reasarch Center for Industrial Development of Biofood Materials, Chonbuk National University, Jeonju, Jeollabuk-do 561- 756, South Korea, 3Department of Animal Science, Institute Agricultural Science and Technology, Chonbuk National University, Jeonju, Jeollabuk-do 561-756, South Korea, 4R&D Center, Seoul Dairy Cooperative, Ansan, South Korea This study was conducted to investigate the effect of Cudrania tricuspidata leaf (CTL) hot water extracts on the growth and fermentation of yogurt starter (YS). Lactic acid bacteria (LABs) as YS were Streptococcus thermophillus (ST) and Lactobacillus bulgaricus (LB). The CTL hot water extracts were added to fermented milk at 0, 0.02, 0.1, 0.2, and 2%. YS was inoculated in pasteurized milk and fermented for 22 hr. The pH, titratable acidity, and number of viable cells of LABs in fermented milk were analyzed. Based on yogurt pH, the fermentation of pasteurized milk added with 0.2% of the CTL hot water extract was finished after 6 hr (pH 4.2~4.26, titratable acidity 0.76~0.80% and 0.7~2.71011 CFU/mL of viable cell counts at 6 hr). However, pasteurized milk without CTL hot water extract was fully fermented after 14 hr (pH 6.43~6.47, titratable acidity 0.15% and 6.6~8.6107 CFU/mL of viable cell counts at 6 hr). These results suggest that 0.2% hot water extract will be effective for the manufacture of the yogurt. Keywords : Cudrania tricuspidata leaf, Streptococcus thermopillus, Lactobacillus burgaricus, yogurt References 1. H. J. Suh and Y. S. Kim, J. food. sci. 26, 144-147 (2006). 2. H. H. Koo and S. H. Chung, J. food. nutr. 7, 45-50 (1994). 3. J. Y. Kim and J. H. Chung, J.technol. sci. 27, 489-496 (2009). 4. H. J. Lee and J. R. Do, J. food. nutr. sci. 40, 982-948 (2011). P0721 Enhancement of Gastrodin Production from Parishin A by Enzymatic Bioconversion Mina KIM1, Nam Keun LEE1,2, Jong Hoon JEONG1, Yong-Seob JEONG1 1Dept. of Food Science and Technology, Chonbuk National University, Jeonju 561-756, 2Research Center for Industrial Development of Biofood Materials, Chonbuk National University, Jeonju 561-756 Gastrodin elata Blume (GE) is used generally against convulsant, vertigo, paralysis, epilepsy, tetanus, asthma and immune dysfunctions in the past in East Asia. Gastrodin (GAS) in particular is known as the major effective component of GE. The purpose of this study is to increase the GAS content from parishin A (PA) by enzyme treatment. The two most effective enzymes (esterase and lipase) out of nine types of enzymes were selected for PA hydrolysis. The both enzymes had different decomposition patterns. Therefore, it was used in combination for synergistic effect. One Factor at a Time (OFAT) method was carried out to confirm the highest point of gastrodin production on each factor (temperature, enzyme ratio, and substrate concentration). Based on these results, the center point of three factors for Response Surface Methodology (RSM) were 35℃, 5:5 (enzyme ratio) and 300 ppm respectively, were used to measure the optimum bioconversion conditions of GAS from PA. Further studies would be carried out by RSM. Keywords : Gastrodin, Parishin A, One factor at a time, Optimum conditions References 1. Zhu, H., Dai, P., Zhang, W., Chen, E., Han, W., Chen, C. and Cui, Y, Enzymic synthesis of gastrodin through microbial transformation and purification of gastrodin biosynthesis enzyme (2010), Biological and Pharmaceutical Bulletin, 33(10), 1680-1684 2. Li, Y.-W. and Ma, C.-L, Improved synthesis of gastrodin, a bioactive component of a traditional Chinese medicine (2014), J. of the Serbian Chemical Society, 79(10), 1205-1212 P0722 Effect of Probiotic Bacteria on the Degradation of Biogenic Amines in Doenjang Boram LIM, Heyjung CHOI, Woohong JOO Department of Biology, Changwon National University, Changwon 641-240 Biogenic amines (BAs) are frequently found in raw and fermented foods. Because of their toxicities, their presence in high concentrations in foods should be avoided. In this study, BA-degrading lactic acid bacteria (LAB) were screened from samples of Daenjang. These strains were then adapted to quantify biogenic amine and monitor the growth of strain during the cultivation period by HPLC and in the usual method, respectively. Some of these bacteria exhibits remarkable biogenic amine-reducing activities. These bacteria exhibits remarkable BAs reducing activities. Three isolates were identified by 16S rRNA sequencing as belonging to Bacillus atrophaeus BCNU 1406, Bacillus methulotrophicus BCNU 1403, Ocenobacillus sojae BCNU 1404, respectively. These strains might have potential for use as functional starter cultures for reducing the presence of BAs, in doenjang making. Keywords : probiotics, biogenic amine, starter culture References 1. S. Shukla, J. Lee, H. Park, J. Yoo, S. Hong, J. Kim and M. Kim. 80 , M1794–M1803(2015). P0723 Degradation of Biogenic Amine by Lactic Acid Bacteria from Kimchi Boram LIM, Hyejung CHOI, Woohong JOO Department of Biology, Changwon National University, Changwon 641-240 Biogenic amines (BAs) are found in many fermented food products and may have adverse effects on the health of consumers. The purpose of this study was to investigate the probiotic properties and BAs reducing activities of Lactic acid bacterial strains isolated from Kimchi. These strains were then adapted to quantify biogenic amine and monitor the growth of strain during the cultivation period by HPLC and in the usual method, respectively. Some of these bacteria exhibits remarkable biogenic amine-reducing activities. These isolates among all isolates were identified by 16S rRNA sequencing as belonging to Leuconostoc mesenteroides BCNU 1420, Lactobacillus arizonensis BCNU 9200, Weissella cibaria BCNU 1401, respectively. These strains might have potential for use as functional starter cultures for reducing the presence of biodenic amines. Keywords : Lactic acid bacteria, Biogenic amine, starter culture References 1. S. Shukla, J. Lee, H. Park, J. Yoo, S. Hong, J. Kim and M. Kim. 80 , M1794–M1803(2015). P0724 Probiotic Bacteria from Jeotgal and Their Use in Reduction of Biogenic Amine Boram LIM, Hyejung CHOI, Woohong JOO Department of Biology, Changwon National University, Changwon 641-240 Biogenic amines (BAs) are found in many fermented food products and may have detrimental effects on the human health. In this study, the probiotic strains were screened from jeotgal. These strains were then adapted to quantify biogenic amine and monitor the growth of strain during the cultivation period by HPLC and in the usual method, respectively. Some of these bacteria exhibits remarkable biogenic amine-reducing activities. These isolates were identified by 16S rRNA sequencing as belonging to staphylococcus equorum BCNU 1405, Bacillus methulotrophicus BCNU 1407, Ocenobacillus sojae BCNU 1408, respectively. These strains might have potential for use as functional starter cultures for reducing BAs in food manufacture. Keywords : jeotgal, Lactic acid bacteria, Biogenic amine References 1. S. Shukla, J. Lee, H. Park, J. Yoo, S. Hong, J. Kim and M. Kim. 80 , M1794–M1803(2015). P0725 Anti-inflammatory and Antitumor Effects of Crude Extracts from Tetragonia tetragonoides Hyejung CHOI, Bora LIM, Woohong JOO Department of Biology, Changwon National University, Changwon 51140 In this study, we examined the anti-inflammatory effect and antitumor activity by the crude extract (methanol and polysaccharide) and solvent fractions (hexane, dichloromethane, ethylacetate, butanol) of Tetragonia tetragonioides which has been known to superior plants for the traditional prevention and treatment of stomach-related diseases. Anti- inflammatory activity of T. tetragonioides extracts and fractions were exuded through the inhibition of lipopolysaccharide (LPS, 1 μg/ml) induced nitric oxide (NO) and interleukin (IL)-1β production. The production of IL-6 and tumor necrosis factor (TNF)-α were also decreased in LPS induced RAW 264.7 cells after treatment with 10 μg/ml of polysaccharide (PS) extract. Furthermore, 10 μg/ml of hexane fraction strongly inhibited the granulocytes macrophage-colony stimulating factor (GM-CSF) production. In ICR mice previously inoculated with Sarcoma 180, median survival were 22 days and 21 days with an intraperitoneal injection of methanol extract and polysaccharide fraction at a dose of 100 mg/kg/day, respectively and life prolongation effects were 27.17% and 21.39%. The obtained results have preliminary important consequence toward the development of effective anti-inflammatory and antitumor agents using T. tetragonioides. Keywords : Anti-inflammatory, antitumor, RAW 264.7 cells, ICR mice, Tetragonia tetragonioides References 1. A. V. Delgado and A. T. McManus, Neuropeptide 37, 355-361 (2003). P0726 Fermentation Condition Comparison of Black Garlic vinegars by Adding Apple Extract Sungmin HA, Hyejung CHOI, Woohong JOO Dept of Biology and Chemistry, Changwon National University, Changwon, 51140 This study was performed to investigate the quality characteristics and antioxidant activity of black garlic vinegar supplemented with or not-supplemented with apple extract during two-step fermentation. The quality characteristic of vinegar including total acidity, pH, ethanol, total sugar, brix, polyphenol and total flavonoid were evaluated. Antioxidant activities of black garlic vinegar were then examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power. During ethanol fermentation, ethanol contents of vinegar increased, but total sugar contents decreased. During acid fermentation, total acidities of vinegar increased, but ethanol content of vinegar decreased. Vinegar not- supplemented with apple extract was observed total polyphenol content of 214mg/100g after 13day of fermentation. Further, total flavonoid contents of vinegar not-supplemented with apple extract was observed 13.32mg/100g, after 13days of fermentation. DPPH radical scavenging activities of vinegar not-supplemented with apple extract was observed 42.54% after 13day of fermentation. Reducing power of vinegar not-supplemented with apple extract was 0.324 after 1day and 1.034 at the end of fermentation. Moreover, Vinegar supplemented with apple extract was observed total polyphenol content of 415mg/100g after 13day of fermentation. Further, total flavonoid contents of vinegar supplemented with apple extract was observed 23.32mg/100g, after 13days of fermentation. DPPH radical scavenging activities of vinegar supplemented with apple extract was observed 55.54% after 13day of fermentation. Reducing power of vinegar supplemented with apple extract was 0.424 after 1day and 1.234 at the end of fermentation. Therefore, Apple extract could be effectively used as a vinegar fermentation. Keywords : vinegar, fermentation, black garlic References 1. Horiuchi J, Kanno T and Kobayashi M, J Biosci Bioeng. 90, 292-293(2000). P0727 Antioxidative Activities of Water and Ethanol Extracts from Stachys sieboldii Da-Jeong CHOE, Hee-Young AHN, Ha-Rin JIN, Tae-Hoon KIM, Young-Wan KIM, Young-Su CHO Department of Biotechnology, Donga-A University, Hadan-2dong Sahagu, Busan 604-714, Korea Stachys sieboldii(S. sieboldii), commonly called crosne, Chinese artichoke, Japanese artichoke and knotroot, is a perennial herbaceous plant of the family labiate, originating from China, Japan and Russia. Its rhizome can be grown and eaten as a root vegetable. The water and ethanol extracts from S. sieboldii were tested by in vitro experimental models of biological activities (DPPH radical scavenging activity, Fe/Cu reducing power, linoleic acid peroxidation using ferric thiocyanate and thiobarbituric acid(TBA) methods and peroxidation of rat liver homogenate using TBARS method) and bioactive materials(phenolic compounds, flavonoids). The water extract was bioactive materials more than ethanol extract. Also, biological activities were stronger in water extract than ethanol extract. Especially, in inhibitions of lipid peroxidation, water extract was nearly 1.5 times stronger than BHT(butylated hydroxytoluene). Overall, the study provides basic data for understanding the antioxidative activities of Stachys sieboldii for the development of functional foods. Keywords : Antioxidative activity, TBARS, Stachys sieboldii 한국생물공학회, 생물공학의 동향 : 2015.10 기능성바이오 및 융합소재공학 P0801 In vitro Selection of Specifically Binding RNA Aptamer to Endothelial Cell-Specific Molecule 1 (ESM-1) Protein for Early Detection of HCC Dae-Young PARK1, Woo-Ri SHIN1, Simranjeet Singh SEKHON1, Dae-Ghon KIM3, Jiho MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University,, 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University,, 3Research Institute of Clinical Medicine of Chonbuk National University Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with the highest incidence rates reported in East Asia. Because necessity of efficient method for detection of HCC is increase nowadays, we choose protein that can use as HCC biomarker, Endothelial cell-specific molecule 1 (ESM-1). Most type of the HCC is hypervascular tumors, and ESM-1 protein promote angiogenesis in HCC occur process. For efficient HCC detection method, we use the aptamers. Aptamers are consist of single-stranded DNA or RNA and they can bind to various targets. Aptamers show very high affinity and specificity and have a number of advantages compare with antibody. Therefore we are used to RNA aptamers for ESM-1 protein detection and RNA aptamers selection with special process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The selection of RNA aptamers that bind to ESM-1 protein is raise the development level of early detection and treatment technology for HCC. This study was supported by Fund of Biomedical Research Institute, Chonbuk National University Hospital. (20120801002) Keywords : aptamer, ESM-1, SELEX References 1. Ozaki K, Toshikuni N, George J, Minato T, Matsue Y, Arisawa T, Tsutsumi M. Serum endocan as a novel prognostic biomarker in patients with hepatocellular carcinoma(2014). J Cancer. 5(3). 221-30. 2. Leroy X, Aubert S, Zini L. et al. Vascular endocan (ESM-1) is markedly overexpressed in clear cell renal cell carcinoma. Histopathology (2010).56(2).180-7. P0802 Screening of DNA Aptamers Specific to PDGF Receptor for Cell Culture Media Supplement Eun-Sil KIM1, Ji-Man CHA1, Simranjeet Singh SEKHON1, Ji-Young AHN1, Jiho Min MIN2, Yang-Hoon KIM1 1Department of Microbiology, College of Natural Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, Chungbuk 362-763, South Korea., 2Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju, Jeonbuk 561-756, South Korea The animal cells culture depends on the culture conditions and requires medium supplemented with fetal bovine serum (FBS). However, fetal bovine serum is expensive and limited. It is also the risk of infection by bovine-derived bacteria, mycobacteria and virus. The cell growth factor including platelet-derived growth factor (PDGF) that promotes cell growth and division by binding protein tyrosine kinase receptors PDGF receptor-α and -β through signal transduction. Aptamers are oligonucleotide that can bind with high affinity and specificity to target molecule. Aptamers are possible to develop at low cost in a short period of time and also safe from bovine-derived bacterial, mycobacterial, viral infection. In this study, DNA aptamers will be generated to specifically bind to PDGFR through an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment). The DNA aptamers specific to PDGF receptor can substitute existing cell culture media supplement. This work was supported by the Technological Innovation R&D Program (S2177590) funded by the Small and Medium Business Administration(SMBA, Korea)" Keywords : aptamer, SELEX, PDGFR References 1. Tan Y, Shi YS, Wu XD, Liang HY, Gao YB, Li SJ, Zhang XM, Wang F, Gao TM. DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro. Acta Pharmacol Sin. 2013 Dec;34(12):1491-8 2. Pitulescu ME, Adams RH. Regulation of signaling interactions and receptor endocytosis in growing blood vessels. Cell Adh Migr. 2014 Jul 4;8(4):366-77 P0803 Production of Mussel Adhesive Protein Fused with Neuropeptides and Evaluation on Nerve Regenerative Capacity Hogyun CHEONG, Bong-Hyuk CHOI, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea Neural regeneration of large defect is one of the most challenging fields of regenerative medicine. There are various attempt to develop a material which not only is biocompatible with but also promotes nerve tissue regeneration. Current nerve regenerative devices are made of collagens or polymer materials; however, their performance and clinical experience are still unsatisfactory in many aspects. In material’s aspect, a back bone material should provide proper biocompatibility to recruit nerve cells quickly; in addition, it should also have functionalities that can promote cell mobilization, growth, maturation, and etc. Mussel adhesive protein is a natural bioadhesive possessing biocompatibility and biodegradability [1]. These unique properties make mussel adhesive protein as a promising protein-based backbone material for versatile tissue engineering applications. In addition, various peptides can be recombinantly introduced to mussel adhesive protein that it is possibly tailored into a biomaterial for a specific tissue. Herein, recombinant fusion proteins of mussel adhesive protein with four distinct neuropeptides are recombinantly produced and evaluated in vitro for their regenerative capacities on nerve using Schwann cells. Keywords : Mussel adhesive protein, neuropeptide, neural regeneration References 1. J.M. Hong, and B.J. Kim, J. of Acta Biomaterialia. 8, 2578-2586 (2012). P0804 Antibacterial Actions of Synthetic Peptides with Repeated Sequence in Drug-resistant Bacteria Nam-Hong KIM, Young-Min KIM, Eun-Ji KIM, Jae-Eun JEONG, Yung-Hoon PARK, Mi-Kyeong JANG, Seong-Cheol PARK* Department of Polymer Science and Engineering, College of Engineering, Sunchon National University, Jeonnam, 540-950, Korea To overcome conventional mechanisms of drug-resistance in pathogens, Antimicrobial peptides (AMPs) have been proposed to be an effective means due to the physical perturbation and disruption of microbial membranes. Present study was designed a series of synthetic α-helical peptide with amphiphilicity. They were comprised of recurring (XWZX)n-NH2 sequences where X: cationic amino acids, W: tryptophan, Z:hydrophobic amino acids, n: number of repeat units. We analyzed alternative modes of action of eight AMPs in drug resistant pseudomonas aeruginosa and taphylococcus aureus, and their cell selectivity according to peptide length or substitution of amino acids. The results might provide useful information in designation of synthetic AMPs as therapeutic candidate with potential to combat antibiotics resistance. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded the Ministry of Education, Science and Technology (2013R1A1A4A01010701). Keywords : peptide, bacterial, helical References 1. H. Nikaido, Multidrug resistance in bacteria (2009), Annu. Rev.Biochem, 78, 119-146 P0805 Synthesis of Isosorbide-dinorfloxacin Using Emzymatic Catalyst Nam-Hong KIM1, Jong-Wan LEE2, Young-Min KIM1, Eun-Ji KIM1, Jae-Eun JEONG1, Seong-Cheol PARK1, Yung-Hoon PARK1, Mi-Kyeong JANG*1 1Department of Polymer Science and Engineering, College of Engineering, Sunchon National University, Jeonnam 540-950, Korea, 2IC Chemical, 80, Yeosusandan 4-Ro, Yeosu, Jeonnam, Korea Norfloxacin is a synthetic antibacterial agent with broad-spectrum and has been used to treat for complicated urinary tract infections. However, the poor water solubility of norfloxacin is a limiting factor in the bioavailability. We synthesized isosobide-dinorfloxacin(ISDN), that carboxyl group of norfloxacin was conjugated on two hydroxyl group of isosobide via esterification by Novozyme 435 (lipase immobilized on acryl resin), resulted in the significantly enhanced water solubility. Antibacterial efficiency of ISDN was maintained in drug-susceptible bacteria, while its minimal inhibitory concentration (MIC) was reduced in drug-resistant bacteria. Interestingly, its cytotoxicity in mammalian cells and resistance-development in bacterial cells were remarkably reduced, comparing with free norfloxacin. In addition, the increased transdermal diffusion capacity and in vivo antibacterial efficacy of ISDN proposed to have potential, being able to develop as a novel prodrug. Keywords : Prodrug, Norfloxacin, Antibacterial References 1. Eggeman T, Verser D. The importance of utility systems in today's biorefineries and a vision for tomorrow,(2006), Appl Biochem Biotechnol , 129-132, 361-381. P0806 Development of Polyvinyl Chloride Composite Having Antimicrobial Effects and Flame Retardancy Ju-Hwan CHOI, Tae-Hee PARK, Se-Ho PARK, Jae-Yeul LEE, Hyeongsu LEE, Yuri PARK, Daesuk BANG, Kwang-Hwan JHEE School of Science and Engineering of Chemical Materials, Kumoh National Institute of Technology, Gumi, Gyeongsangbuk- do 39177, Korea Polyvinyl Chloride (PVC) composite is widely used for various purposes. New properties addition to PVC composite such as antimicrobial and flame retardancy, it will be very beneficial to industrial field [1, 2]. This study was conducted to investigate the antibacterial activity and flame retardancy of PVC composite that was composed with inorganic bacteriocide and aluminum trihydroxide (ATH) mixed with PVC. Inorganic bacteriocide was selected containing zinc, silver and zeolite. ATH was selected considering aspects of environmental and economical material. We confirmed the antimicrobial activity by adopting turbidity assay, shaking flask method, drop-test, double staining assay, and scanning electron microscopy. The growth of Staphylococcus aureus and Klebsiella pneumoniae were significantly inhibited by the composite containing 5 wt% inorganic bacteriocide and ATH each. Also, we assayed the thermogravimetric analysis (TGA) and limiting oxygen index (LOI) for the detection of flame retardancy of PVC composite containing ATH and inorganic bacteriocide. TGA and LOI data showed that the tendency to steady increase of flame retardancy by the increasing of ATH concentration. We can expect that the inorganic bacteriocide and ATH containing PVC composite could be used for biomaterial applications due to their functional abilities to reduce biofilm formation, according to our study. Keywords : Antimicrobial activity, flame retardancy, polyvinyl chloride composite, biofilm formation References 1. W. Zhang, P. K. Chu, J. Ji, Y. Zhang, X. Liu, R. K. Y. Fu, P. C. T. Ha, Q. Yan, Plasma surface modification of poly vinyl chloride for improvement of antibacterial properties (2006), Biomaterials, 27, 44. 2. J. S. Kim, E. Kuk, K. N. Yu, J. H. Kim, S. J. Park, H. J. Lee, S. H. Kim, Y. K. Park, Y. H. Park, C. Y. Hwang, Y. K. Kim, Y. S. Lee, D. H. Jeong, M. H. Cho, Antimicrobial effects of silver nanoparticles (2007), Nanomedicine, 3, 95. P0807 Synthesis and Antimicrobial Activity of Polylactic Acid Composite Containing Silver/Zinc/Zeolite Compound Tae-Hee PARK, Ju-Hwan CHOI, Se-Ho PARK, Jae-Yeul LEE, Hyeongsu LEE, Yuri PARK, Daesuk BANG, Kwang-Hwan JHEE School of Science and Engineering of Chemical Materials, Kumoh National Institute of Technology, Gumi, Gyeongsangbuk- do 39177, Korea Biocompatible and biodegradable polymers became very important and gained a lot of attention from both biomedical and ecological outlooks in the past decade. Polylactic acid, PLA, is one of the most widely used biodegradable/bio-based plastic alternatives to traditional petroleum-based plastics [1]. So, the objectives of this study were to development PLA composite containing silver/zinc/zeolite compound, to add the antimicrobial activity of these composites against Staphylococcus aureus (S. aureus, Gram-positive bacteria) and Klebsiella pneumoniae (K. pneumoniae, Gram-negative bacteria) [2]. PLA composites containing 5, 10, 15 and 20 wt% silver/zinc/zeolite compound were manufactured by using intermeshing co-rotating twin screw extruder. Scanning electron microscopy and energy dispersive spectroscopy were used to examine the morphology and composition of the surfaces of the PLA. The growth of S. aureus and K. pneumoniae were analyzed in the presence of PLA composites as measured by drop-test, shaking flask method. The quantitative assay of antimicrobial activity was used double staining assay using calcein-AM and propidium iodide. The PLA composite exhibits effective antibacterial activity against Gram-negative and Gram-positive bacteria. Taken together, our data indicate the potential of PLA composites for applications in antimicrobial packaging. Keywords : Antibacterial activity, silver/zinc/zeolite compound, polylactic acid, drop-test References 1. I. E. Yuzay, R. Auras, and S. Selke, Poly(lactic acid) and zeolite composites prepared by melt processing: Morphological and physical-mechanical properties (2010), J. Appl. Polym. Sci., 115, 2262. 2. T. Jin, L. Liu, H. Zhang, and K. Hicks, Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes (2009), J. Food Sci. Technol., 44, 322. P0808 Mussel-inspired Adhesive Protein-based Electrospun Nanofibers Reinforced by Fe (III)–DOPA Complexation Sangsik KIM, Dong Soo HWANG School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea Marine mussels utilize multiple bidentate complexes formed by Fe(III) and DOPA in a mussel adhesive protein (fp-1) to reinforce tough and elastic byssal fibers as a specialized underwater adhesive aid. In this study, mussel-inspired electrospun nanofibers were fabricated using a recombinant mussel adhesive protein (rfp-1), Fe(III)–DOPA complexes, and polycaprolactone. The mechanical properties of the fabricated nanofibers were reinforced by the Fe(III)–DOPA complex found in fp-1, which is a key component of the naturally occurring high-performance mussel fiber coating. Experimental results show that the stoichiometry of Fe(III)–DOPA complexes in the nanofibers could be controlled by buffer pH conditions and the stiffness of the nanofiber mat increased linearly with the concentration of the Fe(III)– DOPA complexes, as monitored by resonance Raman spectroscopy. This suggests the potential of Fe(III)–DOPA complexation as an effective strategy for modulating the mechanical properties of nanofibrous biomedical materials by using pH variations. Keywords : Mussel adhesive protein, Electrospun nanofibers, Fe(III)-DOPA complexation References 1. Hongbo Zeng at al, Strong reversible Fe3+ mediated bridging between dopa-containing protein films in water(2010), PNAS, 107(29), 12850 P0809 Selection of Botulinum Toxin Type E-Specific Peptides by Phage Display Ga-Young PARK1, Se Hee LEE1, Gna AHN1, Eunji LEE1, Yae-Eun JEON1, Yang-Hoon KIM1, Hobaek YOON2, Ji-Young AHN1 1Department of Microbiology, Chungbuk National University, Cheong-Ju, Korea, 2National Institute of Animal Science, RDA, Korea Botulinum toxin is known as neurotoxin produced by Clostridium botulinum and is classified into serotype A to G. Especially, Botulinum neurotoxin type E (BoNT-E) cause a food-born botulism in human. Here, we screened short peptides that bind to the serotype E using random phage display technique. BoNT-E-specific peptide candidates were obtained through 4 rounds of selection including negative selection. The high affinity peptide candidates were tested by ELISA and dot blotting assay. Among candidates, 3 peptides were finally selected. For the future work, we will confirm blocking of BoNT-E toxicity by cytotoxicity test. Our peptide can utilize to develop BoNT-E detection and neutralization. This work was supported by "CRP for Agriculture Science & Technology Development (PJ010530)" Rural Development Administration, Republic of Korea. Keywords : Botulinum toxin type E, BoNT-E, phage display, peptide display References 1. Yoshimasa et al., Random Phage Display-Based Screening of Peptides that Bind to Botulinum Neurotoxin Binding Protein, Nontoxic Nonhemagglutinin(2013), Curr Microbiol, 67, pp. 188-192 2. Ullman et al., In vitro methods for peptide display and their applications(2011), BRIEFINGS IN FUNCTIONAL GENOMICS, 10(3), pp. 125-134 P0810 Selection of DNA Aptamer Switched on Signaling of Insulin Receptor Ga-Young PARK, Se Hee LEE, Gna AHN, Eunji LEE, Yang-Hoon KIM, Ji-Young AHN Department of Microbiology, Chungbuk National University, Cheong-Ju, South Korea Insulin plays important role in the regulation of glucose homeostasis and metabolism as cellular growth factor. Interaction between insulin and its receptor (Insulin Receptor, IR) leads to auto-phosphorylation of IR, which induce phosphorylation of other proteins. Cascade signal reactions of IR regulate metabolism, cell division and other gene expressions. Aptamer is single-stranded RNA or ssDNA having high specificity and affinity with target. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a process to select target-specific aptamer. In this study, we carried out SELEX process to obtain DNA aptamers switching on signaling of IR. IR-specific aptamer candidates were acquired through 10 rounds of selection and real-time PCR. For the future work, we will confirm IR signaling by interaction of aptamer and IR through western blotting. The IR-specific aptamer can utilize as a substitute of insulin by adding a free-serum media to incubate mammalian cell. This work was supported by the Technological Innovation R&D Program (S2177590) funded by the Small and Medium Business Administration (SMBA, Korea). Keywords : Insulin receptor, insulin signaling, aptamer, SELEX References 1. JONGSOON LEE and PAUL F.PILCH, The insulin receptor: structure, function, and signaling(1994), the American Physiological Society, pp. C319-C333 2. Regina Stoletenburg, Christine Reinemann and Beate Strehilitz, SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands(2007), Biomolecular Engineering, 24, pp. 381-403 P0811 Bio-Silicification of Alkaline Stable Carbonic Anhydrase for CO2 Sequestration Development Ryeo Gang SON, Ki Ha MIN, Chang Hyun LEE, Mi Ran KI, Seung Pil PACK Department of Biotechnology and Bioinformatics, Korea University, Chungnam 339-700, Korea Carbonic anhydrase (CA) is a biocatalyst for CO2sequestration because of its distinctive ability to accelerate CO2 hydration. In this study, we designed and applied a-type CA from Hahella chejuensis(HCA), which was previously reported as a highly active CA in alkaline conditions, but was mostly expressed as insoluble forms. By designing the signal peptide-removed HCA(SP-), production of a high amount of soluble protein was achieved under a microbial expression system (70 mg/L of culture). In addition, through spermine-mediated biosilicification, HCA(SP-) was encapsulated in silica particles [HCA(SP- )@silica] under mild conditions. HCA(SP-)@silica exhibited high immobilization efficiency and stable activity over a range of temperature and pH. Even after 10 cycles of use, HCA(SP-)@silica retained more than 90% of its residual activity in mild conditions and 80% in pH 10 conditions. Moreover, HCA(SP-)@silica showed better CO2 hydration performances in high pH conditions than BCAII@silica. These results, describing the design of HCA(SP-) and HCA(SP-)@silica with alkaline activity, will be useful in the development and improvement of practical CA-based CO2 sequestration processes. Keywords : biosilicification, CO2 sequestration, alkaline stable carbonic anhydrase References 1. Min, K.-H., et al. Chemosphere (2015) doi: 10.1016/j.chemosphere.2015.07.020. P0812 Hydroxyapatite Anisotropic Growth and Wet Mechanical Properties Enhancement of Dopamine Crosslinked Chitosan- Hydroxyapatite Composite Ekavianty PRAJATELISTIA1, Dong Soo HWANG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea., 2Integrative Bioscience and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. Synthetic hydroxyapatite (HAp) has been used due to its excellent biocompatibility and the chemical similarity to natural bone. Incresing aspect ratio of the synthetic HAp particle is one of the main concerns regarding tissue engineering because natural bone is a composite consisting of HAp nanorods. Here, we found that in the presence of chitosan and dopamine, the HAp particles grew anisotropically in a needle shape with an aspect ratio of ~4.4. The needle-shaped HAp particles were dispersed well in the chitosan matrix, and dopamine-mediated cross-linking enhanced the stiffness and reduced swelling in the presence of water. The composite is too weak for use in hard tissue repair, but could be used for curing dentin sensitivity by blocking and remineralization on dentinal tubules, and in drug-delivery applications. Keywords : Hydroxyapatite, Anisotropic growth, Wet mechanical properties References 1. Prajatelistia, E.; Lim, C.; Oh, D. X.; Jun, S. H.; Hwang, D. S., Engineering in Life Sciences; 15(2);254-261, (2015) 2. Nguyen NK, Leoni M, Maniglio D, Migliaresi C. Journal of biomaterials applications;28:49-61, (2013). 3. Kumar R, Prakash KH, Cheang P, Gower L, Khor KA. Journal of the Royal Society, Interface / the Royal Society.5:427-439, (2008) P0813 Engineering RGD Peptide on Silica Forming Fluorescent Proteins for Imaging and Cargo Delivery Mi Ran KI, Jae Hyeon PARK, Ki Baek YEO, Seung Pil PACK Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-Ro, Sejong 339-700, Korea Silica has a wide range of industrial uses and its nontoxic and highly biocompatible characteristics lend well to applications in biomedical field. Biomimetic silica synthesis using proteins or peptides has attracted much attention as a new paradigm for silica synthesis under ambient condition. Fluorescent protein has been developed as a standard tool for investigating intracellular properties as a biosensor, and monitoring gene expression as a reporter gene. We engineered silicaforming fluorescent protein without disturbing protein function, letting the protein induce silica precipitates in the presence of silica monomer. By this, bright and stable fluorescent silica nanoparticles were provided. To realize the application in bioimaging, cell staining, and drug delivery with bioimaging in vivo, the cell recognition motif (RGD; Arg-Gly-Asp) was genetically fused to C-terminus of silicaforming fluorescent protein. The RGD-modification allowed its silicified form to bind to cells expressing integrin receptors. This might have potential for imaging and cargo delivery for cancer therapy. Keywords : silica forming fluorescent protein, biosilicification, RGD peptide References 1. Ki Mi Ran, Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S- transferase (GST) (2013), Bioprocess and Biosystems Engineering, 36(5), 643-648 P0814 Injectable Hydrogels by Crosslinking Modified Carbohydrate with Protein for Potential Wound Dressing Ju-Eun LEE1, Sang-Hyuk LEE1, Byung-gee KIM1,2 1Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, 151-742, Seoul, South Korea, 2School of Chemical and Biological Engineering, Seoul National University, 151-742, Seoul, South Korea Over the past few years, there has been a growing interest in hydrogels having biocompatible and biodegradable properties. In particular, the application of hydrogels in the biomedical field continues to grow. However, there are many disadvantages such as difficulty handling and tissue damage. Injectable hydrogels have attracted attention in recent years because they have various advantages. For example, the use of injectable hydrogels offers a simple and minimally invasive surgical procedure. In addition, these hydrogels can be readily injected into target locations when used as drug delivery vehicles. In this study, we develop in situ-forming hydrogels by using ionic strength of salt. First, we synthesized modifided carboxymethylcellulose and hyaluronic acid by conjugating with monophenolic substances. And then, the modified carbohydrate was crosslinked with gelatin by enzymes to form the hydrogels. We characterized the mechanical properties, swelling degree and degradation rate of this hydrogel. These results propose promising use of this hydrogel as potential wound dressing. Keywords : hydrogel, injectable, crosslinking, wound dressing References 1. Yuko Ogushi, Shinji Sakai and Koei Kawakami, Synthesis of enzymatically-gellable carboxymethylcellulose for biomedical applications(2007), Journal of Bioscience and Bioengineering, 104, 30 2. Nahum Lee, Eun Jung Kim and Byung-Gee Kim, Regioselective Hydroxylation of trans-Resveratrol via Inhibition of Tyrosinase from Streptomyces avermitilis MA4680(2012), ACS chemical biology, 7, 1687 3. C. Ghobril and M. W. Grinstaff, The chemistry and engineering of polymeric hydrogel adhesives for wound closure: a tutorial(2015), Chemical Society Reviews, 44, 1820 P0815 Biosilicification by New Silica Forming Peptides Derived from Ectocarpus siliculosus Ki Baek YEO, Mi Ran KI, Seung Pil PACK Department of Biotechnology and Bioinformatics, Korea University, Sejong-Ro 2511, Sejong, 339-700 Korea The silaffin polypeptide, highly post translational modified peptide, are responsible for mediating silicification at ambient conditions. And synthetic R5 peptide, the repeat unit of the silaffin, showed similar silica precipitation activity without post translational modifications. But R5 peptide has low silica precipitation activity below pH 7 which is important pH range for biological applications. In this study, we tried to search new SFP (silica forming peptide) which have better silica precipitation activity than R5 peptide. New SFP, named EctP1, EctP2 derived from Ectocarpus siliculosus was discovered by gene database searching. These peptides showed better silicification ability than R5 peptide. Especially at pH 6, EctP1 could form silica deposition while R5 could not. EctP1, EctP2 and R5 fused on to c-terminus of GFP (green fluorescent protein) by SFP fusion vector system we developed. GFP fusion proteins showed silicification ability and EctP1’s pH capability sustained after protein fusion. GFP-EctP2 fusion protein showed better protein expression level than GFP-R5 fusion protein. We expect these new silica forming peptides can be a novel tool for biological applications. Keywords : biosilicification, silica forming peptide, ectocarpus siliculosus References 1. Ki Mi Ran, Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S- transferase (GST) (2013), Bioprocess and Biosystems Engineering, 36(5), 643-648 P0816 Design and Synthesis of Silica-coated Human Ferritin Nanoparticles for Drug Delivery Thi Khoa My NGUYEN, Mi Ran KI, Sung Ho KIM, Ryeo Gang SON, Seung Pil PACK Department of Biotechnology and Bioinformatics, Korea University, Sejong-Ro 2511, Sejong, 339-700 Korea Caged protein nanoparticles possess many desirable features for an ideal drug delivery system. Ferritin is a major iron storage protein composed of 24 subunits, which self-assemble to form a cage-like nanostructure (inner diameter: 8 nm, exterior diameter: 12 nm). Herein, a new hybrid material that contains silica-coated human ferritin cage was developed. By gene technologies, heavy chain of human ferritin (HuFn) was genetically modified to fuse a silica forming peptide (SFP) sequence (Kps peptide derived from phage display library or Kpt peptide derived from putative membrane protein sequences) to N-terminus of ferritin, resulting in presenting SFP moieties on the surface of HuFn (SFP-HuFn). This caged protein is used as template in SFP-mediated biosilicification reaction to control the shape and the size of inorganic-organic hybrid particles (Si-KpsHuFn and Si-KptHuFn) in a homogeneity and monodispersion. This study paves the way for promising further drug delivery application studies. Keywords : human ferritin, silica forming peptide, hybrid nanoparticles, drug delivery References 1. Ki Mi Ran, Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S- transferase (GST) (2013), Bioprocess and Biosystems Engineering, 36(5), 643-648 P0817 Electrospun Gelatin Scaffold with Surface Deposited Biomineral Sung Ho KIM, Mi Ran KI, Ki Baek YEO, Seung Pil PACK Department of Biotechnology and Bioinformatics, Korea University, Sejong-Ro 2511, Sejong, 339-700 Korea Hydroxyapatite (HA) has been widely used for restoring bone defects in osteopathy and dentistry. However, HA has some disadvantages such as low mechanical properties and slow bioresorbility. Calcium carbonate can be an alternative material because it resorbs faster than HA but still provide good biological properties. Silica plays important role in bone formation and calcification of implant. In order to fabricate a better scaffold for bone tissue repairing, we mineralized electrospun gelatin scaffold with calcium carbonate and silica. The morphology and composition of this composite scaffold was analyzed by scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. SEM images showed CaCO3 and silica particles on the surface of the gelatin scaffold. Biological properties were determined by growing MC3T3-E1 cell line over the scaffolds. Cell adhesion and proliferation were observed in the composite scaffold. The composite scaffold made from gelatin, calcium carbonate, and silica developed in this study may be useful for application in bone tissue engineering. Keywords : silica, calcium carbonate, cell adhesion, proliferation References 1. Ki Mi Ran, Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S- transferase (GST) (2013), Bioprocess and Biosystems Engineering, 36(5), 643-648 P0818 Depolymerization of Chitosan Using Solution Plasma Process: A Novel Approach on Polymers Utilization Davoodbasha MUBARAKALI1,2, Seong-Cheol KIM2,3, Sang-Yul LEE2,3, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Centre for Surface Technology and Applications, Korea Aerospace University, Goyang, 412-791, 3Dept. Materials Engineering, Korea Aerospace University, Goyang , 412- 791 Chitosan has been utilized in biomedicine due to its non-toxic, biocompatible, biodegradable, and unique antimicrobial properties. Depolymerization of high molecular weight chitosan to low molecular weight chitosan (LMWC) was attempted using solution plasma process (SPP) without adding hazardous chemicals. Chitosan was subjected to plasma discharge at 800 V, 35 kHz for 0-120 min. SPP effectively triggered the depolymerization process with enhanced water solubility and high yield of water soluble chitosan in much shorter time than other methods. Viscosity and chemical composition of the depolymerized chitosan solution was assessed using a viscometer and IR spectroscopy. GPC analysis indicated that the molecular weight of chitosan (3.0x105 Da) was decreased to 7.8x103 Da within 30 min of plasma treatment. Size and stability of LMWC were assessed by DLS and zeta potentials, which confirmed the formation of nano-sized chitosan. LMWC exhibited antimicrobial activity against E. coli, S. aureus, and C. albicans with MIC of 80, 160 and 300 μg/ml. Furthermore, LMWC showed an excellent antioxidant property (70 %) and swelling ratio of 2 mg/mg. Therefore, SPP seemed to be a novel approach in polymer technology adoptable for facile, economic, and eco-friendly generation of anticipated pure materials for potential applications in nanomedicine. Keywords : Solution plasma porcess, LMW Chitosan, Chitosan, Depolymerization, antimicrobial activity References 1. Sawaguchi, A., Ono, S., Oomura, M., Inami, K., Kumeta, Y., Honda, K, Sameshima-Saito, R., Sakamoto, K., Ando, A., Saito, A. 2015. Chitosan degradation and associated changes in bacterial community structures in two contrasting soils. Soil Sci. Plant Nutri. 61 (in press). 2. Fan, W., Yan, W., Xu, Z., Ni, H. 2012. Erythrocytes load of low molecular weight chitosan nanoparticles as a potential vascular drug delivery system. Colloids Surf. B Biointerfaces 95:258-265. 3. Takai, O. 2008. Solution plasma processing. Pure Appl.Chem. 80: 2003-2011. P0819 Synthesis of Cellulose-coated Nanoceria with Excellent In-vitro Antioxidant Property Davoodbasha MUBARAKALI1,2, Seong-Cheol KIM2,3, Sang-Yul LEE2,3, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Centre for Surface Technology and Applications, Korea Aerospace University, Goyang, 412-791, 3Dept. Materials Engineering, Korea Aerospace University, 412-791 Nanoceria has been demonstrated as a potential antioxidative nano-drug. However, its short residence time in the body and toxic solvents used in the synthesis processes hindered its potential clinical applications. Solution plasma process (SPP) is a physical method used for generation of nanobiomaterials. In this present study, cellulose was used as matrix of nanobiocomposites using SPP. Cellulose-nanoceria (C-NC) biocomposites were synthesized by discharging plasma for 15 min at 800 V, 30 kHz using a unipolar power supply into solutions of cellulose (1 %) and Ce(NO3)2 (1,5 mM). Synthesis and microstructures of the biocomposites were characterized by UV-Vis spectroscopy, FTIR and FE-SEM equipped with EDS. TEM showed cubical nanoceria in the size range of 5-20 nm, which were well-distributed throughout the matrix without agglomeration in C1-NC5. The biocomposites showed an excellent antioxidant property in DPPH, hydroxyl, and superoxide free radical scavenging assays. C1-NC5 showed pH and incubation time-dependent antioxidant property. In vitro cell viability and reactive oxygen species (ROS) scavenging assays proved that the C-NC biocomposites had much more effective antioxidant properties than other biocomposites. Therefore, the C-NC biocomposites synthesized by one-step eco- friendly SPP have potential as antioxidant biomaterials and sustained release to scavenge ROS in the modern medicine. Keywords : Solution plasma porcess, nanoceria, cellulose, biocomposites, antioxidant References 1. MubarakAli, D., S. Lee, S. Kim, and J. Kim. 2015. One-step synthesis of cellulose/silver nanobiocomposites using a solution plasma process and characterization of their broad spectrum antimicrobial efficacy. RSC Adv., 2015, 5, 35052-35060. 2. Karakoti, A.S., N.A. Monteiro-Riviere, R. Aggarwal, J. P. Davis, R. J. Narayan, W. T. Self, J. McGinnis, and S. Seal 2008. Nanoceria as Antioxidant: Synthesis and Biomedical Applications. J. Material. 60:33-37. 3. Takai, O. 2008. Solution plasma processing. Pure Appl.Chem. 80: 2003-2011. P0820 Facile Synthesis of Nanoceria Using Solution Plasma Process Bo-Ram PARK2, Davoodbasha MUBARAKALI1, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Dept. Life Sciences, Incheon National University, Incheon, 406-772 Cerium oxide nanoparticles, popularly known as nanoceria, are efficient free radical scavengers that are considered as a potent therapeutic option for the treatment of reactive oxygen species (ROS) mediated disorders. The capability of nanoceria switching between oxidation states is comparable to that of biological antioxidants. This capability imparts nanoceria with the very important biological property of radical scavenging. Solution plasma process (SPP) is a physical method for facile generation of nanomaterials. Nanoceria (NC) biocomposites were synthesized by discharging plasma in 2 mM Ce(NO3)2 solution at 800 V with frequency of 30 kHz for 0-25 min. Synthesis and structure of the nanoceria particles were characterized using UV-Vis spectroscopy, SEM and XPS. TEM analysis showed spherical shaped nanoceria in the size range of 5-10 nm without agglomeration. Nanoceria showed an excellent antioxidant property by DPPH, hydroxyl and superoxide free radical scavenging assays. The highest scavenging activity of the NC particles was 85% in the hydroxyl radical scavenging assay and 82% in the superoxide radical scavenging assay. Additionally, NC showed pH dependent antioxidant property. Keywords : Solution plasma porcess, nanoceria, antimicrobial activity References 1. MubarakAli, D., S. Lee, S. Kim, and J. Kim. 2015. One-step synthesis of cellulose/silver nanobiocomposites using a solution plasma process and characterization of their broad spectrum antimicrobial efficacy. RSC Adv., 2015, 5, 35052-35060. 2. Karakoti, A.S., N.A. Monteiro-Riviere, R. Aggarwal, J. P. Davis, R. J. Narayan, W. T. Self, J. McGinnis, and S. Seal 2008. Nanoceria as Antioxidant: Synthesis and Biomedical Applications. J. Material. 60:33-37. 3. Takai, O. 2008. Solution plasma processing. Pure Appl.Chem. 80: 2003-2011. 4. Jin, A., S. Kim, S. Lee, and J. Kim. 2014. Synthesis and Characterization of Silver Nanoparticles Using a Solution Plasma Process. J. Nanosci. Nanotech. 14:8094-8097. P0821 Synthesis of Hydroxyapatite/Silver Nanoparticles as Coating Material for Dental Implants Using Solution Plasma Process Yulah JEONG2, Davoodbasha MUBARAKALI1, Jung-Wan KIM1,2 1Div. Bioengineering, Incheon National University, Incheon, 406-772, 2Dept. Life Sciences, Incheon National University, Incheon, 406-772 Hydroxyapatite (HA), Ca10(PO4)6(OH)2, is a synthetic ceramic material that is very similar to natural bones. However, HA has high risk of bacterial accumulation and consequently to biofilm. Silver nanoparticles (AgNPs) could be a remedy for the issue due to its antimicrobial potential. Solution plasma process (SPP) is a rapid green strategy to synthesize nanoparticle. The aim of this study was to synthesize HA/AgNPs via SPP and evaluation of their antimicrobial activity. Ca(NO3)2 and H3PO4 were used as precursors of HA with stoichiometric composition of Ca/P ratio 1.67. Concentration of Ca(NO3)2, H3PO4, and AgNO3 used was 0.1-0.2 M, 0.06-0.12 M, and 1-3 mM respectively. HA/AgNPs solutions were subjected to plasma discharge at 900V for 30 min. The UV/Vis spectra of HA/AgNPs containing 5 mM of AgNO3 showed blue shift of absorbance peak at 400-450 nm, confirming formation of AgNPs in HA. Agar diffusion and CFU reduction assay were performed to investigate their antimicrobial activity against eight species of pathogens including Streptococcus mutans. HA/AgNPs containing 0.1 M of Ca(NO3)2, 0.06 M of H3PO4, and 5 mM of AgNO3 showed the best antimicrobial activity. Its structure and affinity to titanium are to be investigated for its potential in application to dental implant. Keywords : hydroxyapatite, silver nanoparticle, solution plasma process, artificial bone, antimicrobial activity References 1. Sadat-Shojai, M., M. T. Khorasani, E. D. Khoshdargi, and A. Jamshidi. 2013. Synthesis methods for nanosized hydroxyapatite with diverse structures. Acta Biomaterialia. 7591-7621. 2. Ignajatovic, N. and D. Uskokovic. 2004. Synthesis and application of hydroxyapatite/polylactide composite biomaterial. Appl. Surf. Sci. 15:314-319. 3. Sanabani, J. S., A. A. Madfa, and F. A. Al-Sanabani. 2013. Application of Calcium Phosphate Materials in Dentistry. Int. J. Biomater. 876132. P0822 Effects of a Human Originated Adiponectin Peptide on Lipogenesis of an Immortalized Human Sebocytes So Yoon CHA1, Myoungjin OH1, Hyunkyung CHOI1, Changdeok KIM2, Janghee HAHN3, Changseo PARK1 1Department of Chemical Engineering, Dongguk University, Seoul,100-715, 2Department of Dermatology, College of Medicine, Chungnam National University, Daejeoun, 301-747, 3Department of Anatomy and Cell Biology, School of Medicine, Kangwon National University, Gangwon-do, 200-701 Adiponectin has diverse biological functions for example, anti-inflammation, increase of fat oxidation, reduction of glucose production in liver, improving insulin sensitivity, and accelerating wound healing. Recently, adiponectin receptors have been identified from keratinocytes and dermal fibroblasts and found to have distinct effect on epidermal homeostasis [1]. Other studies also showed that adiponectin stimulated the production of hyaluronic acid from human dermal fibroblasts and ceramide biosynthesis was inhibited when the adiponectin level in the serum of a mouse was decreased [2,3]. Adiponectin production in cultured mouse sebocytes was also reported [4]. However not many studies have been conducted to elucidate the biological role of adiponectin in human sebocytes. In this study, a synthetic tripeptide JP derived from adiponectin was evaluated on its effect on lipogenesis using a human immortalized sebocyte cell line, CNU-sebo1. JP slightly increased lipid production of the sebocytes when treated alone. Stimulation of lipogenesis was markedly enhanced when treated with linoleic acid (LA). The expression of fatty acid synthase gene FAS was increased more than 4 folds by co-treatment of LA and the JP while LA and JP alone enhanced FAS expression 1.7 and 2.8 folds respectively. The results suggest that LA and the tripeptide JP work together in a cooperative manner in lipogenesis of the human immortalized sebocytes. The results also will provide an opportunity to develop a cosmetic formulation using the tripeptide to improve xerotic skins of which reduction of sebum production is a major cause. Keywords : adiponectin, sebocyte, tripeptide, lipogenesis References 1. Sayaka Shibata et al, The Journal of Immunology, 189, 3231-3241, (2012). 2. Ezure, T., and Amano, BioFactors, 31, 229-236, , (2007). 3. Takumi Yamane, Kaquo Kobayashi-Hattori and Yuichi Oishi, Mol.Nutr.Food Res., 55, S186-S192, (2011). 4. Yumiko Akazawa et al, Connective Tissue Research, 52:4, 322-328, (2011) P0823 Bioengineered Mussel Glue as an Osteogenic Bone Adhesive for Titanium Surface Yun Kee JO1, Bong-Hyuk CHOI1, Cong ZHOU2, Jin-Soo AHN3, Sang Ho JUN4, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea, 2Department of Medicine, Korea University Graduate School, Seoul 136-705, Korea, 3School of Dentistry, Seoul National University,Seoul 110-749, Korea, 4Department of Dentistry, Anam Hospital, Korea University Medical Center,Seoul 136- 705, Korea Dental implant prosthesis greatly depends on primary stability and biocompatibility through a correct and stable osseointegration between titanium fixture and bone cells for tissue regeneration. The concept of artificial extracellular matrix (ECM) has been the focus of attention with its significant biological activitiy for the enhancement of target cell behaviors such as proliferation and differentiation in tissue engineering. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on inorganic and organic surfaces even in wet environment. Previously, we developed new cell adhesive, MAP-RGD, by fusion of MAP with RGD peptide, one of the major cell adhesion recognition motifs. In the present work, we investigated in vitro and in vivo osteoinductivity of MAP- RGD on titanium mesh surfaces. We found that cellular behaviors of mouse pre-osteoblast MC3T3-E1 cells such as adhesion, proliferation, spreading, and differentiation were significantly increased on MAP-RGD-coated titanium mesh surface. Also, the mRNA expressions of osteogenic differentiation marker genes were up-regulated in MC3T3-E1 cells on MAP-RGD-coated titanium mesh surface. Furthermore, MAP-RGD-coated titanium mesh surface showed improved new bone formation in rat calvarial defect model compared to non-coated surface as a result of the radiographic analysis and histological evaluations. Collectively, RGD peptide-fused MAP can be successfully employed in medical implant prosthesis and bone tissue engineering applications. Keywords : RGD peptide, mussel adhesive proteins, titanium implant, bone tissue engineering, bone regeneration References 1. B. –H. Choi, H. Cheong, J. -S. Ahn, C. Zhou, J. J. Kwon, H. J. Cha and S. H. Jun, J. Mater. Chem., 2015, 3, 546- 555. P0824 Study on the Manufacturing Method of Water-based Acrylic Adhesive Tape for Healthcare In-Ho LEE1,4, Ha-Sun JANG4, Seok-Hwan PARK2, Don-Hee PARK1,2,3 1School of Biological Sciences and Biotechnology, Chonnam National University, Gwangju, 61186, 2Interdisciplinary Program of Graduate School for Bioenergy and Biomaterials, Chonnam National University, Gwangju, 61186, 3Department of Biotechnology & Bioengineering, Chonnam National University, Gwangju, 61186, 4Hosan P&T Co., Ltd, Naju-Si, 58277 Currently, domestic manufacturing of various adhesive tape is fabricated using hazardous solvent materials such as VOCs, endocrine disruptors, carcinogens, etc. These materials may act as toxin causing various side effects in the organism. Therefore, pressure-sensitive adhesive was used to solve this problem and to improve the production process. The pressure- sensitive adhesive tape production process is solvent free system that does not use a conventional solvent which release harmful substance. Safety of the adhesive tape for healthcare through direct contact with the skin is the purpose to improve healthcare using this technology production. Keywords : Adhesive tape, Water-based acrylic, Healthcare P0825 Study on Manufacture and Properties of Antibacterial Coating Materials Hyoung-Min LEE1, Seung-Yong WI1,4, Sung-Hoon LIM3, Si-Min LEE3, Yong Hwan ROH4, Won-Sik KANG5, Seok-Hwan PARK1, Don-Hee PARK1,2,3 1Interdisciplinary Program of Bioenergy and Biomaterials, Chonnam National University, Gwangju, 61186, 2Department of Biotechnology & Bioengineering, Chonnam National University, Gwangju, 61186, 3School of Biological Sciences and Biotechnology, Chonnam National University, Gwangju, 61186, 4TR chemtech Co., Ltd, Incheon, 21695, 5Tae Kang Co., Ltd, Sejong-Si, 30046 In this study, antibacterial property was measured by treated the antibacterial with WO3, MoO3, Li(OH)3. Escherichia coli, Staphlylococcus aureus subsp. aureus, Bacillus cereus, Salmonella enterica subsp. enterica, and Saccharomyces cerevisiae were growth in the culture medium agar plate. Fifteen microliter antibacterial was dropped into agar plate and incubated with the variation condition of temperature 25~35℃, time 1~24h and the concentration of antibacterial 0~100%. Killed bacteria by antibacterial were observed by a change in size of the clean zone. In comparison, 2 mL antibacterial was also dropped into liquid culture medium and incubated with the variation condition of temperature 25~35℃, time 1~5h and the concentration of antibacterial 0~100%. Killed bacteria were measured by 600 nm absorbance. The activity of antibacterial was observed by concentration over 25% in all temperature and higher concentration of antibacterial increased the antibacterial activity. As time increased the antibacterial activity was also increased, but after 5 hours, the activity was not much difference. Keywords : Antibacterial activity, Antibacterial material, Coating material P0826 Immobilization of Hemoglobin on Functionalized Multi-Walled Carbon Nanotubes-Poly-L- Histidine-Zinc Oxide Nanocomposites toward the Detection of Bromate and H2O2 Cheol Hwan KWAK1, A. T. Ezhil VILIAN2, Young-Kyu HAN2, Yun Suk HUH1 1Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 402-751, Republic of Korea, 2Department of Energy and Materials Engineering, Dongguk University-Seoul, Seoul, 100-715, Republic of Korea A novel biocompatible sensing strategy has been developed based on functionalized multi-walled carbon nanotube, poly-L- histine, and ZnO nanocomposite film for the immobilization of hemoglobin. The fabricated electrochemical biosensor based on the immobilized Hb revealed a fast response time (<3 s) with a wide linear range (4-18000 µM and 2-15000 µM) and detection limit (as low as 0.01 µM and 0.30 µM). The proposed third-generation biosensor was successfully applied to milk and urine samples for the detection of H2O2 and bromate. Keywords : Carbon nanotubes, Electrochemistry, Biosensor, Immobilization of Hemoglobin References 1. Hosseinzadeh, R, Moosavi-Movahedi, A. A., & Ghourchian, H., Electrochemistry and molecular modeling of the hemoglobin–benzene interaction with a nanocrystalline mixed metal oxide (2014), RSC Advances, 4 (90), 49128- 49136. P0827 Immobilization of Myoglobin on Au Nanoparticle-Decorated Carbon Nanotube/Polytyramine Composite as a Mediator-Free H2O2 and Nitrite Biosensor Seo Yeong OH1, A.T. Ezhil VILIAN2, Cheol Hwan KWAK1, Young-Kyu HAN2, Yun Suk HUH1 1Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon 402-751, Republic of Korea., 2Department of Energy and Materials Engineering, Dongguk University-Seoul, Seoul 100-715, Republic of Korea A novel composite film was designed for use as a highly selective mediator-free amperometric biosensor, and a method was created for accomplishing direct electrochemistry of myoglobin on a multi-walled carbon nanotube and tyramine-composite decorated with Au nanoparticles. The proposed Mb/Au-PTy-f-MWCNT biofilm exhibited excellent electrocatalytic behavior toward H2O2 and nitrite with linear ranges of 2 to 5000 µM and 1 to 8000 µM and lower detection limits of 0.01 µM and 0.002 µM. This biosensor can be applied to detect H2O2 and nitrite in disinfectant cream, eye drops, pickle juice, and milk samples. Keywords : H2O2, nitrite, biosensor References 1. Lu, X., Zhang, H., Ni, Y., Zhang, Q. & Chen, J. Porous nanosheet-based ZnO microspheres for the construction of direct electrochemical biosensors. Biosens. Bioelectron. 24, 93-98 (2008). P0828 Facile Preparation Strategy of MnO2/Carbon Nanotubes Decorated with a Nanocomposite of Pt Nanoparticles as a New Platform for the Electrochemical Detection of Catechin in Re Jun Yeong KIM1, A.T. EZHIL VILIAN2, Young-Kyu HAN2, Yun Suk HUH1 1Dep. of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 402-751, 2Dep. of Energy and Materials Engineering, Dongguk University-Seoul, Seoul, 100-715 Herein, we describe a simple and facile synthesis strategy of MnO2/carbon nanotubes decorated with a nanocomposite of Pt nanoparticles using a simple electrodeposition method. Notably, we achieved a very low detection limit (under optimized conditions) of catechin ca. 0.02 μM (S/N = 3); the linear range is 2–950 μM with excellent sensitivity. The real time application of catechin in red wine, black tea, and green tea samples with excellent performance. Schematic representation of the preparation of the Pt/MnO2/f-MWCNT film for sensing catechin in green tea and red wine samples. Keywords : carbon nanotube, electrodeposition method, catechin References 1. Wang, X.-G., Li, J. & Fan, Y.-J. Fast detection of catechin in tea beverage using a poly-aspartic acid film based sensor. Microchimica Acta 169, 173-179 (2010). 한국생물공학회, 생물공학의 동향 : 2015.10 생물공정공학 P0901 Production of DagA and Ethanol by Sequential Utilization of Sugars in Mixed-Sugar Medium Mimicking Microalgae Hydrolysate Juyi PARK, Yong Keun CHANG Dept. of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea Microalgal hydrolysate, a potentially low-cost alternative carbon source, could be used as a feedstock for valuable bioproducts production. The acid hydrolysate of Nannochloropsis oceanica contains 7 different sugars of glucose, galactose, xylose, rhamnose, ribose, mannose and fucose. At first, it was tried to produce DagA, a β-agarase by using a recombinant Streptomyces lividans in a mixed-sugar medium mimicking Nannochloropsis oceanica hydrolysate. Glucose, however, in the medium was found to have negative effects on the consumption of the other sugars and DagA biosynthesis causing a low substrate utilization efficiency and DagA productivity. To overcome such difficulties, a new strategy of sequential substrate utilization was developed. In the first step, glucose was consumed by Saccharomyces cerevisiae together with galactose and mannose producing ethanol, and then DagA was produced from the remaining sugars of xylose, rhamnose and ribose. Fucose was not consumed at all. By adopting the two-step process, the overall substrate utilization efficiency was about 3- fold increased with about 2-fold improvement of DagA production, let alone the additional benefit of ethanol production. The unconsumed fucose has a potential to be a high-value-added product with many applications once recovered. Keywords : mixed-sugar medium, microalgal hydrolysate, sequential substrate utilization, DagA, ethanol References 1. J. Park, S. K. Hong and Y. K. Chang, J. Microbiol. Biotechnol. 24(12), 1622-1628 (2014) 2. J. Park, S. K. Hong and Y. K. Chang, Bioresource technology. 191, 414-419 (2015) P0902 Development of EGFR Specific DNA Aptamer for Cell Culture Medium Supplements Soo-Young YOON1, Ji-Man CHA1, Simranjeet Singh SEKHON1, Ji-Young AHN1, Jiho MIN2, Yang-Hoon KIM1 1Dept. of Microbiology, College of Natural Sciences, Chungbuk National University, Cheongju, Chungbuk 362-763, South Korea, 2Graduate School of Semiconductor and Chemical Engineering, Chunbuk National University, Jeonju 561-756, South Korea Serum-containing medium is usually used for mammalian cell culture. Serum is a complex medium and therefore the reproducibility of cell culture product may not guaranteed. Accordingly, serum-free defined medium is used with medium supplements; for example, growth factors, hormones, and adhesion factors. But they have shortcomings such as high cost and unstable production. Growth factor in medium play an essential role in cell growth and differentiation, especially epidermal growth factor (EGF) mediates many intracellular signaling pathways by combine with EGF receptor (EGFR). In the present study, we carried out SELEX to select DNA aptamer that specifically combine with EGFR instead of EGF. The affinity between target and aptamer has been evaluated using surface plasmon resonance (SPR). Aptamers advantages of low cost, easy modification and high stability. EGFR specific DNA aptamer can be used in cell culture medium as an EGF substitute, and is expected to replace the role of EGF in the culture medium. This work was supported by the Technological Innovation R&D Program (S2177590) funded by the Small and Medium Business Administration(SMBA, Korea)" Keywords : EGFR, SELEX, Aptamer, Culture Medium References 1. H. Ogiso, R. Ishitani, O. Nureki, S. Fukai, M. Yamanaka, J.H. Kim, K. Saito, A. Sakamoto, M. Inoue, M. Shirouzu, S. Yokoyama, Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains, Cell 110 (2002) 775–787. 2. Stoltenburg, R., Reinemann, C., Strehlitz, B., 2007, SELEX-a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng. 24(4), 381-403. P0903 Development of Process for Enhanced iIolation of Ampelopsin Fro Dried Fruit of Ampelopsis grossedenta with Cellualse Wa GAO, Jin-Woo LEE Department of Biotechnology, Dong-A University, Busan 604-714, Korea The traditional method for isolation of ampelopsin, one of the most common flavonoid isolated from the plant species of Ampelopsis grossedentata, is the simple hydrothermal extraction around at 100℃. To develop the improved and safe process, the effect of treatment of cellulase on hydrolysis of the dried fruit of A. grossedentata was investigated. Thee treatment of cellulase was found to decrease the temperature and time for extraction of reducing sugars. The conditions of filter press and continuous centrifuge for removal of insoluble materials was optimized after hydrolysis of the dried fruit of A. grossdentata with treatment of cellulase. The recovery yield of ampelopsin from the dried fruit of A. grossedentata was calculated to be 39.4% by chromatographic analysis. The economic and safe process for isolation of ampelopsin at lower temperature with treatment of cellulase was developed in this study. Keywords : Ampelopsin, Ampelopsis grossedentata, Cellulases, Recovery References 1. Yoo, S. M., S. Y. Mun, and J. H. Kim, Recovery and pre-purification of (+)-dihydromyricetin from Hovenia dulcis (2006) Process Biochem. 41: 567-570. P0904 2, 3-Butanediol (2,3-BDO) Production from Nannochloropsis oceanica Hydrolysate by Fermentation Yong Jae KIM Dept. of Chemical and Biomolecular Eng., KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea 2, 3-Butanediol (2,3-BDO) is a valuable chemical feedstock which can be converted into solvent methyl ethyl ketone and 1, 3-butadiene. It was reported that 2,3-BDO could be successfully produced from glucose and xylose present in cellulose and hemicellulose by feremntation. Microalgal biomass which have various carbohydrate or sugar residues could be another promising feedstock for producing 2,3-BDO if a microbial strain with a broad substrate specificity. In this study, 2,3-BDO was produced from a synthetic medium simulating N. oceanica hydrolysate containing 7 different sugars of glucose, galactose, xylose, mannose, ribose, rhamnose, and fucose(10 g/L in total) by using Klebsiella oxytoca at pH 5.5 and 37℃. All the sugars contained in the medium were completely consumed in 9 hours although the uptake of the other sugars was inhibited by glucose lowering the process efficiency. Such inefficiency due to catabolic repression should be resolved to improve the productivity. Fucose was found to begin to be utilized only when all the other sugars had been depleted at about 9 hours. It, however, had no contribution to 2,3-BDO production. The final 2,3-BDO concentration was 2.2 g/L. It was concluded that fermentation could be stopped at 9 hours with no loss of 2,3-BDO production and that the unconverted fucose could be separated and purified from the broth as a high value added chemical compound. Keywords : Klebsiella oxytoca, 2,3-butanediol, sugar mixture, microalgae hydrolysate References 1. Winfield ME. The catalytic dehydration of 2,3-butanediol to 1,3-butadiene. J. Counc. Sci. Ind. Res. 1945;18:412- 423 2. Villet R. Biotechnology for producing chemicals from biomass. Ferment. Chem. from Biomass. Sol. Energy Res. Inst. 1981;2:621-754. 3. Stülke J, Hillen W. Carbon catabolite repression in bacteria. Curr Opin Microbiol 1999;2:195-201. P0905 Effects of Antifoaming Agents in Bacterial Growth and Production Nam-Hong KIM1, Jin-Sun JANG2, Young-Min KIM1, Eun-Ji KIM1, Jae-Eun JEONG1, Seong-Cheol PARK1, Yung-Hoon PARK1, Mi-Kyeong JANG*1 11Department of Polymer Science and Engineering, College of Engineering, Sunchon National University, Jeonnam 540- 950, Korea, 2IC Chemical, 80, Yeosusandan 4-Ro, Yeosu, Jeonnam, Korea As foaming appears as a problem in chemical and fermentation processes that inhibits reactor performance, its mitigation are important factor in reactor performances such as production of biomolecules and microbial growth in fermentation processes. The present study investigated antifoaming ability measured in the macroscopic recirculation foam column, and cell growth and protein expression was evaluated in two bacteria with 12 synthetic antifoaming agents. These findings suggested a useful structure of antifoaming agent in fermentation processes. This work (Grants No. C0276732) was supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2015. Keywords : antifoaming agents , bacterial bioprocess, fermentation processes References 1. ÇalikP, IleriN, ErdinçBI, AydoganN, ArgunM. Novel Antifoam for fermentation Processes: Fluorocarbon- Hydrocarbon Hybrid Unsymmetrical Bolaform Surfactant(2005). Langmuir,21, 8613 P0906 Determination of Optimal Dissolved CO Concentration for Bioreactor Operation Using Thermococcus onnurineus NA1 Strains Nulee JANG, Muhammad YASIN, Yeseul JEONG, Shinyoung PARK, Yeubin LEE, In Seop CHANG, Hyunsoo KANG School of Environmental Science of Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 500-712, Korea Hydrogen (H2) is recognized as an outstanding energy resource because of the higher energy density and no greenhouse gas emissions. Carbon monoxide (CO) which is a key component of synthesis gas is used as a substrate by Thermococcus onnurineus NA1, a hyper-thermophilic archaeon. This strain has a significant potential to produce bio-hydrogen. However, it is important to determine the optimal dissolved CO concentration in the medium due the fact that CO is a non-competitive inhibitor and reduces the specific CO consumption rates The purpose of this research was to determine and compare the intrinsic kinetic parameters (KS, Ki) of Thermococcus onnurineus NA1 strains (Wild type, mutants MC01, MC02 and WTC156T), using substrate inhibition kinetic model by Andrews. KS and Ki values were used to find the optimal dissolved CO concentration. The results showed the maximum specific CO consumption rates of WT, MC01, MC02 and WTC156T can be achieved by maintaining the dissolved CO concentrations at 0.56, 0.52, 0.58 and 0.75 mM respectively. WTC156T has shown the highest CO consumption rate in these strains. Optimal reactor conditions were predicted using kinetic modeling. Keywords : Thermococcus onnurineus NA1, Carbon monoxide, hydrogen, Archaea, Synthesis gas References 1. Bae, S. S., T. W. Kim, et al. H2 production from CO, formate or starch using the hyperthermophilic archaeon, Thermococcus onnurineus, Biotechnology letters 34(1), 75-79 (2012) 2. Kim MS, Bae SS, Kim YJ, Kim TW, Lim JK, Lee SH, Choi AR, Jeon JH, Lee JH, Lee HS, Kang SG. CO- dependent H2 production by genetically engineered Thermococcus onnurineus NA1. Appl Environ. Microbiol. 79(6), 2048-53 (2013) 3. Andrews JF. A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates. Biotechnol. Bioeng. 10(6), 707-23 (1968) P0907 Acute Ecotoxicity of Plant Extract Made by Cinnamomum japonicum and Camptotheca acuminata Using Daphnia Magna In Taek PARK, Seung Hun JEON, Je Jong LEE, Bo A KIM, An Jin WI, Seok Bong YOO, Whoa Shig PARK, Bong Sup SHIM Forest Resources Research Institute, 7 Dado-ro, Sanpo-myeon, Naju city, Jeonnam province 58213, Korea Recently, Development of Environmentally-friendly pesticide has been actively. It is because Agricultural certification with low pesticides will be excluded from environment-friendly agricultural products. This study was to evaluate ecotoxicity of plant extract, prior to the commercialization of materials extracted from Cinnamomum japonicum and Camptothca acuminata by Ethanol solvent, performed using the water flea(Daphnia Magna). The assessment was that EC50 of Cinnamomum japonicum extract after 48 hours is 74.8 ppm, and Camtotheca acuminata extract is 37.1 ppm. These result show that Camtotheca acuminata extract toxicity was stronger than Cinnamomum japonicum extract. In other words, Cinnamomum japonicum extract was more safety in natural ecosystems either directly or indirectly, and has more high commercialization possibility of environment-friendly control product. Keywords : : Acute ecotoxicity, Daphnia Magna, Cinnamomum japonicum, Camtotheca acuminate References 1. Organization for Economic Cooperation and Development(OECD) OECD guideline for the testing of chemicals: fish embryo toxicity(FET) test. 2006[cited 2014 Jun 20]. Available from: http://www.oecd.org/chemiacalsafety/testing/36817070.pdf. 2. Yim EC, Kim HJ, Kim SJ.Acute toxicity assessment of camphor in biopesticides by using Daphnia Magna and Danio rerio. Environ Health Toxicol. 2014;29: e2014008. P0908 Antifungal Activity of Extracts from Korean Plant Species against Causative Pathogens of Prunus serrulata Seung Hun JEON, Bo A KIM, Je Jong LEE, In Taek PARK, Seok Bong YOO, Bong Sup SHIM, An Jin WI, Whoa Shig PARK Forest Resources Research Institute, 7 Dado-ro, Sanpo-myeon, Naju city, Jeonnam province 58213, Korea In this study, to search a natural antifungal compounds, the substances from extracts of seeds of Zanthoxylum ailanthoides and leaves of Camptotheca acuminata which grows naturally in Korea were extracted by two different solvents. Findings made in this study are expected to provide effective extract method to prevent the infection of canker and gray mold. Botryosphaeria dothidea and Botrytis cinerea are to cause the canker and gray mold of Prunus serrulata, respectively. Canker forms yellowing leaves and death of some twigs on P. serrulata. Gray mold forms water soaked spots at leaves on it. B. dothidea and B. cinerea in P. serrulata were measured by agar diffusion method. Z. ailanthoides seeds and C. acuminata leaves were extracted by two solvents which were water and ethanol. Using paper disc method, water extract of seeds of Z. ailanthoides showed an antifungal effect against B. cinerea more than B. dothidea. Then water extract of leaves of C. acuminata rarely indicated an antifungal activity against these fungi. However, the others did not have fungicidal effects against these pathogens. From these results, water extract of seeds of Z. ailanthoides have antifungal activity components. It is suggested that extract of Z. ailanthoides seeds has high possibility to use as a fungicidal material. Also, Z. ailanthoides and C. acuminata growing naturally in Korea are invaluable to develop as a natural antifungal material after isolation and identification. Keywords : Fungicide, Zanthoxylum ailanthoides, Camptotheca acuminata, Prunus serrulata References 1. D. G. Lee, K. Choi and S. H. Lee, GC/MS analysis of volatile constituents from woody plants. CNU Journal of Agricultural Science. Vol. 38, No. 4: 723-730 (2011). 2. J. I. Cho and J. Y. Cho, Isolation of Antifungal Bacterial Strain Bacillus sp. Against Gray Mold infected in Kiwi Fruits and its Disease Control. Korean Journal of Organic Agriculture. Vol. 14, No. 4: 399-410 (2006). P0909 Analysis of Taxifolin Contents under Different Methods of Poria cocos Cultivation Je Jong LEE, Seung Hun JEON, In Taek PARK, Bo A KIM, An Jin WI, Seok Bong YOO, Whoa Shig PARK, Bong Sup SHIM Forest Resources Research Institute, 7 Dado-ro, Sanpo-myeon, Naju city, Jeonnam province 58213, Korea Poria cocos were cultivated using ground cultivation method and underground cultivation method. Cultivation of P. cocos was used Pinus densiflora, Antipathes japonica Brook and Pinus rigida Mill. Taxifolin known as immunity strengthening substances are contained in large amounts in the bark of pines. The ingredients contained in bark of pines cultivation process set a hypothesis that can be absorbed. Cultivated P. cocos was extracted solvent after drying. Pre-treated extract was analyzed HPLC. As a result, the ground cultivated pine P. cocos were confirmed to be contained taxifolin 3.92 ppm. Bark of P. cocos cultivation using pine, Antipathes japonica of each 134.23, 36.78 ppm. Keywords : Poria cocos, cultivation, taxifolin References 1. O. B. Choi, D. B. Cho and D. P. Kim, The components of cultivated Poria cocos, Korean J. Food & Nutr., vol. 9, No. 4, 438 ~ 440 (1996). P0910 Moment Analysis of Lysozyme in Cation Exchange-High Performance Liquid Chromatography (CE-HPLC) KwanYoung KO, InHo KIM Dept. of Chemical Engineering, Chungnam National University, Daejeon, 34134 Parameters of lysozyme separation in CE-HPLC (WCX-300 HENENCHEM) were calculated by moment analysis from experiment data. 30 mM sodium phosphate buffer (pH 9) was used for equilibrium. The buffer containing NaCl (0.5, 0.75, 1.0 M) eluted lysozyme. Concentrations of lysozyme were in the range of 3, 5, and 7 mg/ml. The column is with the size of 4.6*250 mm. Flow velocity was varied from 0.5 to 1.5 mL/min. Moment analysis was conducted using GR (general rate) model. Equilibrium constants (K) read to be 13.8, 12.8, and 11.9 for 0.5, 0.75 and 1.0 M salt. After the molecular diffusivity was obtained by using Wilke-Chang equation, the axial dispersion coefficient was calculated as 4.22 to 10.29 . The external mass transfer coefficients were obtained as 0.131, 0.128, and 0.125 by using Wilson-Geankoplis equation. The intra-particle diffusivity was calculated by comparing theoretical plate number and van Deemter equation. The values were from 1.74 to 8.56 . Influence on overall Htotal by each mass transfer phenomenon was also determined by calculating Hax, Hf, and Hd. Keywords : Moment Analysis, Lysozyme, General Rate Model, Parameter Estimation References 1. D. Y. Choi and K. H. Row, J. Ind. Eng. Chem., 10, 1052 (2004). 2. K. Miyabe, J. Sep. Sci., 32, 757 (2009). 3. K. Miyabe and R. Isogai, J. Chromatotgraphy A., 1218, 6639 (2011). P0911 Stand-Alone Hollow Fiber Membrane Bioreactor (HFMBR) Configurations for Microbial Syngas Utilization: Opportunities and Challenges Muhammad YASIN, Shinyoung PARK, Yeseul JEONG, Nulee JANG, In Seop CHANG School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea Microbial conversion of syngas is a potential technology for the production of biofuels and biochemicals, using coal, lignocellulosic biomass and waste gases from iron works. However, poor gas-liquid mass transfer (GL-MT) limits the scaleup of the technology. The use of hollow fiber membranes (HFMs) has made it possible to achieve a high GL-MT efficiency and an increase in the CO mass transfer rate above the maintenance requirement has resulted in increased cell concentrations (X). Therefore, the further development of HFMBRs has the potential to achieve high X and rapid GL-MT. Indeed, a simple HFMBR capable of producing a high GL-MT without requiring pumping and agitation would be economical at a larger scale. We developed a high GL-MT, HFMBR system, using microporous polyvinyl fluoride (PVDF) -1 HFMs. High CO volumetric mass transfer coefficient (kLa) of 155.16 h was achieved without using pumps and stirrers. Finally, CO/CO2 fermentation was conducted using Eubacterium limosum KIST612, to assess the practicality of the HFMBR system under biotic conditions. Lower residence time of the substrate gases in the fermentation medium is a potential drawback that needs to be addressed. Keywords : HFMBR, Syngas, Carbon monoxide References 1. Munasinghe, P.C., Khanal, S.K. Syngas fermentation to biofuel: evaluation of carbon monoxide mass transfer and analytical modeling using a composite hollow fiber (CHF) membrane bioreactor. Bioresour. Technol. 122, 130– 136 (2012) P0912 Selection of Technical Vocabularies for Teaching of Biotechnology and Bioprocess Engineering Chang-Ho PARK Dept. of Chemical Engineering, Kyung Hee University, Yongin-si, 446-701, South Korea Biotechnology is a hi-tech area requiring multidisciplinary education and research. This study selected keywords for teaching in biotechnology and bioprocess engineering. In order to extract keywords more efficiently a new method of word selection was developed which takes advantage of information in Google Books site. 533 words families were grouped in a list designated as Biotechnology Word List (BTWL). The words in the BTWL were compared with Coxhead (2000)’s Academic Vocabulary List (the AWL) and Wang et al. (2008)’s Medical Academic Word List (the MAWL). The BTWL contained 160 academic word families overlapping with the MAWL. Especially, 68 word families in the BTWL overlapped with both the AWL and the MAWL, and these are the core academic vocabulary in biotechnology. Ninety two words (32.7%) of the 281 words overlapped with the BTWL. These 92 words, even though not included in the AWL, are especially important academic vocabulary for students in the biotechnology field. Two third of words in the BTWL were technical vocabulary to be educated in biotechnology. Keywords : Biotechnology terms, Technical vocabulary, Academic vocabulary, BTWL References 1. Coxhead, A. (2000), A New Academic Word List, TESOL Quarterly, 34, 213-338. 2. Wang, J., Liang, S.-L. & Ge, G.-C. (2008), Establishment of Medical Academic Word List, English for Specific Purposes, 27, 442-458. P0913 High Production of 2,3-Butaniedol from Glycerol Using Raoutlella ornithinolytica B6 without 1,3-Propanediol Formation Taeyeon KIM1,2, Sukhyeong CHO2, Han Min WOO2, Jin-Ho SEO1, Youngsoon UM2 11Interdisciplinary program in agriculture biotechnology, Collage of Agriculture and Life Science, Seoul National University, Seoul 151-742, 22Clean Energy Center, Korea Institute of Science and Technology, Seoul 136-791 Although glycerol is an attractive feedstock for biochemical production, a high production of 2,3-butanediol (2,3-BD) from glycerol has been hampered because of a considerable 1,3-propanediol (1,3-PD) formation [1]. In this study, we report the high production of 2,3-BD from glycerol using Raoultella ornithinolytica B6 without 1,3-PD. To optimize of fermentation condition for 2,3-BD production by R. ornithinolytica B6 from glycerol, the influences of agitation speeds were investigated. In the fed-batch fermentation using glycerol without pH control, the fianl 2,3-BD concentration increased with an agitation speeed (23.8 g/L at 200 rpm, 23.3 g/L at 300 rpm, and 36.4 g/L at 400 rpm) and 2,3-BD productivity also increased (0.22 g/L/h at 200 rpm, 0.47 g/L/h at 300 rpm, 0.74 g/L/h at 400 rpm). Further improvement of 2,3-BD production was achieved by pH control. The pH decreased from pH7.0 to 5.5 during 9 hours of fermentation, and then the pH was maintained above 5.5 with 5 N KOH. Finally, 60.7 g/L 2,3-BD was obtained with 0.85 g/L/h of productivity from glycerol. This is the first report on 2,3-BD production from glycerol by Raoultella genus. Keywords : glycerol, fed-batch fermentation, Raoultella, 2,3-Butanediol References 1. Xiu, Z. L. and A. P. Zeng, Appl. Microbiol. Biotechnol. 78(6), 917 (2008) P0914 Bioreactor Studies for Enhanced Production of cis-cis Muconic Acid with Recombinant Escherichia coli Cells Seong-Ryeoul SEO1, Sun-Ok PARK1, Eung-Soo KIM2, Sang-Jong LEE1, Do-Hoon LEE3, Sang-Young KIM3, Gie-Taek CHUN4 1STR Biotech. Ltd., Chunchon, South Korea, 2Inha University, Inchon, South Korea, 3Korea Institute of Industrial Technology, Chonan, south Korea, 4College of Biomedical Science, Kangwon National University, Chunchon, South Korea Cis-cis muconic acid (MA) utilized as a raw material for new functional resins, pharmaceuticals and agrichemicals is an unsaturated dicarboxylic acid with six carbon atoms. MA was revealed to be easily converted to adipic acid, a valuable commodity chemical for bio-plastics including nylon 6-6 and polyurethane and polyethylene terephthalate (PET). For development of MA high-yielding recombinant Escherichia coli cells, three additional genes from various sources (i.e., both aroZ gene encoding DHS dehydratase from and aroY gene encoding PCA decarboxylase from K. pneumonia, and catA gene encoding catechol 1,2-dioxygenase from A. calcoaceticus) were introduced into aromatic amino acids auxotrophic mutants by use of plasmid expression vectors with various strong promoters. As a first step to enhanced production of MA by the newly-developed E. coli cells, induction strategy was set up at 5L bioreactor level through determination of inducer kind & amount, induction time and inducing medium composition. Especially, intensive studies were conducted to develop an efficient induction method for overcoming catabolite repression phenomenon caused by high level of residual glucose. Notable was the positive role of glycerol added in the production medium, since it contributed to remarkable enhancement in the MA-biosynthetic capability of the recombinant without causing catabolite repression. In addition, fermentation conditions such as agitation speed, dissolved oxygen level and pH-control were also investigated using the 5L stirred tank bioreactors. Fermentation results from the batch and fed-batch bioreactor operations performed with the recombinant E. coli cells will be presented in this paper as well. Keywords : muconic acid, fed-batch, Escherichia coli References 1. Erik W, Anne K, Diana H, Thomas W, Klaus. V., 2012, Appl Microbiol Biotechnol 93:1057–1063 P0915 Bioreactor Studies for Mass Production of cis-cis Muconic Acid Using High-Yielding Recombinant Corynebacterium glutamicum Cells Min-Ji KIM1, Soo-Yeon KIM1, Yun-Hee KIM2, Sun-Ok PARK2, Woo-Shik SHIN3, Eung-Soo KIM4, Sang-Jong LEE2, Do- Hoon LEE3, Sang-Young KIM3, Gie-Taek CHUN1 1College of Biomedical Science, Kangwon National University, Chunchon, Kangwon-do 24341, South Korea, 2STR Biotech. Ltd., Chunchon, Kangwon-do 24232, South Korea, 3Korea Institute of Industrial Technology, Chonan, Chungcheongnam-do 31056, South Korea, 4Inha University, Inchon 22212, South Korea Cis-cis muconic acid (MA), an unsaturated dicarboxylic acid with six carbon atoms was reported to be easily converted to adipic acid, a valuable commodity chemical for bio-plastics including nylon 6-6 and polyurethane and polyethylene terephthalate (PET) [1]. As a first step to mass production of MA, medium optimization was carried out in order to find optimal compositions of both growth- and production-medium. For this purpose, efficient statistical methods were adopted, such as full factorial design (FFD), steepest ascent methods (SAM) followed by response surface methods (RSM). In bioreactor fermentations, C/N ratio of the production medium was observed to have significant influences on the MA productivity of the high-yielding recombinant cells of C. glutamicum. Notably, Status of the inoculated cells (i.e., inoculum age and inoculum amount) transferred to the final production fermentor were revealed to have profound effects on the biosynthetic capability of the producing cells. In addition, physiochemical fermentation conditions such as dissolved oxygen level and pH-control were intensively investigated using 5L stirred tank bioreactors. As a result, approximately 10 fold increase in MA production level was obtained in the 5L bioreactor cultures performed under the optimized conditions, as compared to the shake-flask fermentations. Also, For inhibition of cell growth and over production of MA, vegetable oil was added in production medium for SAM with high concentration of C-source and investigated various vegetable oil concentration. It was found that about 3ml/L of vegetable oil have effect on different production medium than production medium for SAM with low concentration of C-source(called SLP). Keywords : cis-cis munonic acid, recombinant Corynebacterium glutamicum, medium optimization References 1. Hal S. Alper, Metabolic engineering of muconic acid production in Saccharomyces cerevisiae(2013), Metabolic engineering, Vol 15, 55-66 P0916 Acid Extraction of Chlorella pyrenoidosa for 2,3-Butandiol Production by Enterobacter aerogenes Ja Hyun LEE1, Han Suk CHOI1, Dong Sup KIM1, Ju Hun LEE1, Soo Kweon LEE1, Chulhwan PARK2, Seung Wook KIM1 1Department of Chemical and Biological Engineering, Korea University, Seoul, Korea, 2Department of Chemical Engineering, Kwangwoon University, Seoul, Korea This study aimed to utilize Chlorella pyrenoidosa as algal biomass for acid-thermal hydrolysis of fermentable sugars via hydrochloric acids, which were employed as substrate in 2,3-butandiol conversion. To enhance the reducing sugar, concentration of acid and the solid/liquid ratio was investigated under the range of 0.5~10% and 20~140 g/L and was optimized to 2% and 100 g/L, respectively. The maximum hydrolysis efficiency from C. pyrenoiudosa was achieved 96% with the optimal conditions. In addition, the reducing sugars obtained from this acid hydrolysis were employed as algal feedstock of 2,3-BDO fermentation by Enterobacter aerogenes ATCC 29007. Finally, the reducing sugars from C. pyrenoiudosa was utilized as carbon source of 2,3-BDO fermentation and the 2,3-BDO yield was about 99%. Keywords : Algae, Hydrolysis, Reducing sugar, 2,3-Butandiol References 1. Harun, R., Danquah, M.K., Influence of acid pre-treatment on microalgal biomass for bioethanol production (2011), Process Biochem., 46, 304–309 P0917 Improved Ethanol Production by Casein Hydrolysate with Optimization of Fermentation Media for Enterobacter aerogenes ATCC 29007 Ju Hun LEE, Ja Hyun LEE, Han Suk CHOI, Dong Sup KIM, Soo Kweon LEE, Seung Wook KIM Department of Chemical and Biological Engineering, Korea University, 145, Anam-Ro, Seongbuk-Gu, Seoul, 136-701, Korea In recent years, there has been a remarkable increase in the biodiesel production. Also, crude glycerol which is by-product of the biodiesel was rapidly increased in quantity. In this study, ethanol production from glycerol by Enterobacter aerogenes ATCC29007 was carried out under micro aerobic culture condition. Also, the effect of additional source of casein hydrolysate during cultivation was investigated for improved ethanol production. The enhanced ethanol production by casein hydrolysate was identified by expression level of alcohol dehydrogenase (adhE). In addition, the optimization of main media including casein hydrolysate for ethanol production was performed by response surface method (RSM). The major factors for RSM with the optimized values were as follows: 9 g/L peptone, 5.9 g/L (NH4)SO2, 14.12 g/L casein hydrolysate and 37 g/L glycerol. The fermentation conditions were performed at initial pH of 6, 37oC, and 180 rpm for 24 h. Finally, the ethanol concentration was about 0.89 yield under optimized conditions. Keywords : Ethanol, Media optimization, Gene expression level References 1. S. J. Lee, S. B. Kim, S. W. Kang, S. O. Han, C. Park and S. W. Kim, Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes. (2012), Bioprocess Biosystems Engineering, 35, 85-92 2. S. B. Kim, J. H. Lee, K. K. Oh, S. J. Lee, J. Y. Lee, J. S. Kim, and S. W. Kim, Dilute acid pretreatment of barley straw and its saccharification and fermentation. (2011), Biotechnology and Bioprocess Engineering, 16, 725–732 P0918 Pseudo-eukaryotic Nucleus System for mRNA Transcription Seung Won SHIN1, Soong Ho UM1,2 1School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, South Korea, 2SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, South Korea Cell metabolism is a highly functionalized and tightly networked process. Cellular compartments, so called organelles, such as mitochondria, chloroplasts, cell nucleus, etc. are highly optimized to their specific functions of own, and cross-works among them can provide complex cellular metabolism regulation. Whilst there are various kinds of compartments with each of their own specialized function, they all share in common that proteins are used as a major fundamental biological molecule. Information used to synthesize and regulate proteins originates from genes, which are sequences of nucleotides that comprnise DNA, encoding proteins or functional RNAs. Thus, the organelles, such as nucleus, mitochondria and chloroplast, which contain genetic materials have a significant role in cellular regulations. The importance of this mechanism, so-called the central dogma, can be applied to metabolism-mimicking synthetic cell designs in the same context. [1] In this study, we created a novel cell-inhalable pseudo-eukaryote-nucleus (PEN) that can produce mRNA transcripts for protein synthesis. PENs consisted of a linearized plasmid, in which genes for proteins are encoded, and a DNA crosslinker. As a DNA crosslinker, X-shaped DNA nanostructure (X-DNA) was used. These two components come together to form a gene- containing DNA hydrogel structure by T4 DNA ligase enzymatic crosslinkage. We employed closed envelopes in which a synthetic nucleus was generated simultaneously with the ability to produce mRNA transcripts. It is speculated that this new synthetic cell mimicry could be a substitute of natural gene encoder for the application of gene-relevant technologies. Keywords : RNA transcription, Synthetic nucleus, DNA hydrogel References 1. Pier L. Luisi, Pasquale Stano. Synthetic biology: Minimal cell mimicry. Nat. Chem. 3, 755-756 (2011) P0919 Enzymatic Hydrolysis of Spent Coffee Ground for Manno-Oligosaccharides Production Hah Young YOO, Soo Kweon LEE, Ja Hyun LEE, Dong Sup KIM, Ju Hun LEE, Han Suk CHOI, Seung Wook KIM Department of Chemical and Biological Engineering, Korea University, 145, Anam-Ro, Seongbuk-Gu, Seoul, 136-701, Korea Spent coffee grounds (SCG) which is the residual materials obtained from Café or instant coffee industry have been remarked at the integrated biorefineries due to their high levels of heterogeneous polysaccharides content.In our previous work, an extracellular β-mannanase from Streptomyces sp. was purified and the biochemical characterization was investigated. In this study, β-mannanase and cellulase were applied in enzymatic hydrolysis of SCG for manno- oligosaccharides production. The carbohydrates content in SCG were determined by HPLC (10.8% glucan, 21.7% mannan, 8.2% galactan and 3.1% arabinan). The conversion of reducing sugars by β-mannanase (200 U) was about 20% at 50 ºC for 72 h. Finally, the conversion of reducing sugars was about 70% by β-mannanase (200 U) with cellulase (60 FPU). Keywords : Spent coffee grounds, β-mannanase, manno-oligosaccharides, Enzymatic hydrolysis References 1. Hah Young Yoo, G. C. Pradeep, Seung Wook Kim, Don Hee Park, Yun Hee Choi, Joo Won Suh, Jin Cheol Yoo, A novel low-molecular weight alkaline mannanase from Streptomyces tendae (2015), Biotechnology and Bioprocess Engineering, 20(3), 453-461 P0920 Optimization of Alcohol Precipitation and Vacuum Distillation for 2,3-Butanediol Recovery from Fermentation Broth Hee-Geun NAM, Sangjun JEON, Hyohak SONG Research and Development Center, GS Caltex Corporation, Daejeon, 34122 Alcohol precipitation and vacuum distillation were previously suggested to recover 2,3-Butanediol(2,3-BD) from fermentation broth.[1] Although 2,3-BD of high purity can be effectively recovered with this method, the recovery ratio still needs to be improved. Based on mass balance analysis on the whole process, 2,3-BD loss turned out to be occurred mainly through cake waste in precipitation process and heavy residue of distillation process. The waste cake was washed with alcohol and the optimal amount of alcohol was determined by experiment. Because heavy residue from distillation can be easily hardened at low 2,3-BD composition, thus possibly leading operational problems in pipe and pump, the discharge condition of heavy residue was firstly confirmed based on its viscosity characteristics before optimizing recovery ratio. It was decided that about 20% of 2,3-BD among the distillation feed stream should be discharged with heavy residue. In order to recover this 2,3-BD from heavy residue stream, heavy residue was recycled to alcohol precipitation procedure and mixed with concentrated broth and then treated with alcohol again. With these strategies, the recovery ratio of whole process could attain > 90% at purity of 95 wt%. The optimized process condition and scheme can be applied to large scale process for industrial production with proper cake dehydration equipment and energy efficient evaporator. [This work was supported by the Industrial Strategic Technology Development Program (No. 10050407) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).] Keywords : 2,3-Butanediol, Recovery, Alcohol precipitation, Vacuum Distillation References 1. S. Jeon, D. Kim, H. Song, H. J. Lee, S. Park, D. Seung, and Y. K. Chang, J. Biosci. Bioeng. 117, 464 (2014) P0921 Reaction Process Optimization on Production of 16-Hydroxy Palmitic Acid Beom Gi PARK, Eunok JUNG School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea 16-Hydroxy palmitic acid (16-HPA) is one of a valuable chemicals as a key component of adhesives, lubricants, cosmetic intermediates and potential anticancer agents. When it comes to cosmetic intermediates, 16-HPA is known as a precursor of pseudo-ceramide. Pseudo-ceramides is a well-known material for cosmetics because of its water-retaining property. In this work, we used a novel CYP153, well-known ω-hydroxylase of linear fatty acids and alkanes with high region-selectivity. For an over-production of 16-HPA, various approaches can be applied such as metabolic engineering, synthetic biological approaches, protein engineering and bioprocess optimization. A main theme of this work is focused on bioprocess optimization of 16-HPA production. Keywords : CYP153, hydroxy fatty acid, bioprocess, hydroxylase References 1. Bae et al.,fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity, Appl. Microbiol. Biotechnol., 98(21), 8917-8925 P0922 Antibody-Mimicking Peptide for Human Myeloperoxidase from Phage Display Technique Soi YUN, Eun Kyu LEE Dept. of Bionanotechnology, Hanyang University-ERICA, Ansan, Korea 15588 Because whole antibody is expensive and delicate to use for immune-binding applications, peptidesthat can mimic the binding characteristics of an antibody are used as an alternative [1]. Phage display is a technology that can identify a phage that displays a foreign peptide on its surface with exterior molecules/materials interaction. Biopanning, an in vitro selection process, is used to screen and identify the peptide towards a target protein. Our target protein was human myeloperoxidase (hMPO), a biomarker for acute cardiac diseases [2]. In this study,we tried to find anti-hMPO antibody mimicking peptides. From five rounds of biopanning, four phage clones (P2, P3, P5, and P9) showing specific binding affinity to hMPO were identified by ELISA. Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Using ELISA and quartz crystal microbalance (QCM), the immune binding affinity of the synthesized peptides was compared with that of the native antibody [3]. Our results indicate that the anti-hMPO antibody mimicking peptides from phage display technique can be used for immunodiagnostics applications. Keywords : Phage display, human myeloperoxidase, MPO, QCM References 1. T. B. D.Hien et al., J. Biotechnology, 161, 213– 220 (2012). 2. D. Tousouliset al., Curr Med Chem, 19, 2572-2587(2012). 3. M. Bianco et al., Biosensors Bioelectronics, 42, 646–652 (2013). P0923 Supercritical Carbon Dioxide Inactivation of Candida albicans Biofilms Jungwoo YANG1, Hyong Seok PARK2, Hee Jung CHOI2, Kyoung Heon KIM1 1Department of Biotechnology, Graduate School, Korea University, Seoul 136-713, 2Division of Infectious Diseases, Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul 158-710 An alternative sterilization method using supercritical carbon dioxide (SC-CO2) was tested on Candida albicans biofilms. The effect of varying pressure, temperature, and treatment time on the inactivation of C. albicans spores in suspensions and in biofilms were examined. The parameters that led to the complete inactivation of C. albicans biofilms ranged between of 5–20 min, 100–200 bar, and 35–45°C, respectively. Notably, treatment of SC-CO2 at either 100 bar and 40°C or 200 bar and 30°C induced complete inactivation of spores within 5 min. Furthermore, it was found that wet biofilm (0.4%, w/w) had higher sensitivity to SC-CO2 than dried biofilm. Finally, spore inactivation was confirmed by confocal laser scanning microscopy analysis. In this study, the use of a low-temperature SC-CO2 sterilization method was proven to be effective in fungal biofilm inactivation, and the extent of SC-CO2 penetration into the cell membrane was revealed to be the key factor for biofilm inactivation Keywords : Sterilization, Supercritical carbon dioxide, Candida albicans, Biofilm References 1. Park HS et al. Effective inactivation of Candida albicans biofilms by using supercritical carbon dioxide (2015). Bioprocess and Biosystems Engineering 38(9), 1731-1737 P0924 Design of Algicidal Peptides aganist Anti-Harmful Algal Blooms Nam-Hong KIM1, Young-Min KIM1,2, Eun-Ji KIM1, Jae-Eun JEONG1, Yung-Hoon PARK1, Seong-Cheol PARK1, Mi- Kyeong JANG1 1Department of Polymer Science and Engineering, College of Engineering, Sunchon National University, Jeonnam, 540-950, Korea, 2Department of KOREA TESTING & RESEARCH INSTITUTE 13, Yulchonsandan 4-ro, Haeryong-myeon, Suncheon-si, Jeollanam-do, 540-856, Korea Harmful algal blooms (HABs), that cause red tide in marine water system, triggered tremendous economic losses and serious destruction of marine ecosystems and human health. Although previous reports for controlling and preventing HABs have been proposed in treatments of anti-HAB materials and constructions of mechanical systems, the research for anti-HAB mechanisms was limited. The aim of the present study was to understand the mode of algicidal action with synthetic peptides via substitutions of amino acid. We found that the balance between cationicity and hydrophobicity was a factor for designation of anti-HAB material without cytotoxicity in marine organisms. Furthermore, anti-HABs bioassay in scallop indicated that synthetic algicidal peptide is able to control HABs due to direct disrupting cell and chloroplast membrane. Keywords : Algicidal Peptide, Harmful algal blooms, Red tide P0925 Which One is the Optimal Extraction Solvent for the Global Metabolite Profiling of Yeast Il Sub CHOI, Sooah KIM, Kyoung Heon KIM Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 Saccharomyces cerevisiae is one of the most important industrial strains for producing ethanol and valuable bioproducts. Up to now, metabolomics, which is the study of global changes of metabolome in a living organism at a specific time point, has been used to understand metabolisms and regulations related to ethanol and bioproducts production. To obtain meaningful metabolomics data, well optimized metabolome sample preparation is required. Especially, determination of extraction solvents is important for high recovery of metabolites. In this study, for metabolite profiles of S. cerevisiae by using gas chromatography/time-of-flight mass spectrometry (GC/TOF MS), we evaluated extraction solvents including pure methanol, acetonitrile/water mixture, acetonitrile/methanol/water mixture, and boiling ethanol based on various statistical analyses. Among these extraction solvents, acetonitrile/water mixture(1:1, v/v) at −20 °C was found to be a best extraction solvent for metabolite profiles of S. cerevisiae. These results could be applied as standard protocol of metabolome sample preparation for metabolomics or metabolic engineering of yeast. Keywords : Extraction solvent, GC/TOF MS, Metabolite profiling, Metabolomics References 1. Kim, S et al. Evaluation and optimization of metabolome sample preparation methods for Saccharomyces cerevisiae (2013) Analytical chemistry, 85, 2169-2176 P0926 Cold Methanol Quenching vs. Fast Filtration for the Global Metabolite Profiling of Saccharomyces cerevisiae Ju Hwan SONG, Sooah KIM, Kyoung Heon KIM Dept of Biotechnology, Graduate school, Korea University, Seoul 136-713 Saccharomyces cerevisiae produces various metabolites such as primary and secondary metabolites. The abundance of cellular metabolites can represent biological and environmental change of cell. Therefore, metabolomics can be applied as a tool in metabolic and physiological studies of the yeast. To obtain reproducible and reliable data from metabolomics, accurate sample preparation methods should be established. However, there are no studies on the systematic evaluation of quenching methods on metabolite profiles of S. cerevisiae. In this study, we evaluated sampling procedures for S. cerevisiae using gas chromatography-time-of-flight mass spectrometry (GC-TOF MS). A total of 110 metabolites of S. cerevisiae including amines, amino acids, fatty acids, organic acids, phosphate and sugars were identified. The cold methanol quenching, which is the most frequently used quenching method, showed significant losses of intracellular metabolites caused by severe cell membrane damage of yeast. Thus, to prevent the metabolite losses, we suggest the fast filtration method as an alternative procedure which is suitable for the quenching method for yeast. Keywords : Metabolite profiling, GC/TOF MS, Metabolomics References 1. Kim, S et al. Evaluation and Optimization of Metabolome Sample Preparation (2013), Analytical chemistry, 85, 2169-2176 P0927 Biotransformation of 12-Keto Oleic Acid into Ester by Recombinant Corynebacterium glutamicum-Based Biocatalyst Hyeonsoo KIM, Jinwon LEE Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, Republic of Korea Fatty acids of vegetable and animal origin (e.g., oleic acid, ricinoleic acid) have been the most prominent renewable feedstock of chemical industry in the past and in the present. The chemical conversion processes have critical demerits. Typical example is environmental pollution, unwelcomed and dangerous byproduct spun off from chemical process. Therefore expectation for eco-friendly bioprocess using renewable feedstock is rising. The purpose of this study is to produce ester from ricinoleic acid in Corynebacterium glutamicum, which has been used for the industrial production of various amino acids (e.g., lysine, glutamic acid) via whole-cell bioconversion. A recombinant Corynebacterium glutamicum ATCC13032 express a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440. The BVMO were inserted into the E. coli-C. glutamicum shuttle expression vector through In-fusion PCR cloning technology. Product analysis was determined by using GC/MS. Keywords : Biocatalysts, Biotransformation, Corynebacterium glutamicum References 1. Sung Sun Yim et. al., Isolation of Fully Synthetic Promoters for High-Level Gene Expression in Corynebacterium glutamicum (2013), Biotechnology and Bioengineering, Vol. 110, No. 11, November 2. Ji-Won Song, et al., Multistep Enzymatic Synthesis of Long-Chain a,w-Dicarboxylic and ω-Hydroxycarboxylic Acids from Renewable Fatty Acids and Plant Oils(2013), Angew. Chem. Int. Ed. , 52, 2534 –2537 3. Byeonghun Lee, Saebom Lee, Hyeonsoo Kim, Kijun Jeong, Jinbyung Park, Kyungmoon, Park, and Jinwon Lee Whole cell bioconversion of ricinoleic acid to 12-ketooleic acid by recombinant Corynebacterium glutamicum- based biocatalyst. Journal of Microbiology and Biotechnology Volume 37, Issue 5 (2015), Page 1101-1106 P0928 Enhancement of Polyhydroxyalkanoates Production from Gelidium amansii by Batch and Fed- batch Cultivation of Bacillus megaterium KCTC 2194 Bassam ALKOTAINI, Beom Soo KIM Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea Polyhydroxyalkanoates (PHAs) are linear polyesters synthesized by microbial fermentation of various substrates. PHAs are accumulated in the microbial cell in order to store carbon and energy for future use. In the present work, the pretreated seaweed, Gelidium amansii, was used to produce PHAs via batch and fed-batch cultivation of Bacillus megaterium KCTC 2194 in 2.5 L stirred fermentor. Cultivation conditions were: 10 g/L initial sugar concentration, 1 g/L ammonium sulfate, 30°C, pH 7.0, 300 rpm, and aeration rate of 1.0 vvm. In batch culture at constant pH, the strain was able to produce PHAs up to 51.4% of 5.5 g/L dry cell weight (DCW). The biomass was enhanced by fed-batch cultivation using two feeding strategies, pH-stat and intermittent feeding. When pH-stat was employed, the DCW was 8.2 g/L with 53.2% accumulated PHAs. Meanwhile, the intermittent feeding strategy showed the highest DCW of 10.1 g/L accompanied with a slightly increase of the accumulated PHAs up to 54.5%. Keywords : Polyhydroxyalkanoates , PHAs, Gelidium amansii, Bacillus megaterium, batch and fed batch cultivation P0929 Extraction and Isolation of Theasaponin E1 from Green Tea Seeds and Pharmacological Investigation of Antiangiogenic, Anticancer and Anti-Hyperlipidemic Activities. Jong Deog KIM1,2, Muhammad Imran KHAN1, Moon Geon LEE1, Hyo Jin SEO1, Jin Hyuk SHIN1 1Department of Biotechnology, ChonnamNatational University, San96-1, Dun-Duk Dong, Yeosu, Chonnam, 550-749, Korea,, 2Research center on Anti-Obesity and Health Care, Chonnam National University, San96-1, Dun-Duk Dong, Yosu, Chonnam, 550-749, Korea. Camellia sinensis (Green tea) is a potential rich source of many biologically active constituents. Saponins from green tea have pharmacologically great importance due to their multisided therapeutic effects. Among all tea saponins Theasaponin E1 (TSE1) is considered to be the most active with high therapeutic property. Present work is a good attempt towards the investigation of TSE1 as a good antiangiogenic and anti-hyperlipidemic agent. The activeness of TSE1 against angiogenesis correlated with tumor and cancer progression was determined using human umbilical vein endothelial cells (HUVECs). Different concentrations of TSE1 was used and 10 μg/mL was found most effective in controlling tumor metastasis by inhibition of angiogenesis in HUVECs. The molecular mechanism involves the inhibition of VEGFR-2, β-catenin and PI3-K to NF-kB to stop the activation of Akt pathway. The anti-obesity activity of Theasaponin E1 was measured by Oil Red O staining method using 3T3-L1 cells. A good decrease in the lipid droplet formation in 3T3-L1 cells treated with TSE1 was noted. An effective reduction in body weight was observed in the mice fed with TSE1 plus high-fat diet as compared to the control which was fed only with high-fat diet. Hence our results confirm TSE1 as an effective inhibitor of obesity and angiogenesis leads to prevent tumors and cancers. Keywords : Theasaponin E1 , Antiangiogenic, Camellia sinensis, anti-hyperlipidemic agent, 3T3-L1 cells References 1. Fujiki F, Suganuma M, Okabe S, Sueoka N, Komori A, Sueoka E, Kozu T, Tada Y, Suga K, Imai K. Cancer inhibition by green tea. Mutat. Res. 1998; 402:307–310. 2. Han LK, Kimura Y, Kawashima M, Takaku T, Taniyama T, Hayashi T, Zeng YN, Okuda H. Anti-obesity effects in rodents of dietary teasaponin, a lipase inhibitor. Int. J. Obes. 2001; 25:1459–1464. 3. Christiaens V, Lijnen HR. Angiogenesis and development ofadipose tissue. Mol. Cell. Endocrinol. 2010; 318:2–9. P0930 The Inhibitory Effect of Green Tea Seed Extract on Adipose Tissue Accumulation in Mice Fed with Carbohydrates and Fats Rich Food Jong Deog KIM, Muhammad Imran KHAN, Moon Geon LEE, Jin Hyuk SHIN Department of Biotechnology, ChonnamNatational University, San96-1, Dun-Duk Dong, Yeosu, Chonnam, 550-749, Korea, Obesity a metabolic disorder results from the excessive growth and expansion of adipose tissue associated with severe disorders and complications such as diabetes mellitus, hypertension, cancer and heart disease. The present study aims to investigate the anti-hyperlipidemic and antiobesity activities of green tea seed extract (GTE). Saponins from GTE were extracted through HPLC and the antiobesity activity was determined in-vivo using mice fed with high-fat and high- carbohydrate diets. 3T3-L1 cell line was used for cell viability assay through MTT (3-(4, 5-dimethylthiazol- 2-yl)-2, 5- diphenyltetrazolium bromide) assay. Wako Chemicals, Osaka, Japan was used to measure the level of total cholesterol (TC), triacylglycerol (TG), HDL cholesterol, LDL cholesterol, free fatty acid (FFA), glucose, alanine transaminase (ALT) and aspartate transaminase (AST). Expression of genes related to adipogenesis and lipogenesis in liver of mice, was analyzed by RT-PCR. Results showed that Green tea (Camellia sinensis) seeds extract (GTE) inhibit accumulation of adipose tissues through multiple cells signaling pathways such as suppressing products of adipogenic genes, (PPARγ, C/EBPα, SREBP, LPL, aP2 and leptin), inducing insulin and induction of AMP- activated protein kinase (AMPK) a major controller of glucose and lipid metabolism and transport. Teasaponin (96%, C57H90O26, molecular weight 1200, Aladdin Chemistry Co. Ltd., Shanghai, China) was used as standard. Keywords : Saponins, Adipose tissue , Antiobesity, Alanine transaminase, RT-PCR References 1. Fujiki F, Suganuma M, Okabe S, Sueoka N, Komori A, Sueoka E, Kozu T, Tada Y, Suga K, Imai K. Cancer inhibition by green tea. Mutat. Res. 1998; 402:307–310. 2. Han LK, Kimura Y, Kawashima M, Takaku T, Taniyama T, Hayashi T, Zeng YN, Okuda H. Anti-obesity effects in rodents of dietary teasaponin, a lipase inhibitor. Int. J. Obes. 2001; 25:1459–1464. 3. Christiaens V, Lijnen HR. Angiogenesis and development ofadipose tissue. Mol. Cell. Endocrinol. 2010; 318:2–9. 한국생물공학회, 생물공학의 동향 : 2015.10 바이오칩 및 나노바이오공학 P1001 Nanowire Nanoinjector for Electro-Triggered, Quantitative Gene Delivery Seung Min YOO1, Mijeong KANG2, Bongsoo KIM2, Sang Yup LEE1 1Dept. of Chemical & Biomolecular Engineering, KAIST, Daejeon 305-701, 2Dept. of Chemistry, KAIST, Daejeon 305-701 We developed a Au nanowire (NW) nanoinjector that has the thinnest diameter among the DNA delivering devices as well as optimum mechanical properties, minimizing cell damage. Well-defined single-crystalline Au surface and high electric conductivity of a Au NW nanoinjector allow precisely timed and efficient electrochemical release of DNA molecules attached on a Au NW surface. Both linear DNA and plasmid DNA were delivered separately, and showed successful expression. The Au NW nanoinjector would find important biomedical applications in the fields such as gene therapy, DNA vaccination, targeted drug delivery, and probe/control of cell signaling events. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012- C1AAA001-2012M1A2A2026556) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.] Keywords : nanowire, nanoinjector , Gene delivery References 1. Yoo, S.M., Kang, M., Kang, T., Kim, D.M., Lee, S.Y., Kim, B. Electro-triggered, Spatioselective, Quantitative Gene Delivery into a Single Cell Nucleus by Au Nanowire Nanoinjector, Nano Lett. (2013), 13 (6), 2431–2435. P1002 Electrical Detection of Pathogenic Bacteria Using Single Walled Carbon Nanotube Biosensor Seung Min YOO1, Youn-Kyoung BAEK2, SunHaeRa SHIN1, Ju-Hyun KIM3, Hee-Tae JUNG1, Yang-Kyu CHOI3, Sang Yup LEE1 1Dept. of Chemical & Biomolecular Engineering (BK21 plus program), KAIST, Daejeon, 305-701, 2Powder·Ceramic Division, Korea Institute of Materials Science, Changwon, 632-831, 3School of Electrical Engineering and Computer Science, KAIST, Daejeon, 305-701 We developed Single-walled carbon nanotube (SWNT)-based electrical biosensor for the label-free and specific detection of pathogenic bacteria. In this system, an electrical sensor is composed of a pair of gold electrodes and a linearly patterned SWNT network film that bridges the electrodes as a channel material. This biosensor successfully detected DNA from reference strains and real clinical samples, indicating that it could be a useful label-free platform for identifying specific pathogenic bacteria. We believe that our SWNT-based electrical biosensor could be a promising candidate for future use in highly integrated chips and handheld diagnostic electronics. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001- 2012M1A2A2026556).] Keywords : Single-walled carbon nanotube, electrical biosensor , pathogenic bacteria References 1. Baek, Y. K.; Yoo, S.M.; Kim, J.H.; Jung, D.H.; Kim, Y.S.; Choi, Y.K.; Lee, S.Y .; Jung, H.T., The Effect of Network Density on the DNA-Sensing Performance of Single-Walled Carbon Nanotubes (2009) J. Phys. Chem. C, 113(52), 21566-21571. P1003 Multispot Array-based DNA Detection Combined with S1 Nuclease-Mediated Elimination of Unpaired Nucleotides Seung Min YOO1, Dong Min KIM2, Sang Yup LEE1 1Dept. of Chemical and Biomolecular Engineering (BK21 plus program), KAIST, Daejeon 305-701, 2Center for Applied Life Science, Hanbat National University, Daejeon, 305-719 The accurate detection of mismatched base pairs is critical to many DNA hybridization-based applications in basic research and diagnostics. We herein demonstrate that mismatched DNAs on a multispot array can be accurately detected in a multiplexed way by employing the S1 nuclease-based mismatched base pair-specific cleavage system. After the optimization of the reaction condition, mismatched DNAs present in various pathogenic bacteria and genetic disorders could be successfully detected with stable hybridization signals regardless of the position of the fluorescent label relative to the probe- target duplex. This technique of performing S1 nuclease-mediated cleavage on a multispot array offers high specificity and high-throughput detection of mismatched DNAs. It is expected that this assay system will prove useful for single-assay genotyping and/or the diagnosis of various diseases and pathogens. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012- C1AAA001-2012M1A2A2026556).] Keywords : mismatched DNA, S1 nuclease, multispot array References 1. Yoo, S.M.; Kim, D.M.; Lee, S.Y. Multispot Array Combined with S1 Nuclease-Mediated Elimination of Unpaired Nucleotides (2015) BioChip J., 9(2), 156-163 P1004 Biological Syntheis of Metal Oxide Nanoparticles by Microorganism Yoojin CHOI1, Tae Jung PARK2, Sang Yup LEE1,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Dept. of Chemical & Biomolecular Engineering (BK21 plus program), KAIST, Daejeon, 305-701, Republic of Korea, 2Dept. of Chemistry, Chung-Ang University, Seoul, 156-756, Republic of Korea, 3Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea, 4BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of Korea In this research, we developed a recombinant Escherichia coli (E. coli) system expressing PCS and/or MT for the in vivo synthesis of various metal nanoparticles (NPs), including NPs never synthesized before by chemical methods. Based on this research, various metals, including semiconducting (Cd, Se, Zn, Te), alkali-earth (Cs, Sr), magnetic (Fe, Co, Ni, Mn), and noble (Au, Ag) metals and rare-earth fluorides (Pr, Gd), were incubated in assorted combinations with the recombinant E. coli cells for the synthesis of the corresponding metal NPs. The size of the metal NPs could be controlled by adjusting the concentrations of the supplied metal ions. As the high-cell-density culture of E. coli has been well established, the efficient and cost-effective production of various metal NPs would not be a difficult task. The engineered E. coli system reported herein should be broadly applicable to biosynthesis of metal NPs of interest with tailored optical, electronic, chemical, and magnetic properties. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556).] Keywords : Biosynthesis, Metal nanoparticles, Microorganism References 1. Tae Jung Park, Sang Yup Lee,* Nam Su Heo, and Tae Seok Seo,In Vivo Synthesis of Diverse Metal Nanoparticles by Recombinant Escherichia coli (2010), Angew. Chem. Int. Ed. 249, 7019 P1005 Simple, Rapid Detection of Influenza A (H1N1) Viruses Using a Highly Sensitive Peptide-based Molecular Beacon Kyeonghye GUK1,2, Eun-Kyung LIM2,3, Hyeran KIM3, Juyeon JUNG1,2,3 1Department of Nanobiotechnology, University of Science and Technology (UST), Daejeon, 305-350, 2BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, 3BioNano Health Guard Research Center, Korea Research Insititute of Beioscience and Biotechnology (KRIBB), Daejeon, 305-806 A peptide-based molecular beacon (PEP-MB) was prepared for the simple, rapid, and specific detection of H1N1 viruses using a fluorescence resonance energy transfer (FRET) system. PEP-MB exhibited minimal fluorescence in its "closed" hairpin structures. However, in the presence of H1N1 viruses, the specific recognition of the hemagglutinin (HA) protein of H1 strains by PEP-MB causes the beacon to assume an "open" structure that emits strong fluorescence. PEP-MB can straightforwardly detect H1N1 viruses within 15 min or even 5 min and can exhibit strong fluorescence even at low viral concentrations. From these results, we expect this technique to have great potential for use in multiplex assays of influenza viruses in which several fluorophore and peptide sequences allow for the detection of interactions with specific HA proteins. Keywords : Peptide, Molecular beacon, H1N1 viruses, Rapid detection References 1. S. Thurley, L. Roglin and O. Seitz, Hairpin Peptide beacon: Dual-Labeled PNA-peptide-Hybrids for Protein Detection (2007), J. Am. Chem. Soc. , 129, 12693-12695 P1006 Fabrication of Single Cell Chip to Detect Neural Cell Redox State Based on Spectro- Electrochemical Technique Kyeeong-Jun KIM1, Waleed Ahmed EL-SAID2, Tae-Hyung KIM1, Jeong-Woo CHOI1,2 1Department of chemical and Biomolecular engineering, Sogang University, Seoul, 121-742, 2 Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 121-742 Detection of cellular information is promising research field to determine various kinds of cellular signal. In this study, we introduce spectro-electrochemical technique to detect intra-cellular redox state based on single neural cell chip platform. We fabricate microgap electrode which consist of polystyrene assisted gold nanodot array. Single neural cell (PC12) was seeded on microgap electrode. After neural cell seeded on microgap electrode, spectro-electrochemical technique was applied. Cellular oxidation and reduction process was induced by linear sweep voltammetry. During the redox process, surface enhanced Raman scattering was measured to measuring changed cellular signal during redox process. Changed Raman signal from neural cell during redox process was similar with redox signal from dopamine. Therefore, neural cell’s spectro- electrochemical result is mainly related with secreted dopamine from neural cell. In summary, our newly developed single neural cell chip can be apply to label free, in situ monitoring tool for biochemical changes or cellular information in cell. Acknowledgement: This research was supported by the Leading Foreign Research Institute Recruitment Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (MSIP) (2013K1A4A3055268) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A1A10051725). Keywords : Single cell chip, SERS, Linear sweep voltammetry, Spectro-electrochemical technique, Neural cell, Dopamine References 1. W. A. El-Said, T.–H. Kim, Y.-H. Chung and J. W. Choi, Fabrication of New Single Cell chip to Monitor Intracellular and Extracellular Redox State based on Spectroelectrochemical method (2015), Biomaterials, 40, 80 2. T.-H. Kim, K.-B. Lee and J. W. Choi, 3D Graphene Oxide-encapsulated Gold Nanoparticles to Detect Neural Stem Cell Differentiation, (2013), Biomaterials, 34, 8660 P1007 Electrochemical Performance of Indium Tin Oxide Electrode Loaded with Different Amount of Glucose Oxidase for Biofuel Cell Xue WANG1, Chang_Joon KIM1, Sung Bae KIM1, Hyug_Han KIM2 1Dept.of Chemical Engineering, Gyeongsang National University, Jinju, 660-701, 2Department of Chemistry, Dankook University, Cheonam, 330-741 The immobilized glucose oxidase is necessary for the development of enzymatic biofuel cells which can be applied to power sources of implantable devices as well as biosensors. The key issues in the development of biofuel cells are to improve the power densities and lifetimes. This study aims to improve the power density by increasing the loading of glucose oxidase (GOx) on surface of electrode. Carboxylated single walled carbon nanotube (SWCNT) was attached on the surface of indium tin oxide (ITO) electrode. After activation of SWCNT with EDC/NHS, GOx was immobilized on SWCNT-ITO electrode [1]. A kind of osmium redox polymer, polyvinylimidazole-[osmium (bipyridine)2Cl] (PVI-[Os(bpy)2Cl], was attached on the surface of GOx as mediator. Cathode was prepared by immobilizing laccase on the surface of electrode using + same method. Polyvinylimidazole-Os(4,4'-dichloro-2,2'- bipyridine)2Cl (PVI-Os(dCl-bpy)2Cl2) were used as a mediator for cathode. Electrochemical performances of GOx electrodes were evaluated in 100 mM PBS buffer (pH=7.0) with or without 30 mM glucose by using cyclic voltametry and impedance analysis. The loading amount of SWCNT and GOx was significantly influenced on the performance of biofuel cells. The power density was maximum when the loading of GOx was 3.3 mg-GOx/mg-SWCNT. Keywords : ITO electrode, glucose oxidase, Laccast, SWCNT References 1. M. A. Aziz and H. Yang, Bull. Korean Chem. Soc. 28, 1171( 2007). P1008 Antibacterial Activity and Cytoxicity Test of Carbon Nanotubes Decorated with Copper Nanoparticles Youngmin SEO1, Jonghoon CHOI1,2 1Dept. of Bionano Technology, Graduate School, Hanyang University, Seoul, 04763 , 2Dept. of Bionano Engineering, Hanyang University ERICA, Ansan, 15588 Various nano-scale materials including copper nanoparticles have been actively studied for their capacity to effectively prevent a bacterial growth. Although copper nanoparticles have strong antibacterial activity, they were rarely used because of their apparent cytotoxicity and instability to continuous oxidation [1]. A critical challenge is to enhance the antibacterial properties while decreasing cytotoxicity of copper nanoparticles. The conjugation of multiple nanomaterials with different dimensions –- 3D spherical nanoparticles and 1D high aspect ratio nanotubes – may increase the target-specific antibacterial capacity while retaining the minimal toxic properties to the mammalian cells. In this study, multi-walled carbon nanotubes (MWCNTs) were treated with a mixture of acids and then decorated with copper (Cu) nanoparticles via a chemical reduction of Cu cations by NaBH4. The synthesized Cu-MWCNTs complexes were characterized by transmission electron microscopy (TEM), X-ray diffractometery (XRD) and energy-dispersive X-ray spectroscopy (EDS). Cu-MWCNTs indicated effective antibacterial activity against Methylobacterium spp., Sphingomonas spp. and E. coli. evaluated by a paper disk diffusion and a minimum inhibitory concentration (MIC) assay. In addition, the biocompatibility of Cu-MWCNTs were tested on human fibroblast cells with the minimum concentration required to have antibacterial properties. Reported Cu-MWCNTs carrying minimum copper nanoparticles may be desirable as a next generation antibacterial agent effective to various microbes while be biocompatible to mammalian cells. Keywords : carbon nanotube, copper nanoparticle, antibacterial activity, cytotocixity, methylobacterium spp., sphingomonas spp., E. coli. References 1. J. Drelich and D. Hoffman, Appl. Surf. Sci. 257, 9435-9443 (2011) P1009 Development of High-Resolution Microchip-CE Device for Single-Strand Conformation Polymorphism Analysis Giyoung SHIN1, Gyoo Yeol JUNG1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Korea Accurate and fast genetic marker detection is required for molecular diagnosis. Microchip Capillary electrophoresis-single strand conformation polymorphism (μCE-SSCP) is has great potential for genetic variation detection. Double-stranded DNA forms single-strand conformation by heat denaturing and fast cooling. CE-SSCP based detection can discriminate differences in the conformations of single-stranded DNA molecules. However the most critical limitation of μCE-SSCP is low separation resolution result from short separation channel length. In our previous research, PEO-PPO-PEO triblock copolymer matrix was demonstrated that significantly increase the separation resolution on bench-top CE-SSCP. The limitation of μCE-SSCP could be overcome by utilizing the triblock copolymer matrix. As a result, multiplex and quantitative genetic analysis could be possible to realize on CE-SSCP based microchip device for early stage fast and sensitive molecular diagnosis. Keywords : Microchip, Capillary Electrophoresis, Genetic Analysis, CE-SSCP References 1. Shin, G. W., Hwang, H. S., Oh, M. H., Ryu, C. Y., Salvo, C. J., Feldman, S., Doh, J., Jung, G. Y., A novel pathogen detection system based on high-resolution CE-SSCP using a triblock copolymer matrix., J. Sep. Sci. 2010, 33, 1639-1643. 2. Easley, C. J., Karlinesey, J. M., Bienvenue, J. M., Legendre, L. A., Roper, M. G., Feldman, S. H., Hughes, M. A., Hewlett, E. L., Merkel, T. J., Ferrance J. P., Lander, J. P., A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability., Proc. Natl. Acad. Sci. U. S. A. 103, 19272-19277. 3. Shin, G. W., Kim, Y. T., Heo, H. Y., Chung, B., Seo, T. S., Jung, G. Y., Triblock copolymer-based microchip device for rapid analysis of stuffer-free multiplex ligation-dependent probe amplification products., Electrophoresis. 2012, 33, 3574-3578. P1010 Photothermal Effect of Gold Nanoparticle on the Mice Skin Jae-Yeul LEE, Se-Ho PARK, Ju-Hwan CHOI, Tae-Hee PARK, Kyu-Dong SHIM, Bo-Mi KIM, Eue-Soon JANG, Kwang- Hwan JHEE School of Science and Engineering of Chemical Materials, Kumoh National Institute of Technology, Gumi, Gyeongsangbuk- do 39177, Korea Gold nanoparticles (GNPs) have attracted enormous scientific and technological interest due to their ease of synthesis, chemical stability, and unique optical properties [1]. Here, we confirmed that the GNPs can efficiently convert absorbed light energy into heat energy, and this means their possible use for thermo-cosmetics. We consider the effects of GNPs photothermal effect on the temperature increase on the mice skin. Thermal stability of GNPs was investigated morphological changes depending on irradiation times of visible light energy source by using transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS). And we used the fluorescence microscopy to observe the cell morphology and distribution of GNPs inside the cells. We also utilized a dark-field system to verify the optical properties of the intracellular particles. In addition to measure the uptake dosage per cell treated from 10 to 100 µg/ml GNPs using inductively coupled plasma optical emission spectrometer (ICP-OES). In summary, GNPs-treated mice skin resulted in an average temperature increase approximately 3ºC on visible light exposure for 30 min. Our results indicated that there is no toxicity effect on fibroblast cell due to taking GNPs. Taken together, our data support that GNPs could be used for thermo-cosmetic application. Keywords : Gold nanoparticle, thermo-cosmetics, cellular uptake References 1. M. F. Lengke, M. E. Fleet, and G. Southam, Morphology of gold nanoparticles synthesized by filamentous cyanobacteria from gold(I)-thiosulfate and gold(III)-chloride complexes (2006), Langmuir, 22, 2780. 2. S. W. Tsai, J. W. Liaw, Y. C. Kao, M. Y. Huang, C. Y. Lee, L. R. Rau, C. Y. Huang, K. C. Wei, and T. C. Ye, Internalized gold nanoparticles do not affect the osteogenesis and apoptosis of MG63 osteoblast-like cells: A quantitative, In Vitro study (2013), PLOS ONE, 8, 1. P1011 Development of DNA Aptamers for Detection and Growth Inhibition of Streptococcus mutans Kyeong-Ah LEE1, Woo-Ri SHIN1, Simranjeet Singh SEKHON1, Sung-Jin CHO2, Jiho MIN3, Yang-Hoon KIM1 1Dept. of Microbiology, Chungbuk National University, Cheongju, 362-763, 2Dept. of Biology, Chungbuk National University, Cheongju, 362-763, 3Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, Jeonju, 561-756 Streptococcus mutans have been associated with a primary aetiological agent of dental caries in animals and humans, and it causes tooth enamel corrosion, vulnerable to decay. The importance of S. mutans in the development of dental cares has been studied extensively. In this study, we isolated DNA aptamer binding to S. mutans by SPR(Surface Plasmon Resonance) and developed the aptamer–based detection assay such as ELISA(enzyme-immunosorbent assay). Aptamer selected by whole-cell of S. mutans show that high specificity and affinity more binding to S. mutans than to other species. In addition, selected aptamers inhibit cell growth of S. mutans more than control. This assay with high specificity and growth inhibition can be used as an alternative method for the therapy and detection of oral disease. This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Risk Assessment Research and Development of Rapid Diagnostic Method for Biological, Chemical and Environmental Animal Disease, Project No: PJ01052301)" Rural Development Administration, Republic of Korea. Keywords : Streptococcus mutans, aptamer, detection, growth inhibition References 1. Nasa Savory etc. al, Simultaneous improvement of specificity and affinity of aptamers against Streptococcus mutans by in silico maturation for biosensor development (2013), Biotechnology and Bioengineering, DOI: 10.1002/bit.25111 P1012 A Microengraving Method for Single Cell Culture Yeonho JO1, Jonghoon CHOI1,2 1Dept. of Bionano technology, Graduate school, Hanyang University, Seoul, 04763, 2Bionanoengineering, Hanyang University ERICA, Ansan, 15588 Many scientists have studied Mesenchymal stem cells(MSCs) and Induced pluripotent stem cells(iPSCs) as a cell therapy for autoimmune diseases(ADs). Although their high interests, the interaction between stem cells and T cells are not well investigated yet. Bulk assays are not considered as a proper way to study cell to cell contribution because they are possibly affected by other interactions. Thus, we fabricated a platform facilitates single cell culture for cell to cell investigation. Poly-dimethylsiloane(PDMS) is popular material due to its chemical resistance, low cost, rapid fabrication. We made PDMS microwell(diameter 300um) as a platform for single cell culture by soft lithographic microengraving technique. Before implementing stem cell seeding, we demonstrated 3T3s and Human dermal fibroblasts(HDFs) seeding on the wells to verify cells’ growth rate and stability. As a result, we checked single cell level culturing was possible on the wells. In conclusion, this platform is first step toward understanding interaction between stem cells and T cells, and we succeed single cell culture. It will bring further understanding of stem cell and indicate better cell therapy for ADs. Keywords : single cell, microwell, microengraving References 1. Adebola O Ogunniyi, Nature protocols. Vol.,4 NO.5, (2009). P1013 Size-based Nanometer-Scale Particle Separation in Microstructures Eunwon LEE1, Seri CHOI2, Seung Yeon HA2, Soojeong SHIN2, Jong Wook HONG1,2 1Department of Bionano Technology, Graduate School, Hanyang Univerisity, Seoul 133-791, South Korea, 2Department of Bionano Engineering, Hanyang University, Ansan Kyunggi-do 426-791, South Korea Separation of micro and nanoparticles is an important part of macro and nanotechnology field. Because this area is promising for applications in a variety of inorganic particles and biological samples. Also it can be used in various application of nanotechnology field such as point-of-care diagnostics, drug discovery, and chemical or biological agent detection. In this study, we presented picoliter scale fluid control system for separation of nanometer scale particles based on size distribution. For size-based separation processes, this device is designed to have plural inlet and more than twice inlet number of outlets, and unique channel structure which is drain channel to increase efficiency of separation. Also, we intend to pursue efficient size-based separation by controlling flow rate of drainage. Additionally, our design of picoliter scale fluidic system has been further confirmed using simulation for particle tracing. Possible designs of microfluidics system for nanoparticle separation, are presented and discussed, together with results of simulations and particle tracking for these designs. This system can be innovative tool in various application such as separation of quantum dot, electronic nanomaterials like liquid crystal and bio-nanomaterials like exosome. Keywords : Nanoparticle, Separation, Microfluidics system References 1. M. Hamon and J. W. Hong, New tools and new biology: recent miniaturized systems for molecular and cellular biology (2013), Molecules and Cells, 36, 485 2. SJ Shin, Y. J. Yoo and J. W. Hong, Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System (2015), Journal of Nanoscience and Nanotechnology, 15, 1 3. HW Nho and TH Yoon, Enhanced separation of colloidal particles in an AsPFF device with a tilted sidewall and vertical focusing channels (t-AsPFF-v) (2013), Lab on a Chip, 13, 773 4. J. F. Ashley, C. N. Bowman and R. H. Davis, Hydrodynamic separation of particles using pinched-flow fractionation (2013), AIChE Journal, 59, 3444 5. A. Lenshof and T. Laurell, Continuous separation of cells and particles in microfluidic systems (2010), Chemical Society Reviews, 39, 1203 P1014 A Density-based Fine Separation in Micro Structures Seri CHOI1, Soojeong SHIN1, Jangsun HWANG2, Jonghoon CHOI1,2, Jong Wook HONG1,2 1Department of Bionano Engineering, Hanyang University, Ansan Kyunggi-do 426-791, Korea, 2Department of Bionano Technology, Graduate School, Hanyang Univerisity, Seoul 133-791, Korea When the cell experiment, as the cell viability may vary according to cell treat method. If you remove the live cells and dead cells, this time will be reduced by a variable constant viability. We separated by using a small density difference between dead cells and live cells. The device consists of a separation channel having two inlets and two outlets. Cells are separated by the density difference when passing the main channel of the separation channel. Power which cell receive is calculated using stoke's law. Previous density-based separation experiment or the size of the particle size, showed that the particles which have big difference of density or size. Therefore, It was turned out that small molecules(6um-11um) having subtle difference in density can be separated. So, We can enhance differentiation yield separating undifferentiation cell or dead cell when differentiating stem cell to liver cell by showing possibilities of separation real cells having subtle differences. Keywords : density separation, cell separation, microfluidic References 1. Neta Lavon, Ofra Yanuka and Nissim Benvenisty, Differentiation and isolation of hepatic-like cells from human embryonic stem cells(2004), International Society of Differentiation, 72, 230–238 2. Soojeong Shin, Young Je Yoo, and Jong Wook Hong, Microgravity separation of alginate empty capsule from encapsulated pancreatic islets using microfluidic system(2014), Journal of Nanoscience and Nanotechnology, 15, 1-5 P1015 Selective Surface Modification for a Biochip Application in Micro Structure Hoon Deok PARK1, Soo Jeong SHIN1, Seong Woo LEE2, Jong Wook HONG1,2 1Department of Bionano, Hanyang University, Seoul, South Korea, 2Department of Bionano Engineering, Hanyang University, Ansan Kyunggi-do, South Korea Surface modification is considered to be so important technique recently because of its function as a blocking adsorption. Many research which are conducted on a PDMS chip have problem due to adsorption of sample to the PDMS surface. If it were not for the sample to adsorb to the surface of channel, researchers can get wanted sample as expected. So here we performed some surface modification on a biochip to solve this problem and to find out its function as a blocking adsorption. BSA(Bovine Serum Albumin), which is well known for its function as a blocking protein adsorption, was used and SEM images was obtained according to different experiment conditions. BSA coating didn’t give much of help to the prevention of PS(Polystyrene) particle adsorption, but it did give help to prevention of protein sample adsorption. Keywords : Protein adsorption, Particle adsorption, Surface modification P1016 Evolutionary selection of Short Peptide Binders Recognizing Biomarker Toxin Proteins Myung Yi RYU, Jong Min LIM, Jong Pil PARK Dept. of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan, 38610 Cholera toxin (CTX) is a major virulence agent of Vibrio cholera and it can rapidly lead to progress severe dehydration and shock, and thus death can occur within several hours without appropriate clinical therapy. Therefore, there is a growing demand for new diagnostic biosensing platform which is capable of simple and sensitive for the detection and identification of CTX. In this paper, direct and high sensitive detection of the cholera toxin subunit B (ctxB) was performed quantitatively by using unique peptide binders identified through phage display technique. Upon biopanning of M13 phage library, high affinity of peptides was identified and the final lead peptides had an amino acid sequence of VQCRLGPPWCAK. ELISAs were used to evaluate the binding affinity, and the peptide-displayed phages were found to have nanomolar binding affinity for ctxB. More strikingly, the binding interaction was still specific in the presence of fecal bovine serum, as high as 10%. These results suggested the free peptides or peptide-displayed phages may be useful for the use of detection of cholera toxin in point-of-care applications. Keywords : toxin protein, phage display, peptide binder, biosensor References 1. B. Gabryelczyk, G.R. Szilvay, M. Salomkki, P. Laaksonen, M.B. Linder, Selection and characterization of peptides binding to diamond-like carbon, Colloids and Surfaces B: Biointerfaces, 110 (2013) 66-73 2. C.S. Kim, J.H. Seo, H.J. Cha, Functional Interaction Analysis of GM1-Related Carbohydrates and Vibrio cholerae Toxins Using Carbohydrate Microarray, Analytical Chemistry, 84 (2012) 6884-6890. P1017 Biofunctionalized Polydiacetylene (PDA) Vesicles Encapsulated within Hydrogel Matrix Huisoo JANG1,3, Sungho JUNG1,3, Sun Min KIM2,3, Tae-Joon JEON1,3 1Dept. of Biological Engineering, Inha University, 100 Inharo, Nam-gu Incheon 22212, South Korea, 2Dept. of Mechanical Engineering, Inha University, 100 Inharo, Nam-gu Incheon 22212, South Korea, 3Biohybrid Systems Research Center (BSRC), Inha University, 100 Inharo, Nam-gu Incheon 22212, South Korea In this study, polydiacetylene (PDA) based chromatic biosensor, which is immunohydrogel bead, was developed to detect genetically modified (GM) crop. PDA vesicle possesses a unique colorimetric property that is blue-to-red color transition upon external stimuli such as temperature, pH and mechanical stress. It can be recognized by naked eyes. In our chromatic sensor, PDA vesicle was functionalized with target-specific antibody using EDC/NHS coupled reaction to identify target protein. Antibody conjugated PDA vesicle was then encapsulated into Poly(ethylene glycol) diacrylate(PEG-DA) hydrogel at high density to enhance reaction sensitivity. Furthermore, PDA vesicle encapsulating hydrogel was formed in spherical shape by using droplet-microfluidic device to enable transportation. We named this chromatic biosensor as immunohydrogel bead. Phosphinothricin acetyltransferase (PAT) protein was aimed as model target protein that is GM marker protein. Detection of PAT protein from genetically modified organisms (GMO) was attempted for practical use. Using immunohydrogel bead, PAT protein was detected to 20 nM with naked eyes which means that even 1% of GMO can be detected. Thus, we expect that immunohydrogel bead can be used practically not only in GMO but also in other targets. Keywords : Polydiacetylene (PDA), Hydrogel matrix, Biofunctionalization References 1. Jung* and Jang* et al, Chromatic biosensor for detection of phosphinothricin acetyltransferase by use of polydiacetylene vesicles encapsulated within automatically generated immunohydrogel beads(2015), Anal. Chem, 87(4), 2072-2078 P1018 Synthesis and Characterization of Silver Nanoparticle Prepared from Sophorolipid Sung-pil SEO, Birendra Kumar SINGH, Hyang-Bok LEE, Eun-Ki KIM* Department of Biological Engineering, Inha University, National Research Lab of Skinbioactive Materials, 253 Yonghyun- dong, Nam-gu, Incheon 402-751, Korea Silver nanoparticles are the nanoparticle in the range of 1-100nm. The biosynthesis of silver nanoparticle has gained attention in view of excellent biocompatibility and low toxicity. The present study aims to make the silver nanoparticle using biosurfactant called sophorolipid as reducing and capping agent. Characterization of silver nanoparticle was done by using transmission electron microscopy. We studied the effect of temperature and the structure of sophorolipid derived from various fatty acids chain on the size of silver nanoparticles obtained. On increasing the temperature we observe that particle size become smaller with a narrow size distribution. Broth dilution method was used to determine the minimum inhibitory concentration (MIC). The antimicrobial activity of silver nanoparticles was also influenced by the dimensions of the particles; the smaller the particle size, the greater the antimicrobial effect. It was also observed that with increasing chain of sophorolipid, the antimicrobial activity of silver nanoparticle was increased. The study is focused on effective and efficient synthesis of silver nanoparticle while concerning the high antimicrobial activity and low toxicity poses by nanoparticles. Keywords : Sophorolipid, Silver nanoparticle, Antimicrobial activity References 1. Kumar, C. Ganesh, Suman Kumar Mamidyala., Synthesis of Biosurfactant-Based Silver Nanoparticles with Purified Rhamnolipids Isolated from Pseudomonas aeruginosa BS-161R . J. Microbiol. Biotechnol. (2010), 20(7), 1061–1068 2. Kasture M. B, Patel P., Synthesis of silver nanoparticles by sophorolipids: Effect of temperature and sophorolipid structure on the size of particles. J. Chem. Sci. (2008), 120(6), 515-520 P1019 Characterization of Oxanine, a Reactive Modified Nucleobase, and Its Application as a Novel Cross-linker Eui Kyoung JANG, Pham Nhat VAN, Ki Sung PARK, Seung Pil PACK 1Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Sejong 339-700, Korea The oxidative stress forced chronic inflammation cause DNA damages in tissue, in which guanine could be converted to oxanine. Oxanine has a reactivity against nucleophiles thanks to its unique o-acylisourea ring structure. Up to now, oxanine research has been focused on their cytotoxic effect inducing a DNA-protein crosslink in cellular. In spite of their biological importance, the reaction pattern and behavior is still unclear. In this study, we characterized the reactivity of oxanine against a nucleophile by compared with active carboxyl group by crosslinking reagent (e.g. EDC) showing mechanical similarity with oxanine. We introduce oxanine to 3’-end of DNA oligomer and demonstrate its immobilization onto the chip surface as a model of DNA chip. Keywords : Oxanine, EDC, DNA chip References 1. S.P., Pack, et.al. Reactivity of oxanine: efficient fabrication of DNA microarray by using oxanine-containing DNA oligomer as probe molecule (2008)_Nucleic Acids Symp. Ser., 52, 441–442 P1020 Alternative Immobilization Method of Staphylococcus aureus Protein A at Biosilica for Immunoprecipitation Eui Kyoung JANG, Ki Sung PARK, Mi Ran KI, Jung Hoon CHOI, Seung Pil PACK 1Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Sejong 339-700, Korea Staphylococcus aureus Protein A (SpA) offer many advantages as affinity media to immunoglobulins because it has potential ability to binding with Fc region of antibody. This specific binding affinity can be used for one step purification of antibody with high quality of purity. Furthermore, its application is commonly used as an alternative method to target immuno-precipitation (IP) and provide to evidence of protein-protein interaction either in vitro or in vivo. In recent years, recombinant Protein A is produced cross-linked at different bead by chemical process for IP media. We have established new approach to immobilization of Protein A at bio-silica via Silica Forming Peptide (SFP) that is not required chemical process like extreme temperature, pH, and by-products. Also, our group have discovered on new SFP derived from marine microbes such as diatom, sponge what can be formed biosilica on mild conditions. Here, we designed SFP-SpA gene using recombinant system and it was formed to SpA-Biosilica media for immuno-precipitation by biosilicification. We substantiated that SpA completely immobilized at biosilica particle formed by SFP fused to N-terminus of SpA. To determine whether SpA-biosilica can use for immune-precipitation media, we performed Co-immuno-precipitation to Green Fluorescent Protein (GFP) and Anti-GFP antibody. In addition, to demonstrate the use efficiency of SpA-biosilica media, we compared with Protein A-Agarose commercial product. These findings demonstrate that SpA-biosilica displayed higher affinity to antibody and had more high efficiency of sedimentation than Protein A-Agarose. Keywords : Staphylococcus areus Protein A (SpA), Biosilica , Immobilization References 1. Janoschek L, Freiherr von Roman M, Berensmeier S. Protein A affinity precipitation of human immunoglobulin G. (2014) Journal of chromatography B, Analytical technologies in the biomedical and life sciences., 965:72-8. 2. Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier JB, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity (2000) Proceedings of the National Academy of Sciences of the United States of America, 97:5399-404 P1021 Amyloid Precursor Protein Cleavage Nanolithography with β-secretase by Atomic Force Microscope Sung-Woong HAN1, Jung-Hoon KIM1, Taiji ADACHI2, Hoon-Kyu SHIN1 1National Institute for Nanomaterials Technology, Pohang University of Science and Technology, 77, Cheongam-ro, Nam- Gu, Pohang, Gyeongbuk, 790-784, Korea, 2Department of Biomechanics, Institute for Frontier Medical Sciences, Kyoto University Amyloid precursor protein (APP) processing is essential for the enhancing the neurite outgrowth of young neurons differentiated from neural stem cells, and it plays important roles in many neural processes including cellular development, differentiation, and proliferation. APP processing has an alternative pathway that is considered to underlie the pathogenesis of Alzheimer’s disease (AD)[1]. The APP processing pathway is determined by two kinds of proteolytic cleavage processes of different secretases that are α- and β-secretases, and results in the generation of cleaved APP molecules of different lengths. First, APP is cleaved by α-secretases in the amyloid β (Aβ) peptide sequence, releasing a soluble N-terminal a- secreted APP (sAPPa) fragment which induces neurite outgrowth and results in the prevention of Aβ release. Second, APP is cleaved by β-secretase (β-site APP-converting enzyme 1; BACE1) at the N-terminus of the Aβ peptide sequence, yielding a soluble β-secreted APP (sAPPβ) and Aβ to the outside of the cell. Aβ secretion leads to extracellular Aβ aggregation and plaque growth, which is considered to be a critical factor in the pathogenesis of AD[2]. In recent AD therapeutic studies, β- secretase has been focus for the inhibition of Aβ production[1, 2], and many researchers are focused on finding the inhibitors as a therapeutic target for AD. In this study, we developed a sophisticated technology to evaluate the APP cleavage mechanism at the nano-molecular level by atomic force microscopic (AFM) nanolithography. APP was modified on a glass substrate; nanolithography of APP cleavage by β-secretase-modified AFM probe scanning was achieved. APP cleavage was verified by fluorescent immunostaining. The present method will be very useful in understanding the molecular level of the APP cleavage mechanism by β-secretase in vitro; this method will facilitate inhibitor screening for the therapeutic target of AD[3]. Keywords : Amyloid precursor protein, β-Secretase, Nanolithography, Atomic force microscope References 1. M. D. Carter, G. A. Simms, and D. F. Weaver, The development of new therapeutics for Alzheimer's disease (2010), Clin. Pharmacol. Ther., 88(4), 475. 2. R. Postina, Activation of α-secretase cleavage (2012), J. Neurochem. 120, 46. 3. S. W. Han, H. K. Shin and T.Adachi, Nanolithography of amyloid precursor protein cleavage with β-secretase by atomic force microscopy J. Biomed. Nanotechnol. In press. P1022 Behavior Response of C.elegans to Various Physical Stimuli in a Controlled Microfluidic System Sunhee YOON1,2, Hailing PIAO2,3, Tae-Joon JEON1,2, Sun Min KIM2,3 1Department of Biological Engineering, Inha University, Incheon 402-751, South Korea, 2Biohybrid Systems Research Center (BSRC), Inha University, Incheon 402-751, South Korea, 3Department of mechanical Engineering, Inha University, Incheon 402-751, South Korea Caenorhabditis elegans (C.elegans) is an interesting model due to various behavioral properties, such as electrotaxis, thermotaxis, and chemotaxis [1]. Moreover, as C.elegans and human share over 80% of genes including neural circuit genes, studying C.elegans will provide clues for elucidating human neural circuit [2]. Correspondingly, behavioral properties of C.elegans have been studied to investigate its taxis behavior. However, previous researches were only focused on observing C.elegans behavior upon a single physical-stimulus. In this research, we propose a novel device for analyzing behavior of C.elegans with combined physical stimuli (electric field and temperature). We have shown feasibility and novelty of our platform by comparing wild type worms (N2) with four mutant worms (tax-4, ttx-7, unc-54, unc-6) and analyzing response of worms to certain stimulus. We believe that our microfluidic system will be a standard for studying human neural circuit through C.elegans. Keywords : C.elegans, electrotaxis, thermotaxis, microfluidics References 1. Pouya Rezai, et al., Electrotaxis of Caenorhabditis elegans in a microfluidic environment (2010), Lab on a chip, 10, 220-226 2. Titus Kaletta, et al., Finding function in novel targets: C.elegans as a model organism (2006), Nature Reviews Drug Discovery, 5, 387-399 P1023 Guided In-vitro Wound Healing Process in a Microfluidic Device with Nano-Patterns Insu LEE1, Ga Lahm PARK1, Daegyu KIM2, Tae-Joon JEON2,3, Sun Min KIM1,3 1Department of Mechanical Engineering, Inha University, Incheon, Korea, 2Department of Biological Engineering, Inha University, Incheon, Korea, 3Biohybrid Systems Research Center, Inha University, Incheon, Korea In-vivo wound healing process in wounded tissue involves diverse and complex steps of cellular behavior and signaling pathway. But there are still unrevealed mechanisms for wound healing process. In this study we used simply fabricated nano-pattern on biocompatible PDMS (Polydimethylsiloxane) polymer and analyzed the cellular behaviors on the patterned surface, specifically cell orientation, elongation and migration. And also we embedded nano-patterned PDMS into microfluidic system which can mimic wound healing process. Nano-pattern was embedded for various orientations along to channel for 0, 45, 90 degree of angle. Then we cultured 3T3 fibroblast cell in the microfluidic device, and to represent wound like environment to 3T3 fibroblast cell layer, we used layered flow of trypsin/EDTA and DMEM, cell culture medium in the microchannel. To investigate wound healing process on the nano-patterned surface, we acquired time-lapse images of cell migration in wound healing process and measured its closure rate for cell free area. These results would be utilized to study wound healing process and related cellular behavior and pathway and also cell mechanics field. Keywords : Wound healing , Microfluidics, Nano pattern References 1. H. N. Kim, Y. M. Hong, M. S. Kim, S. M. Kim, K. Y. Suh, Biomaterials, 33, 8782-8792 (2012) P1024 Near Infrared Fluorescence-encoded Lipid Supported Polymeric Particle System for Stem Cell In Vivo Imaging Seung Won SHIN1, Jung Hwan PARK1, Ji Soo YUK1, Soong Ho UM1,2 1School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, South Korea, 2SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, South Korea With the advent of nanotechnology, it is become realized to monitoring and tracking the target molecules in vivo and in situ condition. Several signal-coded molecular tracers were evaluated, and a quantum dot or fluorescence-tags may be a good example. As several biological purposes, such as cancer therapeutics and stem cell implantation can be a target of tracing. Especially, stem cell-based therapy is evaluated as a one of the most promising tools for regenerative medicine. Imaging the grafted stem cell and monitoring their migration or differentiation in in vivo condition is important to make better understanding on stem cell therapeutics. Several conventional imaging tools are already developed [1]. However, it is very important to make non-toxic and non-invasive imaging strategy because of the nature of stem cell than the other differentiated cells. In this aspect, we designed a novel near infrared fluorescence color-encoded light-emitting lipid- supported polymeric particulate (NLP). The lipid composition of NLP was maleimide functionalized to make a covalent bond with thiol group on the cell surface proteins, which can minimize the interaction with metabolic components and genomic damages. Core polymeric particulate contains indocyanine green fluorescent dye for efficient in vivo imaging. The size of NLP was less than 250 nm in diameter and no significant hindrance of cell viability, mobility and stemness of stem cell were observed when the NLP coated on the surface of the cell under certain concentration. The fluorescence signal from the NLP-tagged stem cell was maintained at least 2 weeks in vitro, and in vivo. We propose that this NLP-tagging system has a significant potential for stem cell imaging and further applications in stem cell-based therapeutics. Keywords : in vivo imaging, stem cell monitoring, Nanotechnology References 1. J J. L. West, N. J. Halas, Annu. Rev. Biomed. Eng. 2003, 5(1), 285-292 P1025 Detection of miRNAs in Exosomes Using Molecular Beacon and Its Applications in Disease Diagnosis Min Hee KWON, Ji Hye LEE, Won Jong RHEE Division of Bioengineering, Incheon National University In situ single step detection of microRNAs (miRNAs) in a whole exosome has been developed as a novel diagnosis method that can be utilized for various diseases including cancers. Exosomes are nano-sized extracellular vesicles produced by most types of cells and can be found in the most of body fluids including blood, urine, saliva, breast milk, etc. They contain useful biomarkers including miRNAs from their originating cells. This makes exosomal miRNAs from the body fluids an attractive biomarker that can lead to a paradigm shift in the diagnosis of disease. Herein, we have demonstrated that exosomal miRNAs can be detected directly using a nano-sized fluorescent oligonucleotide probe, molecular beacon (MB). As a model system, we have isolated exosomes from cancer cells and detected exosomal miR-375 and miR-574-3p, the candidate miRNA markers for prostate cancer, using MBs. Finally, we have successfully detected exosomal miRNAs from human urine samples indicating that the method can be applied as non-invasive detection of cancer. The method described here is simple, fast, and sensitive, so it will offer great opportunities for the high-throughput diagnosis and prognosis of diseases. Keywords : miRNA, exosome, diagnosis, disease, molecular beacon, cancer References 1. J. H. Lee, J. A. Kim, M. H. Kwon, Ji Yoon Kang, W. J. Rhee, Biomaterials. 54, 116-125 (2015) P1026 Sensitive Assay of Base Excision Repair Enzyme Activity Using Fluorescent G-quadruplex Probe Chang Yeol LEE, Ki Soo PARK, Yujin JUNG, Hyun Gyu PARK Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, Daejeon 305-701, Republic of Korea A sensitive and selective assay of uracil DNA glycosylase (UDG) activity is developed by utilizing G-quadruplex probe incorporating 2-aminopurine (2-AP), based on a novel design that excision reaction promoted by UDG triggers the formation of G-quadruplex structure with significant fluorescence enhancement of 2-AP within the probe.1 Keywords : 2-aminopurine, base excision repair enzyme, G-quadruplex, uracil DNA glycosylase References 1. C. Y. Lee, K. S. Park and H. G. Park, Chem. Commun. 51, 13744 (2015) P1027 A Novel Method for Highly Specific Isothermal Amplification of Oligonucleotides Based on Graphene Oxide Jung Hun PARK, Hyowon JANG, Hyun Gyu PARK Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701 Over the several decades, the methods for isothermal amplification of nucleic acids have been intensively developed. Among the various technologies, exponential amplification reaction (EXPAR) is one of the representative methods to exponentially amplify target oligonucleotides, possessing high amplification efficiency with short reaction time only in 30 minutes [1]. However, EXPAR has a serious drawback which is generation of non-specific amplification. Even in the absence of the target oligonucleotide, the fluorescence signal is increased as the time goes on. Therefore, we have developed a novel method for highly specific exponential isothermal amplification reaction (EXPAR) of oligonucleotides with graphene oxide (GO). In this strategy, an EXPAR template is first mixed with GO. The EXPAR template is then attached on the GO surface to block secondary structure of the EXPAR template. Therefore, non-specific amplification could be efficiently eliminated caused by secondary structure of the EXPAR template. In the presence of the target oligonucleotide, the target could detach the EXPAR template from the GO surface that leads to amplify the target oligonucleotide as the same as original EXPAR. By employing this strategy, we successfully amplify target oligonucleotides without non-specific amplification. This method is possible to eliminate real-time monitoring of isothermal amplification of oligonucleotide, and end-point assay is enough to analyze target oligonucleotide amplification. Keywords : Isothermal amplification, EXPAR, Graphene oxide References 1. Van Ness, J., Van Ness, L. K. and Galas, D. J., P Natl Acad Sci USA, 100, 4504 (2003). P1028 Self-assembly of Laccase-Cu3(PO4)2.3H2O Hybrid Nanoflowers Based on Sonication Treatment and Its Application in Neurotransmitter Detection Bhagwan S. BATULE, Shreedhar GAUTAM, Hyun Gyu PARK Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701 The nanostructured biocatalysis creates great scope for improving stability of enzymes and expanding horizon for biosensors, bioanalytical devices, and industrial biocatalysis. Recently, the different approaches has been utilized to synthesis nanostructured materials. Also, there are several simple and effective methods for production of enzyme immobilized nanoflowers but the time required for synthesis is more. In this study, we reports a simple sonication method for the synthesis of protein-inorganic hybrid nanostructures with flower like shape, using protein as organic components and Cu3(PO4).3H2O as inorganic component [1]. The sonicated laccase nanoflowers have remarkable reusability and stability. We measured oxidative activity of the sonicated laccase nanoflowers using epinephrine, free laccase lost 50% of its initial activity within 8 days when incubated in PBS (pH 7.4) at 25 oC, but under the same conditions, sonicated laccase nanoflowers maintained most of their initial activity (~90%) upto one month. Further we applied sonicated laccase nanoflowers into the visual detection of epinephrine, which is used to treat anaphylactic shock, bronchial asthma, and organic heart disease and is used in cardiac surgery and myocardial infarction [2]. Especially, this works presents a very facile way for the synthesis of sonicated protein-incorporated hybrid nanomaterials for the applications in biosensor, bioanalytical devices, and enzyme catalysis. In addition, it has great application in biotechnology and biomedicine. Keywords : copper phosphate, laccase, epinephrine, hybrid nanomaterials References 1. B.S. Batule, K.S. Park, M.I. Kim, H.G. Park,Ultrafast sonochemical synthesis of protein-inorganic nanoflowers(2015), Int. J. Nanomedicine, 10, 137 2. X. Q. Lu, Y. Y. Li, J. Du, X. B. Zhou, Z. H. Xue, X. H. Liu, Z. H. Wang, A novel nanocomposites sensor for epinephrine detection in the presence of uric acids and ascorbic acids(2011), Electrochim. Acta, 56, 7261) P1029 Smartphone-based Bio-sensing Platform for Hydrogen Peroxide Producing Assays Using Fenton Reaction and Oxidase Activity of Nanoceria Shreedhar GAUTAM, Bhagwan S BATULE, Hyun Gyu PARK Dept. of Biomolecular and Chemical Engineering, KAIST, Daejeon, 305-701 Artificial enzyme mimics have attracted huge interest in various fields because of the various disadvantages that natural enzymes bear such as low stability, expensiveness, and sophisticated purification methods. Recently, we have exploited the oxidative activity of cerium oxide nanoparticles (nanoceria); it shows great potential in bio-sensing since the assay time is very short (less than 1 minute) and cerium oxide nanoparticles are very cheap.1 Nevertheless, it is rarely used and its research is at infant stage. In this paper, we report the first use of nanoceria for the detection of hydrogen peroxide and the assays that produce hydrogen peroxide as the byproduct. The oxidase activity of nanoceria can be tuned by the DNA irrespective of sequence. We have utilized this interaction of nanoceria and DNA coupled with the Fenton reaction for the colorimetric detection of hydrogen peroxide and assays generating hydrogen peroxide. The color-based reaction, which is function of hydrogen peroxide, is quantified by the smartphone with the aid of its application to find the unknown concentration directly. Furthermore, we take the advantage of the advanced functionalities of smartphone such as intuitive touch-display and internet connection to ease the detection strategy and make it more user-friendly. We displayed the information related to the need of hydrogen peroxide detection on the smartphone and the significance of the measured results. Therefore, this strategy coupled with the smartphone based detection system shows promise in the biochemical and clinical use. Keywords : Fenton Reaction, Smartphone, Nanoceria, Oxidase activity References 1. M. I. Kim; K. S. Park; H. G. Park, Ultrafast colorimetric detection of nucleic acids based on the inhibition of the oxidase activity of cerium oxide nanoparticles. Chemical Communications 2014, 50, 9577-9580. P1030 Smart Phone Assisted Paper Device Based Colorimetric Detection of Glucose by Using Silver Nanoprisms Pei LI, Bhagwan BATULE, Hyun Gyu PARK Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea As materials are reduced in size from the bulk to the nanoscale, they begin to exhibit new and unusual chemical and physical properties. Anisotropic Metal Nanoparticles with unique shape-dependent properties and functionalities can be utilized in a number of important applications ranging from catalysis to self-assembly to imaging to sensing to diagnostics to therapy and nano thin film and solar cell fabrication. Among various shapes of anisotropic nanoparticles, metal triangular nanoprisms have gained significant research interest owing to their remarkable optical, electrical and chemical properties. [1] The sharp tip morphology of AgNPRs is sensitive to etching agents (e.g H2O2). In the presence of etching agent, the tips of AgNPRs can be easily etched resulted in the shape transformation of AgNPRs which leads to a large absorbance wavelength shifting or decreasing. By utilizing this interesting characteristic of AgNPRs, various kinds of biological targets which provide etching agent both by themselves and through their biological pathway can be detected. In this study, we focused on a very important and common clinical indicator –glucose. The human body requires glucose for some of its most important functions. Problems with the amount of glucose in the blood result in serious complications that could lead to coma or even death if not corrected quickly. It is also a clinical indicator of diabetes. According to the World Health Organization, over 150 million people in the world were affected with diabetes in the year 2004 and it is expected to climb further to 366 million in 2030. The affected population has to be tested for blood glucose levels daily for an effective treatment. Thus, there is a great need to develop a simple, cost-effective, easy-to-use and disposable glucose sensor. Since glucose oxidase (GOx) catalyzes the oxidation of glucose to hydrogen peroxide and gluconic acid, the generated H2O2 can performed as an etching agent for AgNPRs. Addition of the glucose to the solution containing GOx and AgNPRs leads to the color changing from blue to colorless. By depositing the sample solution on the cellulose paper, the RGB value of the target spot can be analyzed by the assisting of the smart phone. Initially, by using the smart phone to take a picture of the sample spot to be analyzed then up loading the picture by cloud computing , finally the RGB value of the picture can be analyzed by specific software.[2] We believe, besides glucose multiple on-site biotarget analysis can be performed at the same time by utilizing this promising sensing system. Keywords : glucose , AgNPRs, smart phone, paper device, anisotropic nanoparticles References 1. El-Nour, K. M. A.; Eftaiha, A. a.; Al-Warthan, A.; Ammar, R. A., Synthesis and applications of silver nanoparticles. Arabian journal of chemistry 2010, 3 (3), 135-140. 2. Guan-Hua Chen†, Wei-Yu Chen‡, Yu-Chun Yen†, Chia-Wei Wang‡, Huan-Tsung Chang‡, and Chien-Fu Chen*†, Detection of Mercury(II) Ions Using Colorimetric Gold Nanoparticles on Paper-Based Analytical Devices, Anal. Chem., 2014, 86 (14), pp 6843–6849. P1031 Ultrasensitive Detection of Nucleic Acids Based on Poly (Thymine)-Templated Fluorescence CuNPs Kwan Woo PARK, Bhagwan S. BATULE, Hyun Gyu PARK Dept. of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701 Very recently, there has been a great interest in the synthesis of various fluorescence-emitting metal nanoparticles by utilizing DNA as their templates [1-4]. DNA-templated metal nanoparticles have been widely studied due to their future applications to the various bioassays. Especially, these metal nanoparticles have been developed as a promising fluorophores due to their cost-effectiveness compared to conventional organic fluorescent dyes. Among them, copper nanoparticles (CuNPs) have been extensively studied due to their easy synthesis [3, 4]. Utilizing both poly Thymine (T) scaffolds and reducing agent (ascorbate), fluorescence-emitting CuNPs are produced within 5 minutes [4]. Here, we developed ultrasensitive and selective nucleic acids detection utilizing poly (Thymine)-templated fluorescence- emitting CuNPs. This method shows superior sensitivity with a detection limit of 1.3 aM target synthetic nucleic acids under pure condition. It also reveals high selectivity for discriminating differences between similar nucleic acid sequences. This simple, cost-effective and fast strategy could be a potential tool for biomedical research and clinical diagnosis. Keywords : ultrasensitive nucleic acids detection, copper nanoparticles-based biosensor, DNA-templated metal nanoparticle synthesis References 1. H. C. Yeh, J. Sharma, J. J. Han, J. S. Martinez and J. H. Werner, Nano Lett. 10, 3106-3110 (2010) 2. G. Liu, Y. Shao, K. Ma, Q. Cui, F. Wu and S. Xu, Gold Bull. 45, 69-74 (2012) 3. A. Rotaru, S. Dutta, E. Jentzsch, K. Gothelf and A. Mokhir, Angew. Chem. Int. Ed. 49, 5665-5667 (2010) 4. Z. Qing, X. He, D. He, K. Wang, F. Xu, T. Qing, and X. Yang, Angew. Chem. Int. Ed. 52. 9719-9722 (2013) P1032 Fabrication of Facile and Efficient Carbohydrate Microarray for Carbohydrate-Protein Interaction Analysis Hyung Joon CHA1, Chang Sup KIM1,2, Jeong Hyun SEO1,3, Hye Ryoung HEO1 1Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784, 2School of Biotechnology and Graduate School of Biochemistry, Yeungnam University, Gyeongsan, 712-749, 3School of Chemical Engineering, Yeungnam University, Gyeongsan, 712-749 To analyze the carbohydrate-protein interaction functionally, we developed efficient and facile method for fabrication of functional and high-throughput carbohydrate microarray platform whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. By using the carbohydrate microarray, we efficiently analyzed interaction between Vibrio cholera toxin (ctx) and carbohydrates. These studies demonstrated that the functional carbohydrate microarray is suitable for in vitro analyzing diverse glycan-related biomolecule interactions and can be applied as an effective biosensor for toxin detection. In addition, we characterized new glycosyltransferase WbeW from V. cholerae O1 to contribute to prepare a glycan library with various structures and understand the glycosylation process of V. cholerae O1. Keywords : Carbohydrate, microarray, glycotransferase References 1. Kim, C.S., Seo, J.H., and Cha, H.J., Functional Interaction Analysis of GM1-Related Carbohydrates and Vibrio cholerae Toxins Using Carbohydrate Microarray (2012), Anal.Chem, 84, 6884-6890 2. Seo, J.H., Kim, C.S., Hwang, B.H., and Cha, H.J., A functional carbohydrate chip platform for analysis of carbohydrate-protein interaction (2010), Nanotechnology, 21, 215101 P1033 Biological Synthesis of Graphene-Metal Nanocomposites Using Plant Extract Jing WEN, Bipinchandra K.SALUNKE, Hyunseok KOO, Beom Soo KIM Dept. of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk, 28644 Graphene has attracted tremendous attention from both the experimental and theoretical scientific communities in recent years. Graphene has a large specific surface area, high electron mobility, high Young`s modulus, and high transparency. In this study, nanocomposites of silver/graphene (Ag/GNs), gold/graphene (Au/GNs), and manganese dioxide/graphene (MnO2/GNs) were synthesized using Xanthium strumarium plant extract. The nanocomposites were characterized by UV-vis spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, X-ray diffraction, Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), and Thermo-gravimetric analysis. Results showed that Au/GNs demonstrated higher peroxidase-like activities than the individual nanomaterials. Keywords : graphene, metal, nanocomposite P1034 Ag@Au Bimetallic Nanodendrite Substrate Based DNA Point Mutation Sensor Utilizing Toehold-mediated Surface-enhanced Raman Scattering Su-Nam JEONG1, Saetbyeol KIM2, Hoeil CHUNG2, So Young YOO1,3 1BIO-IT Foundry Technology Institute, Pusan National University, Busan, 609-735, 2Department of Chemistry, Hanyang University, Seoul, 133-791, 3Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan 626-770 A simple and effective surface enhanced Raman scattering (SERS) based DNA sensor utilizing toehold-mediated DNA displacement reaction as a target-capturing scheme has been demonstrated for the detection of Avellino Corneal Dystrophy which is caused by a single point mutation of BIGH3 gene. For a SERS substrate, Au-Ag bimetallic nanodendrites able to provide large numbers of hot spots and surface area were electrochemically synthesized and adopted in this study. The incorporation of both Ag and Au was to simultaneously secure demanding sensitivity and stability of the substrate and an optimal composition satisfying the need was determined. A double-strand sensing element composed of ‘a probe DNA (pDNA)’ complementary to ‘a target DNA (tDNA)’ and ‘indicator DNA tagged with a Raman reporter (iDNA-R)’ was conjugated on the optimized substrate. The conjugation made the reporter molecule close to the surface and induced improved generation of Raman signal. A tDNA released the pre-hybridized iDNA from pDNA via toehold-mediated replacement, which was confirmed by increase of percent intensity change with decrease of Raman signal according to the concentrations of tDNA. The concentration correlation was good and linear in the concentration range from 200 fM to 20 nM, and the achieved limit of detection (LOD) was 62.5 fM, which is superior to that of previously reported toehold- mediated DNA displacement reaction sensing schemes using other tagging methods such as fluorescence and electrochemistry. This study provides simple and effective diagnostic SERS platforms for more sensitive and stable detection of various DNA point mutations, leading to possible diagnosis of point mutation-related diseases including Avellino corneal dystrophy reported here. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Government (2013R1A1A3008484 and 2014S1A2A2027641)]. Keywords : DNA sensor , surface enhanced Raman scattering (SERS), Au-Ag bimetallic nanodendrite, DNA point mutation, BIGH3, Avellino Corneal Dystrophy References 1. D. Y. Zhang and G. Seelig, Nat. Chem. 3, 103-113 (2011).