BIO 1: Enzymatic Biodiesel Chairs: H.C

Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 1: Enzymatic Biodiesel Chairs: H.C. Holm, Novozymes A/S, Denmark; and R. Burton, MARC-IV, Inc., USA

Technical Considerations for Biodiesel Production feedstocks. The recent results from a commercial Using Enzyme Catalysis. R. Burton, MARC-IV, Inc., continuous unit for the enzymatic production of Pittsboro, NC, USA. biodiesel using high FFA’s multiple feedstocks will The search for alternative catalysts for the also be presented. production of biodiesel has been of significant interest to industry. Enzyme Catalyzed Biodiesel Using Liquid Lipases. One primary reason to replace conventional P.M. Nielsen, Novozymes A/S, Bagsvaerd, Denmark. alkaline catalysis is to eliminate soap waste streams During the last year the enzymatic in commercial production. Furthermore, utilizing transesterification using liquid lipase (the BioFAME enzymes in the processing of fatty acid esters can process) has been proven in full production scale in eliminate waste water streams, enhance the co- two plants in USA. The learnings from these plants product quality of glycerol, and provide the ability to are shared in this presentation in the discussion of use lower quality feedstocks. These low quality how to control the process to get biodiesel from feedstocks like yellow grease with higher free fatty used cooking oil and DDGS corn oil. The most acid (FFA) content are largely underutilized for important parameters have been: biodiesel due to the difficulty of processing these  Securing correct pretreatment to keep the types of oils. This paper will evaluate the real world enzyme stable experiences of an enzymatic biodiesel plant. This  Controlling the methanol addition rate operation has developed biodiesel production  Recover the heavy phase and re-use the techniques using the immobilized enzymes Candida enzyme Antarctica Lipase B (CALB), as well as liquid enzyme  Downstream processing formulations for the production of fatty acid esters. We will describe the details in controlling the reaction and document the BioFAME process. The A New Immobilized Lipase for Industrial Production robustness for tolerance of changes in FFA will be of Biodiesel. S. Basheer, TransBiodiesel Ltd., Shfar discussed, as well as the perspectives and how to am, Israel. operate with different types of oil qualities. Lipases have been proposed since more than a decade for the production of fatty acid short-chain Effect of Phospholipids on Free Lipase-mediated alkyl esters for use as biodiesel. Either esterification Biodiesel Production. W. Du, Tsinghua University, of free fatty acids and a short-chain alcohol or Beijing, China. transesterification of glycerides and a short chain Free lipase-catalyzed biodiesel has drawn more alcohol, typically methanol, have been and more attentions in recent years because of its predominantly applied for enzymatic production of advantages of lower cost and faster reaction rate. biodiesel. Reported results showed that different Utilizing free lipase to convert low quality oils, such formulations of various native lipases exhibited high as crude vegetable oils and microbial oils, is degree of intolerance to methanol typically used in beneficial to further reduce the cost of biodiesel the esterification/transesterification reactions of production. However, these oils typically contain different oil feedstocks to produce biodiesel. In some amount of phospholipids. Phospholipids were addition to inhibition of the catalytic activity caused found to affect the lipase-catalyzed process and by methanol, the use of commercially available further influence the enzyme’s thermal stability in immobilized lipases for the production of biodiesel biodiesel production process. In this work, free has faced severe mass transfer constraints. This lipase NS81006-mediated biodiesel production from work will present a new developed immobilized oils containing phospholipids at varied temperature enzyme preparation characterized with its high was investigated systematically. It was found that activity for the catalysis of both esterification as well increasing temperature led to a decreased fatty acid as transesterification reactions, simultaneously. methyl esters (FAME) yield and poor reuse stability Based on the developed enzymatic process different of the lipase in converting oils containing pilot model units have been designed and built for phospholipids for biodiesel production. Further the production of biodiesel using high FFA’s multiple study showed that this inhibitory effect of

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

temperature was mainly attributed to the universal concerns for biodiesel producers, such as coexistence of phospholipids and methanol in the feedstock diversity and crude biodiesel refining to system. A novel two-step enzyme-mediated process meet ASTM specs, but because of the sometimes was developed, with which the above-mentioned subtle differences in the enzymatic reaction, unique inhibitory effect was eliminated, and a FAME yield of solutions are required to address these concerns. 95.1% could be obtained with oils containing 10% Specifically, these unit operations include the phospholipids as the feedstock. recovery and reuse of the enzymes to catalyze multiple batches, the reduction of FFA from the Large Scale, Enzyme-catalyzed Methyl Ester crude biodiesel, and proving the use of the lowest Manufacturing—Justification and Execution. B. quality feedstocks. In the research department at Baughman, Blue Sun Biodiesel, Wheat Ridge, CO, Viesel Fuels, each of these processes is at different USA. stages of development, implementation, and Practical focus on the challenges facing optimization. The results presented offer great investors/owners/engineers choosing to engage the promise of making the large scale enzymatic Novozymes enzymatic catalyst in methyl ester biodiesel process both technically and commercially manufacturing. Substance is based on decades of feasible. practical experience designing, building, operating and maintaining industrial manufacturing facilities in Biodiesel Through Enzymatic Transesterification: the oleo and renewable fuels sectors and the recent The Ecological and Economical Alternative for Low commercialization of the Novozymes enzyme Quality Oil Feedstocks. M. Kellens, Desmet Ballestra catalyst at 30 mgy scale. Practical discussion of the Group, Zaventem, Belgium. six pillars of manufacturing: Regulatory Compliance The increasing tendency to use low quality oils Plan, Volume/Logistics Plan, Quality Plan, Capital as feedstock for biodiesel production has triggered Plan, Expense Plan, HR/HR Organizational the development of various chemical routes to make Development Plan. them suitable for the conventional, mostly alkaline based chemical transesterification process. High FFA From Promise to Practice: Real-life Advances in oils are pre-esterified using strong acids, special Enzymatic Biodiesel. K.P. Staller, Patriarch Oleo, Inc., resins, or bound with glycerine, after which they Garrett, IN, USA. then undergo a further transesterification to convert Enzymatic biodiesel was brought to a all fatty acids into their methylester form. commercial reality this past year. Characteristically, The development of robust Lipase enzymes, the path to commercial operation turned out to be able to attack fatty acids in both free and bound far more difficult than anticipated, but this form, has made it possible to convert low quality presentation will show how the obstacles were high acidity feedstocks into biodiesel with minimum overcome. pretreatment. The enzymatic process has been proven to work Utilization of Liquid Lipase on a Commercial Scale. on used cooking oils, fatty acid distillates and high R. Hobden, Viesel Fuel, LLC, Stuart, FL, USA. acidity oils like DGS corn oil. But the main question In the transition for enzymatic biodiesel from to answer is how to make the enzymatic process lab/pilot scale to commercial scale, not only does cost-efficient enough to compete with the other the catalytic reaction need to be evaluated, but the routes. Some comparisons are made and examples supporting unit operations need also be optimized given to illustrate the true potential of the enzymatic to make enzymatic biodiesel a real option for process. biodiesel production. Many of these processes are

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 1.1/S&D 1.1: Merging Biology and Surfactants Chairs: D.G. Hayes, University of Tennessee, USA; G.A. Smith, Huntsman Corp., USA; and D. Solaiman, USDA, ARS, ERRC, USA

Sophorolipids: Microbial Synthesis and and expanding the structure. Biotechnological Opportunities. I.N.A. Van Bogaert Starmerella bombicola glycosylates long-chain and W. Soetaert, Ghent University, Ghent, Belgium. omega-hydroxy fatty acids and it also directly Sophorolipids are surfactants which are glycosylates secondary alcohols. Although it is produced by specific yeast species, such as difficult to directly glycosylate primary alcohols, they Starmerella bombicola. Thanks to the high yields of are easily converted to the corresponding fatty acid. this biological process (over 400 g/L can be obtained) To redirect unconventional substrates toward APG and the environmentally friendly character of these synthesis, the long-chain alcohol oxidation pathway biosurfactants, they caught the attention of several was blocked at the genome level by knocking out the academic institutes and later on companies. Indeed, fatty alcohol oxidase gene. The total gene sequence sophorolipids are one of the few biosurfactants of the S. bombicola AOX1 (2046 bp) was cloned. which are commercially produced and applied. Knock-out mutants were evaluated by fermentation Although certain microbial biosurfactants are in 1-tetradecanol. The mutants produced much already find applications, their usage range could be higher amounts of APG, indicating that the substrate further broadened if more structural variants would had been redirected toward APG synthesis in those be available, in this way creating molecules with strains. different physoco-chemical properties and functions. Yet, creating variation in a biological controlled Incorporation of Membrane Proteins in the process is not as easy as modifying a chemical Bicontinuous Microemulsion Phase of a Winsor-III reaction; rendering the creation of tailored System. D.G. Hayes2, R. Ye2, V.S. Urban1, S.V. biosurfactants quite a challenge. Pingali1, and H. O'Neill1, 1Oak Ridge National We try to tackle this shortcoming by a combined Laboratory, Oak Ridge, TN, USA, 2University of fundamental and applied approach: getting insight Tennessee, Knoxville, TN, USA. into the biosynthetic pathway and its regulation, Membrane proteins (MPs) account for control and engineer it. In order to achieve this, we approximately 33% of proteins encoded by the de novo sequenced the full genome of the yeast and human genome, and are targets for 50-60% of set up extensive transcriptomics and proteomics therapeutical agents. They are difficult to experiments. We will discuss the identification of the characterize due to their poor solubility in aqueous sophorolipid core pathway and will give several media, and therefore account for only 3% of three- examples of how it can be engineered to produce dimensional structures within the Protein Data Bank. tailored glycolipids. We have successfully incorporated several different MPs (e.g., cytochrome c, melittin, phtotosystem II, Identification of Long-chain Alcohol Oxidase AOX1 and light harvesting complex I and II) Iinto the from Starmerella bombicola and Knocking Out the middle, bicontinuous microemulsion (Bicont ME), AOX1 Gene Leads to Improved Alkyl Polyglucoside phase of Winsor-III systems at concentrations > 1 Production. F. Takahashi, K. Igarashi, and H. g/L, formed using Aerosol-OT / 1,3-dioxolane-based Hagihara, Eco-Innovation Research, Kao Corp., alkyl ethoxylate binary surfactant system. Unlike Wakayama-shi, Wakayama, Japan. other surfactant self-assembly systems employed for Alkyl polyglucoside (APG) has good properties as MP solubilization, the Bicont ME phase is optically a cleaner, foamer, and emulsifier, and does not transparent, isotropic, and contains surfactant hydrolyze at an alkaline pH. In addition to its monolayers of near-zero curvature, to reduce advantages as a traditional alkylglucoside surfactant, denaturation. The MP-laden Bicont ME phases have APG is a low-irritant surfactant that is nontoxic and been investigated by circular dichroism to analyze easily degradable in the environment. Thus, APG is for perturbations in the MPs' secondary structure, considered an environmentally friendly surfactant. and by small-angle neutron scattering (SANS) to We have been working to develop new APG determine structural perturbations of the phase's synthesis methods aimed at decreasing the cost as nanostructure. Via SANS we determined that the well as the burden on the environment and facility, incorporation of MPs reduces the average diameter

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

of the Bicont MEs' nanochannels and increases the surface and self-assembling properties. It exhibited a surface area per volume of the MEs. dominantly low CMC (critical micelle concentration) value of 0.0003%. It was also found that the Surfactants Based on Algae Oil. G.A. Smith and M. molecules spontaneously formed giant aggregations Coleman, Huntsman Corporation, The Woodlands, in aqueous solution by freeze-fracture electron TX, USA. microscopy (FFEM). Undoubtedly, it has many Modern day surfactants are based on natural, interesting potential applications in a variety of fields petrochemical, or a combination of natural and such as cosmetics and homecare industries. petrochemical feedstocks. With the recent emphasis on sustainability, surfactants based on natural Production and Antimicrobial Property of Glycolipid feedstocks are of considerable interest. Typically, Biosurfactants. D.K.Y. Solaiman, R.D. Ashby, and J.A. natural surfactants are based coconut or palm oil. Zerkowski, USDA, ARS, ERRC, Wyndmoor, PA, USA. Both of these materials are also used for food Microbial glycolipids such as rhamnolipid (RL) production. An alternative feedstock which is not and sophorolipid (SL) are an important class of used for food is algae. There are thousands of biosurfactants with excellent surface tension- different algae species which can grow in fresh or lowering activity. Besides their surfactant- and salt water. environment-friendly properties, however, Work was performed to optimize the growth additional value-added property such as conditions for chlorella vulgaris algae in bacteriocidal activity is needed to further heighten photobioreactors (PBR). Different light frequencies their commercial interests. We devised a and fertilizer concentrations were varied to achieve straightforward production scheme and HPLC the optimum growth conditions. In order to analysis method for synthesis and characterization maximize the lipid yield, the algae were stressed and of monorhamnolipid (R1L) and sophorolipid (SL) the lipids extracted. The oil was used to make a produced from patent-strain Pseudomonas variety of different nonionic surfactants. Surface chlororaphis NRRL B-30761 bacterium and properties and detergency measurements were Rhodotorula bogoriensis yeast, respectively. R1L and performed. SL were then incorporated into biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Superior Characteristics of Biosurfactant Surfactin (PHBV) films. Tests on acne-causing bacterium (i.e., and Its Application Potentiality. S. Yanagisawa1, T. Propionibacterium acnes) and on tomato-rotting Nagano1, M. Izumida1, T. Imura2, T. Taira2, and D. causative agent (i.e., Pseudomonas corrugate) Kitamoto2, 1New Business Development Division, showed that these glycolipids possessed excellent Kaneka Corporation, Osaka, Japan, 2Research antimicrobial activity. The finding adds to the Institute for Innovation in Sustainable Chemistry, antimicrobial spectrum of RL and SL, thus further National Institute for Advanced Science and increasing the potential commercial values of Technology, Tsukuba, Japan. glycolipid biosurfactants. Surfactin (SF) is one of the most promising biosurfactants produced by fermentation under mild Production of Rhamnolipids from Unique conditions with naturally derived raw materials and Pseudomonads. G. Deluga, D. Derr, N. Lohitharn, D. a type of safe microorganism, Bacillus subtilis. It is MacEachran, J. Ulrich, W. Jordan, and R. Mirani, produced and currently commercially available as Logos Technologies, Ashburn, VA, USA. sodium salt (Kaneka Corporation). This sustainable The promise of biosurfactants has a long and product is both human- and environment-friendly. storied history of having commercial potential (1). Results of skin irritation test show that it is much One of the major classes of biosurfactants is milder than other typical surfactants. In fact, it glycolipids that have the potential to replace a showed no observable skin irritation at generally number of petroleum based surfactants due their used concentrations. Moreover, it was degraded sulfate and phosphate free nature while having almost completely within a week in the superior surfactant properties. This class of biodegradation test. glycolipids are characterized by either a mono or di The structure of SF is unique compared to that rhamnose head group and a 3- of other surfactants. SF has a heptapeptide head (hydroxyalkanoyloxy)alkanoic acid tail that varies in group interlinked with a ß-hydroxy fatty acid. Due to molecular weight according to a number of factors its unique cyclic peptide structure, it shows excellent including feedstock, fermentation time and

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

Pseudomonas strain. We have developed three presentation summarizes our recent progress in 1) strains of Pseudomonads that produce rhamnolipids production of biosurfactants via high-cell density at high titers. fermentation; and 2) enzymatic synthesis of This presentation will discuss the three strains phosphatidyl saccharides for preparation of of Pseudomonads that have been isolated for the nanoliposomes with enhanced stability. overproduction of both mono- and di-rhamnolipids. Sophorolipids have received special attention Each strain has a unique rhamnolipid molecular due to their biodegradability, low toxicity, and weight profile for a specific feedstock. Results will be production from renewable resources. To produce presented discussing how these two variables sophorolipids with high time-space efficiency, a high- interact. Data demonstrating a correlation between cell-density fermentation strategy was applied. The the molecular weight distribution of the rhamnolipid nutrient concentration and physical parameters isolates and the CMC will be presented. These were optimized for enhanced volumetric biosurfactants have the characteristics and can meet productivity. The protocol was implemented in a 10- the cost targets necessary for large scale L fermentor and a remarkably high volumetric commercialization. productivity (>200g isolated sophorolipids/L/day) (1) Desai JD, Banat IM (March 1997). Microbiol. was achieved. Mol. Biol. Rev. 61 (1): 47–64. Derivatization can tailor surface-active lipids to specific applications. Phospholipids are known to Biosynthesis of Surface-active Lipids. M. Falkeborg form liposomes capable of encapsulating sensitive (Honored Student Award Winner and The ingredients; however, drying processes are usually Manuchehr Eijadi Award Winner), R. Gao, S. Song, required and cryoprotectants are needed to stabilize L.Z. Cheong, X. Xu, and Z. Guo, Dept. of Engineering, the liposomes. To overcome this, an enzymatic Aarhus University, Aarhus, Denmark. method for incorporating sugar moieties into Surface-active lipids find applications in food phospholipids was developed. The resulting and pharmaceutical industries and the interest for phosphatidyl saccharides were capable of forming their economically feasible and ecologically friendly liposomes with increased stability during production and derivatization is increasing. This dehydration.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 2: Biocatalysis I Chairs: C.T. Hou, USDA, ARS, NCAUR, USA; and T. Nagao, Osaka Municipal Technical Research Institute, Japan

Extensive Fatty Acid Specificity Analysis of Various capability to synthesize PGE even in organic solvents, Lipases on Hydrolysis Using Randomized and in particular, lipase OF (Candida cylindracea) Triacylglycerol Prepared with Wide Range Variation could synthesize monoester with high selectivity in of Fatty Acids as Substrate. T. Nagao, S. Tanaka, and tert-butyl alcohol. We next tried to optimize some H. Nakano, Osaka Municipal Technical Research reaction conditions to increase the reaction rate. Institute, Osaka, Japan. Then we found that initial water content in the Lipase has been used as a catalyst in many reaction system significantly affected the reaction purposes, such as production of PUFA-rich oils and rate. Consequently, we were able to acquire the purification of PUFAs. Lipases possess several high-purity polyglycerin fatty acid monoester in a substrate specificities, and among these specificities, relatively short reaction time. fatty acid specificity is the most important property for practical applications. We thus evaluated fatty Production of Biodiesel from Acid Oil, a Low Value acid specificity of 11 commercial available lipases on Feedstock via a Two-step Enzymatic their initial hydrolysis reaction using two random Transesterification. N. Choi1,2, J. Baik1,2, S.W. Yoon1,2, esterified triacylglycerols prepared with wide range N.H. Kim1,2, and I.H. Kim1,2, 1Dept. of Food & variation of fatty acids (fatty acids from C4 to C18, Nutrition, Korea University, Seoul, Republic of Korea, and fatty acids from C14 to C24 including PUFAs) as 2Dept. of Public Health Sciences, Graduate School, substrates. Korea University, Seoul, Republic of Korea. Candida rugosa lipase showed weak activity A two-step enzymatic transesterification in a against C4:0, C6:0, GLA, and fatty acids composed of solvent-free system was attempted as a novel C20 and >C20. Rhizomucor miehei and two Rhizopos approach to produce biodiesel using two different lipases acted effectively against fatty acids lipases. This study involved an employment of a low composed of C20 and >C20, but not against C4:0, value feedstock and an appropriate combination of GLA, and DHA. Pseudozyma antarctica (re-classified two different lipases. Acid oil prepared from rice from Candida antarctica) lipases A and B showed bran oil soapstock and ethanol as a renewable acyl strong activity against wide range fatty acids acceptor were used as substrates. Three immobilized including PUFAs except long chain saturated fatty lipases, namely Novozym 435 from Candida acids. Fatty acid specificity of Penicillium roqueforti, antarctica, Lipozyme RM IM from Rhizomucor Alcaligenes sp., Burkholderia cepacia, Pseudomonas miehei, and Lipozyme TL IM from Thermomyces aeruginosa, and pancreatic lipases were also lanuginose were employed as biocatalysts. Firstly, evaluated. the optimum conditions for the three lipases were determined to be 1:5 of the molar ratio (acid oil to Synthesis of Polyglycerin Fatty Acid Esters by Lipase thanol), 30-40oC for the temperature, and 5-10% for Reactions. Y. Nishiyama, Y. Kataoka, H. Uehara, and the enzyme loading. A two-step transesterification Y. Ueda, The Nisshin OilliO Group, Ltd., Yokosuka, was carried out under the optimum conditions of Kanagawa, Japan. individual lipases obtained. For the order of using Polyglycerin fatty acid ester (PGE) has various lipases in this two-step reaction, high cost lipases features depending on its degree of esterification. such as Novozym 435 or Lipozyme RM IM were Generally, PGE available in the market is made by employed at the first step for an extremely short chemical synthesis methods, and it is known that the reaction time (ca. 15 min). Subsequently, the number of fatty acids esterified to a polyglycerin remaining reaction was carried out using Lipozyme molecule varies around some central value. On the TL IM as a low cost lipase. In conclusion, more than other hand, PGE can be synthesized also by lipase 90% yield of biodiesel was obtained successfully reactions, so we may be able to synthesize PGE within only 8 h of reaction time via the two-step whose degree of esterification is narrowly lipase-catalyzed transesterification. distributed via the lipase’s selectivity. First, we carried out the screening of lipases Genetic Engineering of Oil Palm: Challenges and the which could synthesize PGE from polyglycerins and Way Forward. G.K.A. Parveez, A.R. Omar, M.Y. Abdul fatty acids. Then we found that some lipases had the Masani, I.A.M. Dayang, B. Bahariah, S.M. Masura,

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

N.A. Hanin, W.N.W.S. Syuhada, U.S. Ramli, A. were identified as Acinetobacter haemolyticus. The Othman, A.M. Muhammad Arif, I. Zamzuri, A.H. rest of the strains belong to the genus Pseudomonas. Tarnizi, A. Kushairi, S. Ravigadevi, Malaysian Palm Oil Board, Persiaran Institusi, Kajang, Selangor, Enhancing the Thermostability of Engineered Malaysia. Phospholipase D. Y. Iwasaki, J. Damjanovic, and H. Genetic engineering with all the promises and Nakano, Nagoya University, Nagoya, Aichi, Japan. advantages was considered and implemented almost Transphosphatidylation catalyzed by 20 years ago with the objective of keeping the palm phospholipase D (PLD) can be applied for the oil industry remains competitive. Successful synthesis of various phospholipids. Wild-type production of transgenic oil palm using various Streptomyces PLD could not synthesize methods and selection systems in addition to the phosphatidylinositol (PI), but our recent studies have isolation of useful genes and tissue-specific and created mutant PLD having PI-synthesizing activity. constitutive promoters have push forward the The purpose of this study is to enhance the genetic engineering programme towards producing thermostability of the PI-synthesizing PLD for better novel and higher quality palm oil. Among the targets performance in the PI synthesis. for genetic engineering of oil palm are increasing We first employed combinatorial mutagenesis oleic acid, ricinoleic acid, palmitoleic acid and stearic followed by high-throughput screening. Random acid content, enhancing lycopene content and mutations were introduced into selected positions synthesizing biodegradable plastics. In order to having high B-factor. Screening of the libraries under produce the above targeted products using the restricted conditions yielded several single-point isolated genes and promoters, constructions of mutants. Combining these point mutations yielded various combinations of transformation vectors double and triple mutants; one of which, carrying different genes and promoters for specific D40H/T291Y was revealed to be the most efficient in targets have been successfully carried out. the PI synthesis at high temperature. Transformation of oil palm embryogenic calli Next, we employed rational design approach. with the above constructs has been successfully The residue D40 is part of a nine-residue surface carried out and resulted in a number of transgenic loop, which is solvent-exposed and highly palms that have been transferred onto soil in a fluctuating. Thus, we tried to enhance the stability contained greenhouse. Some of the challenges faced by removing this unstable loop. Mutant enzymes during the research and potential solution will be lacking the loop showed better thermostability; the elaborated. best variant showed 11.7 times longer activity half- life at 70°C than that of the parent. The deletion Production of Soy Polyol Oils: Results of Microbial variants showed twofold higher PI yield in high- Screening and Identification of Positive Cultures. temperature PI synthesis, while at the same time C.T. Hou1, D.P. Labeda2, and K. Ray1, 1RPT, NCAUR, produced less of the hydrolytic side-product, USDA, Peoria, IL, USA, 2BFP, NCAUR, USDA, Peoria, phosphatidic acid. IL, USA. Triacylglycerols (TAG) containing hydroxy fatty Liposomes as Drug Delivery Carrier for Manganese acids has many industrial uses such as the Porphyrin-based Anticancer Drug. T. Aikawa1,2, S. manufacture of aviation lubricant, plastic, paint, Ito1, T. Kondo1,2, and M. Yuasa1,2, 1Dept. of Pure and nylons and cosmetics, because of the hydroxyl Applied Chemistry, Faculty of Science and groups on the fatty acid (FA) constituents. Technology, Tokyo University of Science, Noda, Diacylglycerols (DAG) containing hydroxy FA can also Chiba, Japan, 2Research Institute for Science and be used in the above mentioned industries. Soy- Technology, Tokyo University of Science, Noda, polyols (oxygenated TAG) are important starting Chiba, Japan. materials for the manufacture of polymers such as Our group has developed manganese porphyrin polyurethane. Recently we developed a new method derivatives as an anticancer drug based on anti- to screen microorganisms for the direct production oxidation effect, which can eliminate and exploit of polyol oils from soybean oil. Of the 650 cultures reactive oxygen species generated in cancer cell. To screened, 50 were active in converting soybean oil to effectively accumulate the manganese porphyrin- polyol oil and DAGs products. The 11 most active based anticancer drug into targeted tumor, we strains were identified based on the sequences prepared liposome-based drug carrier that can determined for their 16S rRNA genes. Two strains incorporate the porphyrins in its bilayer membrane.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

The liposome composed of 1,2-distearoyl-sn-glycero- diffusion coefficients in PHB polymers derived from 3-phosphoethanolamine (DSPE) conjugated with fermentations containing higher MeOH contents transferrins(Trfs). Since receptors of Trf are demonstrating a reduction in PHB molecular overexpressed in cancer cell, the liposomes modified weights. While new uses for crude glycerine with Trf are expected to accumulate specifically in continue to generate interest, it is important to cancer cell. Size of the liposomes prepared by understand the effect of all molecules present in the conventional sonication method was ~100 nm. Their crude glycerine on the final products. This discussion surface charge was approximately zero (-0.2 –+0.4 will focus on the effects of methanol on the mV). Flow cytometry analysis clearly showed that fermentation-derived PHB biopolymers hepatic cancer cells tended to uptake the liposomes modified with Trfs compared to those without Trfs. Novel Strategy for Lipase-catalyzed Synthesis of In addition, confocal microscopy revealed that the Biodiesel Using Blended Alcohol as an Acyl liposomes were uptaken through endocytosis. In Acceptor. T.T. Zhao1,2, D.S. No1,2, and I.H. Kim*1,2, vivo analysis, the liposome modified with Trf 1Dept. of Food & Nutrition, Korea University, Seoul, significantly reduced the volume of tumor Republic of Korea, 2Dept. of Public Health Sciences, transplanted in mice compared to liposomes without Graduate School, Korea University, Seoul, Republic Trf. The liposome system in this study is expected to of Korea. enhance efficacy of incorporated drugs without side As a novel strategy, blends of methanol and effects. ethanol were used as acyl acceptors for biodiesel synthesis from soybean oil by lipase-catalyzed Poly(3-hydroxybutyrate) Synthesis from Crude transesterification. Based on enzyme screening, Glycerine: The Methanol Effect. R.D. Ashby, D.K.Y. Novozym 435 from Candida antarctica and Lipozyme Solaiman, and G.D. Strahan, USDA, ARS, ERRC, TL IM from Thermomyces lanuginosa were selected Wyndmoor, PA, USA. for the reaction. For optimization of the reaction, Polyhydroxyalkanoates (PHAs; bacterial the effects of the molar proportion of methanol in polyesters) can be synthesized from refined and the blends, temperature, and enzyme loading were crude glycerine under suitable fermentation studied. The two enzymes displayed similar reaction conditions. The composition of crude glycerine trends in the molar proportion of methanol in the generally varies based on reaction efficiency and blends. When temperature was increased from 30 to product recovery but usually contains (in addition to 50°C, no significant different in the yield of biodiesel glycerine), small amounts of partial glycerides, FFAs, was observed with Novozym 435, whereas there was water and residual short-chain alcohol (usually a rapid decrease in the yield of biodiesel with MeOH). Pseudomonas oleovorans NRRL B-14682 Lipozyme TL IM. For enzyme loading, Novozym 435 produces poly(3-hydroxybutyrate) (PHB) from gave the highest yield of biodiesel (=95%) with 5 - glycerine however; the concentration of MeOH 10% (based on the weight of substrate). Meanwhile, present in crude glycerine-based fermentations has Lipozyme TL IM showed a significant increase in the a reductive effect on PHB molecular weights. We yield of biodiesel with higher enzyme loadings and have formulated a series of glycerine-based the highest yield of biodiesel (=95%) was obtained fermentations containing MeOH (conc. from 0 to 1 when enzyme loading was over 15%. Additionally, wt%) and characterized the properties of the the relative consumption rates of methanol and resulting PHB polymers. 1H NMR revealed that ethanol during the transesterification using the MeOH acts as a chain terminating agent causing a blends were investigated. For both enzymes, the premature halt to polymer elongation. Diffusion reactivity of methanol was significantly higher than ordered spectroscopy (DOSY) revealed decreasing that of ethanol.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 3: Biocatalysis II Chairs: S.H. Yoon, KFRI, Korea; and J. Ogawa, Kyoto University, Japan

Application of Cytochrome P450BM-3 for the concentration were 21.10, 13.25 and 3.14%, Conversion of Eicosapentaenoic Acid to Its Epoxide respectively. Hydroxyl radical scavenging activities of Derivatives. J. Ogawa1, C. Ishikawa1, T. Kimura2, S. those samples tested at 1mg/mL concentration were Kishino1,2, and M. Hibi2, 1Division of Applied Life 86.08, 93.30 and 93.17%, respectively. Refining Sciences, Graduate School of Agriculture, Kyoto process seemed to remove the phenolic compound University, Sakyo-ku, Kyoto, Japan, 2Laboratory of during oil processing. Antiradical substances in Industrial Microbiology, Graduate School of perilla and sesame oils responsible for scavenging Agriculture, Kyoto University, Sakyo-ku, Kyoto, DPPH radicals were present in the methanol fraction, Japan. while the antiradical substances in the sunflower oil We screened P450BM-3 mutants for conversion were in the lipid fraction. DPPH scavenging activity of eicosapentaenoic acid (EPA) under aerobic of ME of sesame oil was significantly higher than condition with NADPH. Through the screening, some that of perilla oil. Superoxide anion scavenging mutants were found to produce several products capacity of ME of perilla oils was found to be greater (UK1, 2, 3, 4). Then, these products were purified than that of both sesame and sunflower oils. and identified with LC-MS, NMR and GC-MS. Finally, these products were identified: UK1 was Manipulation of Lipid Synthesis in the 14,15:17,18-diepoxy-eicosatrienoic acid Hydrocarbon-producing Green Alga Botryococcus (14,15:17,18-DEpETr), UK2 was 17,18-epoxy- braunii, a Potential Biodiesel Producer. I.A. eicosatetraenoic acid (17,18-EpETe), UK3 was 14,15- Guschina and J.L. Harwood*, Cardiff University, epoxy-eicosatetraenoic acid (14,15-EpETe), UK4 was Cardiff, Wales, UK. 11,12-epoxy-eicosatetraenoic acid (11,12-EpETe). Botryococcus braunii, a green colonial The reaction conditions were optimized with microalga, is an unusually rich renewable source of P450BM-3 mutants, and under the optimized hydrocarbons (ranging from 15 to 75% of dry conditions, mutant Al4_Ile converted 0.5 mg/ml EPA weight). Three distinct races of B. braunii have been to 0.20 mg/ml 11,12-EpETe (conversion rate: 38.0% described (A, B, and L) based on the hydrocarbons mol/mol). Mutant F87A converted 0.5 mg/ml EPA to accumulated. Such organisms are believed generally 0.19 mg/ml 14,15-EpETe (conversion rate: 36.1% to be similar to those giving rise to the world’s mol/mol). Wild type P450BM-3 converted 0.5 mg/ml petrochemical deposits. EPA to 0.38 mg/ml 17,18-EpETe (conversion rate: A better understanding of the metabolic 72.2% mol/mol). Mutant L7V converted 0.5 mg/ml reactions involved in lipid biosynthesis in B. braunii is EPA to 0.075 mg/ml 14,15:17,18-DEpETr (conversion needed in order to manipulate and direct carbon rate: 13.5% mol/mol). fluxes into increased hydrocarbon production. For this we have used radiolabelled precursors such as Production and Application of Biofunctional Lipids. 14C-acetate, 14C-glycerol and 14C-glucose and S.H. Yoon, Korea Food Research Institute, measured their incorporation into polar Seongnam-si, Kyunggi-Do, Korea. glycerolipids, non-polar triacylglycerols and Stereospecific analysis of fatty acid distribution hydrocarbons. These lipid fractions were separated of perilla oil was carried out. Linolenic acid was the into individual classes and radio-incorporation major fatty acid in the sn-1, 2 and 3 positions. SC- quantified. A series of compounds were then used to CO2 extraction at 420 bar and 50°C and hexane manipulate key reactions involved in the extraction showed significantly higher oil yield than biosynthesis. Both specific and non-specific effects mechanical press extraction. Fatty acid compositions were shown and their consequences will be in the oils were virtually identical regardless of the discussed. These data provide important information extraction methods. SC-CO2-extracted perilla oils on lipid accumulation in B. braunii and, as such, will contained significantly higher contents of be very useful for lipid manipulation and metabolic tocopherols, sterols, and policosanols than the engineering of this potential industrial alga. mechanical press-extracted and hexane-extracted oils. Superoxide anion scavenging activities of ME of Utilization of Seaweed Biomass for Functional Lipid perilla, sesame and sunflower oil tested at 1 mg/mL Production. K. Arafiles, Y. Eramoto, H. Iwasaka, Y.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

Okamura, Y. Matsumura, Y. Nakashimada, and T. DGA2yl expressing strains had tendency to form one Aki*, Hiroshima University, Higashi-Hiroshima, or two large LBs. Similar acyltransferase mutants Japan. with deleted MFE gene (coding key enzyme involved Marine biomass has been paid attention to as a to beta-oxidation) and SUC2 (invertase gene) have sustainable resource for the production of also been constructed. renewable energy and value-added materials. We The work was supported by grants VEGA aimed at producing functional lipids such as 1/0975/12 and APVV-0662-11. polyunsaturated fatty acids, xanthophylls, and terpenoids from seaweed saccharides by using Enhanced Antimicrobial Activity of Monoglyceride marine protists, thraustochytrids, as biocatalyst. of 7,10-dihydroxy-8(E)-octadecenoic Acid. H.R. Kim1, Assimilation test revealed however that C. Dasagrandhi2, H.R. Son1, F. Wang1, and C.T. Hou2, thraustochytrid strains from the genus 1School of Food Science and Biotechnology, Aurantiochytrium do not have ability to directly Kyungpook National University, Daegu, Korea, utilize such algal saccharides for their growth. Thus, 2Renewable Product Technology Research Unit, a number of bacteria and fungi that can degrade and National Center for Agricultural Utilization Research, convert algal saccharides into suitable substrates for ARS, USDA, Peoria, IL, USA. Aurantiochytrium were obtained from culture Structural modification of lipids via chemical collection or isolated from natural environment, to reaction or microbial bioconversion can change their serve as intermediate biocatalysts. By cultivating in properties or even create novel functionalities. media composed of culture supernatant of some Enzymatic oxidation of lipids can be led to formation algal saccharide-assimilating bacteria, the of oxylipin such as hydroxy fatty acids. One of the Aurantiochytrium strains were able to propagate and multi-hydroxy fatty acids, 7,10-dihydroxy-8(E)- accumulate the target lipids. Detailed analysis on the octadecenoic acid (DOD) was produced from oleic fermentation profile will be demonstrated. acid and lipid containing oleic acid by a bacterial strain Pseudomonas aeruginosa PR3. Recently we Storage Lipid Accumulation by Acyltransferase have shown that DOD presented strong Mutants of Yarrowia lipolytica. M. Certik1, P. antimicrobial activity against broad range of Gajdos1, T. Rossignol2, and J.M. Nicaud2, 1Faculty of pathogenic bacteria. In this study, we tried to Chemical and Food Technology, Slovak University of enhance the antimicrobial activity of DOD by Technology, Bratislava, Slovak Republic, 2INRA formation of monoacylglyceride of DOD(DO-MG). UMR1319 Micalis, Jouy-en-Josas, France. DOD-MG was successfully produced with over 80% The yeast Yarrowia lipolytica features active production yield by lipase-catalyzed esterification “oleaginous” metabolic pathways that efficiently under the optimized reaction condition. accumulate storage lipids mainly in the form of Antimicrobial activity of DOD-MG was highly triacylglycerols (TAG) and sterol esters (SE). The final enhanced by ten times compared to that of DOD. step in TAG synthesis is catalyzed by acyltransferases Dga1p, Dga2p and Lro1p, whereas Are1p is involved Enzymatic Treatment of Soybean Meal. A. Al to SE biosynthesis. To study contribution of these Loman, Q. Li, N.V. Callow, S.M.M. Islam, and L.K. Ju*, acyltransferases in lipid storage, mutants of Y. University of Akron, Akron, OH, USA. lipolytica Q4-LRO1yl, Q4-DGA2yl, Q4-DGA1yl and Q4- Soybean meal, on dry basis, contains about 50% ARE1yl were prepared by reintroduction of single proteins and 30-35% carbohydrates, the latter acyltransferase gene under constitutive promoter include soluble and insoluble carbohydrates of into the Q4 strain (deleted in LRO1yl, DGA2yl, various molecular weights and compositions. We DGA1yl and ARE1yl genes). Mutants Q4 and Q4- have been developing an enzyme-based approach ARE1yl were unable to form neither TAG/SE nor lipid for treating soybean meal and separating protein bodies (LB), other mutants stored TAGs in LB with from carbohydrate. We selected strains and diverse fatty acid profiles. Strains Q4-DGA2yl and produced the optimal enzyme mixtures for the Q4-DGA1yl were the most efficient in TAG formation target purpose. The process conditions have also (approx. 2-3 times more than Q4-LRO1yl). Increased been evaluated and kinetic models developed. High copy number of either DGA2yl or DGA1yl resulted in protein recovery and enrichment are achieved and significant elevation of lipid content in cells with carbohydrate separated is explored for further slight change of fatty acid profile. However, yeasts value-added uses. Results of this work will be expressing DGA1yl formed several small LB, while presented and discussed.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

Immobilization of Phospholipase A1 on Polystyrene experiments by reusing the recovered immobilized Resin and Its Application for Production of phospholipase A1 for 28 consecutive batches. Diacylglycerols Through Glycerolysis of Soybean Oil. Y. Wang1, N. Liu2, Z. Zhang1, W. Bai1, and M. Liu1, One-stage and Two-stage Enzymatic Syntheses of 1Dept. of Food Science and Engineering, Jinan Structured Lipids from Extra Virgin Olive Oil for Use University, Guangzhou, Guangdong, China, 2College as Infant Formula Fat Analogs. G. Pande1, J.S.M. of Life Science and Engineering, Shanxi University of Sabir2, N.A. Baeshen2, and C.C. Akoh1,2, 1University of Science and Technology, Xi'an, Shanxi, China. Georgia, Athens, GA, USA, 2King Abdulaziz University, To overcome high cost of some commercial Jeddah, Kingdom of Saudi Arabia. immobilized lipases in production of structured Structured lipids (SLs) with high palmitic acid lipids, a phospholipase A1, was immobilized by content at the sn-2 position enriched with adsorption and the immobilized phospholipase A1 arachidonic acid (ARA) and docosahexaenoic acid was employed to catalyze a glycerolysis reaction of (DHA) were produced using extra virgin olive oil, soybean oil to prepare diacylglycerols (DAG). Firstly, tripalmitin, ARA and DHA single cell oils free fatty the effect of nature of the polystyrene supports, the acids. Four types of SLs were synthesized using kinetic behavior and stability of immobilized immobilized lipases from Candida antarctica and phospholipase A1 were evaluated. Six macroporous Thermomyces lanuginosus, based on one-stage (one- resins has been investigated as the supports. The pot) and two-stage (sequential) syntheses. The SLs adsorption process was modeled by Langmuir and were characterized for fatty acid profile, Freundlich equations, and the experimental data triacylglycerol (TAG) molecular species, melting and was better fit for the former one. Combined crystallization profiles, tocopherols, and phenolic strategies of scanning electron micrograph (SEM), compounds. All the SLs had >50 mol% palmitic acid thermogravimetric analysis (TGA) and fourier at the sn-2 position. The predominant TAGs in all SLs transform infrared (FTIR) spectroscopy were were PPO and OPO. The total tocopherol content of employed to characterize the immobilized SL2-1, SL2-2, SL1-1, and SL1-2 were 70.46, 68.79, phospholipase A1. The immobilized phospholipase 79.64, and 79.31 μg/g, respectively. SL2-2 had the A1 was used as the catalyst for DAG production highest melting completion (42.0°C) and through glycerolysis from soybean oil. In a solvent- crystallization onset (27.6°C) temperatures. The free system, with a reaction of 12 h under the lipases had better reusability in two-stage synthesis optimum conditions, an upper lipid layer of the but one-stage synthesis was a faster reaction and reaction mixture with 53.7 wt% DAG content was also resulted in higher ARA and DHA content than obtained. The immobilized phospholipase A1 two-stage synthesis. All the SLs produced in this demonstrated a remarkable reusability in recycling study may be suitable as infant formula fat analogs.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 4: Biotechnology for Oilseed Improvement Chairs: T.A. McKeon, USDA, ARS, WRRC, USA; and R. Wilson, Oilseeds & Bioscience Consulting, USA

Bringing Back the Castor Plant as a Domestic Crop. linolenic acid and high in stearidonic acid. We have T.A. McKeon1, X. He1, D.L Auld2, and S. Leviatov3, further developed the oil component of this high 1USDA-ARS WRRC, Albany, CA, USA, 2Texas Tech omega-3 fatty acid soybean by creating a set of University, Lubbock, TX, USA, 3Evofuel Ltd., Rehovot, gene-stacks in the crop that leads to the Israel. simultaneous accumulation of the very-long chain The castor plant Ricinus communis L. produces omega-3 fatty acid, eicosapentaenoic acid (EPA), and castor oil, an important chemical feedstock used for the carotenoid, astaxanthin. Greenhouse and small- manufacture of lubricants, detergents, plasticizers, scale field trials conducted with the soybean events polymers, paints and even chocolate. Because of the producing this novel oil revealed the levels of EPA presence of the toxic protein ricin, and the expense and astaxanthin were sustained over generations, of energy needed to detoxify castor seed meal, under both controlled and field conditions, with EPA castor oil production is limited to a few exporting levels reaching approximately 4% and astaxanthin countries. Its cost is generally two to three times the accumulating to 25 µg per gram seed. cost of soy oil, even though it can be produced as cheaply. A low or no-ricin castor crop would support Helping Industry Turn Over a New Leaf: Game- increased cultivation of castor. We have applied a changing Technology Enabling Oil Production in sandwich enzyme-linked immunosorbent assay Leaves. A.G. Green (AAOCS Lipid Research Award (ELISA) for the detection of ricin in castor beans and Winner), T. Vanhercke, J.R. Petrie, and S.P. Singh, screened a collection of castor breeding lines as well CSIRO Plant Industry, Canberra, ACT, Australia. as several commercial varieties using this assay. Plant triglyceride oils offer great technical Although the average content derived by extracting potential to provide much-needed renewable several seeds provided a fair representation of the replacements to petroleum derived fuels and ricin content for a given selection, there was enough chemicals. However to expand plant oil production seed-to-seed variation to justify determining the sufficiently to make major inroads into the high ricin content of individual seeds in order to select global demands for these industrial products will the lowest ricin producers. Ricin levels observed require game-changing technologies. A particularly ranged from 1.16% to 6.25% by weight. We have promising recent development has been the also addressed concerns about castor as an invasive metabolic engineering of leaves to synthesise and weed, and demonstrated that glyphosate is an accumulate commercially significant levels of effective means for eliminating even maturing castor storage oils. Here we report that the coexpression of plants. three genes involved in different aspects of TAG production resulted in the accumulation of more Designing of a Prototype Soybean-based Feedstock than 15% TAG (17% total lipids) by dry weight in for Aquaculture. T. Clemente, University of Nicotiana tabacum (tobacco) leaves, predominantly Nebraska-Lincoln, Lincoln, NE, USA. within the leaf mesophyll cells and without any The aquaculture industry is expected to expand significant alterations to their growth and seed over the coming years as a means to meet the setting properties. If translatable to high biomass growing demand for seafood. A challenge facing this crops, these initial levels would already equate to oil expected expansion in aquaculture is meeting the yields per hectare that exceed those of most existing aquafeed supply needs in a sustainable fashion that oilseed crops. Such highly productive vegetative does not compromise the world’s wild fisheries. An plant oil production platforms could provide a step- avenue to address this challenge is designing change in the bio-based energy and materials agquafeed formulations in which marine-based landscape, opening up prospects of achieving a ingredients are replaced with land-based protein and sustainable, scalable and affordable raw material lipid sources. To this end, we have formulated a feed supply for industry without compromising on supply for the high-quality finfish Seriola rivoliana, with a of food oils from conventional sources. soybean protein concentrate inclusion level of 40%, and half of the lipid component sourced from a high omega-3 fatty acid soybean oil enriched with

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

Engineering Increased Plant Diacylglycerol histidine tagged Brassica napus DGAT1, produced in Acyltransferase Accumulation in Baker’s Yeast. M.S. Saccharomyces cerevisiae. n-Dodecyl-ß-D- Greer (Biotechnology Division Student Award maltopyranoside (DDM) was used to solubilize the Winner) and R.J. Weselake, Agricultural Lipid enzyme from yeast microsomes. Cobalt affinity Biotechnology Program, Dept. of Agricultural, Food chromatography was employed to highly purify and Nutritional Science, University of Alberta, solubilized BnaDGAT1, the identity of which was Edmonton, AB, Canada. verified through in gel tryptic digestion coupled with Recently, there has been great interest in LC-MS/MS sequencing. After tag removal, the increasing triacylglycerol (TAG) accumulation in both enzyme was purified to apparent homogeneity in micro-organisms and plants to provide edible oil active form using FPLC-Superdex 200 13/30. Analysis and/or feedstock for production of biofuels and of gel filtration fractions by SDS-PAGE suggested that other bioproducts. Diacylglycerol acyltransferase different DGAT1 oligomers in detergent micelles (DGAT) catalyzes the acyl-CoA-dependent acylation were resolved during the gel filtration process. A of sn-1,2-diacylglycerol to produce TAG. Expression major symmetrical peak with apparent molecular of modified Brassica napus DGAT1 coding sequences mass of 213 kDa, was used for preliminary in Saccharomyces cerevisiae revealed that the amino crystallization trials. BnaDGAT1 was purified about acid sequence in the N-terminal region of these 70-fold over the solubilized fraction and exhibited a enzymes can alter their in vivo accumulation, specific activity of 11.11±0.32 nmoles TAG/min/mg resulting in up to a two-fold increase in cellular TAG protein. content. Placement of an N-terminal tag, containing poly-histidine and a second epitope, on the various Engineered Oil Seed Crops with Fish Oil DHA Levels. forms of BnaDGAT1 stabilized accumulation of the S.P. Singh and J.R. Petrie, CSIRO Plant Industry, enzymes, irrespective of the amino acid sequence of Canberra, ACT, Australia. their native N-terminal sequence. Microsomal Omega-3 long chain (=C20) polyunsaturated fractions from S. cerevisiae, producing the high- fatty acid (omega-3 LC-PUFA) have critical roles in accumulating enzymes, exhibited DGAT specific human health and development with studies activities which were substantially greater than indicating that deficiencies in these fatty acids can typically reported for recombinant production of increase the risk or severity of cardiovascular and DGATs in yeast. DGAT activity was further assayed inflammatory diseases in particular. In order to meet based on mass spectrometric quantification of TAG the increasing demand for these oils there is an isolated by high temperature gas chromatography. urgent need for an alternative and sustainable DGAT-catalyzed TAG production using this method source of EPA and DHA. This talk will discuss recent was strongly correlated (R2=0.99) with enzyme- progress in the production of the omega-3 LC-PUFA catalyzed TAG production based on the use of DHA, in plant seeds. Groups have reported good radiolabeled acyl-CoA. progress in engineering the C20 EPA with seed fatty acid levels similar to that observed in bulk fish oil Solubilization and Purification of Recombinant (18%) although undesirable omega-6 PUFA levels Brassica napus Diacylglycerol Acyltransferase 1. have also remained high. The conversion of EPA to K.M. Caldo1, M.S. Greer1, G. Chen1, M.J. Lemieux2, the particularly important C22 DHA, however, has and R.J. Weselake1, 1Alberta Innovates Phytola been problematic with many attempts resulting in Centre, Dept. of Agricultural, Food and Nutritional the accumulation of EPA/DPA but only a few percent Science, University of Alberta, Edmonton, AB, of DHA. I will describe the production of fish-oil like Canada, 2Dept. of Biochemistry, University of levels of the C22 fatty acid DHA in seed oils of two Alberta, Edmonton, AB, Canada. oilseed crop species, camelina and canola, with high Diacylglycerol acyltransferase 1 (DGAT1) has omega-3/omega-6 ratios. Importantly, these results been used in lipid biotechnology to modify oil were achieved using a single multi-gene construct deposition in a variety of organisms. Although the which potentially will allow for a simpler pathway for first DGAT gene was identified more than 15 years deregulation and breeding. We consider this to be a ago and despite its importance in lipid biochemistry, breakthrough in the development of sustainable DGAT1 has not been subjected to intensive alternative sources of DHA. structure-function studies due to difficulties in purifying the membrane-bound enzyme. Herein, we describe a method for purifying recombinant poly-

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

Potential Benefits of a Novel Dietary PUFA, Shimizu6, and J. Ogawa1,4, 1Division of Applied Life dihomo-gamma-linolenic acid (DGLA) in Sciences, Graduate School of Agriculture, Kyoto Cardiovascular Disease. H. Gallagher1, I.A. University, Sakyo-ku, Kyoto, Japan, 2Laboratory of Guschina1, I. Khozin-Goldberg2, S. Boussiba2, D. Industrial Microbiology, Graduate School of Ramji1, and J.L. Harwood1, 1Cardiff University, Agriculture, Kyoto University, Sakyo-ku, Kyoto, Cardiff, Wales, UK, 2Ben-Gurion University of the Japan, 3Research Unit for Physiological Chemistry, Negev, Sede-Boqer Campus, Israel. Kyoto University, Sakyo-ku, Kyoto, Japan, 4Research Atherosclerosis, the underlying cause of Division of Microbial Sciences, Kyoto University, myocardial infarction, stroke and peripheral vascular Sakyo-ku, Kyoto, Japan, 5Suntory Global Innovation disease, is responsible for about 34% of all deaths in Center Ltd., Shimamoto-cho, Mishima-gun, Osaka, the Western world. Atherosclerosis is a chronic Japan, 6Faculty of Bioenvironmental Science, Kyoto inflammatory disorder and measures to control Gakuen University, Kameoka, Kyoto, Japan. inflammation offer promising avenues for treatment. Mortierella alpina 1S-4 is capable of producing DGLA, an n-6 PUFA, has been found to reduce triacylglycerols rich in polyunsaturated fatty acids atherosclerotic development in a mouse model of (PUFAs) including arachidonic acid (ARA). On the this disease though the mechanism remains ill- other hand, this fungus accumulates a small amount defined. With the availability of DGLA from a mutant of eicosapentaenoic acid (EPA) with 0.3 g/L of of Parietochloris incisa (a green alga of potential culture broth and 10% in total fatty acids below a commercial importance) the possibility occurred of cultural temperature of 20°C. A ω3-fatty acid investigating DGLA affects at the molecular level – desaturase gene (maw3) which is involved in with the development of new dietary treatments as biosynthesis of n-3 PUFAs such as EPA was an end point. We have used macrophages in a previously isolated from M. alpina 1S-4. In this robust model for atherosclerosis and monitored the research, we investigated the products of MAW3 effect of DGLA in comparison to other fatty acids. catalyzing reaction in the yeast transformant. Two Inflammatory, cytokine and signalling pathway unknown fatty acids de novo synthesized in the yeast responses were monitored. Our data go some way transformant expressing maw3 gene were identified to explaining the effectiveness of DGLA and its as n-4 hexadecadienoic acid (16:29cis,12cis) and n-1 promise as a dietary component to reduce hexadecatrienoic acid (16:39cis,12cis,15) by GC-MS and cardiovascular disease. 1H-NMR analyses. The MAW3 was demonstrated to be a trifunctional ∆12/∆15/ω3-desaturase, exhibiting Development of Enzyme Cascade Reactions for the ∆12-desaturation for 16:19cis, ∆15-desaturation for Conversion of Fatty Acids into Valuable 16- and 18-carbon fatty acids that had a preexisting Oleochemicals. U.T. Bornscheuer1 and J.B. Park2, cis-double bond at ∆12 position, and ω3- 1Institute of Biochemistry, University of Greifswald, desaturation for 20-carbon fatty acids having that at Greifswald, Germany, 2Ewha Womans University, ∆14-position. Seoul, South Korea. In this lecture an enzyme cascade reaction will Lipid Oxidation Can Cause Aberrant Results in be presented. By the successive action of oleate Moisture & Oil Content Determination of Oilseeds hydratase, alcohol dehydrogenase, Baeyer-Villiger Particularly High ω3 Sources. D. Hildebrand1, S. monooxygenase and esterase (all expressed Patel1, M. Ma1, M. Al-Amery1, and P. Armstrong2, recombinantly in E. coli for whole cell 1University of Kentucky, Lexington, KY, USA, 2USDA- biotransformation) we can access dicarboxylic acids ARS, Peoria, IL, USA. and omega-hydroxy carboxylic acids from Seeds generally range from 10 – 15% moisture unsaturated fatty acids. This system is highly flexible at harvest and are often dried further for storage. as the proper choice of enzymes in the cascade can Very long term storage can be achieved at be used to direct the formation of desired products temperatures below freezing and = 7-8% moisture. to meet the needs of the oleochemical industry. This can be achieved by mild heating, freeze drying or with desiccants. To achieve complete removal of Characterization of a Trifunctional Fatty Acid free water and determine the moisture content for Desaturase from Oleaginous Filamentous Fungus calculation of components on a dry weight basis it is Mortierella alpina 1S-4 Using a Yeast Expression standard to heat seeds at 101 – 110°C until the System. H. Kikukawa1, E. Sakuradani1, S. Kishino1, weight stops changing. Oilseeds are mainly valued S.B. Park2, A. Ando3,4, J. Shima4, M. Ochiai5, S. for the oil and protein components. In drying

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

oilseeds with high polyunsaturated fatty acid levels apparently due to oxygen adsorption and then both at 103°C we find oil contents can decrease with moisture and volatile lipid oxidation product loss. drying especially in seeds with high tri-unsaturated Thus drying by standard methods not only can levels. Oil measured by NMR and NIR can nearly render oil content determinations inaccurate but disappear and this is correlated with loss of the di- also make the moisture content measurement and tri-unsaturated fatty acids linoleate and imprecise. This problem is greater with ground than linolenate. During drying we find seed weights can whole seeds. This problem can be avoided by drying actually increase temporarily and then decrease in an atmosphere of nitrogen (N2) gas.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO 5: General Biotechnology Chairs: D.G. Hayes, University of Tennessee, USA; K. Takahashi, Hokkaido University, Japan; and L. Kleiner, Bunge North America, Inc., USA

Lipid Composition of the Liposome Membrane from soil as a potent producer of polyunsaturated Largely Affects the Transport and Absorption of the fatty acids (PUFAs) in our laboratory and used for Liposomes Through Small Intestinal Epithelial Cell commercial production of arachidonic acid (AA, Model. Y. Konishi and K. Takahashi*, Hokkaido 20:4n-6). A host-system for M. alpina 1S-4 was University, Hakodate, Hokkaido, Japan. developed by means of molecular breeding for Mechanisms involved in transportation and improving and modifying PUFAs productivity and uptake of several kinds of omega 3 phospholipid composition. We developed a transformation liposomes through small intestinal epithelial cell systems for this fungus to improve the fatty acid monolayer models (Caco-2 model and M cell composition. In this study, we demonstrate an useful induced model) were examined through using polyunsaturated fatty acid production by oleaginous endocytosis inhibitors (EIPA and chlorpromazine). filamentous fungus M. alpina breeding. Liposomes were prepared using PC obtained from squid, the PS transphosphatidylated from that PC, A Novel Glycerophosphocholine and SQDG obtained from alga. The PC/PS and Cholinephosphodiesterase and PC/SQDG liposomes, both the MLV and SUV show Glycerophosphoethanolamine higher uptake not only in the M cell induced model, Ethanolaminephosphodiesterase from but also in the Caco-2 model. On the other hand, Streptomyces sanglieri. D. Sugimori, K. Okuda, S. they showed only higher transport through M cell Mineta, and J. Ogasawara, Graduate School of induced model. The transports of PC-MLV through Symbiotic Systems Science and Technology, both models were not inhibited by chlorpromazine, Fukushima University, Fukushima, Japan. but on the contrary inhibited by EIPA in the M cell Glycerophosphocholine induced model. Thus PC-MLV is considered to be cholinephosphodiesterase (GPC-CP) and transported through M cell induced model by glycerophosphoethanolamine macropinocytosis. The transports of PC/SQDG-MLV ethanolaminephosphodiesterase (GPE-EP) through both models were not inhibited by the hydrolyzes glycerophosphocholine (GPC) or inhibitors. PC/SQDG-MLV seemed to open tight glycerophosphoethanolamine (GPE) to glycerol and junction. It was considered that PC/PS-MLV may be phosphocholine or phosphoethanolamine, transported by phagocytosis because the inhibitors respectively. We have discovered that Streptomyces did not affect. The uptake of all the liposomes sanglieri strain A14 produces a novel GPC-CP and examined was not inhibited by both inhibitors. It is GPE-EP extracellularly. Here we report considered that PC-MLV, PC/PS-MLV, and PC/SQDG- characterization, gene cloning, and efficient MLV are up taken by phagocytosis. production of these two enzymes. GPC-CP and GPE- EP were metal-ion independent and monomeric Studies of Oleaginous Filamentous Fungus proteins (~70 kDa). GPC-CP preferred GPC and GPE Mortierella alpina for Useful Polyunsaturated Fatty as substrate; however, GPE-EP showed GPE-specific Acid Production. A. Ando1, T. Okuda2, E. Sakuradani2, activity. The amino acid sequences of GPC-CP and J. Shima3, J. Ogawa2, and S. Shimizu2,4, 1Research GPE-EP showed 76% and 85% identity to those of Unit for Physiological Chemistry, Kyoto University, phospholipases C (PLCs) from Streptomyces spp., Kyoto-shi, Kyoto-fu, Japan, 2Division of Applied Life respectively. However, we concluded that GPC-CP Sciences Graduate School of Agriculture, Kyoto and GPE-EP are essentially different from PLC University, Kyoto-shi, Kyoto-fu, Japan, 3Research because of the difference in the substrate Division of Microbial Sciences, Kyoto University, specificities. The amino acid sequence of GPC-CP Kyoto-shi, Kyoto-fu, Japan, 4Dept. of Bioscience and showed 39% identity to that of GPE-EP. GPC-CP and Biotechnology, Faculty of Bioenvironmental Science, GPE-EP were produced as an active protein by Kyoto Gakuen University, Kameoka-shi, Kyoto-fu, Streptomyces lividans or E. coli as a host cell, Japan. respectively. A filamentous fungus, Mortierella alpina 1S-4, belonging to the Zygomycetes, has been isolated

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

The Novel Enzymes for Polyunsaturated Fatty Acid from an actinomycete, Thermocrispum sp. RD4668 Saturation Involved in Conjugated Fatty Acid was found. To identify the enzyme gene, genome Production by Lactic Acid Bacteria. S. Kishino1,2, M. sequencing of strain RD4668 was performed by next- Takeuchi1, S.B. Park2, A. Hirata1, N. Kitamura1, K. generation sequencing and then a predicted ORF Yokozeki2, S. Shimizu1, and J. Ogawa1, 1Division of database was constructed. The LPls-PLD gene (lpls- Applied Life Sciences Graduate School of Agriculture, pld) was identified from the ORF database using its Kyoto University, Kyoto, Japan, 2Laboratory of peptide sequences analyzed by a Xevo QTOF MS Industrial Microbiology, Kyoto University, Kyoto, system. The amino acid sequence of LPls-PLD shows Japan. 66% identity to that of glycerophosphodiester Saturation metabolism of polyunsaturated fatty phosphodiesterase (UniProt accession no. D3Q1U5). acids by gastrointestinal microbes is a detoxifying LPls-PLD was extracellularly produced by metabolism of anaerobic bacteria, such as lactic-acid Streptomyces lividans transformed using an bacteria. This saturation metabolism generates expression vector, pUC702/lpls-pld. LPls-PLD showed characteristic fatty acids, e.g., conjugated fatty acids no activity toward phosphatidylcholine, and trans-fatty acids, which are well known to plasmalogen, sphingomyeline, and glycero-3- present in ruminant-derived foods. phosphocholine, so that the method using LPls-PLD Our analyses on conjugated fatty acid synthesis in and PLA1 is useful for measuring plasmalogen in a representative gut bacteria, the lactic-acid bacteria, blood sample. demonstrated that Lactobacillus plantarum AKU 1009a can transform the cis-9,cis-12 diene structure Pancreatic Lipase Selectively Hydrolyses DPA over of C18 fatty acids into the conjugated diene EPA and DHA Due to Location of Double Bond in the structures cis-9,trans-11 and trans-9,trans-11. In Fatty Acid Rather Than Regioselectivity. T.O. Akanbi addition, this strain can saturate these conjugated (Honored Student Award Winner), A.J. Sinclair, and dienes into the trans-10 monoene. In cell-free C.J. Barrow, Deakin University, Victoria, Australia. extracts from this strain, we identified the enzymes The enzymatic hydrolysis of canola, anchovy and involved in CLA synthesis. Three enzymes, CLA-HY, seal oils with different types and amounts of CLA-DH, and CLA-DC, are necessary for synthesis of polyunsaturated fatty acids were measured using conjugated fatty acids such as CLA. Through genomic porcine pancreatic lipase (PPL) to establish the fatty analysis in L. plantarum WCFS1, we found that cla-dh acid selectivity of PPL. Substrates were subjected to and cla-dc are located in a cluster with another gene, the same conditions of hydrolysis, with percent cla-er. In light of this, we tried to identify the hydrolysis monitored using iatroscan and fatty acid function of the gene product (CLA-ER) together with selectivity monitored using gas chromatography those of CLA-HY, CLA-DH, and CLA-DC and found that (GC). Regardless of their distribution on the glycerol CLA-ER is the key enzyme for polyunsaturated fatty backbone, as monitored by 13C nuclear magnetic acid saturation. resonance (NMR), a-linolenic acid (ALA) and docosapentaenoic acid (DPA) were rapidly cleaved A Novel Lysoplasmalogen-specific Phospholipase D. by PPL while eicosapentaenoic acid (EPA), Y. Matsumoto1, D. Sugimori1, S. Sakasegawa2, and H. docosahexaenoic acid (DHA) and stearidonic acid Matsumoto2, 1Fukushima University, Fukushima-shi, (STA) were hydrolysed more slowly. Results show Fukushima, Japan, 2Asahi Kasei Pharma Corporation, that PPL preferentially hydrolyses ALA and DPA over Izunokuni-shi, Shizuoka, Japan. EPA, DHA and STA, and this selectivity is due to fatty Plasmalogen, a type of phospholipid, is a acid rather than regioselectivity. The primary biomarker for dementia and cancer. At present, the structural factor associated with resistance to PPL amount of plasmalogen in a blood sample is appears to be the distance of the first double bond determined by the HPLC method using 125I; however, from the ester linkage being hydrolysed. These this method requires complicated processes and results may be useful for using this enzyme to extensive analysis time. Here, we aim to develop a concentrate DPA from marine oils, separating it from new enzymatic method for plasmalogen EPA and DHA. determination. We have previously reported that plasmalogen can be hydrolyzed to lysoplasmalogen Modification of Stearidonic Acid Soybean Oil by by phospholipase A1 (PLA1) from Streptomyces Immobilized Rhizomucor miehei Lipase to albidoflavus NA297. A novel enzyme, Incorporate Caprylic Acid. E.A. Ifeduba lysoplasmalogen-specific phospholipase D (LPls-PLD), (Biotechnology Division Student Award Winner) and

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

C.C. Akoh, University of Georgia, Athens, GA, USA. alcohols of a chain length from C2-C18 using a cheap Immobilized sn-1,3 specific Rhizomucor miehei commercial lipase in a non-polar system. Yields in lipase (RML) was used to catalyze the incorporation the range of 79% to 92% within 72h of incubation of caprylic acid (C8:0) into high stearidonic acid (SDA, were reached depending on the alcohol chain C18:4n-3) soybean oil (SDASO) to form structured length. Further investigations with different phenolic lipids (SLs). The effects of type of biocatalyst (Celite-, acids were conducted to explore the influence of octyl-sepharose-, and Duolite-immobilized RML) and structural differences on the esterification using reaction temperature (30, 40, 50, and 60°C) on lipases. The position of the hydroxyl group, a acidolysis and acyl migration were studied. Celite- saturation/unsaturation in the side chain, and the immobilized RML (C-RML) at 50°C maximized C8:0 number of hydroxyl groups all have a significant incorporation and minimized acyl migration influence on the esterification activity. Therefore, compared to other treatments. The optimal reaction conditions need to be optimized individually substrate molar ratio (C8:0 to SDASO), incubation for each phenolic acid. time, and enzyme load for SL synthesis by C-RML at 50°C was determined by response surface Optimized Synthesis of Structured Lipids by the methodology to be 6:1, 24 h, and 20% weight of Interesterification of Flaxseed Oil and Tricaprylin in substrates, respectively. This optimum treatment Solvent-free Medium. M. Khodadadi and S. was scaled-up in hexane or solvent-free reaction Kermasha, McGill University, Sainte-Anne-de- media using SDASO or an SDA-enriched concentrate Bellevue, QC, Canada. as initial triacylglycerol substrates. This yielded Lipase-catalyzed biosynthesis of structured various SLs with C8:0 contents ranging from 17.0 - lipids (SLs) in solvent-free medium (SFM) was carried 32.5 mol% and SDA contents ranging from 20.6 - out by the interesterification of flaxseed oil (FO) and 42.3 mol%. When digested, these SLs may deliver tricaprylin (TC), using Lipozyme TL-IM from C8:0 for quick energy and SDA for heart health Thermomyces lanuginosus, as the biocatalyst. The making them potentially valuable for medical and bioconversion yield (%) of the medium-long-medium nutraceutical applications. type SLs (MLM-SLs), including CLnC (C-caprylic and Ln-linolenic acids), CLaC (La-linoleic acid) and COC Enzymatic Esterification of Phenolic Acids. A. Schär, (O-oleic acid), considered as the desirable S. Gayathri, and L. Nyström, Institute of Food, monitoring responses, was investigated. Multiple Nutrition and Health, ETH Zurich, Zurich, response surface methodology (MRSM) was used to Switzerland. obtain significant models for the responses, on the Phenolic acids are strong antioxidants, which basis of a five level, five variable central composite have to be lipophilized through an esterification for rotatable design. Reaction parameters, including the application in oil-based systems. Until now, with temperature (Tr, 45 to 65°C), TC to FO molar ratio the exception of alkyl gallates, phenolipids are rarely (Mr, 2:1 to 6:1), enzyme concentration (Ec, 5 to 10%, applied in industrial scale. Hydroxycinnamic acid w/w), reaction time (Rt, 4 to 28 h) and agitation derivates with a hydroxyl group in para-position speed (As, 150 to 350 rpm), were selected for the (incl. ferulic acid) are considered to be difficult to be optimization of the process. Significant models for esterified by lipases due to inhibitory effects. An CLnC, CLaC and COC were determined after efficient esterification of ferulic acid would therefore regression analysis, with backward elimination. After lead to a broader availability of ferulic acid esters the verification of the predicted models, multiple- and increase the chance of future investigation on response surface optimization of the monitored safety, antioxidant potential in various systems, and variables, CLnC, CCLn, CLaC, CCLa, COC and CCO, has finally industrial applications. We achieved an been carried out by means of desirability function efficient esterification of ferulic acid with linear and distance approaches.

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

BIO-P: Biotechnology Poster Session Chairs: J. Ogawa, Kyoto University, Japan; and B.H. Kim, Chung-Ang University, South Korea

1. pH-dependency of Activity and Production of a reported chemical synthesis of furan fatty acids Cold-active Lipase from Pichia lynferdii Y-7723. J.H. using chemical catalysts. However, chemical Bae1, M.H. Kwon1, J.Y. Kim1, C.T. Hou2, and H.R. Kim1, synthesis required complicated multisteps. In this 1School of Food Science and Biotechnology, study we developed a simple one step synthesis of a Kyungpook National University, Daegu, Korea, novel furan fatty acid 7,10 epoxy-octadeca-7,9- 2Renewable Product Technology Research Unit, dienoic acid (EODA) from a dihydroxy fatty acid 7,10- National Center for Agricultural Utilization Research, dihydroxy-8(E)-Octadecenoic acid (DOD) by heat ARS, USDA, Peoria, IL, USA. treatment. Structure of EODA was confirmed by Lipases with abnormal functionalities such as GC/MS, NMR, FTIR analysis and maximum high thermostability and optimal activity at extreme production yield under the reaction conditions of at conditions gain special attentions because of their 90°C, 24 hours reached 80%. As expected, EODA applicability in the restricted reaction conditions. In showed antioxidant activity in terms of scavenging particular, cold-active lipase(CAL)s have gained activity. special attentions in various industrial fields such as washer detergent, pharmaceutical catalyst, and 3. Production of trans-10,cis-12 Conjugated Linoleic production of structured lipid. However, production Acid-enriched Triacylglycerols via Two-step Lipase- of CAL is mostly found from psychrophilic catalyzed Esterifications. I. Kang1, I. H. Kim2, H.D. microorganisms. Recently we found a novel cold- Choi3, and B.H. Kim*1, 1Chung-Ang University, active lipase from Pichia lynferdii Y-7723 which is Anseong, Gyeonggi-Do, Republic of Korea, 2Korea mesophilic yeast strain (Production of a Novel Cold- University, Seoul, Republic of Korea, 3Korea Food Active Lipase from Pichia lynferdii Y-7723, Hak-Ryul Research Institute, Seongnam, Gyeonggi-Do, Kim et al., J. Agric. Food Chem. 2010, 58, 1322–- Republic of Korea. 1326). In this study, we investigated about pH- The aim of this study was to synthesize trans- dependency of activity and production of a novel 10,cis-12 conjugated linoleic acid (t10,c12-CLA)- cold-active lipase from P. lynferdii Y-7723. Catalytic enriched triacylglycerols (TAG) with potential anti- activity of lipase Y-7723 was highly dependent on pH obesity effects via two-step enzymatic reactions. and temperature. Production was also highly Commercial CLA mixtures, containing 33.3% t10,c12- influenced by initial medium pH. CLA and 32.0% cis-9,trans-11 CLA (c9,t11-CLA) was esterifed with dodecan-1-ol for selectively removing 2. Production of a Novel Antioxidant Furan Fatty the c9,t11 isomer in the form of dodecyl esters. The Acid from 7,10-dihydroxy-8(E)-octadecenoic Acid. C. reaction was performed in a recirculating packed Dasagrandhi1(Biotechnology Division Student Award bed reactor, using an immobilized lipase from Winneri), H.M. Park1, C.T. Hou2, and H.R. Kim1, Candida rugosa as a biocatalyst. A free fatty acid 1School of Food Science and Biotechnology, fraction containing 54.7% t10,c12-CLA was produced Kyungpook National University, Daegu, Korea, under the best conditions (temperature, 20°C; 2Renewable Product Technology Research Unit, substrate molar ratio (CLA mixtures to dodecan-1- National Center for Agricultural Utilization Research, ol), 1:1; water content, 0%; reaction time, 36 h) ARS, USDA, Peoria, IL, USA. established in this study. The free fatty acid fraction Furan fatty acids (F-acids) gain attention since enriched in t10,c12-CLA was esterified with glycerol they are known to play important roles in biological for preparing t10,c12-CLA-enriched TAG. The systems including human. Specifically F-acids are reaction was performed in a vacuum stirred batch known to have strong antioxidant activity like radical reactor, using an immobilized lipase from C. scavenging activity. Although widely distributed in antarctica as a biocatalyst. The best combination of mot biological systems, F-acids are trace temperature, enzyme loading, and vacuum level was components and their biosynthesis are complicated 60°C, 10% of the total weight of the substrates, and and quite difficult from sources. Based on 0.4 kPa, respectively. The t10,c12-CLA-enriched TAG biochemical study, they are considered to be an of ~95% was successfully synthesized in 12 h. essential nutritional factor for mammals and should be provided through diet. Hence several studies

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

substrate), and pressure of 80 kPa. The maximum 5. Enrichment of Docosahexaenoic Acid from Tuna conversion of phytosteryl ester was ca. 93 mol % at Oil via Lipase-mediated Esterification Under the optimum condition. Pressurized Carbon Dioxide. N. Ma1,2, S.I. Hong1,2, T.T. Zhao1,2, D. S. No1,2, C.T. Kim3, and I.H. Kim1,2, 7. Inhibition of Lipases by γ-Oryzanol. A. Schär, L. 1Dept. of Food & Nutrition, Korea University, Seoul, Schwarz, and L. Nyström, Institute of Food, Nutrition Republic of Korea, 2Dept. of Public Health Sciences, and Health, ETH Zurich, Zurich, Switzerland. Seoul, Republic of Korea, 3Korea Food Research γ-Oryzanol is a mixture of various phytosterols Institute, Seongnam, Republic of Korea. esterified to a ferulic acid. This lipid soluble mixture This study focused on the use of pressurized CO2 of phenolics is found primarily in rice, and in addition as a reaction medium for the enrichment of to its health benefits (cholesterol lowering, anti- docosahexaenoic acid (DHA) from tuna oil fatty acids inflammatory activity) oryzanol is a good antioxidant via lipase-mediated esterification. The DHA in oil-based systems, including high temperature concentration in tuna oil fatty acids used as a applications. The ferulic acid moiety of oryzanol is a substrate was 20.9 wt%. Of the three lipases tested, hydroxycinnamic acid derivate that bears a hydroxyl Lipozyme RM IM from R. miehei was selected for group in para-position and a double bond in the side further study. Enzyme loading, water addition, and chain. This structural combination in some free reaction time were also explored. Near-supercritical phenolic acids has been demonstrated to inhibit CO2, prepared at 25 °C and 8.3 MPa, was the most lipases. Therefore, the aim of this study was to effective reagent tested for enriching DHA from the explore the possible effect of oryzanol on lipase residual fatty acid fraction. In addition to near- activity. Inhibitory effect of oryzanol on for example supercritical CO2, optimal conditions included a lipase from porcine pancreas was demonstrated in addition of 0.2 wt% (based on total substrates) an O/W emulsion by monitoring the formation of water, enzyme loading of 5 wt% (based on total free fatty acids in a pH-stat. However, the effect substrates), and a reaction time of 18 h. The DHA seems to be strongly dependent on the exposure concentration and recovery yield for the residual time of the lipase to the oryzanol. As lipases in fatty acid fraction under these optimal conditions general are activated on the interface of water and were 75.8 wt% and 81.0 wt%, respectively. lipids, amphiphilic molecules like oryzanol are in the vicinity of the substrate and enzyme to participate in 6. Preparation of Phytosteryl Ester Containing the reaction. Therefore, oryzanol has a potential as Pinolenic Acid in a Solvent-free System Using lipase inhibitor, increasing stability of lipids during Immobilized Candida rugosa lipase. D.S. No1,2, T.T. storage, and decreasing their gastrointestinal Zhao1,2, E.J. Lee1,2, and I.H. Kim1,2, 1Dept. of Food & digestion. Nutrition, Korea University, Seoul, Republic of Korea, 2Dept. of Public Health Sciences, Graduate School, 8. Enrichment of Refined Olive Oil with Palmitic Korea University, Seoul, Republic of Korea. Acid and Docosahexaenoic Acid to Produce Human Phytosteryl ester synthesized with pinolenic acid Milk Fat Analogue. R. Li and C.C. Akoh, University of (PLA) from pine nut oil is expected to have features Georgia, Athens, GA, USA. of both phytosterol and PLA. In this study, lipase Refined olive oil was enriched with palmitic acid from Candida rugosa (CRL) was immobilized and (PA) and docosahaxaenoic acid (DHA) via lipase- then used to optimize conditions for synthesis of catalyzed acidolysis reaction using Novozym 435 in phytosteryl ester containing PLA. Lewatit VP OC hexane to increase its nutritive value. The 1600, a macroporous hydrophobic resin, was incorporation reaction was optimized by response selected as the best carrier, and the optimum surface methodology. Three independent variables, condition for the immobilization of CRL was reaction time (12, 18, and 24 h), temperature (50, established. With immobilized CRL prepared, 55, and 60°C), and substrate molar ratio (refined synthesis of phytosteryl ester with fatty acid from olive oil:DHA:PA = 1:1:6, 1:1:9, and 1:1:12) and three pine nut oil was carried out. Parameters investigated responses, total PA and DHA incorporation and PA were temperature, molar ratio (phytosterol to fatty content at sn-2 position were investigated. Results acid), enzyme loading, and vacuum. Optimum showed that PA was incorporated into the conditions for synthesis of phytosteryl ester were a triacylglycerols (TAGs) of refined olive oil at up to temperature of 60°C, molar ratio of 1:4, enzyme 55.8% while incorporation of PA at sn-2 position and loading of 10% (based on the total weight of the total DHA were found to be up to 33.6% and 3.5%,

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014 respectively. Second-order models were generated contain LOX enzymes; however, to date there has for each of the three responses. A chi-square test only been a limited number of plant derived verified that the predicted values from the models enzymes that have been utilized to produce were not significantly different from the observed resolvins, protectins, or analogues of these values using randomly selected conditions. The molecules. prediction power of the models was further We have characterised the activity of a number confirmed by a solvent-free scale-up reaction. The of potentially useful LOX enzymes from readily produced structured lipids have potentials to be available plant materials. The regiochemistry of the used in infant formula products. products formed from biocatalytic oxidation of EPA and DHA, which is a key factor of their biological 9. Production of New Food Flavor from Myrtenol activity, has been identified by a variety of analytical and Myrtenal Using Spodoptera litura as methods. These products have the potential for use Biocatalysts. R. Motooka, A. Usami, and M. as therapeutic agents against chronic inflammatory Miyazawa, Kinki University, Higashiosaka, Osaka, diseases, or as dietary supplements to help prevent Japan. such diseases. In the course of our studies of generating bioactive compounds from natural products by 11. Conversion of Glycerol in Ethanol Stillage to biotransformation. We reported biotransformation 1,3-Propanediol. K. Ratanapariyanuch1, Y.Y. Shim2, of monoterpenoids by the larvae of common M. Haakensen3, and M. Reaney2, 1Dept. of Food and cutworm (Spodoptera litura). Bioproduct Sciences, University of Saskatchewan, (1S)-(+)- and (1R)-(-)-myrtenol (1 and 2), a Saskatoon, SK, Canada, 2Dept. of Plant Sciences, [3.1.1] bicyclic monoterpene alcohol, are known to Saskatoon, SK, Canada, 3Contango Strategies Ltd., be an important and widespread terpenoids. Saskatoon, SK, Canada. Therefore, in this study, we investigated Stillage is a dilute aqueous mixture of organic biotransformation of compound 1 and 2 by the and inorganic molecules including acetic acid, larvae common cutworm (S.litura). As a results, betaine, glycerol, glycerolphosphorylcholine, lactic compound 1 was converted to (+)-myrtenal (1-1), acid, 1,3-propanediol and succinic acid produced as (+)-myrtenic acid (1-2), (+)-(1S,5S,6R)-6-methyl- a product of ethanol fermentation. Bio-ethanol bicyclo[3.1.1]hept-2-ene-2,6-dimethanol (1-3) and production facilities in the USA produce more than (+)-(1S, 4S, 5R)-2-pinene-4,10-diol (1-4). Similarly, 350 billion liters of stillage annually and thus it could compound 2 was converted to (-)-myrtenal (2-1), (-)- be a resource for the recovery of many useful myrtenic acid (2-2) and (-)-(1R,5R,6R)-6-methyl- compounds. It was observed that storing stillage at bicyclo[3.1.1]hept-2-ene-2,6-dimethanol (2-3). warm temperatures led to conversion of the glycerol In summary, S.litura were converted regio- and and lactic acid to 1,3-propanediol and acetic acid, stereoselectively oxidation at C-10 position and respectively. Interestingly, when Lactobacillus panis hydroxylation at C-4, 9 position of (1S)-(+)- and (1R)- strains were isolated from the fermenting stillage (-)-myrtenol. solution, cultured and reintroduced to the stillage, improved conversion of glycerol to 1,3-propanediol 10. Plant Based Enzymatic Synthesis of Resolvin was observed. It was discovered that modifying pH and Protectin Analogues. T.R. Walsh, J.L. Adcock, and oxygen levels, supplementation of additional and C.J. Barrow, Deakin University, Geelong, glycerol and nutrients altered fermentation rate, Victoria, Australia. efficiency, and conversion. The best conversion Resolvins and protectins have recently been condition was under oxygen-depleted conditions at discovered as potent mediators of inflammation. pH 5, with added glucose (0.1 M) and glycerol (1 M). They have potential for use as treatment for chronic Addition of vitamins (B2, B3, and B12) and freeze- inflammatory diseases such as arthritis, dried stillage increased the total 1,3-propanediol cardiovascular disease and asthma. Biosynthesis of yield. These demonstrated that the ethanol industry these molecules is believed to occur in mammals not only holds considerable promise as a source of through biocatalytic oxidation of the omega-3 fatty commodity platform chemicals, but that the yields acids eicosapentaenoic acid (EPA) and and forms of the compounds can be modified for docosahexaenoic acid (DHA), using enzymes such as further profit. lipoxygenase (LOX). A large number of plants are also known to 12. Integration of Anaerobic/Aerobic Microbial

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014

System to Produce Lipids for Fuels. D.L. Fortela, R. and created a disruptant of the gene by homologous Hernandez, M. Zappi, E. Revellame, W. Holmes, S. recombination. The disruptant grew as well as the Dufreche, and R. Subramaniam, Dept. of Chemical parental strain and produced 7DHC instead of Engineering, University of Louisiana, Lafayette, LA, cholesterol. We isolated 7DHC from the cells and USA. confirmed that 7DHC was easily converted to Lipids from enhanced sludge have been shown vitamin D3 via UV radiation. as a promising feedstock in biodiesel production, but In conclusion, we constructed 7DHC producer the relatively high cost of the carbon source strain and it would be a useful for the production of substrates such as glucose and xylose in the lipid non-animal derived vitamin D3. accumulation creates challenge impacting broad commercialization. In this study, the feasibility of 14. Synthesis of Sugar Ester Surfactant Analog and utilizing as substrates the volatile fatty acids (VFAs) Evaluating Its Emulsion Properties. K. Ren and B. such as acetic acid, propionic acid and butyric acid Lamsal*, Iowa State University, Ames, IA, USA. produced from the anaerobic digestion of cellulose A Glucose-palmitic acid ester was synthesized was examined. The envisioned integrated process is with immobilized lipases from Candida Antarctica in the production of lipids from lignocellulosic biomass 20% dimethyl sulfoxide and 80% 2-metyl-2-butanol while minimizing energy inputs to deconstruct the solvent system at 55°C. The major product, latter to yield assimilable carbon sources. The suspected to be a surfactant-analog with a molecular experiments were conducted in 5-L bioreactors. weight of 464 g/mole was identified using thin layer Experimental results show that the concentration of chromatography and LC-MS. The crude product VFAs in the fermentation medium significantly mixtureat at 0.5% w/w was tested for stability and affects the lipid content of the enhanced sludge droplet size distribution in the oil-in-water emulsion from return activated sludge (RAS). Modeling of the at 10:1 ratio of water to canola oil. This was lipid accumulation from VFAs and the anaerobic compared with 0.5% lecithin in emulsion. Emulsion digestion of cellulose were then conducted to ability was taken as spectrophotometer absorbance determine the kinetic equations used in the of 100X diluted emulsion at 500 nm at time 0, and simulation of the integrated processes. emulsion stability as relative slope of decrease in absorbance values for next 2 h. The emulsion activity 13. Production of Non-animal Derived Vitamin D3 of surfactant-analog product mixture was much by Using a Genetically Engineered Microalgae lower at 0.25 absorbance value, compared to 0.91 of Aurantiochytrium sp. strain. T. Ujihara and S. lecithin. However, the emulsion was more stable Mitsuhashi, Kyowa Hakko Bio Co., Ltd., Tsukuba, with sugar-ester analog with a destabilization slope Ibaraki, Japan. of -0.0005, compared to lecithin at -0.002. Melvern Vitamin D3 is a fat-soluble vitamin involved in particle size measurement of emulsion showed that various important physiological functions in the emulsion stabilized with surfactant analog had peak human body such as calcium metabolism, and the droplet size of 40micron compared to 26 for lecithin demand in the nutritional field is growing. system. We suspect purity of analog to be much Vitamin D3 is currently produced by chemical smaller to be comparable with lecithin on equal conversion from animal derived steroid compounds, weight basis. Optimization of esterification reaction, however, due to the risk of viral contamination non- and purification of analog product will be further animal derived vitamin D3 will be required. Although carried out. it is well-known that some fungi produce vitamin D2 (ergosterol), vitamin D3, a natural form of vitamin D 15. An Efficient Bioprocess for Phytosterol in human body, is thought to be produced only in Extraction and Its Ester Derivatives Production by vertebrates. Using Domestic Agricultural Produce as Substrate. In this study, we examined the production of S.W. Chang, Dept. of Medicinal Botanicals and vitamin D3 from non-animal sources. Since vitamin Health Applications, Dayeh University, Dacun, D3 is synthesized from 7-dehydrocholesterol (7DHC), Changhu, Taiwan. a precursor of cholesterol, we screened cholesterol A natural compound contained in various producers in various microorganisms and isolated a cereals and seeds accompany with cholesterol- microalgae strain Aurantiochytrium sp. S442 which lowering and anti-cancer function was called produced a significant amount of cholesterol. We phytosterol. However, it cannot be synthesized by identified a gene for 7DHC reductase in the strain ourself; instead of we should uptake it from normal

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Abstracts 105th AOCS Annual Meeting & Expo May 4-May 7, 2014 diet. Since the lower solubility in oil-soluble foods is palm mesocarp, in particular the differences in gene the major problem for further industrial application, regulation leading to and including de novo fatty a modification process on phytosterol to produce oil- acid biosynthesis, a multi-platform ‘omics’ soluble ester derivatives was therefore developed. technology was used to profile mesocarp transcripts, Plant sterol esters are well-known functional proteins and metabolites during six critical stages of ingredients and have been reported to have fruit development in comparatively high-yielding beneficial effects on plasma cholesterol level for (HY) and low-yielding (LY) oil palm populations. cosmetic, nutraceutical, and pharmaceutical Amino acids were found to be significantly higher in applications. The present work, we planned to HY palms during fruit maturation in concordance combine the traditional oil extraction and with up-regulation of several amino acid metabolism esterification to produce a multiple function edible genes. Nucleotide precursor availability was also oil product using domestic agricultural produce as observed to be higher in HY at weeks 18-22 substrate. The lipase AY was selected as an optimal compared to LY fruits. 127 genes of interest (GOI) biocatalyst with higher competitive activity toward were identified to be key contributors to oil campesterol in acetone. All materials were extracted production in mesocarp with 32% involved in at 27°C for 3 h and the highest conversion yield of ß- glycolysis, fatty acid biosynthesis and lipid assembly. sitosteryl ester (81.6 ± 1.3%), campesteryl ester Levels of metabolites involved in glycolysis revealed (82.7 ± 1.2%) and stigmasteryl ester (65.8 ± 8.3%) interesting divergence of flux towards glycerol-3- was obtained at 27°C, 0.73 g enzyme amount for 24 phosphate, while carbon utilization differences in h using sesame as substrate. the TCA cycle were proven by an increase in malic acid/citric acid ratio. Several proteins in oxidation 16. Omics: Evidence of Nitrogen Assimilation, Lipid phosphorylation and ATP biosynthesis were up- Biosynthesis, and Energy Management for Higher regulated in HY fruits. Apart from insights into the Oil Palm Yield. T.S. Huat1, T.H. Fang1, T.O.E. Keong1, regulation of enhanced lipid production in oil palm, W.Y. Ching1, N.B. Keat1, T.N.L. Mei1, C.F. Tim2, H. these results provide potentially useful metabolite Kulaveerasingam1, and D.R. Appleton1, 1Sime Darby yield markers and genes of interest to assist with Technology Centre, Serdang, Selangor, Malaysia, genetic marker development for use in breeding 2National University of Singapore, Singapore. programmes. To better understand lipid biosynthesis in oil

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