DOCSLIB.ORG

Characterization of Recombinant Laccase from Trametes Versicolor

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of Recombinant Laccase from Trametes Versicolor Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in enzymatic removal of pharmaceuticals from wastewater I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Katarzyna Litwińska geboren am 22.03.1990 in Wrocław, Polen Greifswald, 2020 Dekan: Prof. Dr. Gerald Kerth 1. Gutachter: Prof. Dr. Hans-Joachim Schüller 2. Gutachter: Prof. Dr. Vlada Urlacher Tag der Promotion: 07.07.2020 TABLE OF CONTENTS TABLE OF FIGURES ............................................................................................................. IV TABLE OF TABLES ............................................................................................................... VI LIST OF ABBREVIATIONS ................................................................................................. VII SUMMARY ............................................................................................................................. IX ZUSAMMENFASSUNG ......................................................................................................... XI 1. INTRODUCTION .............................................................................................................. 1 1.1. Pharmaceuticals in the environment ............................................................................ 1 1.2. Removal of pharmaceuticals in wastewater treatment plants ...................................... 8 1.3. Laccases ..................................................................................................................... 14 1.4. Cytochromes P450 ..................................................................................................... 17 1.5. The yeast Arxula adeninivorans ................................................................................ 20 1.6. Aim of the work ......................................................................................................... 23 2. MATERIALS AND METHODS ...................................................................................... 25 2.1. Media and cultivation methods .................................................................................. 25 2.1.1. Cultivation media ................................................................................................ 25 2.1.2. Cultivation of E. coli XL1-Blue.......................................................................... 26 2.1.3. Cultivation of A. adeninivorans .......................................................................... 26 2.1.4. Cultivation of white-rot fungi ............................................................................. 26 2.1.5. Dry cell weight (DCW) measurement ................................................................ 26 2.1.6. Optical density (OD600) measurement ................................................................ 27 2.1.7. Glucose concentration measurement .................................................................. 27 2.1.8. Fed-batch fermentation of A. adeninivorans ...................................................... 27 2.2. Molecular biology techniques .................................................................................... 28 2.2.1. Primers, plasmids, and cloning strategies ........................................................... 28 2.2.2. Polymerase chain reaction (PCR) ....................................................................... 33 2.2.3. Sequencing .......................................................................................................... 34 2.2.4. Reverse transcription polymerase chain reaction (RT-PCR) .............................. 34 2.2.5. DNA and RNA electrophoresis .......................................................................... 34 2.2.6. Determination of nucleic acid concentrations..................................................... 35 2.2.7. DNA cleavage with restriction endonucleases ................................................... 35 2.2.8. DNA fragments’ extraction from agarose gel ..................................................... 35 2.2.9. Gibson Assembly ................................................................................................ 35 I 2.2.10. Plasmid DNA isolation from E. coli .................................................................... 35 2.2.11. Transformation of E. coli ..................................................................................... 36 2.2.12. Transformation of A. adeninivorans .................................................................... 36 2.2.13. Genomic DNA isolation form A. adeninivorans ................................................. 36 2.2.14. RNA isolation ...................................................................................................... 37 2.2.15. cDNA synthesis ................................................................................................... 37 2.2.16. Preparation of A. adeninivorans cell extracts ...................................................... 37 2.2.17. Microsomes isolation methods ............................................................................ 38 2.3. Biochemical techniques .............................................................................................. 40 2.3.1. SDS-PAGE analysis and molecular mass determination .................................... 40 2.3.2. SDS-PAGE analysis of membrane proteins ........................................................ 40 2.3.3. Western blotting analysis ..................................................................................... 41 2.3.4. Protein purification .............................................................................................. 41 2.3.5. Native gel electrophoresis ................................................................................... 41 2.3.6. Glycosylation staining of PAA gels .................................................................... 42 2.3.7. Deglycosylation reaction ..................................................................................... 42 2.3.8. Determination of protein concentration ............................................................... 42 2.3.9. Determination of laccase activity ........................................................................ 42 2.3.10. Determination of kinetic parameters ................................................................... 43 2.3.11. Estimation of temperature optimum and stability ............................................... 43 2.3.12. Estimation of pH optimum and stability .............................................................. 43 2.3.13. Determination of influence of ionic strength ....................................................... 44 2.3.14. Determination of metal ions influence ................................................................ 44 2.3.15. Optimization of laccase production ..................................................................... 44 2.3.16. Determination of CYP450 activity ...................................................................... 45 2.3.17. Laccase immobilization methods ........................................................................ 45 2.3.18. Degradation tests for carbamazepine, diclofenac, sulfamethoxazole .................. 46 2.3.19. Growth of yeast cells on medium supplemented with carbamazepine ................ 47 2.3.20. Determination of laccase mediators influence on pharmaceuticals degradation .......................................................................................................... 47 2.4. Analytical techniques .................................................................................................. 47 2.4.1. Thin layer chromatography ................................................................................. 47 2.5. Bioinformatics tools .................................................................................................... 48 3. RESULTS .......................................................................................................................... 49 II 3.1. Laccase gene expression ............................................................................................ 49 3.2. Analysis of laccases sequences .................................................................................. 57 3.3. Cultivation condition and Tvlcc5 enzyme purification.............................................. 58 3.4. Biochemical characterization of recombinant laccase Tvlcc5 ................................... 64 3.5. Increase of laccase production ................................................................................... 70 3.6. Laccase immobilization ............................................................................................. 75 3.7. Cytochrome P450 gene expression ............................................................................ 78 3.8. Analysis of CYP450 synthesis in A. adeninivorans .................................................. 79 3.9. Degradation of pharmaceuticals................................................................................. 86 3.9.1. Degradation of pharmaceuticals by laccase ........................................................ 86 3.9.2. Degradation of carbamazepine by whole cell catalyst .......................................
Load more

Recommended publications
  • WO 2018/009838 Al 11 January 2018 (11.01.2018) W !P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/009838 Al 11 January 2018 (11.01.2018) W !P O PCT (51) International Patent Classification: Declarations under Rule 4.17: C12N 5/075 (2010.01) — as to applicant's entitlement to apply for and be granted a (21) International Application Number: patent (Rule 4.1 7(H)) PCT/US2017/041 155 — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(Hi)) (22) International Filing Date: 07 July 2017 (07.07.2017) Published: — with international search report (Art. 21(3)) (25) Filing Language: English — before the expiration of the time limit for amending the (26) Publication Langi English claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (30) Priority Data: — with sequence listing part of description (Rule 5.2(a)) 62/359,416 07 July 2016 (07.07.2016) US (71) Applicant: RUBIUS THERAPEUTICS, INC. [US/US]; 620 Memorial Dr #100W, Cambridge, MA 02139 (US). (72) Inventors; and (71) Applicants: HARANDI, Omid [US/US]; 39 Rowena Road, Newton, MA 02459 (US). KHANWALKAR, Ur- jeet [IN/US]; 2 11 Elm Street, Apt. 3, Cambridge, MA 02139 (US). HARIHARAN, Sneha [IN/US]; 18 Hamilton Road, Apt. 407, Arlington, MA 02472 (US). (72) Inventors: KAHVEJIAN, Avak; 2 Beverly Road, Arling ton, MA 02474 (US). MATA-FINK, Jordi; 8 Windsor Rd #1, Somerville, MA 02144 (US).DEANS, Robert, J.; 1609 Ramsgate Court, Riverside, CA 92506 (US).
    [Show full text]
  • Separation and Partial Purification of Collagenolytic Protease
    Biocatalysis and Agricultural Biotechnology 24 (2020) 101509 Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: http://www.elsevier.com/locate/bab Separation and partial purificationof collagenolytic protease from peacock bass (Cichla ocellaris) using different protocol: Precipitation and partitioning approaches Vagne de Melo Oliveira a, Marcia� Nieves Carneiro da Cunha a, Caio Rodrigo Dias de Assis b,f, Juanize Matias da Silva Batista a, Thiago Pajeú Nascimento a, Juliana Ferreira dos Santos c, Carolina de Albuquerque Lima d, Daniela de Araújo Viana Marques e, Ranilson de Souza Bezerra b, Ana Lúcia Figueiredo Porto a,* a Laboratorio� de Tecnologia de Produtos Bioativos, Departamento de Morfologia e Fisiologia Animal, DMFA, Universidade Federal Rural de Pernambuco, Av. Dom Manoel, de Medeiros, s/n, 52171-900, Recife, PE, Brazil b Laboratorio� de Enzimologia, Departamento de Bioquímica, Universidade Federal de Pernambuco, Cidade Universitaria,� 50670-420, Recife, PE, Brazil c Laboratorio� de Aquicultura e Biotecnologia, LABITEC, Unidade Acad^emica de Serra Talhada, Universidade Federal Rural de Pernambuco, Serra Talhada, PE, Brazil d Universidade de Pernambuco, UPE, Rua Capitao~ Pedro Rodrigues, 105, 55294-902, Garanhuns, PE, Brazil e Universidade de Pernambuco, UPE, Instituto de Ci^encias Biologicas� - ICB, Departmento de Parasitologia, Rua Arnobio� Marques, 310 - Santo Amaro, Recife, PE, 50100- 130, Brazil f Laboratorio� de Compostos Organicos^ em Ecossistemas Costeiros e Marinhos, OrganoMAR, Departamento de Oceanografia,Universidade Federal de Pernambuco, Cidade Universitaria,� 50670-420, Recife, PE, Brazil ARTICLE INFO ABSTRACT Keywords: This comparative study provides a useful protocol for the separation and partial purification of collagenolytic ATPS proteases obtained from peacock bass, by using precipitation and partitioning.
    [Show full text]
  • Arxula Adeninivorans
    Biernacki et al. Microb Cell Fact (2017) 16:144 DOI 10.1186/s12934-017-0751-4 Microbial Cell Factories RESEARCH Open Access Enhancement of poly(3‑hydroxybutyrate‑co‑ 3‑hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production Mateusz Biernacki1, Marek Marzec1,6, Thomas Roick2, Reinhard Pätz3, Kim Baronian4, Rüdiger Bode5 and Gotthard Kunze1* Abstract Background: In recent years the production of biobased biodegradable plastics has been of interest of research- ers partly due to the accumulation of non-biodegradable plastics in the environment and to the opportunity for new applications. Commonly investigated are the polyhydroxyalkanoates (PHAs) poly(hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-V). The latter has the advantage of being tougher and less brittle. The production of these polymers in bacteria is well established but production in yeast may have some advantages, e.g. the ability to use a broad spectrum of industrial by-products as a carbon sources. Results: In this study we increased the synthesis of PHB-V in the non-conventional yeast Arxula adeninivorans by stabilization of polymer accumulation via genetic modifcation and optimization of culture conditions. An A. adenini- vorans strain with overexpressed PHA pathway genes for β-ketothiolase, acetoacetyl-CoA reductase, PHAs synthase and the phasin gene was able to accumulate an unexpectedly high level of polymer. It was found that an opti- 1 mized strain cultivated in a shaking incubator is able to produce up to 52.1% of the DCW of PHB-V (10.8 g L− ) with 12.3%mol of PHV fraction. Although further optimization of cultivation conditions in a fed-batch bioreactor led to lower polymer content (15.3% of the DCW of PHB-V), the PHV fraction and total polymer level increased to 23.1%mol 1 and 11.6 g L− respectively.
    [Show full text]
  • Rhodococcus Jostii Strain 8
    Functional characterisation of alkane-degrading monooxygenases in Rhodococcus jostii strain 8 Jindarat Ekprasert A thesis submitted to the School of Environmental Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy September 2014 University of East Anglia Norwich, UK i Contents List of figures viii List of tables xiii Declaration xv Acknowledgements xvi Abbreviations xvii Abstract xxi Chapter 1 introduction 1 1.1. Significance of alkanes in the environment 2 1.1.1. Chemistry of alkanes 2 1.2. The Rhodococcus genus 3 1.2.1. Common characteristics of Rhodococcus spp. 3 1.2.2. Rhodococcus spp. are capable of degrading gaseous alkanes 4 1.2.3. Potential applications of Rhodococcus in biotechnology 5 1.3. Bacterial enzymes responsible for alkane degradation 6 1.3.1. Integral membrane, non-heme iron alkane hydroxylases (AlkB) 6 1.3.2. Soluble di-iron monooxygenases (SDIMO) 8 1.3.2.1. SDIMO classification 10 1.3.2.2. Molecular genetics of SDIMOs 13 1.3.2.3. Mutagenesis of soluble methane monooxygenase 13 1.3.3. Cytochrome P450 alkane hydroxylases 14 3.3.1. Class I P450 14 3.3.2. Class II P450 (CYP52) 15 3.3.3. Class II P450 (CYP2E, CYP4B) 15 1.3.4. Membrane bound copper-containing (and possibly iron-containing) monooxygenases 15 1.4. Alkane metabolisms in Rhodococcus spp. 16 1.4.1. Aerobic metabolism of C2-C4 gaseous alkanes in bacteria 16 1.4.1.1. Ethane (C2H6) metabolism 16 1.4.1.2. Propane (C3H8) metabolism 17 ii 1.4.1.3.
    [Show full text]
  • KEGG Orthology-Based Annotation of the Predicted
    Dunlap et al. BMC Genomics 2013, 14:509 http://www.biomedcentral.com/1471-2164/14/509 DATABASE Open Access KEGG orthology-based annotation of the predicted proteome of Acropora digitifera: ZoophyteBase - an open access and searchable database of a coral genome Walter C Dunlap1,2, Antonio Starcevic4, Damir Baranasic4, Janko Diminic4, Jurica Zucko4, Ranko Gacesa4, Madeleine JH van Oppen1, Daslav Hranueli4, John Cullum5 and Paul F Long2,3* Abstract Background: Contemporary coral reef research has firmly established that a genomic approach is urgently needed to better understand the effects of anthropogenic environmental stress and global climate change on coral holobiont interactions. Here we present KEGG orthology-based annotation of the complete genome sequence of the scleractinian coral Acropora digitifera and provide the first comprehensive view of the genome of a reef-building coral by applying advanced bioinformatics. Description: Sequences from the KEGG database of protein function were used to construct hidden Markov models. These models were used to search the predicted proteome of A. digitifera to establish complete genomic annotation. The annotated dataset is published in ZoophyteBase, an open access format with different options for searching the data. A particularly useful feature is the ability to use a Google-like search engine that links query words to protein attributes. We present features of the annotation that underpin the molecular structure of key processes of coral physiology that include (1) regulatory proteins of
    [Show full text]
  • Transcriptome Analysis of Differential Gene Expression in Dichomitus Squalens
    bioRxiv preprint doi: https://doi.org/10.1101/359646; this version posted June 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Transcriptome analysis of differential gene expression in Dichomitus squalens 2 during interspecific mycelial interactions and the potential link with laccase 3 induction 4 Zixuan Zhong1, Nannan Li1, Binghui He, Yasuo Igarashi, Feng Luo* 5 6 Research Center of Bioenergy and Bioremediation, College of Resources and Environment, 7 Southwest University, Beibei, Chongqing 400715, People’s Republic of China 8 9 *corresponding author: Feng Luo (FL) [email protected] 10 11 1These authors contributed equally to the paper 12 13 ABSTRACT 14 Interspecific mycelial interactions between white rot fungi are always accompanied by increased 15 production of laccase. In this study, the potential of white rot fungi Dichomitus squalens for 16 enhancing laccase production during interaction with two other white rot fungi Trametes 17 versicolor or Pleurotus ostreatus was identified. To probe the mechanism of laccase induction and 18 the role of laccase played during the combative interaction, we analyzed the laccase induction 19 response to stressful conditions during fungal interaction related to the differential gene expression 20 profile. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. 21 We noted that many differential expression genes (DEGs) encoding proteins were involved in 22 xenobiotics detoxification and ROS generation or reduction, including aldo/keto reductase, 23 glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, 24 manganese peroxidase and laccase.
    [Show full text]
  • Modelling of the Melissa Artificial Ecosystem
    ESTEC/CONTRACT 8125/88/NL/‘FG PRF 141315 Modelling of the MELiSSA artificial ecosystem Toward a structured model of the nitrifying compartment - Description of the respiratory chain of nitrifying organisms - Development of assumptions for the reverse electron flow in the respiratory electron transport chain - Stoichiometric description of the nitrification - Determination of KL~ for oxygen transfer limitation TECHNICAL NOTE 23.2 L. Poughon Laboratoire de Genie Chimique Biologique 63177 AUBIERE Cedex, FRANCE April 1995 Technical note 23.2 Toward a structured model of the nitrifying compartment T.N. 23.2: Modelling of the MELiSSA artificial ecosystem TOWARD A STRUCl-URJZD MODEL OF THE NITRIFYING COMPARTMENT L. Poughon. Laboratoire de Genie Chimique Biologique 63177 AUBIERE Cedex. France. INTRODUCTION In the MELiSSA loop the nitrifying compartment has the same function than the nitrifying process in the terrestrial ecosystem (figure 1) which is to provide an edible N-source for plants or micro-organisms (as Spirulines in the case of the MELiSSA loop). The ammoniflcation processes from organic waste (as for example the human waste faeces and urea) are performed in the MELiSSA loop by the 2 first compartments (liquefying and anoxygenic phototrophs compartments). It must be noted that there are some structural differences between the MELiSSA N-loop and the terrestrial ecosystem: l- the MELiSSA loop represent a very simplified part of the N loop encountered on earth; 2- the denitrification process (N mineral -> N2) or the N2 removing (N2 -> N mineral) are not considered 3- the sole N - source is N03- for Spirulina, it is NlQ+ for phototrophs and it is organic N for the crew.
    [Show full text]
  • Relating Metatranscriptomic Profiles to the Micropollutant
    1 Relating Metatranscriptomic Profiles to the 2 Micropollutant Biotransformation Potential of 3 Complex Microbial Communities 4 5 Supporting Information 6 7 Stefan Achermann,1,2 Cresten B. Mansfeldt,1 Marcel Müller,1,3 David R. Johnson,1 Kathrin 8 Fenner*,1,2,4 9 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, 10 Switzerland. 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 11 Zürich, Switzerland. 3Institute of Atmospheric and Climate Science, ETH Zürich, 8092 12 Zürich, Switzerland. 4Department of Chemistry, University of Zürich, 8057 Zürich, 13 Switzerland. 14 *Corresponding author (email: [email protected] ) 15 S.A and C.B.M contributed equally to this work. 16 17 18 19 20 21 This supporting information (SI) is organized in 4 sections (S1-S4) with a total of 10 pages and 22 comprises 7 figures (Figure S1-S7) and 4 tables (Table S1-S4). 23 24 25 S1 26 S1 Data normalization 27 28 29 30 Figure S1. Relative fractions of gene transcripts originating from eukaryotes and bacteria. 31 32 33 Table S1. Relative standard deviation (RSD) for commonly used reference genes across all 34 samples (n=12). EC number mean fraction bacteria (%) RSD (%) RSD bacteria (%) RSD eukaryotes (%) 2.7.7.6 (RNAP) 80 16 6 nda 5.99.1.2 (DNA topoisomerase) 90 11 9 nda 5.99.1.3 (DNA gyrase) 92 16 10 nda 1.2.1.12 (GAPDH) 37 39 6 32 35 and indicates not determined. 36 37 38 39 S2 40 S2 Nitrile hydration 41 42 43 44 Figure S2: Pearson correlation coefficients r for rate constants of bromoxynil and acetamiprid with 45 gene transcripts of ECs describing nucleophilic reactions of water with nitriles.
    [Show full text]
  • Comparison of Physiology and Genome-Wide Expression in Two Nitrosomonas Spp
    Comparison of physiology and genome-wide expression in two Nitrosomonas spp. under batch cultivation By Mohammad Ghashghavi A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In Microbiology and Biotechnology Department of Biological Sciences University of Alberta © Mohammad Ghashghavi, 2014 Abstract: Ammonia oxidizing bacteria (AOB) play a central role in the nitrogen cycle by oxidizing ammonia to nitrite. Nitrosomonas europaea ATCC 19718 has been the single most studied AOB that has contributed to our understanding of chemolithotrophic ammonia oxidation. As a closely related species, Nitrosomonas eutropha C91 has also been extensively studied. Both of these bacteria are involved in wastewater treatment systems and play a crucial part in major losses of ammonium-based fertilizers globally. Although comparative genome analysis studies have been done before, change in genome-wide expression between closely related organisms are scarce. In this study, we compared these two organisms through physiology and transcriptomic experiments during exponential and early stationary growth phase. We found that under batch cultivation, N. europaea produces more N2O while N. eutropha consumes more nitrite. From transcriptomic analysis, we also found that there are selections of motility genes that are highly expressed in N. eutropha during early stationary growth phase and such observation was completely absent in N. europaea. Lastly, principle homologous genes that have been well studied had different patterns of expression in these strains. This study not only gives us a better understanding regarding physiology and genome-wide expression of these two AOB, it also opens a wide array of opportunities to further our knowledge in understanding other closely related species with regards to their evolution, physiology and niche preference.
    [Show full text]
  • Purification and Characterization of Laccase from Basidiomycete Fomitella Fraxinea
    J. Microbiol. Biotechnol. (2008), 18(4), 670–675 Purification and Characterization of Laccase from Basidiomycete Fomitella fraxinea Park, Kyung Mi† and Sang-Shin Park* Department of Biotechnology, Dongguk University, Gyeongju 780-714, Korea Received: August 8, 2007 / Accepted: November 2, 2007 A laccase was isolated from the culture filtrate of the peroxidase (LiP), manganese peroxidase (MnP), and laccase basidiomycete Fomitella fraxinea. The enzyme was purified [33]. Although the production of MnP and laccase is much to electrophoretical homogeneity using ammonium sulfate more common than that of LiP and MnP, the white-rot precipitation, anion-exchange chromatography, and gel- basidiomycete Coriolopsis rigida secretes no detectable filtration chromatography. The enzyme was identified as LiP or MnP, yet a good amount of laccase [28], and this a monomeric protein with a molecular mass of 47 kDa by laccase has been demonstrated to be capable of degrading sodium dodecyl sulfate-polyacrylamide gel electrophoresis lignin, including nonphenolic moieties. Additional studies (SDS-PAGE) and gel-filtration chromatography, and had have also showed that laccase plays a key role in the lignin an isoelectric point of 3.8. The N-terminal amino acid degradation process [22]. sequence for the enzyme was ATXSNXKTLAAD, which had Laccase (benzenediol:oxygen oxidoreductase, E.C. 1.10.3.2) a very low similarity to the sequences previously reported is a polyphenol oxidase that catalyzes the oxidation of for laccases from other basidiomycetes. The optimum pH phenolic compounds and aromatic amines with molecular and temperature for 2,2'-azino-bis(3-ethylbenzothiazoline- oxygen as the electron acceptor [31]. Laccases are typically 6-sulfonate) (ABTS) were 3.0 and 70oC, respectively.
    [Show full text]
  • Design and Fine-Tuning Redox Potentials of Metalloproteins Involved in Electron Transfer in Bioenergetics
    1 Design and Fine-tuning Redox Potentials of Metalloproteins Involved in Electron Transfer in Bioenergetics Parisa Hosseinzadeh and Yi Lu Abstract: 1. Introduction A significant portion of biological processes are involved with providing vital energy sources such as ATP, and controlling the flow of energy through living systems. These bioenergetics processes, the most important of which are photosynthesis and respiration, require electron transfer (ET) between different redox partners. Metalloproteins are one of the most widely used ET centers in biology. They can be classified into three major classes: cupredoxins, which include type 1 copper (T1Cu) proteins and CuA centers [1- 10], cytochromes [10-17], and iron-sulfur (FeS) proteins [10,18-24]. Each class of ET proteins transfer electrons between different redox partners which possess different reduction potentials (E°) (Figure 1). Therefore, the ET centers need to adjust their E° in a way that matches those of their redox partners. No single class of protein can cover the entire range of physiological E°, which is between ~ -1V, at which protons are reduced to H2, and 1V, at which water is oxidized to O2. Cupredoxins usually function at the high end of the E°, while FeS proteins are mostly involved in ET reactions possessing relatively low E° [10]. The E°’s of FeS proteins overlap significantly with those of cytochromes that often have intermediate E° among the three classes of ET proteins. Figure 1. Reduction potential range of metal centers in electron transfer metalloprotein. Adapted from ref. [10] 2 In this review, we first describe the importance of tuning E° of ET centers, including the metalloproteins described above.
    [Show full text]
  • LACCASE from MYCELIOPHTHORA THERMOPHILA EXPRESSED in ASPERGILLUS ORYZAE Chemical and Technical Assessment (Cta) First Draft Prepared by Zofia Olempska-Beer
    Chemical and Technical Assessment 61st JECFA LACCASE FROM MYCELIOPHTHORA THERMOPHILA EXPRESSED IN ASPERGILLUS ORYZAE Chemical and Technical Assessment (Cta) First draft prepared by Zofia Olempska-Beer © FAO 2004 1 Summary Laccase is an enzyme that catalyzes the oxidation of phenolic compounds such as ortho- and para- diphenols to their corresponding quinones with the concomitant reduction of oxygen to water. The laccase described in this document is manufactured by pure culture fermentation of a genetically modified nonpathogenic and nontoxigenic strain of Aspergillus oryzae that contains the laccase gene derived from Myceliophthora thermophila. This production strain was developed from a nonpathogenic A. oryzae strain IFO 4177 (also known as A 1560) using recombinant DNA techniques and traditional mutagenesis. During fermentation, laccase is secreted to the fermentation broth. It is subsequently purified, concentrated, and formulated with appropriate substances. The formulated laccase, referred to below as the laccase preparation, is marketed for use in brewing beer to prevent the formation of off-flavor compounds, such as trans-2-nonenal. Laccase scavenges oxygen which otherwise would react with fatty acids, amino acids, proteins, and alcohols to form off-flavor precursors. The laccase preparation is marketed under a trade name “Flavourstar.” The manufacturer of this product, Novozymes A/S, submitted a dossier to JECFA containing detailed information about laccase and laccase preparation (Novozymes A/S, 2002). This Chemical and Technical Assessment is based on Novozymes’ information as well as published information relevant to the source microorganism, A. oryzae. The A. oryzae production strain, designated as Mt, was developed by transformation of the A. oryzae host strain How B711 (derived from the A 1560 strain) with two plasmids pRaMB17.WT and pToC90.
    [Show full text]