DOCSLIB.ORG

Utilizing Bacteria. 7

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Utilizing Bacteria. 7 BIOCATALYSTS FOR PRODUCTION A thesis submitted for the degree of Doctor of Philosophy of the University of London By Mahmoud Seyed-Mahmoudian Centre for Biotechnology Imperial College of Science, Technology & Medicine London - England July 1989 Abstract An extensive screen for microorganisms capable of growth on olefins was carried out. Over 100 bacterial strains (mesophilic, thermophilic) were isolated in pure culture with gaseous or liquid (internal & terminal) olefins as the sole source of carbon and energy. The organisms included Aero coccus, Aeromonas,Alcaligenes, Flavobacterium, Micrococcus, Moraxella, Nocardia, Pseudomonas, Staphylococcus, Streptomyces and Vibrio spp. and a variety of Gram-negative, Gram-positive and Gram-variable rods/coccobacilli not yet identified. Given the success of isolating many different genera of alkene-utilizing bacteria, it would suggest that the ability to utilize olefin hydrocarbons is more widespread in nature than previously recognized. Initial studies involved testing a collection of strains for i) growth rate on the substrate of isolation and ii) evidence of epoxide accumulation from lower gaseous olefins. All of the 18 ethene- and propene-utilizing bacteria tested, stereospecifically formed R- 1,2-epoxypropane (ee=90-96%), R -l,2-epoxybutane (ee=90- 98%) and trans- (2/?,37?)-epoxybutane (ee=64-88%), as analyzed by chiral complexation glc. A particularly promising strain of Micrococcus sp. M90C was selected to demonstrate: a) Induction of epoxidation activity in carbohydrate-grown cells. b) Optimization of a biotransformation process for production of epoxyethane. c) Involvement of a specific monooxygenase in the formation of chiral epoxides. d) Stereoselectivity of epoxyalkane degradation. e) Substrate specificity toward a range of olefins. Micrococcus sp. M90C stereospecifically formed S-(+)-phenyl glycidyl ether (ee=93%) and /?-(+)- phenyl methyl sulphoxide (ee>98%), as analyzed by chiral-shift NMR and HPLC. The ability to catalyze heteroatom oxygenation as well as epoxidation of a wide range of olefins in high optical and chemical yields, demonstrates its potential as a 'general-purpose' biocatalyst for the production of optically pure oxygenated compounds. Two other promising ethene-utilizing bacteria (M26, M93 A) each catalyzed the stereospecific formation of S-(+)-phenyl glycidyl ether (ee=93%). This has attracted considerable industrial interest and is currently under investigation at ICI for commercial production of optically active./1-blockers. In addition to Micrococcus sp. M90C, the substrate specificities of 6 other organisms were studied in order to probe the active site of the enzymes involved. The pattern of reactivity for the group of 4 ethene- (M26, M90C, M93A, M186) and 2 propene- (M142, Ml 56) utilizers differed from that expected with peracids, suggesting a considerable degree of enzymatic control.The nature of the substrate, however, modulated enzymatic epoxidations in Staphylcoccus sp. M97B. CONTENTS Page No. List of tables i List of figures ii Acknowledgements iii Abbreviations iv Chapter 1 Introduction 1.1. Microbial epoxidation of olefins 4 1.1.1. Alkane-utilizers 6 1.1.2. Methanotrophs 7 1.1.3. Alkene-utilizers 8 1.1.4. Nitrifying bacterium 11 1.1.5. Biochemical role of epoxidation reactions 12 1.1.6. Haloperoxidase and halohydrin epoxidase 16 1.2. Microbial metabolism of epoxyalkanes 17 1.3. Applications of epoxidation reactions in biotechnological processes 20 Chapter 2 Methods 2.1. General techniques 25 2.1.1. Cleaning of glassware 25 2.1.2. Determination of pH 25 2.1.3. Sterility 25 2.1.4. Centrifugation 26 Page No. 2.2. Chemicals 26 2.3. Bacterial strains 27 2.4. Growth conditions 27 2.5. Biomass analyses 28 2.5.1. Cultural turbidity 28 2.5.2. Determination of protein 28 2.5.2.1. Bio-Rad assay 28 2.5.2.2. Lowry method 29 2.5.3. Dry weight measurements 30 2.5.3.1. Filtration 30 2.5.3.2. Centrifugation 30 2.6. Bioconversions 30 2.7. Preparation of cell-free extracts 31 2.8. Product analyses 31 2.8.1. Gas liquid chromatography 31 2.8.2. Mass spectrometry 34 2.9. Chiral analyses 34 2.9.1. Chiral complexation capillary glc 34 2.9.2. Chiral-shiftlK NMR 34 2.9.3. HPLC 35 2.10. Optical rotation 35 2.11. Staining 36 2.11.1. Differential stains 36 2.11.1.1. Gram method 36 2.11.1.2. Ziehl-Neelsen method (Acid-fast stain) 37 Page No. 2.11.2. Special stains 38 2.11.2.1. Spore stain 3 8 2.11.2.2. Capsule stain 39 2.12. Characterization tests 40 2.12.1. Arginine hydrolysis 40 2.12.2. Bile esculin (aesculin) hydrolysis 41 2.12.3. Carbohydrate fermentation 42 2.12.4. Catalase activity 43 2.12.5. Citrate utilization 44 2.12.6. Fluorescein production 45 2.12.7. Gelatin liquefaction 45 2.12.8. Indole production 46 2.12.9. MacConkey agar 46 2.12.10. Methyl red (MR) 47 2.12.11. Motility 48 2.12.12. Nitrate reduction 49 2.12.13. Oxidase activity 50 2.12.14. Oxidation fermentation (OF) 51 2.12.15. Slide culture 54 2.12.16. Starch hydrolysis 54 2.12.17. Triple sugar iron (TSI) agar 55 2.12.18. Urease activity 56 2.12.19. Voges Proskauer (VP) 57 2.13. Appendix: Gas chromatographic methods 59 Chapter 3 Results and Discussion 3.1. Isolation and characterization of alkene-utilizing microorganisms 69 3.1.1. Isolation strategy 69 3.1.2. Selective culture techniques 72 3.1.2.1. Direct plating 72 3.1.2.2. Enrichment 72 3.1.3. Culture purity and maintenance of isolates 73 Page No. 3.1.4. Taxonomic and biochemical characterization 74 3.1.4.1. Group (I): Mycelial-type 75 3.1.4.2. Group (II): Coccoid 77 3.1.4.3. Group (III): Rods and coccobacilli 78 3.1.5. Selection of suitable bacteria 81 3.1.5.1. Gaseous olefins 81 3.1.5.2. Liquid olefins 81 3.1.5.3. Thermophilic bacteria 82 3.2. Micrococcus sp. M90C 84 3.2.1. Characterization 84 3.2.2. Growth on various substrates 84 3.2.3. Epoxidation of gaseous olefins 86 3.2.4. Enantiomeric composition of epoxyalkanes 86 3.2.5. Low temperature storage stability 88 3.2.6. Process optimization 93 3.2.6.1. pH Optimum 93 3.2.6.2. pH Profile for production vs degradation 95 3.2.6.3. Optimization of temperature, buffer and substrate concentration 95 3.2.7. Induction of epoxidation activity 99 3.2.8. Stereoselectivity of epoxyalkane degradation 101 3.2.9. Effect of cell age on product formation 103 3.2.10. Substrate specificity 103 3.2.10.1. Internal and terminal olefins 103 3.2.10.2. Cyclic olefins 105 3.2.10.3. Aryl-substituted olefins 107 3.2.11. The involvement of a specific monooxygenase 114 3.2.12. Asymmetric sulphur oxygenation 117 3.2.12.1. Introduction 117 3.2.12.2. Production of phenyl methyl sulphoxide 118 3.2.12.3. Optical purity of microbial sulphoxidation 122 Page No. 3.3. Biocatalysts for production of chiral epoxides 126 3.3.1. Growth characteristics on hydrocarbons 126 3.3.2. Enantiomeric excess of lower epoxyalkanes 128 3.4. Beta-blockers 131 3.4.1. Bacterial epoxidation of phenyl allyl ether 131 3.4.2. Enantiomeric excess of phenyl glycidyl ether 136 3.5. A comparison of substrate specificities of alkene- utilizing bacteria 142 3.5.1. Internal and terminal olefins 142 3.5.2. Cyclohexene 143 3.5.3. Styrene and phenyl allyl ether 143 3.5.4. 1-Trifluoromethyl, 1-(4'ethoxy phenyl)-ethene 150 3.6. Summary 151 References 158 List of Tables 1. Solubility of paraffins and olefins in water at room temperature. 2. Solubility of cyclic and aromatic hydrocarbons in water at room temperature. 3. Protein content of various subcellular fractions. 4. Carbohydrate utilization pattern for Gram-negative organisms. 5. Carbohydrate utilization pattern for Gram-positive organisms. 6. Gaseous and liquid olefins as isolation substrates for alkene-utilizing bacteria. 7. Taxonomic and biochemical properties of olefin-utilizing Streptomyces spp. 8. Taxonomic and biochemical properties of olefin-utilizing Nocardia spp. 9. Identification of Gram-positive olefin-utilizing bacteria. 10. Identification of ethene-utilizing bacteria. 11. Identification of Gram-negative olefin-utilizing bacteria. 12. Production of epoxyalkanes by Micrococcus sp. M90C. 13. Enantiomeric composition of epoxyalkanes by ethene-grown Micrococcus sp. M90C. 14. pH Profile for degradation of epoxyethane by Micrococcus sp. M90C. 15. A comparison of catalytic activity of various alkane- and alkene-utilizing bacteria for the epoxidation of ethene. 16. Effect of culture age on formation of epoxyethane by Micrococcus sp. M90C. 17. Epoxidation of olefins by ethene-grown Micrococcus sp. M90C. 18. Production of epoxyethane by whole cells and cell-free extracts of Micrococcus sp. M90C. 19. Enantiomeric composition of epoxyalkanes by whole cells and cell-free extracts of Micrococcus sp. M90C. 20. Catalytic activity of Micrococcus sp. M90C for the production of phenyl methyl sulphoxide. 21. Reported optical purities of microbial phenyl methyl sulphoxide. 22. Pattern of hydrocarbon utilization by ethene- and propene-grown bacteria. 23. Enantiomeric composition of microbial epoxidations. 24. Production of phenyl glycidyl ether by various alkene-utilizing bacteria. 25. Optical rotations and enantiomeric compositions of microbial phenyl glycidyl ether. 26. Reported chiralities of phenyl glycidyl ether by various bacteria. 27. A comparison of substrate specificities of various alkene-utilizing bacteria. 28. Summary of the newly isolated bacterial genera on gaseous and liquid olefins. 29. Reported isolation of bacterial genera on gaseous olefins. i List of Figures 1. Reactions involved in microbial epoxidation. 2. The structures of selected polycyclic aromatic hydrocarbons. 3. The pathways utilized by mammals for the metabolism of aromatic hydrocarbons.
Load more

Recommended publications
  • Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam
    Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam El Khawand A thesis submitted to the University of East Anglia in fulfillment of the requirements for the degree of Doctor of Philosophy School of Environmental Sciences November 2014 ©This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derive there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract Isoprene is a climate active gas emitted from natural and anthropogenic sources in quantities equivalent to the global methane flux to the atmosphere. 90 % of the emitted isoprene is produced enzymatically in the chloroplast of terrestrial plants from dimethylallyl pyrophosphate via the methylerythritol pathway. The main role of isoprene emission by plants is to reduce the damage caused by heat stress through stabilizing cellular membranes. Isoprene emission from microbes, animals, and humans has also been reported, albeit less understood than isoprene emission from plants. Despite large emissions, isoprene is present at low concentrations in the atmosphere due to its rapid reactions with other atmospheric components, such as hydroxyl radicals. Isoprene can extend the lifetime of potent greenhouse gases, influence the tropospheric concentrations of ozone, and induce the formation of secondary organic aerosols. While substantial knowledge exists about isoprene production and atmospheric chemistry, our knowledge of isoprene sinks is limited. Soils consume isoprene at a high rate and contain numerous isoprene-utilizing bacteria. However, Rhodococcus sp. AD45 is the only terrestrial isoprene-degrading bacterium characterized in any detail.
    [Show full text]
  • 429 Chem. Listy 97, 363–520 (2003)
    Chem. Listy 97, 363–520 (2003) Posters P117 APPLICATION OF DEGENERATE REFERENCES OLIGONUCLEOTIDE GENE SHUFFLING FOR CONSTRUCTION OF HYBRID 1. Janssen D. B., Pries F., Ploeg J., Kazemier B., Terpstra P., HALOALKANE DEHALOGENASE Witholt B.: J. Bacteriol. 171, 6791 (1989). 2. Nagata Y., Nariya T., Ohtomo R., Fukuda M., Yano K., ANDREA JESENSKÁa, YUJI NAGATAb, Takagi M.: J. Bacteriol. 175, 6403 (1993). and JIŘÍ DAMBORSKÝc 3. Kulakova A. N., Larkin M. J., Kulakov L. A.: Microbio- logy 143, 109 (1997). a National Centre for Biomolecular Research, Faculty of Science, 4. Jesenska A., Bartos M., Czernekova V., Rychlik I., Masaryk University, Kotlářská 2, Brno, 611 37, Czech Republic, Pavlik I., Damborsky V.: Appl. Environ. Microbiol. 68, e-mail: [email protected]; bDepartment of Environmen- 3724 (2002). tal Life Sciences, Graduate School of Life Sciences, Tohoku 5. Gibbs M. D., Nevalainen K. M., Bergquist P. L.: Gene University, Sendai 980-8577, Japan 271, 13 (2001). Keywords: haloalkane dehalogenase, in vitro recombination, P118 KINETICS AND SPECIFICITY construct, substrate specificity OF HALOALKANE DEHALOGENASE LinB FROM Sphingomonas paucimobilis UT26 Halogenated organic compounds constitute one of the ZBYNĚK PROKOPa, MARTA MONINCOVÁa, largest groups of environmental pollutants as a result of their MARTIN KLVAŇAa, RADKA CHALOUPKOVÁa, widespread use in industry and agriculture. Haloalkane de- DICK B. JANSSENb, YUJI NAGATAc, halogenases hydrolytically convert halogenated aliphatic and JIŘÍ DAMBORSKÝa compounds to corresponding alcohols. Enhancement of cata- lytic properties of environmentally important enzymes using aNational Centre for Biomolecular Research, Masaryk Univer- in vitro evolution techniques is one of the obvious goal of bio- sity, Kotlářská 2, 611 37 Brno, Czech Republic; bUniversity of technology.
    [Show full text]
  • Genetic Characterisaton of Rhodococcus Rhodochrous ATCC
    The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes n ow Joni Frederick A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the Departmentty of of MolecularCape and T Cell Biology, Universitysi of Cape Town er UnivSupervisor: Professor B. T. Sewell Co-supervisor: Professor D. Brady February 2013 Keywords Nitrile hydrolysis Biocatalysis Rhodococcus rhodochrous ATCC BAA-870 Genome sequencing Nitrilase Nitrile hydratase n ow ty of Cape T si er Univ ii Keywords Abstract Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economyn than non- selective chemical methods of performing this reaction, suchow as acid or base hydrolysis.
    [Show full text]
  • ENVIS CENTER on ENVIRONMENTAL BIO TECHNOLOGY
    ENVIS CENTER on ENVIRONMENTAL BIO TECHNOLOGY Abstract Vol. XIX Sponsored by MINISTRY OF ENVIRONMENT AND FORESTS GOVERNMENT OF INDIA NEW DELHI Department of Environmental Science University of Kalyani Nadia, West Bengal December, 2011 Published by: Prof. S. C. Santra Co-ordinator ENVIS Centre of Environmental Biotechnology Department of Environmental Science University of Kalyani, Kalyani –741235, Nadia, West Bengal, INDIA Email: [email protected], [email protected] Website: http://www.deskuenvis.nic.in ENVIS CENTRE on ENVIRONMENTAL BIOTECHBNOLOGY Prof. S. C. Santra : Coordinator , ENVIS Centre ENVIS’s Staff 1. Ms. Amrita Saha : Information Officer 2. Shri S. Banerjee : Web Assistant cum Data Entry Operator C O N T E N T S Sl. Title Page No. No. 1. Background 5 2. Abstract format 6 3. General information 7 4. Abbreviation used 10 5. Abstracts Bioaccumulation 13 Bioremediation 15 Biotransformation 27 Biomarker 30 Biofertilizer 32 Biocomposting 32 Biopesticide 37 Biodegradation 42 Biosensor 73 Bioengineering 81 Agricultural Biotechnology 85 Bioenergy 87 6. Name of Journal 110 7. Author Index 113 ENVIS Centre on Environmental Biotechnology BACKGROUND Environmental Information System (ENVIS) is established in the year 1984 as a network of Information Centres. It is planned by the Ministry of Environment and Forest. Aim of this centre is to provide descriptive and environmental subject related numerical data. Now 78 centres are working under this network on various subject areas in the country. The focal point of this network is situated at the Ministry of Environment and Forest, Government of India, New Delhi. This ENVIS Centre is established for studies on Environmental Biotechnology at the Department of Environmental Science, University of Kalyani, Nadia-741235, West Bengal.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Microbial Cycling of Isoprene, the Most Abundantly Produced Biological Volatile Organic Compound on Earth
    The ISME Journal (2018) 12:931–941 https://doi.org/10.1038/s41396-018-0072-6 REVIEW ARTICLE Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth 1 2 3 Terry J. McGenity ● Andrew T. Crombie ● J. Colin Murrell Received: 30 June 2017 / Revised: 26 September 2017 / Accepted: 12 December 2017 / Published online: 20 February 2018 © The Author(s) 2018. This article is published with open access Abstract Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/ marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, 1234567890();,: initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome.
    [Show full text]
  • Dissertation
    Dissertation Homo- and Heterobimetallic Complexes of the PDIxCy ligand system; Synthesis, Characterization and Reactivity Andreas Joseph Hofmann Technische Universität München Fachgebiet Bioanorganische Chemie Homo- and Heterobimetallic Complexes of the PDIxCy ligand system; Synthesis, Characterization and Reactivity Andreas Joseph Hofmann Vollständiger Abdruck der von der Fakultät Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzende(r): Prof. Dr. Roland A. Fischer Prüfer der Dissertation: 1. Prof. Dr. Corinna R. Hess 2. Hon.-Prof. Dr. Richard W. Fischer Die Dissertation wurde am 15.02.2021 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 30.03.2021 angenommen. Die vorliegende Arbeit wurde im Fachgebiet Bioanorganische Chemie der Technischen Universität München in der Zeit von Juni 2017 bis Februar 2021 angefertigt. Teile dieser Arbeit wurden bereits veröffentlicht: Andreas J. Hofmann, Christian Jandl, Corinna R. Hess “Structural Differences and Redox Properties of Unsymmetric Diiron PDIxCy Complexes” European Journal of Inorganic Chemistry 2020, 2020, 499-505. Andreas J. Hofmann, Lukas Niederegger, Corinna R. Hess “Neighbouring effects on catalytic epoxidation by Fe-cyclam in M2-PDIxCy complexes” Dalton Transactions 2020, 49, 17642-17648. Mein besonderer Dank gilt meiner Doktormutter Frau Prof. Dr. Corinna R. Hess für die Aufnahme in ihren Arbeitskreis und die Möglichkeit, an dieser interessanten Themenstellung zu arbeiten. Außerdem möchte ich Frau Prof. Hess für die wissenschaftliche Betreuung und das mir entgegengebrachte Vertrauen danken. 1.1 Danksagung Mein Dank gilt: Lukas Niederegger, der mich in der ganzen Zeit meiner Promotion unterstützt hat, für seine hilfreichen Tipps und sein großes Engagement, das er für jedes Thema aufbrachte.
    [Show full text]
  • Rekombinante Herstellung Und Charakterisierung Phenoloxidierender Enzyme Aus Geobacillus Stearothermophilus Zur Evaluierung Einer Biosensorischen Anwendung
    Rekombinante Herstellung und Charakterisierung phenoloxidierender Enzyme aus Geobacillus stearothermophilus zur Evaluierung einer biosensorischen Anwendung Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch- Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Jäntges Uwe Konrad aus Aachen Berichter: Universitätsprofessor Dr.-Ing. Winfried Hartmeier Universitätsprofessor Dr. rer. nat. Andreas Schäffer Tag der mündlichen Prüfung: 10.02.2006 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar Für meine Eltern: Clemens und Marlene Jäntges Inhaltsverzeichnis 1 EINLEITUNG 1 1.1 Phenol 1 1.2 Enzymatische Umwandlung und biosensorische Anwendung 3 1.3 Phenolhydroxylasen 5 1.4 Phenoloxidierende Enzyme aus G. stearothermophilus 7 1.5 Rekombinante Expression von Enzymen 9 1.6 Immobilisierung 10 1.7 Aufgabenstellung 11 2 MATERIAL UND METHODEN 13 2.1 Material 13 2.1.1 Chemikalien 13 2.1.2 Enzyme, Reaktionskits und Größenmarker 13 2.1.3 Geräte, Software und Datenbanken 13 2.1.4 Mikroorganismen 15 2.1.5 Vektoren 16 2.1.6 Synthetische Oligonukleotide 16 2.1.7 Rekombinante Plasmide 17 2.1.8 Puffer 17 2.2 Analytik 18 2.2.1 Phenolanalytik 18 2.2.2 Messung von gelöstem Sauerstoff 19 2.3 Mikrobiologische Methoden 20 2.3.1 Anlegen von Stammkulturen 20 2.3.2 Kultivierung 20 2.4 Molekularbiologische Methoden 22 2.4.1 Isolation genomischer DNA aus G. stearothermophilus 22 2.4.2 Agarose-Gelelektrophorese von DNA-Molekülen 23 2.4.3 Polymerasekettenreaktion 23 2.4.4 Restriktionsverdau von DNA 23 2.4.5 Extraktion von DNA-Fragmenten aus Agarosegelen 23 2.4.6 Ligation von DNA 24 2.4.7 Isolation von Plasmid-DNA aus E.
    [Show full text]
  • All Enzymes in BRENDA™ the Comprehensive Enzyme Information System
    All enzymes in BRENDA™ The Comprehensive Enzyme Information System http://www.brenda-enzymes.org/index.php4?page=information/all_enzymes.php4 1.1.1.1 alcohol dehydrogenase 1.1.1.B1 D-arabitol-phosphate dehydrogenase 1.1.1.2 alcohol dehydrogenase (NADP+) 1.1.1.B3 (S)-specific secondary alcohol dehydrogenase 1.1.1.3 homoserine dehydrogenase 1.1.1.B4 (R)-specific secondary alcohol dehydrogenase 1.1.1.4 (R,R)-butanediol dehydrogenase 1.1.1.5 acetoin dehydrogenase 1.1.1.B5 NADP-retinol dehydrogenase 1.1.1.6 glycerol dehydrogenase 1.1.1.7 propanediol-phosphate dehydrogenase 1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) 1.1.1.9 D-xylulose reductase 1.1.1.10 L-xylulose reductase 1.1.1.11 D-arabinitol 4-dehydrogenase 1.1.1.12 L-arabinitol 4-dehydrogenase 1.1.1.13 L-arabinitol 2-dehydrogenase 1.1.1.14 L-iditol 2-dehydrogenase 1.1.1.15 D-iditol 2-dehydrogenase 1.1.1.16 galactitol 2-dehydrogenase 1.1.1.17 mannitol-1-phosphate 5-dehydrogenase 1.1.1.18 inositol 2-dehydrogenase 1.1.1.19 glucuronate reductase 1.1.1.20 glucuronolactone reductase 1.1.1.21 aldehyde reductase 1.1.1.22 UDP-glucose 6-dehydrogenase 1.1.1.23 histidinol dehydrogenase 1.1.1.24 quinate dehydrogenase 1.1.1.25 shikimate dehydrogenase 1.1.1.26 glyoxylate reductase 1.1.1.27 L-lactate dehydrogenase 1.1.1.28 D-lactate dehydrogenase 1.1.1.29 glycerate dehydrogenase 1.1.1.30 3-hydroxybutyrate dehydrogenase 1.1.1.31 3-hydroxyisobutyrate dehydrogenase 1.1.1.32 mevaldate reductase 1.1.1.33 mevaldate reductase (NADPH) 1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) 1.1.1.35 3-hydroxyacyl-CoA
    [Show full text]
  • Microbial Processes Influencing the Attenuation and Impacts of Fuel
    Copyright Jie Ma December 3, 2013 Abstract Microbial processes influencing the attenuation and impacts of ethanol blend fuel releases By Jie Ma Fuel releases that impact groundwater are a common occurrence, and the growing use of ethanol as a transportation biofuel is increasing the likelihood of encountering ethanol in such releases. Therefore, it is important to understand how such releases behave and affect public safety and environmental health, and how indigenous microorganisms respond and affect their migration, fate, and overall impacts. Vapor intrusion risk (i.e., methane explosion and enhanced fuel hydrocarbon vapor intrusion) associated with ethanol blend releases is a potential concern. Using both experimental measurements and mathematical model simulations, this thesis shows that methane is unlikely to build up to pose an explosion hazard (5% v:v) if diffusion is the only mass transport pathway through the unsaturated zone. However, if methanogenic activity near the source zone is sufficiently high to cause advective gas transport, the methane indoor concentration may exceed the flammable threshold. As a group of widely distributed microorganisms, methanotrophs can significantly attenuate methane migration through the vadose zone, and thus alleviate the associated explosion risk. However, methane biodegradation could consume soil oxygen that i would otherwise be available to support biodegradation of volatile hydrocarbons, and increase their vapor intrusion potential. The release of an ethanol blend solution (10 % v:v ethanol solution mixed with 50 mg/L benzene and 50 mg/L toluene) experiment into a pilot-scale (8 m3) aquifer tank produced a large amount of volatile fatty acids (VFAs). The accumulation of VFAs (particularly butyric acid) exceeded the secondary maximum contaminant level value for odor, which represents a previously unreported aesthetic impact.
    [Show full text]
  • Desaturation and Related Modifications of Fatty Acids1
    P1: BHR/ary P2: ARS/NBL/dat QC: ARS/abe T1: ARS April 18, 1998 8:46 Annual Reviews AR060-24 Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998. 49:611–41 DESATURATION AND RELATED MODIFICATIONS OF FATTY ACIDS1 John Shanklin and Edgar B. Cahoon Department of Biology, Brookhaven National Laboratory, Upton, New York 11973; e-mail: [email protected] KEY WORDS: unsaturated fatty acid, protein engineering, binuclear iron, nonheme, oxygenase ABSTRACT Desaturation of a fatty acid first involves the enzymatic removal of a hydrogen from a methylene group in an acyl chain, a highly energy-demanding step that requires an activated oxygen intermediate. Two types of desaturases have been identified, one soluble and the other membrane-bound, that have different con- sensus motifs. Database searching for these motifs reveals that these enzymes belong to two distinct multifunctional classes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids or other substrates. The sol- uble class has a consensus motif consisting of carboxylates and histidines that coordinate an active site diiron cluster. The integral membrane class contains a different consensus motif composed of histidines. Biochemical and structural similarities between the integral membrane enzymes suggest that this class also uses a diiron cluster for catalysis. Soluble and membrane enzymes have been successfully re-engineered for substrate specificity and reaction outcome. It is by ALI: Academic Libraries of Indiana on 03/26/13. For personal use only. anticipated that rational design of these enzymes will result in new and desired activities that may form the basis for improved oil crops.
    [Show full text]
  • Research Collection
    Research Collection Doctoral Thesis Specific oxyfunctionalization of pseudocumene by multistep biocatalysis - Mechanism and scale up Author(s): Bühler, Bruno Albert Publication Date: 2003 Permanent Link: https://doi.org/10.3929/ethz-a-004630430 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 15206 SPECIFIC OXYFUNCTIONALIZATION OF PSEUDOCUMENE BY MULTISTEP BIOCATALYSIS – MECHANISM AND SCALE UP A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences presented by BRUNO ALBERT BÜHLER Dipl. Nat. Sci., Swiss Federal Institute of Technology Zurich, Switzerland born December 26, 1973 Citizen of Homburg, TG (CH) Accepted on the recommendation of Prof. Dr. Bernard Witholt, examiner Dr. Andreas Schmid, co-examiner PD Dr. Bernhard Hauer, co-examiner Prof. Dr. Erick M. Carreira, co-examiner Zurich, 2003 II ACKNOWLEDGMENTS First of all, I would like to thank the promoters of my thesis: Prof. Dr. Bernard Witholt and Dr. Andreas Schmid, who enabled me to carry out this work at the IBT. Thanks a lot for always motivating me, for the helpful and interesting discussions, and for teaching me all the smaller and bigger “details” needed to make good science. Andreas, I thank you for always showing me the positive sides, when my critical attitude tended to end up in frustration, and for your excellent supervision. I am most grateful to PD Dr. Bernhard Hauer and Prof. Dr. Erick M. Carreira for being in the committee acting as coreferees.
    [Show full text]