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The Microbial Epoxidation of Internal Alkenes

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The Microbial Epoxidation of Internal Alkenes CENTRE FOR BIOTECHNOLOGY, IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE, UNIVERSITY OF LONDON. THE MICROBIAL EPOXIDATION OF INTERNAL ALKENES BY PETER JONATHAN AIKENS A thesis submitted for the degree of Doctor of Philosophy of the University of London and the Diploma of Membership of the Imperial College June 1990 1 Abstract. Microorganisms capable of growth on sterically hindered internal alkenes were isolated in pure culture using selective microbial enrichment techniques. Two strains were investigated to ascertain the routes used by the microorganisms to oxidise internal alkenes.P. fluorescens P1 appeared to metabolise a hydroperoxide formed from the alkene via free radical autooxidation with no enzymic process being involved in the initial oxidation. Isolate P2 appeared to contain an inducible non-specific alkane monooxygenase which activated hydrocarbons via co-hydroxylation, this enzyme system also possessed an ability to epoxidise terminal alkenes. Epoxides were not degraded by Isolate P2. An organism chosen as a representative of organisms capable of growth on terminal alkenes (via the epoxide),Mycobacterium M156, showed no ability to epoxidise internal (A3 ) alkenes. The a-pinene monoxygenase system fromP. fluorescens NCIMB 11671 was investigated as a system capable of catalysing the epoxidation of internal alkenes. Detection of this enzyme activity in cell free extracts produced from a-pinene grownP. fluorescens 11671 was shown to be dependant on the physiological state of the organism with only a short time window(-2 hours) available for the harvesting of cells which exhibited this enzyme activity. Rapid reactivation of the monooxygenase system was possible withoutde novo protein synthesis on the addition of extra a-pinene to the growth medium. Accumulated acidic metabolites, associated with initial oxidative products, suggested a route for the metabolism of a-pinene to monounsaturated acids. The a-pinene monooxygenase could only oxidise substituted alkenes and heteroatoms, but within these groups it had a very limited substrate range. From a scaled up biotransformation it was shown to catalyse the production of racemic (+)-limonene oxide from (+)-limonene. Partial purification suggested that the enzyme is multicomponent in nature and required both FAD and Fe2+ for catalytic activity. No evidence for classical cytochrome P450 characteristics was found. 2 Acknowledgements. I am very grateful to my supervisor, Dr David Leak, for his continual advice, enthusiasm and encouragement throughout the course of my work. I would like to thank my industrial sponsors ICI Biological Products , especially Dr Steve Taylor, for their assistance, both financial and practical. I would also like to thank Rosalind Chan for typing this thesis. Finally I would like to thank my Parents and Julie Skinner for their support. 3 Contents. Page Title. 1 Abstract. 2 Acknowledgements. 3 Contents. 4 List of tables. 8 List of figures. 9 1 Introduction. 13 1.1.1 Aims. 13 1 .1 .2 Chemical synthesis and ring opening reactions of epoxides. 13 1.1.3 Biomimetic epoxidations. 14 1.1.4 Enzymic biocatalysis. 17 1 .1.5 Enzyme catalysed reactions, examples of regio and stereoselectivity. 18 1.2 Microbial hydrocarbon oxidations. 2 4 1.2.1 Non-specific alkane monooxygenases. 2 4 1 .2 .2 Alkene epoxidation by alkene specific monooxygenases. 3 2 1 .2 .3 Microbial oxidation of internal alkenes. 41 1.3 Factors influencing the regio and stereoselectivity of monooxygenase catalysed oxidations. I. Reactivity of the active oxygen species. 4 5 1.3.1 Cytochrome P450s. 4 6 1 .3 .2 Non haem iron monooxygenases. 4 7 1 .3 .3 Flavin monooxygenases. 4 7 1 .3 .4 Copper monooxygenases. 8 4 1 .4 Factors influencing the regio and stereoselectivity of monooxygenase catalysed oxidations. II. Substrate binding. 4 9 2 . Materials and Methods. 2 5 2 .1.1 Chemicals. 25 2 .1 .2 Alkenes used as carbon sources during isolation procedures. 5 2 2.1.3 Sterilization. 5 2 2.2.1 Isolation procedure I. 5 2 2 .2.2 Isolation procedure II. 5 3 2 .2 .3 Growth of Isolate P1. 5 4 2 .2 .4 Classification methods and media. 5 4 4 Cell growth and protein content estimations. 5 4 Resting cell assays, P1. 5 5 SDS PAGE of whole cells, P1. 5 6 Impurity degradation-resting cells. 5 6 Impurity degradation-growing cells. 5 6 Impurity GC/MS analysis. 5 7 Growth conditions, Mycobacterium M156. 5 7 M156, resting cell assays. 5 7 Chemical synthesis of epoxides. 5 8 Growth conditions-lsolate P2. 5 9 Trans-2,3-epoxybutane disappearance; P2 growing cultures. 5 9 P2 resting cell C4 hydrocarbon metabolism. 5 9 SDS PAGE-P2. 6 0 Organism,P.fluorescens NCIMB 11671. 6 0 Growth medium, 11671. 6 0 Growth of 11671 on a-pinene. 6 0 Growth of 11671 on succinate. 61 Continuous culture on succinate plus a-pinene. 61 Continuous culture on a-pinene. 6 2 Growth conditions, cometabolism. 6 2 Cell harvesting. 6 2 Hydrocarbon substrate preparation. 6 3 Resting cell assays, a-pinene oxidation. 6 3 Resting cell substrate specificity assays. 6 4 Cell disruption. 6 4 Cellular fractionation. 6 5 Cell free extract preparation. 66 Storage of cell free extracts. 66 Protein concentration estimation. 66 Assays of a-pinene monooxygenase activity in cell free extracts. 66 Large scale (+)-limonene oxide production. 6 7 Inhibitor experiments. 68 Acidic metabolite analysis. 68 Antibiotic inhibition (growth). 69 Antibiotic inhibition, restimulation of active parameters. 6 9 SDS PAGE, 11671. 6 9 Spectral analyses. 7 0 Dialysis experiments. 7 0 (NH4)2 S04 fractionation. 7 0 5 2 .4 .2 5 F.P.L.C. gel filtration. 7 0 Results 3. Isolation of organisms capable of growth on hydrocarbons containing internal alkenes. I. 7 2 3.1 Introduction. 72 3.2.1 Isolation Procedure I. 7 2 3 .2 .2 Isolation Procedure II. 27 3 .2 .3 Classification of Isolate P1. 7 4 3 .2 .4 Carbon sources used by Isolate P1; P1 metabolism of fra/7s-2 ,5-dimethylhex-3-ene. 7 4 3 .2 .5 Induction of polypeptides associated with alkene metabolism. 6 7 3 .2 .6 Isolate P1, impurity degradation. 6 7 3 .2 .7 Identification of impurity present in frans-2,5-dimethylhex-3-ene. 81 3 .3 Metabolism of internal alkenes by an organism capable of catalysing the epoxidation of terminal alkenes. 8 4 3 .4 Discussion. 8 8 3.4.1 Toxicity of hydrocarbons to microorganisms. 0 9 3 .4 .2 Production of hydroperoxides from alkenes. 9 3 3 .4 .3 Microbial degradation of hydroperoxides. 9 4 3 .4 .4 Organisms capable of catalysing the epoxidation of terminal alkenes. 9 7 4. Isolation of organisms capable growth on hydrocarbons containing internal alkenes.il. 9 9 4.1.1 Isolation of microorganisms. 9 9 4 .1 .2 Classification of2 .P 9 9 4 .1 .3 Growth substrate range,2 .P 9 9 4 .1 .4 Resting cell hydrocarbon metabolism studies. 1 0 2 4 .1 .5 Induction of polypeptides associated with hydrocarbon metabolism. 1 1 5 4 .2 Discussion. 1 1 5 5 Studies on the growth of P.fluorescens 11671, optimization of the production of a-pinene monooxygenase activity in cell free extracts and investigation of the acidic metabolites produced during the growth of this organism on a-pinene. 1 21 6 5.1 Introduction. 121 5.2.1 a-Pinene monooxygenase, initial studies using whole cells and cell free extracts. 121 5.2.2 Concentration of cellular material. 123 5 .2 .3 Effect of raising a-pinene concentration on cell growth. 1 24 5 .2 .4 Effect of breakage procedure on the ability to detect a-pinene monooxygenase activity. 1 2 6 5.2.5 Continuous culture (succinate). 130 5.2.6 Continuous culture (a-pinene). 133 5 .2 .7 Alteration of growth conditions to enable the detection of a-pinene monooxygenase activity in cell free extracts. 1 3 3 5 .2 .8 Changes in the growth medium used as markers in the harvesting of cells containing an active a-pinene monooxygenase. 13 5 5 .2 .9 Sensitivity to inhibitors of protein synthesis. 1 38 5 .3 Production of acidic metabolites during growth on a-pinene. 138 5 .4 Discussion. 1 49 6 Studies on the a-pinene monooxygenase from P. fluorescens 11671. 155 6.1.1 a-Pinene monooxygenase, characteristics. 155 6. 1.2 Multicomponent nature of the a-pinene monooxygenase system. 1 5 5 6 .1 .3 Basic kinetic analysis of the a-pinene monooxygenase. 1 5 7 6.1 .4 Substrate specificity of a-pinene monooxygenase. I. Whole cells. 1 5 7 6. 1 .5 Substrate specificity of a-pinene monooxygenase. II. Resting cells. 1 5 9 6.1.6 Substrate specificity of a-pinene monooxygenase. III. Cell free extracts. 15 9 6.1.7 Chirality of enzymically produced (+)-limonene oxide. 162 6 .1 .8 Partial purification of the a-pinene monooxygenase. 1 67 6 .1 .9 Induction of polypeptides associated with the a-pinene monooxygenase. 1 71 6 .2 Discussion. 1 73 7 Conclusions. 1 7 9 7.1 Internal epoxidation of alkenes. 1 7 9 7 a-Pinene monooxygenase. 1 80 References. 1 8 2 List of tables. Page Sources of samples used for enrichment cultures. 7 3 Isolates obtained using isolation procedure I. 7 3 Biochemical and morphological characteristics of Isolate P1. 7 5 Hydrocarbons serving as potential sources of sole carbon for Isolate P1. 7 5 Initial rates of epoxide production/degradation of propene grown Mycobacterium M156. 8 5 Biochemical and morphological characteristics of Isolate 2P. 100 Hydrocarbons supplied as a sole carbon source for Isolate P2. 100 Oxygenated derivatives of4 C compounds supplied as a sole carbon source to Isolate2 . P 101 Hydrocarbon metabolism by resting cell preparations of Isolate 2P. 1 04 a-Pinene monooxygenase activity in cells grown using initial conditions.
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