Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/100734
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dc.contributor.advisorPyke, Simon Matthew-
dc.contributor.advisorBell, Stephen G.-
dc.contributor.advisorFord, Christopher Michael-
dc.contributor.authorMunday, Samuel David-
dc.date.issued2016-
dc.identifier.urihttp://hdl.handle.net/2440/100734-
dc.description.abstractThe cytochrome P450 superfamily catalyses the oxidation of a vast array of organic molecules. Most commonly, this oxidation process ensues by the insertion of a single oxygen atom from dioxygen into an unreactive C-H bond. There is a high degree of interest for this reaction type in conventional synthesis, but it is difficult to achieve high levels of selectivity and is often performed under harsh conditions. CYP102A1 or P450Bm3 from Bacillus megaterium however, can perform this oxidative process under physiological conditions and so researchers have a strong interest in exploiting the potential benefits of this enzyme. The natural substrates of P450Bm3 are fatty acids but this thesis will address both modern and classical techniques to improve catalytic performance with a variety of non-natural substrates. The first two results chapters of this thesis (Chapters 3 and 4) describe the effect of decoy molecules on non-natural substrate oxidation with the aim of improving rates of product formation while maintaining the selectivity of the enzyme. Analysis of the oxidation of these substrates by wild-type P450Bm3 and the variant KT2 showed substantial increases in product formation rate while maintaining the regioselectivity. As a rigorous test of regioselectivity, a selection of xylenes were used that have previously been shown to generate multiple products upon P450Bm3 oxidation. Retention of enantioselectivity was also assessed by using prochiral substrates that have stereocentres introduced upon P450Bm3 oxidation. Chiral chromatography analysis of these turnovers showed that in most cases, the enantioselectivity of the enzyme was either maintained or marginally improved. Knowing that xylenes give a range of oxidation products upon P450Bm3 activity, a wider range of disubsituted benzene compounds were also analysed (Chapter 5). These substrates were chosen to resemble potential xenobiotic compounds in order to assess what metabolites may be produced by P450Bm3 and therefore other P450 systems. These substrates were analysed with several P450Bm3 variants and significantly improved rates of product formation were observed, enabling identification of the likely metabolites. Chapter 6 describes an investigation into two potential CYP102 family members from the bacterium Ktedonobacter racemifer DSM44963 (Krac0936 and Krac9955). Their sequenced genes show similarities to P450Bm3, which encouraged the investigation of a range of fatty acid substrates with these two enzymes. Although their product distributions differed, both Krac0936 and Krac9955 were active with straight-chain saturated and unsaturated fatty acids.en
dc.subjectcytochrome P450sen
dc.subjectcatalysisen
dc.subjectdecoy moleculesen
dc.subjectBacillus megateriumen
dc.subjectKtedonobacter racemiferen
dc.titleInvestigations and applications of self-sufficient cytochrome P450 monooxygenasesen
dc.typeThesesen
dc.contributor.schoolSchool of Physical Sciencesen
dc.provenanceThis electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legalsen
dc.description.dissertationThesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2016.en
Appears in Collections:Research Theses

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