2009MurraySAMPhil.pdf (3.5 MB)
Towards robust and specific enzymatic oxidation of hydrocarbons
thesisposted on 2010-04-30, 15:25 authored by Susan Antonia Murray
Selective hydroxylation/epoxidation of hydrocarbons generates useful chiral intermediates for the production of high value chemicals. The use of mono-oxygenase enzymes to add functional groups in a regio-and stereo-selective manner has the benefits of specificity, with yields in high enantiomeric excess. Two enzymes were investigated with a view to developing a robust and specific biocatalyst for the mono-oxidation of hydrocarbons; the membrane bound alkane hydroxylase from Pseudomonas oleovorans and the soluble cytochrome P450 BM3 from Bacillus megaterium. A histidine tag was engineered to the C-terminus of the alkane hydroxylase in an attempt to isolate it for structural characterisation. Despite successful insertion of the his tag and good protein expression solubilisation and isolation proved unsuccessful. Therefore, the strategy was altered to focus on the more robust and soluble cytochrome P450 BM3. Cytochrome P450 BM3 was analysed for activity with various alkenes and styrene and the reaction products were determined by gas chromatography and mass spectrometry (GC/MS). A point mutant, BM3F87G, was then expressed and purified, and analysed with the same substrates. The mutant demonstrated increased activity towards these non-natural substrates and a reduction in regio-selectivity. The mutant also showed an increase in enantio-selectivity with a preference for R-styrene oxide. A number of cysteines were then engineered into the heme domain of P450 BM3 to allow for attachment of photo-sensitive agents to enable light-induced electron transfer. The mutants did not appear to alter the enzymes ability to bind fatty acid; neither did they alter the enzymes redox potential to any large degree. Initial tests showed that three of the mutated cysteines were solvent accessible and available for attachment of photo-sensitive thiol modifying agents.
Supervisor(s)Munro, A.; Scrutton, N.
Date of award2009-12-18
Awarding institutionUniversity of Leicester