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Investigating the industrial application potentials of cytochrome P450 BM-3 and four novel putative cytochrome P450s isolated from Cupriavidus necator H16

AL-NUAEMI, INAS (2018) Investigating the industrial application potentials of cytochrome P450 BM-3 and four novel putative cytochrome P450s isolated from Cupriavidus necator H16. PhD thesis, University of Sheffield.

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Abstract

Cytochrome P450 is a superfamily of heme-dependent monooxygenases. These enzymes play a very important role in drug metabolism and detoxification since they catalyse a broad range of reactions including hydroxylation, dehalogenation, deamination, epoxidation, peroxidation, desulphurisation and dealkylation involving various substrates, such as alkanes, phenols, steroids and fatty acids. This thesis presents results from two P450 hemoprotein investigations: I) targeting study of the most common cytochrome P450 BM-3 from Bacillus megaterium, and II) discovery study of four novel putative cytochrome P450s from Cupriavidus necator H16. In Part II of this project, P450 BM-3 WT and W5F5 variants were expressed and purified, and their activities in three green solvents, 1-butanol, 2-butanol and dimethyl carbonate, were examined to check the tolerance of the wild type and the mutant W5F5 variants towards green solvents used for the first time with this enzyme. The results revealed a new green solvent with lower denaturation effect on P450 BM-3, the wild type and the mutant, with potential application in industrial biosynthesis. Part III focuses on the characterisation of four novel cytochrome P450 proteins of unknown structure and function. The proteins were analysed structurally and the first predicted models were discussed. Optimisations of the expression and purification of these proteins as well as optical properties were analysed. Scanning the changes in light absorbance of the substrate-protein complex with eleven substrates resulting in Type I changes when lauric acid and 3-phenoxytoluene were added to the purified P450-H16-B1743, as well as the evaluation of the catalytic activity of P450-H16-B1279 showed possible catalytic activity towards indol, malonic acid, adipic acid, 3-phenoxytoluene and S-Ethyl-N,N-dipropylthiocarbama (EPTC).

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Engineering (Sheffield) > Chemical and Biological Engineering (Sheffield)
Identification Number/EthosID: uk.bl.ethos.762561
Depositing User: Mrs INAS AL-NUAEMI
Date Deposited: 21 Dec 2018 10:52
Last Modified: 23 Dec 2019 11:04
URI: http://etheses.whiterose.ac.uk/id/eprint/22435

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