Molecular genetics and microbiology of bioremediation using methane-oxidising bacteria

AL-LUAIBI, Yasin Y. Y. (2015). Molecular genetics and microbiology of bioremediation using methane-oxidising bacteria. Doctoral, Sheffield Hallam University.

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Methanotrophic bacteria can grow on methane as their sole source of carbon and energy. Methylosinus trichosporium OB3b is a Gram negative, methanotrophic bacterium, which can convert methane to methanol by either particulate (pMMO) or soluble (sMMO) methane monooxygenase. The sMMO comprises three polypeptides; hydroxylase (αβγ)2, regulator/coupling protein (protein B), and reductase. The hydroxylase contains the diiron active site. The three components of sMMO are found to be indispensable for full enzyme activity. In the present study, the sMMO hydroxylase was purified by anion exchange chromatography and protein B fused with GST was purified by using affinity chromatography. Attempts to purify the reductase were unsuccessful although the reductase was detected during the purification steps. To shorten the long purification protocol of the hydroxylase and minimize the loss of its activity during multiple purification steps, a new system was developed by inserting a His-tag in the wild type hydroxylase β-subunit. The His-tag hydroxylase was then purified in one step by using an affinity column. The new His-tag system yields hydroxylase with detectable activity when it was tested toward propylene. Solid phase microextraction (SPME) was used for the first time in the present study to detect the product propylene oxide from propylene oxidation for the wild type His-tagged hydroxylase. Crystallographic studies have suggested roles for a number of amino acids within and around the active site. The present study used site-directed mutagenesis to create four new mutants in addition to performing further characterization of another two. The mutation C151S preserved activity toward a range of substrates, and indicated that radical chemistry at this position is not xviii essential for monooxygenase activity toward a number of aliphatic and aromatic hydrocarbons. Results from other mutations included stabilising a previously unstable mutant (C151Y) with a secondary mutation to gain the double mutant E114D C151Y. The mutant R98L showed activity toward the monoaromatic substrate ethyl benzene and the diaromatic substrate naphthalene. Mutation of one of the diiron site coordinating residues E114D resulted in a stable hydroxylase with activity toward naphthalene. In terms of the oxidation of the triaromatic hydrocarbons anthracene and phenanthrene, no activity could be detected for the mutants tested in the present study or the wild type.

Item Type: Thesis (Doctoral)
Thesis advisor - Smith, Thomas [0000-0002-4246-5020]
Thesis advisor - Stanley, Karen
Research Institute, Centre or Group - Does NOT include content added after October 2018: Sheffield Hallam Doctoral Theses
Depositing User: Helen Garner
Date Deposited: 17 Nov 2017 15:47
Last Modified: 03 May 2023 02:05

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