Purification and Characterisation of Isoprene Monooxygenase from Rhodococcus sp. AD45

Sims, Leanne (2020) Purification and Characterisation of Isoprene Monooxygenase from Rhodococcus sp. AD45. Doctoral thesis, University of East Anglia.

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Isoprene is a volatile organic compound (VOC) produced by many plants. Isoprene emissions account for one third of total VOCs from biogenic and anthropogenic sources, equal to that of methane. Atmospheric isoprene affects retention of greenhouse gases, air quality and climate. Recently, research has focussed on the role of bacteria in moderating isoprene emissions. Several diverse taxa of bacteria have been isolated capable of using isoprene as a sole carbon and energy source, including the model organism used here, Rhodococcus sp. AD45, a Gram positive bacterium isolated from freshwater sediment.

The ability to utilise isoprene is dependent upon a multistep pathway. The initial step is oxidation of isoprene to epoxy isoprene, catalysed by a four-component soluble di-iron monooxygenase (SDIMO), isoprene monooxygenase (IsoMO). IsoMO is a six protein complex comprising an oxygenase containing the di-iron active site (IsoABE), a Riesketype ferredoxin (IsoC), NADH reductase (IsoF) and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases.

In this thesis, results of whole-cell characterisation of the isoprene monooxygenase within Rhodococcus sp. AD45 will be presented. This includes the kinetics, substrate range and inhibition of the enzyme by alkynes. The isoprene monooxygenase from Rhodococcus sp. AD45 was found to degrade a range of alkene and aromatic compounds, and was not inhibited by acetylene, despite being inhibited by alkynes of a longer carbon chain length.

The individual components of isoprene monooxygenase were also purified and characterised separately using a range of homologous and heterologous expression systems and purification techniques. Following this, reconstitution of the active complex was attempted, and potential avenues for future research were investigated based on the key findings of this research.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Chris White
Date Deposited: 20 Dec 2021 15:03
Last Modified: 20 Dec 2021 15:03
URI: https://ueaeprints.uea.ac.uk/id/eprint/82743

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