Functional characterisation of alkane-degrading monooxygenases in Rhodococcus jostii strain 8

Ekprasert, Jindarat (2014) Functional characterisation of alkane-degrading monooxygenases in Rhodococcus jostii strain 8. Doctoral thesis, University of East Anglia.

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Short-chain alkanes are gaseous hydrocarbons that contribute to photochemical pollution and ozone production in the atmosphere. A number of studies have shown that Rhodococcus species possess the ability to metabolite a wide range of hydrocarbons since they contain multiple hydrocarbon-degrading enzymes such as soluble diiron monooxygenases (SDIMOs) and alkB-type alkane monooxygenases. This study aimed to investigate the role of multiple alkane-degrading enzymes in the metabolism of gaseous alkanes in Rhodococcus jostii strain 8. R. jostii strain 8 was isolated from a propane enrichment culture using petroleum-contaminated soil as an inoculum. R. jostii strain 8 could grow on ethane, propane, butane, octane, naphthalene and some potential intermediates in alkane metabolism. Oxidation studies showed that R. jostii strain 8 is likely to oxidise propane via both terminal and sub-terminal oxidation of propane and that these activities are induced in propane-grown cells. Alcohol dehydrogenase assays were carried out in order to determine cofactor and substrate ranges of these enzymes. Results showed that alcohol dehydrogenases involved in the metabolism of gaseous alkanes are NDMA-dependent. The size of the genome sequence of R. jostii strain 8 is 8.5 Mbp with a G+C content of 67%. The closest relative of R. jostii strain 8, based on 16S rRNA sequence, is Rhodococcus jostii RHA1 with 99% identity. However, growth profiles and a number of catabolic genes in the genome of R. jostii strain 8 clearly indicated that this bacterium is different from R. jostii RHA1. R. jostii strain 8 contains two alkane-degrading enzyme systems – a propane monooxygenase and an alkB-type alkane monooxygenase. Polypeptide analysis on cell-free extracts from cells grown on gaseous alkanes using SDS-PAGE indicated that propane monooxygenase is inducible during growth on propane. Expression studies using RT-qPCR of prmA and alkB showed that prmA was highly expressed during growth on propane. The exact involvement of alkB-type alkane monooxygenase in the degradation of alkanes was still unclear. A gene transfer system for R. jostii strain 8 was established. Marker-exchanged mutagenesis of prmA and alkB was attempted. Construction of mutated-prmA and mutated-alkB plasmids was achieved. Electroporation conditions were successfully optimised in order to transfer linear DNA into R. jostii strain 8. However, mutants lacking active prmA or alkB are still needed to further study their phenotypes and to provide more evidence supporting the role of these two enzymes.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Jackie Webb
Date Deposited: 26 Jun 2015 14:07
Last Modified: 26 Jun 2015 14:07


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