| Summary: | Members of the genus Rhodococcus are well known for their high metabolic capabilities to degrade wide range of organic compounds ranging from simple hydrocarbons to more recalcitrant compounds such as polychlorinated biphenyls. Their ability to display novel enzymatic capabilities for the transformation of many hazardous contaminants in the environment makes them a potential candidate for bioremediation. Rhodococcus UKMP-5M, an actinomycete isolated in Peninsular Malaysia shows great potential towards degradation of cyanide, hydrocarbons and phenolic compounds. In the present study, the capacity of this strain to degrade halogenated compounds was explored. Preliminary investigations have proven that R. UKMP-5M was not able to utilise any of the halogenated compounds tested as sole carbon and energy source, but the resting cells of R. UKMP-5M was able to dechlorinate several compounds which include chloroalkanes, chloroalcohols and chloroacids and the activity was three fold higher when the cells were grown in the presence of 1-Chlorobutane (1-CB). Therefore, 1-CB was chosen as a substrate to unravel the mechanism of dehalogenation in R. UKMP-5M. In contrast to the classic hydrolytic route for the assimilation of 1-CB in many organisms, R. UKMP-5M was able to metabolise and release chloride from 1-CB, but is unable to use the product from 1-CB metabolism as growth substrate. On comparing the protein profiles of the induced and non-induced cells of R. UKMP-5M, two types of monooxygenases were identified in the induced condition, which were not present in the uninduced sample. The strict oxygen requirement for dechlorination of 1-CB and the identification of monooxygenases in the induced protein extract suggests that 1-CB dehalogenation is likely to be catalysed by a monooxygenase. In addition to these monooxygenases, a protein that was later identified as amidohydrolase (Ah) was also found to be induced when the cells were exposed to 1-CB. Therefore, Ah from R. UKMP-5M was cloned and expressed in E. coli to test the ability of the purified Ah to release chloride from 1-CB. The heterologous expression of Ah in E. coli resulted in the formation of inclusion bodies and the western blot analyses further confirmed that no soluble form of Ah was present. Multiple attempts to obtain a soluble and functionally active Ah were not successful. Therefore, on-column refolding was carried out to obtain a biologically active Ah. A 3D model based on structural homology was predicted as a preliminary step to characterize this protein. However, when assayed with 1-CB, Ah was found not to catalyze dehalogenation. All results of this thesis suggest that metabolism of 1-CB by R. UKMP-5M is via γ-butyrolactone which acts as a potent intracellular electrophile that covalently modifies proteins and nucleic acids. The findings from this research are important to determine the metabolic capacity of a Malaysian Rhodoccoccus in dehalogenation of halogenated compounds and its potential application in bioremediation.
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