Published:
[Erscheinungsort nicht ermittelbar]: University of New South Wales. Biotechnology & Biomolecular Sciences, 2021
Language:
English
Origination:
University thesis:
Dissertation, University of New South Wales. Biotechnology & Biomolecular Sciences, 2021
Footnote:
Description:
Organohalides are recalcitrant, toxic environmental pollutants. The presence of these compounds at elevated levels in soil and aquifers has led to a focus on methods to remediate these sites. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds that may be more biodegradable and less toxic. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants and has been successfully applied in bioremediation of contaminated sites. Therefore, an understanding of the structure–function relationship of RDases is of considerable interest. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as the key RDase responsible for this conversion and different strategies for functional expression of recombinant TmrA is the focus of this thesis. There have been several efforts to express recombinant RDases in different host organisms. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl3, Na2S and cobalamin. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. The specific activity estimated for the recombinant TmrA was 11-fold lower than the activity of the native TmrA. In this study different culture conditions were screened to improve the specific activity of the recombinant TmrA produced in B. megaterium. The screenings showed that lowering the concentration of xylose for induction of TmrA expression increased the specific activity. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different chaperone proteins from the original host did not increase the level of soluble expression however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was expressed in a soluble and active form in the H2-oxidizing C. necator H16, a novel host for the expression of RDases. In summary, a range of strategies to successfully produce recombinant TmrA are presented in this thesis.