imprint:
[Erscheinungsort nicht ermittelbar]: University of New South Wales. Biotechnology & Biomolecular Sciences, 2017
Language:
English
Origination:
University thesis:
Dissertation, University of New South Wales. Biotechnology & Biomolecular Sciences, 2017
Footnote:
Description:
Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic and carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide respiring bacteria provide a potential solution to remediate contaminated sites, as they are capable of utilising organohalides as electron acceptors for the generation of cellular energy. At the heart of these processes are reductive dehalogenases (RDase; EC 1.97.1.8), which are membrane bound enzymes that catalyse reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable.Chloroform (CF), primarily used in the production of refrigerants, is very prevalent and recalcitrant organohalide contamination and its improper disposal also caused groundwater pollution at industrial sites in Sydney, Australia. Its hazardous effect on environment and carcinogenic and organo-toxic effects on human health prompts bioremediation research towards its detoxification. Recently, Dehalobacter (Dhb) sp. strain UNSWDHB, which is capable of respiring CF and converting it to dichloromethane has been identified. The UNSWDHB strain was revealed to produce CF-RDase, as termed TmrA, which has been the focus of the research carried out in this thesis.Firstly, the response of Dhb sp. UNSWDHB to the addition of CF was evaluated from a transcriptomic and proteomic perspective. The elevated expressions of TmrABC proteins, key bioenergetics related membrane-associated and cytoplasmic proteins, enzymes associated with functional Wood-Ljungdahl pathway and complete corrinoid de novo synthesis were revealed, providing a broader view on the bioenergetics and general physiology of the Dehalobacter cells actively respiring with CF. Furthermore, TmrA was produced and purified from the membrane fraction of the UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and anion exchange chromatographic purification. This allowed further biochemical characterisation of the purified enzyme. Lastly, an extensive study to heterologously express TmrA was conducted under several expression conditions to address a reportedly challenging issue with soluble, functional expression and purification of a respiratory RDase. The xylose-inducible expression in a corrinoid-producing Bacillus megaterium as an expression host and two liquid chromatographic steps resulted in a generation of a soluble and functional recombinant TmrA.