Portland State University. Environmental Sciences and Resources Ph. D. Program.
Date of Publication
Doctor of Philosophy (Ph.D.) in Environmental Sciences and Resources: Chemistry
Environmental Science and Management
Fumarase, Methanothermobacter thermautotrophicus, Thermophilic bacteria
3, xii, 97 leaves: ill. 28 cm.
Anaerobic fermentation has been an established technology ever since man started treating sewage. Recently this process has received increased attention because of its inherent ability to produce methane gas, which apart from solar energy, is the cleanest, most non-polluting source of energy. Methanobacterium thermoautotrophicum, a thermophilic bacterium, grows on CO(,2) as a source of carbon as well as electron acceptor, using hydrogen as an electron donor. Labeling studies carried out with ('14)C have shown a presence of partial reductive TCA cycle. In this work, the enzyme fumarate reductase, which belongs to this cycle, has been purified to homogeneity using various separation techniques. In keeping with the thermophilic character of the organism, fumarate reductase is extremely heat resistant. Incubation at 75(DEGREES)C for 24 hours led to an increase in purification. In contrast, the enzyme was found to be very sensitive to oxygen. The crude extract, when exposed to air, lost half of its activity within 20 minutes. Reducing agents were helpful in protecting against loss of enzymatic activity provided that a strict anaerobic atmosphere was maintained. For this reason, the entire purification was performed inside a Freter-type anaerobic chamber using reducing agents. The molecular weight of the native fumarate reductase, as determined by Sephacryl S-300 gel exclusion chromatography, was found to be approximately 80,000. SDS polyacrylamide gel electrophoresis data suggested that the enzyme is a tetramer. Treatment with sulfhydyl reagents as well as Cu('++) caused loss in fumarate reductase activity, indicating that the enzyme contains at least one sulfhydryl group which is important to its activity. The UV/Visible spectrum of fumarate reductase did not reveal the presence of a flavin moiety as a cofactor. Both UV/Visible and fluorescence spectra of fumarate reductase from M. thermoautotrophicum instead, indicated the presence of an unusual cofactor, which could be similar to either tetrahydromethanopterin or F(,420).
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Khandekar, Sanjay S., "Purification and characterization of fumarate reductase from Methanobacterium thermoautotrophicum" (1986). Dissertations and Theses. Paper 490.