Formate dehydrogenase from Mycobacterium vaccae (MycFDH) was cloned and expressed from various plasmid constructs that incorporate hexahistidine tags onto the N-and C-termini of the protein. The most successful FDH variant, dual-tagged FDH L-S expressed from pET28a+, was batch-purified using ammonium sulphate precipitation and IMAC to achieve 96% homogeneity. A significant proportion of the expressed protein was insoluble, and the expression protocol did not respond to solubility optimization efforts. Expression of an FDH-NusA fusion variant appeared to be vulnerable to proteolytic degradation in the cell. None of the strains expressing tagged-FDH variants produced clarified lysate activity levels that were consistently as high as those from the original pUC 119 vector. However, it is likely that the protein aggregation problems encountered are due to overloading of the protein production machinery or related causes, rather than to the presence of the tags themselves. A bioengineered FDH protein closely related to MycFDH was also investigated. FDH GAV was immobilized in polyacrylamide gel to create gel discs or hollow cylinder mini-reactors. The apparent Km(formate) for this enzyme was 15.6 ± 3.6 mM in the immobilized state, and 17.2 ± 1.9 mM in aqueous solution. The activity of FDH GAV was reversibly inhibited by the presence of acrylamide monomer but was not affected by ammonium persulfate or TEMED (alone or in combination) after incubation for one minute. The activity of the immobilized enzyme system was determined to be at least partially limited by diffusion. FDH GAV was also included in an in vitro analysis of the Methanol Linear Dissimilation Pathway (MLDP), a three enzyme system of NAD+ - dependent dehydrogenases that oxidize methanol sequentially to CO2. Horse liver alcohol dehydrogenase (EC 1.1.1.1) appeared to be the rate-limiting enzyme under the conditions used in these experiments, most likely due to its limited activity on methanol. The applicability of FDH and the MLDP to industry and bioelectronics is also considered.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/2489 |
| Date | 07 April 2010 |
| Creators | Sanderson, Dan |
| Contributors | Levin, David Bernard |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
Page generated in 0.002 seconds