Biotin synthetase was expressed in <I>E.coli</I> using the <I>bioB </I> gene cloned in a plasmid vector. Biotin production in these cells was determined using a <I>Lactobacillus plantarum </I>bioassay. It was found that overexpression of biotin synthetase led to an increase in both the rate and quantity of biotin produced compared to wild type bacteria. However the rate of biotin produced decreases sharply as the cells grow from logarithmic to stationary phase. An explanation for this phenomenon was sought by supplementing the growth medium with various additives and analysing their effect on biotin production. Biotin production in cell-free extracts was assayed using a radiometric, chromatographic assay technique. Various additives were assayed for their effect on dethiobiotin to biotin conversion in an attempt to define the exact nature of substrates and cofactors required for biotin production <I>in vitro.</I> Various chromatographic methods were performed on cell-free extracts of biotin synthetase overproducing cells in an attempt to purify the enzyme. Results from these experiments point toward the requirement of other hitherto unidentified proteins to produce biotin from dethiobiotin <I>in vitro.</I> The amino acids sequences of three known biotin synthetase enzymes were compared in order to define the contribution of specific amino acid residues to biotin synthetase structure and function. This led to the synthesis of <I>bioB </I>deletions mutants using recombinant DNA technology. The biotin synthetase amino acid sequence was used to probe available protein databases to search for proteins with similar amino acid sequence motifs. Biotin synthetase shows strong homology to a number of proteins but in particular to enzymes involved in lipoic acid and thiamine biosynthesis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642358 |
| Date | January 1993 |
| Creators | Camp, Dominic J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13293 |
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