Studies on sorbitol dehydrogenase in acetobacter suboxydans
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A chemically defined medium containing no amino acids supported cellular growth of A. suboxydans NCIB 9108. The effectiveness of selected carbon sources for the growth of the organism in a carbohydrate / salts medium was investigated. Growth was, in general, stimulated by the addition of vitamins to a sorbitol / salts medium. The role of yeast extract as a supplier of growth factors is described. The product of sorbitol oxidation by whole growing cells of A. suboxydans was found to be sorbose. G.L.C. analysis showed that sorbitol was completely oxidised to sorbose. Induction of sorbitol dehydrogenase activity by A. suboxydans in the presence of various carbon sources was investigated. Production of sorbitol dehydrogenase during the growth of A. suboxydans on all the carbon sources investigated was found to be maximal in the late log phase. Sorbitol dehydrogenase from A. suboxydans was purified by ammonium sulphate fractionation, chromatography on DEAE - Cellulose and bioaffinity chromatography on 5' AMP - Sepharose 4B. The purification of the partially purified enzyme increased 17 fold (after DEAE - Cellulose chromatography) and 80 fold (after 5' AMP - Sepharose 4B bioaffinity chromatography) compared with the crude extract. The enzyme has a pH optimum in the range of 6.5 - 7.0 with a maximum velocity of 5.35 U. mg-1. The Km was found to be 176 mM for fructose and 0.028 mM m for NADH. Inhibition studies involving sorbitol, mannitol, sorbose, nicotinamide, salicylic acid, P-chloromercuribenzoate and o-iodosobenzoate demonstrated competitive inhibition with all inhibitors studied except sorbose which exhibited uncompetitive inhibition. The values were found to be 19 mM, 600 mM, 13 mM, 5 mM, 10.4 mM, 1.3 mM and 3.6 mM respectively.
Thesis, PhD Doctor of Philosophy
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