BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem
Gewählte Publikation:
Nidetzky, B; Helmer, H; Klimacek, M; Lunzer, R; Mayer, G.
(2003):
Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli
CHEM-BIOL INTER. CHEMICO-BIOLOGICAL INTERACTIONS; 143: 533-542.
- Abstract:
- The plasmid-encoded gene of xylitol dehydrogenase from the yeast Galactocandida mastotermitis was expressed in Escherichia coli at 25 degreesC. Recombinant enzyme was isolated in 70% yield using two steps of biomimetic affinity chromatography with the dye ligand Procion Red HE3B immobilized onto Sepharose 4B-CL. Similar to natural enzyme, recombinant xylitol dehydrogenase is a functional homotetramer with a stoichiometric content of catalytic zinc in each 37-kDa subunit. Though lacking bound Mg2+ found in xylitol dehydrogenase isolated from yeast cell extracts, the recombinant enzyme is as active and stable as the native enzyme. Stereo specificity of enzymic hydrogen transfer from NADH has been determined by H-1-NMR and is 4-pro-R. A detailed steady-state kinetic analysis has been carried out for the enzymic reaction, polyol+NAD(+) <---->ketose+NADH+H+, at pH 7.5 and 25 degreesC using xylitol and D-xylulose, the physiological polyol-ketose pair, as well as D-sorbitol and D-fructose. Primary deuterium kinetic isotope effects on steady-state kinetic parameters for oxidation Of D-sorbitol and reduction Of D-fructose have been measured at pH 7.5. Combined results of initial-rate analysis and isotope effect studies suggest that the enzyme utilizes a preferentially ordered kinetic mechanism in which NAD(+) binds before D-sorbitol and D-fructose is released before NADH. Dissociation of NADH appears to be the main rate-limiting step for D-sorbitol oxidation under substrate-saturated reaction conditions. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
- Autor*innen der BOKU Wien:
- Find related publications in this database (Keywords)
- xylose metabolism
- medium-chain dehydrogenase/reductase
- kinetic mechanism
- stereospecificity