Purification and properties of polyol: NADP oxidoreductase from pichia quercuum.

Abstract

In order to explain the fermentation mechanism of xylitol production from D-xylose by Pichia quercuum, enzymatic study was carried out. Three kinds of enzymes that catalyzed the reduction of D-xylose to xylitol were purified from the extract of the yeast cells by ammoni-um sulfate fractionation, Sephadex G-200 gel filtration, hydroxylapatite chromatography and disc electrophoresis. The purification showed 27-fold, 135-fold and 93-fold increases of specific activities of reductase I, IIa and IIb, respectively, over the crude extract. The reductase IIa was homogeneous in disc gel electrophoresis. The activity ratio of reductase I: IIa: IIb in the crude extract was estimated to be approximately 2: 1: 1. The three enzymes were active with a variety of aldoses and had a specific requirement for NADPH. On the basis of the substrate specificity, coenzyme requirement and the stoichiometry of the reaction, the enzymes belong to polyol: NADP oxidoreductase (EC 1.1.1.21, trivial name, aldose reductase). The molecular weights for reductase I, IIa and IIb were estimated to be 160, 000, 61, 000 and 61, 000, respectively, by gel filtration. Disc gel electrophoresis suggested that reductase IIa and IIb were charge isomeric proteins with the same molecular size. Some other properties of the three enzymes were also described.