Purification and properties of the NAD +-xylitol-dehydrogenase from the yeast Pichia stipitis

Abstract

Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD + and NADP +. During the purification step on DEAE-sephadex A-50 a NAD +-dependent xylitol dehydrogenase could be separated from a NADP +-dependent. The NAD +-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD +-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).