Purification and characterisation of xylitol dehydrogenase from Neurospora crassa NCL communication No. 6347.

Abstract

The purification and characterisation of one of the key enzymes in the conversion of xylose to ethanol, xylitol dehydrogenase (XD), from Neurospora crassa is reported. This organism has the unique ability to directly convert biomass to ethanol. The enzyme was purified 81-fold by fractional ammonium sulfate precipitation, column chromatography on DEAE-cellulose, Sephacryl S-200 and β-NAD agarose. The purified enzyme had a native molecular mass of 87 kDa (gel filtration) and was composed of two identical subunits of molecular mass 43.6 kDa (SDS-PAGE). The enzyme was most active at pH 8.4 and at 28°C. It was most stable at pH 7 and at 4°C. It was activated by Mg2+ and stabilised by Ca2+, Mg2+ and Mn2+. It showed activity towards xylitol and sorbitol, the respective Kms being 28.5 and 100 mM and the Km for NAD was 0.7 mM.