Expression and biochemical characterization of a novel NAD+-dependent xylitol dehydrogenase from the plant endophytic fungus Trichoderma gamsii

Xylitol dehydrogenase (XDH; EC 1.1.1.9), encoded by the XYL2 gene, is a key enzyme in the fungal xylose metabolic pathway. In this work, a putative XDH from the plant endophytic fungus Trichoderma gamsii (TgXDH) was hetero-expressed in Escherichia coli BL21(DE3), purified to the homogeneity, and biochemically characterized. Sequence analysis revealed that TgXDH is 363 amino acids long and belongs to the zinc-containing medium-chain alcohol dehydrogenase superfamily. The size-exclusion chromatography analysis and SDS-PAGE showed that the purified recombinant TgXDH had a native molecular mass of similar to 155 kDa and was composed of four identical subunits of molecular mass of similar to 39 kDa. The optimum temperature and pH of this enzyme were 25 degrees C and pH 9.5, respectively. Kinetic analysis showed that it is an NAD(+)-dependent enzyme that has a polyol substrate preference (based on k(cat)/K-m) in the order xylitol > ribitol approximate to D-sorbitol. The Km values for NAD(+) with these three polyols ranged from 0.23 to 0.70 mM. Moreover, TgXDH showed high substrate affinities as compared to most of its homologs. The K-m values for xylitol, ribitol, and D-sorbitol were 5.23 +/- 0.68 mM, 8.01 +/- 1.22 mM, and 12.34 +/- 1.37 mM, respectively. Collectively, the results will contribute to understanding the biochemical properties of a novel XDH from the filamentous fungi and provide a promising XDH for industrial production of ethanol.