Abstract
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 ∼155kDa and was composed of four identical subunits of molecular mass of ∼39kDa. The optimum temperature and pH of this enzyme were 25°C and pH 9.5, respectively. Kinetic analysis showed that it is an NAD+-dependent enzyme that has a polyol substrate preference (based on kcat/Km) in the order xylitol>ribitol≈d-sorbitol. The Km values for NAD+ with these three polyols ranged from 0.23 to 0.70mM. Moreover, TgXDH showed high substrate affinities as compared to most of its homologs. The Km values for xylitol, ribitol, and d-sorbitol were 5.23±0.68mM, 8.01±1.22mM, and 12.34±1.37mM, 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.
Published Version
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