Cloning, overexpression, purification, and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD+ as a cofactor. The recombinant caXDH has a KM of 8.8 mM and 37.7 μM using the substrate xylitol and NAD+, respectively, and its catalytic efficiency is 53,200 min−1 mM−1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD+ to NADP+ in which the KM and kcat/KM towards NADP+ are 119 μM and 26,200 min−1 mM−1, respectively.