plays an important role in the conversion of d-sorbitol to l-sorbose, which is an essential intermediate for the industrial-scale production of vitamin C. In the fermentation process, some d-sorbitol could be converted to d-fructose and other byproducts by uncertain dehydrogenases. Genome sequencing has revealed the presence of diverse genes encoding dehydrogenases in . However, the characteristics of most of these dehydrogenases remain unclear. Therefore, the analyses of these unknown dehydrogenases could be useful for identifying those related to the production of d-fructose and other byproducts. Accordingly, dehydrogenases in WSH-003, an industrial strain used for vitamin C production, were examined. A nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, which was annotated as xylitol dehydrogenase 2, was identified, codon-optimized, and expressed in BL21 (DE3) cells. The enzyme exhibited a high preference for NAD as the cofactor, while no activity with nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, or pyrroloquinoline quinone was noted. Although this enzyme presented high similarity with NAD-dependent xylitol dehydrogenase, it showed high activity to catalyze d-sorbitol to d-fructose. Unlike the optimum temperature and pH for most of the known NAD-dependent xylitol dehydrogenases (30-40 °C and about 6-8, respectively), those for the identified enzyme were 57 °C and 12, respectively. The values of and of the identified dehydrogenase toward l-sorbitol were 4.92 μM and 196.08 μM/min, respectively. Thus, xylitol dehydrogenase 2 can be useful for the cofactor-reduced nicotinamide adenine dinucleotide regeneration under alkaline conditions, or its knockout can improve the conversion ratio of d-sorbitol to l-sorbose.