Identification of NAD-dependent xylitol dehydrogenase fromGluconobacter oxydansWSH-003

Abstract

22Gluconobacter oxydans plays important role in conversion of D-sorbitol to L-sorbose, 23 which is an essential intermediate for industrial-scale production of vitamin C. In the 24 fermentation process, some D-sorbitol could be converted to D-fructose and other 25 byproducts by uncertain dehydrogenases. Genome sequencing has revealed the 26 presence of diverse genes encoding dehydrogenases in G. oxydans. However, the 27 characteristics of most of these dehydrogenases remain unclear. Therefore, analyses of 28 these unknown dehydrogenases could be useful for identifying those related to the 29 production of D-fructose and other byproducts. Accordingly, dehydrogenases in G. 30 oxydans WSH-003, an industrial strain used for vitamin C production, were examined. 31An NAD-dependent dehydrogenase, which was annotated as xylitol dehydrogenase 2, 32 was identified, codon-optimized, and expressed in Escherichia coli BL21 (DE3) cells. 33The enzyme exhibited high preference for NAD + as the cofactor, while no activity 34 with NADP + , FAD, or PQQ was noted. Although this enzyme presented high 35 similarity with NAD-dependent xylitol dehydrogenase, it showed high activity to 36 catalyze D-sorbitol to D-fructose. Unlike the optimum temperature and pH for most of 37 the known NAD-dependent xylitol dehydrogenases (30°C-40°C and about 6-8, 38 respectively), those for the identified enzyme were 57°C and 12, respectively. The K m 39 and V max of the identified dehydrogenase towards L-sorbitol were 4.92 μM and 196.08 40 μM/min, respectively. Thus, xylitol dehydrogenase 2 can be useful for cofactor 41 NADH regeneration under alkaline conditions or its knockout can improve the 42 conversion ratio of D-sorbitol to L-sorbose. 43 44