Cloning and characterization of a novel NAD+ -dependent xylitol dehydrogenase from Gluconobacter oxydans CGMCC 1.637

Abstract:

Abstract: ［Objective］ To clone the xylitol dehydrogenase gene from Gluconobacter oxydans CGMCC 1.637，to characterize enigmatic properties of xylitol dehydrogenase and to investigate the induction abilities of various carbon sources on the oxidative activity of xylitol dehydrogenase and the effect of various carbon sources on the bioconversion of d-xylulose to xylitol in G.oxydans CGMCC 1.637.［Methods］Touch-down polymerase chain reaction (PCR) was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G.oxydans CGMCC 1.637.［Results］ The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids，with the molecular mass of 27. 95 kDa.Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family．Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols，such as darabitol．Km and Vmax with xylitol was 78.97 mmol/L and 40.17 U/mg，respectively．The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg under optimum conditions (pH 10.0，35℃)．However，the activity of its reverse reaction，d-xylulose reduction，reached 255. 55 U/mg under optimum conditions (pH 6.0，30℃)，10-times higher than that of xylitol oxidation． Oxidative activity of xylitol dehydrogenase was induced when G. oxydans CGMCC 1.637was cultivated on d-sorbitol. D-arabitol，which supported a high cell growth，inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G.oxydans CGMCC 1.637.［Conclusions］ The obtained gene from G.oxydans CGMCC 1.637 was a novel gene encoding xylitol dehydrogenase． Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1. 637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on darabitol were inhibited，which provided a valuable clue for further study to increase xylitol yield from d-arabitol.

Key words:Keywords: Gluconobacter oxydans， short-chain dehydrogenase/reductase family，xylitol dehydrogenase，D-xylulose，xylitol，clone