title: Cloning and characterization of a novel NAD+ -dependent xylitol dehydrogenase from Gluconobacter oxydans CGMCC 1. 637.
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 d-arabitol. K(m) and V(max) 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 degrees C). However, the activity of its reverse reaction, d-xylulose reduction, reached 255.55 U/mg under optimum conditions (pH 6.0, 30 degrees C), 10-times higher than that of xylitol oxidation. Oxidative activity of xylitol dehydrogenase was induced when G. oxydans CGMCC 1.637 was 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 d-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from d-arabitol.
authors.affiliation: Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China. linyanqing1031@yanoo.cn
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Lin
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Xie
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhang
authors.forename: Wenna
authors.initials: W
authors.lastname: Bao
authors.forename: Haifeng
authors.initials: H
authors.lastname: Pan
authors.forename: Boyi
authors.initials: B
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D050542:D-Xylulose Reductase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013696:Temperature; D014993:Xylitol / Q000737:chemistry / Q000378:metabolism*; D014996:Xylulose / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014996:Xylulose; D050542:D-Xylulose Reductase; D014993:Xylitol