Optimization Of Xylitol Production Conditions,Clone And Expression Of Key Enzyme Genes From Gluconobacter Oxydans

A highest yield of D-xylulose was achieved in the optimized fermentation conditions, including pH 5.5,30℃of culture temperature,100 mL fermentation medium in a 1000-mL flask of broth content and 5%(v/v) of inoculums size after 24 h fermentation. The investigation of various carbon sources on the bioconversion of D-xylulose to xylitol in G. oxydans CGMCC 1.637 after grown on them revealed that D-xylulose reductive activities of xylitol dehydrogenase was remarkably induced when G. oxydans CGMCC 1.637was cultivated on D-sorbitol. D-arabitol, which supported a high cell growth, remarkly inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. 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. Touch-down PCR was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. 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. Km and Vmax with xylitol was 78.97mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg at optimum conditions (pH 10.0,35℃). However, the activity of its reverse reaction, D-xylulose reduction, reached 255.55 U/mg at optimum conditions (pH 6.0,30℃), 10-times higher than that of xylitol oxidation.