Lin Yanqing, Xie Zhipeng, Zhang Jianguo, Bao Wenna, Pan Haifeng, Li Boyi
Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China.
Wei Sheng Wu Xue Bao. 2012 Jun 4;52(6):726-35.
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.
Touch-down polymerase chain reaction (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. 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.
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.
从氧化葡萄糖酸杆菌CGMCC 1.637中克隆木糖醇脱氢酶基因，表征木糖醇脱氢酶的神秘特性，并研究各种碳源对木糖醇脱氢酶氧化活性的诱导能力以及各种碳源对氧化葡萄糖酸杆菌CGMCC 1.637中D -木酮糖生物转化为木糖醇的影响。
采用降落聚合酶链反应（PCR）从氧化葡萄糖酸杆菌CGMCC 1.637的染色体DNA中克隆木糖醇脱氢酶基因。
木糖醇脱氢酶798 bp的开放阅读框编码一个265个氨基酸的蛋白质，分子量为27.95 kDa。对该推定蛋白质的序列分析表明它是短链脱氢酶/还原酶家族的一员。木糖醇脱氢酶对木糖醇和山梨醇表现出氧化活性，对其他多元醇如D -阿拉伯糖醇无活性。对木糖醇的K(m)和V(max)分别为78.97 mmol/L和40.17 U/mg。在最佳条件（pH 10.0，35℃）下，木糖醇脱氢酶对木糖醇的最高氧化活性仅为23.27 U/mg。然而，其逆反应即D -木酮糖还原的活性在最佳条件（pH 6.0，30℃）下达到255.55 U/mg，比木糖醇氧化活性高10倍。当氧化葡萄糖酸杆菌CGMCC 1.637在D -山梨醇上培养时，木糖醇脱氢酶的氧化活性被诱导。支持高细胞生长的D -阿拉伯糖醇抑制了木糖醇脱氢酶的氧化活性和氧化葡萄糖酸杆菌CGMCC 1.637的生物转化能力。
从氧化葡萄糖酸杆菌CGMCC 1.637中获得的基因是一个编码木糖醇脱氢酶的新基因。氧化葡萄糖酸杆菌CGMCC 1.637中木糖醇脱氢酶的氧化活性以及在D -阿拉伯糖醇上生长后的氧化葡萄糖酸杆菌CGMCC 1.637的生物转化能力受到抑制，这为进一步研究提高从D -阿拉伯糖醇生产木糖醇的产量提供了有价值的线索。
