葡萄糖酸氧化杆菌木糖醇脱氢酶基因的克隆与表达

【目的】获得葡萄糖酸氧化杆菌(Gluconobacter oxydans CGMCC 1.637)的木糖醇脱氢酶基因,研究其酶学性质及碳源特别是D-阿拉伯醇和木糖醇对该酶活性的影响。【方法】通过已报道序列的木糖醇脱氢酶的保守区设计引物,用聚合酶链式反应(polymerase chain reaction,PCR)扩增获得目的基因片段。根据获得的片段序列设计引物克隆目的基因的5’和3’片段,将所获得的片段拼接,获得完整的木糖醇脱氢酶基因。通过构建工程菌获得重组蛋白,并利用氧化还原反应测定重组酶的活性。用含不同碳源的培养基培养G.oxydans CGMCC 1.637,并测定其破胞上清液木糖醇脱氢酶氧化木糖醇的活性;用不同碳源培养的G.oxydans CGMCC 1.637转化木酮糖,用高效液相色谱法测定木糖醇的产量。【结果】获得一个新的798bp的木糖醇脱氢酶基因,所编码的木糖醇脱氢酶含265个氨基酸,属于短链脱氢酶家族。酶学性质研究发现,该木糖醇脱氢酶催化木糖醇氧化的最适合条件为35℃、pH 10.0,最高活性为23.27 U/mg,催化木酮糖还原为木糖醇的最适条件为30℃、pH 6.0。最高活性为255.55 U/mg;该木糖醇脱氢酶的对木糖醇的Km和Vmax分别为78.97 mmol/L和40.17 U/mg。碳源诱导实验表明,d-山梨醇对G.oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。而在转化实验中,用d-甘露糖培养的G.oxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G.oxydans CGMCC 1.637的转化能力,其中,用阿拉伯醇培养的G.oxydans CGMCC 1.637的转化能力最低,仅为对照的35%。【结论】克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶基因是一个新的基因,用阿拉伯醇培养的G.oxydans CGMCC 1.637破胞液木糖醇脱氢酶活性低;且阿拉伯醇对G.oxydans CGMCC 1.637木酮糖的还原能力具有抑制作用。

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

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.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 d-arabitol were inhibited,which provided a valuable clue for further study to increase xylitol yield from d-arabitol.