芽胞杆菌(Bacillus sp.)YX-1耐有机溶剂葡萄糖脱氢酶的基因克隆与酶学性质

Gene cloning and characterization of a solvent-resistant glucose dehydrogenase from Bacillus sp.YX-1

【目的】从芽胞杆菌(Bacillus sp.)YX-1基因组中克隆出一种有机溶剂耐受型的葡萄糖脱氢酶基因,实现了该基因在大肠杆菌中的高效表达,研究了重组蛋白的酶学性质。【方法】依据芽胞杆菌属中葡萄糖脱氢酶氨基酸序列的保守性,设计合理引物,钓取来源于Bacillus sp.YX-1的葡萄糖脱氢酶基因,构建诱导型表达载体pET28a-gdh,于大肠杆菌中进行表达。镍柱亲和层析法纯化重组蛋白,考察了重组蛋白的酶学性质。【结果】葡萄糖脱氢酶基因全长为786 bp,编码261个氨基酸。酶学研究结果表明:该酶最适反应温度为45℃,最适pH值为8.0;具有良好的有机溶剂耐受性,于50%的辛烷、环己烷、癸烷中室温放置1 h后,酶活仍能保持90%以上;具有较宽的底物谱,对多种糖均具有一定的催化活性,其中催化D-葡萄糖的活力最高,产生还原型辅酶因子;对辅酶NADH和NADPH具有相似的依赖性,对NAD+和NADP+的催化比活分别为8.37 U/mg和8.62 U/mg。【结论】利用生物信息学成功地挖掘出Bacillus sp.YX-1一种耐有机溶剂的葡萄糖脱氢酶,为氧化还原酶在有机相反应中的的辅酶再生循环提供了新型的生物催化剂。

[Objective] A gene encoding solvent-resistant glucose dehydrogenase was cloned from Bacillus sp.YX-1 and expressed in Escherichia coli.The recombinant enzyme was then characterized.[Methods] The glucose dehydrogenase gene was amplified from Bacillus sp.YX-1 genome according to its conserved sequences in Bacillus sp.The recombinant enzyme was over-expressed in E.coli and purified by HisTrap HP affinity chromatography.The purified enzyme were characterized.[Results] The glucose dehydrogenase gene contains an open reading frame of 786 bp encoding 261 amino acids.The maximum activity was observed at 45℃ and pH 8.0.The recombinant enzyme was highly resistant to several organic solvents.More than 90% of the activity was maintained when the enzyme was incubated in 50% cyclohexane,octane,decane at home temperature for 1 h.In addition,the enzyme displayed broad substrate spectrum and has catalytic activity for several sugars to afford reduced coenzymes.It exhibits similar capability to regenerate either NADH or NADPH with specific activity of 8.37 U/mg and 8.62 U/mg for NAD＋ and NADP＋.[Conclusion] The organic solvent-tolerant glucose dehydrogenase was explored successfully on the basis of bioinformatics analysis.The work supplied a new biocatalyst for the cofactor-regeneration during the reaction in organic phases catalyzed by oxidoreductases.