山梨醇脱氢酶的克隆、表达及活性检测

目的:从氧化葡糖杆菌H24中克隆山梨醇脱氢酶基因进行表达并检测其活性。方法:以氧化葡糖杆菌H24基因组DNA为模板,PCR扩增包括启动子、结构基因及其后的终止序列在内的山梨醇脱氢酶基因;将PCR产物插入pMD18T载体,转化大肠杆菌DH5α;通过活性电泳检测山梨醇脱氢酶在大肠杆菌中的表达及活性。结果:从氧化葡糖杆菌H24中扩增得到山梨醇脱氢酶基因并在大肠杆菌中实现表达,重组菌株经活性电泳检测具有醇糖转化活性。结论:原核表达的山梨醇脱氢酶具有很强的醇糖转化活性。

Cloning and Expression of D-Sorbiol Dehydrogenase and Analysis of its Enzyme Activity

Objective：To clone the D-sorbiol dehydrogenase（SLDH） gene from Gluconobacter oxydans H24 and to investigate its expression and biological activity in E.coli.Methods：The SLDH gene including promoter,structural gene and terminator sequence was directly amplified using PCR method from G.oxydans H24 genome.Then it was cloned into the cloning vector pMD18T and was transformed into E.coli DH5α.The expression and biological activity of the SLDH in E.coli DH5α was examined by Native-PAGE.Results：The sorbiol dehydrogenase gene was successfully amplified and expressed in E.coli DH5α.The result of Native-PAGE showed that the recombinant strain have the activity of transforming sorbitol into sorbose.Conclusion：It is indicated that the recombinant sorbiol dehydrogenase have a high activity of transforming sorbitol into sorbose.