Gluconobacter oxydans WD膜结合山梨醇脱氢酶基因在E.coli中的克隆表达

本研究构建了四株含有氧化葡萄糖酸杆菌山梨醇脱氢酶基因的重组大肠杆菌,并初步探究SldB和SldA亚基在山梨醇脱氢酶转化甘油反应中的作用。将pET28a、pETduet与PCR扩增的目的基因连接,构建单启动子调控重组质粒pET28a-sldB、pET28a-sldA、pET28a-sldBA和双启动子调控重组质粒pETduet-sldB'-sldA'。只有含pET28a-sldBA和pETduet-sldB'-sldA'的重组菌具有转化甘油的活性,表明G.oxydans WD的山梨醇脱氢酶催化甘油脱氢需要SldB和SldA亚基的共同作用。串联基因sldBA的蛋白表达结果与双启动子控制sldB和sldA基因蛋白表达结果基本相同,表明位于sldB基因末端的sldA的RBS序列可被E.coli C43的核糖体识别。

Molecular cloning and functional expression of membrane-bounding sorbitol dehydrogenase from Gluconobacter oxydans WD in E. coli

Generally, sorbitol dehydrogenase had two subunits SldB and SldA and similar phenomenon was found in Gluconobacter oxydans W-D. In order to study the relationship of the sorbitol dehydrogenase with its subunits, expression vectors including single promoter plasmid pET28a-sldB, pET28a-sldA, pET28a-sldBA and double promoters plasmid pETduet-sldB＇ -sldA＇ were constructed and transformed into E. coli C43. It was found that only C43/pET28a-sldBA and C43/pETduet-sldB＇ -sldA＇ had ability to convert glycerol among the reeombinations and the enzyme function required both subunit SldB and SldA, and the RBS of sldA located in the sldB could be recognized by the ribosomes of E. coli CA3.