丘状乳杆菌中二醇脱水酶激活因子pduGH的克隆与功能鉴定  

Cloning and characterization of genes encoding reactivating factor pduGH from Lactobacillus collinoides

作　　者：梁甜[1] 齐向辉[1] 韦旭钦[1] 韦宇拓[1] 黄日波[1] 

机构地区：[1]广西大学生命科学与技术学院

出　　处：《广西农业生物科学》2007年第B06期12-15,21,共5页Journal of Guangxi Agricultural and Biological Science

基　　金：国家高技术研究发展计划("863"计划)(2006AA020103)

摘　　要：以丘状乳杆菌(Lactobacillus collinoides)基因组DNA为模版,通过PCR方法得到二醇脱水酶激活因子基因pduGH,连接到表达载体pET30a上,得到重组质粒pET-pduGH。将此重组质粒转化到Escherichia.coli Rosseta(DE3)中进行表达,重组菌株SDS-PAGE结果显示有明显的64.5 ku和12.4 ku两条特异性蛋白。表达的目的蛋白大多为不溶性的包涵体,经变性、复性后得到部分可溶性蛋白。在辅酶B12,ATP,Mg2+或Mn2+存在下,以Clostridium pasteurianum甘油脱水酶为对象进行激活实验,结果证实,重组质粒pET-pduGH的表达产物具有甘油脱水酶激活活性。The reactivating factor for coenzyme B12-dependent glycerol dehydratase and diol dehydratase play a key role in 1, 3-propanediol production. In this study, gene pduGH encoding reactivating factor was cloned from Lactobacillus collinoides by PCR, then inserted into expression vector pET30a . The recombinant plasimd pET-pduGH was constructed. The results of SDS-PAGE analysis and protein lacalization assay showed the high expression of pduGH in Escherichia coli rosseta （DE3）. But most of expression protein was insoluble inclusion body. By the protein soluble and refolding, some soluble protein was obtained. In the presence of free adenosylcobalamn, ATP, and Mg^2＋/Mn^2＋, the glycerol dehyadrtase from Clostridiurn pasteurianurn , Which had been inactivated to be enzyrne-cyanocobalarnin complex, was reactivated the reactivating factor pduGH.

关 键 词：丘状乳杆菌 激活因子 1 3-丙二醇 二醇脱水酶 克隆 

分 类 号：Q78[生物学—分子生物学]