Lactobacillus diolivorans二醇脱水酶激活因子基因的克隆、测序与功能鉴定

Cloning,Sequencing and Characterization of Genes Encoding Diol Dehydratase Reactivating Factor of Lactobacillus diolivorans

根据二醇脱水酶与甘油脱水酶的激活因子基因序列同源性分析,设计简并引物从L.diolivorans基因组DNA中扩增出了假定的二醇脱水酶激活因子基因(gldG、gldH)全序列,补全了GenBank中该基因序列的未知部分,构建了表达质粒pSE-gldGH,以E.coliBL21为宿主,进行诱导表达.表达产物的变性与非变性聚丙烯酰胺凝胶电泳分析表明:gldGH表达产生68ku、13ku两个蛋白质,它们经金属螯合亲和层析与凝胶过滤得到共纯化,纯化产物即假定的激活因子为分子质量约325ku的蛋白质聚合体,由这两个蛋白质以等摩尔量组成,因此假定的激活因子可能为α4β4亚基结构.以L.diolivorans二醇脱水酶为对象,进行激活实验,结果证实gldGH表达产物具备二醇脱水酶激活因子的功能.

It was reported that glycerol and 1,2-propanediol was converted to 1,3-propanediol and 1-propanol in Lactobacillus diolivorans （DSM 14421）under the anaerobic condition, respectively. Its putative genes encoding diol dehydratase, a key enzyme in the pathway, were sequenced. However, their reactivating factor （gldG and gldH） was not completely sequenced. Based on several glycerol dehydratase and diol dehydratase reactivating factors sequence alignment, it was amplified from L. diolivorans with degenerated primers. Then it was inserted into expression vector pSE380. A recombinant plasmid pSE-gldGH was constructed and transformed into Escherichia coli BL21. Recombinant gldG and gldH protein were co-purified by metal chelating affinity chromatography and gel filtration from cell-flee extracts of E. coli overexpressing the gldGH genes. They existed a putative reactivating factor, with an apparent molecular mass of 325 000. The protein complex consisted of equimolar amounts of the two subunits with Mr of 68 000 （α） and 13 000 （β）, encoded by the gldG and gldH genes, respectively. Therefore, its subunit structure was most likely α4β4, different from the common structure α2β2 of the other diol or glycerol dehydratase reactivating factors. In the presence of free adenosylcobalamin, ATP, and Mg^2＋, the factor reactivated diol dehyadratase from L. diolivorans, which had been inactivated to be enzyme-cyanocobalamin complex.