弗氏柠檬酸菌甘油脱水酶激活因子基因的克隆、表达与功能鉴定

1,3-丙二醇是一种重要的化工原料，其生物法生产的研究逐渐受到的关注。研究以弗氏柠檬酸菌的总DNA为模板，通过PCR分别扩增出约1.8kb（dhaF）和0.4kb（dhaG）的两个基因片段分别编码甘油脱水酶激活因子大、小亚基， 连接于pMD-18T载体，测序分析显示与GenBank中相关基因的相似性最高为86%。将两基因以多顺反子的方式与pSE380连接构建表达载体，并在大肠杆菌中进行高效表达，表达量占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化，得到电泳纯级的甘油脱水酶激活因子，SDS-PAGE分析显示：大、小亚基分子量约为63kDa和12kDa；非变性胶分析显示：全酶的分子量约为150kDa，经扫描分析推测甘油脱水酶激活因子很有可能是以α2β2方式结合的。以弗氏柠檬酸菌甘油脱水酶为研究对象，进行激活实验，结果证实该激活因子具备甘油脱水酶激活因子的功能，为进一步阐明甘油脱水酶的激活机制及1,3-丙二醇的高效生产奠定了基础。

Molecular Cloning, Co-expression and Characterization of dhaF and dhaG Genes Encoding Glycerol Dehydratase Reactivating Factor of Citrobacter freundii

1,3-propanediol (1,3-PD) is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The genes dhaF and dhaG encoding glycerol dehydratase reactivase were amplified by using the genomic DNA of Citrobacter freundii. Two segment genes about 1.8Kb and 0.4Kb were obtained and inserted into pMD-18T. There had not more than 86% similarity between the cloning genes and other corresponding genes; then dhaF and dhaG were inserted into pSE380 and co-expressed in E.coli. The interesting recombinant reactivase was about 30 per in the whole crude protein and was purified homogeneous. α and β subunits of reactivase had apparent molecular masses about 63 and 12 kDa by SDS-PAGE, nondenaturing PAGE analysis the molecular mass was about 150kDa. Therefore, its subunit structure was most likely α2β2. In the presence of free adenosylcobalamin, ATP, and Mg2+, the factor reactivated glycerol dehydratase from C. freundii, which had been inactivated. This research is help to make clear the mechanism of reactivase from C. freundii and improve the manufacture of 1，3-PD by the biological pathway.