作　　者： ; ; ; ;

机构地区： 江苏大学食品与生物工程学院

出　　处： 《工业微生物》 2009年第5期7-12,共6页

摘　　要： 1,3-丙二醇是一种重要的化工原料,其生物法生产的研究越来越受到广泛的关注。以克雷伯氏菌的总DNA为模板,通过PCR分别扩增出约1.8 kb的gdrA和0.4 kb的gdrB的两个基因片段,随后,将此两基因以多顺反子的方式与pSE380相连构建表达载体,并在大肠杆菌中进行了高效表达,表达量约占总蛋白的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,得到电泳纯级的激活因子,SDS-PAGE分析显示:大、小亚基分子量约为64 KDa和12 kDa;非变性胶分析显示:全酶的分子量约为150 kDa,扫描分析激活因子是以α_2β_2方式结合的。以克雷伯氏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。该研究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。 1,3-pmpanediol （1,3-PD） is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The genes gdrA and gdrB encoding reactivating factor of glycerol dehydratase were amplified by using the genomic DNA of K. pneumoniae . Two segment genes about 1.8 kb and 0.4 kb were obtained and inserted into pMD-18T. Then, gdrA and gdrB were inserted into pSE380 and co-expressed in E. coli. The recombinant reactivating factor of interest was about 30 percent in the whole crude protein and was purified homogeneous. The a and subunits of reactivating factor had apparent molecular masses about 64 km and 12 kDa by SDS-PAGE, nondenaturing PAGE analysis the molecular mass was about 150 kDa. Therefore, its subunit structure was most likely α2β2 In the presence of free adenosylcobalarnin, ATP and Mg^2＋ , the factor reactivated glycerol dehydratase from K. pneurnoniae . This research is help to clarify the mechanism of reactivating factor from K. pneumoniae and improve the manufacture of 1,3-PD by the biological pathway.

关 键 词： 甘油脱水酶 激活因子 协同表达 结构

领　　域： [生物学] [轻工技术与工程] [化学工程]