【作者】 孟晓蕾；

【导师】 黄日波；

【作者基本信息】 广西大学 ， 发酵工程， 2006， 硕士

【摘要】 从天然菌L．diolivorans中纯化了二醇脱水酶。经非变性聚丙烯酰胺凝胶电泳与SDS-PAGE电泳结果显示，该酶分子量约200kDa，由分子量分别为62kDa、35kDa、22kDa的三种亚基组成，酶相对三种主要底物的K_m值分别是，1，2-丙二醇1.3mmol／L，甘油5.8mmol／L，1，2-乙二醇10.3 mmol／L，酶对辅酶B₁₂的K_m值为1.5μmol／L，而辅酶B₁₂是酶催化活力必需的辅助因子。酶反应的最适pH为8.6，最适温度为37℃。以L．diolivorans基因组DNA为模板，PCR扩增得到了二醇脱水酶基因（gldCDE）及其激活因子基因（gldGH），分别构建了重组质粒pSE-gldCDE与pSE-gldGH，两外源基因均在E．coli BL21中得到表达。经SDS-PAGE分析表明，gldCDE基因的表达蛋白包括62kDa、35kDa、22kDa三个亚基，与其基因序列中的三个读码框相符，并与天然菌中纯化所得二醇脱水酶的蛋白质组成相同；gldGH基因的表达蛋白包括68kDa、13kDa两个亚基，与其基因序列中的两个读码框相符。gldGH基因的表达蛋白经金属鏊合亲和层析与凝胶过滤纯化后，在非变性聚丙烯酰胺凝胶电泳分析中显示其分子量约325kDa，因此推测其很可能为大小两种亚基形成的α₄β₄多聚体。在进一步的酶功能鉴定中，重组菌中的二醇脱水酶基因表达产物不具备脱水酶活力，与丘状乳杆菌（Lactobacillus collinoides）二醇脱水酶基因在E．coli中的重组表达情况一致；纯化后的gldGH基因的重组表达产物，以天然菌中纯化得到的二醇脱水酶为对象，经酶功能检测后确认为二醇脱水酶激活因子。更多还原

【Abstract】 Diol dehydratase of L. diolivorans was purified by ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the native complex was estimated to 200kDa by nondenaturing polyacrylamide gel electrophoresis. Three main bands were present after SDS-PAGE with molecular masses of 62, 35 and 22 kDa, respectively. The Km for the three main substrates were 1.3 mmol/L for 1,2-propanediol, 5.8 mmol/L for glycerol and 10.3 mmol/L for 1,2-ethanediol. Km for adenosylcobalamin coenzyme was 1.5μmol/L, which was required for the enzyme catalysis. The optimal reaction conditions of the enzyme were pH8.6 and 37°C.The genes encoding diol dehydratase （gldCDE） and its reactivating factor （gldGH） were amplified from genomic DNA of L. diolivorans by PCR, then cloned in vector pSE380 respectively forming recombinant plasmids pSE-gldCDE and pSE-gldGH. These genes are expressed by induction of IPTG in E. coliBL21 carrying these recombinant plasmids respectively. After SDS-PAGE, three bands were present as gldCDE expression product with molecular masses of 62, 35 and 22kDa, corresponding to the three open reading frames（ORFs） in gldCDE respectively, and the same with the protein composition of diol dehydratase purified from L. diolivorans; two bands were present as gldGH expression product with molecular masses of 68 and 13kDa, corresponding to the two ORFs gldG and gldH respectively. And after purification by metal chelating affinity chromatography and gel filtration, expression product of gldGH formed a native complex estimated to 325kDa by nondenaturing polyacrylamide gel electrophoresis, which probably was anα₄β₄ subunit structure. In the function identification, gldCDE expression product in E. coliBL21 didn’t performed the activity of dehydratase, which was in the case of the recombinant expression of diol dehydratase genes of Lactobacillus collinoides in E. coli; while, the purified gldGH expression product in E. coliBL21 was identified as a diol dehydratase reactivating factor by the test of reactivating the diol dehyadratase purified from L. diolivorans.更多还原