Lactobacillus collinoides二醇脱水酶及其激活因子的功能鉴定
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摘要
甘油脱水酶(EC 4.2.1.30)是生物转化法生产1，3-丙二醇(工业上有重要用途的化工材料)的限速酶，而二醇脱水酶(EC4.2.1.28)是甘油脱水酶的同工酶，所以近年来也成为研究的热点。
以Lactobacillus collinoides基因组DNA为模板，PCR扩增得到了二醇脱水酶基因(pduCDE)及其激活因子基因(pduGH)，分别连接到pSE380和pET30a(+)中，构建了重组质粒pSE-pduCDE与pET-pduGH，两外源基因分别在E.coli JM109和E.coli rosseta(DE3)中得到表达。经SDS-PAGE分析表明，pduCDE基因的表达蛋白包括63kDa、28kDa、22kDa三个亚基，结果与预期的相符；pduGH基因的表达蛋白包括64.5 kDa、12.4 kDa两个亚基，结果与预期的相符。重组菌中的二醇脱水酶基因pduCDE表达产物不具备脱水酶活力，与Lactobacillus diolvorans二醇脱水酶基因在E.coli(BL21)中的重组表达情况一致。重组质粒pET-pduGH的表达蛋白，在E.coli(DE3)中形成不溶性的包涵体，经变性和复性后再经进一步酶功能鉴定，在国内外首次证明，复性后的pET-pduGH表达蛋白能激活Glostridipasteurianum甘油脱水酶um，具有甘油脱水酶激活因子活性。
Glycerol dehydratase (EC4.2.1.30) has turned out to be a research hotspot for being the rate-limited enzyme in biosynthesis of 1,3-propanediol, which is a useful material in chemical industry, Diol dehydratase(EC4.2.1.28) and glycerol dehydrates are isofunctional enzymes ,so diol dehydratase was widely attended.
The genes encoding diol dehydratase (pduCDE) and its reactivating factor (pduGH) were amplified from genomic DNA of Lactobacillus collinoides by PCR, then cloned in vector pSE380 and pET30a(+) respectively forming recombinant plasmids pSE-pduCDE and pET-pduGH. These genes are expressed by induction of IPTG in E. coli JM109 and E. coli rosseta(DE3) carrying these recombinant plasmids respectively. After SDS-PAGE, three bands were present as pduCDE expression product with molecular masses of 63, 28 and 22kDa, corresponding to the prediction and then pduGH expression product with molecular masses of 64.5 and 14.4kDa, corresponding to the prediction. In the function identification, pduCDE expression product in didn't performed the activity of dehydratase, which was in the case of the recombinant expression of diol dehydratase genes of Lactobacillus diovorans in E. coli BL21; the high expression of pduGH in E.coli rosseta(DE3) 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+) (or Mn~(2+)) , the glycerol dehyadrtase from Clostridium pasteurianum , Which had been inactivated to be enzyme-cyanocobalamin complex, was reactivated the reactivating factor PduGH
以Lactobacillus collinoides基因组DNA为模板，PCR扩增得到了二醇脱水酶基因(pduCDE)及其激活因子基因(pduGH)，分别连接到pSE380和pET30a(+)中，构建了重组质粒pSE-pduCDE与pET-pduGH，两外源基因分别在E.coli JM109和E.coli rosseta(DE3)中得到表达。经SDS-PAGE分析表明，pduCDE基因的表达蛋白包括63kDa、28kDa、22kDa三个亚基，结果与预期的相符；pduGH基因的表达蛋白包括64.5 kDa、12.4 kDa两个亚基，结果与预期的相符。重组菌中的二醇脱水酶基因pduCDE表达产物不具备脱水酶活力，与Lactobacillus diolvorans二醇脱水酶基因在E.coli(BL21)中的重组表达情况一致。重组质粒pET-pduGH的表达蛋白，在E.coli(DE3)中形成不溶性的包涵体，经变性和复性后再经进一步酶功能鉴定，在国内外首次证明，复性后的pET-pduGH表达蛋白能激活Glostridipasteurianum甘油脱水酶um，具有甘油脱水酶激活因子活性。
Glycerol dehydratase (EC4.2.1.30) has turned out to be a research hotspot for being the rate-limited enzyme in biosynthesis of 1,3-propanediol, which is a useful material in chemical industry, Diol dehydratase(EC4.2.1.28) and glycerol dehydrates are isofunctional enzymes ,so diol dehydratase was widely attended.
The genes encoding diol dehydratase (pduCDE) and its reactivating factor (pduGH) were amplified from genomic DNA of Lactobacillus collinoides by PCR, then cloned in vector pSE380 and pET30a(+) respectively forming recombinant plasmids pSE-pduCDE and pET-pduGH. These genes are expressed by induction of IPTG in E. coli JM109 and E. coli rosseta(DE3) carrying these recombinant plasmids respectively. After SDS-PAGE, three bands were present as pduCDE expression product with molecular masses of 63, 28 and 22kDa, corresponding to the prediction and then pduGH expression product with molecular masses of 64.5 and 14.4kDa, corresponding to the prediction. In the function identification, pduCDE expression product in didn't performed the activity of dehydratase, which was in the case of the recombinant expression of diol dehydratase genes of Lactobacillus diovorans in E. coli BL21; the high expression of pduGH in E.coli rosseta(DE3) 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+) (or Mn~(2+)) , the glycerol dehyadrtase from Clostridium pasteurianum , Which had been inactivated to be enzyme-cyanocobalamin complex, was reactivated the reactivating factor PduGH
引文
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