甘油脱水酶基因(gldABC)与1,3-丙二醇氧化还原酶基因(dhaT)的共表达

The Coexpression of Glycerol Dehydratase Gene and 1,3-Propanediol Oxidoreductase Gene

将dhaT基因片段以顺反子的形式插入到重组质粒pMD19TS2中gldABC基因的下游,形成重组克隆质粒pMD19TS3,进而将gldABC与dhaT基因以多顺反子的形式亚克隆至pET-28a(+)表达载体上。终浓度为1mmol/L IPTG诱导重组大肠杆菌E.coli/pET-28a(+)/gldABC-dhaT 2h,SDS-PAGE电泳分析结果表明分别在甘油脱水酶三个亚基分子量相对应的61ku、21ku、16ku和1,3-丙二醇氧化还原酶亚基分子量相对应的42 ku的位置上出现了特异性条带。上清液中酶活性分别为甘油脱水酶23.7U/mL,1,3-丙二醇氧化还原酶30.4 U/mL。

The recombined plasmid pMD19TS3 where the dhaT gene was inserted into the downstream of gldABC gene was constructed. Thereafter the gldABC gene and dhaT gene in tandem were subcloned into expression vector pET-28a（＋）. The recombined strain E. coli BL21 （DE3） was induced to express under the condition of 37℃, lmmol/L IPTG, 2h. Expression product was analyzed by SDS-PAGE and the relative molecular weights of the gldABC expression product were about 61KDa, 21ku, 16ku and 42ku for dhaT. The enzymes activities of glycerol dehydratase and 1,3-propanediol oxidoreductase were 23.7U/mL and 30. 4U/ mL respectively in the supernant.