枯草芽孢杆菌脂肪酶表达质粒的构建及其表达

Construction of B.subtilis Expression Plasmid and Expression of Lipase

以枯草芽孢杆菌168(B.subtilis168)染色体为模板PCR扩增出P43启动子,与大肠杆菌-枯草杆菌穿梭质粒pUBC19相连得到表达载体pUBC-P43,然后将枯草芽孢杆菌脂肪酶基因lipA克隆到载体pUBC-P43启动子下游,得到重组质粒pUBCPL并转化B.subtilisTZ10。经中性红油脂平板、酶切和PCR方法鉴定得到重组菌TZ10/pUBCPL。宿主菌TZ10是B.subtilisDB104染色体缺失了lipA基因后获得。重组菌经初步发酵,以橄榄油为底物测定发酵上清液最高脂肪酶活力为49.1 U.L-1,而相应菌株DB104发酵最高酶活力仅为11.4 U.L-1。

The P43 promoter was amplified （PCR method） using B. subtilis 168 chromosome as template. Then,it was connected with E. coli-B, subtilis shuttle vector pUBC19 forming expression vector pUBC-P43. Finally the B. subtilis lipase gene lipA was put under the downstream of P43 promoter of pUBC-P43 so that pUBCPL was obtained. By transforming of pUBCPL into TZ10 and detecting through neutral red-oil solid culture, enzyme analysis and PCR,the transformant TZ10/pUBCPL was got. The host strain of TZ10 was obtained by inserting cat gene into lipA gene of B. subtilis DB104 chromosome. Primary fermentation results showed that the maximum extracellular enzyme activity of TZ10/pUBCPL was 49.1 U · L^-1 using olive oil as substrate, whereas DB104 only 11.4 U · L^-1.