Pseudomonas putida S1海藻糖合成酶表达质粒的构建及诱导条件优化

采用PCR方法从Pseudomonas putida S1中克隆出编码海藻糖合成酶的基因treS，并与质粒pQE30T相连，构建了表达质粒pQE—TS2。将此重组质粒转化宿主菌E．coliM15进行诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶SDS—PAGE电泳结果表明，treS基因在大肠杆菌中获得了高效表达。通过对诱导温度、诱导剂浓度、加诱导剂时间和诱导时间的优化研究，在菌液生长至OD600值为0．6时，加入诱导剂IPTG至终浓度0．01mmol／L，20℃诱导20h，蛋白的表达量达到每克干细胞89mg的蛋白，粗酶液酶活达到19U／mL。

Construction of Pseudomonas putida S1 trehalose synthase expression vector and optimization of induction conditions

TreS gene encoding trehalose synthase from Pseudomonas putida S1 was amplified by PCR and was ligated to pQE30T plasmid as the expression plasmid pQE-TS2. E. coli M15 competent cells were transformed by the recombinant plasmid pQE-TS2 and induced by IPTG. The trehalose synthase gene was confirmed in E. coli by SDS-PAGE. The influences of induction temperature, IPTG concentration, induction time, and induction duration were investigated. When the cells grew into OD60owas 0. 6, IPTG was added to the final concentration of 0.01 mmol/L. After 20 h induction at 20 ℃, the expression of the trehalose synthase reached 89 mg/g（DCW）. The activity of the trehalose synthase in the crude enzyme was detected as 19 U/mL.