假单胞菌zjwp14中LATC水解酶基因的克隆和表达.pdfVIP

假单胞菌zjwp-14 中L-ATC 水解酶基因的克隆和表达 1 2 1* 1 1 吴敏 ，陈蔚青 ，王普 ，何军邀 ，尹江峰 （1 浙江工业大学生物制药研究所，浙江 杭州 310032 2 浙江树人大学生物与环境学院，浙江 杭州 310015） 摘要：构建能高效表达L-ATC水解酶的重组菌。用PCR方法扩增假单胞菌zjwp-14中L-ATC水解酶基因片段， 将该DNA片段插入T载体上测序得到522bp的DNA序列；将此序列插入表达载体pET-28a （+）上，转入大肠杆 菌BL21(DE3)进行表达，重组菌在0.5mmol/LIPTG诱导5h后，SDS电泳检测重组蛋白分子量为20.3kD。 所构建的表达菌株用于转化DL-ATC，以DTNB法检测转化率达83.65%。L-ATC水解酶在大肠杆菌中实现了高效 表达，获得了具有生物转化活性的L-ATC水解酶重组蛋白。 关键词：假单胞菌；L-ATC 水解酶；基因克隆；表达 中图分类号： 文献标识码：A 文章编号： Gene Cloning and Expression of L-ATC hydrolase from Pseudomonas sp. zjwp-14 1 2 1＊ 1 1 Wu Min , Chen Weiqing , Wang Pu , He Junyao , Yin Jiangfeng 1 Institute of Biopharmaceutical Engineering, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China, wangpu@zjut.edu.cn; 2 College of Biological Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, Zhejiang, China Abstract: To construct overexpressive L-ATC hydrolase recombinated bacterium, L-ATC hydrolase gene was amplified by PCR with artificial primer from Pseudomonas sp. zjwp-14. The amplified gene was recombined in cloning vector T and transformed into E. coli DH5α , the L-ATC hydrolase DNA fragments containing 522bp was detected. The L-ATC hydrolase gene was inserted into expression vector pET-28a (+) and transformed into E. coli BL21(DE3) to express. Recombinated bacterium was induced by 0.5mmol/L IPTG for 5h and the product was determined by SDS. The molecular weight of the enzyme expressed by recombinated bacterium was about 20.3kD. The bioconversion efficiency assayed by DTNB method was 83.65%. L-ATC hydrolase gene was high expressed in E. coli, a L-ATC hydrolase recombinated proteins with ability of active