产GL-7-ACA酰化酶重组大肠杆菌的构建和表达  

Cloning and Expression of GL-7-ACA Acylase in E.coli

作　　者：罗晖[1] 胡晓佳[1] 周航[1] 童忆舟[1] 于慧敏[1] 李强[1] 沈忠耀[1] 

机构地区：[1]清华大学化工系生物化学研究所,北京100084

出　　处：《微生物学通报》2004年第4期38-42,共5页Microbiology China

基　　金：全国优秀博士学位论文作者专项资金项目 (No 2 0 0 3 45 )

摘　　要：为了实现GL 7 ACA酰化酶在大肠杆菌中的成功表达 ,将GL 7 ACA酰化酶基因用PCR的方法去除其信号肽序列 ,并将其连接到质粒pET 2 8a ,通过筛选得到了表达GL 7 ACA酰化酶的重组菌BL2 1 (DE3) /pET ACY。分别考察了诱导温度、菌浓 (OD60 0 )、诱导剂IPTG的用量等因素对重组菌表达GL 7 ACA酰化酶的影响。在优化条件下 ,GL 7 ACA酰化酶酶活可达 2 66U/L。GL 7 ACA酰化酶经一步DEAE Sepharose纯化即可达到 80 %的纯度 ,酶活收率为 5 0 %。To facilitate the expression of GL 7 ACA acylase gene in a recombinant E coli , a fragment of the gene, in which the signal peptide was deleted by PCR method, was inserted into a prokaryotic expression vector, pET 28a By colony PCR method screening, a recombinant plasmid pET ACY was obtained and then transformed into the expression host BL21 (DE3) The influences of induction conditions such as IPTG concentration, the time of induction and the induction temperature on the expression of the recombinant protein were investigated Under optimal condition, the enzyme activity could reach 266 U/L Finally, the recombinant GL 7 ACA acylase can be easily isolated to a purity of about 80% by a simple anion ion exchange chromatography with enzyme activity recovery of 50%

关 键 词：GL-7-ACA酰化酶 大肠杆菌 表达 

分 类 号：Q933[生物学—微生物学]