CPC酰化酶基因的人工合成与重组表达  

Artificial synthesis and expression of the CPC acylase gene in recombinant E.coli

作　　者：安明[1] 于慧敏[1] 罗晖[2] 宋文斯[1] 沈忠耀[1] 

机构地区：[1]清华大学化学工程系生物化工研究所,北京100084 [2]北京科技大学生物科学与技术系,北京100083

出　　处：《清华大学学报（自然科学版）》2008年第9期1499-1503,共5页Journal of Tsinghua University(Science and Technology)

基　　金：全国百篇优秀博士学位论文作者专项(200345);北京市科技新星计划项目(2006B22)

摘　　要：7-氨基头孢烷酸(7-ACA)是头孢菌素类抗生素的中间体,在医药工业中具有重要的应用价值。该文通过人工设计、合成并在重组大肠杆菌中表达头孢菌素C(CPC)酰化酶基因来实现7-ACA的一步酶法催化合成。以Pseudomonassp.SE83菌株的CPC酰化酶蛋白质序列为模板,对CPC酰化酶基因分两段进行了全局优化设计,包括密码子替换、鸟嘌呤和胞嘧啶含量调整以及酶切位点修改等。通过装配聚合酶链式反应方法最终获得了目标基因,并克隆至表达载体pET-28a,成功构建了诱导型重组大肠杆菌E.coli BL21(DE3)/pET28-acy。经异丙基-β-D-硫代半乳糖苷诱导,在Luria-Bertani培养基中,新建重组大肠杆菌所表达的可溶CPC酰化酶占菌体总蛋白的75%以上;经优化培养基摇瓶发酵,重组菌的CPC酰化酶酶活高达2956U/L。采用上述方法获得的CPC酰化酶,在一步酶法合成7-ACA的工业化生产中具有广阔的应用前景。7-amino cephalosporin acid （7-ACA）, an important intermediate of cephalosporinic antibiotics, is of great value in the medical industry. The cephalosporin C （CPC） acylase gene was synthesized and expressed in recombinant E. coli to develop an efficient single step enzymatic bioconversion of 7-ACA from CPC by using CPC acylase. A CPC acylase gene was designed in two fragments based on the protein sequence of cephalosporin acylase in Pseudornonas sp. SE83 for artificial gene synthesis after global optimization of the rare codon usage frequency, reduction of （GC） content and modification of restriction sites, etc. The full length target gene of CPC acylase was successfully achieved by assembly polymerase chain reaction （PCR） and further cloned into an inducible expression vector of pET 28a. A recombinant E. coli BL21（DE3）/pET28-acy was constructed to implement induced over expression of the synthesized CPC acylase gene. With IPTG induction in Luria-Bertani （LB） medium, the recombinant expressed a large amount of soluble CPC acylase accounting for 〉 75% of the total cellular protein. The CPC acylase activity accumulated in the recombinant E. coli BL21（DE3）/pET28-acy in an optimal flask culture reached as high as 2 956 U/L. This CPC acylase production is a promising industrial one-step enzymatic production method for 7-ACA.

关 键 词：7-氨基头孢烷酸 头孢菌素C酰化酶 重组大肠杆菌 基因合成 装配聚合酶链式反应 高表达 

分 类 号：Q781[生物学—分子生物学] Q786