头孢菌素酰化酶基因在大肠杆菌中的表达规律  

Expression of Gene Encoding GL-7ACA Acylase in Escherichia coli

作　　者：王恩多[1] 郑勇刚[1] 李勇[1] 姜卫红[2] 杨蕴刘[2] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室,上海200031 [2]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《生物化学与生物物理学报》2002年第4期526-531,共6页

基　　金：国家高技术"863"计划资助项目 (No .10 3 12 0 2 0 1)~~

摘　　要：假单孢菌sp .130头孢菌素酰化酶催化戊二酰 7 氨基头孢烷酸的水解反应 ,生成 7 氨基头孢烷酸。 7 氨基头孢烷酸是医药工业合成大多数头孢菌素衍生物的起始原料。在 6种大肠杆菌表达质粒上构建了表达该酶的不同载体 ,得到了不同表达结果的大肠杆菌转化子。这些质粒有各自的特点 ,适用于不同的场合。Glutaryl 7 amino cephalosporanic acid acylase (GL 7ACA acylase) from Pseudomonas sp.130 catalyzes hydrolysis of glutaryl 7 amino cephalosporanic acid to produce 7 amino cephalosporanic acid (7 ACA). 7 ACA is the starting material for the industrial production of most cephalosparonic derivatives. Six plasmids for expression of GL 7ACA acylase were constructed and these recombinant plasmids presented different expression characteristics in Escherichia coli . The acylase gene from plasmid pKK CA1 was inserted into plasmid pMFT7 5 and the resulting plasmid pMFT7 CA1 has higher expression in E.coli. The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7 CA1 was near 5 u/g, so the overproduced enzyme was easily purified by a single step anion exchange column chromatography. The enzyme could be purified by immobilized ion affinity chromatography after fused by 6×His in the N terminal of its α subunit. Because plasmid pSML CA1 brings tc R and p15A origin, it is special useful plasmid in fermentation. Two secretory expression plasmids, pSU CA1S and pET CA1 pelB, could secrete the acylase to periplasmic space of bacteria. The whole cells containing the secretory expression plasmid may be used for production of 7 ACA directly.

关 键 词：头孢菌素酰化酶基因 大肠杆菌 表达规律 重组质粒 纯化 假单胞菌 

分 类 号：Q786[生物学—分子生物学]