雷氏普罗威登斯菌青霉素G酰化酶基因在大肠杆菌中的克隆与表达  

Cloning and expression of penicillin G acylase gene from Providencia rettgeri in Escherichia coli

作　　者：周政[1] 张爱晖[2] 周丽萍[3] 杨晟[1] 姜涌明[2] 袁中一[1] 

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,上海200031 [2]扬州大学生物科学与技术学院 [3]江苏大学

出　　处：《工业微生物》2002年第3期1-5,共5页Industrial Microbiology

基　　金：国家自然科学基金(No.30100029);"863"国家高科技资助项目(No.2001AA235081)

摘　　要：利用PCR和分子克隆技术从雷氏普罗威登斯菌(Providencia rettgeri)(ATCC29944)的基因组DNA中获得一个青霉素G酰化酶(penicillin G acylase,PGA)基因并将其装入表达质粒pET24a。携带有重组质粒pETPGA的Escherichia coli基因工程菌BL21(DE3)/pETPGA实现了PGA的高效表达。对发酵条件的研究表明基因工程菌在24℃、添加5g/L甘油条件下以1.0mmol/L IPTG诱导1.5h酶活力即达到993.4U/L,比野生菌酶活力(15U/L)提高了66倍。Based on PCR techniques, a gene encoding penicillin G acylase(PGA) was obtained from genomic DNA of Providencia rettgeri(A.TCC29944). The pET24a vector combined with the PGA gene was transformed into BL21(DE3) and the resulting recombinant Escherichia coli gave a high level expression of PGA. Several factors, including IPTG concentration, temperature and carbon source, were optimized for enzyme overproduction. The optimization significantly increased PGA expression by 66-fold compared to the native expression (15U/L) in P. rettgeri. After inducing with 1. 0mmol/L IPTG at 24! and fermentation in medium with additive of 5g/L glycerol for 1.5 hour, The enzyme expression of recombinant E. coli reached 993.4U/L.

关 键 词：雷氏普罗威登斯菌 青霉素G 酰化酶 基因 大肠杆菌 克隆 表达 

分 类 号：TQ465[化学工程—制药化工]