Expression of gene encoding GL-7ACA acylase in Escherichia coli.
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 recombin ant plasmids presented different expression characteristics in Escherichia coli. The acylase gene from plasmid pKKCA1 was inserted into plasmid pMFT7-5 and the resulting plasmid pMFT7CA1 has higher expression in E.coli. The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7CA1 was near 5 u/g, so the over produced 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 6xHis in the N-terminal of its alpha-subunit. Because plasmid pSMLCA1 brings tc(R) and p15A origin, it is special useful plasmid in fermentation. Two secretory expression plasmids, pSUCA1S and pETCA1pelB, 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.
Wang
En-Duo
ED
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. wed@server.shcnc.ac.cn
Zheng
Yong-Gang
YG
Li
Yong
Y
Jiang
Wei-Hong
WH
Yang
Yun-Liu
YL
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase