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 recombinant plasmids presented different expression characteristics in Escherichia coli. The a-cylase 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)/pMFTCA1 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 x His in the N-terminal of its α-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.