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

WANG En-Duo^*, ZHENG Yong-Gang, LI Yong, JIANG Wei-Hong¹, YANG Yun-Liu¹
( 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; ¹Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China )

Abstract    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 6×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.
Key words    GL-7ACA acylase; recombinant plasmid; expression and purification; Pseudomonas; Escherichia coli

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