Secretory Expression of GL-7-ACA Acylase in E. coli.
The gene of beta-subunit of GL-7-ACA acylase [7beta-(4-carboxybutanamido) cephalosporanic acid acylase] was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B. Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp. into pTrcCA1B. Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235. pTrcCA1S and pKKCA1S were allowed to transform TG1. These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria. As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24. And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.