Studies On Recombinant Expression And Catalysis Properties Of CPC Acylase In E.coli

Cephalosporin C acylase (CCA) is the key enzyme in the one step enzymatic production of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA), which is a starting compound in the manufacture of semi-synthetic cephalosporin antibiotics. This dissertation focused on the construction of the recombinant strains to express the CCA efficently by means of the genetic engineering technics. Then, the catalysis properties of CCA were studied.We got a CCA gene (cpca), synthesized by Sangon. This optimized CCA gene from P. diminuta N176 can express CCA in system BL21(DE3)plysS/ pET24Δ. The evolved acylase represents new substrate specificity and indicates a ~90% conversion of CPC to 7-ACA. The constitutive expression systems J-KTHPR- fcpca,T-KToriP-cpca were constructed. But no CCA activity was found due to the complicated DNA structure of cpca.Cpca was inserted into the inducible plasmid pET28 and pET30 (with T7 promoter). Compared to recombinant plasmid pET28-cpca, pET30-cpca expressed less inclusion body in E.coli JM109(DE3). pET30-cpca realized the CCA activity first after it was transformed into the host cell E.coli. BL21(DE3)plysS. Another recombinant plasmid pET30Δ-cpca was constructed by knocking out the lacI gene. These recombinant plasmids were transformed into different E.coli. B-pET30-cpca was chosen a better espression system induced by lactose in 28℃.Because of a special His-tag sequence in the 3'terminal of cpca gene, CPC acylase could be specifically purified by the mmobilized metal affinity chromatography (IMAC). The studies of catalysis properties of CCA showed that, the acylase catalysis activity reached highest in pH 10 and 55℃. It is better to keep the enzyme in lower temperature. The catalyzed process of CCA showed that the CCP was sensitive to inhibition by the product.The research work of this dissertation laid a foundation for highly expression of CPC acylase and its final industrialized production.