Expression And Immobilization Of A Recombinant Cephalosporin-C Deacetylase

Cephalosporin-C deacetylase (CAH, cephalosporin-C acetylhydrolase) is an ideal catalyst in producing D-7-ACA (deacetyl7-aminocephalosporanic acid), an important intermediate for semisynthetic cephalosporins. Such enzyme activities are found in different organisms, among which the CAH from Bacillus subtilis has been fully studied and successfully applied to commercial production.In this work, a recombinant E. coli DH5a-pCAH, with trp promoter expressing CAH, was constructed. After28hours fermentation,2L medium in7L-fermentor at37℃, the recombinant E. coli produced313kU per liter, with OD600reached to27. The amount of CAH was approximately70%of the total cellular protein.The recombinant CAH crude extract was purified by ammonium sulphate precipitation and ultrafiltration to homogeneity in SDS-PAGE with a yield of56%, purification factor of1.44.The purified CAH was immobilized on the epoxy carrier LX-1000EP(c) covalently. After optimizing the immobilizing conditions, we got an immobilized CAH with activity reached to443U/g higher than the FLAG product (CE), and the activity yield27%of free enzyme. The optimal temperature shifted from35℃to55℃after immobilization as the temperature stability improved. The immobilized CAH activity was not reduced after repeated use for100times in50ml5%7-ACA.