Glutaryl-7-aminocephalosporanic acid(GL-7-ACA) acylase is one of the key enzymes received considerable recognition as a biocatalyst for two-step enzymatic production of 7-ACA. A new recombinant Escherichia coli, E.coli JM105/pMKC-ACY, was constructed and used for the overexpression studies of the GL-7-ACAacylase. The superior culture conditions were figured out. Fed-batch culture of the recombinant strain was further accomplished under the optimized conditions in a 5L fermentor. The GL-7-ACAacylase activity increased to as high as 6668.9 U/L with the highest productivity of 275. 5U/( L·h), which was highly promising for the scale-up enzymatic production of 7-ACA in industry.