The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a recombinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA.