The two enzymes in two disparate forms, D-amino acid oxidase (DAAO) in the permeabilized Pichia pastoris cells and immobilized glutaryl-7-aminocephalosporanic acid acylase (GA) on support, were employed to convert cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) in a single reactor. As a catalyst used in the reaction, DAAO in the permeabilized cells was relatively stable and its half-life was up to 14.5 days at 30 degrees C. In this study, CPC could be converted to 90.9% 7-ACA, 5% alpha-ketoadipyl-7-ACA (AKA-7-ACA) and 4.1% unidentified by-product within 2.5 h. During the reaction process, the loss of DAAO activity in the reactor was at an average rate of 0.07 U min(-1), but it could be compensated by continuous addition of the new permeabilized cell suspension. At the end of reaction, the stable immobilized GA was intercepted in a specially designed reactor, and reused for the next conversions. The permeabilized cells were separated outside the reactor by centrifugation and reused. Thus,, the consecutive production of 7-ACA from CPC in a single reactor is achieved by a fed-batch strategy. In the reaction system, the used permeabilized cells and immobilized GA were renewed every four cycles and every seven cycles, respectively. The yield of 7-ACA reached around 90% at each reaction cycle. (c) 2006 Elsevier Inc. All rights reserved.