The use of penicillin G acylase (PGA) requires efficient methods for immobilizing the enzyme to yield a biocatalyst with high activity and stability for industrial application. This study constructs the immobilized metal affinity membrane (IMAM) via a series of chemical reactions to sequentially couple epichlorohydrin (EPI), iminodiacetic acid (IDA), and copper ions on the regenerated cellulose membrane. The feasibility of direct PGA immobilization on IMAM was examined. Various conditions were tested for constructing IMAM for PGA immobilization. Under the optimal condition, a copper ion of 492.8 mu mol/g with a PGA activity of 11.76 U/g was obtained for IMAM as compared to those for the commercial immobilized metal affinity resin (299.8 mu mol/g and 2.99 U/g). The immobilized PGA membrane was stable in the pH range from 7 to 9 and for temperatures from 40 to 60 degrees C. In addition, 99% of the residual activity could be retained via a 16-times repeated use. Only little activity loss was observed after a 40 days of storage. This study also determines the kinetic parameters V(max), K(m), and the energy of activation (E(a)) for the immobilized PGA membrane. (C) 2011 Elsevier B.V. All rights reserved.