In this study, the bifunctional membrane (epoxy and immobilized copper ion) was constructed to study the effect on penicillin G acylase (PGA) purification and immobilization. In the procedures, PGA was firstly chelated with the immobilized copper on the membrane at 18 'C, for 12 h. Then, it was covalently bound to the membrane via epoxy group under the incubation conditions (i.e., pH 10.0, 18 degrees C, 76 h). In the construction of bifunctional membrane, iminodiacetic acid disodium (IDA) concentration critically affected the bifunctional group ratio in the membrane. When IDA concentration was higher than 0.500 M, the chelated copper ion in membrane reached its a saturation of 52.2 mu mol/disc. Meanwhile, when IDA concentration was 0.050 M, the chelated copper ion would reach 29.9 mu mol/disc, nearly 58% of its saturation. The membrane prepared by 0.05 M IDA was employed to simultaneously purify and immobilize PGA. The resulting PGA membrane was stable even when the chelated copper ion was eluted with ethylenediaminetetraacetic acid disodium salt dehydrate. It was noted that 96.3% of the PGA activity could be retained under 26 times' reactions within more than 2 months. With this stable property, this immobilization approach could be further developed and applied for industrial processes. (c) 2007 Elsevier B.V. All rights reserved.