In this study, an approach to prepare immobilized metal affinity membrane (IMAM) with high metal ions and protein adsorption capacities was developed. In the process of coupling epichlorohydrin (EPI) to the regenerated cellulose membrane (RC membrane), NaOH concentration is found to be the most critical. With a lower NaOH concentration, only a minimal amount of EPI reacted to the RC membrane. When NaOH concentration was higher, the membrane was distorted, which caused a significant pressure drop in flow-through operation. To optimize the IMAM performance, an objective function was defined as the ratio of the model protein, penicillin G acylase (PGA), activity adsorbed on the membrane to the transmembrane pressure drop. According to the criterion, the optimal reaction conditions were found as follows: one RC membrane immersed in 20 ml, 1.4 M NaOH, 5 ml EPI and operated at 24 degrees C, 150 rpm for 14 h. Under this condition, the copper ions and PGA in IMAM were significantly increased to 75.5 +/- 0.25 mu mol/disc and 1.8 U/disc respectively. The adsorption for lysozyme on the prepared IMAM reached 1044 mu g/cm(2), the highest in the literature. (C) 2009 Elsevier Ltd. All rights reserved.