In this study, an immobilized metal ion affinity membrane (IMAM) was prepared and used to immobilize penicillin G acylase (PGA). The stability of storage of the immobilized PGA membrane (IPM) was tested. When stored in deionized water (DI) at 4 degrees C, only 19% residual activity of IPM was retained for 10 days. However, when stored in 10 mM phosphate buffer (PB, pH 8) with 0.1% NaN3, the IPM can retain 99% of its activity for a 10-day reaction and storage test. The feasibility of the IPM regeneration after a long period of reaction was investigated. When using 100 mM EDTA as the stripping solution, the re-immobilized capacity for PGA is only 52% of the original one after 4 regenerations. When the regeneration process was modified by using the stripping solution (300 mM NaCl, 25 mM EDTA, 20 mM PB, pH 8) for 30 min, then immersed in 0.5 M HCl and 0.5 M NaOH respectively for 10 min, the resulting IMAM can reserve 99% PGA immobilized activity after 5 regenerations. (C) 2012 Elsevier B.V. All rights reserved.