The different ionic molecules/compounds were used as a ligand for the immobilization of penicillin G acylase on the highly porous cellulose-based polymeric membrane having buffer flux 1,746 LMH (L m(-2) h(-1)) at 0.5 bar pressure. The immobilized enzyme activity around 250 U-App was obtained with the ligand such as proline, tryptophan, casein acid hydrolysate, and brilliant green. Comparatively, proline showed less IMY% (percentage immobilization yield-58) but higher RTA% (percentage of activity retention-71) and specific activity (145 U-App g(-1)). However, the crosslinked preparation of brilliant green obtained using glutaraldehyde showed 82 +/- 2.7% immobilized enzyme activity after the completion of successive five cycles. In comparison with the free enzyme, the enzyme immobilized on the brilliant green coupled membrane showed around 2.4-fold increase in K-m value (47.4 mM) as well as similar optimum pH (7.2) and temperature (40 degrees C). The immobilized enzyme retained almost 50% activity after 107 days and 50 cycles of operation. Almost 50% decrease in buffer flux after enzyme immobilization was observed. At the end of the 30 cycles, flux pattern shows around 38% decrease in buffer flux however, after 16 cycles of operation flux moves closer towards the steady state.