In order to establish a simple and economic purification scheme for penicillin G acylase (PGA) (EC 3.5.1.11) from a mutant strain of Escherichia coli 11105 with high yield, various protein extraction methods were employed including osmotic and chloroform shocks, and osmotic shock after lysozyme and cetyltrimethylammonium bromide (CTAB) pretreatments of E. coli cells. A purification factor of 7.82 and recovery of 53 % were obtained with osmotic shock when sucrose and EDTA were used at 20 % and 1.22 % concentrations, respectively. Various treatment times of osmotic shock and lysozyme and CTAB pretreatments did not affect the yield of released PGA. The yield of released PGA was found to be lower using chloroform shock than when osmotic shock was employed. Two steps purification procedure developed in this research was satisfactory to obtain the high specific activity required for the production of immobilized PGA.