Penicillin is the most widely used antibiotics, due to their great spectrum of antibacterial activity, low toxicity, and inhibitory effect on bacterial cell wall synthesis and it shares about 19 % of the estimated worldwide antibiotic market Therefore, penicillin G acylase (PGA) that catalyzes the hydrolysis of penicillin G to produce 6- aminopenicillanic acid (6-APA) is very essential for the production of semisynthetic penicillin. It is also important to create efficient methods for immobilization of PGA for high activity and stability to make it viable for industrial applications. Immobilization of PGA on immobilized metal affinity membrane (IMAM) showed more PGA activity compared to those immobilized metal affinity resin. Immobilization gave stability to PGA in the pH range from 7 to 9 and for temperatures from 40 to 60 °C. Immobilization also allowed the enzyme to be reused 16 times and caused very little activity loss after 40 days of storage