The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu 2 + was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH 4 Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH 4 Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH 4 Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97-100%).