Adsorption of penicillin acylase (PA) from a crude aqueous extract of Escherichia coli on chitosan beads and N-benzamide and N-phenylacetamide derivatives of chitosan was investigated. Penicillin acylase adsorbed specifically on the derivatized chitosan beads, with a strong interaction with the ligand groups. The interaction of the active site of PA with the affinity ligands was simulated by molecular modeling. The adsorbent was further used for immobilization of PA by using glutaraldehyde as the activating agent. The immobilized enzyme retained the same activity even after 20 weeks.