The aim of this work was to test a chromatographic support containing 4-amino-antipyrine for its ability to purify penicillin acylase in crude extract and pure penicillin acylase. First, methacryloyl antipyrine (MAAP) as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Poly(ethylene glycol dimethacrylate-methacryloyl antipyrine) [poly(EGDMA-MAAP)] monolith was prepared by an in situ polymerization method. Poly (EGDMA-MAAP) monolith was characterized by elemental analysis, scanning electron microscope, swelling tests and surface area measurements. Swelling ratio of poly(EGDMA-MAAP) monolith was 56%. According to the elemental analysis results MAAP incorporation into polymer structure was found as 58.9 mu mol/g. Specific surface area and average pore size of the monolith was found to be 112.4 m(2)/g and 8 10 nm, respectively. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) monolith was found to be 98.0 mg/g at pH 5.0. Adsorption capacity increased with increasing temperature and salt concentration and decreased with increasing flow-rate. Chromatography with crude feedstock gave 10.3-fold purification with a recovery of 90% from Penicillium chrysogenum (NRRL 1951) and 35.5-fold purification and 89% recovery from Penicillium Purpurogenum crude extracts with LOM NaOH. (c) 2006 Elsevier B.V. All rights reserved.