A mutant strain with high penicillin G acylase activity was derived from Escherichia coli ATCC 11105 by chemical mutagenesis, using N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Penicillin acylase was extracted from the mutant strain and highly purified by DEAE‐cellulose and hydroxyapatite column chromatography followed by preliminary precipitation steps. Vm and Km values of the enzyme (specific activity: 24·81 U mg−1, protein concentration: 0.56 mg cm−3) were found to be 22.73 U cm−3 mm−1 and 3.18 mmold m−3 penicillin G, respectively. The enzyme was shown to be uncompetitively inhibited by excess of substrate. The inhibition by phenylacetic acid was found to be competitive, and 6‐aminopenicillanic acid to be noncompetitive. Inhibition constants for excess of penicillin G, phenylacetic acid and 6‐aminopenicillanic acid were estimated as 74.20, 18.74 and 15.00 mmol dm−3 respectively, at pH 8.0 and 40°C. The activation energy of the enzymatic hydrolysis reaction of penicillin G was found to be 10.78 kcal mol−1. Optimal pH and temperature values of the enzyme were determined as 8.0 and 60°C. Complete loss of the enzyme stability occurred when the enzyme was incubated at pH 10.0 for 2 days or at 50°C for 2 h.