PURIFICATION AND KINETICS OF PENICILLIN-G ACYLASE FROM A MUTANT STRAIN OF ESCHERICHIA-COLI ATCC-11105

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. V(m) and K(m) 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 min-1 and 3.18 mmol dm-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-degrees-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-degrees-C. Complete loss of the enzyme stability occurred when the enzyme was incubated at pH 10.0 for 2 days or at 50-degrees-C for 2 h.