6-Aminopenicillanic acid. VI. Formation of 6-aminopenicillanic acid from penicillin by enzymic hydrolysis.

Abstract

Penicillin amidase activity, resulting in the hydrolysis of phenoxymethylpenicillin to give 6-aminopenicillanic acid, was found to be widespread among micro-organisms. Partial purification of the enzyme, which was essentially exocellular, was obtained by precipitation with ammonium sulphate, or acetone, followed by dialysis. The pH optimum with phenoxymethylpenicillin as substrate was found to be approximately 9.0 with maximum enzyme stability at approximately pH 10.0. Over a relatively short period of reaction time the optimum temperature was approximately 50 °C. The K m value with phenoxymethylpenicillin was found to be 4.0 mg/ml. Using as substrate a concentration of 1 % phenoxymethylpenicillin, a conversion to 6-aminopenicillanic acid of 56 % of theoretical was obtained in the enzyme reaction mixture. Isolation of pure 6-aminopenicillanic acid from this reaction mixture was obtained in 50 % yield. 6-aminopenicillanic acid markedly inhibited the enzyme activity at a concentration of 4.0 mg/ml. Phenoxyacetic acid at this concentration was without effect. At higher concentrations, incubation of 6-aminopenicillanic acid and phenoxyacetic acid with the enzyme resulted in the synthesis of phenoxymethylpenicillin. The pH optimum for enzyme activity in the direction of synthesis was 5.0 to 5.5 compared with approximately 9.0 for hydrolysis.