Kinetic investigation of penicillin g acylase from a mutant strain of Escherichia coli ATCC 11105 immobilized on oxirane–acrylic beads

Abstract

Highly purified penicillin G acylase from a mutant derivative of Escherichia coli ATCC 11105 was immobilized on oxirane–acrylic beads by covalent binding via oxirane groups. The highest specific activity, (322 U g^−‐1 dry matrix at 40°C and at pH 8·0) was obtained by using an enzyme solution having 13·8 U mg^−‐1 specific activity and 72·73 mg total protein. The efficiency of immobilization was 95·8%. Kinetic parameters of immobilized penicillin G acylase were determined at the same pH and temperature by a preparation having 8·1 mg bound protein. The K_m value and substrate inhibition constant of the enzyme were found to be 11·36 mmol dm^−‐3 and 680 mmol dm^−‐3 penicillin G respectively. Phenylacetic acid and 6‐aminopenicillanic acid were estimated as the competitive and non‐competitive inhibitors of the enzyme and their inhibition constants were found to be 90 mmol dm^−‐3 phenylacetic acid and 76·1 mmol dm^−‐3 for 6‐aminopenicillanic acid. The activation energy of the hydrolytic reaction was calculated to be 7·75 kcal mol^−‐1. The immobilized enzyme showed highest activity at pH 8·0 and at 55°C. The enzyme was stable when incubated at 4°C for one day at a pH range of 5·0 to 9·0. Thermal stability (over 30 min) was observed up to 40°C but decreased at higher temperatures and was almost absent at 60°C. A 95% conversion rate was observed at 28°C and at 40°C with 60 and 30 min operation times respectively. Operational stability of the enzyme was improved further with dithiothreitol treatment. Activity loss was around 5% following 20 cycles of repeated use of the enzyme at 40°C. No significant loss of activity was observed at 28°C when the enzyme was used,for 20 cycles. 6‐Aminopenicillanic acid in the reaction mixture was observed to be stable during conversion reactions which were carried out at both temperutures.