KINETIC INVESTIGATION OF PENICILLIN-G ACYLASE FROM A MUTANT STRAIN OF ESCHERICHIA-COLI ATCC-11105 IMMOBILIZED ON OXIRANE ACRYLIC BEADS

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-degrees-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-degrees-C. The enzyme was stable when incubated at 4-degrees-C for one day at a pH range of 5.0 to 9.0. Thermal stability (over 30 min) was observed up to 40-degrees-C but decreased at higher temperatures and was almost absent at 60-degrees-C. A 95% conversion rate was observed at 28-degrees-C and at 40-degrees-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-degrees-C. No significant loss of activity was observed at 28-degrees-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 temperatures.