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 Km 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.
Scientific and Technical Research Council of Turkey, Marmara Scientific and Industrial Research Center, Gebze-Kocaell.
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Erarslan
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Güray
D000179:Acrylates / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005027:Ethylene Oxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010397:Penicillanic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D010399:Penicillin Amidase / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism; D013816:Thermodynamics
D000179:Acrylates; D004800:Enzymes, Immobilized; D010397:Penicillanic Acid; D010399:Penicillin Amidase; D005027:Ethylene Oxide; D010400:Penicillin G; C004408:aminopenicillanic acid