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.
Erarslan
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Scientific and Technical Research Council of Turkey, Marmara Scientific and Industrial Research Center, Gebze-Kocaell.
Güray
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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