Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase.
A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.
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Istituto di Biocatalisi e Riconoscimento Molecolare, C. N. R., Via Mario Bianco 9, 20131 Milano, Italy.
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Riva
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Baldaro
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D000581:Amidohydrolases / Q000737:chemistry*; D001709:Biotechnology; D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010399:Penicillin Amidase; D010710:Phosphates / Q000494:pharmacology; D013696:Temperature
D002511:Cephalosporins; D010710:Phosphates; C030735:7-aminocephalosporanic acid; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase