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Characterization of an industrial biocatalyst: Immobilized glutaryl‐7‐ACA acylase
Author(s) -
Monti Daniela,
Carrea Giacomo,
Riva Sergio,
Baldaro Eva,
Frare Giovanni
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20001020)70:2<239::aid-bit13>3.0.co;2-i
Subject(s) - biocatalysis , chemistry , cephalosporin c , biotransformation , immobilized enzyme , d amino acid oxidase , product inhibition , enzyme , chromatography , catalysis , yield (engineering) , nuclear chemistry , oxidase test , organic chemistry , biochemistry , reaction mechanism , cephalosporin , non competitive inhibition , materials science , antibiotics , metallurgy
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°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°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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 239–244, 2000.