Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates |
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Authors: | Marcelo PA Ribeiro Andrea LO Ferreira Raquel LC Giordano Roberto C Giordano |
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Institution: | Departamento de Engenharia Química, Universidade Federal de São Carlos, c.p. 676, 13.565-905, São Carlos, Brazil |
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Abstract: | Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d-phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack. |
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Keywords: | Ampicillin synthesis selectivity Penicillin G acylase 6-Aminopenicillanic acid Synthesis/hydrolysis ratio |
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