Selectivity of the enzymatic synthesis of ampicillin by E-coli PGA in the presence of high concentrations of substrates

intermediate (the acyl-enzyme complex). When used for the kinetically controlled synthesis of P-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 200mM of 6-APA and 500mM of PGME), using soluble PGA, at 25 degrees 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. (c) 2005 Elsevier B.V. All rights reserved.