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

摘要

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 beta-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 deg 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.