Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in water-methanol mixtures: Effect of cosolvent content and chemical nature of substrate on reaction rates and yields

Abstract

The synthesis of four beta-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed by Escherichia coli penicillin acylase has been investigated in water-methanol mixtures, The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid and D-alpha-phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl accepters. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v)) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values.