A simplified kinetic model for the side reactions occurring during the enzymatic synthesis of ampicillin

Abstract

This work presents a kinetic study of the side reactions of the ampicillin enzymatic synthesis, from phenylglycine methyl ester and 6-aminopenicillanic acid using penicillin G acylase immobilized on agarose. A Michaelis-Menten model with competitive inhibition was fitted to initial rates of ester and antibiotic hydrolysis, at pH 6.5 and 25ºC. Inherent kinetic parameters were estimated for low enzymatic loads, to assure that diffusional resistance was not important. It was observed that ampicillin inhibits the hydrolysis of PGME, but the inhibitory effect of the ester on ampicillin hydrolysis was almost negligible. The obtained parameters were: k cat1= 0.025 mM/UI min, Km1 = 155.4mM, K AE = 16.18mM, k cat2= 4.67x10-3 mM/UI min, Km2 = 11.47, K EA = 0.68 mM. Parameter values are in the range reported in the literature, except for Km1, which is much higher. The large confidence interval for this parameter denotes that the model presents low sensitivity with respect to it

Improving selectivity and productivity of the enzymatic synthesis of ampicillin with immobilized penicillin G acylase

“…The rate of hydrolysis of ampicillin also decreased when PGME was present, but the inhibitory effects were very different in each case: the inhibitory effect of PGME on hydrolysis of ampicillin was much higher than that of ampicillin on hydrolysis of PGME. Consequently, productivity increased for higher concentrations of PGME, but the results of Ferreira et al (2000) on the selectivity of the reaction were not conclusive. The catalyst used in this work had the enzyme immobilized on agarose gel.…”

“…In all assays, initial PGME concentration was 50mM, based on the conclusions of Ferreira et al (2000): high concentrations of PGME inhibited the hydrolysis of ampicillin, but decreased the selectivity of the reaction. PGME concentration was not changed in the experimental planning because the most important factor in selectivity is the 6-APA/PGME ratio, which was modified when the initial 6-APA concentration was changed.…”

“…Kasche (1986) reported that the antibiotic synthesis requires adsorption of 6-APA on the enzyme active site before the substrate-enzyme complex is formed. Ferreira et al (2000) studied the side reactions that compete with the enzymatic synthesis of ampicillin and concluded that the rate of hydrolysis of PGME decreased when this reaction was carried out in the presence of ampicillin. The rate of hydrolysis of ampicillin also decreased when PGME was present, but the inhibitory effects were very different in each case: the inhibitory effect of PGME on hydrolysis of ampicillin was much higher than that of ampicillin on hydrolysis of PGME.…”

Variational calculus (optimal control) applied to the optimization of the enzymatic synthesis of ampicillin

“…Kinetic models Ferreira et al(2000) presented a semi-empirical kinetic model to the enzymatic synthesis of ampicillin. The reaction rates proposed by the authors are:…”

“…The kinetic parameters has been fitted by Ferreira et al, 2000, at pH 6.5 and temperature at 25ºC. Thus, the reaction rates to each component are:…”