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Biotechnology and Applied Biochemistry (2003) 38, (77–85) (Printed in Great Britain)

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Inhibitory effects in the side reactions occurring during the enzymic synthesis of amoxicillin: p-hydroxyphenylglycine methyl ester and amoxicillin hydrolysis

Luciana R. B. Gonçalves*¹, Roberto Fernandez-Lafuente†, Jose M. Guisan†, Raquel L. C. Giordano‡ and Roberto C. Giordano‡

*Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici s/n, Bloco 709, 60455-760, Fortaleza, Ceará, Brazil, †Instituto de Catálisis y Petoleoquímica, CSIC, Madrid, Spain, and ‡Departamento de Engenharia Química, Universidade Federal de São Carlos, Rod. Washington Luiz, km 235, C.P. 676 CEP, São Carlos, SP, Brazil

Key words: antibiotics, penicillin G acylase, kinetic model.

Abbreviations used: 6-APA, 6-aminopenicillanic acid; KCS, kinetically controlled synthesis; PGA, penicillin G acylase; POHPG, p-hydroxyphenylglycine; POHPGME, p-hydroxyphenylglycine methyl ester.

¹To whom correspondence should be addressed (e-mail lrg@ufc.br).

Penicillin G acylase immobilized on glyoxyl-agarose is used to catalyse the reaction between p-hydroxyphenylglycine methyl ester (POHPGME) and 6-aminopenicillanic acid (6-APA). Inhibitory effects affecting the side reactions that occur during the synthesis of amoxicillin have been reported and need to be considered when proposing a kinetic model for the enzymic synthesis. In this work, we present a semi-empirical kinetic model that successively includes different inhibitory effects in the rate equations. The model performance was always compared with experimental data on amoxicillin synthesis. Enzyme load and stirring rate were chosen to prevent diffusional effects. Our results indicate that POHPGME and amoxicillin were competitive inhibitors of the hydrolysis of amoxicillin and POHPGME, respectively. 6-APA was a competitive inhibitor of the hydrolysis of amoxicillin. POHPG was a competitive inhibitor and methanol a non-competitive inhibitor of the hydrolysis of both ester and antibiotic, but the action of methanol was only noticeable at very high concentrations. Adding inhibitory effects to the kinetic model led to a significant increase in the accuracy of the simulations of the overall process of synthesis.

Received 24 January 2003/24 March 2003; accepted 11 April 2003

Published as Immediate Publication 11 April 2003, DOI 10.1042/BA20030016

© 2003 Portland Press Ltd