Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures.

David Gómez-Ríos,Rigoberto Ríos-Estepa,Howard Ramírez-Malule,Peter Neubauer,Stefan Junne

doi:10.3390/antibiotics8010006

David Gómez-Ríos, Rigoberto Ríos-Estepa + Show 3 more

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https://doi.org/10.3390/antibiotics8010006

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Abstract

Clavulanic acid (CA) is a β-lactam antibiotic inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (−80, −20, 4, and 25 °C) and pH 6.8 in chemically-defined fermentation broths is presented. Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours. A reversible-irreversible kinetic model was applied to explain the degradation rate of CA, its dependence on temperature and concentration. Kinetic parameters for the equilibrium and irreversible reactions were calculated and the proposed kinetic model was validated with experimental data of CA degradation ranging 16.3 mg/L to 127.0 mg/L. Degradation of the chromophore CA-imidazole, which is commonly used for quantifications by High Performance Liquid Chromatography, was also studied at 4 °C and 25 °C, showing a rapid rate of degradation according to irreversible first-order kinetics. A hydrolysis reaction mechanism is proposed as the cause of CA-imidazole loss in aqueous solutions.

Highlights

Clavulanic acid (CA) is a soft β-lactam antibiotic with a strong inhibitory effect on β-lactamase enzymes, which confer resistance to bacteria against several known broad-spectrum antibiotics
The kinetics of degradation of CA produced by S. clavuligerus DSM 41826 in a chemical defined medium was satisfactorily represented by two reaction models: one equilibrium reaction for intermediate formation and one irreversible first-order reaction for the degradation product formation
The samples of CA in fermentation broth exhibited a fast decline of concentration during the first 5 h followed by a slower but stable reaction rate in the subsequent hours, which had been observed in previous works

Summary

Introduction

Clavulanic acid (CA) is a soft β-lactam antibiotic with a strong inhibitory effect on β-lactamase enzymes, which confer resistance to bacteria against several known broad-spectrum antibiotics. CA is produced by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus) as secondary metabolite related to the clavams pathway derived from arginine and glycerol metabolism [1,2]. The Streptomyces genus is a typical source of essential antimicrobial compounds; their production is commonly carried out in liquid cultures where expression of secondary metabolite mycelia occurs [3,4]. CA production is usually performed in submerged cultures of S. clavuligerus under aerobic conditions with glycerol as carbon source, maintaining controlled conditions of pH and temperature [5,6,7,8,9]. Previous studies have shown that CA stability increases at slightly acidic pH values [10,11]; several authors found a pH of 6.8 as favorable for obtaining high titers of CA in submerged cultures [5,7,9,12,13,14,15,16].

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