Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures

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.

Comparative Transcriptome Analysis of Streptomyces Clavuligerus in Response to Favorable and Restrictive Nutritional Conditions

“…CA was determined by HPLC Agilent 1200 (Agilent Technologies, Waldbrom, Germany) equipped with a Diode Array Detector (Agilent Technologies, Palo Alto, CA, USA) at 312 nm, using a reverse phase ZORBAX Eclipse XDB-C 18 (4.6 × 150 mm, 18μm Agilent Technologies, Palo Alto, CA, USA) column; 96% v/v KH 2 PO 4 (50 mM, pH 3,2) and a 6% v/v methanol was used as mobile phase at 1 mL/min. CA was imidazole-derivatized at a ratio 1:3; the reaction was kept at 30 °C for 30 min [63]. Phosphate quantification was carried out as molybdivanadophosphoric acid by a colorimetric technique, as described elsewhere [64].…”

Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production

“…Shear conditions in CA production has been previously explored in airlifts [3] and stirred tank reactors [4]; however, the physiological response of S. clavuligerus to shear forces and its relationship with CA secretion is not completely understood. Many efforts have been carried out to increase the yield of CA since not only nutritional, but also environmental and degradation factors compromise the product yield in submerged cultivations of S. clavuligerus [5,6]. Several studies focused on the effect of nutrient concentrations and environmental cultivation conditions on CA biosynthesis, as Ser et al [7] reviewed.…”

Data of clavulanic acid and clavulanate-imidazole stability at low temperatures

“…(4)) with kinetic rate constant k 2 . In this irreversible step, the formed intermediate I* reacts with a CA molecule to form the final degradation product (D), as follows [4], [5], [6], [7]:CA⇆I∗r1=k1true[CAtrue]–k−1true[I∗true]Keq=k1k−1I∗+CA→Dr2=k2true[CAtrue]…”

“…(3). The values of the kinetic rate constants are available in the literature [5]. The ordinary differential equation (ODE) system was solved by using the deterministic method LSODA for stiff and non-stiff ODEs [8].…”