title: Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples.
abstract: We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ (max)=390 nm) is converted to a red product (λ (max)=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A (390)/A (486) ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L(-1) and 50 μg L(-1) to 10 mg L(-1), respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
authors.forename: Xida
authors.initials: X
authors.lastname: Dai
authors.forename: Sihai
authors.initials: S
authors.lastname: Xiang
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gao
authors.forename: Keqian
authors.initials: K
authors.lastname: Yang
mesh_terms: D002511:Cephalosporins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019818:Clavulanic Acid / Q000032:analysis* / Q000494:pharmacology; D003124:Colorimetry / Q000379:methods*; D017077:Culture Media, Conditioned / Q000737:chemistry*; D009154:Mutation; D015203:Reproducibility of Results; D013302:Streptomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D017077:Culture Media, Conditioned; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D001618:beta-Lactamases; C021720:nitrocefin