Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples |
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Authors: | XiDa Dai SiHai Xiang Jia Li Qiang Gao KeQian Yang |
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Institution: | (1) Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China;(2) State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China; |
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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. |
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