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.
Highlights
The fermentation titer of a metabolite is a key factor for its successful production
We recently reported a double-reporter guided mutant selection method to generate Clavulanic acid (CA) over-producing mutants of S. clavuligerus [2]
The cell suspension was sonicated on ice (4×15 s at 30-s intervals) and centrifuged to obtain the cell-free extract, which was used as the crude enzyme for the nitrocefin assay
Summary
The fermentation titer of a metabolite is a key factor for its successful production. A bioassay-HPLC method based on -lactamase-catalyzed hydrolysis of penicillin G was developed to measure the levels of CA in culture broth of S. clavuligerus [4,5]. Another improved -lactamase inhibition-based method for CA detection [6] involved the use of a chromogenic substrate to replace the colorless penicillin G. We report a similar colorimetric, -lactamase inhibition-based assay developed to detect CA in S. clavuligerus culture broth. The absorption of both substrate (A390) and product (A486) is measured, and the A390/A486 ratio is used to calculate the CA concentration These improvements greatly increase the CA detection window, compared with those of previously reported assays. The assay can be readily adapted for use in a microplate format to screen a large number of samples
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