Dissertation > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > General issues

Effect of Urea on Clavulanic Acid Production and a Colorimetric Assay for High Throughput Detection of Clavulanic Acid

Author DaiXiDa
Tutor GaoQiang
School Tianjin University of Science and Technology
Course Fermentation Engineering
Keywords Clavulanic acid Urea Urease Nitrocefin Colorimetric assay Streptomycesclavuligerus
Type Master's thesis
Year 2012
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Clavulanic acid (CA) is a p-lactamase inhibitor produced by Streptomyces clavuligerus. Since urea is a byproduct of CA biosynthesis, we investigated the potential inhibitory effect of urea on CA biosynthesis. Addition of urea inhibited CA production in the wild type, and the production was completely abolished when the concentration of urea reached20mmol/L. To prevent urea hydrolysis, the genes encoding urease were disrupted and the corresponding mutant was analyzed: which accumulated higher concentration of urea, reaching10mmol/L, but this did not inhibit CA production. Therefore, urea itself was excluded as the factor inhibiting CA biosynthesis. So the inhibitory effect of urea on CA production in the wild type may be due to the elevated ammonium concentration or pH resulting from urea hydrolysis by urease. To test these possibilities, ammonium salt were added in the fermentation medium, which showed no inhibitory effect on CA production. Overall, pH increase due to urea hydrolysis was identified as the real reason to inhibit CA production in the wild type strain, it may accelerate CA degradation as reported before. This study laid the foundation for further elucidation of the regulatory mechanisms controlling CA biosynthesis in S. clavuligerus.To facilitate CA over-producing mutants screening, we developed a colorimetric microtiter plate assay for the quantitative measurement of CA titer in culture broths of S. clavuligerus. The assay is based on the reaction that β-lactamase could hydrolyze a yellow substrate nitrocefin (λmax=390nm) into a red product (Xmax=486nm). Since CA can irreversibly inhibit β-lactamase, the level of CA in a sample could be measured by the absorbance ratio of the substrate and the product at their characteristic wavelengths (OD390/OD486). The sensitivity of the assay was determined to be50μg/L and the detection window for CA was from50μg/L to10mg/L. The reliability of the assay was verified by comparing the results with those obtained by HPLC. Eventually, the assay was used to screen a pool of65mutants and the identified over-producing mutants were further evaluated and verified by time-course investigation. Difference in CA titer between two strains can also be qualitatively determined by direct visual detection of the color changes in the assay, which is more convenient than instrument reading. Overall, the microtiter plate assay is quick, reliable and convenient for CA detection, and it is suitable for high throughput screening of CA over-producing strains.

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