论文部分内容阅读
【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。
【Objective】 Clavulanic acid, a β-lactamase inhibitor produced by Streptomyces clavuligerus, is a byproduct urea produced during its synthesis, and aims to explore the effect of urea on the synthesis of clavulanic acid. 【Method】 The effects of urea on the synthesis of clavulanic acid were investigated by urea and ammonium addition during the fermentation process, and the inhibition of urease activity and pH gradient. 【Result】 The results of urea addition experiment showed that the low concentration of urea reduced the yield of clavulanic acid and completely inhibited the synthesis of clavulanic acid when the concentration of urea was added to 20 mmol / L. Since urease hydrolyzes urea to ammonium ions, resulting in an increase in ammonium ion concentration and pH, the effect of urea on the synthesis of clavulanic acid can be more accurately reflected by blocking the activity of C. albicans urease. The results showed that a large amount of urea accumulated in the urease knockout strain fermentation broth at a concentration as high as 10 mmol / L, but the yield of clavulanic acid did not decrease significantly, indicating that urea could not inhibit the synthesis of clavulanic acid at this concentration. Adding urea reduced the yield of wild clavulanic acid, probably due to the urea being hydrolyzed to ammonium ions or to the pH changes it caused. However, the results of adding ammonium salt in clavulanic acid fermentation broth showed that ammonium ion had no inhibitory effect on the yield of clavulanic acid. In addition, pH gradient experiments confirmed that different pH had a significant impact on clavulanic acid yield. 【Conclusion】 The inhibitory effect of urea and ammonium ion on the synthesis of clavulanic acid was eliminated. It was confirmed that the increase of pH caused by urease hydrolysis was the real reason for the decrease of the yield of wild clavulanic acid with urea addition, which provided a basis for elucidating the mechanism of clavulanic acid synthesis .