Optimization Medium of CMP-sialic Acid Synthase Fermented by Streptococcus agalactiae
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摘要: 为获得低成本高酶活的产CMP-唾液酸合成酶(Cytidine 5'-monophosphate N-acetylneuraminic acid synthetase,NmCSS)培养基,对无乳链球菌(Streptococcus agalactiae)产CMP-唾液酸合成酶发酵生产培养基进行优化。通过单因素试验筛选发酵温度、碳源、氮源、pH,确定对NmCSS酶活具有明显影响的因子及水平值;采用响应面法(Box-Behnken)确定以氮源浓度、碳源浓度、pH为三个主要影响因子的最适条件。结果表明,当培养温度为34℃时,蔗糖浓度为0.10%,氮源浓度为2.50%,pH为7.5、Na2HPO4 2.5 g/L、NaCl 5.0 g/L时,NmCSS酶活为(5.895±0.005)×107 U/mol,较基础发酵培养基的酶活(0.507±0.002)×107 U/mol提高了10.627倍,显示出了良好的优化效果。响应面方法优化得到的低成本高酶活培养基为无乳链球菌产NmCSS及其应用奠定了基础。
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关键词:
- 无乳链球菌 /
- CMP-唾液酸合成酶 /
- 响应面设计 /
- 培养基优化
Abstract: To optimize the fermentation medium of CMP-sialic acid synthase(NmCSS)fermented by Streptococcus agalactiae with low cost and high enzyme activity,the ferment temperature,carbon source,nitrogen source and pH were selected by single factor experiment to determine the main factors and level values which had significant influence on the enzyme activity of NmCSS. Box-Behnken response surface design was used to determine the optimal concentration for nitrogen source,carbon source and pH of medium. The results showed that the enzyme activity of NmCSS could be up to(5.895±0.005)×107 U/mol when the temperature was 34 ℃,sucrose concentration was 0.10%,nitrogen source concentration was 2.50%,pH values was 7.5,Na2HPO4 was 2.5 g/L and NaCl was 5.0 g/L. After fermentation verification,the enzyme activity of NmCSS was 10.627 times higher than the enzyme activity of(0.507±0.002)×107 U/mol under basic fermentation medium conditions. The low cost and high enzyme activity medium were optimized by response surface method,which laid a foundation for the production and application of NmCSS from Streptococcus agalactiae.
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