大孔树脂吸附法高通量快速提取微生物次生代谢产物
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贵州省科学技术基金项目(黔科合J字[2008]2088号)资助


Rapid and High Throughput Extraction of Secondary Metabolites from Microorganisms Using Macro-Porous Adsorption Resin
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    摘要:

    为建立一种适合药物高通量筛选的快速提取微生物代谢产物中小分子化合物的方法,采用弱极性AB-8和中等极性ADS-17大孔吸附树脂从细菌发酵液吸附小分子代谢产物,并用50%乙醇溶液将吸附的小分子化合物洗脱下来,干燥后用二甲基亚砜溶解备用;应用流感病毒神经氨酸酶作为抗流感病毒药物筛选的靶标,MUNANA作为底物,测定发酵液中活性物质对神经氨酸酶的抑制活性。优化了神经氨酸酶抑制试验条件,底物500倍稀释、病毒用量1 μL、pH 6.0为最佳反应条件。证实了大孔吸附树脂吸附法纯化的微生物发酵液中小分子化合物粗提物具有不同程度的流感病毒神经氨酸酶抑制活性。与直接使用发酵液及用有机溶剂乙酸乙酯提取法比较,用大孔吸附树脂吸附法制备的细菌代谢产物的抗流感病毒神经氨酸酶活性明显好于直接使用发酵液。该方法与乙酸乙酯提取法效果相当,但制备更简便,能实现高通量筛选。

    Abstract:

    Macro-porous adsorption resins,AB-8 and ADS-17,were used to extract small chemical molecules from bacterial fermentation liquid and the absorbed small molecules were eluted with 50% ethanol.The neuraminidase inhibition activities of the extracts were determined with a sensitive fluorometric assay using the substrate sodium (2′-O-4-methylumbelliferyl N-acetylneuraminic acid,MUNANA),the reaction was carried out at pH 6.0 and in the presence of 1.0 μL of viral solution.The results showed that resin purification method is much better than using fermentation liquid directly.The small molecules can be greatly enriched and the amount of fermentation liquid required in resin purification method is much less than that of organic reagent extraction.Extracts of resin-purification method is equivalent to ethyl acetate extraction in activity against neuraminidase of influenza virus H1N1.But the cost of time and reagent is much less,indicating that the resin purification method is suitable for high throughput preparation of microbial metabolites.

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王俊波,曹文涛.大孔树脂吸附法高通量快速提取微生物次生代谢产物[J].华中农业大学学报,2010,29(6):732-736

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  • 收稿日期:2010-03-08
  • 最后修改日期:2010-07-16
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