枯草芽孢杆菌CF-3抑菌蛋白的分离与鉴定

Purification and Identification of Fungistatic Protein from Bacillus subtilis CF-3

本研究采用硫酸铵分级盐析、交联葡聚糖凝胶分子层析（Sephadex G-75）、聚丙烯酰胺凝胶电泳（SDS-PAGE）等方法对生防菌枯草芽孢杆菌CF-3无菌发酵液对复端孢霉F中的主要抑菌蛋白进行了分离与鉴定。结果表明：CF-3无菌发酵液中抑菌蛋白的硫酸铵最佳沉淀饱和度为60%~70%,蛋白质沉淀量为149.03μg/mL,极显著高于其他浓度（p≤0.01）;利用60%~70%饱和度的硫酸铵提取的粗蛋白质经Sephadex G-75凝胶柱层析后获得2个吸收峰,8管收集液,其中第2管峰收集液抑菌活性显著高于其他管（p≤0.05）;将流分2收集液进一步分离纯化后,将抑菌效果最好的2.3号流分的两条蛋白条带割胶回收,利用生物质谱技术鉴定为γ-谷氨酰转肽酶和胞内丝氨酸蛋白酶,分子量分别约为42.5 ku和33.9 ku。以上结果可为该菌株及其抑菌蛋白的开发应用奠定基础。

Ammonium sulfate precipitation, Sephadex G-75 gel column chromatography, and sodium dodecylsulfate polyacrylamide gel electrophoresis（SDS-PAGE） were used to purify and identify the primary fungistatic proteins in Cephalothecium fungi that act on the biocontrol agent Bacillus subtilis CF-3. The results demonstrated that the active protein could be optimally precipitated with 60~70% ammonium sulfate with a content of 149.03 μg/mL, which was significantly higher than the yield obtained from other ammonium sulfate concentrations（p≤0.01）. Crude proteins separated by ammonium sulfate at a saturation of 60~70% were further purified by Sephadex G-75 gel column chromatography, yielding two absorption peaks and eight fractions. The second fraction had a significantly greater fungistatic effect than the other fractions（p≤0.05） and was further purified. Band 2.3 of the flowthrough had the highest fungistatic activity and was cut from the gel for collection. Biological mass spectrometry was used to identify the bands as g-glutamyl transpeptidase and intracellular serine protease, with molecular masses of 42.5 ku and 33.9 ku, respectively. These results laid the foundation for the application of this bacterial strain and these fungistatic proteins.