解淀粉芽胞杆菌HRH317抗菌蛋白的分离纯化及其抗菌作用

为探究解淀粉芽胞杆菌Bacillus amyloliquefaciens HRH317菌株所产抗菌蛋白物质对禾谷镰孢菌Fusarium graminearum的抑制机理,采用硫酸铵分级沉淀菌株发酵液,DEAE Sepharose Fast Flow柱和Sephadex G-200柱层析分离纯化抗菌蛋白,聚丙烯酰胺凝胶电泳检测抗菌蛋白分子量;并采用平板计数法和扫描电子显微镜观察病原菌孢子萌发和菌丝生长情况。结果表明,硫酸铵饱和度为50%时HRH317菌株发酵液的抑菌活性最大,抑菌圈直径可达2.02 cm,显著大于其它饱和度处理;2次柱层析可纯化出抗菌蛋白,分子量约为36.0 k D。禾谷镰孢菌经该抗菌蛋白处理后12h,病原菌孢子萌发率为55.8%,显著低于对照,且2~12 h间其孢子萌发率平均下降了57.3%;该抗菌蛋白还可造成病原菌菌丝变细、畸形与扭曲,且末端膨大等现象。表明解淀粉芽胞杆菌HRH317菌株产生的抗菌蛋白对禾谷镰孢菌孢子萌发和菌丝发育具有较强的抑制作用。

Isolation, purification and inhibitive effect of antifungal protein of Bacillus amyloliquefaciens HRH317

In order to investigate the inhibitive mechanism of the antifungal protein produced by Bacillus amyloliquefaciens HRH317 against the fungus Fusarium graminearum, the antifungal protein was precipitated fractionally by ammonium sulphate after fermentation, and purified by sequential column chromatography using DEAE Sepharose Fast Flow and Sephadex G-200 as column filling matrix. Molecular weight of the purified protein was determined by SDS-PAGE. The growth of hyphae and germination of spores were observed with scanning electron microscopy and checked by plate counting. The results showed that the protein precipitated by ammonium sulphate of 50% saturation degree exhibited the highest inhibitive activity with an inhibitive circle of 2.02 cm in diameter, which was significantly stronger than proteins harvested at other saturation degrees and later being purified through sequential column chromatography. SDS-PAGE results showed that the molecular weight of the purified antifungal protein was 36.0 kD. The rate of spore germination of pathogens was 55.8% when F. graminearum were treated by antifungal protein for 12 h; the average rate of spore germination was decreased by 57.3%, which was significantly lower than the control in 2-12 h. Hyphae of F. graminearum were caused to be withered, distorted, with swollen ends by the antifungal protein. The results indicated that the antifungal protein produced by B. amyloliquefaciens HRH317 had a high capacity to inhibit the spore germination and hyphal growth of F. graminearum.