枯草芽孢杆菌B25抗真菌作用及抗菌蛋白的分离纯化

Studies on Antifungal Activity and Purification of Antifungal Substance from Bacillus subtilis B25 Strain

枯草芽孢杆菌B25是对香蕉枯萎病镰刀菌有较好拮抗作用的一株生防菌。采用70%饱和(NH_4)_2SO_4沉淀法获得的B25的发酵液粗提物作用于病原菌,能使菌丝或孢子肿大、扭曲或畸形。粗提物质通过DEAE-Sepharose fast flow离子交换层析与Sephadex G-100凝胶层析进行分离纯化,经SDS-PAGE电泳检测得到了单一的条带,再通过Nano LC-ESI-MS/MS质谱分析,获得了17个肽段的氨基酸序列。NCBI数据库检索,与之序列相似性最高的仅为37%,可能是一种新的抗真菌蛋白。

Bacillus subtilis B25 was confirmed to have stronger antagonism against Fusarium oxysporum f. sp.cubense. The antagonistic substance could be extracted from cell-free culture broth supernatants by 70%（w/v）（NH_4）_2SO_4 saturation. The examination of antagonistic mechanism under light microscope showed that the antifungal protein of B25 appeared to inhibit pathogens by leading to mycelium and spores tumescence,distortion,abnormality. Coarse extraction material purificated further by ion exchange chromatography on DEAE-Sephadex fast flow and gel filtration chromatography on Sephadex G-100. The purified material showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. The active fraction was identified by Nano LC-ESI-MS/MS. The amino acid sequences of 17 peptides segments were obtained. The result of sequence alignment showed that the similarity was 37%,and it might be an unknown protein through indexing from the NCBI data base.