枯草芽胞杆菌NCD-2菌株抑菌蛋白的分离及鉴定

Isolation and identification of an antifungal protein produced by Bacillus subtilis NCD-2

枯草芽胞杆菌NCD-2菌株对灰葡萄孢菌具有较强的抑菌活性,抑菌蛋白是其产生的抑菌物质之一。本研究为明确NCD-2菌株所产抑菌蛋白的作用方式和特性,采用牛津杯法测定抑菌蛋白的抑菌活性及其对病原菌菌丝生长的影响,采用混合培养法测定其对病原菌分生孢子萌发的影响,同时采用阴离子交换层析和非变性聚丙烯酰胺凝胶电泳技术对其进行分离纯化,并利用MALDI-TOF-MS进行鉴定。结果表明,NCD-2菌株产生的粗蛋白能够显著抑制灰葡萄孢菌分生孢子的萌发和菌丝生长,并导致病原菌菌丝分枝增多、局部膨大肿胀。通过分离纯化获得具抑菌活性的蛋白组分D1-3,经鉴定该蛋白的肽指纹图谱与leucyl aminopeptidase[Bacillus subtilis](WP_041057629.1)的氨基酸序列匹配率最高,达到65%,相对分子质量为53.936 k Da,功能分析表明组分D1-3可能是一种新的抑菌蛋白。

Bacillus subtilis NCD-2 exhibits an effective inhibitory activity against mycelial growth of Botrytis cinerea,which caused tomato gray mold. The antifungal protein was one of the major antifungal active compounds produced by this strain. To clarify the action mode and the characteristics of the antifungal protein,the inhibitory activity of the crude protein against mycelial growth and conidial germination of B. cinerea were evaluated with the Oxford-cup test method and the dual culture method,respectively. The anion exchange chromatography and native PAGE were employed for further purification of the protein,and the active fraction of purified proteins against B. cinerea was identified by MALDI-TOF-MS. The crude proteins showed the significant inhibition against mycelial growth and conidial germination of B. cinerea and led to the abnormal growth of the mycelia with ramification and swellings. Among the fractions of the purified proteins,only the fraction D1-3 showed antifungal activity against this pathogen. MALDI-TOF-MS revealed that fraction D1-3 with a relative molecular mass of 53.936 kDa had a high homology（ 65%） with the leucyl aminopeptidase produced by Bacillus subtilis（ WP-041057629.1）. It is suggested that fraction D1-3 is a novel antifungal protein.