枯草芽孢杆菌菌株BAB-1表面活性素的分离纯化及性质分析

Purification and characterization of surfactin produced by Bacillus subtilis strain BAB-1

枯草芽孢杆菌菌株BAB-1对番茄灰霉病菌有较强的抑制作用,为了明确其抑菌活性相关成分,通过盐酸沉淀、甲醇抽提法,从该菌株发酵上清液中分离得到了一种脂肽类化合物。经HPLC分析纯化后,发现其保留时间与表面活性素标准品基本一致。TLC层析结果表明该菌株能够产生与表面活性素标准品Rf值一致的成分。进一步通过电喷雾质谱分析,得到其[M+H]+值分别为m/z 1 009.3、1 023.4、1 037.4的成分,与已知的表面活性素3种同系物的[M+H]+值一致。最后采用PCR扩增特异性片段sfp的方法证实该菌株有产生表面活性素的同源基因。综合分析表明,枯草芽孢杆菌菌株BAB-1能够产生表面活性素,且提纯后的表面活性素具有明显的溶血和排油活性,并通过显微观察确定了其十字晶体状结构。

Bacillus subtilis strain BAB-1 exhibited an effective inhibitory activity against Botrytis cinerea which caused tomato gray mold.To clarify the chemical nature of the antagonistic compounds,lipopeptides were isolated from the cultural supernatant of strain BAB-1 by the methods of hydrochloric acid precipitation and methanol extraction.The lipopeptide compound showed a similar retention time to the standard sample of surfactin in HPLC and TLC analyses.Furthermore,ESI/MS analysis of the purified surfactant revealed 3 [M＋H]^＋ ion peaks at m/z 1 009.3,m/z 1 023.4 and m/z 1 037.4 respectively,which were in agreement with the 3 homologous compounds of standard surfactin.Specific PCR amplification of the sfp gene fragment confirmed the existence of the surfactin biosynthetic genes in strain BAB-1.In addition,the purified surfactin had hemolytic and oil expansion activities,and its crystals appeared a cruciate structure under light microscope.