高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物

Isolation and identification of lipopeptides produced by Bacillus subtilis using high performance liquid chromatography and electrospray ionization mass spectrometry

枯草芽孢杆菌JA因产生多种脂肽类化合物而具有广阔的开发前景。JA发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类化合物的粗提物。将粗提物溶于流动相,采用反相高效液相色谱分离,对收集的洗脱峰组分进行电喷雾质谱(ESI-MS)分析。根据质荷比推断JA菌株产生的脂肽类化合物属于3个家族,分别为surfactin,iturin和fengycin,是枯草芽孢杆菌合成的重要生物表面活性素。对一级质谱中的主成分进行串联质谱分析,进一步确定了3种脂肽类化合物的分子结构。实验证明ESI-MS是一种鉴定脂肽类化合物及其同系物的可靠方法。

Bacillus subtilis JA produces a broad spectrum of bioactive lipopeptides with great potential applications in agriculture and industry. Crude lipopeptides were extracted with 100% methanol from the precipitate which was obtained by adding 6 mol/L HCl to the cell-free culture broth. The crude extract was separated on a Diamonsil C18 column （5 μm, 250 mm×4.6 mm） in reversed-phase high performance liquid chromatography （HPLC） system to separate the lipopeptide homologues. Five peaks were eluted from HPLC. Electrospray ionization mass spectrometry （ESI-MS） was used to analyze each HPLC fraction. The results showed three series of ion peaks. According to the values of m/z, the three series of ions were classified into surfactin, iturin and fengycin homologues, which were well-known biosurfactants produced by B. subtilis strains. The major ions were structurally characterized using tandem mass spectrometry. This study suggested a reasonable method for the isolation and identification of lipopeptides produced by B. subtilis strains.