枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

Purification and identification of iturin A from Bacillus subtilis JA by electrospray ionization mass spectrometry

摘要:

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性，明确抗生素的种类是进一步研究的基础。用6mol/L盐酸沉淀JA菌株的去菌体培养基，再用甲醇抽提获得抗生素的粗提物。利用反相HPLC系统，将粗提物过Diamonsil C18柱，收集有抗小麦赤霉病等病原真菌活性的化合物1、2。运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D。再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片，结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln (βAA为14个碳原子的氨基脂肪酸)，属于脂肽类抗生素iturin A。化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物。研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法。

Abstract:

Lipopeptides produced by Bacillus subtilis JA antagonized a broad spectrum of plant fungal pathogens. The purification and identification of the lipopeptide antibiotics plays an important role for further research. Crude lipopeptides were extracted with methanol from the precipitate, which was obtained by adding 6mol/L HCl to the cell-free culture broth and then stored at 4°C overnight. The crude extract was run on reversed-phase HPLC system with a Diamonsil C18 column (250 mm × 4.6 mm, Dikma) to separate the lipopeptides. Two antifungal compounds, which had strong inhibitory activity against various plant fungal pathogens, such as Fusarium graminearum, were purified. The molecular weights of two compounds were determined by electrospray ionization mass spectrometry (ESI/MS). Two compounds, with molecular weights of 1042.4 Da and 1056.5 Da, were homologues differed by a structure of –CH2 (CH2 = 14 Da). ESI collision induced dissociation mass spectrometry analysis was used to sequence the structure of purified compounds. Typical b- and y- type fragments showed that compound 1 (with a molecular weight of 1042.4 Da) had a primary structure of Pro – Asn – Tyr – βAA – Asn – Tyr – Asn – Gln (βAA represented β-amino acid), which was consistent with lipopeptide iturin A. Compound 2 was a homologue of iturin A.