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

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.