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

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 microm, 250 mm x 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.

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

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 degrees C overnight. The crude extract was run on reversed-phase HPLC system with a Diamonsil C18 column (250 mm x 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. 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-PAA-Asn-Tyr-Asn-Gln (PAA represented beta-amino acid), which was consistent with lipopeptide iturin A. Compound 2 was a homologue of iturin A.

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC.

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.