Structural Characterization of Lipopeptides Isolated from Bacillus Globigii Spores

摘要

Several species of Bacillus spores produce surface-active antibiotic and antifungal lipopeptides, phospholipids, and polypeptides. Some of these are detected as biomarkers when intact spores are analyzed by Matrix Assisted Laser Desorption/Ionization time-of-flight (MALDI-TOF) mass spectrometry. Bacillus globigil spores, grown in new sporulation media (NSM), were suspended and then analyzed using a MALDI-TOF mass spectrometer to screen for biomarkers with 4- methoxycinnamic acid as matrix. Spores, vegetative cells, and the culture supernatant show a biomarker cluster composed of six molecules and centered at a molecular mass of 1478 Da. The biomarker was isolated from the cell-free culture supernatant by solid phase extraction to yield a crude product. Its components were concentrated, separated by reverse-phase high-pressure liquid chromatography, and then eluted as a cluster of three peaks. The three peaks were collected separately and preliminary analysis was conducted with a MALDI- TOF. A Fourier Transform Mass Spectrometer (MALDI source) instrument was used to determine the monoisotopic protonated masses to be 1463.8,1477.8, and 1505.8 Da, respectively to their order of elution. The mass differences of 14 and 28 Da, suggest that they are homologous molecules. Acid hydrolysis of each molecule showed it to contain an ester linkage. Methyl esterification, using methanolic HCl, of the open form of the molecule at m/z 1477.8 resulted in a product 56 Da higher, indicating the presence of four carboxylic acid moieties. A quadripole- time-of-flight instrument with nanospray source (Q-star) was used to sequence the reaction products. The purified compounds have identical amino acid sequences to previously identified lipopeptides in Bacillus subtilis.