Purification and structure determination of antimicrobial peptides from Bacillus subtilis TG26

Two groups of antifungal components, the S1–S5 and the S_TG peptides, were purified from Bacillus subtilis TG26 using a series of chromatographic steps. The peptides were characterized using mass spectra analysis, amino acid analysis, protein sequencing, and two-dimensional nuclear magnetic resonance.; The S1–S5 peptides are found to be closely related in structure. S2 is equal to S1 + CH₂; S3 is S1 + K+; S4 is S1 + 2 CH₂; and S5 is S2 + CH₂ + Na+. The S1–S5 peptides may have a cyclic structure. The S1 and S4 peptides were subjected to amino acid composition analysis, which showed that they have identical composition, with three Glx, one Ala, one Pro, one Ile, two Tyr, and two unknowns, X and Y. X is estimated as an isomer of Thr (Thr*), and Y is proposed to be Orn. However, a complete structure could not be obtained due to their unusual structure.; The S_TG peptide is another active component produced by B. subtilis TG26. The molecular weight of this peptide is 1021 Da. It contains two Asx, two Ser, one Glx, one Thr, one Tyr, and another unusual amino acid, AMX. The structure of AMX was derived from mass data as an unusual amino acid with a long alkyl chain. The S_TG peptide may also have a cyclic structure. The primary structure of the S_TG peptide is shown below:*; The S_TG peptide has amphipathic properties and exhibits strong inhibitory activity against Fusarium graminearum. At high concentrations, it is toxic to mammalian cells. It inhibits the growth of Xanthomonas campestris 97-1 and X. campestris campestris . However, it has almost no activity against Erwinia carotovora carotovora, E. carotovora atroseptica 9, Escherichia coli 102, and Pseudomonas syringae phoseolicola 56. The results of scanning electron microscopy indicate that the peptide may act on the cell membrane by lipid-lipid interactions.; The S1–S5 peptides and the S_TG peptide may be synthesized by multienzyme complexes: peptide synthetases. The possible strategies in cloning the peptide synthetase genes are discussed.; *Please refer to dissertation for diagram.