Isolation and partial chemical characterization of an antimicrobial peptide produced by Bacillus subtilis

An endophytic bacterium (Bacillus subtilis EN 63-1) isolated from apple fruit produces an antibiotic which has been purified to homogeneity and partially characterized. It consists of an acidic peptide containing asparagine/aspartate, glutamine/glutamate, serine, glycine, alanine, proline, valine, and leucine in a ratio of 2:3:1:1:1:1:1:4. Based on its infrared spectrum, the peptide antibiotic contains an acyl chain and has a lactone bond in its structure. Its molecular weight, estimated by SDS-polyacrylamide gel electrophoresis, is 1.5 kDa; however, it forms aggregates in excess of 20 kDa. The peptide antibiotic is similar to a number of lipopeptide antibiotics often termed "biosurfactants" and classified as fatty acid containing peptolides. The antibiotic has a broad spectrum of antimicrobial activity against Gram negative bacteria, shows little activity against Gram positive bacteria, and displays some anti-fungal activity. Studies on the effect of the peptide on Agrobacterium vitis cells suggest a bactericidal mode of action rather than a bacteriostatic effect; however, cell death is not associated with cell lysis. Antimicrobial activity is associated with the active growth of the producing organism rather than with the stationary phase. The activity of the peptide antibiotic is stable over a fairly wide range of pH and temperature. Peptide activity begins to decrease at alkaline pH, but is stable at neutral and acidic pH. Complete loss of activity is observed above pH 10. At an acidic pH (pH 3.0) the peptide begins to lose activity above 60°C, demonstrating a complete loss of activity at 100°C; however, at neutral pH the peptide remains stable up to 80°C and a complete loss of activity is not observed at 100°C.Land and Food Systems, Faculty ofGraduat