Abstract
Antimicrobial peptides (AMPs) are a diverse class of bioactive molecules that exert their effects through multiple mechanisms, including membrane disruption, inhibition of cell wall synthesis, interference with intracellular targets, and binding to nucleic acids or proteins. They exhibit broad-spectrum antimicrobial activity and a low propensity to induce drug resistance, positioning them as promising candidates for combating multidrug-resistant infections. However, their clinical translation faces significant challenges, including protease susceptibility, potential cytotoxicity, high production costs, and suboptimal pharmacokinetic properties. In this study, a strain of Bacillus licheniformis SAM-D318 isolated from Hu sheep intestine was used as the peptide-producing strain; genomic analysis confirmed the absence of classical virulence toxin genes despite intrinsic multidrug resistance. A previously unreported cationic α-helical antimicrobial peptide, SAM-KB40 (4176.92 Da, pI 10.886), was obtained from the fermentation supernatant of B. licheniformis SAM-D318 and purified using cation-exchange chromatography and C18 reversed-phase chromatography, achieving a 70.45-fold increase in specific activity. Structural prediction by PEP-FOLD4 suggested an α-helical propensity in the C-terminal region, while circular dichroism spectroscopy indicated that SAM-KB40 adopts a predominantly random coil conformation in aqueous solution. The peptide exhibited potent antibacterial activity against Clostridium perfringens (MIC = 0.5 µg/mL, MBC = 4 µg/mL) and showed selective inhibition against Staphylococcus aureus and Listeria monocytogenes. SAM-KB40 demonstrated exceptional thermal stability (> 97% activity retained at 100 °C for 60 min), good tolerance to acidic conditions (pH 3-4) and bile salts (up to 0.50%), and remarkably low hemolytic activity (1.96% at 1000 µg/mL). Notably, the peptide neutralized recombinant C. perfringens α-toxin at 16 µg/mL in a Vero cell-based assay and exhibited no cytotoxicity at concentrations up to 128 µg/mL, demonstrating a favorable in vitro safety profile.Sequence homology analysis against UniProt, NCBI, APD, and BACTIBASE databases confirmed that SAM-KB40 has not been previously reported. While the peptide shows promising in vitro dual antibacterial and toxin-neutralizing capacity, its high sensitivity to proteinase K (39.78% residual activity) limits oral application. These findings position SAM-KB40 as a promising lead candidate for further development as a feed additive or therapeutic agent against C. perfringens-associated diseases, pending subsequent in vivo validation.