Bioactivity-Guided Isolation of Putative Bioactive Peptides from Bacillus spizizenii ATCC 6633

The search for new antimicrobial agents has highlighted Bacillus species as important sources of bioactive molecules. In this study, a bioactivity-guided fractionation approach combined with antimicrobial screening and de novo mass spectrometry was used to investigate peptide candidates produced by Bacillus spizizenii ATCC 6633. RP-HPLC fractionation of cell-free supernatants yielded fractions with potent activity against a panel of Gram-negative and Gram-positive bacteria such as Escherichia coli SBS363, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, as well as partial activity against yeast Pichia kudriavzevii IOC 4559. Notably, fraction FC14 exhibited significant antibiofilm activity against P. aeruginosa and over 80% growth inhibition against P. kudriavzevii . Mass spectrometry analysis of active fractions revealed a cluster of molecular features within the 500–1300 Da range, consistent with short peptide molecules. De novo MS/MS sequencing enabled the identification of three primary candidate sequences: FATTL (551.30 Da), KDSmEEY (916.35 Da), and ANKKEEENEGS (1233.55 Da). These peptide candidates showed low similarity to previously annotated antimicrobial peptides in public databases, suggesting they represent novel putative antimicrobial candidates. Overall, this study highlights the utility of bioactivity-guided fractionation coupled with de novo sequencing to identify and characterize specific peptide candidates within complex bacterial secretomes.