Vol. 9, Special Issue 12, Part H (2025)

Bowman-Birk inhibitors (BBIs) are small, cysteine-rich serine protease inhibitors widespread in legumes, characterized by their remarkable stability and dual inhibitory activity against trypsin and chymotrypsin. In this study, representative BBI sequences were retrieved from UniProtKB for nine legume species, Arachis hypogaea, Arachis duranensis, Cicer arietinum, Cajanuscajan, Glycine max, Vigna radiata, Phaseolus angularis, Pisum sativumand Medicago sativa. Multiple sequence alignment (MSA), domain architecture mapping, phylogenetic analysis, and physicochemical characterization were conducted to investigate structural conservation, reactive loop diversity, and evolutionary adaptation. Results revealed highly conserved cysteine frameworks forming canonical disulfide linkages essential for β-trefoil fold stability, while reactive loop motifs (CTKS/CTRS) exhibited lineage-specific divergence correlating with protease target specificity. Phylogenetic and pairwise identity analyses delineated clear evolutionary clusters corresponding to legume subfamilies. Biochemical property analysis demonstrated that dual-loop inhibitors possess higher molecular weight and moderate stability, whereas single-loop forms are smaller and more stable, reflecting functional specialization. These findings underscore the modular evolution of BBIs from ancestral single-domain to double-headed forms, balancing structural rigidity and adaptive protease inhibition.