Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10⁻⁹ mol·L⁻¹. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 μmol·L⁻¹, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Inga cylindrica, a tropical fruit tree of the Fabaceae family (subfamily Mimosoideae), is native to South America. The seeds from this family are an essential source of trypsin inhibitors, which display promising bioactivity for increasing host defense against pathogens. The aim of the present study was to characterize the antimicrobial and antibiofilm activities of the trypsin inhibitor extracted from I. cylindrica seeds, ICTI. ICTI demonstrated antifungal activity with a minimum inhibitory concentration (MIC) of 32.11 μmol.L^-1, and a minimum fungicidal concentration (MFC) of 32.1 μmol.L^-1, against Cryptococcus gattii, Candida albicans, Candida glabrata and Candida guilliermondii. Combining ICTI with Amphotericin B had a significant synergistic effect, reducing the concentration of the antibiotic by 75% for C. albicans and 94% for C. gatti. The significant increase (16 x) in activity observed with ergosterol (1.01 mol.L^-1) for C. albicans and C. gatti, and the lack of activity against bacterial strains, suggests that ICTI interferes with the integrity of the fungal cell membrane. Treatment with ICTI at 10 x MIC resulted in a 51% reduction in biofilm formation and a 56% decrease in mature biofilm colonies for C. albicans. Finally, ICTI displayed no toxicity in the in vivo Galleria mellonella model. Given its antifungal and antibiofilm properties, ICTI could be a promising candidate for the development of new antimicrobial drug prototypes.