PROPRIEDADES ANTIMICROBIANA DO INIBIDOR DE TRIPSINA DE SEMENTES DE Enterolobium gummiferum

Abstract

Plant peptidase inhibitors (PIs) play crucial roles in plant physiological processes. In this study, we isolated and characterized a new trypsin inhibitor from the Kunitz family, from seeds of Enterolobium gummiferum (Fabaceae, Mimosoideae), called EgPI (E. gummiferum peptidase inhibitor). The purification process involves gel filtration chromatography and hydrophobicity, resulting in a molecule with a high degree of purity. EgPI exhibited a single band of ~20 kDa on polyacrylamide electrophoresis (SDS-PAGE). Under reducing conditions, two polypeptide chains were observed, with 15 kDa and 5 kDa. Functional characterization revealed that EgPI presented a molar inhibition stoichiometry in the ratio of 1:1 against bovine trypsin, providing a direct interaction between the reactive site of the inhibitor and the active site of the enzyme. A dissociation constant (Ki) of 8.4x10-9 µmol/L was determined, demonstrating a high layer between EgPI and trypsin. Amino-terminal sequencing of EgPI determined the first 25 amino acid residues, which revealed the homology of EgPI with Kunitz family inhibitors. Circular dichroism analysis provided information about the secondary structure of EgPI under different conditions. Under native conditions, the inhibitor presents a theoretical increase in β-sheets (21.6 %) and disordered structures (45.7 %). Heated thermal stability studies showed that EgPI maintained the conformation necessary for its inhibitory activity up to 70 °C. Its secondary conformation was stable over a wide pH range, detailed between 2 and 10. Regarding its antimicrobial properties, EgPI showed antibiofilm activity against mature fungal biofilms, at concentrations lower than those tested for the commercial drug, amphotericin B. Characterization of EgPI contributes to our understanding of the functional, structural, and biological properties of Kunitz inhibitors. The high layer of EgPI for trypsin allows for future studies with potential applications in various fields of research, including agriculture, antimicrobial therapy, and research in various areas of health. Further investigation into other biological activities of EgPI increases our knowledge of its applications and broadens its scope in biotechnology research.