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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 \u00d7 10\u22129 mol\u00b7L\u22121. 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 \u00b0C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 \u03bcmol\u00b7L\u22121, 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.<\/jats:p>","DOI":"10.3390\/molecules29163777","type":"journal-article","created":{"date-parts":[[2024,8,10]],"date-time":"2024-08-10T06:25:24Z","timestamp":1723271124000},"page":"3777","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts"],"prefix":"10.3390","volume":"29","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-8791-2304","authenticated-orcid":false,"given":"Matheus M.","family":"da Silva","sequence":"first","affiliation":[{"name":"Laborat\u00f3rio de Purifica\u00e7\u00e3o de Prote\u00ednas e suas Fun\u00e7\u00f5es Biol\u00f3gicas, Faculdade de Ci\u00eancias Farmac\u00eauticas, Alimentos e Nutri\u00e7\u00e3o, Universidade Federal de Mato Grosso do Sul, Campo Grande 79070-900, MS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9549-4848","authenticated-orcid":false,"given":"Caio F. 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