Laboratório de Purificação de Proteínas e suas Funções Biológicas, Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande 79070-900, MS, Brazil.
Instituto Federal de Mato Grosso, Campus Guarantã do Norte, Guarantã do Norte 78520-000, MT, Brazil.
Molecules. 2024 Aug 9;29(16):3777. doi: 10.3390/molecules29163777.
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 seeds named EgPI ( 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 mol·L. 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, and EgPI reduced 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.
植物肽酶抑制剂在植物防御机制和生理过程中发挥着关键作用。在本研究中，我们从种子中分离并鉴定了一种名为 EgPI（肽酶抑制剂）的 Kunitz 胰蛋白酶抑制剂。纯化过程涉及两步色谱分离，分别使用分子筛和疏水树脂，得到高纯度和高得率的抑制剂。EgPI 在 SDS-PAGE 中呈现出约 20 kDa 的单一条带。在还原条件下，抑制剂表现出两条多肽链，分子量分别为 15 和 5 kDa。功能表征表明，EgPI 对胰蛋白酶的抑制作用呈现 1:1 的抑制计量比，解离常数为 8.4×10 mol·L。EgPI 的氨基末端测序揭示了其与 Kunitz 抑制剂的同源性。圆二色性分析提供了 EgPI 二级结构的信息，显示出典型的 Kunitz 抑制剂特征。稳定性研究表明，EgPI 在 70°C 以下和 pH 值为 2 至 8 的范围内保持其二级结构，从而保持其抑制活性。微生物筛选表明，EgPI 在 1.125 μmol·L 时对致病性酵母具有抗生物膜特性，并且 EgPI 抑制了 82.7%的生物膜形成。EgPI 对胰蛋白酶的高亲和力表明其在各个领域具有潜在的应用价值。此外，其抗生物膜特性表明它在农业和抗菌治疗研究方面的有用性，突显了我们研究的实际意义。
