Laboratório de Fisiologia E Bioquímica de Microrganismos, Centro de Biociências E Biotecnologia, Universidade Estadual Do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego, 2000, Campos dos Goytacazes, RJ, Brazil.
Laboratório de Bioquímica Marinha, Departamento de Engenharia de Pesca, Universidade Federal Do Ceará, Fortaleza, CE, Brazil.
Probiotics Antimicrob Proteins. 2021 Jun;13(3):862-872. doi: 10.1007/s12602-020-09739-3. Epub 2021 Jan 17.
Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.
抗菌肽 (AMPs) 存在于多种生命形式中，对致病微生物具有广谱抑制活性，是对抗多药耐药病原体的有前途的替代品。本工作的目的是从辣椒果实中鉴定和表征 AMPs，并评估它们对属念珠菌的酵母和α-淀粉酶的抑制活性。最初，从果实中提取蛋白质后，将提取物进行阴离子交换层析，得到两个馏分。馏分 D1 进一步通过分子筛层析进行分级，得到三个馏分。这些馏分显示出低分子量的肽，并且在馏分 F3 中仅观察到两个约 6.5 kDa 的蛋白质条带。通过质谱分析，我们鉴定出馏分 F3 中最低分子量的蛋白质条带与植物防御素家族的 AMPs 具有相似性。我们将该肽命名为 CcDef3（辣椒 defensin 3）。这些馏分的抗真菌活性针对属念珠菌的酵母进行了分析。在 200μg/mL 时，馏分 F1 抑制 C. tropicalis 的生长 26%，馏分 F2 抑制 C. buinensis 的生长 35%，馏分 F3 抑制所有测试的酵母，对 C. albicans（86%）的生长抑制活性更大随后是 C. buinensis（69%）和 C. tropicalis（21%）。馏分 F1 和 F2 促进了所有测试酵母的细胞膜通透性，并分别增加了 C. buinensis 和 C. tropicalis 中内源性活性氧 (ROS) 的诱导。我们还观察到，浓度为 50μg/mL 的馏分 F3 抑制了 10 龄黄粉虫幼虫和人唾液的α-淀粉酶活性 96%和 100%。因此，我们的结果表明，含有 CcDef3 的馏分 F3 是一种非常有前途的蛋白质馏分，因为它具有抗真菌潜力，并且能够抑制不同的α-淀粉酶酶的活性。
