Key Laboratory of Geriatric Nutrition and Health of Ministry of Education, Beijing Technology and Business University, Beijing 100048, China; Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China.
Key Laboratory of Agro-Products Primary Processing, Academy of Agricultural Planning and Engineering, Ministry of Agriculture and Rural Affairs, Beijing 100125, China.
Int J Biol Macromol. 2023 Sep 1;248:125932. doi: 10.1016/j.ijbiomac.2023.125932. Epub 2023 Jul 21.
The present study aimed to purify and characterize a novel low-molecular-weight antimicrobial peptide (AMP) named as PNMGL2 produced by Lactiplantibacillus plantarum NMGL2. The AMP was effectively separated and purified by ethyl acetate extraction and DEAE-Sepharose anion exchange chromatography. Tricine-SDS-PAGE of the purified AMP showed a major protein band below 1.7 kDa, which was identified by MALDI-TOF MS to be a hexapeptide LNFLKK (761.95 Da), and structurally characterized to be combination of helixes and random coil by a PEP-FOLD 3 De novo approach. The antimicrobial activity of LNFLKK was confirmed by chemical synthesis of the peptide that showed clear inhibition (MIC 7.8 mg/mL) against both Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes), and Gram-negative bacteria (Enterobacter sakazakii, Escherichia coli and Shigella flexneri). PNMGL2 was pH resistant (pH 2-9), heat stable (121 °C, 30 min), and protease sensitive. Treatment of UV rays, sodium chloride and organic solvents did not decrease the activity. Sequencing of the whole genome of L. plantarum NMGL2 revealed presence of a bacteriocin gene cluster with two putative bacteriocin genes (ORF4 and ORF5) that were not expressed, confirming the significance of PNMGL2 contributing the antimicrobial activity of the strain. This study demonstrated the low-molecular-weight AMP that was uncharacterized in the relevant available databases, suggesting its potential application as a novel natural food preservative.
本研究旨在纯化和鉴定一种新型的低分子量抗菌肽（AMP），命名为 PNMGL2，由植物乳杆菌 NMGL2 产生。该 AMP 通过乙酸乙酯萃取和 DEAE-Sepharose 阴离子交换层析有效分离和纯化。纯化 AMP 的 Tricine-SDS-PAGE 显示出一条主要的蛋白带，分子量低于 1.7 kDa，通过 MALDI-TOF MS 鉴定为六肽 LNFLKK（761.95 Da），通过 PEP-FOLD 3 De novo 方法确定其结构为螺旋和无规卷曲的组合。通过化学合成肽证实了 LNFLKK 的抗菌活性，该肽显示出对革兰氏阳性菌（金黄色葡萄球菌和单核细胞增生李斯特菌）和革兰氏阴性菌（阪崎肠杆菌、大肠杆菌和福氏志贺菌）的明显抑制作用（MIC 7.8 mg/mL）。PNMGL2 对 pH 具有抗性（pH 2-9），热稳定（121°C，30 min），且对蛋白酶敏感。紫外线、氯化钠和有机溶剂处理不会降低其活性。植物乳杆菌 NMGL2 的全基因组测序显示存在一个细菌素基因簇，其中包含两个假定的细菌素基因（ORF4 和 ORF5），它们没有表达，证实了 PNMGL2 对该菌株的抗菌活性有重要贡献。本研究表明，该低分子量 AMP 在相关可用数据库中未被描述，表明其作为新型天然食品防腐剂具有潜在应用价值。
