α-螺旋抗菌肽YHX-1的从头设计及抑菌活性研究  

De Novo Design and Antibacterial Activity ofα-Helical Antimicrobial Peptide YHX-1

作　　者：李剑勋 步雨珊 刘银雪 刘伊索 印伯星 周炜 易华西[1,2] LI Jianxun;BU Yushan;LIU Yinxue;LIU Yisuo;YIN Boxing;ZHOU Wei;YI Huaxi(College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;Yangzhou Yangda Kangyuan Dairy Co Ltd, Yangzhou 225000, China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003 [2]扬州市扬大康源乳业有限公司,江苏扬州225000

出　　处：《食品科学技术学报》2022年第3期98-104,111,共8页Journal of Food Science and Technology

基　　金：国家重点研发计划项目(2018YFC1604305);山东省自然科学基金重点项目(No.ZR2020KC009);江苏省“双创人才”项目(JSSCRC2021399)。

摘　　要：抗菌肽因其高效的抗菌活性和独特的抗菌机制,不仅可作为生物防腐剂预防食品加工贮藏中的微生物腐败,还有望缓解抗生素滥用引起的耐药性问题。为得到一种高效安全的新型抗菌肽,以现有抗菌肽数据库为基础,确定优化了新型抗菌肽的序列长度、电荷数、氨基酸组成,并结合生物信息学预测及分析工具,从头设计得到了序列为GKLLSKLLKKLLK的新型抗菌肽YHX-1;通过琼脂扩散法、最小抑菌浓度(MIC)、最小杀菌浓度及杀菌动力学曲线评价了其对革兰氏阳性菌和革兰氏阴性菌的抑菌活性,并进一步测定了YHX-1在盐离子、血浆和微酸性环境中的稳定性、二级结构、溶血活性及细胞毒性。结果表明:YHX-1对6株指示菌均有良好抑菌活性,其中对单增李斯特菌和鼠伤寒沙门氏菌的MIC为4μg/mL,在2 h内对单增李斯特菌的致死率为99.99%。YHX-1在盐离子(200 mmol/L Na^(+)、2 mmol/L Ca^(2+))和10%~25%血浆环境中均表现出一定的敏感性,MIC增至原来的2~8倍,而微酸性环境(pH值5.4~7.4)对YHX-1的MIC不会产生显著影响。圆二色光谱表明,YHX-1在细胞膜模拟环境中呈现典型的α-螺旋构象。溶血活性和细胞毒性实验表明,YHX-1具有较好的细胞选择性。YHX-1不仅抑菌谱广、抑菌活性明显,而且具有良好的安全性,有望作为新型抗菌剂应用于食品防腐保鲜,其从头设计策略旨在为新型抗菌肽的发掘提供参考。Antimicrobial peptides can not only be used as biological preservatives to prevent microbial corruption in food processing and storage,but also be expected to alleviate the problem of drug resistance caused by antibiotic abuse because of its high antibacterial activity and unique antibacterial mechanism.To obtain a novel antimicrobial peptide,the sequence length,charge numbers and amino acid composition of this antimicrobial peptides were determined and optimized based on the existing database of antimicrobial peptides.According to the bioinformatics prediction,analysis tools and rational de novo design,the novel antimicrobial peptide YHX-1 was designed with the sequence of GKLLSKLLKKLLK.The antibacterial activity of YHX-1 against gram-positive and gram-negative bacteria was evaluated by agar diffusion method,minimum inhibitory concentration(MIC),minimum bactericidal concentration and bactericidal kinetic curves.Its stability in salt ion,plasma and slightly acidic environment,secondary structure,hemolytic activity and cytotoxicity were also determined.The results showed that YHX-1 showed good antibacterial activity against six kinds of pathogenic bacteria with 4μg/mL of MIC against Listeria monocytogenes and Salmonella typhimurium,and it could cause a mortality rate of more than 99.99%of Listeria monocytogenes within 2 h.YHX-1 showed a certain sensitivity for salt ions(200 mmol/L Na^(+)or 2 mmol/L Ca^(2+))and 10%-25%plasma which the MIC increased by 2 to 8 times.Furthermore,the MIC of YHX-1 was not significantly affected by slightly acidic environment(pH 5.4-7.4).The results of circular dichroism spectrum showed a typicalα-helix structure of YHX-1.Hemolytic activity and cytotoxicity test showed that YHX-1 had good cell selectivity.The newly designed antibacterial peptide YHX-1 was efficient broad-spectrum bactericidal,highly bactericidal efficient and safe which could be used as a new antibacterial agent for food preservation.The de novo design strategy could also provide a reference for designing antimicrobial

关 键 词：抗菌肽 从头设计 生物信息学 抗菌活性 安全性 

分 类 号：TS201[轻工技术与工程—食品科学] Q939.92[轻工技术与工程—食品科学与工程]