一种猪溶菌酶来源的抗菌六肽的分离鉴定及其性质  

Purification, identification and characterization of an anti-microbial hexapeptide from Sus scrofa lysozyme

作　　者：朱德伟[1,2,3] 蔡国林[1,2,3] 陆健[1,2,3] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2017年第6期1046-1056,共11页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2013CB733602);中央高校基本科研业务费专项资金资助(No.JUSRP51302A);江苏高校优势学科建设工程项目资助~~

摘　　要：为提高猪溶菌酶(Sus scrofa lysozyme,SSL)的抗革兰氏阴性菌活性,将其进行了不同蛋白酶的水解,选择抗革兰氏阴性菌效果最好的水解产物,利用凝胶过滤色谱和反相制备色谱进行分离,对其功能成分进行液质联用鉴定。对分离得到的物质进行抗菌活性验证和生物信息学的分析,并在此基础上对抗菌物质的杀菌机理进行了探讨。结果表明,胰蛋白酶的水解产物具有较高的杀灭革兰氏阴性菌的活性,进一步分离纯化得到了具有抗革兰氏阴性菌活性的六肽A-W-V-A-W-K。经化学合成验证,该六肽既保留了SSL的部分抗菌活性,也具备杀灭多种革兰氏阴性菌的能力。进一步分析发现其位于SSL分子C端的一个螺旋-回环-螺旋的结构中,并由此推测其杀菌机理是通过改变细胞膜的渗透性,进而使细胞内溶物流出而造成细胞死亡,而抗菌实验也验证了这一推测。该抗菌肽的发现为后续提高SSL的抗菌活性提供了理论依据。Sus scrofa lysozyme （SSL） was digested by different proteases to find peptides with enhanced antibacterial activity against gram-negative bacteria. Hydrolysate with the highest anti-bacterial activity was loaded onto a gel filtration chromatography column followed by a reversed-phase one. The obtained substance was identified by？liquid chromatography-mass spectrometry, synthesized to test its antibacterial spectrum and analyzed for bioinformatics. The hydrolysate of trypsin showed the highest antibacterial activity. By purification and identification, the functional peptide with sequence of A-W-V-A-W-K was obtained. The peptide was synthesized and proved to retain partial function of SSL and had activity against gram-negative bacteria. By bioinformatics analysis, the peptide was found to locate in a helix-loop-helix structure, suggesting that the peptide may kill cells by penetrating cell membrane and cause the outflow of cell contents. The discovery of the peptide could lay the foundation for improving the antibacterial activity of SSL.

关 键 词：猪溶菌酶 大肠杆菌 抗菌肽 抗生素 膜渗透性 

分 类 号：Q51[生物学—生物化学]