猪血浆半胱氨酸蛋白酶抑制肽的筛选及其机制  

Screening of Pig Plasma Cysteine Protease Inhibitor Peptides and Their Mechanisms

作　　者：王嘉敏 王逐鹿 常思佳 刘汉雄 王震宇 WANG Jiamin;WANG Zhulu;CHANG Sijia;LIU Hanxiong;WANG Zhenyu(School of Food Science and Technology,National Engineering Research Center of Seafood,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学食品学院,国家海洋食品工程技术研究中心,辽宁大连116034

出　　处：《食品安全导刊》2022年第36期43-49,共7页China Food Safety Magazine

基　　金：国家自然科学基金青年项目,鱼糜凝胶形成过程中外源半胱氨酸蛋白酶抑制剂的作用机制研究(31901762)。

摘　　要：本研究对猪血浆中半胱氨酸蛋白酶抑制肽进行了制备筛选,并对其抑制动力学和作用机制进行了研究。通过胰蛋白酶酶解制备的猪血浆多肽中,分子量在0~3 kDa的多肽组分抑制率为35.15%,其抑制活性最高;利用UPLC-Q-TOF联用质谱鉴定,从猪血浆多肽组分中鉴定多肽氨基酸序列,分析得到的多肽氨基酸序列有35条,根据目标蛋白来源和细胞定位,选择其中两条氨基酸序列,并命名其为PP1、PP2,PP1为EAVLGLWGKVNVDEVGGEALGR、长度为22 aa,分子量为2267.2 Da,其蛋白来源为血红蛋白,PP2为VILGAHEEYHLGEGVQEIDVSK,长度为22 aa,分子量为2421.2 Da,其蛋白来源为纤溶酶原;然后将抑制肽PP1、PP2与组织蛋白酶L模拟分子对接,分析可知抑制肽与组织蛋白酶L可以通过活性中心紧密结合,分子间作用力键长范围为1.96104~5.01880?,其中PP1作用位点为H3、H4、H6、H14、N15、GLY20、GLN21和GLY23,组织蛋白酶L的作用位点为H166、H234、H239、H244、H256、H313、LYS117、SER158和GLU159,分子间作用力主要为氢键,极少部分为静电力,推测其主要通过竞争性抑制酶与底物的结合。上述研究结果可为产品加工的改进提供重要的理论依据,也为研究蛋白酶抑制肽的生理功能提供重要的理论参考。In this study,the preparation and screening of cysteine protease inhibitors in pig plasma were carried out,and the inhibition kinetics and mechanism of action were studied.Through the trypsin digestion of pig plasma polypeptides,the inhibition rate of polypeptides with molecular weight range of 0~3 kDa was 35.15%,which was the highest inhibitory activity.The polypeptide amino acid sequences from the components of pig plasma polypeptides were sing UPLC-Q-TOF combined with mass spectrometry to identify 35 polypeptide amino acid sequences were analyzed.According to the target protein source and cell localization,two peptides were selected and named PP1 and PP2.PP1 was EAVLGLWGKVNVDEVGGEALGR,with a length of 22 aa and a molecular weight of 2267.2 Da,and its protein source was hemoglobin.PP2 was VILGAHEEYHLGEGVQEIDVSK,with a length of 22 aa and a molecular weight of 2421.2 Da,and its protein source was fibrinolytic enzyme.Then,the inhibitor peptide PP1 and PP2 were docked with the cathepsin L,and it was found that the inhibitor peptide and the cathepsin L could be tightly bound through the active center,and the intermolecular force bond length range was 1.96104~5.01880Å.The active sites of PP1 were H3,H4,H6,H14,N15,GLY20,GLN21 and GLY23,and the active sites of tissue protease L were H166,H234,H239,H244,H256,H313,LYS117,SER158 and GLU159.The intermolecular force was mainly hydrogen bond,and a very small part was electrostatic force.The results suggested that it mainly inhibited the binding of enzyme and substrate by competitive inhibition.The above research results can provide important theoretical basis for the improvement of product processing,and also provide important theoretical reference for the study of physiological functions of protease inhibitors.

关 键 词：猪血浆蛋白 半胱氨酸蛋白酶抑制肽 分离纯化 分子对接 

分 类 号：TS251.93[轻工技术与工程—农产品加工及贮藏工程]