圆二色谱法分析金属离子对鲍鱼脯氨酰内肽酶的作用  

Effect of Metal Ions on the Activity of a Prolyl Endopeptidase from Haliotis discus hannai Using Circular Dichroism Spectrum

作　　者：李越 颜龙杰 翁凌 章骞 曹敏杰 LI Yue;YAN Longjie;WENG Ling;ZHANG Qian;CAO Minjie(College of Food and Biological Engineering,Jimei University,Xiamen 361021,China;National & Local Joint Engineering Research Center of Processing Technology for Aquatic Products,Xiamen 361021,China)

机构地区：[1]集美大学食品与生物工程学院,福建厦门361021 [2]水产品深加工技术国家地方联合工程研究中心,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2019年第3期167-173,共7页Journal of Jimei University：Natural Science

基　　金：国家自然科学基金项目(31471640,31701676);农业部贝类产业技术体系项目(CARS-49);福建省科技计划项目(2017N5011)

摘　　要：使用原核表达系统表达了皱纹盘鲍( Haliotis discus hannai )脯氨酰内肽酶(prolyl endopeptidase,PEP),并对其进行高度纯化。SDS-PAGE结果表明,该酶的相对分子质量约为85 ku,其二级结构主要由α-螺旋(28.3%)、反向平行结构(17.4%)、平行结构(8.70%)、β转角(19.0%)、无规卷曲(28.5%)组成。选取PEP特异性荧光底物Suc-Gly-Pro-MCA测定其活性,并选取了多种常见金属盐溶液对其进行抑制作用分析。结果发现,Al^3+、Cu^2+、 Zn^2+、Ag^+和 Pb^2+能够抑制PEP的活性。利用圆二色谱法,对金属离子作用下PEP的二级结构变化进行分析和计算。分析结果表明,不同金属离子对PEP的作用效果不同:Al^3+、Cu^2+和Zn^2+在影响PEP结构时也影响其活性;Ca^2+、Mg^2+对PEP的结构和活性均不产生明显影响,而Ag^+和 Pb^2+可在不影响PEP二级结构的前提下抑制其活性。Purified recombinant PEP from abalone (Haliotis discus hannai) was obtained using a prokaryotic expression system,with a relative molecular mass of 85 ku.The secondary structure of PEP mainly contained of alpha-helix-28.3%,antiparallel-17.4%,parallel-8.70%,beta-turn-19.0% and random coil-28.5%.The measurement of enzyme activity was based on a specific fluorogenic substrate Suc-Gly-Pro-MCA and a variety of common metal salts solution was selected for the inhibition assay.The results showed that the enzyme activity of PEP was strongly inhibited by Al^3+,Cu^2+,Zn^2+,Ag^+ and Pb^2+.The secondary structure of PEP treated with metal ions was analyzed using circular dichroism spectrum.The influence of various metal ions on the PEP activity was different.In comparison,Al^3+,Cu^2+ and Zn^2+ had an impact on both enzyme activity and the secondary structure,while Ca^2+ and Mg^+ did not influence the secondary structure and activity obviously.Ag^+ as well as Pb^2+ revealed a strong inhibition on enzyme activity while less influence on the secondary structure.

关 键 词：脯氨酰内肽酶 皱纹盘鲍 金属离子 圆二色谱 抑制剂 

分 类 号：Q51[生物学—生物化学]