重组鲍鱼肌肉脯氨酰内肽酶的性质研究  

Study on the Properties of Recombinant Prolyl Endopeptidase from Abalone(Haliotis discus hannai)Muscle

作　　者：李婉玉 李越 翁凌 陈守峰 陈玉磊 刘光明 曹敏杰 LI Wanyu;LI Yue;WENG Ling;CHEN Shoufeng;CHEN Yulei;LIU Guangming;CAO Minjie(College of Food and Biological Engineering,Jimei University,Xiamen 361021,China;Collaborative Innovation Center of Marine Food Deep Processing,Dalian Polytechnic University,Dalian 116034,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]海洋食品深加工协同创新中心,辽宁大连116034

出　　处：《食品工业科技》2021年第7期129-135,共7页Science and Technology of Food Industry

基　　金：国家重点研发计划项目(2018YFD0901004);国家自然科学基金(31772049)。

摘　　要：为研究皱纹盘鲍(Haliotis discus hannai)中脯氨酰内肽酶(Prolyl endopeptidase,Hdh-PEP)的酶学特性与结构特性,利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa,在pH2~6、温度20~60℃条件下,Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明,HdhPEP催化结构域中有三个高度保守的氨基酸序列:Seq 1:K-D-G-T-K/R-I-P、Seq 2:Y-G-Y-G-G-F和Seq 3:I-RG-G-E-Y/F。酶动力学研究表明,Hdh-PEP的米氏常数Km为5.32μmol/L,催化常数kcat值为15.7 s-1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用,丝氨酸蛋白酶抑制(PMSF)对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体,可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。In order to study the enzymatic characteristics and structure of prolyl endopeptidase from Haliotis discus hannai(Hdh-PEP), recombinant PEP was cloned and highly expressed in E.coli. Hdh-PEP with molecular weight of 85 kDa was successfully purified and its surface hydrophobicity was greatly affected by pH at low value(pH2~6) and temperature(20~60 ℃). Amino acid sequence homology analysis showed that there were three highly conserved sequences in the catalytic domain of Hdh-PEP: Seq 1: K-D-G-T-K/R-I-P, Seq 2: Y-G-Y-G-G-F and Seq 3: I-R-G-G-E-Y/F. Kinetic study revealed that the Km and kcat of Hdh-PEP were 5.32 μmol/L and 15.7 s-1, respectively. Specific inhibitors SUAM-14746 and ZPP of PEP had strong inhibition on Hdh-PEP activity, and serine protease inhibitor(PMSF) also exhibited inhibition. A high specific polyclonal antibody toward Hdh-PEP was prepared which could be applied for the detection of native PEP in abalone muscle. The successful in vitro expression of Hdh-PEP and preparation of a specific polyclonal antibody against Hdh-PEP provided an important reference for subsequent investigation on Hdh-PEP.

关 键 词：脯氨酰内肽酶 皱纹盘鲍 表面疏水性 多克隆抗体 

分 类 号：Q556.3[生物学—生物化学]