重组鲍鱼肌肉脯氨酰内肽酶的性质研究

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，Hdh-PEP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-R-G-G-E-Y/F。酶动力学研究表明，Hdh-PEP的米氏常数K_m为5.32 μmol/L，催化常数k_cat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。

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

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.