皱纹盘鲍脯氨酰内肽酶的分离纯化及性质  

Purification and characterization of a prolyl endopeptidase from Haliotis discus hannai

作　　者：陈守峰 杨汝晴 陈玉磊 孙乐常[1,2] 张凌晶 刘光明[1,2] 曹敏杰 CHEN Shoufeng;YANG Ruqing;CHEN Yulei;SUN Lechang;ZHANG Lingjing;LIU Guangming;CAO Minjie(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;Collaborative Innovation Center of Marine Food Deep Processing,Dalian 116034,China)

机构地区：[1]集美大学海洋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：《水产学报》2022年第12期2432-2442,共11页Journal of Fisheries of China

基　　金：国家重点研发计划(2018YFD0901004);国家自然科学基金(31772049);国家现代农业产业技术体系专项(CARS-49)。

摘　　要：为探究脯氨酰内肽酶(PEP)在皱纹盘鲍性腺发育过程中的作用,实验采用荧光定量PCR(qRT-PCR)技术分析皱纹盘鲍不同组织中PEP的表达情况。通过硫酸铵分级沉淀和连续柱层析,从皱纹盘鲍性腺中分离纯化得到高纯度天然PEP。利用液相色谱串联质谱法(LC-MS/MS)鉴定肽片段序列,以Suc-Gly-Pro-MCA为底物分析PEP的酶学性质。利用圆二色谱分析温度对PEP结构的影响;采用免疫印迹(Western blot)和qRT-PCR分析鲍性腺在不同发育时期PEP的蛋白和基因表达水平。结果显示,皱纹盘鲍的性腺中,PEP基因表达量和酶活性最高。利用硫酸铵分级沉淀和连续柱层析,从性腺中分离纯化得到分子量约为82 ku,等电点约为5.5的PEP,LC-MS/MS分析得到502个氨基酸残基,与NCBI中皱纹盘鲍脯氨酰内肽酶(KY214290)一致。PEP的最适温度和最适pH分别为25℃和6.0,在15~25℃和pH 5~8条件下能够保持较高的活性。随着温度升高,PEP的二级结构有明显的改变,拟合热变性温度为(51.4±0.2)℃。PEP在雄性和雌性性腺的各个生长期均能被检出,在雄性性腺的成熟中期和雌性性腺的成熟后期表达量最高。研究表明,PEP在鲍性腺不同发育时期表达量的差异表明其可能参与性腺发育的过程。Prolyl endopeptidase(PEP)is a serine protease which has been implicated in many biological processes,such as learning and memory,cell proliferation and differentiation,and glucose metabolism.A small number of reports have also suggested PEP participates in mammalian reproductive process by specifically degrading peptide hormones and gonadal hormones containing proline residues.Compared with mammals,research on PEP of molluscs is relatively scarce at home and abroad.This observation led us to investigate PEP and whether it is related to reproductive development of mollusk.We analyzed the natural PEP of Haliotis discus hannai and the protein expression level changes during gonad development.Fluorescence quantitative PCR(qRT-PCR)and enzyme activity analysis were used to detect the expression of the PEP in tissues of H.discus hannai.The enzyme activity and the expression level of PEP were the highest in H.discus hannai gonad,followed by the muscle.PEP was purified from the gonad by column chromatography with a molecular weight of 82 ku.The isoelectric point of PEP was determined to be 5.5 by two dimensional electrophoresis,and it was similar to the isoelectric point of predicted PEP of abalone.The 182 peptides were obtained by liquid chromatography tandem mass spectrometry(LCMS/MS)analysis,which were consistent with the H.discus hannai prolyl endopeptidase of NCBI(KY214290).The analysis of enzymatic properties showed that the optimum temperature and pH of PEP were 25℃and 6.0,respectively,and high enzyme activity can be maintained at 15-25℃and pH 5-8.Circular dichroism was used to analyze the effect of temperature on structure of PEP.The results showed that the secondary structure of PEP changed significantly as the temperature increased.When the temperature increases from 25℃to 95℃,the content ofα-helix structure decreases from 12.5%to 5.8%,and the random coil increases from 24.4%to 28.0%.When the temperature was lowered from 95℃to its optimum temperature(25℃),the content ofα-helical structure decreased

关 键 词：皱纹盘鲍 脯氨酰内肽酶 纯化 性质分析 性腺发育 

分 类 号：Q786[生物学—分子生物学] TS254.4[轻工技术与工程—水产品加工及贮藏工程]