鲈鱼脯氨酰内肽酶的分离纯化、性质分析及分子克隆

doi:10.7506/spkx1002-6630-20200501-004

食品科学 ›› 2021, Vol. 42 ›› Issue (14): 78-85.doi: 10.7506/spkx1002-6630-20200501-004

鲈鱼脯氨酰内肽酶的分离纯化、性质分析及分子克隆

杨汝晴，陈守峰，肖琳琳，陈玉磊，孙乐常，张凌晶，刘光明，曹敏杰

（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）

发布日期:2021-07-27
基金资助:
“十三五”国家重点研发计划重点专项（2018YFD0901004）；国家自然科学基金面上项目（31772049）

Prolyl Endopeptidase from Sea Bass (Lateolabrax japonicus): Purification, Characterization and Molecular Cloning

YANG Ruqing, CHEN Shoufeng, XIAO Linlin, CHEN Yulei, SUN Lechang, ZHANG Lingjing, LIU Guangming, CAO Minjie

(1. College of Food and Biological Engineering, Jimei University, Xiamen 361021, China; 2. Collaborative Innovation Center of Marine Food Deep Processing, Dalian 116034, China)

Published:2021-07-27

摘要/Abstract

摘要： 采用(NH4)2SO4盐析和连续柱层析法从鲈鱼（Lateolabrax japonicus）骨骼肌中分离纯化了一种脯氨酰内肽酶（prolyl endopeptidase，PEP）。通过液相色谱-串联质谱分析，得到43 个与红鳍东方鲀的PEP高度一致的肽片段，证实该酶为PEP。以琥珀酰-甘氨酰-脯氨酸-4-甲基-7-香豆素为底物，确定PEP的最适pH 6.0和最适温度35 ℃。温度变化对PEP活力的影响较大，通过圆二色谱检测显示加热对PEP的二级结构有较为明显的影响，且热变性不可逆。通过分子克隆技术获得PEP全长cDNA开放阅读框含2 226 个核苷酸，编码741 个氨基酸残基，预测分子质量为84.12 kDa。通过同源建模得到鲈鱼PEP的分子结构，PEP为典型的α/β水解酶折叠排列，其催化三联体（His-711、Asp-672、Ser-585）被β-折叠形成的螺旋桨区域的中心通道所覆盖。催化活性中心空间构象稳定对维持PEP活力至关重要，而其独特的分子结构是底物特异性的基础。

关键词: 鲈鱼；脯氨酰内肽酶；纯化；性质分析；圆二色谱；同源建模

Abstract: A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of sea bass (Lateolabrax japonicus) by ammonium sulfate fractionation followed by column chromatography. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), 43 peptide fragments were obtained highly identical to the sequence of prolyl endopeptidase from Takifugu rubripes, confirming the purified protein to be a prolyl endopeptidase. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 ℃, respectively. Circular dichroism (CD) spectroscopy showed that heating had a significant effect on the secondary structures of PEP, causing irreversible denaturation. The full-length cDNA sequence of PEP was cloned, which contained an open reading frame of 2 226 nucleotides, encoding a protein of 741 amino acid residues with a deduced molecular mass of 84.12 kDa. The molecular structure of PEP was obtained by homologous modeling. PEP exhibited a typical α/β hydrolase folding arrangement. Its catalytic triplets (His-711, Asp-672, and Ser-585) were covered with the central channel of the propeller region formed by β-folding. The stability of the catalytic domain of PEP could be essential for maintaining its activity and its unique structure could be the basis for its substrate specificity.

Key words: sea bass; prolyl endopeptidase; purification; characterization; circular dichroism; homology modeling

中图分类号:

Q556.3

杨汝晴，陈守峰，肖琳琳，陈玉磊，孙乐常，张凌晶，刘光明，曹敏杰. 鲈鱼脯氨酰内肽酶的分离纯化、性质分析及分子克隆[J]. 食品科学, 2021, 42(14): 78-85.

YANG Ruqing, CHEN Shoufeng, XIAO Linlin, CHEN Yulei, SUN Lechang, ZHANG Lingjing, LIU Guangming, CAO Minjie. Prolyl Endopeptidase from Sea Bass (Lateolabrax japonicus): Purification, Characterization and Molecular Cloning[J]. FOOD SCIENCE, 2021, 42(14): 78-85.

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鲈鱼脯氨酰内肽酶的分离纯化、性质分析及分子克隆

Prolyl Endopeptidase from Sea Bass (Lateolabrax japonicus): Purification, Characterization and Molecular Cloning

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