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

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2226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。

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 chromatographytandem 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-ProMCA 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 2226 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 typicalα/βhydrolase folding arrangement.Its catalytic triplets(His-711,Asp-672,and Ser-585)were covered with the central channel of the propeller region formed byβ-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.