草鱼肌肉脯氨酸内肽酶的研究  

Study on a Prolyl Endopeptidase from the Skeletal Muscle of Grass Carp(Ctenopharyngodon idellus)

作　　者：翁凌[1,2] 林韵[1] 张玲[1] 张凌晶[1,2] 颜龙杰 刘光明[1,2] 曹敏杰[1,2] 

机构地区：[1]集美大学食品与生物工程学院,福建厦门361021 [2]福建省水产品深加工工程研究中心,福建厦门361021

出　　处：《中国食品学报》2015年第9期23-31,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31071519);十二五国家科技支撑计划(2012BAD38B09)

摘　　要：脯氨酸内肽酶（Prolylendopeptidase，PEP）是一类能够特异性水解低分子质量多肽链中脯氨酸残基羧基端肽键的内切酶，与胶原蛋白的后期降解有密切关系。以淡水鱼草鱼为原料。采用硫酸铵分级沉淀。DEAE—Sephacel阴离子交换，Phenyl—Sepharose疏水层析和羟基磷灰石层析等方法从草鱼肌肉中分离纯化得到一种脯氨酸内肽酶。SDS—PAGE结果显示，草鱼PEP的分子质量为75ku。酶学性质分析表明，其最适pH为6．0，在pH5．5～7．5间有较好的稳定性。酶的最适温度为30℃，其热稳定性较差。PEP能够特异性分解肽链羧基端为脯氨酸残基的荧光底物，PEP的特异性抑制剂SUAM以及丝氨酸蛋白酶抑制剂苯甲基磺酰氟对其有强烈的抑制作用。酶动力学研究表明，草鱼PEP的k为19．23μmol／L，kca为2．5s-1。肽指纹质谱分析结果表明，草鱼PEP与青锵鱼、罗非鱼、斑马鱼PEP的序列同源性分别为98-3％．96．1％和94．4％。Prolyl endopeptidase （PEP） is a kind of endopeptidase which specifically cleaves low molecular weight peptides on the carboxyl side of proline residues, plays an important role in further degradation of collagens. In the pre- sent study, a prolyl endopeptidase was purified to homogeneity from grass carp muscle by ammonium sulfate fractiona- tion, column chromatographies including DEAE-Sephacel, Phenyl-Sepharose and Hydroxyapatite and the biochemicalcharacteristics were investigated. SDS-PAGE showed that the molecular weight of PEP was 75 ku. Optimal pH of the pu- rified enzyme was 6.0, and it was stable at pH between 5.5 to 7.5. The optimal temperature of the enzyme was 30 ℃ while with poor thermal stability. The enzyme activity was strongly inhibited by SUAM, a competitive and specific in- hibitor of prolyl endopeptidases. PEP showed it specifically cleaved fluorogenic substrates having proline residue on the carboxyl sides, strongly suggesting that enzyme is a kind of prolyl endopeptidase. The Km and kcat of the enzyme was 19.23 μmol/L and 2.5 s-1. MALDI-TOF-MS analysis revealed that the sequence of grass carp PEP shared 98.3%, 96.1% and 94.4% identities to PEPs from Oryzias latipes, Oreochromis niloticus and Danio rerio, respectively.

关 键 词：脯氨酸内肽酶 草鱼 纯化 性质分析 

分 类 号：TS201.25[轻工技术与工程—食品科学]