罗非鱼脯氨酸内肽酶的分离及抑制剂对其的作用机制-《集美大学学报（自然科学版）》

文章信息/Info

Title:: Purification of a Prolyl Endopeptidase from the Skeletal Muscle of Tilapia(Oreochromis spp)and Its Inhibitory Mechanism

作者:: 肖琳琳1; 翁凌1; 2; 钟婵1; 刘光明1; 2; 曹敏杰1; 2; （1.集美大学食品与生物工程学院,福建厦门 361021；2.水产品深加工技术国家地方联合工程研究中心，福建厦门 361021）

Author(s):: XIAO Linlin1; WENG Ling1; 2; ZHONG Chan1; LIU Guangming1; 2; CAO Minjie1; 2; (1.College of Food and Biological Engineering,Jimei University,Xiamen 361021,China；2.National andLocal Joint Engineering Research Center of Processing Technology for Aquatic Products,Xiamen 361021,China)

Keywords:: tilapia; prolyl endopeptidase(PEP); purification; dynamic analysis

摘要:: 采用硫酸铵盐析及柱层析技术，从罗非鱼肌肉中分离纯化得到分子质量约为85 ku的脯氨酸内肽酶（prolyl endopeptidase,PEP)。通过肽质量指纹质谱分析，获得13个肽片段，含128个氨基酸残基，结果显示，与伯氏朴丽鱼（Haplochromis burtoni）的PEP完全一致。该酶特异分解荧光底物Suc-Gly-Pro-〖JP〗MCA和Suc-Gly-Pro-Leu-Gly-Pro-MCA，PEP特异性抑制剂SUAM-14746和丝氨酸蛋白酶抑制剂PMSF可以抑制该酶的活性。PEP催化Suc-Gly-Pro-MCA水解反应的活化能（Ea）为47.42 kJ/mol。SUAM-14746对PEP表现为竞争性抑制作用，抑制常数（KI）为1.91 μmol/L。金属离子Zn2+和Cu2+对PEP的抑制类型均为混合型抑制，其中对游离酶的抑制常数（KI）分别为1.80 mmol/L和0.07 mmol/L，对酶－底物络合物的抑制常数（KIS）分别为2.33 mmol/L和1.17 mmol/L。

Abstract:: A PEP was purified from the skeletal muscle of tilapia(Oreochromis spp)by ammonium sulfate fractionation and a series of column chromatographies.PEP was in homogeneity with molecular weight of approximately 85 ku on SDS-PAGE.Peptide mass fingerprinting(PMF)of PEP obtained 13 fragments including 128 amino acid residues which was identical to a PEP from Haplochromis burtoni,suggesting this enzyme is a prolyl endopeptidase.The enzyme specifically hydrolyzed fluorescent substrates Suc-Gly-Pro-MCA and Suc-Gly-Pro-Leu-Gly-Pro-MCA.Using Suc-Gly-Pro-MCA as substrate,the effect of proteinase inhibitors on PEP was investigated.The results showed that SUAM-14746,a specific inhibitor of prolyl endopeptidase could strongly inhibit its activity while other proteinase inhibitors did not show any effects. Activation energy(Ea)of the enzyme was 47.42 kJ/mol.SUAM-14746 competitively inhibited its activity with KI of 1.91 μmol/L.Metal ions Zn2+and Cu2+ both revealed mixed inhibition to PEP,and their inhibition constants(KI)were 1.80 mmol/L and 0.07 mmol/L,while enzyme substrate complexes(KIS)were 2.33 mmol/L and 1.17 mmol/L,respectively.