日本鳗鲡胰蛋白酶的分离纯化及性质分析

通过硫酸铵分级盐析、DEAE-Sepharose阴离子交换柱层析、Phenyl-Sepharose疏水柱层析、Sephacryl S-200凝胶过滤柱层析等方法，从日本鳗鲡（Anguilla japonica）肝胰腺中分离纯化得到胰蛋白酶，SDS-PAGE显示其分子质量为21.5 ku。以Boc-Phe-Ser-Arg-MCA为底物，胰蛋白酶最适温度和最适pH值分别为40 ℃和8.5。动力学实验表明，Km值和kcat值分别为3.1 μmol/L和59.9 s-1。丝氨酸蛋白酶抑制剂Pefabloc SC、PMSF、benzamidine、STI等对其有特异抑制效果。底物特异性结果显示，该酶特异分解Arg和Lys残基的C端。肽质量指纹图谱获得5个片段，共76个氨基酸残基，与基因文库中的日本鳗鲡胰蛋白酶氨基酸序列完全相同。说明本研究所纯化的蛋白质为胰蛋白酶。

Purification and Characterization of a Trypsin from the Hepatopancreas of Japanese Eel(Anguilla japonica)

A trypsin was purified to homogeneity from the hepatopancreas of Japanese eel(Anguilla japonica) by ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose anion-exchange,Phenyl-Sepharose hydrophobic interaction and Sephacryl S-200 gel-filtration.SDS-PAGE revealed that the molecular weight of the trypsin is about 21.5 ku.Using Boc-Phe-Ser-Arg-MCA as substrate,the optimal temperature and pH of trypsin were 40 ℃ and 8.5,respectively.Kinetic parameters of Km and kcat were 3.1 μmol/L and 59.9 s-1,respectively.Serine proteinase inhibitors of Pefabloc SC,PMSF,benzamidine,and STI specifically inhibited the activity of the enzyme.Substrate specificity revealed that it specifically cleaved at the carboxyl sites of Arg and Lys residues.Peptide mass fingerprinting obtained 5 peptide fragments containing 76 amino acid residues which were identical to the sequence of a Japanese eel trypsin reported in the GenBank.All the above results strongly suggested that the purified protein is a trypsin.