海参肠组织蛋白酶D的提取及酶学特性研究

郭晓坤; 李傲婷; 韩佳润; 杜椅楠; 郭天民; 于翠平; 唐越; 吴海涛

doi:10.13386/j.issn1002-0306.2017.18.026

海参肠组织蛋白酶D的提取及酶学特性研究

Extraction and characterization of cathepsin D from sea cucumber (Stichopus japonicus) guts

摘要

摘要: 本研究以海参肠为原料,采用p H3.0 50 mmol/L甘氨酸-盐酸缓冲体系,按1∶6（w/v）的料液比,在4℃浸提1 h,获得海参肠组织蛋白酶D的粗酶液。以酸变性牛血红蛋白为底物,进行酶活测定,并研究了该酶的酶学性质。结果表明,海参肠组织蛋白酶D粗酶的最适p H为3.0,在p H3.0⁷.0之间稳定性较好;最适反应温度为50℃,在4⁴0℃具有较高稳定性。5 mmol/L的金属离子Mg²⁺、Ca²⁺、Ni²⁺、Zn²⁺、Cd²⁺和K⁺对该酶有激活作用,而Fe³⁺和Fe²⁺则对其有抑制作用。天冬氨酸蛋白酶抑制剂Pepstatin A对该酶活性有较强的抑制作用。上述结果说明,在本研究条件下提取获得的海参肠组织蛋白酶D粗酶是一种酸性蛋白酶,其组成以天冬氨酸蛋白酶为主。

Abstract: The crude cathepsin D was obtained from sea cucumber ( Stichopus japonicus) guts in this paper.The crude cathepsin D was extracted by using 50 mmol/L Glycine-HCl buffer ( p H3.0) with material to solvent ratio of 1∶ 6 ( w/v) at 4 ℃ for 1 h.The activity and characterization of the enzyme were investigated by using acid denatured hemoglobin as a substrate.The results showed that the optimum p H of the crude cathepsin D was p H3.0 and was stable between p H3.0 ~ 7.0. The crude cathepsin D exhibited its optimal temperature at 50 ℃ and was stable at 4 ~ 40 ℃. The crude cathepsin D was activated by metal ions of Mg²⁺, Ca²⁺, Ni²⁺, Zn²⁺, Cd²⁺and K⁺, while inhibited by Fe³⁺ and Fe²⁺at concentration of 5 mmol/L.The crude cathepsin D was strongly inhibited by aspartic protease inhibitor pepstatin A. These results suggested that the crude cathepsin D obtained from S.japonicus guts was acid protease maily composed of aspartic protease under the condition of present study.