金鲳鱼内脏酸性蛋白酶的分离纯化及酶学性质研究

Isolation,purification and enzymatic characterization of acid protease from golden pompano viscera

以金鲳鱼内脏为原料,依次采用p H7.0的磷酸钠缓冲液浸提,0~55%硫酸铵沉淀,Sephadex G-100和Sephadex G-50凝胶过滤法得到纯化的酸性蛋白酶,并研究了其酶学性质。结果表明,纯化酶的分子量为18.5 ku,最适p H为4.0,最适温度为35℃,具有较好的酸稳定性和耐盐能力;以血红蛋白为底物时,该蛋白酶的米氏常数Km为10.13 g/L;胃蛋白酶抑制剂(pepstatin A)对该酶活性有抑制作用,而乙二胺四乙酸(EDTA)、反-环氧丁二酰基-L-亮氨酰胺基(4-胍基)丁烷(E-64)和苯甲基磺酰氟(PMSF)对此酶活性基本无影响,据此推断该蛋白酶是一种天冬氨酸蛋白酶;此酶对罗非鱼鱼皮明胶的酶解能力强于猪胃蛋白酶,与木瓜蛋白酶和碱性蛋白酶的能力相近。

Golden pompano viscera was taken as raw material in the present study.Acid protease was obtained by homogenation of tissue in sodium phosphate buffer（ pH7.0）, precipitation by 0-55% ammonium sulfate, Sephadex G-100 gel and Sephadex G-50 gel.Then the enzymatic characterization was also studied.The experimental results showed that the molecular weight of purification enzyme was 18.5 ku, the optimum pH was 4.0 and the most suitable temperature was 35 ℃ .It had good acid stability and salt tolerance. Using hemoglobin as a substrate, the Km was 10.13 g/L. Pepstatin A inhibited the enzyme, while EDTA, E-64 and PMSF had no influence on the enzyme, which indicated that this enzyme was probably aspartic proteinase. It had the similar hydrolysis ability of gelatin of tilapia fish skin to papain and alkaline protease, and stronger than porcine pepsin.