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以大菱鲆肠道为材料,对大菱鲆消化生理中起重要作用的肠蛋白酶的分离纯化条件和理化性质进行了研究。经Tris-HCl缓冲液抽提,硫酸铵分级分离,DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-100凝胶过滤层析分离纯化,获得大菱鲆肠蛋白酶Ⅰ的电泳纯制品,并对该酶的性质进行了研究。结果显示:纯酶的分子量约为58kD。纯酶最适反应pH为9.0,最适反应温度50℃;pH稳定范围6.0~11.0,30℃以下酶活性稳定;Mn2+可激活大菱鲆肠蛋白酶Ⅰ,Cu2+、Zn2+、Ag+、Fe3+则对酶活性有抑制作用;巯基蛋白酶抑制剂显著抑制大菱鲆肠蛋白酶Ⅰ的活性,金属蛋白酶抑制剂对大菱鲆肠蛋白酶Ⅰ无影响。50℃、pH9.0条件下以双倒数作图法得大菱鲆肠蛋白酶Ⅰ催化酪蛋白水解的Km值为2.92g.L-1。
The intestine of turbot was used as experimental material to study the conditions of purification and physicochemical properties of intestine protease which plays an important role in the digestive physiology of turbot. Purified by Tris-HCl buffer, ammonium sulfate fractionation, DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephadex G-100 gel filtration chromatography to obtain the pure product of enterotactinase Ⅰ The nature of the enzyme was studied. The results showed that: pure enzyme molecular weight of about 58kD. The optimum reaction pH of pure enzyme was 9.0, and the optimal reaction temperature was 50 ℃. The pH range of stability was 6.0 ~ 11.0, and the enzyme activity was stable at 30 ℃. Enzymes Ⅰ, Cu2 +, Zn2 +, Ag + and Fe3 + activated by turbidity of Mn2 + Activity inhibition; thiol protease inhibitor significantly inhibited turbot the activity of intestinal protease Ⅰ, metalloproteinase inhibitor had no effect on the turbot intestinal proteinase Ⅰ. 50 ℃, pH9.0 under the conditions of double reciprocal mapping turbot catfish enterase Ⅰ catalytic casein hydrolysis Km value of 2.92g.L-1.