青鱼胰蛋白酶的分离纯化及部分性质研究

Study on the Isolation and Purification of Trypsinase from Mylopharyngodon piceus and Its Properties

[目的]为青鱼的养殖和综合利用提供试验依据。[方法]对青鱼胰蛋白酶进行分离纯化,测定青鱼胰蛋白酶的分子量、热稳定性和酸碱稳定性,探讨pH值、温度、金属离子和EDTA对青鱼胰蛋白酶活力的影响。[结果]经SDS-PAGE和凝胶过滤,测得青鱼胰蛋白酶的分子量分别为44和43.5 kD。青鱼胰蛋白酶具有一定碱性耐受性,在pH值为8～10时活性较高,其最适pH值约为9,最适反应温度为50℃。以酪蛋白为底物,在pH值8.5、37℃时测得该酶Km值为4.6×103 mg/L。Mg2+对该酶有明显促进作用,而Cu2+、Mn2+、Pb2+和EDTA对青鱼胰蛋白酶的抑制作用很大。[结论]EDTA能抑制青鱼胰蛋白酶的作用,说明该酶可能为金属蛋白酶。

[Objective] The aim of the research was to provide the experimental basis for the culture and the comprehensive utilization of Mylopharyngodon piceus. [Method] Trypsinase was isolated and purified from M. piceus to determine the molecular weight, the heat stability and pH stability of trypsinase from M. piceus. And the effects of pH value, temperature, metal ions and EDTA on the activity of trypsinase from M. piceus were discussed. [Result] The molecular weight of trypsinase from M. piceus was determined as 44 and 43.5 kD resp. through SDS-PAGE and gel filtration. Trypsinase from M. piceus had certain alkalinetoleranee and its activity was higher at pH value of 8-10. The optimum pH value and the optimum reaction temperature were 9 and 50 ℃ resp.Under the conditions of taking casein as substrate with pH value of 8.5 at 37 ℃, Km value of this enzyme was determined as 4.6× 10^3 mg/L. Mg^2＋ had an obvious promotion to this enzyme, while Cu^2＋, Mn^2＋, Pb^2＋ and EDTA had greater inhibition on trypsinase from M. piceus.[Conclusion] EDTA could inhibit the action of trypsinase from M. piceus, which indicated that this enzyme was probably metalloproteinase.