摘要
本试验测定了pH、温度和EDTA对纯化的绵羊的瘤胃微生物胞外蛋白酶活性的影响,并用一已知氨基酸排列顺序的肽段(由15个氨基酸组成)作为底物,进一步测定了蛋白酶的酶切位点。结果表明,(1)纯化蛋白酶的最适pH在6.0～6.5之间,最适温度为40℃左右。(2)不同浓度的EDTA(浓度分别为1,10,25,50,75和100mM)对蛋白酶活性没有影响,因而该酶属于丝氨酸蛋白酶类。(3)纯化蛋白酶是一种内肽酶,其酶切位点在组氨酸—酪氨酸(?)、天冬氨酸—丙氨酸(?)、亮氨酸—赖氨酸(?)和(或)缬氨酸—赖氨酸(?)相连的肽键。(4)胰蛋白酶和纯化的蛋白酶虽同属丝氨酸蛋白酶类,但前者对底物具有高度的专一性,后者对底物的专一性不强。
The effects of pH, temperature, and EDTA on the activity of the purified extracellular protease from sheep ruminal microorganisms were tested. Further study was also carried out to determine the protease cleavage site by using a polypeptide chain of known linear sequence as substrate(consist of 15 amino acids).Results showed that: (1)the optimal pH for the protease was between 6.0 and 6.5,and the optimal temperature was at about 40℃.(2)the activity of the pro- tease was not affected by various concentrations of EDTA(1, 10, 25, 50, 75 and 100mM, respec- tively) at optimal conditions thus the purified protease belonged to the serine protease type.(3)the purified protease was an endopeptidase, and had the exact cleavage sites on His-Tyr,Asp-Ala Leu-Lys. and/or Val-Lys of the substrate. (4)one of the main reasons that proteins were degraded exstensively in the rumen was associated with many cleavage sites of the extracellular pro- tease from microorganisms.
