香菇酸性蛋白酶纯化及其动力学

PURIFICATION AND KINETIC STUDY OF ACID PROTEASE FROM Lentinus edodes

香菇酸性蛋白酶在 pH 3.5的条件下提取最佳,提取液经 DEAE-Sephadex A50和Sephadex G75层析可以得到纯酸性蛋白酶,用 SDS 凝胶电泳和 Sephadex G200凝胶过滤法可测得该酶是由分子量为40 000道尔顿的单亚基所组成。Mn^(2+)对该酶具有正协同性激活作用,Hill 系数为2.62,其活性中心与天门冬氨酸残基有关,自由酶的 pKa 为3.1,与底物结合后转移到2.5。S-PI 对该酶表现出可逆的抛物线型竞争抑制,它和酶有2个以上的结合位点。

Acid protease was extracted optimally at pH 3.5 and purified enzyme was ob- tained through DEAD-Sephadex A50 and Sephadex G75 chromatography.This enzyme was composed of a single polypeptid chain with a molecular weight of 40 kDa as determined by sodium dodecyl sulfate polyacrylamid gel electrophoresis and Sephadex G200 filter chro- matography.Mn^(2+) can activate the enzyme in positive synergistic way and Hill coefficient is 2.62.Its active center must be correlative to one or more aspartic acid residues. The pKa of free enzyme is 3.1 and moves to 2.5 after binding to substrate.Pepsin in- hibitor S-PI can rapidly inhibit the activity of enzyme in reversible parabolic competitive inhi- bition model that means S-PI binds to more than one active sites of enzyme.