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采用甲苯自溶法从鲜酵母中提取了蔗糖酶,并用乙醇分级及DEAE-纤维素柱层析进行了纯化,用PAG凝胶电泳作了纯度鉴定,在pH5.0,30℃条件下进行了酶反应,用双倒数作图法测出其Km=2.1×10-2mol/L,Vmax=0.26(每分钟的光密度值).在此系统中,加入不同浓度的抗坏血酸(Vit.C),发现其具有激活作用并存在量效关系.双倒数作图显示:酶的表观Vmax(Vp)随抗坏血酸浓度的增加而增大,但其表观Km(Kp)不变(Kp=Km).经实验结果分析,推论出抗坏血酸激活作用的酶促反应方程式,并推导出反应速度公式
Sucrase was extracted from fresh yeast by toluene autolysis method and purified by ethanol fractionation and DEAE-cellulose column chromatography. The purity was determined by PAG gel electrophoresis. The purity was determined at pH 5.0 and 30 ℃ Enzyme reaction, measured by double reciprocal mapping Km = 2.1 × 10-2mol / L, Vmax = 0.26 (optical density value per minute). In this system, adding different concentrations of ascorbic acid (Vit.C), found that it has an activation and there is a dose-effect relationship. The double reciprocal plot shows that the apparent Vmax (Vp) of the enzyme increases with increasing ascorbic acid concentration, but its apparent Km (Kp) does not change (Kp = Km). Through the analysis of experimental results, deduced the enzymatic reaction equation of ascorbic acid activation and deduced the formula of reaction rate