白玉菇酸性磷酸酯酶的分离纯化及酶学性质研究

Separation and Purification of Acid Phosphatese and Enzymic Properties of White Hypsizygus marmoreus

[目的]对白玉菇酸性磷酸酯酶进行分离纯化,并研究其酶学性质。[方法]以白玉菇为材料提取酸性磷酸酯酶(ACPase,EC.3.1.3.2),进一步将原酶液盐析、层析2种常用的蛋白纯化技术,分离得到酸性磷酸酯酶,并对该酶的酶学性质进行研究。[结果]该酶分子量约为70 kD,水解对硝基苯磷酸二钠(pNPP)最适pH为4.5,等电点为5.5,最适温度为40℃,该酶在50℃时有热激活效应;紫外吸收光谱测定结果表明,酶有2个吸收峰,分别在220和280 nm处;耐热时间表明在40℃下耐热保温40 min后,酶活力为最大活力的60.79%,并在保温20 min时酶活力最大,50℃时酶活力随着保温时间的延长而降低,保温20 min后,酶活力下降为最初酶活力的20.77%;Hg^(2+)、Pb^(2+)、Ag+、Cd^(2+)4种金属离子对酶活性均有抑制作用,Ag+抑制作用最强。酶的动力学参数Km为0.031 mmol/L、Vmax为0.204 mmol/(L·min)、酶反应初速度为18.66×10^(-3)μmol/L。[结论]酸性磷酸酯酶的活性受温度、pH、金属离子等外界条件的影响,因此在栽培过程中需要控制环境温度及培养基酸碱度等条件。

[Objective]To isolate and purify the acid phosphatase from white Hypsizygus marmoreus and study its enzymatic properties.[Method]The acid phosphatase(ACPase,EC.3.1.3.2)was extracted from white Hypsizygus marmoreus as the material,and the original enzyme solution was further salted out and chromatographed through two commonly used protein purification techniques to isolate the acid phosphatase,and the enzymatic properties of the enzyme were studied.[Result]The molecular mass of the enzyme was 70 kD,the optimum pH of ACPase was 4.5 when pNPP was used as substrate,isoelectric point was 5.5,and the optimum temperature was 40℃,and could be activated at 50℃.The results of UV absorption spectroscopy showed that the enzyme had two absorption peaks at 220 and 280 nm respectively.The heat-resisting time showed that after heat-resisting at 40℃for 40 min,the enzyme activity was 60.79%of the maximum activity,and the enzyme activity was the largest at 20 min.At 50℃,the enzyme activity decreased with the extension of the incubation time.After 20 minutes of incubation,the enzyme activity decreased to 20.77%of the original enzyme activity.The four metal ions of Hg^(2+),Pb^(2+),Ag+,Cd^(2+)had inhibitory effects on enzyme activity,and Ag+had the strongest inhibitory effect.The enzyme kinetic parameters Km was 0.031 mmol/L,Vmax was 0.204 mmol/(L·min),and the initial reaction rate of the enzyme was 18.66×10^(-3)μmol/L.[Conclusion]The activity of acid phosphatase is affected by external conditions such as temperature,pH,metal ions,etc.Therefore,it is necessary to control the environment temperature and the pH of the medium during the cultivation process.