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通过分步盐析,DEAE-Cellulose-52阴离子交换层析和Sephadex G-100凝胶柱层析,对双孢蘑菇中的多酚氧化酶(PPO)进行了分离纯化,并对在分离纯化过程中蛋白得率及酶活性进行了测定。最终得到纯化倍数为103.2,比活为566.57U·mg-1,蛋白得率为0.47%的酶液。经SDS-PAGE电泳检验,所得到的PPO呈单一蛋白带,分子质量为25.5kD。利用扫描探针显微镜对该酶的分子形态进行了观察,发现双孢蘑菇中的PPO分子呈椭球状,分子高度在8~12nm之间。
The polyphenol oxidase (PPO) from Agaricus bisporus was isolated and purified by fractional salting-out, DEAE-Cellulose-52 anion exchange chromatography and Sephadex G-100 gel column chromatography. Protein yield and enzyme activity were determined. Finally, the purification yield was 103.2, the specific activity was 566.57U · mg-1, the protein yield was 0.47% enzyme solution. After SDS-PAGE electrophoresis, the resulting PPO was a single protein band with a molecular mass of 25.5kD. The molecular morphology of the enzyme was observed by scanning probe microscope and found that the PPO molecules in Agaricus bisporus were ellipsoid with the molecular height of 8 ~ 12nm.