麻疯树过氧化氢酶的分离纯化

Purification and characterization of catalase from Jatropha curcas L.

麻疯树种仁经抽提、硫酸铵盐析、Phenyl Sepharose CL-4B层析、DEAE-Sepharose F.F.层析,获得了纯化的麻疯树过氧化氢酶(CAT).该酶比活力为69.23 U/mg,纯化倍数为37.63倍.经Sephacryl S-300 HR测定,该酶表观分子量为232 kD,SDS-PAGE显示为一条分子量为59 kD的条带,表明该酶是由四个相同的分子量为59 kD的亚基组成.经等电聚焦测定该酶的等电点为4.7.经过温度和pH考察,该酶最适温度为30℃,最适pH为7.0.Mn^(2+)对酶活有促进作用,Zn^(2+)、Cd^(2+)、Mg^(2+)、Cu^(2+)、Fe^(3+)、Co^(2+)有抑制作用.6×10^(-3)mmol/L NaN_3以及0.9 mmol/L KCN能使酶活丧失.该酶的V_(max)和K_m分别为5 U/mL·min,1.5 mmol/L.

Catalase（CAT） was purified from the powder of Jatropha curcas L. kernels using saline extraction, saturation of ammonium sulfate precipitation, Phenyl Sepharose CL-4B chromatography and DEAE-Sepharose F.F. chromatography. The specific activity was 69.23U/mg and the purification fold was 37.63. The molecular weight of the CAT determined by Sephacryl S-300 HR was 232 kDa and the single band examined by SDS-PAGE suggested that the CAT is composed of four subunits of equal size （59 kD）. The isoelectric point is at pH 4.7. Studies on the properties of the CAT showed that the optimum temperature and pH value were 30℃ and 7. 0 respectively. Mn^2＋ enhanced the enzyme activity whereas Zn^2＋ Cd^2＋ Mg^2＋ Cu^2＋ Fe^3＋Co^2＋ inhibited the activity. The NaN3 and KCN with concentration of 6 × 10^-3 mmol/L and 0.9 mmol/L respectively caused the enzyme activity to be lost. Maximun velocity （Vmax） was 5 U/mL· min and michaelis-Menten constant （Km） was 1.5 mmol/L.