东乡野生稻过氧化物酶分离纯化及其酶学特性研究

Peroxidase Purification and Its Enzymatic Kinetics of Dongxiang Wild Rice

过氧化物酶与植物体内活性氧的清除和逆境适应性反应密切相关,为了深入研究过氧化物酶在植物抗逆反应中的重要作用,以东乡野生稻叶片组织为材料,采用浸提、盐析、透析和柱色谱法分离纯化POD蛋白,采用聚丙烯凝胶活性电泳检测蛋白,利用愈创木酚法研究POD的活性和酶学特性。结果表明,经磷酸缓冲液浸提、40%~80%硫酸铵盐析、再经Sephadex G-100凝胶柱层析等步骤,从东乡野生稻叶片组织分离制备得到POD1同工酶组分,酶蛋白的纯化倍数为16.49,比活力为568.80 U/mg。聚丙烯凝胶活性电泳检测仅呈单一POD1同工酶带,属于单一组分。POD酶Km为1.26 mmol/(L·min),最大反应速度Vmax为17.51 mmol/(L·min),最适宜的pH 5.0,最适宜温度35℃~45℃,Cu^2+和Zn^2+对POD有较强的激活作用,而Fe^3+对POD活性有一定的抑制作用,POD酶活性受20%的甲醇、乙醇和丙酮等有机试剂抑制。

Peroxidase is closely related to the scavenging of reactive oxygen species(ROS)and the adaptive response to stresses in plants.To further explore the role of peroxidase in stress resistance of crops,the leaves of Dongxiang wild rice were used as materials.POD protein was isolated and purified by extraction,salting out,dialysis and column chromatography,the protein was detected by active gel electrophoresis with polyacrylamide gel.The activities and the enzymatic characteristics of POD were studied by the guaiacol method.The results showed that a component of POD1 isozyme was obtained from leaves of Dongxiang wild rice via extraction with phosphate buffer,precipitation by ammonium sulfate of 40%-80% saturation and separation by Sephadex G-100 chromatography.The purification multiple of the protease was 16.49,specific activity was 568.80 U/mg.Polyacrylamide gel electrophoresis(PAGE)test showed a homogeneous component.POD enzyme had Km of 1.26 mmol/(L·min),Vmax of 17.51 mmol/(L·min),the optimum pH value of 5.0 and optimum temperature of 35℃-45℃.Cu^2+ and Zn^2+ had strong activating effects on POD activity,while Fe^3+ had inhibition effect,and POD activity was inhibited by 20% of methanol,ethanol and acetone.