鼠伤寒沙门菌超氧化物歧化酶SodA基因的表达及其酶活性的测定

Expression of Salmonella typhimurium SodA gene and determination of activities of the recombinant SodA

为了了解鼠伤寒沙门菌超氧化物歧化酶SodA的生物学功能,本试验经过克隆后表达、纯化而获得了重组SodA蛋白,克隆的SodA基因的序列长度为621 bp,重组SodA蛋白的分子质量为23.7 ku。通过邻苯三酚自氧化法测定了SodA酶比活性。测定结果显示,SodA的最适温度为40℃,最适pH为7.0;Mn^(2+)、Zn^(2+)、Cu^(2+)可以提升SodA酶活性,SDS、氯仿-乙醇、Fe^(3+)能抑制SodA催化效率,H_2O_2、Mg^(2+)、Ca^(2+)、Ba^(2+)、Na^+、K^+对蛋白活性无影响;SodA催化邻苯三酚的νmax为2.03 U/min,κm为0.801 mmol/L。过氧化氢酶CAT对SodA活性的影响研究结果显示,在CAT存在条件下,SodA的耐酸碱、抗乙醇能力得到增强,温度稳定性无明显变化,说明CAT可以增强SodA活性。上述研究结果为深入研究鼠伤寒沙门菌超氧化物歧化酶SodA的功能奠定了基础。

To investigate the biological functions of superoxide dismutase SodA of Salmonella typhimurium,superoxide dismutase SodA gene was cloned from Salmonella typhimurium and expressed in E.coli.The length of the SodA gene was 621 bp,and the molecular weight of the protein was 23.7 ku according to SDS-PAGE.Optimal temperature and pH for the enzymatic activities of SodA were 40 ℃ and 7.0,respectively.The enzymatic activity of SodA was elevated by Mn^（2＋）,Zn^（2＋）and Cu^（2＋）,and inhibited by SDS, chloroformethanol,and Fe^（3＋）,but not affected by the addition of H_2O_2,Mg^（2＋）,Ca^（2＋）,Ba^（2＋）,Na＋and K＋.The kinetic parameters νmaxand κmin pyrogallol were 2.03 U/min and 0.801 mmol/L,respectively.Moreover,the capacity of SodA were strengthened in acid and alkali-resistance and ethanol-tolerance in the presence of the catalase CAT.The above-mentioned results provided a solid basis for researches concerning the functions of SodA in Salmonella typhimurium in future.