青梅果超氧化物歧化酶粗酶的提取及活性研究

摘要

[目的] 测定青梅果超氧化物歧化酶(SOD)活性,筛选青梅果SOD粗酶提取最有效的方法.[方法] 以重庆綦江青梅果为原料,采取不同料液比提取青梅SOD粗酶,以Sevage法除去杂蛋白,硫酸铵分级沉淀.同时对各步骤所得SOD粗酶活性、蛋白量、比活的检测结果进行比较.[结果] H3PO4缓冲液的用量对SOD粗酶的提取有直接影响.当H3PO4缓冲液与梅果的比例为 3∶ 1时,测得酶的比活最高,为 25.34 U/mg蛋白;粗酶液按3∶ 1体积比加入的氯仿-乙醇混合液除去杂蛋白效果明显;粗酶液添加(NH4)2SO4 至90%饱和度沉淀SOD效果好,比活较提取液上升51.43 U/mg蛋白. [结论] 青梅果SOD粗酶提取的最佳条件:H3PO4缓冲液与青梅比例为 3∶ 1,温度为25～30 ℃,pH值为7.8, Sevage法除去杂蛋白,(NH4)2SO4盐析从35%添加至90%饱和度沉淀SOD.该条件下,总蛋白为215.13 mg,酶总活力为5 415.74 U/mg蛋白,比活为25.34 U/mg蛋白.%[Objective] The research aimed to determine the activity of superoxide dismutase(SOD)in Vatica numgachampoi and screen out the most effective method of extracting crude SOD from V.numgachampoi.[Method] With V.numgachampoi from Qijiang of Chongqing as raw materials, Different material-liquid ratio was used to extract crude SOD from V.numgachampoi.Highly purified samples of SOD were obtained by ammonium sulfate precipitation and Sevage method.The detection results of the activity, protein content and specific activity of crude SOD obtained from different steps were compared.[Result] The dosage of H3PO4 buffer had direct influences on the extraction of crude SOD.When the volume ratio of H3PO4 buffer to V.numgachampoi was 3∶ 1, the specific activity of SOD was highest(25.34 U/mg protein).When the mixed solution of chloroform and ethanol was added to crude enzyme solution at the proportion of 3∶ 1, the protein removal effect was obvious.When the saturation of added (NH4)2SO4 to crude enzyme solution reached 90%, the precipitation effect of SOD was better and the specific activity was increased 51.43 U/mg protein than the extract.[Conclusion] The optimum extraction conditions of crude SOD from V.numgachampoi were as follows: the volume ratio of H3PO4 buffer to V.numgachampoi of 3∶ 1, at 25-30 ℃, pH value of 7.8, removing protein by using Sevage method and the saturation of (NH4)2SO4 for precipitating SOD increased from 35% to 90%.Under these conditions, total protein was 215.13 mg, total enzyme activity was 5 415.74 U/mg protein, the specific activity was 25.34 U/mg protein.