【作者】 李豪；

【导师】 车振明；

【作者基本信息】 西华大学 ， 食品科学， 2006， 硕士

【摘要】 超氧化物歧化酶是一种金属酶,它能够使超氧阴离子自由基发生歧化反应,生成氧气和过氧化氢,从而消除超氧阴离子自由基对机体的危害,广泛存在于各种生物体内。目前已从细菌、藻类、霉菌、高等植物、昆虫、鱼类、鸟类和哺乳动物等各种生物体内分离得到,并通过多种方法应用到实际中,如化妆品、医药、食品、人及动植物许多疾病的检测与治疗方面。但是大多是从动物体内提取,因动物体内SOD含量多,活性高,相对来说免疫原性较小;而植物体内相对含量少,活性低。我国养殖业发展迅速,猪血资源丰富,但利用率不高,附加值很低,绝大部分直接排放到环境中,既造成资源的浪费,又污染了环境。猪血超氧化物歧化酶具有非常的活性,性能比较稳定,从猪血中分离纯化超氧化物歧化酶,可以物尽其用,变费为宝,有很高的社会经济效益。基于以上几方面的原因,本文探讨了从猪血中分离纯化SOD的方法并对其稳定性进行了研究。现将研究结果报告如下: 1.分离纯化研究 以新鲜猪血为材料,在低温条件下,高速离心除去血浆后,用0.9%的NaCl溶液洗涤红血球。将干净的红血球中加入0.6%的Triton X-100低温下冻融处理2小时,得到溶血液;用有机溶剂(乙醇-氯仿)处理溶血液,低温下离心除去血红蛋白,收集上清液,得到粗酶液;将粗酶液依次进行磷酸盐萃取、丙酮沉淀和适当的热处理,得到比活力比较高的酶液;再经过DEAE Sephadex A-50柱层析,得到纯化SOD。本课题对热变性法和丙酮沉淀法的最佳工艺参数进行更多还原

【Abstract】 Superoxide dismutase is a kind of metal enzyme that could catalyze the reaction of superoxide anion free radical, and create oxygen and catalyses. Every kind of living creature has superoxide dismutase. At present,superoxide dismutase has been acquired from germs, algae, fungi, plant, insect, fish, birds and animal, and it has been extensively applied to the daily life, such as the cosmetics, medicine, food and the examination of some diseases. Because of the low immunogenic, high content and activity of SOD in animal, it is isolated and purified from animal’s blood. With the development of the livestock breeding,the resource of procine blood is becoming more and more. However, most of the procine blood can’t be used, which waste the resource and pollute the surrounding. SOD is produced from swine blood, which is a good thing that can make good profits. According to these reasons, Superoxide dismutase(SOD), which plays a very inportant role in protecting organisms from oxygen toxicity, was purified from porcine erythrocyte in this paper. The stability of SOD was also studied in this paper. 1 、 Separation and purificationFirst, erythrocyte membrances were disintegrated via freeze-thraw methods in the presence of Triton X-100. Second, porcine eythrocyte coarse CuZn-SOD wasobtained by several steps, which were about removing hemoglobin by ethanol and chloroform firstly, extraction by dipotassium hydrogen phosphate secondly, precipitation by acetone thirdly, heat denaturation fourthly, etc. In the end, through DEAE Sephadex A-50, it was completely purified. Hie yield and the specific activity of SOD after this process were 57% and 5863U/mg, respectively. Purified SOD was testified by PAGE electrophoresis. 2, Stability studyTemperature and pH value are two important factors that affect the activity of. SOD and SOD has a good stability on the condition of them. The enzymatic activity is comparatively stable below 60 "C, for example, the crude SOD solution being kept in 60°C for 30 minute, the enzymatic activity only reduces 24%;however the temperature raised higher than 70 °C, the activity reduces obviously, such as reducing 59% on the condition of 70"C for 30 minute and 74% on the condition of 80V for 30 minutes. The enzymatic activity is stable within pH 5~9;Onee exceeding this range, it reduces deeply. For example, the rest activity is 60% on the condition of pH 4.0 and 58% on the condition of pH 10.0. The activity is nothing on the condition of pH 12.更多还原