【作者】 谢岩黎；

【导师】 李元瑞；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2003， 硕士

【摘要】 大蒜含有丰富的超氧化物歧化酶（Superoxide Dismutase，SOD），超氧化物歧化酶具有清除超氧自由基（O₂^-·）功能，有效地预防O₂^-·对机体的毒害作用。本研究采用热变性、聚乙二醇6000（PEG）沉淀作用、DEAE-纤维素柱层析三种方法对大蒜细胞溶质中的超氧化物歧化酶进行了分离纯化；利用改良的连苯三酚自氧化法测定了酶活力，同时研究了酶对温度、pH的稳定性；并对酶的类型、在紫外光区的吸收光谱等性质进行了研究。研究结果如下： 1．大蒜细胞溶质中超氧化物歧化酶粗酶液中存在一种干扰酶活性发挥的物质，该物质对热不稳定，而大蒜细胞溶质中超氧化物歧化酶对热较稳定，利用超氧化物歧化酶、干扰物质、杂蛋白的热稳定差异，用热变性法进行初步分离。实验表明在60℃20min进行热变性，钝化了抑制酶活性发挥的干扰物质，并使酶液中总蛋白浓度从24mg／mL下降到15.8mg／mL，得到比活为15.9U／mg的粗酶。 2．聚乙二醇6000（PEG）是一种水溶性非离子聚合物，可使蛋白质发生沉淀作用，并且其对被沉淀的蛋白质有较好的选择性；当溶液中PEG的浓度为16％～26％，上清液中超氧化物歧化酶活力保持不变；当PEG浓度提高至28％时，上清液中酶活力开始下降，至PEG浓度为40％时，上清液中酶活力为零，表明酶液中超氧化物歧化酶从溶液中完全析出，得到了比活为129.3U／mg的酶蛋白。 3．PEG沉淀后的酶蛋白经DEAE-纤维素（DE-52）柱层析，利用pH7.8 0.05mol／L磷酸缓冲液（PB）+1.0mol／L NaCl梯度洗脱，使超氧化物歧化酶蛋白和杂蛋白有效地分开。层析最佳的条件是：流速为1.2mL／min、柱高比为1:10、进样量为6mL；超氧化物歧化酶被完全被洗脱的时间为33min，洗脱体积为40mL，杂蛋白被洗脱下来洗脱时间为66min，体积为80mL，表明DEAE-纤维素（DE-52）柱层析具有较高的柱效和分辨率，得到了比活为3145.5U／mg超氧化物歧化酶蛋白。 4．大蒜细胞溶质中超氧化物歧化酶是一种对热和酸碱度稳定性较好的酶，低于60℃酶活力较为稳定，60℃30min酶活力仅损失19.1％；高于70℃酶活力损失明显，70℃30min酶活力损失56.6％，80℃30min酶活力损失70.8％；pH4～9范围内酶活力保持稳定，pH＜4和pH＞9时，超氧化物歧化酶活力下降明显，pH2.0时剩余酶活力为21％，pH10时，剩余酶活力49.1％，pH=12时，酶活力完全损失。 5．大蒜细胞溶质中超氧化物歧化酶对H₂O₂和氰化物敏感，经鉴定酶的类型是Cu·Zn-SOD；在紫外光区的最大吸收峰是258nm，表明其是含有酪氨酸和色氨酸较少的一类酶蛋白。 6．经过三步分离纯化，从500g大蒜中得到15.6mg产品，比活为3145.5U／mg，纯化倍数为197.8，回收率39.2％。更多还原

【Abstract】 Allium Sativum contains a quantity of Superoxide Dismutase(SOD) having the function of removing the superoxide free radical (O2-).so it can prevent O2- from harming the organism effectively. In this research three methods are used to separate and purify SOD from Alliun Sativum cell plasma , which is thermodenaturation, precipitation of Polyethylene glycol(PEG) and the column chromatography of DEAE-cellulose. Enzymes activity is measured with improved Pyragallol autoxidation, meanwhile the stabilities of enzyme towards temperature and pH, the type of enzyme and absorption spectrum in ultraviolet region are studied. So the results of study are following.1.There is something unknown to interfere SOD activity in the crude extract of Allium Sativum cell plasma. This material is unstable to heat, but SOD is opposite. So the primary method of purification is based on the different heat stability among SOD, interfering material, heteroprotein. When the crude extract is keeping in 60℃ for 20 min ,it can reduce the total protein concentration from 24mg/ml to 15.8mg/ml, and disactivate the interfering material from Allium Sativum cell plasma. The enzyme protein can be obtained which specific activity is 15.9U/mg.2.Polyethylene glycol 6000(PEG) is a kind of water soluble and non-ionic polymer.lt can make protein to precipitate and has a better alternative to precipitation of protein. For example, the concentration of PEG within 16%-28%, the SOD activity of supernatant fluid is steady; while it raises to 28%, the activity begins to reduce; At the point of 40%, the SOD activity of supernatant fluid becomes zero. It indicates that SOD is precipitated completely. The SOD activity can reach 129.3U/mg by utilizing gradient elution of PEG precipitation.3.SOD precipitated with PEG is separated effectively from heteroprotein by the column chromatography of DEAE-cellulose(DE-52) and eluted gradualy by pH7.8 0.05mol/L Phosphoric acid(PB) and 1.0mol/L Nacl. The best column chromatography conditions: the flow rate is 1.2ml/min, the rate of diameter to height is 1:10,the added quantity is 6 ml;The time is 33 minute and the elution volume is 40ml when SOD is completely eluted, and the time is 66 minute and the volume is 80 ml when the most heteroprotein are eluted. So the column chromatography of DEAE-cellulose (DE-52) is high effective and differential. Finally, SOD which specific activity is 3145.5U/mg can be obtained.4.SOD has a good stability to heat and pH. The enzymatic activity iscomparatively stable below 60℃, for example the exact being kept in 60℃ for 30 minute, the enzymatic activity only reduces 19.1%;however the temperature raised higher than 70℃, the activity reduces obviously, such as reducing 56.6% on the condition of 70℃ for 30 minute and 70.8% on the condition of 80℃ for 30 minute. The enzymatic activity is stable within pH 4-9; Once exceeding this range , it reduces deeply. For example, the rest activity is 21% on the condition of pH 2 and 49.1% of pH 10. The activity is nothing on the condition of pH 12.5.The SOD from Allium Sativum cell plasma is sensitive to H2O2 and cyanide, so it indicates this type of SOD is copper/zinc-SOD.The absorption apex exhibits at 258nm. it demonstrates the enzyme contains few Tyrosine and Tryptophan.6.The product weighted 15.6mg can be obtained from 500g Allium Sativum thought three methods .The specific activity of purified SOD is 3145.5U/mg, purification times is 197.8 and yield is 39.2% .更多还原