Purification and characterization of superoxide dismutase from garlic
Purification and characterization of superoxide dismutase from garlic
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大蒜超氧化物歧化酶的纯化及表征
DOI：
10.1016/j.fbp.2010.07.003
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发表时间：
2011-10-01
影响因子：
4.6
通讯作者：
Cui, Zhanfeng
中科院分区：
文献类型：
--
作者：
Liu, Jianguo;Wang, Jingbing;Cui, Zhanfeng
提出了一种高效、简易的从大蒜中分离超氧化物歧化酶的方法。分离纯化过程包括磷酸盐缓冲液萃取、热处理和两级超滤过程。该酶经纯化得到139倍，比活性为2867 U/mg蛋白，产率为91%。用快速蛋白液相色谱法测定超氧化物歧化酶的天然分子量为28 kDa。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示在14 kDa附近有一条单带，表明天然酶为同型二聚体。酶活性的最佳pH为7.0，在此pH下，酶在50℃、50 mM磷酸钠缓冲液中表现出最大活性。在各种金属离子中，Cu2+和Zn2+对超氧化物歧化酶活性有积极影响，而He是强抑制剂。最终纯化的酶的等电点为5.1-5.4，片含量为46%，与文献值一致。这表明纯化后的SOD具有合理的二级结构。(C) 2010中国化学工程师学会。Elsevier B.V.版权所有。
An efficient and easily scaled up method to isolate superoxide dismutase from garlic is proposed. The separation and purification procedure consists of phosphate buffer extraction, heat treatment and a two-stage ultrafiltration process. The enzyme was purified 139-fold with a specific activity of 2867 U/mg protein and a yield of 91%. The native molecular mass of superoxide dismutase estimated by fast protein liquid chromatography on a Superose 6 column was 28 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a single band near 14 kDa, suggesting that native enzyme was homo-dimeric. The optimal pH for enzyme activity was found to be 7.0, and at this pH the enzyme exhibited maximum activity at 50 C in 50 mM sodium phosphate buffer. Among various metal ions examined, Cu2+ and Zn2+ exerted a positive effect on superoxide dismutase activity, whereas He was found to be a strong inhibitor. The final purified enzyme had an isoelectric point of 5.1-5.4 and a sheet content of 46%, consistent with the literature values. This shows that the purified SOD folded with a reasonable secondary structure. (C) 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.