National Changhua University of Education Institutional Repository : Item 987654321/16775
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题名: Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis
作者: Ken, Chuian-Fu;Lin, Chi-Tsai;Wen, Yu-Der;Wu, Jen-Leih
贡献者: 生物學系
关键词: Cu/Zn superoxide dismutase;I-Mutant 2.0;Oxidative stress;Site-direct mutagenesis;Thermostability
日期: 2007-05
上传时间: 2013-06-05T08:48:16Z
出版者: Springer Science+Business Media, Inc.
摘要: Zebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first β-strand with lower thermostability. We predicted the stability would be increased with single-point mutation at 70°C via the I-Mutant 2.0 server, and generated a mutant SOD with replacement of the free Cys to Ala (ZSODC7A) by site-directed mutagenesis. The mutant was expressed and purified from the Escherichia coli strain AD494(DE3)pLysS and the yield was 2 mg from 0.4 L of culture. The ZSODC7A was heated at 90°C. In a time-dependent assay, the time interval for 50% inactivation was 32 min, and its thermal inactivation rate constant K d was 2 × 10−2 min−1. The mutant was still activated in broad pH range (2.3–12), and had only a moderate effect under sodium dodecyl sulfate treatment. The calculated specific activity of the mutant was 3980 U/mg, twice that of wild-type ZSOD1. In addition, we soaked fish larva with equal enzyme units of either ZSOD1 or ZSODC7A for 2 h, and then stressed them with 100 ppm of paraquat to induce oxidative injury. The survival rate was significant.
關聯: Marine Biotechnology, 9(3): 335-342
显示于类别:[生物學系] 期刊論文

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