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题名: Replace buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhance its stability by site-directed mutagenesis.
作者: Chuian-Fu Ken
Chi-Tsai Lin
Yu-Der Wen
Jen-Leih Wu
贡献者: 國立臺灣海洋大學:生命科學暨生物科技學系
关键词: thermostability
Cu/Zn superoxide dismutase
I-Mutant 2.0
oxidative stress
site-direct
mutagenesis
日期: 2007
上传时间: 2016-09-06T06:41:02Z
出版者: Marine Biotechnology
摘要: Abstract: 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 Kd 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: 9(3)
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/38452
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