National Changhua University of Education Institutional Repository : Item 987654321/1835
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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: Ken CF;Lin CT;Wen YD;Wu JL.
贡献者: 生物學系
日期: 2007
上传时间: 2010-11-15T07:02:50Z
摘要: Zebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first beta-strand with lower thermostability. We predicted the stability would be increased with single-point mutation at 70 degrees 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 degrees 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 x 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):335-42. (SCI)
显示于类别:[生物學系] 期刊論文

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