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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/27082

Title: Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis
Authors: Chuian-Fu Ken;Chi-Tsai Lin;Yu-Der Wen;Jen-Leih Wu
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: Cu/Zn superoxide dismutase;I-Mutant 2.0;oxidative stress;site-direct mutagenesis;thermostability
Date: 2007-05
Issue Date: 2011-10-21T02:22:34Z
Publisher: Marine Biotechnology
Abstract: 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 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.
Relation: 9(3), pp.335-342
URI: http://ntour.ntou.edu.tw/handle/987654321/27082
Appears in Collections:[生命科學暨生物科技學系] 期刊論文

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