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Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis

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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.

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Acknowledgments

This work was supported by the National Science Council of the Republic of China under grant NSC 92-2313-B-019-023 and NSC 93-2313-B-018-003 to C-F. Ken. We thank Dr. Nady Shaw for providing information on I-Mutant 2.0.

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Authors and Affiliations

  1. Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan

    Chuian-Fu Ken

  2. Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan

    Chi-Tsai Lin

  3. Department of Biology, National Changhua University of Education, Changhua, Taiwan

    Yu-Der Wen

  4. Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan

    Jen-Leih Wu

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  1. Chuian-Fu Ken
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  2. Chi-Tsai Lin
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Correspondence to Jen-Leih Wu.

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Chuian-Fu Ken and Chi-Tsai Lin contributed equally to this article.

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Ken, CF., Lin, CT., Wen, YD. et al. Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis. Mar Biotechnol 9, 335–342 (2007). https://doi.org/10.1007/s10126-006-0143-y

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