Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis
Ken, C.-F.; Lin, C.-T.; Wen, Y.-D.; Wu, J.-L. (2007). Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis. Mar. Biotechnol. 9(3): 335-342. https://dx.doi.org/10.1007/s10126-006-0143-y
In: Marine Biotechnology. Springer-Verlag: New York. ISSN 1436-2228; e-ISSN 1436-2236, meer
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| Author keywords |
Cu/Zn superoxide dismutase; I-Mutant 2.0; oxidative stress; site-directmutagenesis; thermostability |
| Auteurs | | Top |
- Ken, C.-F.
- Lin, C.-T.
- Wen, Y.-D.
- Wu, J.-L.
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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 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. |
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