Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis

Chuian-Fu Ken, Chi Tsai Lin, Yu-Der Wen, Jen Leih Wu

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)335-342
Number of pages8
JournalMarine Biotechnology
Volume9
Issue number3
DOIs
Publication statusPublished - 2007 May 1

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Aquatic Science

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