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

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site-directed mutagenesis
Zebrafish
Site-Directed Mutagenesis
Danio rerio
Cysteine
cysteine
superoxide dismutase
replacement
mutants
Paraquat
Point Mutation
Sodium Dodecyl Sulfate
Larva
Fishes
Hot Temperature
Escherichia coli
Wounds and Injuries
Enzymes
paraquat
heat inactivation

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Aquatic Science

Cite this

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title = "Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis",
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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Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis. / Ken, Chuian-Fu; Lin, Chi Tsai; Wen, Yu-Der; Wu, Jen Leih.

In: Marine Biotechnology, Vol. 9, No. 3, 01.05.2007, p. 335-342.

Research output: Contribution to journalArticle

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