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 language | English |
|---|---|
| Pages (from-to) | 335-342 |
| Number of pages | 8 |
| Journal | Marine Biotechnology |
| Volume | 9 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2007 May 1 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Aquatic Science
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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 journal › Article
TY - JOUR
T1 - Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis
AU - Ken, Chuian-Fu
AU - Lin, Chi Tsai
AU - Wen, Yu-Der
AU - Wu, Jen Leih
PY - 2007/5/1
Y1 - 2007/5/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=34249946977&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34249946977&partnerID=8YFLogxK
U2 - 10.1007/s10126-006-0143-y
DO - 10.1007/s10126-006-0143-y
M3 - Article
C2 - 17549562
AN - SCOPUS:34249946977
VL - 9
SP - 335
EP - 342
JO - Marine Biotechnology
JF - Marine Biotechnology
SN - 1436-2228
IS - 3
ER -