Abstract
A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21 (DE3)pLysS. The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni2+-nitrilotriacetic acid Sepharose. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 °C for 10 min. The inactivation rate constant (kd) was 6.64 x 10-2 min-1 at 60 °C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.
| Original language | English |
|---|---|
| Pages (from-to) | 5688-5694 |
| Number of pages | 7 |
| Journal | Journal of Agricultural and Food Chemistry |
| Volume | 51 |
| Issue number | 19 |
| DOIs | |
| Publication status | Published - 2003 Sep 10 |
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All Science Journal Classification (ASJC) codes
- Chemistry(all)
- Agricultural and Biological Sciences(all)
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Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property. / Ken, Chuian Fu; Cheng, Yu Feng; Chang, Ching Fong; Lin, Chi Tsai.
In: Journal of Agricultural and Food Chemistry, Vol. 51, No. 19, 10.09.2003, p. 5688-5694.Research output: Contribution to journal › Article
TY - JOUR
T1 - Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property
AU - Ken, Chuian Fu
AU - Cheng, Yu Feng
AU - Chang, Ching Fong
AU - Lin, Chi Tsai
PY - 2003/9/10
Y1 - 2003/9/10
N2 - A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21 (DE3)pLysS. The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni2+-nitrilotriacetic acid Sepharose. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 °C for 10 min. The inactivation rate constant (kd) was 6.64 x 10-2 min-1 at 60 °C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.
AB - A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21 (DE3)pLysS. The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni2+-nitrilotriacetic acid Sepharose. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 °C for 10 min. The inactivation rate constant (kd) was 6.64 x 10-2 min-1 at 60 °C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.
UR - http://www.scopus.com/inward/record.url?scp=0041333146&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0041333146&partnerID=8YFLogxK
U2 - 10.1021/jf030162p
DO - 10.1021/jf030162p
M3 - Article
C2 - 12952420
AN - SCOPUS:0041333146
VL - 51
SP - 5688
EP - 5694
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
SN - 0021-8561
IS - 19
ER -