Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property

Chuian Fu Ken, Yu Feng Cheng, Ching Fong Chang, Chi Tsai Lin

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 languageEnglish
Pages (from-to)5688-5694
Number of pages7
JournalJournal of Agricultural and Food Chemistry
Volume51
Issue number19
DOIs
Publication statusPublished - 2003 Sep 10

Fingerprint

Epinephelus malabaricus
Superoxide Dismutase
Zinc
Copper
superoxide dismutase
Enzyme activity
Complementary DNA
copper
zinc
Enzymes
enzymes
Pollution
Nitrilotriacetic Acid
Proteolysis
Amino Acids
enzyme activity
Polymerase chain reaction
Biomarkers
Transcription
Sodium Dodecyl Sulfate

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Cite this

Ken, Chuian Fu ; Cheng, Yu Feng ; Chang, Ching Fong ; Lin, Chi Tsai. / Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property. In: Journal of Agricultural and Food Chemistry. 2003 ; Vol. 51, No. 19. pp. 5688-5694.
@article{73c40415d7f44d90a26840ed152a9f06,
title = "Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property",
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.",
author = "Ken, {Chuian Fu} and Cheng, {Yu Feng} and Chang, {Ching Fong} and Lin, {Chi Tsai}",
year = "2003",
month = "9",
day = "10",
doi = "10.1021/jf030162p",
language = "English",
volume = "51",
pages = "5688--5694",
journal = "Journal of Agricultural and Food Chemistry",
issn = "0021-8561",
publisher = "American Chemical Society",
number = "19",

}

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 journalArticle

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 -