Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability

Chuian Fu Ken, De Feng Weng, Kow Jen Duan, Chi Tsai Lin

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR 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 (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni2+nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (kd) was 9.69 × 10-2 min-1 at 70°C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.

Original languageEnglish
Pages (from-to)784-789
Number of pages6
JournalJournal of Agricultural and Food Chemistry
Volume50
Issue number4
DOIs
Publication statusPublished - 2002 Feb 13

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enzyme stability
Enzyme Stability
Pagrus major
Superoxide Dismutase
Zinc
Copper
superoxide dismutase
Complementary DNA
copper
zinc
Enzyme activity
Enzymes
Nitrilotriacetic Acid
Proteolysis
Amino Acids
Dimerization
Disulfides
Dimers
Sepharose
Escherichia coli

All Science Journal Classification (ASJC) codes

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

Cite this

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title = "Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability",
abstract = "A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR 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 (53-91{\%}) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni2+nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (kd) was 9.69 × 10-2 min-1 at 70°C. The enzyme activity was not significantly affected under 4{\%} SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.",
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Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability. / Ken, Chuian Fu; Weng, De Feng; Duan, Kow Jen; Lin, Chi Tsai.

In: Journal of Agricultural and Food Chemistry, Vol. 50, No. 4, 13.02.2002, p. 784-789.

Research output: Contribution to journalArticle

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T1 - Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability

AU - Ken, Chuian Fu

AU - Weng, De Feng

AU - Duan, Kow Jen

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