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

All Science Journal Classification (ASJC) codes

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

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