Characterization of Copper/Zinc-Superoxide Dismutase from Pagrus major cDNA and Enzyme Stability

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• 作者：Chuian-Fu Ken ; De-Feng Weng ; Kow-Jen Duan ; and Chi-Tsai Lin
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2002
• 出版时间：February 13, 2002
• 年：2002
• 卷：50
• 期：4
• 页码：784 - 789
• 全文大小：135K
• 年卷期：v.50,no.4(February 13, 2002)
• ISSN：1520-5118

文摘

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 cDNAclone 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 and147) 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 anexpression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expressionof 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). Thehalf-life was 8.6 min and the inactivation rate constant (k_d) was 9.69 × 10^-2 min^-1 at 70 C. Theenzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme wasresistant to proteolysis by both trypsin and chymotrypsin.Keywords: Pagrus major; expression; Escherichia coli; PCR; pET-20b(+)