National Changhua University of Education Institutional Repository : Item 987654321/16771
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题名: Copper/Zinc−Superoxide Dismutase from Epinephelus malabaricus cDNA and Enzyme Property
作者: Ken, Chuian-Fu;Cheng, Yu-Feng;Chang, Ching-Fong;Lin, Chi-Tsai
贡献者: 生物學系
关键词: Epinephelus malabaricus;Copper/zinc-superoxide dismutase (Cu/Zn-SOD);Expression;Echerichia coli;PCR;pET-20b(+)
日期: 2003-09
上传时间: 2013-06-05T08:48:11Z
出版者: American Chemical Society
摘要: 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 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.
關聯: Journal of Agricultural and Food Chemistry, 51(19): 5688-5694
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