National Changhua University of Education Institutional Repository : Item 987654321/16781
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題名: Cloning, Expression, and Characterization of an Enzyme Possessing both Glutaredoxin and Dehydroascorbate Reductase Activity from Taiwanofungus Camphorata
作者: Ken, Chuian-Fu;Lin, Choa-Yi;Jiang, Yu-Chi;Wen, Lisa;Lin, Chi-Tsai
貢獻者: 生物學系
關鍵詞: Taiwanofungus camphorata;Glutaredoxin;Three-dimension homology structure (3Dhomology structure);Expression;β-hydroxyethyl disulfide [HED, (HOCH2CH2)2S2];Dehydroascorbate (DHA)
日期: 2009-11
上傳時間: 2013-06-05T08:48:23Z
出版者: American Chemical Society
摘要: Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli. Functional TcGrx was expressed and purified by Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of 15 kDa on 15% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (Km) values for β-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 °C was 8.5 min, and its thermal inactivation rate constant Kd was 6.52 × 10−2 min−1. The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.
關聯: Journal of Agricultural and Food Chemistry, 57(21): 10357-10362
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