Cloning, Expression, and Characterization of an Enzyme Possessing both Glutaredoxin and Dehydroascorbate Reductase Activity from Taiwanofungus camphorata
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- 作者：Chuian-Fu Ken ; Choa-Yi Lin ; Yu-Chi Jiang ; Lisa Wen ; Chi-Tsai Lin
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2009
- 出版时间：November 11, 2009
- 年：2009
- 卷：57
- 期：21
- 页码：10357-10362
- 全文大小：872K
- 年卷期：v.57,no.21(November 11, 2009)
- ISSN：1520-5118
文摘
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.