Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata

Chuian Fu Ken, Choa Yi Lin, Yu Chi Jiang, Lisa Wen, Chi Tsai Lin

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Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU 193660) 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 Ni 2+-nitrllotriacetic 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 x 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.

Original languageEnglish
Pages (from-to)10357-10362
Number of pages6
JournalJournal of Agricultural and Food Chemistry
Issue number21
Publication statusPublished - 2009 Nov 11


All Science Journal Classification (ASJC) codes

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

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