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

Chuian F. Ken, Choa Y. Lin, Yu C. Jiang, Lisa Wen, Chi Tsai Lin

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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
Volume57
Issue number21
DOIs
Publication statusPublished - 2009 Nov 11

Fingerprint

Taiwanofungus
glutathione dehydrogenase (ascorbate)
Glutaredoxins
Cloning
Organism Cloning
molecular cloning
Enzymes
enzymes
sulfides
Disulfides
Denaturation
imidazoles
reducing agents
heat inactivation
Reducing Agents
Electrophoresis
denaturation
Sodium Dodecyl Sulfate
Sepharose
Escherichia coli

All Science Journal Classification (ASJC) codes

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

Cite this

@article{2ba2f63e0d9847d4b541085a9d5af276,
title = "Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata",
abstract = "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.",
author = "Ken, {Chuian F.} and Lin, {Choa Y.} and Jiang, {Yu C.} and Lisa Wen and Lin, {Chi Tsai}",
year = "2009",
month = "11",
day = "11",
doi = "10.1021/jf9021256",
language = "English",
volume = "57",
pages = "10357--10362",
journal = "Journal of Agricultural and Food Chemistry",
issn = "0021-8561",
publisher = "American Chemical Society",
number = "21",

}

Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata. / Ken, Chuian F.; Lin, Choa Y.; Jiang, Yu C.; Wen, Lisa; Lin, Chi Tsai.

In: Journal of Agricultural and Food Chemistry, Vol. 57, No. 21, 11.11.2009, p. 10357-10362.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata

AU - Ken, Chuian F.

AU - Lin, Choa Y.

AU - Jiang, Yu C.

AU - Wen, Lisa

AU - Lin, Chi Tsai

PY - 2009/11/11

Y1 - 2009/11/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=70449101120&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70449101120&partnerID=8YFLogxK

U2 - 10.1021/jf9021256

DO - 10.1021/jf9021256

M3 - Article

C2 - 19886686

AN - SCOPUS:70449101120

VL - 57

SP - 10357

EP - 10362

JO - Journal of Agricultural and Food Chemistry

JF - Journal of Agricultural and Food Chemistry

SN - 0021-8561

IS - 21

ER -