Molecular and biochemical characterizations of dehydroascorbate reductase from sesame (Sesamum indicum L.) hairy root cultures.
Dehydroascorbate reductase (DHAR) is a biotechnologically or physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction for most higher plants. A DHAR cDNA was isolated from sesame (Sesamum indicum L.) hairy roots, and its structure and biochemical properties were characterized to provide some information about its expressional and biochemical profiles in the hairy root cultures. The cDNA contained a catalytic motif CXXS, which may be indicative of a thiol-dependent redox function. A fusion DHAR expressed in an Escherichia coli expression system was purified with four purification steps until a homogeneous single band signal was seen in an acrylamide gel, and its antibody was prepared for Western blot analyses. The biochemical results showed that the purified recombinant DHAR had an optimal pH of around 6.0, which was different from those (pH 7.8-8.2) of other plant species. The temperature optimal for the DHAR activity was in a relatively wide range of 30-60 degrees C. It was proved by a real-time RT-PCR technique that the transcription activity of the DHAR was about 2-5-fold higher during the first 3 week cultures than during the latter 3 week ones. The highest activity of the sesame DHAR was detected in the 4 week cultures of the hairy roots, after which its activity was rapidly decreased to approximately 80%, suggesting that the most active DHAR occurred in this culture period. Western blot analyses confirmed that the presence of DHAR enzyme was identified in both cultures of the fused E. coli and the sesame hairy roots.
Chun
Jae-An
JA
Departments of Biotechnology and Environmental Biotechnology, Dong-A University, Busan 604-714, South Korea.
Lee
Woo-Hyup
WH
Han
Mi-Ok
MO
Lee
Jin-Woo
JW
Yi
Young-Byung
YB
Goo
Young-Min
YM
Lee
Shin-Woo
SW
Bae
Shin-Chul
SC
Cho
Kang-Jin
KJ
Chung
Chung-Han
CH
D018744:DNA, Plant / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D031685:Sesamum / Q000201:enzymology*; D013696:Temperature; D046509:Tissue Culture Techniques
D018744:DNA, Plant; D011994:Recombinant Proteins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate)