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 °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. Keywords: Dehydroascorbate reductase; differential expression; hairy root cultures; protein purification; real-time RT-PCR; recombinant DHAR; Sesamum indicum L.