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The effects of various abiotic factors, including polyethylene glycol (PEG), silver nitrate (AgNO3) and potassium phosphate (KH2PO4) on biomass growth, extracellular production of recombinant fungal phytase and its transcription activity by realtime RT-PCR were examined with transformed sesame hairy roots. The PEG treatments decreased both biomass growth (80.5≈82.3%) and phytase production (82.1≈96.4%) at all concentrations tested, except 1.0 g/L PEG, which increased biomass growth to 112.4% of that of the control. The AgNO3 treatments also resulted in reduced biomass growth (77≈92%) and phytase production (84.4≈96.3%) at all concentrations applied except 1.5 mg/L AgNO3, at which biomass was increased to 109.6% of that of untreated roots. The potassium phosphate treatments increased biomass growth, production of recombinant fungal phytase and its transcription until a concentration of 340 mg/L was attained, and at 510 mg/L a rapid decrease was observed in all of the aforementioned parameters. Combined treatments of PEG (1 g/L) and AgNO3 (1.5 mg/L) exhibited both positive and negative influences on biomass growth and production and transcription of the recombinant fungal phytase when medium containing 170 and 340 mg/L of potassium phosphate were used, respectively. The addition of PEG alone to culture medium containing potassium phosphate at both of the aforementioned concentrations decreased biomass while increasing production and transcription of recombinant fungal phytase. Conversely, the addition of AgNO3 alone to culture medium increased biomass but decreased production and transcription of fungal phytase. Throughout these experiments, the most effective treatment for enhanced biomass growth, and enhanced production and transcription of recombinant fungal phytase was attained when the 1 g/L PEG and 1.5 mg/L AgNO3 were combined in medium containing 340 mg/L potassium phosphate.

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.