Abstract
Tertiary structure of the recombinant glutamate dehydrogenase from Thermococcus kodakaraensis KOD1 ( Tk -rGDH) converts into an intact form induced by the heat treatment. This phenomenon, heat-induced structural maturation, means that high temperature plays an important role in the proper folding and oligomerization of Tk -rGDH. In this work, we analyzed the heat-induced structural maturation of Tk -rGDH by differential scanning microcalorimetry (DSC), circular dichroism (CD), and activity measurements. In DSC measurements, the peak of adsorption of non-heated Tk -rGDH (nh- Tk -rGDH) was two times smaller than that of Tk -rGDH heated at 70°C for 30 min (h- Tk -rGDH). The transition temperature ( T m ) of h- Tk -rGDH was 115°C, which was about 3°C higher than that of nh- Tk -rGDH. In the presence of 0.5 M NaCl, the nh- Tk -rGDH showed two peaks at 107°C and 114°C, while the h- Tk -rGDH showed a single peak at 115.7°C. The heat-induced conformational change process was monitored by changes in CD intensity at 222 nm, and the result showed that heat-induced structural maturation is irreversible. The heat treatment at 70°C showed the highest enhancement in activity, which was 15% larger than that of heat-treated Tk -rGDH at 40°C. The results indicate that heat-induced structural maturation involves an irreversible process, transforming the non-heated form to the stable and active form.
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