Analysis and improvement of hyperthermostable endo-glucanase from Archaea

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In this study, I tried to analyze and improve the function of the hyperthermostable cellulose from hyperthermophilic archaea Pyrococcus horikoshii. I examined the function and role of the unique hyperthermostable cellulase that containing the membrane binding site with protein engineering method. As a result, I succeeded in mass expression and solubilization of the enzyme by improving the C terminus residue of the enzyme. Furthermore, I succeeded in structure and a functional analysis of the catalytic site of the enzyme and succeeded in identification of the unknown function protein in the cellulose gene with protein engineering method. The detailed role of the protein is in progress. Furthermore, I succeeded in preparation of the crystals of the enzyme by improving N or C terminus of the enzyme. By changing the crystallization condition, high quality crystals of the enzyme were obtained. Using molecular replacement method, I solved the crystal structure of the enzyme. The refinement of the structural analysis is in progress. From the present structural data, it was elucidated that the enzyme exhibits the TIM barrel structure and catalytic residues of the enzyme are similar to those of the well known cellulase, but there is a big difference in the substrate recognition loop structures among the cellulases. This loop structure seems to be related to substrate specificity. I feel that the activity toward the crystalline cellulose can be improved by analyzing the detailed part. Using this structural data, I succeeded in preparation of new chimera enzyme between this cellulose and hyperthermophilic chitinase from Pyrococcus fuiosus. It was elucidated this chimera enzyme exhibited the activity of two times as high as that of the cellulose.