| ○要約 (任意): | □ | (英) Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) showed high thermostability, but thermal aggregation was observed on heating above 85 °C. In previous studies, we suggested that substitutions/deletion of several residues increased the thermostability of Tth PPase; (1) Stable hexameric formation by the improvement of intertrimer interface (T138H/A141D(HD)), (2) The decrease of flexibility by the deletion of Ala144-Lys145(Del), (3) The enhancement of structural integrity by the decrease of volume of Cys168 in C-terminal region (C168A). In this study, we constructed three multi-mutants (HDDel, HDC168A and HDDelC168A) by combination of above thermostabilizing factors, and investigated these thermostabilities in terms of the enzyme activity, conformation and quaternary structure. The results revealed that the thermostabilities of all the three multi-mutants are much more stable than wild type at any criterion. In particular, HDdelC168A showed the highest thermostability of them. After heating at 85ºC for 1h, 70% of its enzymatic activity and quaternary structure were remained, and the change of fluorescence spectra was not observed. Hence, the combination of thermostabilizing factors may be effective on the stabilization of enzymatic activity and conformation, and suppression of thermal aggregation. Therefore, it was suggested that the microenvironment in Thr138, Ala141, Ala144, Lys145 and Cys168 would play a key role in the stable conformation of Tth PPase. (日)
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