Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus |
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Authors: | Chon Hyongi Nakano Rikita Ohtani Naoto Haruki Mitsuru Takano Kazufumi Morikawa Masaaki Kanaya Shigenori |
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Institution: | Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 5665-0871, Japan. |
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Abstract: | The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII. |
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