title: Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus.
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.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 5665-0871, Japan.
authors.forename: Hyongi
authors.initials: H
authors.lastname: Chon
authors.forename: Rikita
authors.initials: R
authors.lastname: Nakano
authors.forename: Naoto
authors.initials: N
authors.lastname: Ohtani
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Masaaki
authors.initials: M
authors.lastname: Morikawa
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; C117079:ribonuclease HIII