Scientific Reports of the Faculty of Agriculture, Okayama University
Published by the Faculty of Agriculture, Okayama University
ONLINE ISSN : 2186-7755

好熱性細菌Thermus sp.O-3-1由来耐熱性アミダーゼの精製及び性質検討

Kobayashi, Fumiaki
Aomine, Hiroki
Mizunashi, Wataru
Yu, Fujio
Inagaki, Kenji Kaken ID researchmap
The gene encoding a thermostable amidase (EC from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally, as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.
thermostable enzyme
D-amino acid specific amidase
原著論文 (Original paper)