Identification of a thermostable and enantioselective amidase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7.
We have characterized an amidase expressed from the putative amidase gene (ST0478) selected from the total genome analysis from the thermoacidophilic archaeon, Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as an insoluble aggregated 6 x His-tagged fusion protein in Escherichia coli. The protein was purified with denaturing, refolding on affinity column chromatography, size exclusion filtration, and heat treatment. The enzyme exhibited high thermostability and the optimum activity for amide cleavage against benzamide was observed at around 75 degrees C and pH 7.0-8.0. It also showed enantioselectivity for (R,S)-2-phenylpropionamide and preferentially hydrolyzed the S-enantiomer. This novel enzyme is the second characterized archaeal amidase.
Suzuki
Yoichi
Y
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Ohta
Hiromichi
H
D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D008968:Molecular Conformation; D015394:Molecular Structure; D016366:Open Reading Frames; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature
D019843:Archaeal Proteins; D010666:Phenylpropionates; D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases