title: Identification, purification, and characterization of a thermophilic imidase from pig liver.
abstract: This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.
authors.affiliation: Institute of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: T M
authors.initials: TM
authors.lastname: Su
authors.forename: Y S
authors.initials: YS
authors.lastname: Yang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D002384:Catalysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry*; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000581:Amidohydrolases; C020047:dihydropyrimidinase