Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution.
D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.
Bakke
Mikio
M
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037, Japan. mbakke@mail.kikkoman.co.jp
Setoyama
Chiaki
C
Miura
Retsu
R
Kajiyama
Naoki
N
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013552:Swine; D013696:Temperature; D014162:Transfection
D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase