Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution.
We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.
Sakaue
Ryoichi
R
Research and Development Division, Kikkoman Corporation, Noda City, Chiba Prefecture 278-0037, Japan. rsakaue@mail.kikkoman.co.jp
Kajiyama
Naoki
N
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C103012:amadoriase