Accéder au contenu
Merck

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution.

Applied and environmental microbiology (2003-01-07)
Ryoichi Sakaue, Naoki Kajiyama
RÉSUMÉ

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.

MATÉRIAUX
Référence du produit
Marque
Description du produit

Sigma-Aldrich
Fructosyl-Amino Acid Oxidase from Corynebacterium sp., recombinant, expressed in E. coli, lyophilized powder, ≥0.45 units/mg protein
Référence
Conditionnement
Disponibilité
Prix
Quantité