title: Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions.
abstract: Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O(2). Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O(2) as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl-(α)                         N-valyl-histidine (f-(α)Val-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-(α)Val-His in the physiological target range in air.
authors: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan.
authors: Seungsu
authors: S
authors: Kim
authors: Eri
authors: E
authors: Nibe
authors: Wakako
authors: W
authors: Tsugawa
authors: Katsuhiro
authors: K
authors: Kojima
authors: Stefano
authors: S
authors: Ferri
authors: Koji
authors: K
authors: Sode
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D006442:Glycated Hemoglobin / Q000032:analysis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000594:Amino Acid Oxidoreductases; D006442:Glycated Hemoglobin; D011994:Recombinant Proteins; C517652:hemoglobin A1c protein, human; C520096:fructosyl-peptide oxidase