Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions |
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Authors: | Seungsu Kim Eri Nibe Wakako Tsugawa Katsuhiro Kojima Stefano Ferri Koji Sode |
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Institution: | (1) Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan;(2) Ultizyme International Ltd, 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan; |
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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 O2. 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 O2 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-αVal-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-αVal-His in the physiological target range in air. |
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