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.
Similar content being viewed by others
References
Ferri S, Kim S, Tsugawa W, Sode K (2009) Review of fructosyl amino acid oxidase engineering research: a glimpse into the future of hemoglobin A1c biosensing. J Diabetes Sci Technol 3:585–592
Finot PA, Viani R, Bricout J, Mauron J (1969) Detection and identification of pyridoxine, a second lysine derivative obtained upon acid hydrolysis of heated milk. Experientia 25:134–135
Hirokawa K, Nakamura K, Kajiyama N (2004) Enzymes used for the determination of HbA1C. FEMS Microbiol Lett 235:157–162
Kim S, Ferri S, Tsugawa W, Mori K, Sode K (2010a) Motif-based search for a novel fructosyl peptide oxidase from genome databases. Biotechnol Bioeng 15:358–366
Kim S, Nibe E, Ferri S, Tsugawa W, Sode K (2010b) Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system. Biotechnol Lett 32:1123–1129
Kouzuma T, Usami T, Yamakoshi M, Takahashi M, Imamura S (2002) An enzymatic method for the measurement of glycated albumin in biological samples. Clin Chim Acta 324:61–71
Liu LM, Hood S, Wang Y, Bezverkov R, Dou C, Datta A, Yuan C (2008) Direct enzymatic assay for %HbA1c in human whole blood samples. Clin Biochem 41:576–583
Sakurabayashi I, Watano T, Yonehara S, Ishimaru K, Hirai K, Komori T, Yagi M (2003) New enzymatic assay for glycohemoglobin. Clin Chem 49:269–274
Yamaguchi M, Kambe S, Eto T, Yamakoshi M, Kouzuma T, Suzuki N (2005) Point of care testing system via enzymatic method for the rapid, efficient assay of glycated albumin. Biosens Bioelectron 21:426–432
Yoshida N, Sakai Y, Isogai A, Fukuya H, Yagi M, Tani Y, Kato N (1996) Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins. Eur J Biochem 242:499–505
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kim, S., Nibe, E., Tsugawa, W. et al. Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions. Biotechnol Lett 34, 491–497 (2012). https://doi.org/10.1007/s10529-011-0787-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-011-0787-1