title: Enzymes used for the determination of HbA1C.
abstract: To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized. Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His. The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay. Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli. Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase. We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).
authors.affiliation: Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-city, Chiba Pref. 278-0037, Japan. khirokawa@mail.kikkoman.co.jp
authors.forename: Kozo
authors.initials: K
authors.lastname: Hirokawa
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nakamura
authors.forename: Naoki
authors.initials: N
authors.lastname: Kajiyama
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology; D001435:Bacteriophages / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004796:Clinical Enzyme Tests / Q000379:methods*; D003920:Diabetes Mellitus / Q000175:diagnosis*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006442:Glycated Hemoglobin / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000594:Amino Acid Oxidoreductases; D010450:Endopeptidases; D006442:Glycated Hemoglobin; D010455:Peptides; D011994:Recombinant Proteins; C103012:amadoriase