Enzymes used for the determination of HbA1C.
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).
Hirokawa
Kozo
K
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-city, Chiba Pref. 278-0037, Japan. khirokawa@mail.kikkoman.co.jp
Nakamura
Kazuo
K
Kajiyama
Naoki
N
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
D000594:Amino Acid Oxidoreductases; D010450:Endopeptidases; D006442:Glycated Hemoglobin; D010455:Peptides; D011994:Recombinant Proteins; C103012:amadoriase