To develop an enzymatic measurement of HbA1C, 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 α-glycated molecules, therefore it seemed suitable for the HbA1C 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 β-chain of HbA1C 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 HbA1C. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Hirokawa, K., Nakamura, K., & Kajiyama, N. (2004). Enzymes used for the determination of HbA1C. FEMS Microbiology Letters, 235(1), 157–162. https://doi.org/10.1016/j.femsle.2004.04.027