Enzymes used for the determination of HbA1C

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.