Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan.
Biotechnol Lett. 2012 Mar;34(3):491-7. doi: 10.1007/s10529-011-0787-1. Epub 2011 Nov 4.
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 O(2). 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 O(2) 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-(α)Val-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-(α)Val-His in the physiological target range in air.
目前用于糖化蛋白分析的酶法分析使用黄素酶，催化果糖肽的氧化去糖化，称为果糖基肽氧化酶（FPOXs）。然而，由于 FPOXs 是氧化酶，因此基于它们的电子介体型电化学监测所产生的信号会受到溶解氧（O2）的影响。因此，开展了 FPOX 中 Asn56 的染料介导脱氢酶活性的改进及其在酶电极构建中的应用研究。对来自 Phaeosphaeria nodorum 的 FPOX 的 Asn56 进行饱和诱变研究，产生了突变体，这些突变体对将 O2 用作电子受体的催化能力明显降低，同时与亲本酶相比，显示出更高的染料介导的使用人工电子受体的脱氢酶活性。因此，构建了实际上的果糖基肽脱氢酶，Asn56Ala，然后将其应用于产生用于测量糖化（α）N-缬氨酰组氨酸（f-（α）Val-His）的酶电极，该物质是 HbA1c 的蛋白酶消化产物。酶电极可以在空气中测量生理靶标范围内的 f-（α）Val-His。
