Development of the 2.5th Generation Biosensor for HbA1c Using Engineered Fructosyl Peptide Oxidase

Abstract

Introduction Haemoglobin A1c (HbA1c) is an important glycaemic control marker for diabetes mellitus. The level of HbA1c reflects the average blood glucose level over the prior 2–3 months. Recently an enzymatic assay has been introduced, which employs fructosyl peptide oxidase (FPOx). These methods have high accuracy, however, they are time-consuming, expensive, and require complicated analytical procedures. There is an increasing need for simple, rapid and miniaturized point-of-care testing (POCT). Our research group has been engaged in the development of novel electrochemical methods for glycated proteins, including HbA1c. We previously reported on the isolation and molecular engineering of FPOx derived from Phaeosphaeria nodorum (PnFPOx), which catalyzes the oxidative deglycosylation of fructosyl peptide and yield glucosone and free peptide, and it is specific to α-fructosyl amino acid (Kim et al., 2010). We reported the engineering of this enzyme to yield the mutant FPOx, Asn56Ala (N56A) with minimized oxidase activity whereas its dye-mediated dehydrogenase activity is higher than the wild-type, and its application for the electrochemical measurement of HbA1c (Kim et al., 2012). The X-ray structure of both of wild-type and mutant PnFPOxs were elucidated, which revealed the existence of the channel where oxygen may access to FAD (Shimasaki et al., 2017). Considering that the protease digestion pretreatment to release the fructoseyl peptide is necessary for the current FPOx based HbA1c assay, flow injection analysis (FIA) would be ideal platform for HbA1c electrochemical sensing system. To avoid the impact of oxygen (1st generation) and the use of electron mediator (2nd generation) in the carrier solution, direct electron transfer principle, the 3rd generation principle, is ideal for FIA based enzyme electrochemical sensing. However, FPOx is not able to transfer electron directly to electrode. In this study, we report our challenge in the development of enzyme electrochemical measurement of HbA1c by FIA system using engineered FPOx based on 2.5th generation principle. We recently reported the electrochemical sensor using mediator-modified enzyme, which doesn’t need external electron acceptor (Hatada et al., 2018), designated as 2.5th generation principle. We first engineered PnFPOx mutant to construct mediator-modified PnFPOx and evaluated the electrochemical behavior of mediator modified-PnFPOx for further application for the FIA system. Method The recombinantly prepared PnFPOx wild type and mutants were modified with amine reactive phenazine ethosulfate (arPES), according to our previous study. The modification by PES was confirmed by dye mediated dehydrogenase activity using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as electron acceptors. Fructosyl valine (FV) was used as the substrate. Result and discussion Most of FAD dependent oxidoreductases, including FPOx, cannot utilize MTT as the primary external electron acceptor, therefore, successful modification of PES at the residues proximate to FAD, can be confirmed by the dye-mediate dehydrogenase activity using MTT as the color indicating dye, which will be occurred by the intra-molecular electron transfer between FAD and covalently bound PES, and by the electron transfer from reduced PES to external MTT. Regarding the PES modification of wild type and N56A mutant according to our previous protocol, no dye-mediated dehydrogenase activity was observed when MTT was used as the electron acceptor. However, cyclic voltammetry (CV) measurements of PES modified wild type and N56A mutant showed typical redox peaks corresponding to PES, revealing that PES molecules were modified on the surface of wild type and N56A mutant FPOxs, but not at the residue(s) proximate to FAD. Therefore, Lys residues were introduced by substituting adequate residue(s) at the proximate of FAD, where no significant impact on structural nor activity would be predicted. As the results, the PES modified lysine introduced mutant showed the dye-mediated dehydrogenase activity with MTT without losing catalytic activity. Accordingly, the double mutant with N56A and lysine, was constructed, which showed the highest dye-mediated dehydrogenase activity with MTT with negligible oxidase activity. This constructed PES-modified wild type and engineered FPOx were then immobilized on the gold electrode using self-assembled monolayer (SAM), and their electrochemical properties were investigated. CV analysis revealed that electrodes with PES-modified lysine introduced mutant enzymes showed catalytic current in the presence of enzyme substrate, FV, but the electrodes with wild type nor N56A did not. The characterization of FIA system will be also reported. References Kim et al., Biotechnol. Bioeng. 106 (3), 358-366 (2010)Kim et al., Biotechnol. Lett. 34, 491-497 (2012)Shimasaki et al., Sci. Rep. 7, 2790 (2017)Hatada et al., Bioelectrochemistry 121, 185-190 (2018)