Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for SMBG sensors due to their superior substrate specificity and insensitivity to oxygen. The 3D structure of AfGDH was already elucidated, revealing that the redox cofactor FAD is buried deeply within the FADGDH protein molecule. Therefore, direct electron transfer (DET) principle is hardly available for FADGDH. Recently, we have reported on the construction of a fusion enzyme with FADGDH with heme b domain derived from extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH). However, to prepare soluble and active fusion protein, the employment of eukaryotic host microorganisms is inevitable considering the structure and nature of heme b domain derived from CDH. Besides, we previously reported that Escherichia coli derived cytochrome b562 (cytb562) served a stable and versatile electron acceptor for variety of oxidoreductase. In this study, we designed a fusion protein between FADGDH and cyt b562, to create a DET type GDH being recombinantly produced using E.coli as the host microorganisms. The designed fusion enzyme was successively produced in E.coli as the soluble protein, showing dye-mediated glucose dehydrogenase activity, with expected molecular weight, showing intramolecular electron transfer between FAD and fused cyt b562. The constructed fusion FADGDH was then immobilized on the glassy carbon electrode to investigate its electrochemical properties. The enzyme electrode with this fusion FADGDH clearly showed rapid increase in the catalytic current toward the addition of glucose, with glucose concentration dependent manner. The same electrode configuration, but with wild type FADGDH without cyt b562 did not result any current increase, though. These results demonstrated that the creation of a DET type FADGDH was achieved, which can be produced using E.coli as the host microorganisms, with the same substrate specificity as the current commercially available glucose sensor.

Construction of fungus FADGDH harboring electron transfer domain and its application for direct electron transfer type enzyme sensor

2019 Spring National Meeting

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