Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain

Abstract

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b₅₆₂ (cyt b₅₆₂) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b₅₆₂ at either the N- or C-terminus of fungal FADGDH, (cyt b₅₆₂-GDH or GDH-cyt b₅₆₂), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b₅₆₂ specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b₅₆₂. Consequently, both cyt b₅₆₂-GDH and GDH-cyt b₅₆₂ showed DET abilities toward electrode. Interestingly, cyt b₅₆₂-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b₅₆₂. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Keywords: 3rd generation glucose sensor; Cytochrome b(562); Direct electron transfer; Enzyme electrode; FAD-Dependent glucose dehydrogenase; Fusion enzymes.