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The Expression and Electroochemical Characterization of Fructose Dehydrogenase
The Expression and Electroochemical Characterization of Fructose Dehydrogenase
Wednesday, May 14, 2014
Grand Foyer, Lobby Level (Hilton Orlando Bonnet Creek)
Redox enzymes and proteins are related to the many vital processes, such as tricarboxylic cycle and respiratory chain. There are only a limited number of redox enzymes and proteins that enable to couple enzyme reaction and electrode reaction without any mediators. In other words, they can transfer the electron to (or from) electrodes directly. This reaction is called direct electron transfer (DET)-type bioelectrocatalysis reaction. DET reaction has received considerable attention for construction of electrochemical applications, such as biosensors and biofuel cells. There must be several factors governing the DET reaction, but the details have not yet clearly elucidated.
D-Fructose dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter japonicus NCBR 3260 is a heterotrimeric membrane-bound enzyme. Subunits I and II have covalently bound flavin adenine dinucleotide (FAD) and three heme C moieties, respectively. FDH shows strict substrate specificity to D-fructose and is used in diagnosis and food analysis. FDH is one of the DET-type enzymes. FDH has high DET activity. DET reaction of FDH occurs at a large variety of electrodes. The FAD in subunit I is the catalytic site to accept electrons form the substrate, and the electrons are transferred to the electrode through the heme C in subunit II.
For the first step to explore the mechanisms of the DET reaction of FDH, we sequenced the genes encoding each subunit of the FDH complex from G. japonicus NBRC3260 and constructed an expression system to highly produce FDH in a G. oxydans strain. We also constructed derivatives modified in subunit I/III subcomplex (ΔcFDH) lacking of the heme C subunit. The recombinant FDH reacts with electrode directly. However, ΔcFDH catalyzes the oxidation of D-fructose with several artificial electron acceptors, but loses the DET ability. The formal potentials (E˚') of the three heme C moieties of FDH have been determined to be –10 ± 4, 60 ± 8 and 150 ± 4 mV (vs. Ag|AgCl|sat. KCl) at pH 5.0, while the onset potential of FDH-catalyzed DET-type bioelectrocatalytic wave is –100 mV. Judging from these results, we conclude that FDH communicates electrochemically with electrodes via the heme C.
D-Fructose dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter japonicus NCBR 3260 is a heterotrimeric membrane-bound enzyme. Subunits I and II have covalently bound flavin adenine dinucleotide (FAD) and three heme C moieties, respectively. FDH shows strict substrate specificity to D-fructose and is used in diagnosis and food analysis. FDH is one of the DET-type enzymes. FDH has high DET activity. DET reaction of FDH occurs at a large variety of electrodes. The FAD in subunit I is the catalytic site to accept electrons form the substrate, and the electrons are transferred to the electrode through the heme C in subunit II.
For the first step to explore the mechanisms of the DET reaction of FDH, we sequenced the genes encoding each subunit of the FDH complex from G. japonicus NBRC3260 and constructed an expression system to highly produce FDH in a G. oxydans strain. We also constructed derivatives modified in subunit I/III subcomplex (ΔcFDH) lacking of the heme C subunit. The recombinant FDH reacts with electrode directly. However, ΔcFDH catalyzes the oxidation of D-fructose with several artificial electron acceptors, but loses the DET ability. The formal potentials (E˚') of the three heme C moieties of FDH have been determined to be –10 ± 4, 60 ± 8 and 150 ± 4 mV (vs. Ag|AgCl|sat. KCl) at pH 5.0, while the onset potential of FDH-catalyzed DET-type bioelectrocatalytic wave is –100 mV. Judging from these results, we conclude that FDH communicates electrochemically with electrodes via the heme C.