Enzyme, enzyme electrode, biosensor, bioreactor, and biofuel cell

Abstract

The enzyme electrode 1 comprises: an electrode base material 6; a first catalyst layer 8 comprising an enzyme (a1) 2 capable of catalyzing the oxidation-reduction reaction of the redox pair NAD(P)H 5 and NAD(P) 4 and directly exchanging electrons accompanying the oxidation-reduction reaction with the electrode base material 6; and a second catalyst layer 9 comprising an NAD(P)H- or NAD(P)-dependent oxidoreductase (b) 3 and NAD(P)H 5 and / or NAD(P) 4. The first catalyst layer 8 and the second catalyst layer 9 are stacked in this order on the electrode base material 6.

Description

Enzymes, enzyme electrodes, biosensors, bioreactors, and biofuel cells

[0001] The present invention relates to an enzyme, an enzyme electrode, a biosensor, a bioreactor, and a biofuel cell.

[0002] In recent years, as interest in energy issues has grown, biofuel cells that use bio-related substances such as sugars and alcohols as fuel have been attracting attention. Biofuel cells use enzymes as electrode catalysts and can generate electricity by combining a fuel oxidation reaction at the anode with a reduction reaction of oxygen or other substances at the cathode.

[0003] At the anode of a biofuel cell, electrons extracted from the fuel by the catalytic function of enzymes are transferred to the electrode. Nicotinamide adenine dinucleotide (NAD)-dependent oxidase and nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidase, which use free nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzymes, are commonly used to oxidize the fuel at the anode. However, the NADH and NADPH produced by these catalytic reactions have very high overvoltages due to direct electrolysis, necessitating the use of catalysts such as redox dyes and o-quinones.

[0004] To address the above-mentioned issues, a method has been proposed in which a substance called an electron mediator is used to catalyze electron transfer between the enzyme and the electrode. An electrode reaction using such an electron mediator is called a mediated electron transfer (MET) enzyme-functional electrode reaction. For example, Patent Documents 1 to 5 disclose biofuel cells that combine an NAD(P)H- or NAD(P)-dependent oxidoreductase with a MET enzyme-functional electrode reaction.

[0005] JP 2004-71559 A JP 2012-151130 A JP 2012-178335 A JP 2009-69085 A JP 2018-68287 A

[0006] As mentioned above, MET-type biofuel cells face the challenge of requiring the electron mediator itself to be immobilized to prevent dissipation from the enzyme-immobilized electrode. In contrast, electrode reactions in which the enzyme can directly transfer electrons to the electrode and do not require an electron transfer mediator to mediate electron transfer between the enzyme and the electrode are called direct electron transfer (DET) enzyme electrode reactions. These reactions do not require the immobilization of an electron mediator and are attracting attention as ideal reaction systems.

[0007] The present invention has been made in view of the above-mentioned current situation, and aims to provide an enzyme electrode that can directly transfer electrons involved in an oxidation-reduction reaction between an electrode substrate and an enzyme.

[0008] The present inventors have conducted extensive research to solve the above problems, and have found that in an electrode comprising an electrode substrate on which a first catalyst layer containing an enzyme that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P) and can directly exchange electrons associated with the redox reaction with the electrode substrate, and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), electrons associated with the redox reaction are directly exchanged between the electrode substrate and the enzyme. This led to the discovery that the above problems can be successfully solved, and has resulted in the present invention.

[0009] The present invention is an enzyme-containing electrode, which comprises an electrode substrate, a first catalyst layer containing an enzyme (a1) that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P) and is capable of directly donating and receiving electrons associated with the redox reaction between the electrode substrate and the first catalyst layer, and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), and the first catalyst layer and the second catalyst layer are laminated in this order on the electrode substrate.

[0010] The present invention also relates to an enzyme that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P), the enzyme having one flavin mononucleotide and two Fe—S clusters, the distance between the flavin mononucleotide and at least one of the Fe—S clusters being within 2 nm, and at least one of the Fe—S clusters being present within 2 nm from the surface of the enzyme.

[0011] The enzyme electrode of the present invention has the above-mentioned configuration and can directly transfer electrons involved in an oxidation-reduction reaction between the electrode substrate and the enzyme, and therefore can be suitably used in biofuel cells, biosensors, bioreactors, etc.

[0012] FIG. 1 is a schematic diagram of one embodiment of the enzyme electrode of the present invention. FIG. 2 is a diagram showing the distance between cofactors in rFoDH1 obtained in Preparation Example 1. FIG. 3 is a diagram showing the distance from the cofactor to the enzyme surface in rFoDH1 obtained in Preparation Example 1. FIG. 4 is a diagram showing the distance from the cofactor to the enzyme surface in rFoDH1β(Me) obtained in Preparation Example 2. FIG. 5 is a diagram showing voltammograms in cyclic voltammetry (CV) measurements using enzyme electrodes 1 and 2 obtained in Preparation Examples 4 and 5. FIG. 6 is a diagram showing a voltammogram in CV measurements performed on enzyme electrode 2 obtained in Preparation Example 5 using a measurement solution containing NAD-dependent glucose dehydrogenase and glucose. FIG. 7 is a diagram showing a voltammogram in CV measurements performed on enzyme electrode 2 obtained in Preparation Example 5 using a measurement solution containing NAD-dependent glycerol dehydrogenase and dihydroxyacetone. FIG. 8 is a diagram showing a voltammogram in CV measurements on enzyme electrode 3 obtained in Preparation Example 6. Fig. 9 is a diagram showing a voltammogram in CV measurement of the enzyme electrode 4 obtained in Preparation Example 7. Fig. 10 is a diagram showing the results of chronoamperometry (CA) measurement of the enzyme electrode 5 obtained in Preparation Example 8. Fig. 11 is a diagram showing voltammograms in CV measurement of the enzyme electrodes 6 and 7 obtained in Preparation Examples 9 and 10.

[0013] The enzyme electrode, enzyme, biosensor, bioreactor, and biofuel cell of the present invention will be described below. However, the present invention is not limited to the following configurations, and can be appropriately modified and applied within the scope of the present invention. Note that a combination of two or more of the individual preferred configurations of the present invention described below also constitutes the present invention.

[0014] [Enzyme Electrode] The enzyme electrode of the present invention comprises an electrode substrate and a first catalyst layer containing an enzyme (a1) (hereinafter also referred to as enzyme (a1)) that catalyzes the redox reaction of a redox pair between the oxidized and reduced forms of nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate and can directly exchange electrons associated with the redox reaction with the electrode substrate. In this specification, the oxidized form of nicotinamide adenine dinucleotide is also referred to as NAD, and its reduced form is also referred to as NADH. The oxidized form of nicotinamide adenine dinucleotide phosphate is also referred to as NADP, and its reduced form is also referred to as NADPH. Furthermore, NAD(P)H means NADH or NADPH, and NAD(P) means NAD or NADP. The enzyme electrode of the present invention further comprises a second catalyst layer containing NAD(P)H or NAD(P)-dependent oxidoreductase (b) (hereinafter also referred to as enzyme (b)) and NAD(P)H and / or NAD(P), and the first catalyst layer and the second catalyst layer are laminated in this order on the electrode substrate.

[0015] The enzyme electrode of the present invention is suitable for use as an anode in a biofuel cell, and NAD(P) receives electrons generated by oxidation of a substrate by the oxidoreductase (b) to form NAD(P)H, which is then oxidized by the enzyme (a1), and the generated electrons can be directly transferred by the enzyme (a1) to an electrode substrate. When used as an anode in a biofuel cell, the enzyme electrode of the present invention does not require an electron mediator for electron transfer between the electrode substrate and the enzyme (a1).

[0016] The enzyme electrode of the present invention may or may not contain an electron mediator, but preferably does not substantially contain one. The content of the electron mediator is preferably 200 mol% or less relative to 100 mol% of the enzyme (a1). More preferably, it is 50 mol% or less, even more preferably 10 mol% or less, particularly preferably 1 mol% or less, and most preferably 0 mol%. The electron mediator refers to a substance that can donate and accept electrons with an enzyme or coenzyme and can also donate and accept electrons with a conductive substrate. However, in this specification, NAD(P)H and NAD(P) are not included in the electron mediator.

[0017] The electron mediator is not particularly limited, and examples thereof include metal elements such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, and W, or metal complexes having an ion of such a metal as a central metal (alkali metal ferricyanides such as ferrocene, potassium ferricyanide, lithium ferricyanide, and sodium ferricyanide, or alkyl-substituted derivatives thereof (methyl-substituted, ethyl-substituted, propyl-substituted, and the like), potassium octacyanotungstate, and the like); quinones such as quinone, benzoquinone, anthraquinone, and naphthoquinone; heterocyclic compounds such as viologen, methyl viologen, benzyl viologen, phenazine methosulfate, phenazine ethosulfate, bipyridine, and derivatives thereof; and 2,6-dichlorophenol indophenol, methylene blue, potassium β-naphthoquinone-4-sulfonate, and vitamin K.

[0018] The enzyme electrode of the present invention is characterized in that the first catalytic layer and the second catalytic layer are laminated in this order on an electrode substrate. The first catalytic layer may contain the enzyme (a1), but may also contain a solvent or a buffer component contained in a buffer solution. Examples of the solvent include aqueous solvents such as water and ethanol. Water is preferred. Examples of the buffer solution component include phosphates such as potassium phosphate and sodium phosphate, imidazole, carbonates, borates, tartrates, citrates, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), and 3-morpholinopropanesulfonic acid (MOPS).

[0019] The second catalyst layer may contain the enzyme (b) and NAD(P)H and / or NAD(P), or may contain a solvent or a buffer component contained in a buffer solution, such as those described for the first catalyst layer.

[0020] In the enzyme electrode of the present invention, the method for laminating the first catalytic layer and the second catalytic layer on the electrode substrate is not particularly limited. Examples of the method for laminating the first catalytic layer and the second catalytic layer on the electrode substrate include a method of applying a composition containing the enzyme (a1) (hereinafter also referred to as the enzyme (a1)-containing composition) to the electrode substrate and drying it. The application method may be any commonly used method, such as spin coating, spraying, screen coating, dip coating, or blade coating.

[0021] The enzyme (a1)-containing composition may contain the enzyme (a1), and may also contain a solvent or a buffer component. The enzyme (a1)-containing composition may also contain an immobilizing agent such as a polymer or a crosslinking agent, and the enzyme (a1) may be more strongly immobilized to the electrode substrate by the immobilizing agent.

[0022] The polymer is not particularly limited, but examples thereof include polyvinylimidazole (PVI), polyallylamine, polyamino acids (e.g., polylysine, etc.), polypyrrole, polyacrylic acid, polyvinyl alcohol, a graft copolymer of polypropylene and maleic anhydride, a copolymer of methyl vinyl ether and maleic anhydride, orthocresol novolac epoxy resin, etc. The crosslinking agent is not particularly limited, but examples thereof include polyethylene glycol diglycidyl ether (PEGDGE), glutaraldehyde, disuccinimide suberate, succinimide-4-(p-maleimidophenyl)butyrate, etc.

[0023] The method for laminating the second catalyst layer on the electrode substrate is not particularly limited, and examples thereof include a method of applying a composition containing the enzyme (b) and NAD(P)H and / or NAD(P) (hereinafter also referred to as the enzyme (b)-containing composition) to the electrode substrate on which the first catalyst layer has been laminated, and then drying the applied composition. The method for applying the enzyme (b)-containing composition is not particularly limited, and examples thereof include a method of applying the enzyme (a1)-containing composition described above.

[0024] The enzyme (b)-containing composition may contain the enzyme (b) and NAD(P)H and / or NAD(P), and may also contain the above-mentioned solvent or buffer component. The enzyme (a1)-containing composition may also contain an immobilizing agent such as the above-mentioned polymer or crosslinking agent, and the enzyme (b) may be more strongly immobilized to the electrode substrate by the immobilizing agent.

[0025] <First catalyst layer> The enzyme (a1) may be any enzyme that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P) and directly transfers electrons associated with the redox reaction between the enzyme (a1) and the electrode substrate, but preferably contains one flavin mononucleotide (hereinafter also referred to as FMN) and at least one Fe—S cluster. FMN and the Fe—S cluster are capable of transferring electrons, and a preferred embodiment of the present invention is one in which the enzyme (a1) contains FMN and the Fe—S cluster as cofactors.

[0026] The number of Fe—S clusters contained in the enzyme (a1) is not particularly limited, but is preferably 1 to 10 per molecule. The number per molecule is more preferably 2 to 8, and even more preferably 3 to 5. In this specification, the number of Fe—S clusters per enzyme molecule can be measured using an atomic absorption spectrometer utilizing the acetylene combustion method.

[0027] The Fe—S cluster is composed of an iron atom and a sulfur atom, and a cysteine ​​residue in the polypeptide is primarily coordinated to the iron atom of the Fe—S cluster. Specific examples of Fe—S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] types. When the enzyme (a1) contains at least one Fe—S cluster, the type is not particularly limited, but it preferably contains a [2Fe-2S] and / or a [4Fe-4S] Fe—S cluster. A form containing a [2Fe-2S] Fe—S cluster is more preferred, a form containing a [2Fe-2S] Fe—S cluster and a [4Fe-4S] Fe—S cluster is even more preferred, and a form containing one each of a [2Fe-2S] and a [4Fe-4S] Fe—S cluster is particularly preferred. In this specification, the number of Fe—S clusters and the number of FMNs, which will be described later, contained in an enzyme refer to the number per enzyme molecule.

[0028] When the enzyme (a1) has FMN and an Fe—S cluster, it is preferable that electrons are transferred from NAD(P)H to the electrode substrate via the FMN and at least one Fe—S cluster in the enzyme (a1). The distance between the FMN and the Fe—S cluster is not particularly limited, but it is preferable that the distance between the FMN and at least one Fe—S cluster is within 2 nm. The distance between the FMN and the Fe—S cluster is the distance between the nearest atoms in both the FMN and the Fe—S cluster. In this specification, the distance between atoms in the enzyme can be calculated based on 3D structure information. Furthermore, 3D structure analysis of the enzyme can be performed by cryo-electron microscopy. Information on the 3D structure of the enzyme may be obtained from a database such as PDB.

[0029] When the enzyme (a1) has two or more Fe—S clusters, the distance between the FMN and the nearest Fe—S cluster is preferably within 2 nm. More preferably, the distance between the FMN and a [2Fe-2S] type Fe—S cluster is within 2 nm. Even more preferably, the distance between the FMN and each of the two Fe—S clusters is within 2 nm.

[0030] The distance between the FMN and the Fe—S cluster is more preferably within 1.5 nm, and in one embodiment, even more preferably within 1 nm.

[0031] When the enzyme (a1) has two or more Fe—S clusters, the distance between at least two Fe—S clusters is preferably within 3 nm, more preferably within 2.5 nm.

[0032] In the enzyme (a1), at least one Fe—S cluster is preferably present within 2 nm from the surface of the enzyme (a1). This shortens the distance between the redox center in the enzyme and the electrode, thereby improving the rate of interfacial electron transfer between the enzyme and the electrode. As used herein, the distance from the surface of the enzyme to an Fe—S cluster or the like refers to the distance between the α-carbon of an amino acid residue present on the surface of the enzyme and an atom of the Fe—S cluster or the like. As used herein, the amino acid residue present on the enzyme surface refers to an amino acid residue in the enzyme (a1) that is accessible to the solvent based on the PDBePISA program (http: / / www.ebi.ac.uk / msd-srv / prot_int / ).

[0033] When the enzyme (a1) has two or more Fe—S clusters, it is preferable that at least two of the Fe—S clusters are present within 2 nm from the surface of the enzyme (a1).An embodiment in which all of the Fe—S clusters contained in the enzyme (a1) are present within 2 nm from the surface of the enzyme (a1) is also one of the preferred embodiments of the present invention.

[0034] A preferred embodiment of the present invention is one in which the enzyme (a1) has one flavin mononucleotide and one or more Fe-S clusters, the distance between the flavin mononucleotide and at least one Fe-S cluster is within 2 nm, and at least one Fe-S cluster is present within 2 nm from the surface of the enzyme (a1). Another preferred embodiment of the present invention is one in which the enzyme (a1) has a [2Fe-2S] type Fe-S cluster within 2 nm from the surface of the enzyme (a1). The Fe-S cluster within 2 nm from FMN and the Fe-S cluster within 2 nm from the enzyme surface may be the same or different, but are preferably the same.

[0035] The enzyme (a1) preferably has a β subunit having one flavin mononucleotide and one or more Fe—S clusters.

[0036] The enzyme (a1) preferably has two Fe—S clusters in the β subunit, more preferably a [2Fe-2S] type Fe—S cluster and a [4Fe-4S] type Fe—S cluster in the β subunit.

[0037] The enzyme (a1) preferably contains the β subunit of formate dehydrogenase derived from a methanol-utilizing bacterium. The methanol-utilizing bacterium is not particularly limited, but is preferably a bacterium of the genus Methylorubrum, more preferably Methylorubrum extorquens, and even more preferably Methylorubrum extorquens AM1. The amino acid sequence of the α subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 is shown in SEQ ID NO: 1. The amino acid sequence of the β subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 is shown in SEQ ID NO: 2.

[0038] The enzyme (a1) may further have an α subunit having at least one Fe—S cluster. The type of Fe—S cluster possessed by the α subunit is not particularly limited, but is preferably a [4Fe-4S] type and / or a [2Fe-2S] type. The number of Fe—S clusters possessed by the α subunit is not particularly limited, but is preferably 1 to 5, more preferably 1 to 4, particularly preferably 2 to 4, and most preferably 4. An embodiment in which the α subunit has three [4Fe-4S] type Fe—S clusters and one [2Fe-2S] type Fe—S cluster is one of the preferred embodiments of the present invention.

[0039] Preferably, the α subunit further comprises tungsten pterin, which is a pterin complex containing tungsten as a central metal. Tungsten pterin can serve as an active center that catalyzes an oxidation-reduction reaction using, for example, formic acid or carbon dioxide as a substrate. When the enzyme (a1) comprises tungsten pterin, it can transfer electrons obtained from a substrate such as formic acid to an electrode substrate, or transfer electrons transferred from the electrode substrate to a substrate such as carbon dioxide.

[0040] When the enzyme (a1) has a β subunit and an α subunit, the positional relationship between them is not particularly limited, but the distance between at least one Fe—S cluster in the β subunit and at least one Fe—S cluster in the α subunit is preferably within 2 nm, more preferably within 1 nm.

[0041] The enzyme (a1) may or may not contain the α subunit of formate dehydrogenase derived from a methanol-utilizing bacterium.

[0042] The enzyme (a1) preferably has an average molecular weight of 50,000 or more and 500,000 or less. This can further improve the electron transfer rate between the enzyme (a1) and the electrode substrate. When the enzyme (a1) is composed of only the β subunit, the average molecular weight is more preferably 50,000 or more and 100,000 or less, and even more preferably 60,000 or more and 80,000 or less. When the enzyme (a1) contains the α subunit, the average molecular weight is more preferably 150,000 or more and 300,000 or less, and even more preferably 160,000 or more and 200,000 or less. The average molecular weight of the enzyme can be measured by gel filtration chromatography.

[0043] The enzyme (a1) preferably has an amino acid sequence that has 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2. The sequence identity is more preferably 80% or more, and even more preferably 90% or more. The sequence identity of the amino acid sequence can be calculated using the BLAST-P program provided by NCBI.

[0044] The surface charge of the enzyme (a1) is not particularly limited, but it is preferable that the portion located within 2 nm from the active center be positively or negatively charged. In this case, for example, by modifying the surface of the electrode substrate with a surface modification group having a charge opposite to that of the enzyme (a1), the enzyme (a1) can be immobilized so that the active center of the enzyme (a1) is oriented toward the electrode substrate. This can further increase the electron transfer rate between the enzyme (a1) and the electrode substrate. More preferably, the surface charge of the portion of the surface of the enzyme (a1) located within 2 nm from the active center is negative. Particularly preferably, the surface charge of the portion located within 2 nm from the Fe—S cluster contained in the enzyme (a1) is negative.

[0045] The enzyme (a1) may be any enzyme that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P), but is preferably an enzyme that has catalytic activity in the oxidation reaction of NAD(P)H. The enzyme activity in the oxidation reaction of NAD(P)H is not particularly limited, but is preferably 0.1 units or more per milligram of enzyme. The enzyme activity is more preferably 0.5 units or more, even more preferably 1.0 units or more, and particularly preferably 1.5 units or more. The enzyme activity can be measured by a solution enzyme activity evaluation method using NADH as a substrate.

[0046] The amount of the enzyme (a1) contained in the first catalyst layer is not particularly limited, but is preferably 0.05 pmol / cm 2 relative to the surface area of ​​the electrode substrate. ２ Above, 1000 pmol / cm ２ It is preferably 0.5 pmol / cm or less, and more preferably 0.5 pmol / cm ２ Above, 100 pmol / cm ２ More preferably, it is 5 pmol / cm or less. ２ Above, 10 pmol / cm ２ The following is the result.

[0047] The enzyme (a1) is not particularly limited in terms of origin, so long as it has the catalytic function. For example, it may be a biological molecule extracted from animals, plants, microorganisms, etc., or may be synthesized by genetic engineering or chemical means. It may be a naturally occurring molecule prepared by appropriate protein isolation and purification techniques from any organism, such as naturally occurring bacteria, yeast, or animals and plants, or may be produced as a recombinant organism by genetic engineering techniques, or may be chemically synthesized. Alternatively, the first catalyst layer may be made of an organism itself, such as a microorganism, organelle, or cell, containing the enzyme (a1). Alternatively, a crude product from such an organism may be used in the first catalyst layer.

[0048] When the enzyme (a1) is produced as a recombinant by genetic engineering techniques, methods commonly used in the art can be used, such as synthesizing an appropriate probe DNA based on the amino acid sequence of formate dehydrogenase derived from Methylorubrum extorquens AM1 (the amino acid sequences shown in SEQ ID NOs: 1 and 2) and using this to select a formate dehydrogenase gene from a chromosomal DNA or cDNA library, or preparing appropriate primer DNA based on the amino acid sequence and amplifying DNA containing a target gene fragment by a suitable polymerase chain reaction (PCR) such as 5' RACE or 3' RACE, and then ligating these DNA fragments to obtain DNA containing the full-length of the target gene.

[0049] The gene encoding the enzyme (a1) may be ligated or inserted into various vectors, or may be incorporated into a chromosome or genome. When a vector is used, commercially available kits such as TA Cloning Kit (Invitrogen) and In-Fusion HD Cloning Kit (Clontech) can be used for cloning into the vector; commercially available plasmid vector DNAs such as pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio), pBluescript SK+ (Stratagene), pYES2 / CT (Invitrogen), and pET21a(+); and commercially available bacteriophage vector DNAs such as λEMBL3 (Stratagene). Alternatively, vectors for homologous recombination such as pK18mobsacB (Schaefer et al., Gene, vol. 45, pp. 69-73 (1994)) and pCM1682 (H. Iguchi et al., Environ. Microbiol. Rep. 10 (2018) 634-643) can be used. Using the recombinant DNA obtained in this manner, host organisms such as Methylorubrum extorquens, E. coli DE3 strain, E. coli JM109 strain (Takara Bio Inc.), E. coli DH5α strain (Takara Bio Inc.) and other Escherichia coli can be transformed.

[0050] The transformant obtained as described above is preferably cultured under conditions that allow expression of the introduced gene, and the enzyme (a1) is isolated and purified from the transformant culture. The method for isolating and purifying the enzyme (a1) is not particularly limited, and conventional protein isolation and purification methods can be used. Examples of isolation and purification methods include known isolation and purification techniques, such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, and various types of chromatography, such as hydrophobic, anionic, cationic, and affinity chromatography, which can be used alone or in appropriate combinations. In particular, when affinity chromatography is used, it is also preferable to express the enzyme (a1) as a fusion protein with a tag peptide, such as a histidine tag (His-Tag), and utilize its affinity for such tag peptide.

[0051] Furthermore, when the enzyme (a1) is produced as a recombinant, a mutation may be introduced into the amino acid sequence of formate dehydrogenase derived from Methylorubrum extorquens AM1 shown in SEQ ID NO: 1. When introducing a mutation, a person skilled in the art can predict a mutation that will alter the surface charge while maintaining the enzyme activity, based on information on the three-dimensional structure of the formate dehydrogenase and the properties of the amino acids.

[0052] <Second catalytic layer> The NAD(P)H- or NAD(P)-dependent oxidoreductase (b) (hereinafter also referred to as enzyme (b)) contained in the second catalytic layer may be an enzyme that catalyzes an oxidation-reduction reaction of a substrate using NAD(P)H or NAD(P) as an electron acceptor. When the electrode of the present invention is an anode, it may be an oxidase (dehydrogenase) of the substrate, and when the electrode of the present invention is a cathode, it may be a reductase of the substrate.

[0053] The substrate is not particularly limited, and examples thereof include alcohols, sugars, fats, polyamino acids such as peptides and proteins, organic acids, etc. One or more of these may be used.

[0054] The alcohol is not particularly limited, but examples thereof include monohydric alcohols such as methanol, ethanol, n-propyl alcohol, iso-propyl alcohol, n-butyl alcohol, sec-butyl alcohol, and tert-butyl alcohol, and polyhydric alcohols such as ethylene glycol, diethylene glycol, and glycerol (glycerin).

[0055] The sugars are not particularly limited, but include glucose, glucose-1, D-glucose, L-glucose, glucose-6-phosphate, lactate, lactate-6-phosphate, D-lactate, L-lactate, fructose, galactose-1, galactose, aldose, sorbose, mannose, and the like.

[0056] The organic acid is not particularly limited, and examples thereof include glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvic acid, pyruvic acid, acetyl-CoA, citric acid, cis-aconitic acid, isocitric acid, oxalosuccinic acid, 2-oxoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, L-malic acid, and oxaloacetic acid, as well as intermediates in sugar metabolism.

[0057] The fats are not particularly limited as long as they are esters of fatty acids and glycerin, and the fatty acids constituting the fats are also not particularly limited, and may be saturated fatty acids, monounsaturated fatty acids, or polyunsaturated fatty acids.

[0058] The substrate is more preferably an alcohol or a sugar, and even more preferably glycerol.

[0059] The enzyme (b) is preferably an oxidase. More preferred examples of the enzyme (b) include glycerol dehydrogenase, glucose dehydrogenase, a series of enzymes in the electron transport system, ATP synthase, and enzymes involved in sugar metabolism (e.g., hexokinase, glucose phosphate isomerase, phosphofructokinase, fructose bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate mutase, phosphopyruvate hydratase, pyruvate kinase, L-lactate dehydrogenase, D-lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malonate dehydrogenase). One or more of these enzymes can be used. Among these, glycerol dehydrogenase and glucose dehydrogenase are preferred.

[0060] In one embodiment, the second catalyst layer may contain a plurality of types of the enzyme (b), and the substrate can be decomposed in stages by the plurality of enzymes (b).

[0061] The enzyme (b) is not particularly limited in terms of origin, etc., as long as it has the catalytic function. It may be a naturally occurring enzyme purified from a naturally occurring organism by an appropriate protein isolation and purification technique, or it may be a recombinant enzyme produced by genetic engineering techniques or a chemically synthesized enzyme. Commercially available products may also be used. Examples of methods for producing a recombinant enzyme by genetic engineering techniques include methods similar to those for producing enzyme (a1). Alternatively, organisms themselves, such as microorganisms, organelles, and cells, containing enzyme (b), may be used in the second catalyst layer. Alternatively, crude products from these organisms may be used in the second catalyst layer.

[0062] The amount of the enzyme (b) contained in the second catalyst layer is not particularly limited, but is preferably 0.01 mol % or more and 10,000 mol % or less, more preferably 0.1 mol % or more and 1,000 mol % or less, and even more preferably 1 mol % or more and 100 mol % or less, relative to 100 mol % of the enzyme (a1).

[0063] The amount of NAD(P)H or NAD(P) contained in the second catalyst layer is not particularly limited, but the total amount of NAD(P)H and NAD(P) is preferably 0.5 mol % or more and 50,000 mol % or less, more preferably 5 mol % or more and 5,000 mol % or less, and even more preferably 50 mol % or more and 500 mol % or less, relative to 100 mol % of the enzyme (a1).

[0064] <Electrode substrate> The electrode substrate provided in the enzyme electrode of the present invention is a conductive substrate that can be connected to an external circuit and can transfer electrons. The electrode substrate is not particularly limited in material or shape, as long as it has the relevant properties. The material of the electrode substrate may be a conductive material, for example, carbon materials such as carbon cloth, carbon paper, graphite, glassy carbon, activated carbon, carbon black, and carbon nanotubes; metals or alloys such as gold, platinum, copper, palladium, titanium, aluminum, silver, and nickel; SnO ２ , In ２ O ３ , W.O. ３ , TiO ２ The electrode substrate may be composed of a single layer of one of these, or may be composed of a laminated structure of two or more layers.

[0065] The carbon material is preferably activated carbon, carbon black, or carbon nanotubes. The use of a carbon material as a conductive material can further improve conductivity with the electrode. The carbon material is preferably in particulate form. In one preferred embodiment of the present invention, the enzyme electrode of the present invention comprises at least one type of carbon particle selected from the group consisting of activated carbon, carbon black, and carbon nanotubes.

[0066] The metal is preferably gold, platinum, copper, palladium, titanium, etc. In one preferred embodiment of the present invention, the enzyme electrode of the present invention contains at least one metal selected from the group consisting of gold, platinum, copper, palladium, and titanium.

[0067] In the electrode substrate, for example, when two or more of the conductive materials are used, a binder such as a polymer may be used. The polymer is not particularly limited, but may include fluorine-containing polymers such as polyvinylidene fluoride (PVDF) and polyvinyl fluoride (PVF), copolymers thereof, and copolymers of these monomers with ethylene or styrene. Other examples include polymers such as polystyrene, polyethylene, and polypropylene, as well as hydrophilic polymers such as polyacrylic acid, polylysine, and carboxymethyl cellulose, and conductive polymers such as polyaniline, polypyrrole, and their derivatives, polyaniline sulfonic acid. Among these, conductive polymers are preferred, and polyaniline and polypyrrole are more preferred. A preferred embodiment of the present invention is an enzyme electrode of the present invention that includes at least one selected from the group consisting of polyaniline and polypyrrole.

[0068] The enzyme electrode of the present invention preferably contains carbon particles, a metal, or a conductive polymer as a conductive electrode material. In one preferred embodiment of the present invention, the enzyme electrode of the present invention contains at least one material selected from the group consisting of carbon particles, a metal, and a conductive polymer.

[0069] The electrode base material may have a flat substrate surface or may have irregularities or pores on the substrate surface, but preferably has pores. This allows the first catalytic layer and the second catalytic layer to be sufficiently fixed by the electrode base material. The size of the pores is not particularly limited, but is preferably 1 nm or more and 100 nm or less, and more preferably 2 nm or more and 10 nm or less.

[0070] The electrode substrate may or may not have a substrate surface coated with a surface-modifying group, but a coated form is preferred. The surface-modifying group is preferably one that can interact with the enzyme (a1) through intermolecular forces, hydrogen bonds, Coulomb forces, etc., or one that can be covalently bonded to the enzyme (a1). The surface-modifying group is more preferably one that interacts with the enzyme (a1) through Coulomb forces, and even more preferably one that has a charge opposite to the surface charge of the enzyme (a1). When the enzyme (a1) has a positive surface charge, the surface-modifying group is preferably anionic. When the enzyme (a1) has a negative surface charge, the surface-modifying group is preferably cationic. Particularly preferably, the surface-modifying group has a charge opposite to the surface charge of the portion of the enzyme (a1) that is located within 2 nm of the Fe—S cluster contained in the enzyme (a1).

[0071] The surface modifying group is not particularly limited, and examples thereof include functional groups or reactive groups such as amine groups, sulfonic acid groups, sulfate groups, phosphate groups, sulfhydryl groups, carboxyl groups, and salt groups thereof, thiol groups, hydroxyl groups, azido groups, azo groups, nitro groups, nitrile groups, cyano groups, allene groups, isonitrile groups, urea groups, aldehyde groups, ketone groups, NHS esters, imidoesters, maleimides, pyridyldithiols, allyl azides, haloacetates, isocyanates, carbodiimides, allyl azides, diazirines, hydrazides, psoralens, iodides, pyridine disulfides, and vinyl sulfones. The surface modifying group is preferably one having an anionic group or a cationic group. The anionic group is a group having an anion or a group capable of releasing a hydrogen ion to form an anion, and examples thereof include carboxyl groups, sulfonic acid groups, sulfate groups, phosphate groups, and salt groups thereof. The cationic group is a group having a cation or a group capable of accepting a hydrogen ion to form a cation, and examples thereof include amine groups such as primary to tertiary amino groups, neutralized products of primary to tertiary amino groups with acids such as hydrochloric acid and acetic acid, and quaternary ammonium salt groups.

[0072] The surface-modifying group-containing compound used to modify the surface of the electrode substrate is preferably a compound having an anionic group or a cationic group and a structure capable of interacting with the surface of the electrode substrate. When the surface of the electrode substrate is a carbon material, the compound preferably has a structure capable of π-π interaction with the carbon material. Examples of structures capable of π-π interaction include structures derived from aromatic compounds and heterocyclic compounds. The aromatic compound may have a heteroatom. A polycyclic aromatic compound is more preferred as the aromatic compound. The surface-modifying group-containing compound is preferably an aromatic compound having an anionic group or a cationic group, or a heterocyclic compound having an anionic group or a cationic group.

[0073] Examples of the polycyclic aromatic compound include pyrene, coronene, chrysene, naphthacene, pentacene, picene, perylene, anthracene, phenanthrene, fluorene, naphthalene, fluoranthene, acenaphthene, acenaphthylene, triphenylene, and derivatives thereof.

[0074] Examples of aromatic compounds or heterocyclic compounds having a heteroatom include terthiophene, tetraphenylbenzidine, tetraphenylnaphthacene, benzothiophene, thiophene, pyrrole, carbazole, phenanthroline, phenylpyridine, quinoline, triphenylamine, diphenylamine, oxazole, oxadiazole, quinacridone, fulcrenone, phthalocyanine, spiropyran, viologen, spiroperimidine, benzoic acid, benzophenone, phenylamine, diphenyl ether, diphenyl sulfide, diphenyl sulfone, bisphenol, anthraquinone, phosphonium compounds, fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof.

[0075] The surface-modifying group-containing compound is preferably a pyrene derivative. Examples of pyrene derivatives include those having the functional group or reactive group described above, and more preferably those having an anionic group or cationic group. Specific examples include 1-pyrenemethylamine, 1-aminopyrene, dimethyl-pyren-1-yl-methylamine, and diethyl-pyren-1-yl-amine. The functional group or reactive group and the pyrene may be separated by a spacer such as an alkyl group or polyethylene glycol. Modifications such as these functional groups, reactive groups, and spacers may be bonded to any carbon atom of the pyrene.

[0076] The amount of the surface modifying group-containing compound used is not particularly limited, but is preferably 0.05 pmol / cm relative to the surface area of ​​the electrode substrate. ２ Above, 1000 pmol / cm ２ It is preferably 0.5 pmol / cm or less, and more preferably 0.5 pmol / cm ２ Above, 100 pmol / cm ２ More preferably, it is 5 pmol / cm or less. ２ Above, 10 pmol / cm ２ The following is the result.

[0077] Hereinafter, an embodiment of the enzyme electrode of the present invention will be described with reference to the drawings.

[0078] 1 is a schematic diagram of one embodiment of the enzyme electrode of the present invention. The enzyme electrode 1 comprises an electrode substrate 6, a first catalyst layer 8 containing an enzyme (a1)2 that catalyzes the redox reaction of a redox pair of NAD(P)H5 and NAD(P)4 and can directly exchange electrons associated with the redox reaction with the electrode substrate 6, and a second catalyst layer 9 containing an enzyme (b)3 and NAD(P)H5 and / or NAD(P)4. When the enzyme electrode 1 of the present invention serves as an anode, electrons generated by oxidation of a substrate 7 by the enzyme (b)3 are received by NAD(P)4 to form NAD(P)H5, which is then oxidized by the enzyme (a1)2, and the generated electrons can be directly transferred to the electrode substrate 6 by the enzyme (a1)2.

[0079] [Oxidoreductase (a2)] The present invention also provides an enzyme that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P), the enzyme having one flavin mononucleotide and two Fe—S clusters, the distance between the flavin mononucleotide and at least one of the Fe—S clusters being within 2 nm, and at least one of the Fe—S clusters being present within 2 nm from the surface of the enzyme (hereinafter also referred to as enzyme (a2)).

[0080] The enzyme (a2) is not particularly limited as long as it has the above-mentioned configuration, but it is preferable that when used in an electrode, it can directly exchange electrons with the electrode substrate in the oxidation-reduction reaction.

[0081] The types of the two Fe—S clusters in the enzyme (a2) are not particularly limited, but preferably contain a [2Fe-2S] type and / or a [4Fe-4S] type Fe—S cluster, more preferably a [2Fe-2S] type Fe—S cluster, and even more preferably a form containing one each of a [2Fe-2S] type and a [4Fe-4S] type Fe—S cluster.

[0082] In the enzyme (a2), the distance between the FMN and the nearest Fe—S cluster is preferably within 2 nm. More preferably, the distance between the FMN and the [2Fe-2S] type Fe—S cluster is within 2 nm. Even more preferably, the distance between the FMN and each of the two Fe—S clusters is within 2 nm.

[0083] The average molecular weight of the enzyme (a2) is not particularly limited, but is preferably 50,000 or more and 100,000 or less. This further improves the electron transfer rate between the enzyme (a2) and the electrode substrate when the enzyme (a2) is used in an electrode. The average molecular weight of the enzyme (a2) is more preferably 60,000 or more and 80,000 or less.

[0084] The enzyme (a2) preferably has a sequence identity of 70% or more with the amino acid sequence shown in SEQ ID NO: 2. The sequence identity is more preferably 80% or more, and even more preferably 90% or more. The sequence identity of the amino acid sequence can be calculated using the BLAST-P program provided by NCBI.

[0085] The surface charge of the enzyme (a2) is not particularly limited, but it is preferable that the portion within 2 nm from the active center is positively or negatively charged. In this case, when the enzyme (a2) is used in an electrode, for example, the surface of the electrode substrate can be modified with a surface modification group having a charge opposite to the surface charge of the enzyme (a2), thereby immobilizing the enzyme (a2) so that the active center of the enzyme (a2) is oriented toward the electrode substrate. This can further increase the electron transfer rate between the enzyme (a2) and the electrode substrate. More preferably, the surface charge of the portion of the surface of the enzyme (a2) within 2 nm from the active center is negative. Particularly preferably, the surface charge of the portion within 2 nm from the Fe—S cluster contained in the enzyme (a2) is negative.

[0086] The enzyme (a2) is not particularly limited in terms of origin, etc., as long as it has the above-mentioned configuration. It may be a naturally occurring enzyme purified from a naturally occurring organism by an appropriate protein isolation and purification technique, or it may be a recombinant enzyme produced by genetic engineering techniques or a chemically synthesized enzyme.

[0087] When the enzyme (a2) is produced as a recombinant by genetic engineering techniques, it can be produced, for example, using a method similar to the method for producing the enzyme (a1) described above, based on the amino acid sequence of the β subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 (the amino acid sequence shown in SEQ ID NO: 2). When the enzyme (a2) is used in electrodes, etc., organisms themselves, such as microorganisms, organelles, and cells, containing the enzyme (a2), may be used. Alternatively, crude products from these organisms may be used.

[0088] When the enzyme (a2) is produced as a recombinant, a mutation may be introduced, for example, into the amino acid sequence shown in SEQ ID NO: 2 of the β subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1, as described for the enzyme (a1).

[0089] The enzyme (a2) can be suitably used as an electrode for a fuel cell, etc. An embodiment in which the enzyme (a1) in the enzyme electrode of the present invention is the enzyme (a2) is also one of the suitable embodiments of the present invention.

[0090] [Biosensor] The present invention also relates to a biosensor equipped with the enzyme electrode of the present invention. The biosensor of the present invention preferably comprises the enzyme electrode of the present invention as a working electrode and a counter electrode. Measurement using the biosensor is performed by contacting a measurement sample with the biosensor, causing an oxidation-reduction reaction between the enzyme (b) contained in the enzyme electrode of the present invention and the substance to be measured, and detecting the resulting current. The presence or absence or concentration of a substrate in a sample can be determined based on the response current value.

[0091] Measurement methods using the biosensor of the present invention include commonly used methods such as chronoamperometry, which measures oxidation current or reduction current, or coulometry and cyclic voltammetry.

[0092] [Bioreactor] The present invention also relates to a bioreactor equipped with the enzyme electrode of the present invention. The bioreactor of the present invention is not particularly limited as long as the enzyme electrode of the present invention acts as a reaction site with the reactant, but in one embodiment, it is preferable that the enzyme electrode of the present invention is installed in a column reactor. In the above embodiment, when a solution containing the reactant is passed through the column reactor and brought into contact with the enzyme electrode, a product is obtained from the reactant through an enzymatic reaction caused by the enzyme (b) contained in the enzyme electrode of the present invention. The reactant applied to the bioreactor is not particularly limited, and may be a substrate that can be oxidized or reduced by the enzyme (b). Specific examples include the substrates described above.

[0093] [Biofuel Cell] The present invention also relates to a biofuel cell comprising the enzyme electrode of the present invention. The enzyme electrode of the present invention in the biofuel cell may be either an anode or a cathode, but is preferably an anode. The biofuel cell of the present invention is not particularly limited as long as it comprises the enzyme electrode of the present invention and the anode and cathode are connected by an external circuit, but it is preferable that it is configured to include a diaphragm separating the anode and the cathode.

[0094] In one embodiment, the biofuel cell of the present invention preferably comprises an anode made of the enzyme electrode of the present invention, a cathode, and a diaphragm separating the anode and the cathode.

[0095] The cathode in a biofuel cell according to one embodiment of the present invention can be an enzyme catalyst, such as a multi-copper enzyme (e.g., pyruvate oxidase, ascorbate oxidase, or laccase), or a metal catalyst (e.g., platinum). When an enzyme catalytic mechanism is used for the reaction on the cathode side, the enzyme may be immobilized on an electrode substrate, or may be supplied to an appropriate electrode substrate as an enzyme solution without being immobilized. In this case, the electrode substrate can be the same as the electrode substrate described above for the enzyme electrode of the present invention.

[0096] The diaphragm may be made of any material or have any shape, so long as it has ionic conductivity that allows it to pass through protons and other ions, while preventing the passage of components on the negative electrode side and components on the positive electrode side other than ions such as protons. For example, a cellulose membrane or a solid electrolyte membrane may be used. Examples of solid electrolyte membranes include solid membranes with ion exchange functions, such as organic polymers having strong acid groups such as sulfo groups, phosphate groups, phosphonic groups, and phosphine groups, weak acid groups such as carboxy groups, and polar groups, but are not limited thereto. Specifically, cellulose membranes and perfluorocarbon sulfonic acid (PFS)-based resin membranes, such as Nafion®, a copolymer of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinyl ether], can be used.

[0097] The present specification discloses the following:

[0098] <1> An enzyme-containing electrode, the electrode comprising: an electrode substrate; a first catalyst layer containing an enzyme (a1) that catalyzes a redox reaction of a redox pair of NAD(P)H and NAD(P) and is capable of directly donating and receiving electrons associated with the redox reaction between the electrode substrate and the first catalyst layer; and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), the first catalyst layer and the second catalyst layer being laminated in this order on the electrode substrate.

[0099] <2> The enzyme electrode according to <1>, wherein the enzyme (a1) has one flavin mononucleotide and one or more Fe—S clusters, the distance between the flavin mononucleotide and at least one Fe—S cluster is within 2 nm, and the at least one Fe—S cluster is present within 2 nm from the surface of the enzyme (a1).

[0100] <3> The enzyme electrode according to <1> or <2>, wherein the enzyme (a1) includes a β subunit of formate dehydrogenase derived from a methanol-utilizing bacterium.

[0101] <4> The enzyme electrode according to any one of <1> to <3>, wherein the surface of the electrode base material is coated with a surface modifying group, and the surface modifying group has a charge opposite to the surface charge of a portion of the surface of the enzyme (a1) that is located within 2 nm from an Fe—S cluster contained in the enzyme (a1).

[0102] <5> The enzyme electrode according to any one of <1> to <4>, wherein the enzyme electrode comprises at least one selected from the group consisting of carbon particles, metals, and conductive polymers, the carbon particles being at least one selected from the group consisting of activated carbon, carbon black, and carbon nanotubes, the metal being at least one selected from the group consisting of gold, platinum, copper, palladium, and titanium, and the conductive polymer being at least one selected from the group consisting of polyaniline and polypyrrole.

[0103] <6> The enzyme electrode according to any one of <1> to <5>, wherein the substrate of the NAD(P)H or NAD(P)-dependent oxidoreductase (b) is glycerol.

[0104] <7> A biosensor comprising the enzyme electrode according to any one of <1> to <6>.

[0105] <8> A bioreactor comprising the enzyme electrode according to any one of <1> to <6>.

[0106] <9> A biofuel cell comprising the enzyme electrode according to any one of <1> to <6>.

[0107] <10> An enzyme that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P), the enzyme having one flavin mononucleotide and two Fe—S clusters, the distance between the flavin mononucleotide and at least one of the Fe—S clusters being within 2 nm, and at least one of the Fe—S clusters being present within 2 nm from the surface of the enzyme.

[0108] <11> The enzyme according to <10>, which, when used in an electrode, can directly exchange electrons with an electrode substrate during the oxidation-reduction reaction.

[0109] <12> The enzyme according to <10> or <11>, having an average molecular weight of 50,000 to 100,000.

[0110] <13> The enzyme according to any one of <10> to <12>, which is a β subunit of formate dehydrogenase derived from a methanol-utilizing bacterium.

[0111] EXAMPLES The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0112] Preparation Example 1: Enzyme (a1) (rFoDH1) A recombinant formate dehydrogenase derived from Methylorubrum extorquens AM1 (hereinafter also referred to as rFoDH1) was prepared as follows. A formate dehydrogenase gene-disrupted strain of Methylorubrum extorquens AM1 was prepared. The formate dehydrogenase gene, the methanol dehydrogenase subunit 1 precursor gene (promoter), and a His-tag sequence for enzyme purification were inserted into a genomic DNA transformation plasmid (pCM1682). The resulting plasmid and the disrupted strain were mixed and transformed by electroporation. After culturing the transformed strain, the cells were disrupted and the resulting suspension was centrifuged. The resulting supernatant was applied to a Ni-NTA Agarose (QIAGEN) column to purify the enzyme, yielding a recombinant (rFoDH1).

[0113] Based on the above-described three-dimensional structural analysis of rFoDH1, the distances between cofactors in rFoDH1 are shown in Figure 2, and the distances from each cofactor to the protein surface are shown in Figure 3. In Figures 2 and 3, the [4Fe-4S] Fe-S clusters in the α subunit of rFoDH1 are represented as A1, A2, and A3, and the [2Fe-2S] Fe-S cluster is represented as A4. In Figures 2 to 4, the [4Fe-4S] Fe-S cluster in the β subunit of rFoDH1 or FoDH1B is represented as B1, and the [2Fe-2S] Fe-S cluster is represented as B2. The numerical values ​​in Figures 2 to 4 are in angstroms.

[0114] Preparation Example 2: Enzyme (a2) (rFoDH1β(Me)) The β subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 (hereinafter also referred to as FoDH1B) was prepared as follows. A formate dehydrogenase gene-disrupted strain of Methylorubrum extorquens AM1 was prepared. The formate dehydrogenase β subunit gene, methanol dehydrogenase subunit 1 precursor gene, and His-tag sequence were inserted into a genomic DNA transformation plasmid (pCM1682), and the resulting plasmid and the disrupted strain were mixed and transformed by electroporation. After culturing the transformed strain, the enzyme was purified in the same manner as in Preparation Example 1 to obtain a recombinant (rFoDH1β(Me)).

[0115] Preparation Example 3: Enzyme (a2) (rFoDH1β(Ec)) The β subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 (hereinafter also referred to as rFoDH1β(Ec)) was prepared by heterologous expression in Escherichia coli as follows. CAT was added to the 5' end of the gene encoding FoDH1B, and the stop codon at the 3' end was replaced with CTCGAG. This was then introduced into the pET21a(+) vector using restriction enzymes NdeI and XhoI to obtain the plasmid pET21a-FoDHIβ. The resulting pET21a-FoDHIβ was introduced into the protein expression Escherichia coli ArcticExpress(DE3) strain (Agilent 230192) according to the attached protocol to construct a plasmid-introduced strain. As a preculture, the above-mentioned plasmid-introduced strain was inoculated into 5 ml of TB medium (containing antibiotics (100 μg / ml ampicillin + 20 μg / ml gentamicin)) containing iron citrate at a final concentration of 0.3 mM and cultured overnight at 37°C, 200 rpm. The resulting culture was diluted 100-fold into 20 ml of TB medium (containing antibiotics and 0.3 mM iron citrate) in a 50 ml unbaffled flask and cultured at 37°C, 200 rpm for 3.5 hours. After that, the culture flask was cooled in ice water, and isopropyl-β-D(-)-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, followed by overnight culture at 15°C, 200 rpm. The resulting culture medium was subjected to cell disruption, and the resulting suspension was centrifuged. The resulting supernatant was purified by immobilized metal affinity chromatography (TALON (registered trademark) 2 ml Disposable Gravity Column, manufactured by Takara Bio Inc.), and the buffer was exchanged with 100 mM Tris-HCl pH 8.0 using a centrifugal ultrafiltration filter unit Vivaspin (registered trademark) Turbo 15 10,000 MWCO (Sartorius) to obtain rFoDH1β(Ec).

[0116] <Measurement of NADH oxidation activity> The NADH oxidation activity of rFoDH1, rFoDH1β(Me), and rFoDH1β(Ec) obtained in Preparation Examples 1 and 2 was measured by the following method. Using a cuvette with an optical path length of 1 cm, the measurement was carried out at room temperature (25±2°C) in 100 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM 2,6-Dichlorophenolindophenol sodium salt (manufactured by MP Biomedical Co., Ltd., hereinafter also referred to as DCIP) and 1 mM NADH. The change in absorbance at 600 nm accompanying the reduction of DCIP was measured, and the molar extinction coefficient at 600 nm (20.6 mM －１ cm －１ Enzyme activity was calculated from the results of the analysis at pH 7.0 (J. McD. Armstrong, Biochim. Biophys. Acta 86 (1964) 194-197). Protein quantification was performed using a BCA Protein Assay (Thermo). NADH oxidation measurements revealed that the specific activities of rFoDH1, rFoDH1β(Me), and rFoDH1β(Ec) were 4.1±0.6 U / mg, 3.0±0.7 U / mg, and 24.7±1.7 U / mg, respectively.

[0117] Preparation Example 4: Enzyme Electrode 1 Containing Enzyme (a1) (rFoDH1) A glassy carbon electrode (3 mm diameter, manufactured by BAS, hereinafter also referred to as GCE) was polished with alumina particles having an outer diameter of 1.0 μm and 0.05 μm, and then ultrasonically cleaned with ultrapure water. Next, 10 mg of multi-walled carbon nanotubes (manufactured by Sigma-Aldrich, outer diameter: 10±1 nm, length: 3-6 μm, hereinafter also referred to as MWCNT) were added to 1-methylpyrrolidone (NMP), and the suspension was subjected to ultrasonic treatment for 2 hours. 10 μL of the uniformly dispersed MWCNT dispersion was added dropwise to the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was then dissolved in 10 mM 1-pyrenemethylamine hydrochloride (manufactured by Sigma-Aldrich, hereinafter also referred to as PyNH ２ The sample was immersed in an N,N-dimethylformamide (DMF) solution containing PyNH ２ The PyNH / CNT / GCE electrode was obtained. ２After washing the / CNT / GCE with DMF and ultrapure water, 15 μL of a 1 mg / mL rFoDH1 solution (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was dropped onto the electrode, and the electrode was kept at 4°C for 1 hour under water vapor saturated conditions to immobilize the enzyme, thereby obtaining enzyme electrode 1.

[0118] Preparation Example 5 Enzyme Electrode 2 Containing Enzyme (a2) (rFoDH1β(Me)) The enzyme electrode 2 was obtained in the same manner as in Preparation Example 3, except that a 1 mg / mL FoDH1B solution (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was used instead of the rFoDH1 solution.

[0119] Test Example 1: CV Measurement 1 Cyclic voltammetry (CV) measurements were carried out using the enzyme electrodes 1 and 2 obtained in Preparation Examples 4 and 5. The measurement device and measurement conditions were as follows: Measurement device: Electrochemical analyzer ALS660E (manufactured by BAS) Solution: 1 M potassium phosphate buffer (pH 8.0), 50 mM NADH, 50 mM NAD ＋ Temperature: 25°C Atmosphere: Ar Rotation speed: 100 rpm Potential sweep rate: 5 mVs １ The measurement results are shown in Figure 5. In Figure 5, the results for enzyme electrode 1 are shown by a broken line, and the results for enzyme electrode 2 are shown by a solid line. ＋ The dotted line shows the results measured under conditions that did not include

[0120] In CV measurement 1, oxidation and reduction waves were observed at enzyme electrodes 1 and 2, confirming a DET-type reaction. A larger catalytic current density was obtained at enzyme electrode 2 using FoDH1B. This is thought to be due to an increase in the effective enzyme adsorption amount due to the enzyme size.

[0121] Test Example 2: CV Measurement 2 Cyclic voltammetry (CV) measurements were performed using the electrode 2 obtained in Preparation Example 5. Measurements were performed using the same measurement equipment and under the same conditions as in Test Example 1, except that the following measurement solution was used: Measurement solution: 1 M potassium phosphate buffer (pH 8.0), 1 mM NADH, 10 mM glucose, 1 mg / mL NAD-dependent glucose dehydrogenase (manufactured by Toyobo Co., Ltd., EC1.1.1.47, 250 U / mg, hereinafter also referred to as GDH). The measurement results are shown in Figure 6 (solid line). The dashed line shows the results of measurements performed under conditions that did not contain glucose or GDH, and the dotted line shows the results of measurements performed under conditions that did not contain NADH, glucose, or GDH.

[0122] In CV measurement 2, the addition of GDH and glucose increased the oxidation catalyst current and reduced the reduction peak, indicating that a coupled system between the NAD / NADH regeneration system and the oxidation reaction of glucose by GDH was established.

[0123] Test Example 3: CV Measurement 3 Cyclic voltammetry (CV) measurement was carried out using the electrode 2 obtained in Preparation Example 5. The measurement was carried out using the same measuring device and under the same conditions as in Test Example 1, except that the following measurement solution was used: Measurement solution: 1 M potassium phosphate buffer (pH 8.0), 1 mM NAD ＋ The measurement results are shown in Figure 7 (solid line). The results measured under conditions without DHA and GIDH are shown by a dashed line. ＋ The results measured under conditions that did not contain DHA or GIDH are shown by the dotted line.

[0124] In CV measurement 3, the addition of GIDH and DHA increased the catalytic current on the reduction side and reduced the peak on the oxidation side, indicating that a conjugated system was formed between the NAD / NADH regeneration system and the reduction reaction of DHA by GIDH.

[0125] Preparation Example 6: Enzyme Electrode 3 Containing Enzyme (a2) (rFoDH1β(Ec)) A glassy carbon electrode (GCE) was polished with alumina particles having outer diameters of 1.0 μm and 0.05 μm, and then ultrasonically cleaned with ultrapure water. Next, 10 mg of multi-walled carbon nanotubes (MWCNT) were added to 10 mL of 1-methylpyrrolidone (NMP), and the suspension was subjected to ultrasonic treatment for 2 hours. 10 μL of the uniformly dispersed MWCNT dispersion was added dropwise to the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was then dissolved in 10 mM 1-pyrenemethylamine hydrochloride (PyNH ２ ) in N,N-dimethylformamide (DMF) solution and left at room temperature for 1 hour. ２ The PyNH / CNT / GCE electrode was obtained. ２ After washing the / CNT / GCE with DMF and ultrapure water, 30 μL of a 1 mg / mL rFoDH1β(Ec) solution (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was dropped onto the electrode, and the electrode was kept at 4°C for 1 hour under water vapor-saturated conditions to allow the enzyme to adsorb onto the electrode surface, yielding enzyme electrode 3. The excess rFoDH1β(Ec) solution was removed and used in the next procedure.

[0126] Preparation Example 7: Enzyme Electrode 4 Containing Enzyme (a2) (rFoDH1β(Ec)) and Enzyme (b) (GDH) The enzyme electrode 3 was immersed in 1.5 mL of 1 M potassium phosphate buffer (pH 8.0) containing 1 M glucose. 200 μL of a 10 mg / mL solution of glucose dehydrogenase (GDH, Toyobo Co., Ltd., GLD-311) as NAD(P)H or NAD(P)-dependent oxidoreductase (b) (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added to the buffer, and NADH (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added to a final concentration of 10 mM. The resulting solution was adsorbed onto the surface of the enzyme electrode 3, yielding an enzyme electrode 4 containing enzyme (a2), enzyme (b), and NADH. The cyclic voltammetry (CV) measurements described below were performed while the enzyme electrode 4 was immersed in the buffer. Therefore, the above buffer solution contains GDH in addition to excess NADH, but since the CV and CA measurements described below are performed in a static solution, it can be assumed that the excess NADH and GDH that are not present near the electrode, i.e., not immobilized and present in the portion that can be considered as the bulk solution, do not affect the electrochemical reaction.

[0127] Preparation Example 8: Enzyme electrode 5 containing enzyme (a2) (rFoDH1β(Ec)) and enzyme (b) (GDH) The enzyme electrode 3 was prepared by adding 50 mM NADH, 50 mM NAD ＋ The cells were immersed in 1.5 mL of 1 M potassium phosphate buffer (pH 8.0) containing NADH, NAD ＋ was adsorbed onto the electrode surface of the enzyme electrode 3. Next, 150 μL of a 10 mg / mL solution of glucose dehydrogenase (GDH, GLD-311 manufactured by Toyobo Co., Ltd.) as NAD(P)H or NAD(P)-dependent oxidoreductase (b) (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added to the buffer solution and adsorbed onto the electrode surface of the enzyme electrode 3, thereby obtaining an enzyme electrode 5 containing the enzyme (a2), the enzyme (b), and NADH. In the chronoamperometry (CA) measurement described below, the enzyme electrode 5 is used while immersed in the buffer solution. Glucose was added stepwise to the buffer solution in which the enzyme electrode 5 was immersed, and the catalytic current value due to glucose oxidation was observed.

[0128] Preparation Example 9 (Comparative Example): Enzyme Electrode 6 Containing Enzyme (b) (GDH) Only A glassy carbon electrode (GCE) was polished with alumina particles having outer diameters of 1.0 μm and 0.05 μm, and then ultrasonically cleaned with ultrapure water. Next, 10 mg of multi-walled carbon nanotubes (MWCNT) were added to 10 mL of 1-methylpyrrolidone (NMP), and the suspension was subjected to ultrasonic treatment for 2 hours. 10 μL of the uniformly dispersed MWCNT dispersion was added dropwise to the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was then dissolved in 10 mM 1-pyrenemethylamine hydrochloride (PyNH ２ ) in N,N-dimethylformamide (DMF) solution and left at room temperature for 1 hour. ２ The PyNH / CNT / GCE electrode was obtained. ２ After washing the / CNT / GCE with DMF and ultrapure water, 30 μL of a solution containing GDH (0.4 mg / ml), NADH (10 mM), and glutaraldehyde (5%) (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was dropped onto the electrode, and the electrode was left at room temperature for 1 h to allow the enzyme to be adsorbed onto the electrode surface, thereby obtaining enzyme electrode 6.

[0129] Preparation Example 10 (Comparative Example): Enzyme Electrode 7 with Enzyme (a2) (rFoDH1β(Ec)) Adsorbed on Enzyme Electrode 6 The enzyme electrode 6 was immersed in 1.5 mL of 1 M potassium phosphate buffer (pH 8.0) containing 1 M glucose. 500 μL of a 1 mg / mL rFoDH1β(Ec) solution (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added to the buffer to obtain the enzyme electrode 7 with rFoDH1β(Ec) adsorbed on the electrode surface of the enzyme electrode 6. The cyclic voltammetry (CV) measurements described below were performed while the enzyme electrode 7 was immersed in the buffer. Therefore, although the buffer also contains excess rFoDH1β(Ec), since the CV measurements described below were performed in a static solution, it can be assumed that the excess rFoDH1β(Ec) not present near the electrode, i.e., not immobilized and present in the bulk solution, does not affect the electrochemical reaction.

[0130] Test Example 4: CV Measurement 4 Cyclic voltammetry (CV) measurement was carried out using the enzyme electrode 3 obtained in Preparation Example 6. The measurement device and measurement conditions are as follows: Measurement device: Electrochemical analyzer ALS660E (manufactured by BAS) Solution: 1 M potassium phosphate buffer (pH 8.0), 50 mM NADH, 50 mM NAD ＋ Temperature: 25°C Atmosphere: Ar Potential sweep rate: 5 mVs －１ The measurement results are shown in FIG.

[0131] In CV measurement 4, oxidation and reduction waves were observed at enzyme electrode 3, confirming a DET-type reaction. Enzyme electrode 3 using rFoDH1β(Ec) expressed in E. coli yielded a higher catalytic current density than the enzyme expressed in the same strain. This is thought to be due to the increased activity of rFoDH1β(Ec) when expressed in E. coli.

[0132] Test Example 5: CV Measurement 5 Cyclic voltammetry (CV) measurement was performed using the enzyme electrode 4 obtained in Preparation Example 7. The measurement was performed using the same measurement device and under the same conditions as in Test Example 1, except that the following measurement solution was used. Measurement solution: 1 M potassium phosphate buffer (pH 8.0), 10 mM NADH, 1 M glucose, 1 mg / mL NAD-dependent glucose dehydrogenase (GDH). The measurement results are shown in Figure 9 (solid line). The results measured under conditions without GDH are shown with a dashed line, and the results measured under conditions without NADH or GDH are shown with a dotted line.

[0133] In CV measurement 5, the oxidation catalytic current increased when GDH and NADH were adsorbed onto the electrode containing rFoDH1β(Ec), indicating that a coupled system between the NAD / NADH regeneration system by rFoDH1β(Ec) and the glucose oxidation reaction by GDH was established.

[0134] Test Example 6: CA Measurement 6 Chronoamperometry (CA) measurement was performed using the enzyme electrode 5 obtained in Preparation Example 8. The measurement was performed using the same measurement device and under the same conditions as in Test Example 4, except that the following measurement solution was used: Measurement solution: 1 M potassium phosphate buffer (pH 8.0), 50 mM NADH, 1 mg / mL NAD-dependent glucose dehydrogenase The measurement results are shown in Figure 10 (solid line).

[0135] In CA measurement 6, the current value 2 minutes after the addition of GDH decreased to the extrapolated line for the case without GDH, but did not return to the extrapolated line after the addition of glucose. Furthermore, the current value increased depending on the amount of glucose added, which clearly indicates that this is a current associated with the oxidation of glucose.

[0136] Test Example 7 (Comparative Example): CV Measurement 7 Cyclic voltammetry (CV) measurements were performed using the enzyme electrode 6 obtained in Preparation Example 9 and the enzyme electrode 7 obtained in Preparation Example 10. The measurements were performed using the same measurement equipment and conditions as in Test Example 4, except that the following measurement solution was used: Measurement solution: 1 M potassium phosphate buffer (pH 8.0), 10 mM NADH, 1 M glucose, 0.25 mg / mL rFoDH1β. The measurement results are shown in FIG. 11. The results for enzyme electrode 6 are shown by the dashed line, and the results for enzyme electrode 7 are shown by the solid line. No increase in catalytic current was observed in either enzyme electrode 6 or 7. The reason why enzyme electrode 6 did not show an increase in catalytic current is that it contains only GDH and does not contain rFoDH1β(Ec). The results for enzyme electrode 7 are due to the fact that, although it contains rFoDH1β(Ec) and GDH, a first catalytic layer containing rFoDH1β(Ec) was layered on top of a second catalytic layer containing GDH on the electrode substrate. From the above, it can be said that the technical significance of laminating the first catalyst layer and the second catalyst layer in this order on the electrode substrate in the enzyme electrode of the present invention has become clear.

[0137] 1 Enzyme electrode 2 Enzyme (a1) 3 Enzyme (b) 4 NAD(P) 5 NAD(P)H 6 Electrode base material 7 Substrate 8 First catalyst layer 9 Second catalyst layer

Claims

1. An electrode comprising an enzyme, The electrode comprises an electrode substrate, a first catalyst layer containing an enzyme (a1) that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P) and is capable of directly donating and receiving electrons involved in the redox reaction between the electrode substrate and the first catalyst layer, and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), an enzyme electrode, wherein the first catalyst layer and the second catalyst layer are laminated in this order on the electrode substrate;

2. 2. The enzyme electrode according to claim 1, wherein the enzyme (a1) has one flavin mononucleotide and one or more Fe—S clusters, the distance between the flavin mononucleotide and at least one Fe—S cluster is within 2 nm, and the at least one Fe—S cluster is present within 2 nm from the surface of the enzyme (a1).

3. 3. The enzyme electrode according to claim 1, wherein the enzyme (a1) comprises a β subunit of formate dehydrogenase derived from a methanol-utilizing bacterium.

4. the surface of the electrode substrate is coated with a surface modifying group, The enzyme electrode according to claim 1 or 2, wherein the surface modifying group has a charge opposite to the surface charge of a portion of the surface of the enzyme (a1) that is located within 2 nm from the Fe—S cluster contained in the enzyme (a1).

5. the enzyme electrode contains at least one material selected from the group consisting of carbon particles, metals, and conductive polymers; the carbon particles are at least one selected from the group consisting of activated carbon, carbon black, and carbon nanotubes; the metal is at least one selected from the group consisting of gold, platinum, copper, palladium, and titanium; 3. The enzyme electrode according to claim 1, wherein the conductive polymer is at least one selected from the group consisting of polyaniline and polypyrrole.

6. 3. The enzyme electrode according to claim 1, wherein the substrate of the NAD(P)H or NAD(P)-dependent oxidoreductase (b) is glycerol.

7. A biosensor comprising the enzyme electrode according to claim 1 or 2.

8. A bioreactor comprising the enzyme electrode according to claim 1 or 2.

9. A biofuel cell comprising the enzyme electrode according to claim 1 or 2.