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

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

[Technical Field]

[0001] The present invention relates to enzymes, enzyme electrodes, biosensors, bioreactors, and biofuel cells. [Background technology]

[0002] In recent years, with growing concern over energy issues, biofuel cells that use bio-related substances such as sugars and alcohols as fuel have attracted attention. Biofuel cells use enzymes as electrode catalysts and can generate electricity by combining the oxidation reaction of the fuel at the anode with the reduction reaction of oxygen and other substances at the cathode.

[0003] In the anode of a biofuel cell, electrons extracted from the fuel by the catalytic function of enzymes are transferred to the electrodes. As enzymes that carry out the oxidation reaction of the fuel at the anode, nicotinamide adenine dinucleotide (NAD)-dependent oxidases and nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidases, which use free nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzymes, are commonly used. However, the NADH and NADPH produced by these catalytic reactions have very high overpotentials due to direct electrolysis, so it was necessary to use catalysts such as redox dyes or o-quinones.

[0004] To address the above challenges, a method has been proposed that uses a substance called an electron mediator to catalyze electron transfer between enzymes and electrodes. Electrode reactions using such electron mediators are called mediator electron transfer (MET) type enzyme-functional electrode reactions. For example, Patent Documents 1 to 5 disclose biofuel cells that combine NAD(P)H or NAD(P)-dependent oxidoreductase with MET-type enzyme-functional electrode reactions. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2004-71559 [Patent Document 2] Japanese Patent Publication No. 2012-151130 [Patent Document 3] Japanese Patent Publication No. 2012-178335 [Patent Document 4] Japanese Patent Publication No. 2009-69085 [Patent Document 5] Japanese Patent Publication No. 2018-68287 [Overview of the project] [Problems that the invention aims to solve]

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

[0007] This invention has been made in view of the above-mentioned circumstances, and aims to provide an enzyme electrode that can directly transfer electrons between an electrode substrate and an enzyme in connection with a redox reaction. [Means for solving the problem]

[0008] The present inventors have diligently researched to solve the above problems and have discovered that in an electrode in 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 transfer electrons with respect to the redox reaction with respect to the electrode substrate, and a second catalyst layer containing NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P) are laminated in this order on an electrode substrate, electron transfer in relation to the redox reaction can be directly carried out between the electrode substrate and the enzyme, and thus the above problems can be successfully solved, leading to the present invention.

[0009] The present invention relates to an enzyme-containing electrode, the electrode comprising an electrode substrate, a first catalyst layer containing an enzyme (a1) that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P) and can directly transfer electrons with the electrode substrate in connection with the redox reaction, and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), wherein the first catalyst layer and the second catalyst layer are stacked in this order on the electrode substrate.

[0010] The present invention relates to an enzyme that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P), wherein the enzyme has one flavin mononucleotide and two Fe-S clusters, the distance between the flavin mononucleotide and at least one of the Fe-S clusters is within 2 nm, and at least one of the Fe-S clusters is located within 2 nm of the surface of the enzyme. [Effects of the Invention]

[0011] The enzyme electrode of the present invention has the above-described configuration and can directly transfer electrons between the electrode substrate and the enzyme in connection with oxidation-reduction reactions, making it suitable for use in biofuel cells, biosensors, bioreactors, and the like. [Brief explanation of the drawing]

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

Mode for Carrying Out the Invention

[0013] Hereinafter, the enzyme electrode, enzyme, biosensor, bioreactor, and biofuel cell of the present invention will be described. However, the present invention is not limited to the following configurations and can be modified and applied as appropriate without altering the essence of the invention. Furthermore, combinations of two or more of the preferred configurations of the present invention described below also constitute 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 of oxidized and reduced forms of nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and that can directly transfer electrons with the electrode substrate in connection with the redox reaction. In this specification, the oxidized form of nicotinamide adenine dinucleotide is also referred to as NAD, and its reduced form as NADH; the oxidized form of nicotinamide adenine dinucleotide phosphate is also referred to as NADP, and its reduced form 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), wherein 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 the anode of a biofuel cell. When the substrate is oxidized by the oxidoreductase (b), electrons are received by NAD(P) to become NAD(P)H, and this NAD(P)H is oxidized by enzyme (a1), and the resulting electrons can be directly transferred by enzyme (a1) to the electrode substrate. The enzyme electrode of the present invention, when used as an anode in a biofuel cell, eliminates the need for an electron mediator in 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 it is preferable that it is substantially free of one. The electron mediator content is preferably 200 mol% or less relative to 100 mol% of enzyme (a1). More preferably 50 mol% or less, even more preferably 10 mol% or less, particularly preferably 1 mol% or less, and most preferably 0 mol%. An electron mediator is a substance that can exchange electrons with an enzyme or coenzyme, and further with a conductive substrate. However, in this specification, NAD(P)H and NAD(P) are not included in the definition of an electron mediator.

[0017] The above-mentioned electron mediators are not particularly limited, but examples include metal elements such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, and W, or metal complexes with ions of these metals as the central metal (alkali metal ferricyanides such as ferrocene, potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, or alkyl substituted versions thereof (methyl substituted, ethyl substituted, propyl substituted, etc.), potassium octacyanotungstate, etc.); quinones such as quinone, benzoquinone, anthraquinone, and naphthoquinone; heterocyclic compounds such as viologen, methyl viologen, benzyl viologen, phenazine methosulfate, phenazine ethosulfate, bipyridine, or derivatives thereof; and others such as 2,6-dichlorophenolindophenol, methylene blue, potassium β-naphthoquinone-4-sulfonate, and vitamin K.

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

[0019] The above-mentioned second catalyst layer may contain the above-mentioned enzyme (b) and NAD(P)H and / or NAD(P), but may also contain buffer components contained in the solvent or buffer. The solvents and buffer components mentioned above are the same as those described for the first catalyst layer.

[0020] In the enzyme electrode of the present invention, there are no particular limitations on the method of laminating the first catalyst layer and the second catalyst layer on the electrode substrate. A method for laminating the first catalyst layer and the second catalyst layer on the electrode substrate is, for example, a method of coating the electrode substrate with a composition containing enzyme (a1) (hereinafter also referred to as the enzyme (a1)-containing composition) and drying it. Any commonly used coating method is acceptable, such as spin coating, spraying, screening, dip coating, or blade coating.

[0021] The above enzyme (a1)-containing composition may contain enzyme (a1), but may also contain solvents or buffer components. Furthermore, the above enzyme (a1)-containing composition may contain immobilizing agents such as polymers or crosslinking agents, and the enzyme (a1) may be strongly immobilized on the electrode substrate by the immobilizing agent.

[0022] The polymers mentioned above are not particularly limited, but examples include polyvinylimidazole (PVI), polyallylamine, polyamino acids (e.g., polylysine), polypyrrole, polyacrylic acid, polyvinyl alcohol, graft copolymers of polypropylene and maleic anhydride, copolymers of methyl vinyl ether and maleic anhydride, and orthocresol novolac type epoxy resins. The above crosslinking agents are not particularly limited, but examples include polyethylene glycol diglycidyl ether (PEGDGE), glutaraldehyde, disuccimidyl suberate, succimidyl-4-(p-maleimidophenyl)butyrate, etc.

[0023] The method for laminating the second catalyst layer onto the electrode substrate is not particularly limited, and examples include applying a composition containing 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 it. The method for applying the enzyme (b)-containing composition is not particularly limited, and examples include applying the enzyme (a1)-containing composition described above.

[0024] The enzyme (b)-containing composition may contain enzyme (b) and NAD(P)H and / or NAD(P), but may also contain the aforementioned solvent and buffer components. Furthermore, the enzyme (a1)-containing composition may contain the aforementioned polymer or immobilizing agent such as a crosslinking agent, and enzyme (b) may be strongly immobilized on the electrode substrate by the immobilizing agent.

[0025] <First catalyst layer> The enzyme (a1) described above can catalyze the redox reaction between the redox pairs of NAD(P)H and NAD(P) and directly transfer electrons with the electrode substrate in connection with the redox reaction, but it is preferable that it has one flavin mononucleotide (hereinafter also referred to as FMN) and at least one Fe-S cluster. FMN and Fe-S clusters can transfer electrons, and a form in which the enzyme (a1) has FMN and Fe-S clusters as cofactors is one of the preferred embodiments of the present invention.

[0026] The number of Fe-S clusters in the enzyme (a1) described above is not particularly limited, but it is preferably 1 to 10 per molecule. More preferably, the number is 2 to 8 per molecule, 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 employing the acetylene combustion method.

[0027] The Fe-S cluster described above is composed of iron atoms and sulfur atoms, with cysteine ​​residues in the polypeptide primarily coordinating to the iron atoms of the Fe-S cluster. Specifically, Fe-S clusters include the [2Fe-2S] type, the [3Fe-4S] type, and the [4Fe-4S] type. If the enzyme (a1) contains at least one Fe-S cluster, the type is not particularly limited, but it is preferable that it contains [2Fe-2S] type and / or [4Fe-4S] type Fe-S clusters. More preferably, it is a form containing a [2Fe-2S] type Fe-S cluster, even more preferably a form containing a [2Fe-2S] type Fe-S cluster and a [4Fe-4S] type Fe-S cluster, and particularly preferably a form containing one [2Fe-2S] type and one [4Fe-4S] type Fe-S cluster. In this specification, the number of Fe-S clusters and the number of FMNs (described later) in an enzyme refer to the number per enzyme molecule.

[0028] If the enzyme (a1) has FMN and Fe-S clusters, 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 mentioned above is the distance between the nearest neighbor atoms in both the FMN and the Fe-S cluster. In this specification, the interatomic distances in an enzyme can be calculated based on information about its three-dimensional structure. Furthermore, the three-dimensional structure of enzymes can be analyzed by cryo-electron microscopy. Information on the enzyme's three-dimensional structure can also be obtained from databases such as the PDB.

[0029] When the enzyme (a1) has two or more Fe-S clusters, it is preferable that the distance between the FMN and the nearest Fe-S cluster is 2 nm or less. More preferably, the distance between the FMN and the [2Fe-2S] type Fe-S cluster is 2 nm or less. More preferably, the distance between the FMN and 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. In one embodiment, it is more preferably within 1 nm.

[0031] If the enzyme (a1) described above has two or more Fe-S clusters, it is preferable that the distance between at least two Fe-S clusters is 3 nm or less. More preferably, it is 2.5 nm or less.

[0032] In the enzyme (a1) described above, it is preferable that at least one Fe-S cluster is located within 2 nm of the surface of the enzyme (a1). This shortens the distance between the redox center within the enzyme and the electrode, thereby improving the electron transfer rate at the enzyme-electrode interface. In this specification, the distance from the surface of the enzyme to Fe-S clusters, etc., means the distance between the α-carbon of an amino acid residue present on the surface of the enzyme and the atom of the Fe-S cluster, etc. In this specification, amino acid residues present on the enzyme surface refer to amino acid residues in enzyme (a1) that are accessible to the solvent, based on the PDBePISA program (http: / / www.ebi.ac.uk / msd-srv / prot_int / ).

[0033] If the enzyme (a1) has two or more Fe-S clusters, it is preferable that at least two Fe-S clusters are located within 2 nm of the surface of the enzyme (a1). In which all Fe-S clusters of the enzyme (a1) are located within 2 nm of the surface of the enzyme (a1), this is also one of the preferred embodiments of the present invention.

[0034] One preferred embodiment of the present invention is a form 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 located within 2 nm of the surface of the enzyme (a1). Another preferred embodiment of the present invention is a form in which the enzyme (a1) has a [2Fe-2S] type Fe-S cluster within 2 nm from the surface of the enzyme (a1). Fe-S clusters within 2 nm of the FMN and Fe-S clusters within 2 nm of the enzyme surface may be the same or different, but are preferably the same.

[0035] The enzyme (a1) described above preferably has a β subunit comprising one flavin mononucleotide and one or more Fe-S clusters.

[0036] The enzyme (a1) described above preferably has two Fe-S clusters in its β subunit. More preferably, it has a form in which the β subunit has a [2Fe-2S] type Fe-S cluster and a [4Fe-4S] type Fe-S cluster.

[0037] The enzyme (a1) described above preferably contains the β-subunit of formate dehydrogenase derived from methanol-utilizing bacteria. While there are no particular restrictions on methanol-assimilating bacteria, they are preferably 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 Sequence ID No. 1. The amino acid sequence of the β-subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1 is shown in Sequence ID No. 2.

[0038] The enzyme (a1) described above 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 it is preferably of the [4Fe-4S] type and / or the [2Fe-2S] type. The number of Fe-S clusters in 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. A configuration in which the above-mentioned 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 above α subunit further comprises tungstenpterin, which is a pterin complex containing tungsten as the central metal. Tungsten pterin can, for example, act as an active center that catalyzes redox reactions using substrates such as formic acid and carbon dioxide. If the enzyme (a1) contains tungsten pterin, it can transfer electrons obtained from a substrate such as formic acid to an electrode substrate, and can also transfer electrons transferred from the electrode substrate to a substrate such as carbon dioxide.

[0040] When the enzyme (a1) described above has a β subunit and an α subunit, their relative positions are not particularly limited, but it is preferable that 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 within 2 nm. More preferably, it is within 1 nm.

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

[0042] The enzyme (a1) described above preferably has an average molecular weight of 50,000 or more and 500,000 or less. This makes it possible to further improve the electron transfer rate between the enzyme (a1) and the electrode substrate. When the enzyme (a1) consists only of 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 above enzyme (a1) contains the above 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 above enzymes can be measured by gel filtration chromatography.

[0043] The enzyme (a1) described above preferably has an amino acid sequence that has 70% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2. The sequence identity is more preferably 80% or more, and even more preferably 90% or more. The sequence identity of the above amino acid sequences 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 within 2 nm of the active site is positively or negatively charged. In this case, for example, by modifying the surface of the electrode substrate with a surface modifying group having a charge opposite to that of the enzyme (a1), the enzyme (a1) can be immobilized so that its active site is oriented toward the electrode substrate. This makes it possible to further increase the electron transfer rate between the enzyme (a1) and the electrode substrate. More preferably, the surface charge of the portion of the enzyme (a1) at a distance of 2 nm or less from the active site is negative. Particularly preferred is the surface charge of the portion of the enzyme (a1) at a distance of 2 nm or less from the Fe-S cluster.

[0045] The enzyme (a1) described above can be any enzyme that catalyzes the redox reaction between the redox pairs of NAD(P)H and NAD(P), but it 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 it 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 unit or more, and particularly preferably 1.5 units or more. The above enzyme activity can be measured by a solution enzyme activity evaluation method using NADH as a substrate.

[0046] The amount of enzyme (a1) contained in the first catalyst layer described above is not particularly limited, but it is recommended to use 0.05 pmol / cm³ relative to the surface area of ​​the electrode substrate. 2 More than 1000pmol / cm 2 Preferably, the following: More preferably 0.5 pmol / cm³ 2 More than 100pmol / cm 2 The following is more preferably 5 pmol / cm³ 2 More than 10 pmol / cm 2 The following applies:

[0047] The enzyme (a1) described above is not particularly limited in its origin, as long as it possesses the catalytic function described above. For example, it may be a bio-derived molecule extracted from plants, animals, or microorganisms, or it may be genetically engineered or chemically synthesized. It may be naturally occurring, prepared by appropriate protein isolation and purification techniques from any organism such as naturally occurring bacteria, yeast, and plants, or it may be manufactured as a recombinant by genetic engineering or chemically synthesized. Furthermore, the organism itself, such as microorganisms containing enzyme (a1), organelles, and cells, may be used in the first catalyst layer. Alternatively, crude products derived from these organisms may be used in the first catalyst layer.

[0048] When producing the above enzyme (a1) as a recombinant by genetic engineering techniques, methods commonly used in the art can be used. For example, a suitable probe DNA can be synthesized based on the amino acid sequence of formate dehydrogenase derived from Methylorubrum extorquens AM1 (the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2), and this can be used to select the formate dehydrogenase gene from a library of chromosomal DNA or cDNA. Alternatively, a suitable primer DNA can be prepared based on the amino acid sequence, and the DNA containing the target gene fragment can be amplified by a suitable polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method. These DNA fragments can then be ligated to obtain DNA containing the full length of the target gene.

[0049] The gene encoding the enzyme (a1) described above may be ligated or inserted into various vectors, or incorporated into chromosomes or genomes. When using vectors, commercially available kits such as the TA Cloning Kit (Invitrogen) or the 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 DNA such as λEMBL3 (Stratagene) can be used. Additionally, homologous recombination vectors such as pK18mobsacB (Schaefer et al., Gene, vol. 45, p. 69-73 (1994)) and pCM1682 (H. Iguchi et al., Environ. Microbiol. Rep. 10 (2018) 634-643) can also be used. The recombinant DNA obtained in this way can be used to transform host organisms, such as Methylorubrum extorquens, Escherichia coli DE3 strain, Escherichia coli JM109 strain (Takara Bio Inc.), and Escherichia coli DH5α strain (Takara Bio Inc.).

[0050] It is preferable to culture the transformed organism obtained as described above under conditions that enable the expression of the introduced gene, and then isolate and purify the enzyme (a1) from the culture of the transformed organism. The isolation and purification methods for enzyme (a1) are not particularly limited, and standard protein isolation and purification methods can be used. For isolation and purification, known isolation and purification techniques such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, hydrophobicity, anion chromatography, cation chromatography, and affinity chromatography can be used individually or in appropriate combinations. In particular, when using affinity chromatography, it is 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 a tag peptide.

[0051] Furthermore, when producing enzyme (a1) as a recombinant, for example, a mutation may be introduced in the amino acid sequence shown in Sequence ID No. 1 of the formate dehydrogenase derived from Methylorubrum extorquens AM1. When introducing mutations, those skilled in the art can predict mutations that alter the surface charge while maintaining enzyme activity, based on information about the three-dimensional structure of the formate dehydrogenase and the properties of the amino acids.

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

[0053] The above substrates are not particularly limited, but examples include alcohols, sugars, fats, polyamino acids such as peptides and proteins, and organic acids. One or more of these can be used.

[0054] The above alcohols are not particularly limited, but examples 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 above sugars are not particularly limited, but examples 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, etc.

[0056] The above organic acids are not particularly limited, but examples include glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvic acid, pyruvate, acetyl-CoA, citric acid, cis-aconitic acid, isocitrate, oxalosuccinate, 2-oxoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, L-malic acid, oxaloacetate, and intermediate products of sugar metabolism.

[0057] The above-mentioned fats are not particularly limited as long as they are esters of fatty acids and glycerol, and the fatty acids that make up the fats are 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) above is preferably an oxidase. More preferably, the enzyme (b) above includes glycerol dehydrogenase, glucose dehydrogenase, a series of enzymes in the electron transport chain, ATP synthase, and enzymes involved in carbohydrate metabolism (e.g., hexokinase, glucose phosphate isomerase, phosphofructokinase, fructose diphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglyceromtase, 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, malonate dehydrogenase, etc.). One or more of these can be used. Among these, glycerol dehydrogenase and glucose dehydrogenase are particularly preferred.

[0060] In one embodiment, the second catalyst layer may contain multiple types of enzyme (b), and the substrate can be progressively degraded by multiple enzymes (b).

[0061] The enzyme (b) described above is not particularly limited in its origin, as long as it possesses the catalytic function described above. It may be naturally derived, purified by appropriate protein isolation and purification techniques from naturally occurring organisms, or it may be a recombinant product manufactured by genetic engineering or chemically synthesized. Commercially available products may also be used. Methods for producing recombinant organisms using genetic engineering techniques include those similar to those used for producing enzyme (a1). Furthermore, the organism itself, such as microorganisms containing enzyme (b), organelles, and cells, may be used in the second catalyst layer. Alternatively, crude products derived from these organisms may be used in the second catalyst layer.

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

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

[0064] <Electrode base material> The electrode substrate of the enzyme electrode of the present invention is a conductive substrate that can be connected to an external circuit and can transfer electrons. As long as the electrode substrate has the above-mentioned properties, there are no particular restrictions on the material, shape, etc. The material of the electrode substrate may be any conductive material, such as carbon materials like carbon cloth, carbon paper, graphite, and glassy carbon, activated carbon, carbon black, and carbon nanotubes; metals or alloys such as gold, platinum, copper, palladium, titanium, aluminum, silver, and nickel; and conductive oxides such as SnO2, In2O3, WO3, and TiO2. The electrode substrate described above may consist of a single layer of one of these materials, or it may consist of a laminated structure of two or more layers of the same material.

[0065] Preferred carbon materials include activated carbon, carbon black, and carbon nanotubes. By using carbon material as the conductive material, the conductivity with the electrodes can be further improved. The carbon material described above is preferably in particulate form. One preferred embodiment of the present invention is a form in which the enzyme electrode of the present invention includes at least one carbon particle selected from the group consisting of activated carbon, carbon black, and carbon nanotubes.

[0066] Preferred metals include gold, platinum, copper, palladium, and titanium. A form of the enzyme electrode of the present invention that includes at least one selected from the group consisting of gold, platinum, copper, palladium, and titanium is one of the preferred embodiments of the present invention.

[0067] In the above electrode substrate, for example, if two or more types of conductive materials are used, a binder such as a polymer may be used. The polymers mentioned above are not particularly limited, but polymers containing fluorine atoms such as polyvinylidene fluoride (PVDF) and polyvinyl fluoride (PVF), copolymers thereof, and polymers of copolymers of these monomers with ethylene or styrene may be used. Furthermore, in addition to polymers such as polystyrene, polyethylene, and polypropylene, hydrophilic polymers such as polyacrylic acid, polylysine, and carboxymethylcellulose, and conductive polymers such as polyaniline, polypyrrole, and their derivatives such as polyaniline sulfonic acid are also used. Among these, conductive polymers are preferred, and polyaniline and polypyrrole are more preferred. A form of the enzyme electrode of the present invention that includes at least one selected from the group consisting of polyaniline and polypyrrole is one of the preferred embodiments of the present invention.

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

[0069] The electrode substrate may have a flat surface or may have irregularities or pores on its surface, but it is preferable that it has pores. This ensures that the first catalyst layer and the second catalyst layer are sufficiently fixed by the electrode substrate. The size of the pores is not particularly limited, but it is preferably between 1 nm and 100 nm. More preferably between 2 nm and 10 nm.

[0070] The electrode substrate described above may or may not have its surface coated with surface-modifying groups, but a coated form is preferred. The surface modification groups described above are preferably those that can interact with enzyme (a1) through intermolecular forces, hydrogen bonds, Coulomb forces, etc., or that can form covalent bonds with enzyme (a1). More preferably, the surface modification group described above interacts with enzyme (a1) by Coulomb force and has a charge opposite to the surface charge of enzyme (a1). If enzyme (a1) has a positive surface charge, the surface modification group is preferably anionic. If enzyme (a1) has a negative surface charge, the surface modification group is preferably cationic. Particularly preferable is that the surface of the enzyme (a1) has a charge opposite to the surface charge of the portion located within 2 nm of the Fe-S cluster contained in the enzyme (a1).

[0071] Surface modifying groups are not particularly limited, but examples include functional or reactive groups such as amine groups, sulfonic acid groups, sulfate groups, phosphate groups, sulfhydryl groups, carboxyl groups and their salts, thiol groups, hydroxyl groups, azi groups, azo groups, nitro groups, nitrile groups, cyano groups, allene groups, isonitrile groups, urea groups, aldehyde groups, ketone groups, NHS esters, imide esters, maleimides, pyridyl dithiols, allyl azides, haloacetates, isocyanates, carbodiimides, allyl azides, diaziline, hydrazides, psoralens, iodines, pyridine disulfide, and vinyl sulfones. The surface modification group is preferably an anionic group or a cationic group. The above-mentioned anionic groups are groups that have anions or groups that can release hydrogen ions to form an anion, and include carboxyl groups, sulfonic acid groups, sulfate groups, phosphate groups, and salts thereof. The cationic group described above is a group that has a cation or a group that can accept a hydrogen ion to form a cation. Examples include primary to tertiary amino groups, neutralized products of primary to tertiary amino groups with acids such as hydrochloric acid and acetic acid, and amine groups such as quaternary ammonium bases.

[0072] The surface-modifying group-containing compound used for modifying the electrode substrate surface is preferably a compound having an anionic group or a cationic group and a structure that can interact with the electrode substrate surface. When the electrode substrate surface is made of carbon material, it is preferable that the electrode substrate has a structure that can interact with the carbon material in a π-π relationship. Examples of structures capable of π-π interaction include aromatic compounds and structures derived from heterocyclic compounds. Aromatic compounds may also contain heteroatoms. More preferably, the aromatic compound is a polycyclic aromatic compound. Preferred surface-modifying group-containing compounds include aromatic compounds having anionic or cationic groups, and heterocyclic compounds having anionic or cationic groups.

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

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

[0075] The surface modification group-containing compound is preferably a pyrene derivative. The pyrene derivative preferably has, for example, the above functional group or reactive group, and more preferably has an anionic group or a cationic group. Specific examples include 1-pyrenemethylamine, 1-aminopyrene, dimethyl-pyren-1-yl-methylamine, or diethyl-pyren-1-yl-amine. Also, an alkyl group, polyethylene glycol, or the like may be interposed as a spacer between the functional group or reactive group and pyrene. Further, the modification of these functional groups, spacers, etc. may be bonded to any carbon position of pyrene.

[0076] The amount of the surface modification group-containing compound used is not particularly limited, but is preferably 0.05 pmol / cm 2 or more and 1000 pmol / cm 2 or less with respect to the surface area of the electrode substrate. More preferably, it is 0.5 pmol / cm 2 or more and 100 pmol / cm 2 or less, and still more preferably 5 pmol / cm 2 or more and 10 pmol / cm 2 or less.

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

[0078] Figure 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 enzyme (a1)2 which catalyzes the redox reaction of the redox pair of NAD(P)H5 and NAD(P)4 and can directly transfer electrons with the electrode substrate 6 in connection with the redox reaction, and a second catalyst layer 9 containing enzyme (b)3 and NAD(P)H5 and / or NAD(P)4. When the enzyme electrode 1 of the present invention is the anode, NAD(P)4 receives electrons generated by the oxidation of substrate 7 by enzyme (b)3 to become NAD(P)H5, and this NAD(P)H5 is oxidized by enzyme (a1)2, and the resulting electrons can be directly transferred by enzyme (a1)2 to the electrode substrate 6.

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

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

[0081] The types of the two Fe-S clusters in the enzyme (a2) described above are not particularly limited, but it is preferable that it contains [2Fe-2S] type and / or [4Fe-4S] type Fe-S clusters. More preferably, it contains [2Fe-2S] type Fe-S clusters, and even more preferably, it contains one [2Fe-2S] type and one [4Fe-4S] type Fe-S cluster.

[0082] In the enzyme (a2) described above, 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. More preferably, the distance between the FMN and the two Fe-S clusters is within 2 nm.

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

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

[0085] The surface charge of the enzyme (a2) described above is not particularly limited, but it is preferable that the portion within a distance of 2 nm from the active site is positively or negatively charged. In this case, when the enzyme (a2) is used as an electrode, for example, the enzyme (a2) can be fixed so that its active site is oriented toward the electrode substrate by modifying the electrode substrate surface with a surface modifying group having a charge opposite to that of the enzyme (a2). This makes it possible to further increase the electron transfer rate between the enzyme (a2) and the electrode substrate. More preferably, the surface charge of the portion of the enzyme (a2) at a distance of 2 nm or less from the active site is negative. Particularly preferred is the surface charge of the portion of the enzyme (a2) at a distance of 2 nm or less from the Fe-S cluster.

[0086] The enzyme (a2) described above is not particularly limited in its origin, as long as it has the above composition. It may be of natural origin, purified by appropriate protein isolation and purification techniques from naturally occurring organisms, or it may be manufactured as a recombinant by genetic engineering or chemically synthesized.

[0087] When producing the above enzyme (a2) as a recombinant by genetic engineering, for example, it can be produced using the same method as the method for producing enzyme (a1) 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 Sequence ID No. 2). Furthermore, when using the above enzyme (a2) in electrodes or the like, the organism itself, such as microorganisms, organelles, and cells containing the enzyme (a2), may be used. Alternatively, crude products from these organisms may be used.

[0088] When producing the above enzyme (a2) as a recombinant, as described for enzyme (a1), for example, a mutation may be introduced in the amino acid sequence shown in Sequence ID No. 2 of the β-subunit of formate dehydrogenase derived from Methylorubrum extorquens AM1.

[0089] The above enzyme (a2) can be suitably used as an electrode for fuel cells and the like. In the present invention, the form in which enzyme (a1) is enzyme (a2) is also one of the preferred embodiments of the present invention.

[0090] [Biosensor] The present invention is also a biosensor equipped with the enzyme electrode of the present invention. The biosensor of the present invention is preferably configured to include the enzyme electrode of the present invention as the working electrode and a counter electrode thereof. Measurement using the biosensor described above is performed by bringing the sample to be measured into contact with the biosensor, which causes an oxidation-reduction reaction between the enzyme (b) contained in the enzyme electrode of the present invention and the substance to be measured, and by detecting the electric current generated by this reaction. The presence or absence or concentration of the substrate in the sample can be measured using this response current value.

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

[0092] [Bioreactor] The present invention also includes 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 reactants, but in one embodiment, it is preferable that the enzyme electrode of the present invention is installed in a column reactor. In one embodiment described above, when a solution containing the reactants is passed through a column reactor and brought into contact with an enzyme electrode, a product is obtained from the reactants by an enzymatic reaction mediated by enzyme (b) contained in the enzyme electrode of the present invention. The reactants applied to the bioreactor described above are not particularly limited and can be any substrate that can be oxidized or reduced by enzyme (b). Specifically, the substrates mentioned above are examples.

[0093] [Biofuel cell] The present invention also relates to a biofuel cell equipped with the enzyme electrode of the present invention. The enzyme electrode of the present invention in the above-mentioned biofuel cell may be an anode or a cathode, but it 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 that separates the anode and cathode.

[0094] In one embodiment, the biofuel cell of the present invention is preferably configured to include an anode made of the enzyme electrode of the present invention, a cathode, and a diaphragm that separates the anode and the cathode.

[0095] As the cathode in the biofuel cell of one embodiment of the present invention, for example, an enzyme catalyst such as a multi-copper enzyme such as pyruvate oxidase, ascorbate oxidase, or laccase, or a metal catalyst such as platinum can be used. When an enzyme catalytic mechanism is used for the reaction on the cathode side, it is preferable that the enzyme be fixed to the electrode substrate, or supplied onto a suitable electrode substrate as an enzyme solution without being fixed. In this case, the electrode substrate can be the same as the electrode substrate described for the enzyme electrode of the present invention.

[0096] The above-mentioned diaphragm has ionic conductivity that allows protons to pass through, and does not allow components on the negative electrode side or positive electrode side other than ions such as protons to pass through, but there are no restrictions on its material or shape. For example, a cellulose membrane can be used, and a solid electrolyte membrane can also be used. Examples of solid electrolyte membranes include solid membranes with ion exchange function, such as organic polymers having strong acid groups such as sulfo groups, phosphate groups, phosphone groups, and phosphine groups, weak acid groups such as carboxyl groups, and polar groups, but the invention is not limited to these. Specifically, cellulose membranes and perfluorocarbon sulfonic acid (PFS)-based resin membranes such as Nafion®, which is a copolymer of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinyl ether], can be used.

[0097] This specification discloses the following:

[0098] <1> An enzyme electrode comprising an electrode substrate, a first catalyst layer containing an enzyme (a1) that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P) and can directly transfer electrons with the electrode substrate in connection with the redox reaction, and a second catalyst layer containing an NAD(P)H or NAD(P)-dependent oxidoreductase (b) and NAD(P)H and / or NAD(P), wherein the first catalyst layer and the second catalyst layer are stacked in this order on the electrode substrate.

[0099] <2> The enzyme (a1) described above 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 located within 2 nm of the surface of the enzyme (a1). <1> The enzyme electrode described above.

[0100] <3> The above enzyme (a1) contains the β subunit of formate dehydrogenase derived from methanol-utilizing bacteria. <1> or <2> The enzyme electrode described above.

[0101] <4> The surface of the electrode substrate is coated with a surface modifying group, and the surface modifying group has a charge opposite to the surface charge of the portion of the enzyme (a1) that is within 2 nm of the Fe-S cluster contained in the enzyme (a1). <1> ~ <3> An enzyme electrode as described in any of the following.

[0102] <5> The above enzyme electrode comprises at least one selected from the group consisting of carbon particles, a metal, and a conductive polymer, wherein 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, and the conductive polymer is at least one selected from the group consisting of polyaniline and polypyrrole. <1> ~ <4> An enzyme electrode as described in any of the following.

[0103] <6> The substrate of NAD(P)H or NAD(P)-dependent oxidoreductase (b) is glycerol. <1> ~ <5> An enzyme electrode as described in any of the following.

[0104] <7> <1> ~ <6> A biosensor comprising an enzyme electrode as described in any of the following.

[0105] <8> <1> ~ <6> A bioreactor comprising an enzyme electrode as described in any of the following.

[0106] <9> <1> ~ <6> A biofuel cell comprising an enzyme electrode as described in any of the following.

[0107] <10> An enzyme that catalyzes the redox reaction of a redox pair of NAD(P)H and NAD(P), wherein the enzyme has one flavin mononucleotide and two Fe-S clusters, the distance between the flavin mononucleotide and at least one of the Fe-S clusters is within 2 nm, and at least one of the Fe-S clusters is located within 2 nm of the surface of the enzyme.

[0108] <11> When the above enzyme is used as an electrode, it can directly transfer electrons with the electrode substrate in connection with the above oxidation-reduction reaction. <10> The enzyme described.

[0109] <12> The average molecular weight is 50,000 to 100,000. <10> or <11> The enzyme described.

[0110] <13> The β-subunit of formate dehydrogenase derived from methanol-utilizing bacteria, <10> ~ <12> An enzyme listed in any of the following. [Examples]

[0111] The following are examples illustrating the present invention in more detail. However, 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 knockout strain of the formate dehydrogenase gene in Methylorubrum extorquens AM1 was prepared. The formate dehydrogenase gene, the methanol dehydrogenase subunit 1 precursor gene (the promoter), and a His-tag sequence for enzyme purification were inserted into a genomic DNA transformation plasmid (pCM1682). The resulting plasmid was mixed with the knockout strain and transformed by electroporation. After culturing the transformed strain, the cells were disrupted, and the resulting suspension was centrifuged. The supernatant was then purified by sieving on a Ni-NTA Agarose (QIAGEN) column to obtain recombinant enzyme (rFoDH1).

[0113] Based on the three-dimensional structural analysis of rFoDH1 described above, Figure 2 shows the distances between the cofactors of rFoDH1, and Figure 3 shows the distance from each cofactor to the protein surface. In Figures 2 and 3, the [4Fe-4S] type Fe-S clusters in the α subunit of rFoDH1 are represented as A1, A2, and A3, and the [2Fe-2S] type Fe-S cluster is represented as A4. In Figures 2-4, the [4Fe-4S] type Fe-S cluster in the β subunit or FoDH1B of rFoDH1 is represented as B1, and the [2Fe-2S] type Fe-S cluster is represented as B2. The units of the numerical values ​​in Figures 2-4 are 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 knockout strain of the formate dehydrogenase gene in Methylorubrum extorquens AM1 was prepared. The gene for the β subunit of formate dehydrogenase, the methanol dehydrogenase subunit 1 precursor gene, and a His-tag sequence were inserted into a genomic DNA transformation plasmid (pCM1682). The resulting plasmid was mixed with the knockout strain and transformed by electroporation. After culturing the transformed strain, the recombinant (rFoDH1β(Me)) was obtained by enzyme purification in the same manner as in Preparation Example 1.

[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 described below. The gene encoding FoDH1B was modified by adding CAT to its 5' end and substituting the 3' stop codon with CTCGAG. This modified gene was then introduced into a pET21a(+) vector using restriction enzymes NdeI and XhoI to obtain the plasmid pET21a-FoDHIβ. The obtained pET21a-FoDHIβ was then introduced into a protein-expressing Escherichia coli Arctic Express (DE3) strain (Agilent 230192) according to the provided protocol to construct a plasmid-transformed strain. As a pre-culture, the plasmid-introduced strain was inoculated into 5 ml of TB medium containing 0.3 mM ferric citrate (containing antibiotics (100 μg / ml ampicillin + 20 μg / ml gentamicin)) and cultured overnight at 37°C and 200 rpm. The resulting culture was diluted 100-fold in 20 ml of TB medium (containing antibiotics and 0.3 mM ferric citrate) in a 50 ml baffle-less flask, cultured at 37°C and 200 rpm for 3.5 hours, then the culture was cooled in the flask in ice water, and isopropyl-β-D(-)-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM. The culture was then cultured overnight at 15°C and 200 rpm. The obtained culture solution was centrifuged after cell disruption to obtain a suspension, and the obtained supernatant was purified by immobilized metal affinity chromatography (manufactured by Takara Bio Inc., TALON (registered trademark) 2 ml Disposable Gravity Column), and buffer exchange was performed using a centrifugal ultrafiltration filter unit Vivaspin (registered trademark) Turbo15 10,000 MWCO (Sartorius) with 100 mM Tris-HCl pH 8.0 to obtain rFoDH1β(Ec).

[0116] <NADH Oxidation Activity Measurement> The NADH oxidation activities of rFoDH1, rFoDH1β(Me), and rFoDH1β(Ec) obtained in Preparation Examples 1 and 2 were measured by the following method. Using a cuvette with an optical path length of 1 cm, it 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 Biomedicals, 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 enzyme activity was calculated from the molar absorption coefficient at 600 nm (20.6 mM -1 cm -1 at pH 7.0 (J.McD. Armstrong, Biochim. Biophys. Acta 86 (1964) 194-197)). For protein quantification, BCA Protein Assay (manufactured by Thermo) was used. As a result of the NADH oxidation measurement, 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)> Glassy carbon electrodes (3 mmΦ, manufactured by BAS, hereinafter also referred to as GCE) were polished with alumina particles with 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 (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 ultrasonically treated for 2 hours to uniformly disperse the MWCNTs. 10 μL of the resulting MWCNT dispersion was dropped onto the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was immersed in an N,N-dimethylformamide (DMF) solution containing 10 mM 1-pyrenemethylamine hydrochloride (manufactured by Sigma-Aldrich, hereinafter also referred to as PyNH2), left at room temperature for 1 hour, and a PyNH2 / CNT / GCE electrode was obtained. After washing the prepared PyNH2 / CNT / GCE with DMF and ultrapure water, 15 μL of 1 mg / mL rFoDH1 solution (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added dropwise to the electrode, and the enzyme was fixed by holding it at 4°C for 1 hour under water vapor saturation conditions to obtain enzyme electrode 1.

[0118] <Preparation Example 5: Enzyme electrode 2 containing enzyme (a2) (rFoDH1β(Me))> 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 rFoDH1 solution.

[0119] <Test Example 1: CV Measurement 1> Cyclic voltammetry (CV) measurements were performed using enzyme electrodes 1 and 2 obtained in preparation examples 4 and 5. The measuring device and measurement conditions are as follows: Measurement device: Electrochemical analyzer ALS660E (manufactured by BAS Corporation) Solution: 1M potassium phosphate buffer (pH 8.0), 50mM NADH, 50mM NAD + Temperature: 25℃ Atmosphere: Ar Rotation speed: 100 rpm Potential sweep speed: 5mVs- 1 The measurement results are shown in Figure 5. In Figure 5, the results for enzyme electrode 1 are shown by a dashed line, and the results for enzyme electrode 2 are shown by a solid line. Furthermore, for electrode 2, NADH and NAD + The results measured under conditions that did not include [the specified condition] are shown by the dotted line.

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

[0121] <Test Example 2: CV Measurement 2> Cyclic voltammetry (CV) measurements were performed using electrode 2 obtained in Preparation Example 5. The same measurement equipment and conditions as in Test Example 1 were used, except for the measurement solution described below. Measurement solution: 1M potassium phosphate buffer (pH 8.0), 1mM NADH, 10mM glucose, 1mg / mL NAD-dependent glucose dehydrogenase (Toyobo Co., Ltd., EC 1.1.1.47, 250U / mg, hereafter also referred to as GDH) The measurement results are shown in Figure 6 (solid lines). Results measured under conditions without glucose and GDH are shown with dashed lines, and results measured under conditions without NADH, glucose, and GDH are shown with dotted lines.

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

[0123] <Test Example 3: CV Measurement 3> Cyclic voltammetry (CV) was performed using electrode 2 obtained in preparation example 5. The measurement was carried out using the same measuring apparatus and conditions as in Test Example 1, except for the use of the following measurement solutions. Measurement solution: 1M potassium phosphate buffer (pH 8.0), 1mM NAD +10 mM dihydroxyacetone (DHA), 1 mg / mL NAD-dependent glycerol dehydrogenase (Toyobo Co., Ltd., EC1.1.1.6, 50 U / mg, hereinafter also referred to as GIDH) The measurement results are shown in Figure 7 (solid line). The results measured under conditions that did not include DHA and GIDH are shown by the dashed line, and NAD + The results measured under conditions that did not include DHA and 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 coupled system exists 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))> Glassy carbon electrodes (GCE) were polished with alumina particles with 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 (MWCNTs) were added to 10 mL of 1-methylpyrrolidone (NMP), and the suspension was ultrasonically treated for 2 hours to uniformly disperse the MWCNTs. 10 μL of the resulting MWCNT dispersion was added dropwise to the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was immersed in an N,N-dimethylformamide (DMF) solution containing 10 mM 1-pyrenemethylamine hydrochloride (PyNH2) and left at room temperature for 1 hour to obtain a PyNH2 / CNT / GCE electrode. The prepared PyNH2 / CNT / GCE was washed with DMF and ultrapure water. Then, 30 μL of 1 mg / mL rFoDH1β(Ec) solution (dissolved in 1M potassium phosphate buffer (pH 8.0)) was added dropwise to the electrode. The electrode was then held at 4°C for 1 hour under water vapor saturation conditions to adsorb the enzyme onto the electrode surface, obtaining enzyme electrode 3. Excess rFoDH1β(Ec) solution was removed before being 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 described above was immersed in 1.5 mL of 1 M potassium phosphate buffer (pH 8.0) containing 1 M glucose. To the buffer, 200 μL of a 10 mg / mL solution of NAD(P)H or glucose dehydrogenase (GDH, GLD-311, manufactured by Toyobo Co., Ltd.) as NAD(P)-dependent oxidoreductase (b) (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added. Further, NADH (dissolved in 1 M potassium phosphate buffer (pH 8.0)) was added to a final concentration of 10 mM, and these were adsorbed onto the electrode surface of enzyme electrode 3 to obtain enzyme electrode 4 containing enzyme (a2), enzyme (b), and NADH. The cyclic voltammetry (CV) measurement described below uses the enzyme electrode 4 while immersed in the buffer solution. Therefore, although the buffer solution contains excess NADH as well as GDH, the CV and CA measurements below are performed in a static solution, so the excess NADH and GDH present in the bulk solution, which are not immobilized and can be considered as such, 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 above is treated with 50 mM NADH and 50 mM NAD. + Immerse in 1.5 mL of 1 M potassium phosphate buffer (pH 8.0) containing NADH, NAD + The enzyme (a2), enzyme (b), and NADH were adsorbed onto the electrode surface of enzyme electrode 3. Next, 150 μL of a 10 mg / mL solution of NAD(P)H or glucose dehydrogenase (GDH, GLD-311, manufactured by Toyobo Co., Ltd.) as NAD(P)-dependent oxidoreductase (b) (dissolved in 1M potassium phosphate buffer (pH 8.0)) was added to the buffer and adsorbed onto the electrode surface of enzyme electrode 3 to obtain enzyme electrode 5 containing enzyme (a2), enzyme (b), and NADH. The chronoamperometry (CA) measurement described below was performed with enzyme electrode 5 immersed in the buffer. Glucose was added stepwise to the buffer in which 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 only enzyme (b) (GDH)> Glassy carbon electrodes (GCE) were polished with alumina particles with 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 (MWCNTs) were added to 10 mL of 1-methylpyrrolidone (NMP), and the suspension was ultrasonically treated for 2 hours to uniformly disperse the MWCNTs. 10 μL of the resulting MWCNT dispersion was added dropwise to the GCE and dried at 70°C to obtain a CNT / GCE electrode. The CNT / GCE was immersed in an N,N-dimethylformamide (DMF) solution containing 10 mM 1-pyrenemethylamine hydrochloride (PyNH2) and left at room temperature for 1 hour to obtain a PyNH2 / CNT / GCE electrode. After washing the prepared PyNH2 / 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 added dropwise, and the mixture was left at room temperature for 1 hour to adsorb the enzyme onto the electrode surface, thereby obtaining enzyme electrode 6.

[0129] <Preparation Example 10 (Comparative Example): Enzyme electrode 7 with enzyme (a2)(rFoDH1β(Ec)) adsorbed onto enzyme electrode 6> The enzyme electrode 6 described above 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 enzyme electrode 7, which was adsorbed onto the electrode surface of enzyme electrode 6. The cyclic voltammetry (CV) measurement described below was performed with enzyme electrode 7 immersed in the buffer. Therefore, although the buffer contains excess rFoDH1β(Ec), since the CV measurement below is performed in a static solution, the excess rFoDH1β(Ec) present in the bulk solution, which is not immobilized and is not located near the electrode, can be considered as not affecting the electrochemical reaction.

[0130] <Test Example 4: CV Measurement 4> Cyclic voltammetry (CV) was performed using the enzyme electrode 3 obtained in preparation example 6. The measuring device and measurement conditions are as follows: Measurement device: Electrochemical analyzer ALS660E (manufactured by BAS Corporation) Solution: 1M potassium phosphate buffer (pH 8.0), 50mM NADH, 50mM NAD + Temperature: 25℃ Atmosphere: Ar Potential sweep speed: 5mVs -1 The measurement results are shown in Figure 8.

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

[0132] <Test Example 5: CV Measurement 5> Cyclic voltammetry (CV) measurements were performed using the enzyme electrode 4 obtained in Preparation Example 7. The same measurement apparatus and conditions as in Test Example 1 were used, except for the measurement solution described below. Measurement solution: 1M potassium phosphate buffer (pH 8.0), 10mM NADH, 1M glucose, 1mg / 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 and GDH are shown with a dotted line.

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

[0134] <Test Example 6: CA Measurement 6> Chronoamperometry (CA) was performed using the enzyme electrode 5 obtained in Preparation Example 8. Except for the use of the following measurement solutions, the measurement was carried out using the same measuring apparatus and conditions as in Test Example 4. Measurement solution: 1M potassium phosphate buffer (pH 8.0), 50mM NADH, 1mg / 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 GDH addition decreased to the extrapolation line for the case without GDH, but did not return to the extrapolation line after glucose was added. Furthermore, since the current value increased depending on the amount added, it is clear that this current is due to the oxidation of glucose.

[0136] <Test Example 7 (Comparative Example): CV Measurement 7> Cyclic voltammetry (CV) measurements were performed using enzyme electrode 6 obtained in Preparation Example 9 and enzyme electrode 7 obtained in Preparation Example 10. The same measurement apparatus and conditions as in Test Example 4 were used, except for the measurement solutions described below. Measurement solution: 1M potassium phosphate buffer (pH 8.0), 10mM NADH, 1M glucose, 0.25 mg / mL rFoDH1β The measurement results are shown in Figure 11. The results for enzyme electrode 6 are shown by a dashed line, and the results for enzyme electrode 7 are shown by a solid line. No increase in catalytic current was observed for either enzyme electrode 6 or 7. The absence of an increase in catalytic current in enzyme electrode 6 is due to the fact that it contains only GDH and does not contain rFoDH1β(Ec). The results for enzyme electrode 7, although containing rFoDH1β(Ec) and GDH, are due to the first catalyst layer containing rFoDH1β(Ec) being laminated on top of the second catalyst layer containing GDH on the electrode substrate. Therefore, the technical significance of the first and second catalyst layers being laminated in this order on the electrode substrate in the enzyme electrode of the present invention has been clarified. [Explanation of Symbols]

[0137] 1. Enzyme electrode 2. Enzyme (a1) 3. Enzymes (b) 4 NAD(P) 5 NAD(P)H 6 Electrode substrate 7. Matrix 8. First Catalyst Layer 9. Second Catalyst Layer

Claims

1. An electrode containing an enzyme, The electrode comprises an electrode substrate, a first catalyst layer containing an enzyme (a1) that catalyzes the redox reaction between the redox pair of NAD(P)H and NAD(P) and can directly transfer electrons with the electrode substrate in connection with the redox reaction, 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 in which the first catalyst layer and the second catalyst layer are laminated on the electrode substrate in that order.

2. The enzyme electrode according to claim 1, wherein the enzyme (a1) comprises 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 located within 2 nm of the surface of the enzyme (a1).

3. The enzyme electrode according to claim 1 or 2, wherein the enzyme (a1) comprises a β-subunit of formate dehydrogenase derived from methanol-utilizing bacteria.

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 the portion of the enzyme (a1) that is within 2 nm of the Fe-S cluster contained in the enzyme (a1).

5. The enzyme electrode comprises at least one 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. The enzyme electrode according to claim 1 or 2, wherein the conductive polymer is at least one selected from the group consisting of polyaniline and polypyrrole.

6. The enzyme electrode according to claim 1 or 2, 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.

10. An enzyme that catalyzes the redox reaction of the redox pair of NAD(P)H and NAD(P), The enzyme has one flavin mononucleotide and two Fe-S clusters. An enzyme in which 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 located within 2 nm of the surface of the enzyme.

11. The enzyme according to claim 10, which, when used as an electrode, can directly transfer electrons with respect to the oxidation-reduction reaction between the enzyme and the electrode substrate.

12. The enzyme according to claim 10 or 11, wherein the average molecular weight is 50,000 to 100,000.

13. The enzyme according to claim 10 or 11, which is a β-subunit of formate dehydrogenase derived from methanol-utilizing bacteria.

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