Biofuel cell using enzyme electrodes

Abstract

To construct a biofuel cell capable of maximally producing electrical energy from fuel through efficient and stable electron transfer between an enzyme / electrode substrate by optimizing an electron mediator in the biofuel cell using an enzyme electrode.SOLUTION: In a biofuel cell including an enzyme electrode as an anode, the enzyme electrode includes an electrode base material, a NAD(P)-dependent fuel oxidase as an electrocatalyst for fuel oxidation, a NAD(P) flavin reductase that reduces flavin in the presence of reduced NAD(P) that is produced along with fuel oxidation by the fuel oxidase, and a flavin at concentration ranging from 0.5 mM to 100 mM. The flavin is one or more selected from FAD and its derivatives, and FMN and its derivatives.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a biofuel cell using an enzyme electrode, and more particularly to a biofuel cell that utilizes a flavin reductase and a flavin redox reaction system to improve the efficiency of electron transfer between the enzyme and the electrode substrate. [Background technology]

[0002] In recent years, biofuel cells that utilize the energy conversion systems of enzymes and microorganisms have been developed. Biofuel cells that utilize bioprocesses have mild conditions and high selectivity, and use various organic substances present in the environment, such as sugars and alcohols, as fuel. Organic substances have the advantage of being able to reduce the weight of the battery because of their high weight energy density, and are also excellent from the perspective of sustainability. Biofuel cells that use such organic substances as an energy source are lightweight, safe, and have a small environmental impact, and are therefore expected to be further developed as power sources for small electronic devices such as drones, portable devices, and implantable devices.

[0003] In the case of a biofuel cell using an enzyme as an electrode catalyst, it generally includes an anode (enzyme electrode) where an enzyme-induced oxidation reaction of fuel takes place, and a cathode where a reduction reaction takes place, and the anode and cathode are connected by an external circuit, and if necessary, a diaphragm is included to separate the anode and cathode. As the enzyme that performs the fuel oxidation reaction at the anode, a nicotinamide adenine dinucleotide (hereinafter sometimes abbreviated as "NAD")-dependent fuel oxidase or a nicotinamide adenine dinucleotide phosphate (hereinafter sometimes abbreviated as "NADP")-dependent fuel oxidase is commonly used (hereinafter, "NAD" and "NADP" may be collectively referred to as "NAD(P)"). In the anode using the NAD(P)-dependent fuel oxidase, the electrons extracted from the fuel by the catalytic function of the NAD(P)-dependent fuel oxidase are converted into oxidized NAD(P) (hereinafter, "NAD(P)"). + ") and NAD(P) +is reduced to reduced NAD(P) (hereinafter sometimes abbreviated as "NAD(P)H"). Electrons are transferred from the generated NAD(P)H to the electrode, but the efficiency of electron transfer from NAD(P)H to the electrode substrate is low, so that sufficient battery output cannot be obtained at present.

[0004] Therefore, with the aim of improving the performance of biofuel cells, research and development is being conducted from the viewpoint of, for example, increasing the efficiency of electron transfer between the enzyme and the electrode substrate. For example, a biofuel cell using a coenzyme oxidase has been reported in order to increase the efficiency of electron transfer between the enzyme and the electrode substrate (see, for example, Patent Document 1, etc.). Patent Document 1 relates to a biofuel cell that uses, as the anode, an enzyme electrode in which an NAD(P)-dependent oxidoreductase is immobilized as an electrode catalyst on an electrode substrate. In the examples, the anode of the biofuel cell is a fuel (glucose), a fuel oxidase (NAD-dependent glucose dehydrogenase), an oxidized coenzyme (NAD + ), coenzyme oxidase (diaphorase), electron transfer substance (2-amino-1,4-naphthoquinone (hereinafter sometimes abbreviated as "ANQ")), and electrode in that order. In other words, the biofuel cell is configured so that electrons extracted from the fuel by the catalytic function of the fuel oxidizing enzyme are transferred to the electrode substrate via diaphorase and ANQ. With this configuration, a current of 45 mA / cm at -0.3 V (vs. Ag / AgCl) can be obtained. 2 It has been reported that a current density of 1000 times higher than that ...

[0005] Furthermore, a technique has been reported in which electrons are transferred between the enzyme and the electrode substrate by utilizing the oxidation-reduction reaction of flavin (see, for example, Non-Patent Documents 1 and 2). Non-Patent Document 1 relates to a biosensor for detecting flavins, which uses, as a working electrode, an enzyme electrode in which NAD(P)-dependent flavin reductase is immobilized on an electrode substrate as an electrode catalyst. At the working electrode of the biosensor, NAD(P)H and H are converted by the catalytic function of the flavin reductase. + and oxidized flavins, NAD(P)+ and is converted to a reduced flavin, i.e., dihydroflavin, which is configured to transfer electrons to the electrode substrate.

[0006] Non-Patent Document 2 relates to a biosensor for detecting lactate, which uses an enzyme electrode having NAD(P)-dependent lactate dehydrogenase immobilized on an electrode substrate as an electrode catalyst as a working electrode. + The biosensor is configured so that electrons are transferred in the following order: NAD(P)-dependent flavin reductase, riboflavin, and the electrode. In other words, the biosensor is configured so that electrons extracted from lactic acid, which is a detection target substance, by the catalytic function of lactate dehydrogenase are transferred to the electrode substrate via flavin reductase and riboflavin.

[0007] Here, flavin is a general term for compounds having an isoalloxazine skeleton, and representative examples include riboflavin, flavin adenine dinucleotide (hereinafter sometimes abbreviated as "FAD"), and flavin mononucleotide (hereinafter sometimes abbreviated as "FMN"). In Non-Patent Document 1, when the above three types of flavin were compared, it was confirmed that the highest current value was obtained when riboflavin was used. However, since riboflavin has a lower solubility in water than FAD and FMN, it cannot be supplied to the enzyme electrode at a high concentration. Therefore, in both the biosensors in Non-Patent Document 1 and Non-Patent Document 2, flavin is used at a concentration of 0.1 mM or less. On the other hand, when constructing a biofuel cell, which generally requires a higher current density than a biosensor, it becomes necessary to increase the amount of flavin, which is an electron transfer substance, in order to improve the electron transfer efficiency between the enzyme / electrode substrate. However, it is expected that the low solubility of riboflavin will be an obstacle.

[0008] In addition, reduced flavin in the aqueous solution is quickly oxidized by oxygen and converted to oxidized flavin. Therefore, in the presence of oxygen, it is expected that the efficiency of electron transfer to the electrode substrate will decrease. In view of this technical common sense, the measurements using the biosensors in Non-Patent Documents 1 and 2 are performed under an argon atmosphere. However, in order to maintain an anaerobic environment on the anode side of a biofuel cell, it becomes necessary to purge the fuel, etc. with an inert gas such as argon, which is expected to require complicated steps in the production and operation of the biofuel cell and to increase costs.

[0009] In view of these circumstances, there have been no reports to date of the application of flavin reductase and a flavin redox reaction system to an electron transport system between an enzyme and an electrode substrate in a biofuel cell. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent Publication No. 2010-92667 [Non-patent literature]

[0011] [Non-Patent Document 1] Serge C. et al., “A Poly(amphiphilic pyrrole)-Flavin Reductase Electrode for Amperometric Detemination of Flavins”, Anal. Chem. (1997), Vol. 69, No. 15, pp. 3095-3099. [Non-Patent Document 2] Serge C. et al., "An Original Electroenzymatic System: Flavin Reductase-Riboflavin for the Improvement of Dehydrogenase-Based Biosensors. Application to the Amperometric Detection of Lactate", Electroanalysis (1997), Vol. 9, No. 9, pp. 685-688 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0012] As described above, various techniques for efficient electron transfer between an enzyme and an electrode substrate have been reported in the past, but they have not fully met expectations in terms of cell performance, such as the cell output of a biofuel cell.

[0013] Therefore, the present invention aims to construct a biofuel cell using an enzyme electrode that can generate maximum electrical energy from fuel through efficient and stable electron transfer between the enzyme and electrode substrate by optimizing the electron transfer substance. [Means for solving the problem]

[0014] Therefore, the inventors conducted intensive research to achieve the above-mentioned object, and as a result, discovered that by using a flavin reductase and a specific type and concentration range of flavin for electron transfer between the enzyme and the electrode substrate in a biofuel cell, efficient electron transfer between the enzyme and the electrode substrate can be achieved and cell output can be improved, and the present invention was completed based on this finding.

[0015] That is, the present invention provides the following [1] to [4].

[0016] [1] A biofuel cell comprising an enzyme electrode as an anode, The enzyme electrode is An electrode substrate; Nicotinamide adenine dinucleotide-dependent fuel oxidase or nicotinamide adenine dinucleotide phosphate-dependent fuel oxidase as an electrode catalyst for oxidizing a fuel; a nicotinamide adenine dinucleotide-dependent flavin reductase or a nicotinamide adenine dinucleotide phosphate-dependent flavin reductase that reduces flavin in the presence of reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate produced as a result of the oxidation of a fuel by the fuel oxidizing enzyme; and a flavin in a concentration range of 0.5 mM to 100 mM; In this biofuel cell, the flavin is one or more selected from flavin adenine dinucleotide and derivatives thereof, and flavin mononucleotide and derivatives thereof.

[0017] According to the above configuration [1], a biofuel cell capable of efficient and stable electron transfer between the enzyme and the electrode substrate can be provided by using a flavin reductase and a specific type and concentration range of flavin. This makes it possible to efficiently and stably convert the chemical energy of fuels such as sugars, alcohols, and organic acids into electrical energy, thereby enabling the biofuel cell to have a high output. Conventionally, electrons extracted from the fuel by the catalytic function of the NAD(P)-dependent fuel oxidase, which is an electrode catalyst, are converted into electrical energy by the NAD(P)-dependent fuel oxidase. + When the reduction of NAD(P)H was used to transfer the electrons to the electrode substrate, sufficient cell output could not be obtained due to the low efficiency of electron transfer from NAD(P)H to the electrode substrate. The biofuel cell with this configuration has a high efficiency of electron transfer between the enzyme and the electrode substrate, enabling output comparable to that of lithium-ion batteries, and is expected to be applied to various technical fields such as electronics, medicine, food, and the environment, including providing power to small devices such as drone batteries.

[0018] [2] The biofuel cell according to [1] above, wherein the flavin is flavin adenine dinucleotide or flavin mononucleotide.

[0019] According to the above configuration [2], flavin adenine dinucleotide or flavin mononucleotide is easy to handle and obtain, and is inexpensive, and is therefore particularly advantageous in terms of industrial mass production of biofuel cells and in terms of economy, etc.

[0020] [3] The biofuel cell according to [1] or [2] above, wherein the flavin is in a concentration range of 5 mM to 50 mM.

[0021] According to the above configuration [3], the concentration range of flavin is further optimized, so that a biofuel cell capable of particularly efficient and stable electron transfer between the enzyme and the electrode substrate can be provided.

[0022] [4] The biofuel cell according to any one of the above [1] to [3], wherein the nicotinamide adenine dinucleotide-dependent fuel oxidase or the nicotinamide adenine dinucleotide phosphate-dependent fuel oxidase is glucose dehydrogenase.

[0023] The above configuration [4] is particularly advantageous in terms of industrial mass production of biofuel cells and economics, since it is easy to handle and obtain, and can use inexpensive glucose as fuel. [Brief description of the drawings]

[0024] [Figure 1] FIG. 2 is a schematic diagram of the electrode configuration of the anode of a biofuel cell according to one embodiment of the present invention. [Diagram 2] 1 is a graph showing the results of Example 3-1 in which the electron transfer function of flavin reductase was evaluated, in which a phosphate buffer was used as the pH buffer solution. [Diagram 3] 1 is a graph showing the results of Example 3-2 in which the electron transfer function of flavin reductase was evaluated, in which an imidazole buffer solution was used as the pH buffer solution. [Figure 4] FIG. 13 is a graph showing the results of Example 3-3 in which the electron transfer function of flavin reductase was evaluated, in which diaphorase was used as a comparison with the case in which flavin reductase was used. [Diagram 5] FIG. 1 is a graph showing the results of Example 3-4 in which the electron transfer function of flavin reductase was evaluated, comparing the results when FAD, FMN, and riboflavin were used as flavins. [Figure 6] FIG. 4 is a graph showing the results of Example 4-1 in which the concentration dependence of flavin and flavin reductase was evaluated, and showing the results of evaluating the effect on electrode performance using flavin at various concentrations. [Figure 7] FIG. 4 is a graph showing the results of Example 4-2 in which the concentration dependence of flavin and flavin reductase was evaluated, and shows the results of evaluating the effect on electrode performance using flavin reductase at various concentrations. [Figure 8] 1 is a graph showing the results of Example 5 in which the electrode performance of an electrode using glucose as an electron donor was evaluated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In one embodiment of the biofuel cell, at least the anode is configured as an enzyme electrode containing an enzyme as an electrode catalyst. The enzyme electrode of the anode is configured to use an enzyme containing an NAD(P)-dependent fuel oxidase as an electrode catalyst, and to use an NAD(P)-dependent flavin reductase and a specific type and concentration range of flavin for electron transfer between the enzyme and the electrode substrate.

[0026] [anode] The electrode configuration of the anode of a biofuel cell according to one embodiment of the present invention is shown in Fig. 1. The anode is configured to include an NAD(P)-dependent fuel oxidase, a fuel, NAD(P), an NAD(P)-dependent flavin reductase, a specific type of flavin in a specific concentration range, and an electrode substrate. Here, the NAD(P)-dependent fuel oxidase is NAD(P) +It is an electrode catalyst having a catalytic function of oxidizing fuel in the presence of []. NAD(P)-dependent flavin reductase is an enzyme having a catalytic function of reducing oxidized flavin in the presence of NAD(P)H generated along with the oxidation of fuel by NAD(P)-dependent fuel oxidase. Flavin is a substrate of the flavin reductase. Hereinafter, each component will be described in detail, but it is not limited to the description and can be appropriately modified.

[0027] <NAD(P)-dependent fuel oxidase> NAD(P)-dependent fuel oxidase is an electrode catalyst of the anode of a biofuel cell according to an embodiment of the present invention. It catalyzes the oxidation reaction of fuel in the presence of NAD(P), and at the same time, NAD(P) is reduced by two-electron reduction accompanying the oxidation reaction of the fuel. + is used to convert it to NAD(P)H. That is, as shown in the following reaction formula, NAD(P)-dependent fuel oxidase + catalyzes the reaction of reversibly converting fuel and NAD(P) + into the oxidation product of fuel, NAD(P)H, and H Fuel + NAD(P) + = Oxidation product of fuel + NAD(P)H + H +

[0028] The NAD(P)-dependent fuel oxidase used in the biofuel cell according to an embodiment of the present invention is not particularly limited as long as it has the above catalytic function. Therefore, NAD(P) +Enzymes capable of decomposing organic substances such as sugars, alcohols, and organic acids in the presence of the enzyme can be preferably used. For example, oxidoreductases can be used, and in particular, dehydrogenases and oxidases can be used. Specifically, glucose dehydrogenase, fructose dehydrogenase, alcohol dehydrogenase, gluconate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, pyruvate dehydrogenase, aldehyde dehydrogenase, glucose oxidase, fructose oxidase, alcohol oxidase, lactate oxidase, glutamate oxidase, pyruvate oxidase, aldehyde oxidase, and the like can be exemplified, and in particular, glucose dehydrogenase and the like used in Example 5 can be preferably used. However, the enzymes are not limited to these, and can be appropriately selected based on the type of fuel supplied to the biofuel cell, and the like.

[0029] The NAD(P)-dependent fuel oxidase may be used alone or in combination with multiple enzymes. When multiple enzymes are combined, they may catalyze the same reaction, or they may catalyze different reactions. Thus, for example, an arbitrary enzyme may be used in combination with another type of enzyme that can function in conjunction with the enzyme. In addition, an arbitrary enzyme may be combined with another enzyme that catalyzes a subsequent reaction of the catalytic reaction by the enzyme to construct a multi-step reaction system.

[0030] The NAD(P)-dependent fuel oxidase may be immobilized on the electrode substrate of the anode, or may be supplied to the anode as an enzyme solution dissolved in a suitable solvent. It may also be supplied in the form of a gel or the like. The solvent is an aqueous medium, and may be distilled water or a suitable buffer solution. Examples of buffer components contained in the buffer solution 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). These may be used alone, or two or more of them may be used in combination.

[0031] The NAD(P)-dependent fuel oxidase may require other coenzymes or cofactors in addition to NAD(P). For example, cofactors include metal ions such as copper, iron, manganese, selenium, magnesium, zinc, and calcium. However, the cofactors are not limited to these, and a substance required for the expression of the catalytic function of the enzyme is appropriately selected depending on the type of enzyme. The form of the enzyme may be either an apo form or a holo form, but is configured so as to be able to exert its catalytic function during the electrode reaction. For example, when the enzyme is immobilized on the electrode substrate in the apo form, a means for converting the enzyme into an active holo form is appropriately provided, such as supplying a coenzyme and a cofactor to the immobilized enzyme during the operation of the enzyme electrode.

[0032] The NAD(P)-dependent fuel oxidase is not particularly limited in terms of origin, so long as it has the above-mentioned catalytic function. Therefore, 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. Furthermore, a commercially available product may be used.

[0033] Furthermore, so long as it has the above-mentioned desired catalytic function, it may be an organism itself, such as a microorganism, an organelle, or a cell, or it may be a crude product from such an organism.

[0034] The amount of the NAD(P)-dependent fuel oxidase can be appropriately changed according to the purpose of use of the biofuel cell, the type of fuel, the type and form of the enzyme, etc.

[0035] <NAD(P)-dependent flavin reductase> The NAD(P)-dependent flavin reductase is used in the biofuel cell according to an embodiment of the present invention as a catalyst for the reduction reaction of flavin in the presence of NAD(P)H generated along with the oxidation reaction of the fuel of the NAD(P)-dependent fuel oxidase. The flavin reductase catalyzes the reaction of reducing oxidized flavin to reduced flavin. At the same time, along with the reduction reaction, NAD(P)H generated along with the oxidation reaction of the fuel of the NAD(P)-dependent fuel oxidase is oxidized to convert it into NAD(P) + to convert. That is, the NAD(P)-dependent flavin reductase has a function of reversibly converting oxidized flavin, NAD(P)H, and H + to reduced flavin and NAD(P) + to convert. Oxidized flavin + NAD(P)H + H + = Reduced flavin + NAD(P) +

[0036] The NAD(P)-dependent flavin reductase used in the biofuel cell according to an embodiment of the present invention is not particularly limited as long as it has the above catalytic function. Therefore, the flavin reductase prepared in Example 1 etc. can be exemplified, but it is not limited thereto. It can be appropriately selected based on the configuration of the biofuel cell.

[0037] The NAD(P)-dependent flavin reductase can be used alone or in combination of a plurality of enzymes. When combining a plurality of enzymes, the enzymes may be those that catalyze the same reaction or enzymes that catalyze different reactions may be combined.

[0038] The NAD(P)-dependent flavin reductase may be immobilized on an electrode substrate of the anode, or may be dissolved in a suitable solvent and supplied to the anode as an enzyme solution. It may also be supplied in the form of a gel or the like. The solvent is an aqueous medium, and may be distilled water or a suitable buffer solution. The buffer components contained in the buffer solution are as described above.

[0039] The NAD(P)-dependent flavin reductase may require other coenzymes or cofactors in addition to NAD(P). Examples of cofactors include metal ions such as copper, iron, manganese, selenium, magnesium, zinc, and calcium. However, they are not limited to these, and a substance required for the expression of the catalytic function of the enzyme is appropriately selected depending on the type of enzyme. The enzyme may be in either an apo form or a holo form, and is configured so as to be able to exert its catalytic function during the electrode reaction. In detail, it is configured in the same manner as the NAD(P)-dependent fuel oxidase described above.

[0040] The NAD(P)-dependent flavin reductase is not particularly limited in terms of origin, etc., so long as it has the above-mentioned catalytic function, and can therefore be prepared in the same manner as the above-mentioned NAD(P)-dependent fuel oxidase.

[0041] The amount of NAD(P)-dependent flavin reductase used can be appropriately changed depending on the purpose of use of the biofuel cell, the type of flavin, the type and form of the enzyme, etc. For example, it can be used in a concentration range of 1 mg / mL to 200 mg / mL, preferably 5 mg / mL to 50 mg / mL.

[0042] <Flavin> In a biofuel cell according to one embodiment of the present invention, flavin functions as an electron transfer substance that transfers electrons to an electrode substrate. Flavin is a general term for compounds having an isoalloxazine skeleton, and is a compound that can be a substrate for the above-mentioned NAD(P)-dependent flavin reductase.

[0043] The flavin used in the biofuel cell of one embodiment of the present invention is used in a concentration range of 0.5 mM or more, preferably 0.5 mM to 300 mM, more preferably 0.5 mM to 100 mM, and particularly preferably 5 mM to 50 mM.

[0044] The flavin is configured to be able to come into contact with the NAD(P)-dependent flavin reductase in the above concentration range. The flavin may be immobilized on an electrode substrate, or may be supplied to the anode as a solution dissolved in a suitable solvent. The flavin may also be supplied in the form of a gel or the like. The solvent is an aqueous medium, and may be distilled water or a suitable buffer solution. The buffer components contained in the buffer solution are as described above. For example, when the flavin is contained in the fuel solution and supplied, the final concentration of the flavin in the fuel solution when contacted with the NAD(P)-dependent flavin reductase is prepared to be within the above concentration range. Similarly, when the flavin is prepared as a solution separate from the fuel solution, the final concentration of the flavin when contacted with the NAD(P)-dependent flavin reductase is prepared to be within the above concentration range. Therefore, the flavin is preferably a substrate for the NAD(P)-dependent flavin reductase and has a solubility that allows it to be prepared in the above concentration range as an aqueous solution.

[0045] Specifically, the flavin is one or more selected from flavin adenine dinucleotide (hereinafter sometimes abbreviated as "FAD") and derivatives of FAD, flavin mononucleotide (hereinafter sometimes abbreviated as "FMN") and derivatives of FMN. Note that riboflavin, which is widely used as a flavin, is not suitable for use in the biofuel cell of one embodiment of the present invention in terms of its solubility in aqueous media.

[0046] There are no particular limitations on the FAD derivatives and FMN derivatives as long as they have the above properties, similar to FAD and FMN, respectively. FAD derivatives and FMN derivatives refer to compounds in which any part of the FAD and FMN molecules has been modified by any chemical reaction, such as introduction or substitution of a substituent at any position of FAD and FMN, or oxidation or reduction. In this case, there are no particular limitations on the type, number, and introduction or substitution position of the substituent. In addition, FAD derivatives and FMN derivatives also include salt forms in which FAD and FMN form salts with any metal ion such as sodium, and FAD and FMN in hydrate forms containing water molecules.

[0047] Examples of FAD derivatives and FMN derivatives include compounds in which hydrogen atoms at any position (e.g., the nitrogen atom at position 3 and the carbon atoms at positions 6 to 9) of the isoalloxazine ring of FAD and FMN are substituted with any substituent, and compounds in which substituents on the isoalloxazine skeleton (e.g., the methyl groups on the carbon atoms at positions 7 and 8 and the hydrogen atoms of the methyl groups) are substituted with other substituents. Other examples include compounds in which hydrogen atoms at any position of the ribitol moiety are substituted with any substituent. Other examples include compounds in which hydrogen atoms at any position (e.g., the carbon atoms at positions 2 and 8) of the purine ring of the adenine moiety of FAD are substituted with any substituent, and compounds in which substituents on the purine ring (e.g., the amino group on the carbon atom at position 6 and the hydrogen atoms of the amino group) are substituted with other substituents. Further examples include compounds in which the hydrogen atoms at any position (e.g., the positions of the 2nd to 5th carbon atoms) of the tetrahydrofuran ring of the ribose portion of FAD are substituted with any substituent, and compounds in which the substituents on the tetrahydrofuran ring (e.g., the 3rd and 4th hydroxyl groups and the hydrogen atoms of the hydroxyl groups) are substituted with other substituents.

[0048] Examples of the substituent include, but are not limited to, a hydrogen atom, a halogen group, a hydroxy group, a nitro group, a cyano group, a carboxy group, an amino group, a sulfo group, a formyl group, a sulfanyl group, a phosphate group, an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an alicyclic heterocyclic group, an aromatic hydrocarbon group, or an aromatic heterocyclic group.

[0049] The halogen group is selected from a fluoro group, a chloro group, a bromo group, an iodo group, and the like.

[0050] The aliphatic hydrocarbon group may be linear or branched, and may be saturated or unsaturated. There is also no particular restriction on the chain length. Examples of the aliphatic hydrocarbon group include, but are not limited to, alkyl groups, alkenyl groups, and alkynyl groups each having 1 to 10 carbon atoms, and more preferably having 1 to 5 carbon atoms.

[0051] In the alicyclic hydrocarbon group, the bond between the ring constituent atoms may be saturated or unsaturated, and there is no particular restriction on the number of ring members. In addition to a single ring, it also includes those having a ring assembly such as a condensed ring or a spiro ring. Examples of the alicyclic hydrocarbon group include, but are not limited to, a cycloalkyl group having preferably 3 to 10 carbon atoms, particularly preferably 3 to 7 carbon atoms, and a cycloalkenyl group having preferably 4 to 10 carbon atoms, particularly preferably 4 to 7 carbon atoms.

[0052] The alicyclic heterocyclic group is a non-aromatic heterocyclic group having one or more heteroatoms as ring-constituting atoms. It includes not only single rings but also ring assemblies such as condensed rings and spiro rings. The bond between the ring-constituting atoms may be saturated or unsaturated, and there is no particular restriction on the number of ring members. Preferred examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, but there is no particular restriction on the type of heteroatom, and there is no particular restriction on the number and introduction position of the heteroatom. For example, there can be mentioned an alicyclic heterocyclic group having preferably 2 to 7 carbon atoms, particularly preferably 2 to 5 carbon atoms, and preferably 1 to 5 heteroatoms, particularly preferably 1 to 3 heteroatoms. In addition, when there are multiple heteroatoms, they may be the same type of atoms or different types of atoms.

[0053] The aromatic hydrocarbon group (aryl group) is not particularly limited as long as it has an aromatic ring. It includes not only a single ring but also a ring assembly such as a condensed ring or a spiro ring. There is also no particular limit to the number of ring members. For example, an aromatic hydrocarbon group having preferably 6 to 22 carbon atoms, particularly preferably 6 to 14 carbon atoms, can be mentioned.

[0054] The aromatic heterocyclic group is an aromatic heterocyclic group having one or more heteroatoms as ring-constituting atoms. It includes not only single rings but also ring assemblies such as condensed rings and spiro rings. There is no particular limitation on the number of ring members. Preferred examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, but there is no particular limitation on the type of heteroatom, and there is no particular limitation on the number and introduction position of heteroatoms. For example, there can be mentioned aromatic heterocyclic groups having preferably 1 to 5 carbon atoms, particularly preferably 3 to 5 carbon atoms, and preferably 1 to 4 heteroatoms, particularly preferably 1 to 3 heteroatoms. In addition, when there are multiple heteroatoms, they may be the same type of atoms or different types of atoms.

[0055] These substituents may be further substituted with an appropriate substituent, and therefore include any combination of the above-mentioned substituents, such as, but not limited to, alkyloxy groups, aryloxy groups, alkylamino groups, arylamino groups, alkylthio groups, arylthio groups, halogenated alkyl groups, halogenated aryl groups, alkylcarboxy groups, and arylcarboxy groups.

[0056] <Electrode base material> The electrode substrate is a conductive substrate that can be connected to an external circuit and can transmit electrons. Therefore, as long as it has the relevant properties, there is no particular limitation on the material or shape. Examples of the conductive substrate include carbon materials such as carbon cloth, carbon paper, graphite, and glassy carbon, metals or alloys such as aluminum, copper, gold, platinum, silver, nickel, and palladium, and conductive oxides such as SnO2, In2O3, WO3, and TiO2. However, the conductive substrate is not limited to these, and any conductive substrate made of a material known in the art can be used.

[0057] In addition, it may be configured as a single layer or a laminated structure of two or more layers. In addition, in order to improve the conductivity, commercially available carbon black such as Ketjenblack, conductive carbon fine particles such as activated carbon powder, etc. may be applied to the substrate. In this case, a binder such as PVDF may be used. The size and shape of the electrode substrate are not particularly limited and can be appropriately adjusted depending on the purpose of use. Therefore, it can be configured as a microelectrode with an electrode area reduced to the order of micrometers.

[0058] Preferably, NAD(P)-dependent fuel oxidase and NAD(P)-dependent flavin reductase are immobilized on the electrode substrate, but they may be configured to be supplied onto the electrode substrate as an enzyme solution or the like without being immobilized. Alternatively, only one of the enzymes may be immobilized and the other may be supplied as an enzyme solution or the like, or only a portion of the enzyme may be immobilized and the remaining enzyme may be supplied as an enzyme solution or the like. When immobilizing an enzyme on an electrode substrate, it can be performed by a method known in the art, such as a carrier binding method in which the enzyme is immobilized via physical adsorption, ionic bond, or covalent bond. In addition, a crosslinking method in which the enzyme is immobilized by crosslinking with a crosslinking reagent having a bivalent functional group such as glutaraldehyde can also be used. Furthermore, an entrapment method in which the enzyme is encapsulated and immobilized in a semipermeable membrane such as a polysaccharide such as alginic acid or carrageenan, a polymer having a mesh structure such as a conductive polymer, a redox polymer, or a photocrosslinkable polymer can also be used. These methods may also be used in combination. For example, when immobilizing an enzyme on an electrode substrate by physical adsorption, the enzyme can be easily immobilized by contacting the electrode substrate with an enzyme solution and drying it.

[0059] In addition, when immobilizing, the NAD(P)-dependent fuel oxidase and the NAD(P)-dependent flavin reductase are immobilized so that they can exert their catalytic activity. Therefore, the enzymes may be immobilized in a holo form containing coenzymes and cofactors necessary for exerting their catalytic activity, or in an apo form not containing them. When immobilized in an apo form, the coenzymes and cofactors may be immobilized in the same or different layers. In addition, other substances necessary for the expression of the catalytic function of the enzymes may be immobilized, and may be immobilized in the same or different layers as the enzymes. For example, flavins responsible for electron transfer to the electrode substrate may be immobilized on the electrode substrate, and may be immobilized in the same or different layers as the enzymes.

[0060] <Anode reaction mechanism> In the anode of the biofuel cell of one embodiment of the present invention, as shown in FIG. 1, the NAD(P)-dependent fuel oxidase is + In the presence of NAD(P)+, fuel is oxidized and NAD(P)+ is reduced to NAD(P)H. Then, in the presence of NAD(P)H, NAD(P)-dependent flavin reductase reduces oxidized flavin to reduced flavin, and NAD(P)H is converted to NAD(P). + The resulting reduced flavin transfers electrons to the electrode substrate and is converted to oxidized flavin. By repeating this cycle, electrons extracted from the fuel are efficiently and stably transferred to the electrode substrate.

[0061] [Biofuel Cell] A biofuel cell according to an embodiment of the present invention is configured to include an anode configured as described above. The biofuel cell includes an anode that performs an oxidation reaction and a cathode that performs a reduction reaction, the anode and the cathode are connected by an external circuit, and a diaphragm that separates the anode and the cathode as necessary. In the biofuel cell, electrons generated by the oxidation of the fuel are extracted to the electrode substrate and protons are generated. Then, as shown in the above-mentioned anode reaction mechanism, these electrons are transferred to the electrode substrate of the anode. Then, an electric current is generated by transferring the electrons from the anode to the cathode through the external circuit. Meanwhile, the protons generated on the anode side pass through the diaphragm and move to the cathode side, and are used for water generation together with the electrons that have moved from the anode side through the external circuit. The cathode is preferably configured by fixing a catalyst that can transfer electrons by reducing an oxidizing agent such as oxygen or hydrogen peroxide, and is configured to generate water by reacting the protons generated on the anode side with oxygen.

[0062] 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, the enzyme is preferably immobilized on a suitable electrode substrate, but may be configured to be supplied to a suitable electrode substrate as an enzyme solution without being immobilized. In this case, the electrode substrate can be similarly one that is exemplified as the electrode substrate for the anode.

[0063] The fuel in the biofuel cell of one embodiment of the present invention is appropriately set depending on the type of NAD(P)-dependent fuel oxidase, and can be a compound that serves as a substrate for NAD(P)-dependent fuel oxidation. For example, the fuel can be sugars such as glucose, fructose, and sucrose, alcohols such as methanol, and organic acids such as gluconic acid, lactic acid, glutamic acid, and pyruvic acid.

[0064] The fuel may be supplied in any form as long as it is supplied so as to come into contact with the NAD(P)-dependent fuel oxidase. The fuel is preferably supplied as a fuel solution dissolved in a suitable solvent, but may be supplied in the form of a gel or the like. The solvent is an aqueous medium, and may be distilled water or a suitable buffer solution. The buffer components contained in the buffer solution are as described above.

[0065] The fuel solution may be supplied containing NAD(P)-dependent fuel oxidase, NAD(P)-dependent flavin reductase, flavin, etc. Also, the NAD(P)-dependent fuel oxidase, NAD(P)-dependent flavin reductase, flavin, etc. may be supplied in a form dissolved in an appropriate buffer solution as a solution separate from the fuel solution.

[0066] Furthermore, when the NAD(P)-dependent fuel oxidase and NAD(P)-dependent flavin reductase are prepared in the form of an apoenzyme that does not contain coenzymes or cofactors necessary for the expression of catalytic activity, the fuel solution may be prepared by including these coenzymes or cofactors. Furthermore, these coenzymes and cofactors may be supplied as a solution separate from the fuel solution, dissolved in an appropriate buffer solution. Furthermore, other substances necessary for the expression of the catalytic activity of the enzymes may be prepared by including them in the fuel solution, or may be prepared as a separate solution.

[0067] The membrane used in the biofuel cell of one embodiment of the present invention is not limited in its material and shape as long as it has ion conductivity that allows protons and the like to pass through and has the property of not allowing 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 the like can be used, and a solid electrolyte membrane can also be used. Examples of solid electrolyte membranes include solid membranes having ion exchange functions such as strong acid groups such as sulfo groups, phosphate groups, phosphonic groups, and phosphine groups, weak acid groups such as carboxy groups, and organic polymers having polar groups, but are not limited to these. Specifically, a cellulose membrane and a perfluorocarbon sulfonic acid (PFS)-based resin membrane such as Nafion (registered trademark), which is a copolymer of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinyl ether], can be used.

[0068] In the biofuel cell of one embodiment of the present invention, since flavin functions as an electron transfer substance that transfers electrons between the enzyme and the electrode substrate, an additional electron transfer substance is not particularly required. However, this does not preclude the inclusion of an additional electron transfer substance. Therefore, for example, known electron transfer substances such as 2,6-dichloroindophenol (DCIP), ferricyanide, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), benzoquinone derivatives, osmium complexes, and ruthenium complexes may be included as necessary.

[0069] With this configuration, a biofuel cell capable of efficient and stable electron transfer between the enzyme and the electrode substrate can be provided using a flavin reductase and a specific type and concentration range of flavin. This allows the chemical energy of fuels such as sugars, alcohols, and organic acids to be efficiently and stably converted into electrical energy, thereby enabling the biofuel cell to have a high output. Conventionally, electrons extracted from the fuel by the catalytic function of the NAD(P)-dependent fuel oxidase, which is an electrode catalyst, are converted into electrical energy by the NAD(P)-dependent fuel oxidase.+ When the reduction of NAD(P)H is transferred to the electrode substrate via NAD(P)H, sufficient cell output cannot be obtained because the efficiency of electron transfer of NAD(P)H to the electrode substrate is low. The biofuel cell of one embodiment of the present invention has a high efficiency of electron transfer between the enzyme and the electrode substrate, and therefore can provide an output comparable to that of a lithium ion battery. In addition, a significant decrease in the efficiency of electron transfer between the enzyme and the electrode substrate due to the presence of oxygen was not observed. The production does not require complicated steps or expensive reagents, and is very advantageous from an economic perspective. Thus, the biofuel cell of one embodiment of the present invention is expected to be applied to various technical fields, such as providing a power source for small devices such as drone batteries. EXAMPLES

[0070] The present invention will be described in more detail below with reference to examples. However, it should be clearly understood that the present invention is not limited to the following examples and that various embodiments are possible.

[0071] Example 1. Enzyme production In this example, NAD(P)-dependent flavin reductase, a component of a biofuel cell, was produced by genetic engineering.

[0072] In this example, four types of NAD(P)-dependent flavin reductases shown in Table 1 were produced.

[0073] [Table 1]

[0074] Nucleic acid molecules encoding each NAD(P)-dependent flavin reductase (FRD-1 to 4) were prepared by artificial synthesis (Genscript), and each nucleic acid molecule was inserted into the pET-22b(+) (Novagen) vector via the Nde I-Xho I site so that a His-tag was added to the C-terminus of the enzyme. The E. coli BL21(DE3) strain was used as the expression host and transformed with the above vector. The transformed BL21(DE3) strain was cultured with shaking in LB medium. The cells were cultured at 37°C until OD600nm reached 0.4 to 0.6, and then 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, followed by culture at 28°C for 16 hours. After the culture was completed, the cells were lysed using BugBuster (Millipore: 71456-3), and the target enzyme was purified from the soluble fraction to obtain the enzyme sample. The enzyme was purified using TALON (TaKaRa: Z5501N) resin, and the resulting enzyme sample was used in the following examples.

[0075] Example 2. Electrode performance evaluation In the following examples, the electron transfer function to an electrode substrate by the oxidation-reduction reaction system of NAD(P)-dependent flavin reductase and flavin was evaluated. In these examples, a method for evaluating the performance of an electrode will be described.

[0076] (Preparation of reaction solution) The reaction solution was prepared by adding NADH (Roche: 10107735001) and FAD (Tokyo Chemical Industry: F0014), FMN (Fujifilm Wako Pure Chemical Industries: 065-00171), or riboflavin (Tokyo Chemical Industry: R0020) as flavin to the pH buffer solution, and each NAD(P)-dependent flavin reductase prepared in the above examples. The pH buffer solution used was a phosphate buffer solution (pH 7.0) with a final concentration of 0.5 M or a 2 M imidazole buffer solution (pH 7.0).

[0077] (Measurement of electrode performance) The electrode performance was measured by cyclic voltammetry (CV) using an electrochemical analyzer (BAS600). The working electrode was a carbon cloth (Nippon Carbon: GF-20-S9) punched into a circle with a diameter of 7 mm, and a platinum wire (BAS: 002233) for current collection. The reference electrode was a silver-silver chloride electrode (BAS: 013393), and the counter electrode was a plastic tube covered with cellophane (Futamura Chemical: ordinary cellophane P-5, size 300, grade A) filled with the same buffer solution as the reaction solution, into which a platinum wire (BAS: 002222) was inserted. CV measurements under each condition were performed at room temperature in the range of -0.55 V to -0.25 V at a sweep rate of 0.05 V / s after dropping 40 μL of the reaction solution prepared above onto the carbon cloth.

[0078] Example 3. Evaluation of electron transfer function of NAD(P)-dependent flavin reductase In this example, based on the electrode performance evaluation method of Example 2, the electron transfer function to the electrode substrate by the oxidation-reduction reaction system of NAD(P)-dependent flavin reductase and flavin was evaluated.

[0079] 3-1. Composition of reaction solution: NADH, FAD, NAD(P)-dependent flavin reductase, phosphate buffer Phosphate buffer (pH 7.0) was used as the pH buffer, and electrode performance was evaluated by CV measurement in a reaction solution containing 0.1 M NADH, 0.5 mM FAD, and 5 mg / mL of each NAD(P)-dependent flavin reductase (FRD-1 to 4). In addition, a similar evaluation was performed with a reaction solution that did not contain NAD(P)-dependent flavin reductase. The results are shown in Figure 2. As a result, an oxidation current was confirmed when using a reaction solution containing any of the NAD(P)-dependent flavin reductases FRD-1 to 4, compared to when no NAD(P)-dependent flavin reductase was used. The oxidation current at a potential of -0.3 V was highest in the order of FRD-1>FRD-2>FRD-3>FRD-4.

[0080] 3-2. Composition of reaction solution: NADH, FAD, NAD(P)-dependent flavin reductase, imidazole buffer solution When imidazole buffer solution (pH 7.0) was used as the pH buffer solution, electrode performance evaluation was performed by CV measurement in the same manner as in 3-1 above. The results are shown in FIG. 3. As a result, as in the case of using phosphate buffer solution (pH 7.0), oxidation current was confirmed when using reaction solutions containing any of the NAD(P)-dependent flavin reductases FRD-1 to 4. The oxidation current at a potential of -0.3V was high in the order of FRD-3>FRD-1>FRD-4>FRD-2, which was different from the case of using phosphate buffer solution. In addition, the current value was generally lower than when phosphate buffer solution was used, and the current value was constant at a potential of -0.35V or higher.

[0081] 3-3. Composition of reaction solution: NADH, FAD, diaphorase, 0.5M phosphate buffer The diaphorase used in Patent Document 1, which is exemplified as a prior art document above, is an enzyme that is frequently used in the technical field for the oxidation of NAD(P)H. The electron transfer function to the electrode substrate when such diaphorase is used was also evaluated in the same manner. Here, two types of diaphorase (Toyobo: DAD-301 and DAD-311) were evaluated. In detail, except that diaphorase was added to a reaction solution using 0.5M phosphate buffer as a pH buffer instead of NAD(P)-dependent flavin reductase, the electrode performance was evaluated by CV measurement in the same manner as in the above examples. The results are shown in FIG. 4. As a result, unlike the NAD(P)-dependent flavin reductase used in 3-1 and 3-2, no clear oxidation current was observed. From this, it is considered that diaphorase does not have the function of reducing flavin, and it can be determined that the oxidation current observed in 3-1 and 3-2 above is due to NAD(P)-dependent flavin reductase.

[0082] 3-4. Composition of reaction solution: NADH, FAD, FMN or riboflavin, NAD(P)-dependent flavin reductase, 0.5M imidazole In addition to FAD, when FMN and riboflavin were used as flavins, electrode performance evaluation was performed by CV measurement in the same manner as in the above examples. FMN was measured at a final concentration of 0.5 mM like FAD, but riboflavin had low solubility and could not be prepared at the same concentration, so it was measured at a final concentration of 0.1 mM. The results are shown in Figure 5. As a result, FMN showed an oxidation current of the same magnitude as FAD, and riboflavin also showed a clear oxidation current, but the current value was small. Therefore, although any flavin can be used as an electron transfer substance for NAD(P)-dependent flavin reductase, it is considered preferable to use FAD or FMN from the viewpoint of its electron transfer efficiency. In particular, when a relatively high current value is required, such as in a biofuel cell, it is considered preferable to use FAD or FMN.

[0083] Example 4. Evaluation of concentration dependence of flavin and NAD(P)-dependent flavin reductase In this example, based on the electrode performance evaluation method of Example 2, the electron transfer function to the electrode substrate by the redox reaction system of NAD(P)-dependent flavin reductase and flavin was evaluated from the viewpoint of the concentration dependence of flavin and NAD(P)-dependent flavin reductase.

[0084] 4-1. Flavin concentration dependence The electrode performance evaluation in the reaction solution with varying FAD concentrations was carried out by CV measurement in the same manner as in the above examples. The reaction solution was prepared using an imidazole buffer (pH 7.0) as the pH buffer, containing 0.2 M NADH, 0.5, 1, 5 or 10 mM FAD, and 10 mg / mL NAD(P)-dependent flavin reductase (FRD-3). The results are shown in Figure 6. As a result, the oxidation current also increased with increasing FAD concentration, and the oxidation current at a potential of -0.3 V at an FAD concentration of 10 mM was 80 mA / cm. 2 A high current value exceeding 10 ...

[0085] 4-2. Concentration dependence of NAD(P)-dependent flavin reductase The electrode performance was evaluated by CV measurement in the same manner as in the above examples, using reaction solutions with different concentrations of NAD(P)-dependent flavin reductase. The reaction solutions were prepared using imidazole buffer (pH 7.0) as the pH buffer, containing 0.2 M NADH, 10 mM FAD, and 10, 20, or 30 mg / mL NAD(P)-dependent flavin reductase (FRD-3). The results are shown in Figure 7. As a result, at all FRD-3 concentrations, the oxidation current increased with increasing concentration, and at an FRD-3 concentration of 30 mg / mL, the oxidation current at a potential of -0.3 V was 150 mA / cm. 2 Thus, a very high current value was obtained.

[0086] Example 5. Evaluation of electrode performance using glucose as an electron donor In this example, based on the electrode performance evaluation method of Example 2, glucose was used as the electron donor instead of NAD(P)H, and the electron transfer function to the electrode substrate by the redox reaction system of NAD(P)-dependent flavin reductase and flavin was evaluated.

[0087] (electrode configuration) Usually, in biofuel cells, the electron donor is not NAD(P)H but the fuel for the biofuel cell. Therefore, we evaluated the electron transfer function to the electrode substrate by the redox reaction system of NAD(P)-dependent flavin reductase and flavin in an electrode using glucose as the electron donor and NAD(P)-dependent glucose dehydrogenase as the fuel oxidase that extracts electrons from glucose. The electrode configuration is as shown in Figure 1, where glucose corresponds to the fuel and NAD(P)-dependent glucose dehydrogenase corresponds to the NAD(P)-dependent fuel oxidase.

[0088] (Preparation of reaction solution) The reaction solution was prepared by adding 0.6 M glucose, 20 mg / mL glucose dehydrogenase (Amano Enzyme: GLUCDH "Amano" NA), 25 mM NAD (Roche: 10127965001), 10 mM FAD, and 30 mg / mL NAD(P)-dependent flavin reductase (FRD-3) prepared in Example 1 to the pH buffer. The pH buffer used was an imidazole buffer (pH 7.0) with a final concentration of 2 M.

[0089] (Measurement of electrode performance) The electrode performance was measured by CV measurement in the same manner as in the above example. The results are shown in Figure 8. As a result, a higher oxidation current was obtained than when NADH was used as the electron donor. The oxidation current at a potential of -0.3 V was 200 mA / cm. 2 As described above, a very high current density was achieved. Since it is possible to generate a high current at such a low potential, the biofuel cell according to one embodiment of the present invention, which uses an NAD(P)-dependent flavin reductase and a specific type and concentration range of flavin for electron transfer on the anode side electrode substrate, can be constructed as a high-output cell. [Industrial Applicability]

[0090] The present invention relates to a biofuel cell using an enzyme electrode, which can achieve efficient and stable electron transfer between the enzyme and the electrode substrate, and can provide a high-output biofuel cell. The present invention can be used in all fields where biofuel cells are required, for example, in various industrial fields such as electronics, medicine, food, and the environment. In particular, the present invention can be applied to power sources for small electronic devices such as portable devices such as drones and desktop calculators, and implantable devices such as cardiac pacemakers.

Claims

1. A biofuel cell comprising an enzyme electrode as an anode, The enzyme electrode is An electrode substrate; Nicotinamide adenine dinucleotide-dependent fuel oxidase or nicotinamide adenine dinucleotide phosphate-dependent fuel oxidase as an electrode catalyst for oxidizing a fuel; a nicotinamide adenine dinucleotide-dependent flavin reductase or a nicotinamide adenine dinucleotide phosphate-dependent flavin reductase that reduces flavin in the presence of reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate produced as a result of the oxidation of a fuel by the fuel oxidizing enzyme; and a flavin in a concentration range of 0.5 mM to 100 mM; In this biofuel cell, the flavin is one or more selected from flavin adenine dinucleotide and derivatives thereof, and flavin mononucleotide and derivatives thereof.

2. The biofuel cell of claim 1 , wherein the flavin is flavin adenine dinucleotide or flavin mononucleotide.

3. The biofuel cell of claim 1 or 2, wherein the flavin is in a concentration range of 5 mM to 50 mM.

4. The biofuel cell according to any one of claims 1 to 3, wherein the nicotinamide adenine dinucleotide-dependent fuel oxidase or the nicotinamide adenine dinucleotide phosphate-dependent fuel oxidase is glucose dehydrogenase.

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