Working electrode for enzyme reaction for generating malic acid

Abstract

To provide a working electrode useful as a working electrode to be used for an enzyme reaction for generating malic acid.SOLUTION: A working electrode to be used for an enzyme reaction for generating malic acid from oxaloacetic acid is disclosed. The working electrode includes nano-carbon and malate dehydrogenase, and a cofactor of malate dehydrogenase is present while being attached or in proximity to the working electrode.SELECTED DRAWING: None

Description

[Technical field]

[0001] A technique is disclosed relating to a working electrode for an enzyme reaction that produces malic acid. [Background technology]

[0002] Bicarbonate ions are a significant indicator of electrolyte dispersion and anion deficiency, and are useful in the medical diagnosis of acid-base imbalance in the respiratory and metabolic systems. Toyobo Co., Ltd. has released a bicarbonate ion measurement kit called "Diacolor CO2." The product's package insert (Non-Patent Document 1) describes the measurement principle as follows: bicarbonate ions in a sample react with phosphoenolpyruvate carboxylase and Mg 2+ It is described that in the presence of , it reacts with phosphoenolpyruvic acid to produce oxaloacetic acid and inorganic phosphorus, and the oxaloacetic acid is reduced to malic acid by malate dehydrogenase, and at the same time, acetylnicotinamide adenine dinucleotide (reduced form) is oxidized. Here, the decrease in absorbance of acetylnicotinamide adenine dinucleotide (reduced form) is proportional to the bicarbonate ion concentration in the sample, so the bicarbonate ion concentration in the sample can be determined by measuring the change in absorbance. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Package insert for "Diacolor CO2" manufactured by Toyobo Co., Ltd. Summary of the Invention [Problem to be solved by the invention]

[0004] One object is to provide a working electrode that is useful as a working electrode for use in an enzymatic reaction that produces malic acid. [Means for solving the problem]

[0005] As a result of intensive research aimed at solving such problems, it was found that the enzyme reaction of producing malic acid from oxaloacetate can be promoted by using a working electrode containing nanocarbon and malate dehydrogenase, and having a cofactor of malate dehydrogenase attached or present in the vicinity thereof. It was also found that the enzyme reaction of producing malic acid from bicarbonate ions can be promoted by using a working electrode containing nanocarbon and malate dehydrogenase, and having a cofactor of malate dehydrogenase, phosphoenolpyruvate or a salt thereof, and phosphoenolpyruvate carboxylase attached or present in the vicinity thereof. Based on such findings, further research was conducted, and the present invention, as exemplified below, was completed.

[0006] [Section 1] A working electrode for use in an enzyme reaction for producing malic acid from oxaloacetic acid, Contains nanocarbon and malate dehydrogenase, A working electrode to which a cofactor for malate dehydrogenase is attached or in close proximity. [Section 2] A working electrode for use in an enzyme reaction for producing malic acid from bicarbonate ions, Contains nanocarbon and malate dehydrogenase, A working electrode having attached thereto or in the vicinity thereof a cofactor for malate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate and / or a salt thereof. [Section 3] Item 3. The working electrode according to item 1 or 2, wherein a mediator is attached to or present in the vicinity of the working electrode. [Section 4] Item 4. The working electrode according to Item 3, wherein the mediator is a phenazinium salt. [Section 5] Item 5. The working electrode according to any one of Items 1 to 4, wherein the nanocarbon is a carbon nanotube. [Section 6] 6. The working electrode according to any one of items 1 to 5, wherein a compound having an aromatic ring skeleton is attached to or present in the vicinity of the working electrode. [Section 7] Compounds having an aromatic ring skeleton include thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, toluene, 5-hydroxyindole, aniline, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol, and 2-tert-butyl-5-methylphenol. Item 7. The working electrode according to item 6, wherein the active material is at least one selected from the group consisting of t-butyl-4-ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, 2-isopropyl-5-methylanisole, o-cresol, m-cresol, and p-cresol. [Section 8] 8. The working electrode according to any one of items 1 to 7, wherein the cofactor of malate dehydrogenase is reduced nicotinamide adenine dinucleotide, reduced 3-acetylpyridine adenine dinucleotide, or reduced nicotinamide adenine dinucleotide phosphate. [Section 9] Item 9. The working electrode according to any one of Items 2 to 8, wherein the phosphoenolpyruvate carboxylase is derived from an acetic acid bacterium. [Section 10] Item 10. The working electrode according to Item 9, wherein the phosphoenolpyruvate carboxylase derived from an acetic acid bacterium is at least one selected from the group consisting of phosphoenolpyruvate carboxylase derived from Acetobacter pasteurianus, phosphoenolpyruvate carboxylase derived from Acetobacter aceti, phosphoenolpyruvate carboxylase derived from Acetobacter zylinum, phosphoenolpyruvate carboxylase derived from Acetobacter hansenii, and phosphoenolpyruvate carboxylase derived from Gluconobacter oxydans. [Section 11] Item 11. A bicarbonate ion measurement sensor comprising the working electrode and a counter electrode according to any one of Items 2 to 10. [Section 12] Item 12. The bicarbonate ion measuring sensor according to item 11, further comprising a reference electrode. [Section 13] 11. A biofuel cell comprising the working electrode according to any one of items 1 to 10 as a cathode and a counter electrode as an anode. Effect of the Invention

[0007] A working electrode is provided that is useful as a working electrode for use in an enzymatic reaction that produces malic acid. [Brief description of the drawings]

[0008] [Figure 1] 1 shows cyclic voltammograms measured by immobilizing single-walled carbon nanotubes and malate dehydrogenase on a working electrode. [Diagram 2] The cyclic voltammogram measured by fixing 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) to the working electrode of FIG. 1 is shown. [Diagram 3] The cyclic voltammogram measured by fixing 1-methoxy-5-ethylphenazinium ethyl sulfate (1-methoxy PES) to the working electrode of FIG. 1 is shown. [Figure 4]1 shows cyclic voltammograms measured in an enzyme reaction using phosphoenolpyruvate carboxylase and malate dehydrogenase with single-walled carbon nanotubes and malate dehydrogenase immobilized on a working electrode. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In one embodiment, the working electrode for use in the enzymatic reaction of producing malic acid from oxaloacetate contains nanocarbon and malate dehydrogenase, and preferably has a cofactor of malate dehydrogenase attached thereto or present in the vicinity thereof. This embodiment includes the case where the working electrode contains a cofactor of malate dehydrogenase in addition to nanocarbon and malate dehydrogenase, and the case where the working electrode part containing nanocarbon and malate dehydrogenase is immersed in a liquid medium containing a cofactor of malate dehydrogenase. Oxaloacetate may be a reaction product obtained by reacting phosphoenolpyruvic acid and / or a salt thereof with bicarbonate ions in the presence of phosphoenolpyruvate carboxylase. Thus, in another embodiment, the working electrode for use in the enzymatic reaction of producing malic acid from bicarbonate ions contains nanocarbon and malate dehydrogenase, and preferably has a cofactor of malate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvic acid and / or a salt thereof attached thereto or present in the vicinity thereof. This embodiment includes the case where the working electrode contains, in addition to nanocarbon and malate dehydrogenase, a cofactor of malate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvic acid and / or a salt thereof, and the case where the working electrode portion containing nanocarbon, malate dehydrogenase, and a cofactor of malate dehydrogenase is immersed in a liquid medium containing phosphoenolpyruvate carboxylase, and phosphoenolpyruvic acid and / or a salt thereof. The bicarbonate ion may be generated by dissolving a precursor such as sodium carbonate or carbon dioxide in water.

[0010] The nanocarbon is not particularly limited as long as it is a substance that has an electron transfer function and is recognized as a nanocarbon. Such substances refer to carbon materials that are mainly composed of carbon, including, for example, carbon nanotubes, carbon nanohorns, carbon nanotwists, cocoons, carbon nanocoils, graphene, and fullerenes. The carbon nanotubes may be single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes. In one embodiment, the nanocarbon is preferably a carbon nanotube, and more preferably a single-walled carbon nanotube. The nanocarbon may be used alone or in combination of two or more kinds.

[0011] Malate dehydrogenase is not particularly limited as long as it has the activity of catalyzing the conversion of oxaloacetate to malic acid. Examples of malate dehydrogenase include EC 1.1.1.37, EC 1.1.1.38, EC 1.1.1.39, and EC 1.1.1.40. In one embodiment, the malate dehydrogenase is preferably EC 1.1.1.37. One type of malate dehydrogenase can be used alone or two or more types can be used in combination. Malate dehydrogenase usually requires a cofactor to express its activity.

[0012] The cofactor (sometimes called coenzyme, etc.) of malate dehydrogenase is not particularly limited as long as it has electron donating properties. Examples of the cofactor include reduced nicotinamide adenine dinucleotide (NADH), reduced 3-acetylpyridine adenine dinucleotide (acetyl NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and analogs thereof (e.g., nicotinamide guanine dinucleotide, nicotinamide hypoxanthine dinucleotide). Among these, NADH, acetyl NADH, or NADPH is preferred. The cofactor can be used alone or in combination of two or more.

[0013] The amount of the cofactor attached to or brought close to the working electrode is not particularly limited. The amount of the cofactor is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, even more preferably 1 part by mass or more, and even more preferably 10 parts by mass or more, relative to 100 parts by mass of malate dehydrogenase. The amount of the cofactor is, for example, 1,000,000 parts by mass or less, preferably 100,000 parts by mass or less, more preferably 10,000 parts by mass or less, even more preferably 1000 parts by mass or less, and even more preferably 100 parts by mass or less, relative to 100 parts by mass of malate dehydrogenase. The lower limit and upper limit can be combined arbitrarily.

[0014] Phosphoenolpyruvate carboxylase (PEPC) is not particularly limited as long as it has the activity of catalyzing the conversion of bicarbonate ions to oxaloacetate. In one embodiment, PEPC is preferably PEPC derived from acetic acid bacteria. Examples of PEPC derived from acetic acid bacteria include PEPC derived from the genus Acetobacter, Gluconobacter, Fraturia, or Acidomonas. Suitable examples include PEPC derived from Acetobacter pasteurianus, PEPC derived from Acetobacter acetii, PEPC derived from Acetobacter zylinum, PEPC derived from Acetobacter hansenii, and PEPC derived from Gluconobacter oxydans. Phosphoenolpyruvate carboxylase can be used alone or in combination of two or more.

[0015] Examples of phosphoenolpyruvic acid and / or a salt thereof include phosphoenolpyruvic acid (free form); alkali metal salts of phosphoenolpyruvic acid (including hydrates) such as potassium phosphoenolpyruvate and sodium phosphoenolpyruvate; and combinations thereof.

[0016] The amount of phosphoenolpyruvic acid and / or its salt attached to or adjacent to the working electrode is not particularly limited. The amount of phosphoenolpyruvic acid and / or its salt is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, and even more preferably 1 part by mass or more, relative to 100 parts by mass of PEPC. The amount of phosphoenolpyruvic acid and / or its salt is, for example, 1,000,000 parts by mass or less, preferably 100,000 parts by mass or less, more preferably 10,000 parts by mass or less, and even more preferably 1,000 parts by mass or less, relative to 100 parts by mass of PEPC. The lower limit and upper limit can be combined arbitrarily.

[0017] In one embodiment, a mediator is preferably attached to or present in proximity to the working electrode, including cases where the working electrode includes a mediator in addition to the nanocarbon and malate dehydrogenase, and cases where the portion of the working electrode including the nanocarbon and malate dehydrogenase is immersed in a liquid medium including the mediator.

[0018] The mediator is not particularly limited as long as it has electron transfer ability. Examples of the mediator include phenazinium salts such as phenazine methosulfate, phenazine ethosulfate, 1-methoxy-5-methylphenazinium methyl sulfate (1-methoxy PMS), and 1-methoxy-5-ethylphenazinium ethyl sulfate (1-methoxy PES); ferrocyanides such as potassium ferrocyanide; ferrocene, dimethylferrocene, and other ferrocenes; thionine; viologens such as benzyl viologen and methyl viologen; dyes such as Meldola Blue, methylene blue, and neutral red; phenols such as catecholamine, p-aminophenol, 2,6-dichloroindophenol, and hydroquinone; benzoquinones such as 1,4-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone; osmium complexes; and diaphorase. Among these, from the viewpoint of stability, phenazinium salts are preferred, and 1-methoxy PMS or 1-methoxy PES is particularly preferred. The mediators can be used alone or in combination of two or more.

[0019] The amount of the mediator attached to or placed close to the working electrode is not particularly limited. The amount of the mediator is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, and even more preferably 1 part by mass or more, relative to 100 parts by mass of the nanocarbon. The amount of the mediator is, for example, 1,000,000 parts by mass or less, preferably 100,000 parts by mass or less, more preferably 10,000 parts by mass or less, and even more preferably 1,000 parts by mass or less, relative to 100 parts by mass of the nanocarbon. The lower limit and the upper limit can be arbitrarily combined. The amount of the mediator is, for example, 0.00001 parts by mass or more, preferably 0.0001 parts by mass or more, and even more preferably 0.001 parts by mass or more, relative to 100 parts by mass of the malate dehydrogenase. The amount of the mediator is, for example, 10,000 parts by mass or less, preferably 1,000 parts by mass or less, and more preferably 100 parts by mass or less, relative to 100 parts by mass of malate dehydrogenase. The above lower limit and upper limit can be combined in any manner.

[0020] In one embodiment, a compound having an aromatic ring skeleton is preferably attached to or present in the vicinity of the working electrode. This embodiment includes a case where the working electrode contains a compound having an aromatic ring skeleton in addition to the nanocarbon and malate dehydrogenase, and a case where the working electrode portion containing the nanocarbon and malate dehydrogenase is immersed in a liquid medium containing a compound having an aromatic ring skeleton.

[0021] The compound having an aromatic ring skeleton is not particularly limited as long as it is an electron transfer promoter that promotes electron transfer of nanocarbon. The compound having an aromatic ring skeleton may be a low molecular weight compound having a molecular weight of 1000 or less, or a high molecular weight compound having a molecular weight of more than 1000. The number of ring-constituting atoms of the aromatic ring skeleton is, for example, 5 to 18, preferably 5 to 16, and more preferably 5 to 14. The aromatic ring skeleton includes a skeleton consisting of one benzene ring, a skeleton consisting of two or more (for example, 2 to 4) benzene rings (naphthalene skeleton, anthracene skeleton, etc.), a skeleton consisting of a condensed ring of a benzene ring and another aromatic ring (nitrogen-containing aromatic ring, oxygen-containing aromatic ring, sulfur-containing aromatic ring, etc.) (phenanthroline skeleton, benzofuran skeleton, benzimidazole skeleton, carbazole skeleton, etc.), and a skeleton consisting of an aromatic ring consisting of carbon and another element (nitrogen, oxygen, sulfur, etc.) (triazine skeleton, triazole skeleton, pyridine skeleton, etc.). The compound having an aromatic ring skeleton is preferably a compound that does not function as a mediator by itself.

[0022] In one embodiment, the compound having an aromatic ring skeleton preferably has an electron-donating substituent. The electron-donating substituent includes a hydroxy group, an amino group, and a methyl group. A preferred electron-donating substituent is a hydroxy group. Examples of the compound having an electron-donating substituent and an aromatic ring skeleton include a compound having a benzene ring substituted with a hydroxy group (e.g., thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, 5-hydroxyindole, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol, 2-tert-butyl-4 -ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, and 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, o-cresol, m-cresol, p-cresol), a compound having a benzene ring substituted with an amino group (e.g., aniline), and a compound having a benzene ring substituted with a methyl group (e.g., toluene, 2-isopropyl-5-methylanisole). Among these compounds, thymol, phenol, or carvacrol are preferred. In one embodiment, the compound having an aromatic ring skeleton is preferably not a mediator. The compound having an aromatic ring skeleton can be used alone or in combination of two or more.

[0023] The amount of the compound having an aromatic ring skeleton attached to or placed close to the working electrode is not particularly limited. The amount of the compound having an aromatic ring skeleton is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, even more preferably 1 part by mass or more, and even more preferably 10 parts by mass or more, relative to 100 parts by mass of the nanocarbon. The amount of the compound having an aromatic ring skeleton is, for example, 1,000,000 parts by mass or less, preferably 100,000 parts by mass or less, and even more preferably 10,000 parts by mass or less, relative to 100 parts by mass of the nanocarbon. The lower limit and upper limit can be combined arbitrarily.

[0024] In one embodiment, the working electrode may further contain a dispersant. The dispersant is not particularly limited as long as it is a substance capable of suppressing aggregation of nanocarbon and dispersing it. Examples of the dispersant include sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide, and octylphenol ethoxylate. Among these, sodium cholate or sodium deoxycholate is preferred. The dispersant may be used alone or in combination of two or more.

[0025] The shape of the working electrode is not particularly limited as long as it can be used as an electrode, and may be, for example, a wire, coil, net, rod, film, or plate. In one embodiment, the working electrode preferably has a substrate suitable for an electrode on which an enzyme used in a biosensor is immobilized, and the substrate is preferably an insulating substrate on which a metal film (e.g., a metal thin film) is formed. For example, a glass substrate or a plastic substrate (e.g., a PET substrate) can be used as the insulating substrate. The type of metal that forms the metal film is not particularly limited as long as it is used for an electrode, and examples of the metal that can be used include gold, platinum, and titanium. In addition, the substrate may be an insulating substrate on which a carbon film (e.g., a carbon thin film made of carbon paste) is formed instead of a metal film.

[0026] In one embodiment, the working electrode is preferably a substrate on which nanocarbon and malate dehydrogenase (and optionally at least one selected from a cofactor of malate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate and / or a salt thereof) are loaded. The method of loading these substances is not particularly limited. For example, a dispersion liquid in which each of these substances is dispersed or a solution in which each of these substances is dissolved is prepared, and the solution is dropped in sequence on a predetermined portion of the substrate (in the case where the substrate is an insulating substrate on which a metal thin film is formed, the portion on which the metal thin film is formed), and then dried. This operation is repeated to load the substances. Alternatively, after preparing a dispersion liquid in which each of these substances is dispersed or a solution in which each of these substances is dissolved, these are mixed together, dropped on a predetermined portion of the substrate, and then dried, so that the substances can be loaded. The dispersion medium or solvent is not particularly limited, and examples thereof include water, alcohol-based solvents (e.g., ethanol), ketone-based solvents (e.g., acetone), and combinations thereof.

[0027] In one embodiment, it is preferable to mix a dispersant in the dispersion liquid containing nanocarbon. The mixing ratio of the dispersant is arbitrary, but is preferably 0.2 to 2.5% (w / v), for example. The mixing ratio of the nanocarbon is also arbitrary, but is preferably 0.05 to 0.5% (w / v), for example.

[0028] In one embodiment, each substance contained in the working electrode may be immobilized on a substrate. The immobilization can be performed by appropriately selecting a known method. For example, a liquid in which a substance suitable for immobilization, such as tetrafluoroethylene / perfluoro[2-(fluorosulfonylethoxy)polyvinyl ether] copolymer (e.g., Nafion (trademark)) or carboxyl methyl cellulose, is dissolved is dropped onto the portion of the substrate on which each of the substances is loaded, and then dried to immobilize the substances.

[0029] In one embodiment, it is preferable to immerse the working electrode part containing the nanocarbon and malate dehydrogenase in a liquid medium. The liquid medium is typically a buffer solution, examples of which include acetate buffer solution, citrate buffer solution, phosphate buffer solution, borate buffer solution, and Tris-HCl buffer solution. In this embodiment, the working electrode part is preferably a part where the nanocarbon and malate dehydrogenase are loaded on a substrate, and oxaloacetic acid and / or bicarbonate ion or its precursor (sodium carbonate, carbon dioxide, etc.) may be added to the liquid medium in which the working electrode part is immersed. In addition, in this embodiment, it is preferable that the working electrode part and / or the liquid medium further contain at least one selected from a cofactor of malate dehydrogenase, a mediator, and a compound having an aromatic ring skeleton. Furthermore, in this embodiment, it is preferable that the liquid medium to which the bicarbonate ion or its precursor can be added contains phosphoenolpyruvate carboxylase, and phosphoenolpyruvic acid and / or a salt thereof.

[0030] In one embodiment, the bicarbonate ion measuring sensor preferably includes the above-mentioned working electrode and counter electrode. The shape of the counter electrode is not particularly limited as long as it can be used as an electrode, and may be, for example, a wire, a coil, a mesh, a rod, a film, or a plate. In one embodiment, the counter electrode may be a metal wire such as a platinum wire. In another embodiment, the counter electrode may have a substrate suitable for an electrode, and the substrate may be an insulating substrate on which a metal film (e.g., a metal thin film) is formed. The insulating substrate may be, for example, a glass substrate or a plastic substrate (e.g., a PET substrate). The type of metal that forms the metal film is not particularly limited as long as it is used for an electrode, and examples of the metal that can be used include gold, platinum, and titanium.

[0031] The bicarbonate ion measuring sensor may or may not include a reference electrode. The bicarbonate ion measuring sensor may further include components that are usually included in biosensors, such as a potentiostat and a current detection circuit. The specific components of the counter electrode, potentiostat, and current detection circuit are arbitrary as long as the sensor can perform the intended measurement, and can be designed by appropriately selecting from means known in the art.

[0032] The present invention includes a biofuel cell comprising the above-mentioned working electrode as a cathode and a counter electrode as an anode. EXAMPLES

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] [Example 1] An electrode with gold printed on the working electrode (round gold electrode DEP-ER-P, Bio Device Technology, Inc., working electrode area: 3.67 mm 2 ) was dropped 2μL of an aqueous dispersion containing 2% (w / v) sodium cholate and 0.15% (w / v) single-walled carbon nanotubes (SuperPureTubes, NanoIntegris, outer diameter 1.1-1.7 nm) onto the working electrode of the electrode, and then dried. After the carbon nanotube dispersion was dried, 2μL of 1% (w / v) thymol dissolved in 50% (v / v) ethanol was dropped onto the working electrode, and then dried. After the thymol solution was dried, 2μL of malate dehydrogenase (MAD-211, Toyobo Co., Ltd.; 20U / μL) dissolved in 100mM potassium phosphate buffer (pH 7.5) was dropped onto the working electrode, and then dried. After the malate dehydrogenase solution was dried, 2μL of 600mM β-NADH (β-nicotinamide adenine dinucleotide (reduced form)) dissolved in ultrapure water was dropped onto the working electrode, and then dried. After the β-NADH solution was dried, any one of the following (1) to (3) was performed on the working electrode site. (1) No operation (no electronic mediator added) (2) 2 μL of 1-methoxy PMS (Dojindo Laboratories; 6 mM) was added dropwise and dried. (3) 2 μL of 1-methoxy PES (Dojindo Laboratories; 6 mM) was dropped and dried.

[0035] After drying, 2 μL of 2% (w / v) Nafion solution was dropped onto the working electrode and dried to immobilize the carbon nanotubes and malate dehydrogenase on the working electrode.

[0036] The working electrode of the electrode chip prepared above was set on the working electrode of an electrochemical analyzer (ALS 760E, manufactured by BAS Co., Ltd.), a silver / silver chloride electrode was set on the reference electrode, and a platinum wire was set on the counter electrode. The working electrode and reference electrode were immersed in 60 mM Tris-HCl buffer (pH 8.0), and the counter electrode was immersed in hydrogen peroxide solution (16 mM hydrogen peroxide / 60 mM Tris-HCl buffer (pH 8.0)) in a sample holder (BAS Co., Ltd.). The sample holder with the counter electrode immersed in it was immersed in 60 mM Tris-HCl buffer (pH 8.0) together with the working electrode and reference electrode. Ion-permeable glass was attached to the tip of the sample holder, allowing ions to pass freely.

[0037] Oxaloacetic acid was added to the solution in which the working electrode was immersed, and measurements were performed by cyclic voltammetry. Cyclic voltammograms measured at oxaloacetic acid concentrations of 0 mM, 0.001 mM, and 0.1 mM are shown in Figures 1 to 3. Figure 1 shows the case where no electron mediator was added, Figure 2 shows the case where 1-methoxy PMS was dripped onto the working electrode and dried, and Figure 3 shows the case where 1-methoxy PES was dripped onto the working electrode and dried.

[0038] In this cyclic voltammogram, the current value at -2.4 V when sweeping from 0.8 V to -0.8 V was as shown in Table 1 below.

[0039] [Table 1]

[0040] From the results of Example 1, it was found that the current value of the enzyme electrode changes depending on the concentration of oxaloacetic acid. This suggests that the enzyme reaction in which oxaloacetic acid is produced from carbon dioxide (which dissolves in water to produce bicarbonate ions) by phosphoenolpyruvate carboxylase and malic acid is produced from the oxaloacetic acid as a raw material can be applied to the cathode of an enzyme fuel cell. It was also found that the amount of change in the current value that changes depending on the concentration of oxaloacetic acid increases by adding an electron mediator.

[0041] [Example 2] An electrode with gold printed on the working electrode (round gold electrode DEP-ER-P, Bio Device Technology, Inc., working electrode area: 3.67 mm 2 ) was dropped 2μL of an aqueous dispersion containing 2% (w / v) sodium cholate and 0.15% (w / v) single-walled carbon nanotubes (SuperPureTubes, NanoIntegris, outer diameter 1.1-1.7 nm) onto the working electrode of the electrode, and then dried. After the carbon nanotube dispersion was dried, 2μL of 1% (w / v) thymol dissolved in 50% (v / v) ethanol was dropped onto the working electrode, and then dried. After the thymol solution was dried, 2μL of malate dehydrogenase (MAD-211, Toyobo Co., Ltd.; 20U / μL) dissolved in 100mM potassium phosphate buffer (pH 7.5) was dropped onto the working electrode, and then dried. After the malate dehydrogenase solution was dried, 2μL of 600mM β-NADH (β-nicotinamide adenine dinucleotide (reduced form)) dissolved in ultrapure water was dropped onto the working electrode, and then dried. After drying the β-NADH solution, 2 μL of 1-methoxy PMS (Dojindo Laboratories; 6 mM) was dropped onto the working electrode and dried. After drying, 2 μL of 2% (w / v) Nafion solution was dropped onto the working electrode and dried, and carbon nanotubes and malate dehydrogenase were immobilized on the working electrode.

[0042] The working electrode of the electrode chip prepared above was set on the working electrode of an electrochemical analyzer (ALS 760E, manufactured by BAS Co., Ltd.), a silver / silver chloride electrode was set on the reference electrode, and a platinum wire was set on the counter electrode. The working electrode and the reference electrode were immersed in a phosphoenolpyruvate carboxylase (PPC-301, manufactured by Toyobo Co., Ltd.; 10 U / mL) solution (60 mM Tris-HCl buffer (pH 8.0) / 3.2 mM potassium phosphoenolpyruvate / 10 mM magnesium sulfate), and the counter electrode was immersed in hydrogen peroxide (16 mM hydrogen peroxide / 60 mM Tris-HCl buffer (pH 8.0)) in a sample holder (manufactured by BAS Co., Ltd.). The sample holder with the counter electrode immersed in it was immersed in the phosphoenolpyruvate carboxylase solution together with the working electrode and the reference electrode. Ion-permeable glass was attached to the tip of the sample holder, allowing ions to pass freely.

[0043] Sodium carbonate was added to the solution in which the working electrode was immersed, and measurements were performed by cyclic voltammetry. Figure 4 shows the cyclic voltammograms measured at sodium carbonate concentrations of 0 mM, 0.01 mM, and 0.1 mM.

[0044] In this cyclic voltammogram, the current value at -2.4 V when sweeping from 0.8 V to -0.8 V was as shown in Table 2 below.

[0045] [Table 2]

[0046] From the results of Example 2, it was found that the current value of the enzyme electrode changes depending on the concentration of bicarbonate ions (which are generated when sodium carbonate dissolves in water). This suggests that the enzyme reaction that produces malic acid from carbon dioxide (which dissolves in water to produce bicarbonate ions) can be applied to the cathode of an enzyme fuel cell.

Claims

1. A working electrode for use in an enzyme reaction for producing malic acid from oxaloacetic acid, Contains nanocarbon and malate dehydrogenase, A working electrode to which a cofactor for malate dehydrogenase is attached or in close proximity.

2. A working electrode for use in an enzyme reaction for producing malic acid from bicarbonate ions, Contains nanocarbon and malate dehydrogenase, A working electrode having attached thereto or in the vicinity thereof a cofactor for malate dehydrogenase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate and / or a salt thereof.

3. The working electrode according to claim 1 or 2, wherein a mediator is attached to or present in close proximity to the working electrode.

4. 4. The working electrode according to claim 3, wherein the mediator is a phenazinium salt.

5. The working electrode according to claim 1 or 2, wherein the nanocarbon is a carbon nanotube.

6. The working electrode according to claim 1 or 2, wherein a compound having an aromatic ring skeleton is attached to or present in the vicinity of the working electrode.

7. The compound having an aromatic ring skeleton is thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, toluene, 5-hydroxyindole, aniline, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol, 2-tert butyl-4-ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, 2-isopropyl-5-methylanisole, o-cresol, m-cresol, and p-cresol. The working electrode according to claim 6, which is at least one selected from the group consisting of cresol.

8. 3. The working electrode according to claim 1, wherein the cofactor for malate dehydrogenase is reduced nicotinamide adenine dinucleotide, reduced 3-acetylpyridine adenine dinucleotide, or reduced nicotinamide adenine dinucleotide phosphate.

9. 3. The working electrode according to claim 2, wherein the phosphoenolpyruvate carboxylase is derived from an acetic acid bacterium.

10. The working electrode according to claim 9, wherein the phosphoenolpyruvate carboxylase derived from an acetic acid bacterium is at least one selected from the group consisting of phosphoenolpyruvate carboxylase derived from Acetobacter pasteurianus, phosphoenolpyruvate carboxylase derived from Acetobacter aceti, phosphoenolpyruvate carboxylase derived from Acetobacter zylinum, phosphoenolpyruvate carboxylase derived from Acetobacter hansenii, and phosphoenolpyruvate carboxylase derived from Gluconobacter oxydans.

11. A bicarbonate ion measuring sensor comprising the working electrode according to claim 2 and a counter electrode.

12. The bicarbonate ion measuring sensor according to claim 11 , further comprising a reference electrode.

13. A biofuel cell comprising the working electrode according to claim 1 or 2 as a cathode and a counter electrode as an anode.

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