JP2004071559A - Fuel cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve a large current density, while making use of biometabolism. <P>SOLUTION: A fuel cell achieves a large current density, while making use of biometabolism. While a fuel is decomposed stepwise by a plurality of enzymes, electrons generated with oxidation are transferred to an electrode. In the plurality of enzymes decomposing the fuel, the enzyme activity of decomposing enzymes in a former step is equal to or below that in a latter step. In the case of coenzyme being involved therein, the enzyme activity of oxidizing enzymes, which oxidize the coenzymes, is equal to or higher than the sum of the enzyme activities of an enzyme group taking part in yielding the reduced forms of the coenzymes among the plurality of enzymes stepwisely decomposing the fuel. <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to a fuel cell utilizing biological metabolism.

A fuel cell basically includes a fuel electrode, an oxidizer electrode (air electrode), and an electrolyte, and its operating principle is based on a reverse operation of electrolysis of water. That is, the fuel cell generates water (H ₂ O) and extracts electricity by feeding hydrogen and oxygen, that is, performs power generation. More specifically, the fuel (hydrogen) supplied to the fuel electrode is oxidized and separated into electrons and protons (H ⁺ ), and this H ⁺ moves to the air electrode via the electrolyte, and the H ⁺ moves to the air electrode. H ₂ O is produced by reacting with the supplied oxygen.

Fuel cells function as high-efficiency power generators that directly convert the energy of fuel into electric energy, and the energy of fossil energy, such as natural gas, oil, and coal, is high, regardless of where or when it is used. It can be extracted as electric energy at the conversion efficiency.

There has been active research and development of fuel cells for large-scale power generation applications.For example, a fuel cell is mounted on a space shuttle, which can supply crew water simultaneously with electric power, and a clean power generator. We have a proven track record.

In recent years, fuel cells exhibiting a relatively low operating temperature range from room temperature to about 90 ° C., such as polymer solid electrolyte fuel cells, have been developed and are receiving attention. For this reason, applications are being sought not only for large-scale power generation, but also for small systems such as power supplies for driving automobiles and portable power supplies such as personal computers and mobile devices.

However, although the polymer electrolyte fuel cell has the advantage of exhibiting a low operating temperature range as described above, there are still many problems to be solved. Specifically, catalyst poisoning by CO when methanol is used as the fuel and operated at around room temperature, the need for a catalyst using an expensive noble metal such as Pt, generation of energy loss due to crossover, This is because it is difficult to handle hydrogen using hydrogen.

Therefore, attention has been paid to the fact that biological metabolism performed in living organisms is a highly efficient energy conversion mechanism, and proposals have been made to apply this to a fuel cell. The biological metabolism here includes respiration, photosynthesis, and the like performed in microorganisms and cells. Biological metabolism has the characteristics that the power generation efficiency is extremely high and the reaction proceeds under mild conditions at about room temperature.

For example, respiration takes in nutrients such as sugars, fats and proteins into microorganisms or cells, and converts these chemical energies into glycolytic and tricarboxylic acid (hereinafter referred to as TCA) circuits having a number of enzymatic reaction steps. Reduces nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ) in the process of producing carbon dioxide (CO ₂ ) via redox, such as reduced nicotinamide adenine dinucleotide (NADH) It is a mechanism that converts water into energy, that is, electrical energy, and further converts the electrical energy of these NADHs directly into electrical energy of a proton gradient in an electron transfer system, reduces oxygen, and generates water. The electric energy obtained here generates ATP from ADP via the ATP synthase, and this ATP is used for reactions necessary for the growth of microorganisms and cells. Such energy conversion occurs in the cytosol and mitochondria.

In photosynthesis, reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP ⁺ ) is reduced by taking in light energy and reducing nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP ⁺ ) through an electron transfer system. It is a mechanism that oxidizes water to generate oxygen in the process of converting it to electrical energy such as NADPH). This electric energy captures CO ₂ and is used for a carbon fixation reaction, and is used for carbohydrate synthesis.

生成 The formation reaction of NADH, which occupies an important position in biological metabolism, is represented by the following formula (3).

Several hundred types of dehydrogenases (dehydrogenases) have been known so far, and play an important role in catalyzing the conversion of various substrates to products with high selectivity. Such reaction selectivity of the enzyme is due to the enzyme having a unique three-dimensional structure because it is a protein molecule. For this reason, in the living body, several tens of types of dehydrogenases and the like react in order with the taken-in fuel, and oxidation proceeds until CO ₂ is finally formed.

As a technique for utilizing the metabolism of a living body as described above in a fuel cell, a microbial cell in which electric energy generated in the microbe is extracted outside the microbe through an electron mediator and the electrons are passed to an electrode to obtain an electric current has been reported. (See, for example, Patent Document 1).

However, microorganisms and cells have many unnecessary functions other than the above-mentioned reaction such as conversion of chemical energy into electric energy, and thus the above-described method consumes enough electric energy for undesired reactions. Energy conversion efficiency is not exhibited.
JP-A-2000-133297

Therefore, there has been proposed a fuel cell configuration in which enzymes and electron mediators involved in the reaction are taken out of microorganisms and cells and an appropriate environment is reproduced to perform only a desired reaction.

However, the fuel cell having such a configuration has a problem that the reaction rate of the enzyme is slow, and the actually obtained current density is extremely small.

Therefore, the present invention has been proposed to solve such a conventional problem, and an object of the present invention is to provide a fuel cell capable of realizing a large current density while utilizing biological metabolism. I do.

In order to achieve the above object, a fuel cell according to the present invention is a fuel cell that decomposes fuel by a stepwise reaction by a plurality of enzymes and transfers electrons generated by an oxidation reaction to an electrode. When the enzyme activity of the enzyme 1 when the decomposition product 1 is generated by the decomposition reaction by the enzyme 1 is U (E1), and the sum of the enzyme activities of the enzyme group 2 that decomposes the decomposition product 1 is U (E2), U (E1) ≦ U (E2).

複合 In a complex enzyme reaction in which fuel is decomposed stepwise by a plurality of enzymes, intermediate products that impair enzyme activity must be decomposed promptly. In the present invention, the enzyme activity of each enzyme is set stepwise so that the enzyme activity of the enzyme group that performs the subsequent decomposition reaction is greater than the enzyme activity of the enzyme 1 that performs the previous decomposition reaction. , The fuel is quickly decomposed.

In the present invention, the enzyme 1 is an oxidase, the decomposition reaction by the enzyme 1 is an oxidation reaction, and the fuel cell transfers electrons to a coenzyme by the oxidation reaction. And a coenzyme oxidase that produces an oxidized form of the coenzyme. The enzyme activity of the coenzyme oxidase is defined as U (Co). Assuming that the sum of the enzyme activities of the involved enzyme groups is U (E), U (Co) ≧ U (E).

In a fuel cell in which electrons are transferred to a coenzyme by an oxidation reaction of the enzyme 1, if the coenzyme oxidase that oxidizes the coenzyme is insufficient for the production rate of the reduced form of the coenzyme, the enzyme reaction of the coenzyme oxidase is insufficient. Becomes rate-limiting. In the present invention, the enzyme activity U (Co) of the coenzyme oxidase that oxidizes the coenzyme is equal to or more than the sum of the enzyme activities U (E) of the enzymes involved in the oxidation of the fuel (that is, the production of the reduced form of the coenzyme). Therefore, the enzyme reaction of the coenzyme oxidase is not limited, and the reduced form of the coenzyme is quickly oxidized, and the electrons generated at this time are transferred to the electrode via the electron mediator.

More specifically, the case where methanol is used as a fuel will be described in more detail. For example, a fuel cell using methanol as a fuel includes a fuel electrode, an air electrode, and a proton interposed between the fuel electrode and the air electrode. It has a conductive membrane, an enzyme solution capable of transferring electrons to and from the fuel electrode, and the enzyme solution contains alcohol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, diaphorase, and an electron mediator. Here, the enzyme activities of the alcohol dehydrogenase, the formaldehyde dehydrogenase, the formate dehydrogenase, and the diaphorase are referred to as U (ADH), U (FalDH), U (FateDH), and U (DI), respectively.

First, assuming that alcohol dehydrogenase is enzyme 1, U (ADH) corresponding to the enzyme activity U (E1) of enzyme 1 is the sum of the enzyme activities of a group of enzymes (formaldehyde dehydrogenase and formate dehydrogenase) that decompose a degradation product (formaldehyde). For U (E2) = U (FalDH) + U (FateDH), it is necessary that U (E1) = U (ADH) ≦ U (E2) = U (FalDH) + U (FateDH). Next, assuming that formaldehyde dehydrogenase is enzyme 1, U (FalDH) corresponding to the enzyme activity U (E1) of enzyme 1 is the sum of the enzyme activities U (formate dehydrogenase) of the enzyme group (formate dehydrogenase) which decomposes the degradation product (formate). For E2) = U (FateDH), it is necessary that U (E1) = U (FalDH) ≦ U (E2) = U (FateDH). In consideration of these, it is preferable that the fuel cell using methanol as a fuel satisfies the following expression (1).
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) ... Equation (1)

Next, in the above fuel cell, the coenzyme oxidase is diaphorase, the enzyme activity U (DI) of which corresponds to U (Co), and alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase are all coenzyme enzymes. Participates in the formation of reductants. Therefore, it is necessary to satisfy the following expression (2).
U (ADH) + U (FalDH) + U (FateDH) ≦ U (DI) ... Equation (2)

In the fuel cell configured as described above, alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase generate a total of three molecules of NADH in the process of catalyzing the oxidation reaction from methanol as fuel to CO ₂ . The diaphorase transfers two electrons from the generated NADH to the fuel electrode via an electron mediator. H ⁺ generated in these processes reaches the air electrode via the enzyme solution and the proton conducting membrane. At the air electrode, water is generated from H ⁺ , oxygen (O ₂ ), and electrons from an external circuit.

Then, the enzyme solution is prepared so that the enzyme activity of each enzyme is increased according to the order of oxidizing methanol, that is, the order of alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. Generation speed becomes large.

Also, since the enzyme solution is prepared so that the enzyme activity of diaphorase is higher than the sum of the enzyme activities of alcohol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, the transfer rate of electrons from NADH to the fuel electrode can be reduced without saturating diaphorase. Can be improved.

燃料 The fuel cell according to the present invention further transfers electrons from a coenzyme to an electron mediator, wherein the electron mediator is vitamin K3.

For example, in the case of a fuel cell using methanol as a fuel, a fuel electrode, an air electrode, a proton conductive membrane interposed between the fuel electrode and the air electrode, and an enzyme solution capable of transferring electrons to and from the fuel electrode A fuel cell using methanol as a fuel, wherein the enzyme solution contains alcohol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, diaphorase, and an electron mediator, and the electron mediator is vitamin K3. There is a feature.

Since vitamin K3 has an equilibrium redox potential relatively close to a coenzyme oxidase (eg, diaphorase) that oxidizes a coenzyme, it has good compatibility with diaphorase and electron transfer, and improves electron transfer speed when used as an electron mediator. .

As is clear from the above description, the fuel cell according to the present invention uses the enzyme of each enzyme so that the reaction of decomposing the fuel and the reaction of the coenzyme (for example, NADH) transferring electrons to the fuel electrode do not stop. The level of activity is regulated. Alternatively, in the fuel cell according to the present invention, vitamin K3 that is compatible with dehydrogenase (for example, DI) is selected as an electron mediator in order to increase the electron transfer speed. Therefore, according to the present invention, it is possible to provide a fuel cell in which the reaction speed is improved and a higher current density is realized.

Hereinafter, a fuel cell to which the present invention is applied will be described in detail with reference to the drawings.
The fuel cell of the present invention utilizes biological metabolism, and as shown in FIG. 1, a fuel electrode, an air electrode, a proton conductive membrane separating the fuel electrode and the air electrode, an enzyme described later, An enzyme solution in which an enzyme, an electron mediator or the like is dissolved is a basic component. The enzyme solution is held in the fuel electrode chamber so as to be in contact with the fuel electrode. Then, the fuel is continuously supplied to the enzyme solution in the fuel electrode chamber.

In this fuel cell, when one or more NAD ⁺ -dependent dehydrogenases described below as complex dehydrogenase oxidize a fuel (eg, methanol) to CO ₂ through a plurality of steps in an enzyme solution, a coenzyme Generate NADH from NAD ⁺ . The generated NADH transfers two electrons to the fuel electrode via diaphorase via an electron mediator. Then, when the electrons reach the air electrode through an external circuit, a current is generated. Further, H ⁺ generated in the above-described process moves to the air electrode through the proton conductive membrane or the enzyme solution without the membrane. At the air electrode, water is generated from the reached H ⁺ , the two electrons supplied from the external circuit, and oxygen.

In the present invention, as described below, by optimizing the enzymatic activity of an enzyme such as NAD ⁺ -dependent dehydrogenase, selecting an optimal electron mediator, or combining them, The electron movement speed is increased, and the current density is improved. Therefore, the reaction of transferring electrons from the enzyme solution to the fuel electrode in the reaction shown in FIG. 1 will be described in more detail with reference to FIG.

In the enzyme solution, alcohol dehydrogenase (hereinafter, referred to as ADH) for producing formaldehyde and NADH from methanol as a fuel and formaldehyde dehydrogenase for producing formic acid and NADH from formaldehyde are used as NAD ⁺ -dependent dehydrogenase. : less FalDH referred to as), formate dehydrogenase to produce CO ₂ and NADH from formic acid (formate dehydrogenase:. is dissolved three following FateDH referred to as).. In addition, NADH dehydrogenase which oxidizes NADH to decompose it into NAD ⁺ and H ⁺ , that is, diaphorase (hereinafter referred to as DI) is dissolved in the enzyme solution. In the enzyme solution, an electron mediator that receives two electrons from NADH via DI and transfers it to the fuel electrode is dissolved. Also, the coenzyme NAD ⁺ required for the reaction by the NAD ⁺ -dependent dehydrogenase is dissolved in the enzyme solution.

Here, the enzyme activities of a plurality of enzymes that decompose the fuel contained in the enzyme solution must satisfy the following conditions. First, assuming that alcohol dehydrogenase is enzyme 1, U (ADH) corresponding to the enzyme activity U (E1) of enzyme 1 is the sum of the enzyme activities of a group of enzymes (formaldehyde dehydrogenase and formate dehydrogenase) that decompose a degradation product (formaldehyde). For U (E2) = U (FalDH) + U (FateDH), it is necessary that U (E1) = U (ADH) ≦ U (E2) = U (FalDH) + U (FateDH). Next, assuming that formaldehyde dehydrogenase is enzyme 1, U (FalDH) corresponding to the enzyme activity U (E1) of enzyme 1 is the sum of the enzyme activities U (formate dehydrogenase) of the enzyme group (formate dehydrogenase) which decomposes the degradation product (formate). For E2) = U (FateDH), it is necessary that U (E1) = U (FalDH) ≦ U (E2) = U (FateDH). In consideration of these, it is preferable that the fuel cell using methanol as a fuel satisfies the following expression (1).
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) ... Equation (1)

By selecting the three optimal NAD ⁺ -dependent dehydrogenases described above and increasing the ratio of their enzymatic activities in the order of decomposition of methanol, methanol can be converted to CO ₂ without accumulating intermediate products such as formaldehyde and formic acid. , And the production rate of NADH can be sufficiently increased.

Further, the enzyme activity of DI involved in transfer of electrons from NADH to the fuel electrode needs to be greater than the sum of the enzyme activities of three types of NAD ⁺ -dependent dehydrogenase involved in the production of NADH. In addition, the moving speed of electrons from the generated NADH to the fuel electrode can be increased. This is expressed by the following formula (2), where the enzyme activity of DI is U (DI).
U (ADH) + U (FalDH) + U (FateDH) ≦ U (DI) ... Equation (2)

When the enzymatic activity of DI is less than the sum of the enzymatic activities of NAD ⁺ -dependent dehydrogenase, the enzymatic reaction of DI becomes rate-determining, and the electron transfer rate becomes slow, resulting in insufficient current density.

Ｕ Here, U (unit) is one index indicating enzyme activity, and indicates the degree to which 1 μmol of a substrate reacts per minute at a certain temperature and pH.

電 圧 The voltage of the fuel cell is set by controlling the oxidation-reduction potential of the electron mediator used for each electrode. That is, in order to obtain a higher voltage, it is preferable to select an electron mediator having a more negative potential on the fuel electrode side and an electron mediator having a more positive potential on the air electrode side. However, consideration must also be given to the reaction affinity of the electron mediator for the enzyme, the rate of electron exchange with the electrode, and the structural stability against inhibitory factors (light, oxygen, etc.).

From such a total point of view, it is presently preferable to select vitamin K3 (2-methyl-1,4-naphthoquinone, hereinafter referred to as VK3) as an electron mediator acting on the fuel electrode. VK3 has an equilibrium redox potential (in a solution of pH 7.0) of −210 mV (vsAg / AgCl), and has an equilibrium redox potential of DI having an active site of flavin mononucleotide (hereinafter, referred to as FMN). The potential is slightly positive compared to about -380 mV (vsAg / AgCl), which is in a pH 7.0 solution. As a result, the exchange rate of electrons between the DI and the electron mediator becomes moderately high, and a large current density can be obtained. At the same time, a relatively large voltage can be obtained when a battery is configured in combination with the air electrode. it can.

In contrast, when, for example, 1,4-benzoquinone (+93 mV (vsAg / AgCl)), which has a more positive potential than VK3, is used as an electron mediator, the effect of diffusion of the electron mediator molecule in the solution increases. The current density hardly changes compared to VK3, and the expected effect cannot be obtained. In addition, there is the problem that the battery voltage will be lower. Further, when an anthraquinone-2-sulfonate (−549 mV (vsAg / AgCl)), which has a further negative potential than VK3 and a slightly more positive potential than DI, is used as an electron mediator, the current density is inversely higher. At the same time, the battery voltage increases. In this case, the current density of the battery can be improved to some extent by increasing the reaction electrode area by devising the electrode structure and constructing the reaction field three-dimensionally at a high density. Thus, it is also possible to select an electron mediator having an appropriate oxidation-reduction potential in addition to VK3. In addition, for example, a compound having a quinone skeleton, a metal complex such as Os, Ru, Fe, and Co, a viologen compound such as benzyl viologen, a compound having a nicotinamide structure, a compound having a riboflavin structure, and a compound having a nucleotide-phosphate structure Can be used as an electron mediator acting on the fuel electrode.

As the electron mediator acting on the air electrode, ABTS [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate)]), metal complexes such as Os, Ru, Fe, Co, etc. may be used. it can.

In this system, power is generated as follows. First, when methanol as a fuel is supplied to the enzyme solution, ADH in the enzyme solution catalyzes the oxidation of methanol to formaldehyde. At this time, 2H ⁺ and two electrons are removed from methanol to produce NADH, which is a reduced form of NAD ⁺ , and H ⁺ .

Next, FalDH adds H ₂ O to formaldehyde and removes 2H ⁺ and two electrons to produce formic acid. At this time, NADH and H ⁺ are generated.

Next, FateDH is to remove the 2H ⁺ and 2 electrons from formic acid, to produce a CO ₂ as the final product. At this time, NADH and H ⁺ are generated. If the final product, CO _2, is removed from the enzyme solution system (usually as a gas), it does not significantly change the pH of the enzyme solution. For this reason, a decrease in enzyme activity is suppressed.

Then, the DI is oxidized from the NADH generated in the above-described process, and electrons are transferred to the oxidized form of the electron mediator to be a reduced form of the electron mediator. When the electron mediator is VK3, the oxidized form of VK3 receives two electrons and 2H ⁺ and becomes an oxidized form of VK3. The reduced form of the electron mediator then passes the electrons to the fuel electrode and returns to the oxidized form of the electron mediator. The NADH oxidized by DI becomes NAD ⁺ and H ⁺ , and the generated NAD ⁺ is reused in the above-mentioned process of decomposing methanol by the NAD ⁺ -dependent dehydrogenase. As a result, two electrons are transferred from one molecule of NADH to the fuel electrode, and a direct current can be extracted.

As described above, a total of three molecules of NADH are produced during the process in which three types of NAD ⁺ -dependent dehydrogenase decompose one molecule of methanol to CO ₂ . At this stage, in the process of extracting each H ⁺ from the fuel having H ⁺ having a different energy state, the chemical energy is converted to the same substance called NADH. By utilizing the chemical energy of NADH, that is, by passing the electrons of NADH to the fuel electrode, a fuel cell with high energy conversion efficiency can be realized.

In order to smoothly transfer electrons from NADH to the fuel electrode, it is preferable that the electron mediator be sufficiently present in the enzyme solution for the enzyme activity of DI.

In order for the above enzyme to react efficiently and constantly, it is preferable that the pH of the enzyme solution is maintained at, for example, around pH 7 by a buffer such as Tris buffer or phosphate buffer. Further, the temperature of the enzyme solution is preferably maintained at, for example, around 40 ° C. by a temperature control system. Further, the ionic strength (Ion Strength: hereinafter referred to as IS) of the enzyme solution has an adverse effect on the enzyme activity if it is too high or too low. The strength is preferably, for example, about 0.3. However, the above-mentioned pH, temperature and ionic strength have optimum values for the respective enzymes to be used, and are not limited to the above-mentioned values.

Furthermore, the various enzymes described above, coenzymes and electron mediators are used by being dissolved in an enzyme solution, and at least one of these is placed on an electrode or by a general method proposed in the field of biosensors and the like. It may be used by being fixed near the electrode. For example, by using an electrode in which a material having a large surface area such as activated carbon is three-dimensionally arranged at a high density as a fuel electrode, the effective reaction electrode area of the electron mediator is expanded, and the current density is further improved. Can be. Further, by immobilizing the enzyme on the electrode surface at a high density by crosslinking with glutaraldehyde, for example, the transfer of electrons from the enzyme to the electron mediator near the electrode surface becomes more efficient, and the current density can be improved.

The enzyme used in the present invention is not limited to the above-mentioned enzyme, and may be other enzymes. ADH, FalDH, FateDH, and DI described above may be relatively stable to pH or an inhibitor by mutation. Further, as the enzyme for the air electrode, a known enzyme such as laccase or bilirubin oxidase may be used.

As fuels for the fuel cell of the present invention, in addition to methanol, alcohols such as ethanol, sugars such as glucose, fats, proteins, and organic acids such as intermediate products of sugar metabolism (glucose-6-phosphate, fructose-6). -Phosphate, fructose-1,6-bisphosphate, triosephosphate isomerase, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, acetyl-CoA, Citric acid, cis-aconitic acid, isocitric acid, oxalosuccinic acid, 2-oxoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, L-malic acid, oxaloacetic acid, and the like, and mixtures thereof may be used. Among them, glucose, ethanol, intermediate products of sugar metabolism and the like, together with the above-mentioned enzymes alone or in combination using a plurality of appropriate types, particularly by using a plurality of enzymes involved in the TCA cycle, by optimizing environmental conditions, A system in which the fuel is oxidized to CO ₂ can be realized in the same manner as the above-described system in which the fuel is methanol. In particular, glucose is a preferable fuel because it is an extremely easy-to-handle material.

In this case, it is preferable to select and use the most suitable enzyme according to the fuel. For example, enzymes used for the fuel electrode include glucose dehydrogenase, a series of enzymes in the electron transfer system, ATP synthase, enzymes involved in sugar metabolism (eg, hexokinase, glucose phosphate isomerase, phosphofructokinase, fructose diphosphate aldolase, Triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglyceromutase, phosphopyruvate hydratase, pyruvate kinase, L-lactate dehydrogenase, D-lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, 2- Oxoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, malonate dehydrogenase, etc.) Mention may be made of the enzyme.

FIG. 3 shows a complex enzyme reaction when ethanol is used as a fuel. In the case of ethanol, in the first step, ethanol is oxidized to acetaldehyde by the action of alcohol dehydrogenase (ADH), and in the second step, acetaldehyde is oxidized to acetic acid by the action of aldehyde dehydrogenase (AlDH). In each step, NAD ⁺ (oxidized form) is reduced to produce NADH (reduced form). The transfer of electrons via the electron mediator is the same as in the case of methanol shown in FIG. Here, assuming that the enzyme activities of ADH and AlDH are U (ADH) and U (AlDH), respectively, U (ADH) corresponding to U (E1) and U (AlDH) corresponding to U (E2) are 0 < U (ADH) ≦ U (AlDH). The sum of these U (ADH) and U (AlDH) is less than or equal to the enzyme activity of DI U (DI), and U (ADH) + U (AlDH) ≦ U (DI).

FIG. 4 shows a complex enzyme reaction when glucose is used as a fuel. In the case of glucose, in the first stage oxidation reaction, β-D-glucose is decomposed into D-glucono-δ-lactone by the action of glucose dehydrogenase (GDH). D-glucono-δ-lactone is converted to D-gluconate by hydrolysis, and D-gluconate hydrolyzes adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and phosphate in the presence of gluconokinase. Decomposition results in phosphorylation to 6-phospho-D-gluconate. This 6-phospho-D-gluconate is oxidized to 2-keto-6-phospho-D-gluconate by the action of phosphogluconate dehydrogenase (PhGDH) in the second stage oxidation reaction. In each oxidation reaction, NAD ⁺ (oxidized form) is reduced to produce NADH (reduced form). The transfer of electrons via the electron mediator is the same as in the case of methanol shown in FIG. Here, assuming that the enzyme activities of GDH and PhGDH are U (GDH) and U (PhGDH), respectively, U (GDH) corresponding to U (E1) and U (PhGDH) corresponding to U (E2) are 0 < U (GDH) ≦ U (PhGDH). The sum of these U (GDH) and U (PhGDH) is not more than the enzyme activity of DI U (DI), and U (GDH) + U (PhGDH) ≤ U (DI).

Although it is possible to extract two NADH molecules per one molecule of ethanol and glucose, it is necessary to further improve the energy density of the fuel. Therefore, as a method of decomposing glucose into CO _2, it is necessary to utilize sugar metabolism. Ethanol must be converted to acetyl-Co-A with acetaldehyde dehydrogenase (AalDH) and then passed to the TCA cycle.
In addition, when glucose is used as a fuel, sugar metabolism can be applied. Complex enzyme reactions utilizing glucose metabolism are roughly divided into glycolysis and pyruvate production by the glycolytic system, and the citric acid cycle. The glycolytic system and the citric acid cycle are widely known reaction systems. Therefore, the description is omitted here.

カ ー ボ ン In the fuel cell of the present invention, carbon such as glassy carbon, Pt, Au or the like can be used for the fuel electrode. As the air electrode, for example, a material in which carbon carrying a catalyst such as Pt or the like is bonded with a fluororesin or the like can be used. The air electrode may further contain an oxidoreductase such as laccase. As the proton conductive membrane, for example, a fluorine-based resin such as Nafion117 (trade name, manufactured by DuPont, USA) is suitable.

As described above, in the fuel cell of the present invention, the ratio of the enzyme activity of each enzyme is increased in a later stage, and is defined, for example, as in the above formulas (1) and (2). The reaction for decomposing the fuel (for example, methanol) to CO ₂ and producing NADH and H ⁺ , and the reaction for decomposing the produced NADH can be rapidly advanced. Also, by selecting VK3 as the electron mediator, the speed of electron transfer from NADH to the fuel electrode can be increased. By using the above methods alone or in combination, it is possible to realize an unprecedentedly large current density in a fuel cell utilizing biometabolism. In particular, it is preferable to use two methods in combination, whereby the reaction rate is further improved and a higher current density is obtained.

In addition, the fuel cell of the present invention generates electric power by utilizing biological metabolism, which is a high-efficiency energy conversion mechanism, and therefore has excellent stable operation at room temperature, can be reduced in size and weight, and can handle fuel. Also has the advantage of being extremely easy.

Furthermore, the enzymes that contribute to the cell reaction can be obtained by culturing cells or microorganisms that produce the target enzyme, and extracting and purifying the cells by a conventional method, so that the cost of the fuel cell can be reduced. Become.

The present invention is not limited to the above description, and can be appropriately modified without departing from the gist of the present invention.

Hereinafter, specific examples to which the present invention is applied will be described based on experimental results. In the following Comparative Examples and Examples, enzymes, coenzymes, electron mediators, fuels, etc. are added to the solution as needed.However, each of them is first dissolved in the solution, and the added amount is controlled to be very small, on the order of several μl. The experiment was conducted so that the decrease in the substance concentration before dropping and the change in solution temperature could be ignored.

<Experiment 1>
First, the ratio of the optimal enzyme activity of NAD ⁺ -dependent dehydrogenase was examined by examining the production rate of NADH.

Example 1
To 3 ml of 0.1 M Tris-HCl buffer (pH 7.0, IS = 0.3), 5 mM of NAD ⁺ and 1 M of methanol were added, and the solution was stirred and purged with argon gas. Further, 25 units of ADH, 50 units of FalDH and 75 units of FateDH were added. From the time when these NAD ⁺ -dependent dehydrogenases were added to the solution, the absorbance was measured over time using an ultraviolet-visible spectrophotometer. The optical path length is 1 cm. A measurement wavelength of 340 nm characteristic of NADH was selected, and the concentration of NADH generated from the change in absorbance was determined. The temperature of the enzyme solution was controlled so as to fall within a range of 40 ± 1 ° C.

Example 2
The NADH concentration was measured in the same manner as in Example 1, except that 25 units of ADH, 100 units of FalDH, and 200 units of FateDH were added as NAD ⁺ -dependent dehydrogenase.

Comparative Example 1
The NADH concentration was measured in the same manner as in Example 1 except that 25 units of ADH was added as the NAD ⁺ -dependent dehydrogenase, and FalDH and FateDH were not added.

Comparative Example 2
The NADH concentration was measured in the same manner as in Example 1 except that 25 units of ADH, 50 units of FalDH were added, and FateDH was not added as the NAD ⁺ -dependent dehydrogenase.

Comparative Example 3
As an NAD ⁺ -dependent dehydrogenase, 25 units of ADH were added, measurement was started without adding FalDH and FateDH, 50 units of FalDH was added 7.8 hours after the start of the measurement, and further 9.0 hours after the start of the measurement The NADH concentration was measured in the same manner as in Example 1 except that 75 units of FatDH were added later.

Comparative Example 4
The NADH concentration was measured in the same manner as in Example 1 except that 25 units of ADH, 20 units of FalDH, and 15 units of FateDH were added as NAD ⁺ -dependent dehydrogenase.

FIG. 5 shows the change of the NADH concentration with respect to the measurement time of Example 1, Example 2, and Comparative Examples 1 to 4 measured as described above. Table 1 below shows the amounts of NAD ⁺ -dependent dehydrogenase used in Example 1, Example 2, and Comparative Examples 1 to 4. In Table 1, (+) in Comparative Example 3 indicates that the enzyme was not added from the beginning of the measurement but was added during the measurement.

From FIG. 5, it was found that in Example 1, NADH increased steadily with time, and that NADH was higher in Comparative Example 1 and Comparative Example 2, which contained only one or two types of NAD ⁺ -dependent dehydrogenase. It can be seen that the generation rate is extremely high.

In addition, in Example 2, in which the ratio of enzyme activity increasing in the order of ADH, FalDH, and FateDH was further increased as compared with Example 1, an improvement in the NADH generation rate was observed as compared with Example 1.

On the other hand, in Comparative Example 1 in which only ADH was added as the NAD ⁺ -dependent dehydrogenase, the production of NADH almost leveled off 7 hours after the measurement. Further, in Comparative Example 2 to which ADH and FalDH were added as NADH ⁺ -dependent dehydrogenases, although the NADH generation rate was improved as compared with Comparative Example 1, the production of NADH also leveled off. The cause is considered to be that methanol was not decomposed to CO ₂ and was accumulated in the enzyme solution as formaldehyde or formic acid, which caused a change in pH or the like and reduced the enzyme activity. Although not shown in FIG. 5, in Comparative Example 1, no significant increase in the NADH concentration was observed even after 7.8 hours from the start of the measurement.

Further, in Comparative Example 3, the NADH generation rate was as low as in Comparative Example 1 until the addition of FalDH, but the addition rate of FalDH and FateDH rapidly increased the NADH generation rate at each time point. This may be because formaldehyde had accumulated for a while after the start of the measurement, but the addition of FalDH and FateDH caused the decomposition to formic acid and further to CO ₂ .

Comparative Example 4 contained three types of NAD ⁺ -dependent dehydrogenases, but the ADH, FalDH, and FatDH enzyme activity ratios were sequentially reduced in reverse to Example 1, and the NADH generation rate reached a peak. It became. In Comparative Example 4, the enzyme activities of FalDH and FateDH were insufficient compared with the enzyme activities of ADH, and the decomposition of formaldehyde and formic acid as intermediate products was stagnated. It is considered that this phenomenon was caused.

From the results of the above Experiment 1, it is necessary to rapidly decompose an intermediate product that reduces the enzyme activity when methanol is decomposed into CO _2. For this purpose, ADH, FalDH, and FateDH are used as NAD ⁺ -dependent dehydrogenases. It has been found that three enzymes must be present such that the ratio of their enzymatic activities increases in this order.

<Experiment 2>
Next, an open circuit (OCV) measurement was carried out using a three-electrode cell, and the optimal enzyme activity ratio of NAD ⁺ -dependent dehydrogenase and DI was examined. The three-electrode cell uses glassy carbon (diameter 3 mm) as a working electrode, a Pt line as a counter electrode, and Ag / AgCl as a reference electrode. The temperature of the three-electrode cell is controlled so as to fall within a range of 40 ± 1 ° C.

Example 3
In Example 3, OCV was measured when 200 units of DI were added to an enzyme solution prepared by adding VK3 to the enzyme solution of Example 1 in Experiment 1.

And OCV measurement was performed as follows. Specifically, 5 mM of VK3 (oxidized form), 5 mM of NAD ⁺ , and 1 M of methanol were added to 1 ml of Tris-HCl buffer (pH 7.0) (IS = 0.3), and the solution was stirred. And purged with argon gas. Here, the concentration of VK3 (5 mM) is sufficient for the enzyme activity of DI. Next, 25 units of ADH, 50 units of FalDH and 75 units of FateDH were added, and OCV measurement was performed. When the OCV became stable, 200 units of DI were further added, and the OCV was measured over time using this time as a measurement start time.

Example 4
The OCV was measured in the same manner as in Example 3 except that 400 units of DI were added.

Comparative Example 5
OCV was measured in the same manner as in Example 3 except that 25 units of ADH was added as a NAD ⁺ -dependent dehydrogenase, and that FalDH and FateDH were not added.

Comparative Example 6
OCV was measured in the same manner as in Example 3 except that 25 units of ADH and 50 units of FalDH were added as NAD ⁺ -dependent dehydrogenase, and that FateDH was not added.

Comparative Example 7
As an NAD ⁺ -dependent dehydrogenase, 25 units of ADH were added, measurement was started without adding FalDH and FateDH, 50 units of FalDH was added 28.1 minutes after the OCV measurement was started, and 37.3 after the start of the measurement OCV was measured in the same manner as in Example 3 except that 75 units of FateDH were added after one minute.

Comparative Example 8
The OCV was measured in the same manner as in Example 3 except that 100 units of DI were added.

FIG. 6 shows the change in the OCV of Example 3, Example 4, and Comparative Examples 5 to 8 measured as described above. Table 2 below shows the amounts (enzyme activities) of NAD ⁺ -dependent dehydrogenase and DI used in Example 3, Example 4, and Comparative Examples 5 to 8. In Table 2, (+) in Comparative Example 7 indicates that the enzyme was not added from the beginning of the measurement but was added during the measurement.

As is evident from FIG. 6, in all cases, the enzymatic reaction proceeded from the start of the OCV measurement to generate a reduced form of VK3, and a decrease in OCV was observed. However, the results showed that the rate of decrease in OCV was different depending on the amount of the added enzyme activity. For example, in Example 3, a sufficient amount of DI was present with respect to NADH generated by NAD ⁺ -dependent dehydrogenase, and the OCV reduction rate was higher than in Comparative Examples 5 to 8 described below. In Example 4 in which the enzyme activity of DI was further increased, the transfer of electrons from NADH to VK3 was accelerated, the rate of decrease in OCV was increased, and the equilibrium oxidation-reduction potential of VK3 was −0.21 V (vsAg / AgCl). From these facts, it can be said that DI in Example 3 and Example 4 was sufficient for the amount of NADH generated.

On the other hand, in Comparative Example 8 in which the enzyme activity of DI was insufficient compared with the production rate of NADH, the decrease rate of OCV at the beginning of the measurement was sufficient, but the decrease rate of OCV decreased with time. did. The reason for this is considered to be that the enzyme reaction of DI became rate-limiting and the amount of NADH increased.

In Comparative Examples 5 to 7, it is considered that the enzyme activity of DI was sufficient, but the enzyme activity of NAD ⁺ -dependent dehydrogenase was insufficient, the rate of NADH generation was limited, and the rate of decrease in OCV was reduced. . Although not shown in FIG. 6, in Comparative Example 5, no remarkable decrease in OCV was observed even after 28.1 minutes after the start of the measurement.

From the results of Experiment 2 above, it was found that the enzyme activity of DI that oxidizes NADH must be greater than the sum of the enzyme activities of NAD ⁺ -dependent dehydrogenase that produces NADH.

実 験 In the above-mentioned Experiment 2, an example was shown in which glassy carbon was used as the working electrode, that is, the fuel electrode, but the same operation was confirmed when Pt and Au were used.

In the above Experiment 2, an example using a three-electrode cell was shown. However, when a fuel cell using a Pt catalyst as an air electrode and a Nafion membrane as a proton conducting membrane was operated, the same results as in Experiment 2 were obtained. Was obtained.

FIG. 3 is a schematic diagram for explaining an outline of a reaction of the fuel cell to which the present invention is applied. FIG. 2 is a schematic diagram for explaining in detail the reaction on the fuel electrode side in FIG. 1. It is a figure which shows the complex enzyme reaction when ethanol is used as a fuel. It is a figure which shows the complex enzyme reaction when glucose is used as fuel. FIG. 4 is a characteristic diagram showing a change over time of NADH in Experiment 1. FIG. 9 is a characteristic diagram showing a change with time of OCV in Experiment 2.

Claims (14)

A fuel cell that decomposes fuel by a stepwise reaction by a plurality of enzymes and transfers electrons generated by an oxidation reaction to an electrode,
When the enzyme activity of the enzyme 1 when the decomposition product 1 is produced by the decomposition reaction with the enzyme 1 is U (E1), and the total enzyme activity of the enzyme group 2 that decomposes the decomposition product 1 is U (E2), U (E1) ≦ U (E2). 燃料 The fuel cell according to claim 1, wherein the enzyme 1 is an oxidase. 2. The fuel cell according to claim 1, wherein the decomposition reaction by the enzyme 1 is an oxidation reaction, and electrons are transferred to the coenzyme by the oxidation reaction. Further, a coenzyme oxidase that produces an oxidized form of the coenzyme,
When the enzyme activity of the coenzyme oxidase is defined as U (Co), and among the plurality of enzymes, the sum of the enzyme activities of the enzymes involved in the generation of the reduced form of the coenzyme is defined as U (E), 4. The fuel cell according to claim 3, wherein (Co) ≧ U (E). 4. The fuel cell according to claim 3, wherein the oxidized form of the coenzyme is NAD ⁺ and the reduced form is NADH. The fuel cell according to claim 4, wherein the coenzyme oxidase that produces the oxidized form of the coenzyme is diaphorase. 4. The fuel cell according to claim 3, wherein electrons are further transferred from the coenzyme to an electron mediator. 8. The fuel cell according to claim 7, wherein the electronic mediator is vitamin K3. The fuel cell according to claim 1, wherein the fuel is at least one selected from alcohols, sugars, fats, proteins, and organic acids. The fuel cell according to claim 8, wherein the fuel is at least one selected from methanol, ethanol, and glucose. (4) The fuel cell according to (1), wherein the fuel is methanol, and the plurality of enzymes that decompose the fuel stepwise are alcohol dehydrogenase (ADH), formaldehyde dehydrogenase, and formate dehydrogenase. (2) The fuel cell according to (1), wherein the fuel is ethanol, and the plurality of enzymes that decompose the fuel stepwise are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH). 2. The fuel according to claim 1, wherein the fuel is glucose, and the plurality of enzymes that degrade the fuel in a stepwise manner are glucose dehydrogenase (GDH), gluconokinase, and phosphogluconate dehydrogenase (PhGDH). Fuel cell. The fuel is methanol, a plurality of enzymes that gradually decompose the fuel are alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase, and the dehydrogenase that oxidizes the coenzyme is diaphorase,
When the enzyme activities of the alcohol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, and diaphorase are U (ADH), U (FalDH), U (FateDH), and U (DI), respectively, the following formulas (1) and (2) 4. The fuel cell according to claim 3, wherein the following condition is satisfied.
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) Expression (1)
U (ADH) + U (FalDH) + U (FateDH) ≦ U (DI) Expression (2)

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