JP5207576B2 - Fuel cells, portable power supplies and electronic equipment - Google Patents

Description

  The present invention relates to a fuel cell using biological metabolism.

A fuel cell basically includes a fuel electrode, an oxidant electrode (air electrode), and an electrolyte, and its operating principle is based on the reverse operation of water electrolysis. That is, the fuel cell generates water (H ₂ O) and extracts electricity by generating hydrogen and oxygen, that is, generates power. 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, H ₂ O is generated by reacting with the supplied oxygen.

  The fuel cell functions as a highly efficient power generation device that directly converts the energy of the fuel into electric energy, and the energy of fossil energy such as natural gas, oil, coal, etc. is high regardless of where and when it is used It can be taken out as electrical energy at the conversion efficiency.

  Conventionally, research and development of fuel cells for large-scale power generation has been actively conducted. For example, fuel cells are installed in space shuttles and can supply crew members' water at the same time as electric power. There is a proven track record.

  In recent years, a fuel cell having a relatively low operating temperature range from room temperature to about 90 ° C., such as a polymer solid electrolyte fuel cell, has been developed and attracts attention. For this reason, the application to not only large-scale power generation but also small systems such as a power source for driving automobiles and a portable power source for personal computers and mobile devices is being sought.

  However, although the polymer electrolyte fuel cell has the advantage of exhibiting a low operating temperature range as described above, many problems to be solved remain. Specifically, when methanol is used as the fuel and the catalyst is poisoned by CO when operated near room temperature, a catalyst using an expensive noble metal such as Pt is necessary, generation of energy loss due to crossover, It is difficult to handle when using hydrogen as a fuel.

  Accordingly, 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 fuel cells. 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 of about room temperature.

For example, in respiration, nutrients such as sugars, fats, and proteins are taken into microorganisms or cells, and these chemical energies are glycolytic and tricarboxylic acid (hereinafter referred to as TCA) circuits having a number of enzymatic reaction steps. In the process of producing carbon dioxide (CO ₂ ) via nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ), redox like reduced nicotinamide adenine dinucleotide (NADH) This is a mechanism that converts water into electric energy, that is, electric energy, and directly converts the electric energy of NADH into electric energy of proton gradient in the electron transfer system and reduces water by generating oxygen. The electrical energy obtained here produces ATP from ADP via 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.

Photosynthesis takes in light energy and reduces nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP ⁺ ) via an electron transfer system to reduce nicotinamide adenine dinucleotide phosphate ( This is a mechanism that oxidizes water to produce oxygen in the process of conversion into electrical energy such as NADPH. This electric energy takes in CO ₂ and is used for carbon fixation reaction, and is used for carbohydrate synthesis.

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

Hundreds of types of dehydrogenases have been known so far, and play an important role in highly selectively catalyzing the conversion of various substrates into products. This reaction selectivity of the enzyme is due to the fact that the enzyme is a protein molecule and thus has a unique three-dimensional structure. For this reason, in the living body, dozens of types of dehydrogenase and the like react in order with the taken-in fuel, and oxidation proceeds until it finally becomes CO ₂ .

  As a technique for utilizing the above-described biometabolism for a fuel cell, a microbial cell has been reported in which electric energy generated in a microorganism is taken out of the microorganism through an electron mediator and an electric current is obtained by passing the electrons to an electrode. (For example, see Patent Document 1).

However, since microorganisms and cells have many unnecessary functions in addition to the intended reaction such as conversion from chemical energy to electrical energy as described above, the above-described method consumes electrical energy for unwanted reactions. Energy conversion efficiency is not demonstrated.
JP 2000-133297 A

  Therefore, a configuration of a fuel cell is proposed in which only a desired reaction is performed by taking out an enzyme or an electron mediator involved in the reaction from microorganisms or cells and reproducing an appropriate environment.

  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 thereof is to provide a fuel cell capable of realizing a large current density while utilizing biological metabolism. To do.

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

  In a complex enzyme reaction in which fuel is decomposed stepwise by a plurality of enzymes, an intermediate product that impairs enzyme activity needs to be rapidly decomposed. 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 enzyme 1 that performs the previous decomposition reaction. The fuel is quickly decomposed.

  Further, in the present invention, in the fuel cell in which the enzyme 1 is an oxidase, the decomposition reaction by the enzyme 1 is an oxidation reaction, and electrons are transferred to the coenzyme by the oxidation reaction, in addition to the above requirements, Furthermore, it has a coenzyme oxidase that produces an oxidant of the coenzyme, the enzyme activity of the coenzyme oxidase is U (Co), and among the plurality of enzymes, it produces a reduced form of the coenzyme When the sum of the enzyme activities of the enzyme groups involved is U (E), U (Co) ≧ U (E).

  In a fuel cell in which electrons are transferred to a coenzyme by an oxidation reaction by enzyme 1, if there is a shortage of coenzyme oxidase that oxidizes the coenzyme reductant, the enzyme reaction of the coenzyme oxidase Becomes rate limiting. In the present invention, the enzyme activity U (Co) of the coenzyme oxidase that oxidizes the coenzyme is equal to or greater than the sum U (E) of the enzyme activities of the enzyme group involved in the oxidation of the fuel (that is, the production of a reduced form of the coenzyme). Therefore, the enzyme reaction of the coenzyme oxidase does not become rate-determining, the reduced form of the coenzyme is rapidly oxidized, and the electrons generated at this time are transferred to the electrode via the electron mediator.

  More specifically, the case of using methanol as a fuel will be described in more detail. A fuel cell using methanol as a fuel includes, for example, 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 U (ADH), U (FalDH), U (FateDH), and U (DI), respectively.

First, when alcohol dehydrogenase is enzyme 1, U (ADH) corresponding to enzyme activity U (E1) of enzyme 1 is the sum of enzyme activities of enzyme groups (formaldehyde dehydrogenase and formate dehydrogenase) that decompose degradation products (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 enzyme activity U (E1) of enzyme 1 is the sum of enzyme activities U (formic acid dehydrogenase) of enzyme group (formate dehydrogenase) that decomposes decomposition products (formic acid). For E2) = U (FateDH), it is necessary that U (E1) = U (FalDH) ≦ U (E2) = U (FateDH). Taking these into consideration, it is preferable that the fuel cell using methanol as the fuel satisfies the following expression (1).
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) (1)

Next, in the fuel cell, the coenzyme oxidase is diaphorase, the enzyme activity U (DI) corresponds to U (Co), and alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase are all coenzymes. Involved in the production of reductants. Therefore, it is necessary to satisfy the relationship of the following formula (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 . Diaphorase passes two electrons from the generated NADH to the fuel electrode via the electron mediator. H ⁺ generated in these processes reaches the air electrode through the enzyme solution and the proton conducting membrane. In the air electrode, water is generated from H ⁺ , oxygen (O ₂ ), and electrons from the external circuit.

  Then, the enzyme solution is prepared so that the enzymatic 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. The generation speed of is increased.

  In addition, 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 increased without diaphorase being saturated. Can be improved.

  The fuel cell according to the present invention is further characterized in that in the fuel cell in which electrons are transferred from the coenzyme to the electron mediator, 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 passing electrons to and from the fuel electrode The enzyme solution contains alcohol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, diaphorase, and electron mediator, and the electron mediator is vitamin K3. It is characterized by being.

  Since vitamin K3 is relatively close to a coenzyme oxidase (eg, diaphorase) that oxidizes coenzymes, vitamin K3 has a good compatibility with diaphorase and electron transfer, and the electron transfer speed is improved by using it as an electron mediator. .

  As is clear from the above description, the fuel cell according to the present invention has an enzyme of each enzyme so that the reaction of decomposing the fuel and the reaction of coenzyme (for example, NADH) passing electrons to the fuel electrode are not delayed. The level of activity is regulated. Alternatively, in the fuel cell according to the present invention, vitamin K3 having a good compatibility with a dehydrogenase (for example, DI) is selected as an electron mediator in order to increase the movement speed of electrons. Therefore, according to the present invention, it is possible to improve the reaction rate and provide a fuel cell that realizes a higher current density.

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 that separates the fuel electrode and the air electrode, an enzyme described later, and a complement. An enzyme solution in which an enzyme, an electron mediator and the like are dissolved is a basic constituent element. 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 kinds of NAD ⁺ dependent dehydrogenases, which will be described later as complex dehydrogenases, oxidize fuel (for example, methanol) to CO _{2 in a} plurality of stages in an enzyme solution, a coenzyme is used. Generate NADH from NAD ⁺ . The generated NADH passes two electrons to the fuel electrode via the electron mediator by diaphorase. And an electric current generate | occur | produces when an electron reaches | attains an air electrode through an external circuit. 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. In 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 enzyme activity of an enzyme such as NAD ⁺ dependent dehydrogenase, selecting an optimal electron mediator, or a combination thereof, The movement speed of electrons is increased, and the current density is improved. Therefore, the reaction for 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.

The enzyme solution includes alcohol dehydrogenase that generates formaldehyde and NADH from methanol as a fuel (alcohol dehydrogenase: hereinafter referred to as ADH) and formaldehyde dehydrogenase that generates formic acid and NADH from formaldehyde as NAD ⁺ -dependent dehydrogenases. : Formal acid dehydrogenase (hereinafter referred to as FateDH), which generates CO ₂ and NADH from formic acid. In addition, NADH dehydrogenase that 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 passes them to the fuel electrode is dissolved. In addition, coenzyme NAD ⁺ required for the reaction of NAD ⁺ dependent dehydrogenase is also 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, when alcohol dehydrogenase is enzyme 1, U (ADH) corresponding to enzyme activity U (E1) of enzyme 1 is the sum of enzyme activities of enzyme groups (formaldehyde dehydrogenase and formate dehydrogenase) that decompose degradation products (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 enzyme activity U (E1) of enzyme 1 is the sum of enzyme activities U (formic acid dehydrogenase) of enzyme group (formate dehydrogenase) that decomposes decomposition products (formic acid). For E2) = U (FateDH), it is necessary that U (E1) = U (FalDH) ≦ U (E2) = U (FateDH). Taking these into consideration, it is preferable that the fuel cell using methanol as the fuel satisfies the following expression (1).
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) (1)

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

In addition, the enzyme activity of DI involved in the transfer of electrons from NADH to the fuel electrode needs to be made larger than the sum of the enzyme activities of the three types of NAD ⁺ dependent dehydrogenases involved in the production of NADH. The moving speed of electrons from the generated NADH to the fuel electrode can be increased. This is represented 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 enzyme activity of DI is less than the sum of the enzyme activities of NAD ⁺ -dependent dehydrogenase, the enzyme reaction of DI becomes rate-determined, and the electron transfer rate becomes slow, resulting in insufficient current density.

  In addition, U (unit) said here is one parameter | index which shows enzyme activity, and shows the degree to which 1 micromol substrate reacts per minute at a certain temperature and pH.

  The voltage of the fuel cell is set by controlling the redox 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, the reaction affinity of the electron mediator with respect to the enzyme, the rate of electron exchange with the electrode, the structural stability with respect to inhibitory factors (light, oxygen, etc.) must also be considered.

  From such a total point of view, it is currently preferable to select vitamin K3 (2-methyl-1,4-naphthoquinone, Vitamin K3: hereinafter referred to as VK3) as the electron mediator acting on the fuel electrode. VK3 has an equilibrium oxidation-reduction potential (in a solution of pH 7.0) of −210 mV (vsAg / AgCl), and an equilibrium oxidation-reduction potential of DI (flavin mononucleotide: hereinafter referred to as FMN) as an active site ( 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 the battery is configured in combination with the air electrode. it can.

  On the other hand, when, for example, 1,4-benzoquinone (+93 mV (vsAg / AgCl)), which is more positive than VK3, is used as the electron mediator, the influence of diffusion of the electron mediator molecule in the solution is increased. The current density hardly changes compared with VK3, and the expected effect cannot be obtained. Not only that, there arises a problem that the battery voltage becomes smaller. Further, when, for example, anthraquinone-2-sulfonate (−549 mV (vsAg / AgCl)), which has a more negative potential than VK3 and a little more positive than DI, is used as an electron mediator, the current density is reversed. Although it decreases, the battery voltage improves. In this case, the current density as a 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 redox potential in addition to VK3. Other examples include compounds having a quinone skeleton, metal complexes such as Os, Ru, Fe and Co, viologen compounds such as benzyl viologen, compounds having a nicotinamide structure, compounds having a riboflavin structure, compounds having a nucleotide-phosphate structure Can be used as an electron mediator acting on the fuel electrode.

  In addition, as an electron mediator that acts on the air electrode, mainly metal complexes such as ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate)]), Os, Ru, Fe, Co, etc. are used. it can.

In this system, power generation is performed 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 produce formaldehyde. At this time, 2H ⁺ and 2 electrons are removed from methanol, and NADH which is a reduced form of NAD ⁺ and H ⁺ are generated.

Next, FalDH adds H ₂ O to formaldehyde and removes 2H ⁺ and 2 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. The final product, CO ₂ , will not significantly change the pH of the enzyme solution if it is removed from the enzyme solution system (usually as a gas). For this reason, the fall of enzyme activity is suppressed.

Next, DI oxidizes NADH generated in the above-described process, and passes electrons to the oxidized form of the electron mediator to form a reduced form of the electron mediator. When the electron mediator is VK3, the oxidant of VK3 receives two electrons and 2H ⁺ and becomes a reductant of VK3. Next, the reductant of the electron mediator passes the electrons to the fuel electrode and returns to the oxidant of the electron mediator. NADH is oxidized by DI to become NAD ⁺ and H ⁺ , and the generated NAD ⁺ is reused in the process of decomposing methanol by the NAD ⁺ -dependent dehydrogenase described above. As a result, two electrons are passed from one molecule of NADH to the fuel electrode, and a direct current can be taken out.

As described above, a total of three molecules of NADH are produced in the process in which three types of NAD ⁺ dependent dehydrogenases decompose one molecule of methanol to CO ₂ . In this stage, in the process of extracting each H ⁺ from the fuel having H ⁺ having different energy states, the chemical energy is converted into the same substance called NADH. By using the chemical energy possessed by NADH, that is, by passing the electrons possessed by 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 an electron mediator is sufficiently present in the enzyme solution for the enzyme activity of DI.

  In addition, in order for the above enzyme to react efficiently and constantly, the enzyme solution is preferably maintained at a pH of, for example, around pH 7 with a buffer solution such as a Tris buffer solution or a phosphate buffer solution. Also, the temperature of the enzyme solution is preferably maintained at, for example, around 40 ° C. by the temperature control system. Furthermore, the ionic strength (hereinafter referred to as “I.S.”) of the enzyme solution has an adverse effect on the enzyme activity if it is too large or too small. The strength is preferably about 0.3, for example. However, the above pH, temperature, and ionic strength have optimum values for each of the enzymes used, and are not limited to the values described above.

  Further, the various enzymes, coenzymes, and electron mediators described above are used by being dissolved in an enzyme solution, and at least one of them is on an electrode or a general method proposed in the field of biosensors or the like. It may be used by being fixed in the vicinity of 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 as a fuel electrode, the effective reaction electrode area of the electron mediator is expanded and the current density is further improved. Can do. Further, for example, by immobilizing the enzyme on the electrode surface with high density by cross-linking with glutaraldehyde, the transfer of electrons from the enzyme to the electron mediator in the vicinity of the electrode surface becomes more efficient, and the current density can also be improved.

  In addition, the enzyme used for this invention is not limited to the enzyme mentioned above, Other enzymes may be used. In addition, the above-described ADH, FalDH, FateDH, and DI may be relatively stable with respect to pH and inhibitory substances by mutation. Furthermore, known enzymes such as laccase and bilirubin oxidase may be used as the enzyme for the air electrode.

As fuel for the fuel cell of the present invention, methanol, alcohols such as ethanol, sugars such as glucose, fats, proteins, 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, etc.), mixtures thereof, and the like may be used. Among them, glucose, ethanol, intermediate products of sugar metabolism, etc. are combined with the above-mentioned enzymes alone or using a plurality of appropriate types, and in particular, by using a plurality of enzymes involved in the TCA circuit, by optimizing environmental conditions, A system in which the fuel is oxidized to CO ₂ can be realized in the same manner as the system using methanol as the fuel described above. In particular, glucose is a preferred fuel because it is a material that is extremely easy to handle.

  In this case, it is preferable to select and use the optimal enzyme according to a fuel. For example, enzymes used in the fuel electrode include glucose dehydrogenase, a series of enzymes in the electron transport system, ATP synthase, enzymes involved in sugar metabolism (for example, 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 stage, ethanol is oxidized to acetaldehyde by the action of alcohol dehydrogenase (ADH), and in the second stage, acetaldehyde is oxidized to acetic acid by the action of aldehyde dehydrogenase (AlDH). In each stage, NAD ⁺ (oxidant) is reduced to produce NADH (reductant). The delivery of electrons through the electron mediator is the same as in the case of methanol shown in FIG. Here, when 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 U (ADH) and U (AlDH) is equal to or less than the enzyme activity U (DI) of 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. It is phosphorylated by decomposition and becomes 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 ⁺ (oxidant) is reduced to produce NADH (reductant). The delivery of electrons through the electron mediator is the same as in the case of methanol shown in FIG. Here, when the enzyme activities of GDH and PhGDH are U (GDH) and U (PhGDH), U (GDH) corresponding to U (E1) and U (PhGDH) corresponding to U (E2) are 0 < U (GDH) ≦ U (PhGDH). The sum of U (GDH) and U (PhGDH) is equal to or less than the enzyme activity U (DI) of DI, and U (GDH) + U (PhGDH) ≦ U (DI).

  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 carbon carrying a catalyst such as Pt joined with a fluorine resin 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 Nafion 117 manufactured by DuPont, USA is suitable.

As described above, in the fuel cell of the present invention, by increasing the ratio of the enzyme activity of each enzyme as it is in the later stage, for example, as defined in the above formulas (1) and (2), A reaction that decomposes fuel (for example, methanol) to CO ₂ and generates NADH and H ⁺ and a reaction that decomposes the generated NADH can be rapidly advanced. Further, by selecting VK3 as the electron mediator, the moving speed of electrons from NADH to the fuel electrode can be increased. By using the above methods alone or in combination, it is possible to achieve a current density that is unprecedented in a fuel cell using biological metabolism. In particular, it is preferable to use two methods in combination, whereby the reaction rate can be further improved and a larger current density can be obtained.

  In addition, since the fuel cell of the present invention generates power using biological metabolism, which is a highly efficient energy conversion mechanism, it has excellent stability at room temperature, can be reduced in size and weight, and can handle fuel. It also has the advantage of being extremely easy.

  Furthermore, since the enzyme that contributes to the battery reaction is obtained by culturing cells, microorganisms, etc. that produce the target enzyme, and extracting and purifying by a conventional method, it is possible to reduce the cost of the fuel cell. Become.

  In addition, this invention is not limited to the above-mentioned description, In the range which does not deviate from the summary of this invention, it can change suitably.

  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, but each is first dissolved in the solution, and the amount added is controlled to a very small order of several μl. The experiment was conducted so that the decrease in the substance concentration before the dropping and the change in the solution temperature were negligible.

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

Example 1
5 ml of NAD ⁺ and 1M of methanol were added to 3 ml of 0.1 M Tris-HCl buffer (pH 7.0, IS = 0.3), and this solution was stirred and purged with argon gas. Furthermore, 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. The measurement wavelength was 340 nm, which is characteristic of NADH, and the concentration of NADH generated from the change in absorbance was determined. The temperature of the enzyme solution was controlled so as to be within the 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 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 and 50 units of FalDH were added as NAD ⁺ dependent dehydrogenase, and FateDH was not added.

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 were added 7.8 hours after the start of measurement, and 9.0 hours after the start of measurement. The NADH concentration was measured in the same manner as in Example 1 except that 75 units of FateDH 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 in NADH concentration with respect to the measurement time of Example 1, Example 2, and Comparative Examples 1 to 4 measured as described above. In addition, Table 1 below shows the amount of NAD ⁺ dependent dehydrogenase used in Example 1, Example 2, and Comparative Examples 1 to 4. In Table 1, (+) of Comparative Example 3 indicates that the enzyme was added during the measurement, not the enzyme from the beginning of the measurement.

From FIG. 5, in Example 1, NADH increases steadily with the passage of time, and compared with Comparative Example 1 and Comparative Example 2 containing only one type or two types of NAD ⁺ dependent dehydrogenase. It can be seen that the production rate is extremely high.

  Further, 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 production rate of NADH was observed as compared with Example 1.

In contrast, in Comparative Example 1 in which only ADH was added as the NAD ⁺ -dependent dehydrogenase, the production of NADH almost reached its peak after 7 hours of measurement. Further, in Comparative Example 2 in which ADH and FalDH were added as NAD ⁺ -dependent dehydrogenases, the NADH production rate was improved as compared with Comparative Example 1, but the production of NADH reached a peak. This is thought to be because methanol was not decomposed to CO ₂ but accumulated in the enzyme solution as formaldehyde or formic acid, which caused a change in pH and the like to reduce the enzyme activity. Although not shown in FIG. 5, in Comparative Example 1, no significant increase in NADH concentration was observed even after 7.8 hours from the start of measurement.

In Comparative Example 3, the NADH production rate was as low as that in Comparative Example 1 until the addition of FalDH, but the NADH production rate rapidly increased at each time point due to the addition of FalDH and FateDH. This is probably because formaldehyde was accumulated for a while after the start of measurement, but decomposition progressed to formic acid and further CO ₂ by the addition of FalDH and FateDH.

Comparative Example 4 contains three types of NAD ⁺ -dependent dehydrogenases, but the enzyme activity ratios of ADH, FalDH, and FateDH were sequentially decreased as opposed to Example 1, and the NADH production rate peaked out. It became. In Comparative Example 4, the enzymatic activities of FalDH and FateDH are deficient compared to the enzymatic activity of ADH, and the decomposition of formaldehyde and formic acid, which are intermediate products, is stagnant, so that Comparative Examples 1 and 2 are the same. It is thought that this phenomenon was caused.

From the results of the above experiment 1, it is necessary to quickly decompose an intermediate product that reduces the enzyme activity when decomposing methanol into CO ₂ , and for this purpose, NAD ⁺ -dependent dehydrogenase is ADH, FalDH, FateDH. It has been found that these three enzymes must be present so that the ratio of these enzyme activities increases in this order.

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

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

And OCV measurement was performed as follows. Specifically, 5 mM of VK3 (oxidized substance), 5 mM of NAD ⁺ and 1 M of methanol are added to 1 ml of Tris-HCl buffer (pH 7.0) (IS = 0.3), and this solution is stirred. And purging 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 OCV was stabilized, 200 units of DI were further added, and OCV was measured over time with this point as the measurement start time.

Example 4
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 FalDH and FateDH were not added.

Comparative Example 6
As an NAD ⁺ dependent dehydrogenase, OCV was measured in the same manner as in Example 3 except that 25 units of ADH and 50 units of FalDH were added and 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 start of OCV measurement, and 37.3 after the start of measurement. OCV was measured in the same manner as in Example 3 except that 75 units of FateDH were added after a minute.

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

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

As is clear from FIG. 6, in any of the examples, the enzymatic reaction proceeded after the OCV measurement was started to produce a reduced form of VK3, and a decrease in OCV was observed. However, the results showed that the rate of decrease in OCV differs depending on the amount of enzyme activity added. For example, in Example 3, a sufficient amount of DI was present relative to NADH produced by NAD ⁺ dependent dehydrogenase, and the rate of decrease in OCV was greater than 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 −0.21 V (vsAg) which is the equilibrium redox potential of VK3. / AgCl) was quickly reached. From these things, it can be said that DI of Example 3 and Example 4 was sufficient with respect to the amount of NADH produced | generated.

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

In Comparative Examples 5 to 7, the enzyme activity of DI was sufficient, but the enzyme activity of NAD ⁺ dependent dehydrogenase was insufficient, the NADH production rate became rate-limiting, and the decrease rate of OCV was considered to be small. . Although not shown in FIG. 6, in Comparative Example 5, no significant decrease in OCV was observed after 28.1 minutes after the start of 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.

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

  Further, in the above-described experiment 2, an example using a triode cell was shown, but the same result as in the experiment 2 was obtained when a fuel cell using a Pt catalyst as an air electrode and a Nafion membrane as a proton conducting membrane was operated. It was confirmed that

It is a schematic diagram for demonstrating the outline | summary of reaction of the fuel cell to which this invention is applied. In FIG. 1, it is a schematic diagram for demonstrating in detail the reaction by the side of a fuel electrode. It is a figure which shows the complex enzyme reaction at the time of making ethanol into a fuel. It is a figure which shows the complex enzyme reaction at the time of making glucose into a fuel. FIG. 6 is a characteristic diagram showing a change with time of NADH in Experiment 1. FIG. 6 is a characteristic diagram showing a change with time of OCV in Experiment 2.

Claims (9)

A plurality of enzymes that decompose the fuel in stages and simultaneously generate a reduced form of the coenzyme are denoted as E1, E2, and E3, and their enzyme activities are U (E1), U (E2), and U (E3). 0 <U (E1) ≦ U (E2) ≦ U (E3), and the sum of the enzyme activities of the enzyme group involved in the production of the reduced form of the coenzyme (U (E1) + U (E2) + U (E3) ) Is U (E), the enzyme activity U (Co) of the coenzyme oxidase that produces an oxidant of the coenzyme is U (Co) ≧ U (E), and further from the coenzyme, an electron A fuel cell that delivers electrons to the mediator. The fuel cell according to claim 1, wherein the oxidant of the coenzyme is NAD ⁺ and the reductant is NADH.   The fuel cell according to claim 1, wherein the coenzyme oxidase that produces an oxidant of the coenzyme is diaphorase. The fuel cell according to claim 1, wherein the electron mediator is a compound having a quinone skeleton. The fuel cell according to claim 1, wherein the fuel is methanol, and the plurality of enzymes that decompose the fuel in stages are alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. The fuel is methanol, the plurality of enzymes that decompose the fuel stepwise 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) The fuel cell according to claim 1, which satisfies the following relationship:
0 <U (ADH) ≦ U (FalDH) ≦ U (FateDH) (1)
U (ADH) + U (FalDH) + U (FateDH) ≦ U (DI) (2) The fuel cell according to claim 1, wherein the plurality of enzymes are immobilized on or in the vicinity of the electrode. Using fuel cells,
The fuel cell is
A plurality of enzymes that decompose the fuel in stages and simultaneously generate a reduced form of the coenzyme are denoted as E1, E2, and E3, and their enzyme activities are U (E1), U (E2), and U (E3). 0 <U (E1) ≦ U (E2) ≦ U (E3), and the sum of the enzyme activities of the enzyme group involved in the production of the reduced form of the coenzyme (U (E1) + U (E2) + U (E3) ) Is U (E), the enzyme activity U (Co) of the coenzyme oxidase that produces an oxidant of the coenzyme is U (Co) ≧ U (E), and further from the coenzyme, an electron A portable power supply that delivers electrons to the mediator. Using fuel cells,
The fuel cell is
A plurality of enzymes that decompose the fuel in stages and simultaneously generate a reduced form of the coenzyme are denoted as E1, E2, and E3, and their enzyme activities are U (E1), U (E2), and U (E3). 0 <U (E1) ≦ U (E2) ≦ U (E3), and the sum of the enzyme activities of the enzyme group involved in the production of the reduced form of the coenzyme (U (E1) + U (E2) + U (E3) ) Is U (E), the enzyme activity U (Co) of the coenzyme oxidase that produces an oxidant of the coenzyme is U (Co) ≧ U (E), and further from the coenzyme, an electron An electronic device that delivers electrons to a mediator.

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