WO2010061822A1 - Method for immobilizing enzyme on electrode for fuel cell, fuel cell, method for manufacturing fuel cell, electrode for fuel cell, and method for manufacturing electrode for fuel cell - Google Patents

Abstract

Provided are an enzyme immobilizing method, a fuel cell and an electrode for the fuel cell which employ the enzyme immobilizing method, and a method for manufacturing the fuel cell and the electrode. The enzyme immobilizing method prevents reduction in enzyme activity when the enzyme is immobilized on the electrode, so as to make it possible to obtain a high catalyst current value. In the method for immobilizing an enzyme on the electrode used in the fuel cell, an enzyme variant with at least one amino acid residue being deleted, substituted, added, or inserted in a wild type amino acid sequence is used as the enzyme, and the enzyme variant increases in activity through heat treatment. The immobilization is performed within a temperature range which makes it possible to increase the activity of the enzyme variant.

Description

Enzyme immobilization method on fuel cell electrode, fuel cell and production method thereof, and fuel cell electrode and production method thereof

The present invention relates to an enzyme immobilization method on a fuel cell electrode. More specifically, the present invention relates to an enzyme immobilization method in which an enzyme whose activity can be increased by heat treatment is immobilized on an electrode in a temperature range where the activity can be increased. Furthermore, the present invention relates to a fuel cell using the enzyme immobilization method, a fuel cell electrode, and a method for producing them.

The fuel cell has a structure in which a positive electrode (oxidant electrode) and a negative electrode (fuel electrode) face each other with an electrolyte (proton conductor) interposed therebetween. In the conventional fuel cell, the fuel (hydrogen) supplied to the negative electrode is oxidized and separated into electrons and protons (H ⁺ ), the electrons are transferred to the negative electrode, and H ⁺ moves through the electrolyte to the positive electrode. In the positive electrode, this H ⁺ reacts with oxygen supplied from the outside and electrons sent from the negative electrode through the external circuit to generate water (H ₂ O).

A fuel cell is a highly efficient power generation device that directly converts the chemical energy of fuel into electrical energy, and converts the chemical energy of fossil fuels such as natural gas, oil, and coal, regardless of where and when they are used. It can be extracted as electrical energy with efficiency. For this reason, a wide range of applications such as large-scale power generation, automobile drive power supplies, portable power supplies such as personal computers and mobile devices are currently being attempted.

However, when gas is used as the fuel, it is necessary to be careful in handling. In particular, when the fuel is heated to a high temperature, a safety problem arises. Moreover, since an expensive noble metal catalyst such as platinum (Pt) is required, there is a problem in manufacturing cost.

Therefore, in recent years, focusing on the fact that biological metabolism carried out in living organisms is a highly efficient energy conversion mechanism, fuel cells that use enzymes to separate fuel into H + soot and electrons (“biofuel cells” and The development of “enzyme battery” has also been made (see Patent Documents 1 and 2).

In the enzyme battery, alcohols such as methanol and ethanol or saccharides such as glucose can be used as fuel, so that the above safety problems can be solved. In addition, by utilizing the excellent catalytic activity of the enzyme, it becomes possible to produce a fuel cell at a low cost without using a noble metal catalyst.

For the practical application of enzyme batteries, it is necessary to establish a technique for stably obtaining sufficient output, and at present, the enzyme fixed to the electrode works more stably against environmental changes. The development of technology for maintaining high activity has been actively conducted.

JP 2004-71559 A JP 2005-13210 A

In order to obtain a high output in an enzyme battery, it is necessary to efficiently capture electrons generated by the enzyme reaction into the electrode by immobilizing the enzyme on the electrode at a high density.

Usually, when an enzyme is immobilized on an electrode, the enzyme is immobilized on the electrode material by immersing a carbon-based material or the like in an enzyme solution and then evaporating water by drying.

Generally, enzymes have the lowest stability in solution and are easily deactivated. Therefore, after the electrode material is immersed in the enzyme solution, it is necessary to quickly dry and evaporate the water, thereby preventing the enzyme from being deactivated. On the other hand, since the enzyme is easily denatured by heat, it is necessary to perform drying at room temperature or under a low temperature condition of about 30 ° C. even when a dryer is used.

For this reason, conventionally, it is difficult to quickly dry an electrode after being immersed in an enzyme solution, and a decrease in enzyme activity when the electrode is fixed has been an obstacle to obtaining a high catalyst current value and a high output in an enzyme battery. It was.

Accordingly, the present invention provides a method for immobilizing an enzyme on a fuel cell electrode that can obtain an electrode exhibiting a high catalyst current value without causing a decrease in enzyme activity when the enzyme is immobilized on the electrode. The main purpose.
Another object of the present invention is to provide a fuel cell using the enzyme immobilization method of the present invention and a manufacturing method thereof, and an electrode for a fuel cell and a manufacturing method thereof.

In order to solve the above problems, the present invention is a method for immobilizing an enzyme on an electrode used in a fuel cell, wherein at least one amino acid residue is deleted from the wild-type amino acid sequence as the enzyme. An enzyme characterized by immobilizing a mutant enzyme that is a substituted, added, or inserted mutant enzyme and has a property that the activity can be increased by heat treatment in a temperature range that can increase the activity. An immobilization method is provided.
In this enzyme immobilization method, at least one of the wild-type amino acid sequences of SEQ ID NO: 1 of mutant thermotolerant bilirubin oxidase, in particular, bilirubin oxidase derived from imperfect filamentous fungus Myrothecium verrucari, is used as the enzyme immobilized on the positive electrode. A mutant thermotolerant bilirubin oxidase in which the amino acid residues are deleted, substituted, added or inserted can be used.
Further, in this case, in the wild type amino acid sequence, the 225th phenylalanine from the N-terminal is replaced with valine (F225V), the 322nd aspartic acid is replaced with asparagine (D322N), and the 468th methionine is replaced with valine. (M468V) Mutant bilirubin oxidase shown in SEQ ID NO: 2, 225th phenylalanine from the N-terminal is replaced with valine (F225V), 370th aspartic acid is replaced with tyrosine (D370Y), 476th leucine is replaced with proline Substituted (L476P) mutant bilirubin oxidase shown in SEQ ID NO: 3, 264th alanine from valine replaced with valine (A264V), 418th alanine replaced with threonine (A418T), 476th leucine replaced with proline Substituted (L476P) mutated bilirubin oxy represented by SEQ ID NO: 4 Derase, 264th alanine from the N-terminus replaced with valine (A264V), 437th arginine replaced with histidine (R437H), 476th leucine replaced with proline (L476P), mutant bilirubin shown in SEQ ID NO: 5 The oxidase and / or the 103rd alanine from the N-terminus is replaced with proline (A103P), the 264th alanine from the N-terminus is replaced with valine (A264V), and the 270th tyrosine is replaced with aspartic acid (Y270D), 476 It is preferable to perform immobilization in a temperature range of 20 ° C. or more and 65 ° C. or less using a mutant thermostable bilirubin oxidase represented by SEQ ID NO: 6 in which the second leucine is substituted with proline (L476P).

The present invention also relates to a method for producing a fuel cell having a structure in which electrodes face each other via a proton conductor, wherein at least one amino acid residue is deleted, substituted, added or inserted in the wild-type amino acid sequence. And a step of immobilizing the mutated enzyme having the property of increasing the activity by heat treatment in the temperature range where the activity can be increased. A battery manufacturing method and a fuel cell obtained by the manufacturing method are provided.
Furthermore, it is a method for producing an electrode used in a fuel cell, which is a mutant enzyme in which at least one amino acid residue is deleted, substituted, added or inserted in a wild-type amino acid sequence, and is activated by heat treatment And a method for producing a fuel cell electrode, comprising the step of immobilizing a mutant enzyme having a characteristic capable of increasing the activity to the electrode in a temperature range capable of increasing the activity. A fuel cell electrode is also provided.

In the present invention, “Bilirubin oxidase” is an enzyme that catalyzes a reaction that oxidizes bilirubin to biliverdin, and is a kind of enzyme belonging to multi-body oxidase (generic name for enzymes having a plurality of copper ions as active centers). It is.

According to the present invention, when an enzyme is immobilized on an electrode, it is possible to obtain an electrode exhibiting a high catalyst current value without causing a decrease in enzyme activity, and a significant reduction in time and efficiency of the electrode preparation process can be achieved. A possible enzyme immobilization method is provided. In addition, according to this enzyme immobilization method, even if a low-purity or unpurified enzyme is used for immobilization, other than the target enzyme can be inactivated by heat treatment during immobilization. It is possible to obtain an electrode exhibiting a high catalyst current value by preventing hindrance and stability reduction.

It is a figure (one hour of reaction start) which shows an example of heat-resistant screening and has shown the mode of color development of ABTS. It is a figure which shows the UV-vis spectrum of mutant type | mold recombinant BO. 10 is a diagram showing an example of a voltammogram (electrode 1) measured in Example 5. FIG.

In the enzyme immobilization method according to the present invention, an enzyme exemplified below, which has a characteristic that its activity can be increased by heat treatment, is used.

The enzyme fixed to the positive electrode is an oxidase, for example, multi-copper oxidase such as bilirubin oxidase, laccase, ascorbate oxidase.

The enzyme immobilized on the negative electrode is selected according to the fuel used. For example, when a monosaccharide such as glucose is used as the fuel, an oxidase that promotes and decomposes the oxidation of the monosaccharide is used. Usually, in addition to this, a coenzyme oxidase which returns a coenzyme reduced by an oxidase to an oxidant is used. By the action of the coenzyme oxidase, electrons are generated when the coenzyme returns to the oxidized form, and the electrons are transferred from the coenzyme oxidase to the electrode via the electron mediator.

For example, NAD-dependent glucose dehydrogenase such as glucose dehydrogenase (GDH) is used as the oxidase, and NADH oxidoreductase such as diaphorase is used as the coenzyme oxidase.

In addition, when polysaccharides are used as fuel, in addition to the above oxidase and coenzyme oxidase, degradation of polysaccharides, such as hydrolysis, is promoted and degrading enzymes that produce monosaccharides such as glucose are also immobilized. Is done. Here, the polysaccharide means a polysaccharide in a broad sense and refers to all carbohydrates that generate two or more monosaccharides by hydrolysis, and includes oligosaccharides such as disaccharides, trisaccharides, and tetrasaccharides. .

Examples of degrading enzymes include amylase, glucosidase, dextrinase, sucrase, lactase, and cellulase.

Among these enzymes, in particular, an enzyme having a characteristic that its activity is increased by heat treatment is artificially mutated by expressing a gene sequence encoding each enzyme protein in cells such as E. coli. It can be obtained by preparing a mutant enzyme in which at least one amino acid residue is deleted, substituted, added or inserted in the wild-type amino acid sequence, and conducting screening. The modification of the base sequence of the gene can be performed by a technique capable of introducing a gene mutation at random, such as error-prone PCR. A mutant enzyme can be obtained by introducing a modified gene into a host cell by a conventionally known method, extracting a protein from the cell, and then purifying it by affinity column chromatography. The obtained mutant enzyme is screened by a heat resistance test (see Examples) to obtain a mutant enzyme having the above-mentioned characteristics.

In the enzyme immobilization method according to the present invention, the mutant enzyme obtained by screening is immobilized on the electrode in a temperature range where the enzyme activity can be increased. The upper limit of the temperature range is appropriately set for each mutant enzyme (mutant) based on the result of the heat resistance test. The lower limit of the temperature range is 20 ° C. or higher, which is a normal environmental temperature (room temperature), more preferably 30 ° C., and even more preferably 40 ° C.

The immobilization of the mutant enzyme to the electrode can be performed by preparing a mutant enzyme solution according to the conventional method, immersing the electrode in this, and then drying using a dryer or the like. In the enzyme immobilization method according to the present invention, this dry immobilization can be performed in the above temperature range. The lower limit of the temperature range is as described above, but the moisture in the enzyme solution attached to the electrode can be evaporated more rapidly as the drying and fixing is performed at a higher temperature. The time for performing the drying is appropriately set in order to sufficiently evaporate the water in the solution within the heat treatment time in which it was confirmed by the heat resistance test that the enzyme activity of the mutant can be increased. For example, according to the example shown in the examples, the temperature and time of the heat treatment include 60 ° C. within 1 hour or 65 ° C. within 30 minutes.

The mutant enzyme may be immobilized by dropping, spraying, and applying a solution to the electrode, and similarly performing dry fixation. Various immobilizing agents may be used for immobilization, and preferably polycations such as poly-L-lysine (PLL) or salts thereof and polyacrylic acid (for example, sodium polyacrylate (PAAcNa)). ) And other polyanions or salts thereof can be used.

Thus, according to the enzyme immobilization method of the present invention, the water in the enzyme solution can be quickly evaporated, so that the time during which the enzyme is in an unstable solution state is shortened, and the enzyme is deactivated. It becomes possible to prevent. In addition, unlike normally used enzymes, the mutated enzyme used in the present invention has increased enzyme activity due to heat treatment, so that the enzyme after dry fixation expresses higher enzyme activity than before fixation. Will be. Therefore, a high catalyst current value can be obtained with the electrode after enzyme immobilization.

Specifically, the present inventors introduced random mutation into the wild-type amino acid sequence (see SEQ ID NO: 1) of bilirubin oxidase derived from the imperfect filamentous fungus Myrothecium verrucaria (hereinafter, M. verrucaria), and expressed and purified it. The obtained mutant bilirubin oxidase was screened by a heat resistance test, and the following mutants were obtained as mutant bilirubin oxidase whose activity can be increased by heat treatment.
In the above wild-type amino acid sequence, the 225th phenylalanine from the N-terminal is replaced with valine (F225V), the 322nd aspartic acid is replaced with asparagine (D322N), and the 468th methionine is replaced with valine (M468V). 2. Mutant bilirubin oxidase shown in 2.
-The 225th phenylalanine from the N-terminal is replaced with valine (F225V), the 370th aspartic acid is replaced with tyrosine (D370Y), and the 476th leucine is replaced with proline (L476P). Bilirubin oxidase.
-The 264th alanine from the N-terminal is replaced with valine (A264V), the 418th alanine is replaced with threonine (A418T), and the 476th leucine is replaced with proline (L476P). .
-The 264th alanine from the N-terminal is replaced with valine (A264V), the 437th arginine is replaced with histidine (R437H), the 476th leucine is replaced with proline (L476P), and the mutant bilirubin oxidase shown in SEQ ID NO: 5・ Substitute 103th alanine from N-terminal with proline (A103P), 264th alanine from N-terminal with valine (A264V), 270th tyrosine with aspartic acid (Y270D), 476th leucine with proline A mutant bilirubin oxidase represented by SEQ ID NO: 6 substituted (L476P).

These mutant bilirubin oxidases retain a high level of enzyme activity even when heat-treated at 60 ° C. or 65 ° C., exhibit excellent heat resistance, and at the same time, are significantly different from wild-type enzymes when heat-treated at the same temperature. High enzyme activity was expressed.

Therefore, when immobilizing these mutant enzymes on the electrode, a higher catalyst current value than that of the wild-type enzyme can be obtained by fixing at a temperature range of 65 ° C. or lower, more preferably 60 ° C. or lower. It becomes possible. The fixation can be performed under a temperature condition higher than 60 ° C. or 65 ° C. as long as the activity of the mutant enzyme can be increased.

Conventionally known materials such as a carbon-based material can be used as an electrode material for manufacturing an electrode and a fuel cell by the above enzyme immobilization method. In addition, a porous conductive material including a skeleton made of a porous material and a material mainly composed of a carbon-based material that covers at least a part of the surface of the skeleton can be used.

This porous conductive material can be obtained by coating at least a part of the surface of a skeleton made of a porous material with a material mainly composed of a carbon-based material. The porous material constituting the skeleton of the porous conductive material may be basically any material as long as the skeleton can be stably maintained even if the porosity is high. It does not matter whether or not there is sex. As the porous material, a material having high porosity and high conductivity is preferably used. As such a porous material having a high porosity and high conductivity, specifically, a metal material (metal or alloy), a carbon material with a strong skeleton, or the like can be used. When a metal material is used as the porous material, various options are conceivable because the metal material has different state stability depending on the usage environment such as pH and potential of the solution. For example, nickel, copper, silver, gold Foam metal or foam alloy such as nickel-chromium alloy and stainless steel is one of readily available materials. As the porous material, a resin material (for example, a sponge-like material) can be used in addition to the metal material and the carbon-based material. The porosity and pore diameter (minimum pore diameter) of this porous material are in balance with the thickness of the material mainly composed of a carbon-based material that is coated on the surface of the skeleton made of this porous material. Is determined according to the required porosity and pore diameter. The pore diameter of the porous material is generally 10 nm to 1 mm, typically 10 nm to 600 μm.

On the other hand, the material for covering the surface of the skeleton needs to be conductive and stable at the assumed operating potential. Here, a material mainly composed of a carbon-based material is used as such a material. Carbon-based materials generally have a wide potential window, and many are chemically stable. Specifically, the carbon-based material is mainly composed of a carbon-based material, and the carbon-based material is the main component, and the secondary material is selected according to the characteristics required for the porous conductive material. Some materials contain a small amount of material. Specific examples of the latter material include a material whose electrical conductivity has been improved by adding a metal or other highly conductive material to the carbon-based material, or a polytetrafluoroethylene-based material or the like added to the carbon-based material. Thus, it is a material imparted with a function other than conductivity, such as imparting surface water repellency. There are various types of carbon-based materials, but any carbon-based material may be used. In addition to carbon alone, carbon may be added with other elements. The carbon-based material is particularly preferably a fine powder carbon material having high conductivity and a high surface area. Specific examples of the carbon-based material include materials imparted with high conductivity such as KB (Ketjen Black) and functional carbon materials such as carbon nanotubes and fullerenes.

As a coating method of the material mainly composed of the carbon-based material, any coating method can be used as long as the surface of the skeleton made of the porous material can be coated by using an appropriate binder as necessary. Also good. The pore diameter of the porous conductive material is selected so that a solution containing a substrate or the like can easily enter and exit through the pores, and is generally 9 nm to 1 mm, more generally 1 μm to 1 mm, and more generally Specifically, it is 1 to 600 μm. A state in which at least a part of the surface of the skeleton made of a porous material is coated with a material mainly composed of a carbon-based material, or a surface of at least a part of the skeleton made of a porous material is mainly composed of a carbon-based material In the state coated with the material, it is desirable to prevent all the holes from communicating with each other or clogging with a material mainly composed of a carbon-based material.

Electron mediators fixed to the electrodes are 2-methyl-1,4-naphthoquinone (VK3), 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), 2-amino-1,4- Naphthoquinone (ANQ) or the like can be used, and potassium hexacyanoferrate or the like can be used for the positive electrode, if necessary.

As the fuel, when polysaccharide is used, for example, starch, amylose, amylopectin, glycogen, cellulose, maltose, sucrose, lactose and the like can be used. These are a combination of two or more monosaccharides, and any polysaccharide contains glucose as a monosaccharide of the binding unit. Note that amylose and amylopectin are components contained in starch, and starch is a mixture of amylose and amylopectin. When glucoamylase is used as a polysaccharide-degrading enzyme and glucose dehydrogenase is used as an oxidase that degrades monosaccharides, polysaccharides that can be degraded to glucose by glucoamylase, such as starch, amylose, amylopectin, glycogen If it contains any of maltose, it can be used as fuel. Glucoamylase is a degrading enzyme that hydrolyzes α-glucan such as starch to produce glucose, and glucose dehydrogenase is an oxidase that oxidizes β-D-glucose to D-glucono-δ-lactone.

Examples of the proton conductor include electrolytes containing a buffer substance such as dihydrogen phosphate ion (H ₂ PO ₄ ⁻ ), 2-amino-2-hydroxymethyl-1,3-propanediol (abbreviated to Tris), 2- (N-morpholino) ethanesulfonic acid (MES), cacodylic acid, carbonic acid (H ₂ CO ₃ ), hydrogen citrate ion, N- (2-acetamido) iminodiacetic acid (ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid (HEPES), N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid (HEPPS), N- [tri (Hydroxymethyl) methyl] glycine (abbreviated as tricine), glycylglycine, N, can be used N- bis (2-hydroxyethyl) glycine (abbreviated as bicine) electrolyte including. Buffer substances, in general, as long as _a pK _a of 6 to 9, may be used What. In order to obtain a sufficient buffer capacity at the time of high output operation and to fully exhibit the inherent ability of the enzyme, the concentration of the buffer substance contained in the electrolyte is 0.2 M or more. It is effective to be 5M or less, preferably 0.2M to 2M, more preferably 0.4M to 2M, and even more preferably 0.8M to 1.2M. The pH of the electrolyte is preferably around 7, but may be any of 1 to 14 in general.

In the electrode manufacturing method and the fuel cell manufacturing method according to the present invention, an electrode and a fuel cell are obtained by arbitrarily selecting the specific examples exemplified in each of the above configurations using the enzyme immobilization method described above. Can do. In this electrode and fuel cell, it is possible to obtain a high catalyst current value and a high output based on the high enzyme activity of the immobilized enzyme.

(Example 1): cDNA cloning of M. verrucaria-derived bilirubin oxidase (BO) 1-1: Cultivation of M. verrucaria and isolation of messenger RNA M. verrucaria NBRC (IFO) 6113 strain used in this example is independent. Received sales from Biotechnology Headquarters, National Institute of Product Evaluation Technology. The obtained dried cells were suspended in a condensate (polypeptone: 0.5%, yeast extract: 0.3%, MgSO ₄ .7H ₂ O: 0.1%), and this suspension was potato dextrose agar ( PDA) plates (potato dextrose: 2.4%, agarose: 1.5%). When this was cultured at room temperature for 5 to 7 days, the surface of the PDA plate was covered with white mycelia. This was collected with a spatula and stored at -80 ° C. The yield of the bacterial cells was 50 to 60 mg (wet weight) per PDA plate (diameter 9 cm).

Messenger RNA (hereinafter referred to as mRNA) was extracted as total RNA (mixture of mRNA, ribosomal RNA, and transfer RNA). 100 μg (quantified by UV absorption) of total RNA was obtained from about 100 mg of M.verrucaria frozen cell powder, and a quarter of the total RNA was used as template RNA for one reaction of the next reverse transcription PCR.

1-2: Preparation of BO gene fragment by reverse transcription PCR.
Reverse transcription PCR was performed using OneStep RT-PCR Kit (Qiagen) using the total RNA as a template. PCR primers used for reverse transcription PCR were designed as shown in the following “Table 1” based on the base sequence of the previously reported circular DNA (cDNA) of BO.

As a result of agarose gel electrophoresis of the obtained PCR product, a strong band was confirmed around 1700 bp. Since this 1700 bp size was assumed to be an amplified fragment containing the target BO gene, this fragment was excised from an agarose gel slab and used in the next step.

1-3: Integration of BO gene fragment into pYES2 / CT vector The obtained 1700 bp amplified fragment was digested with restriction enzymes HindIII and XbaI and then ligated with pYES2 / CT plasmid vector (Invitrogen) digested with the same enzymes. At this time, Calf intestine-derived alkaline phosphatase (Takara Bio) is used for dephosphorylation of the 5 ′ overhang of the pYES2 / CT vector by restriction enzyme treatment, and T4 DNA ligase is used for the ligation reaction between the inserted fragment and the pYES2 / CT vector. (Takara Bio) was used.

The E.coli TOP10 strain (Invitrogen) was transformed with the reaction product obtained here and inoculated on LB / Amp agar plate medium (see Table 2 for composition). After overnight culture, transformant colonies having drug resistance to ampicillin were obtained. This was cultured overnight in 3 ml of LB / Amp medium, and a plasmid vector was isolated from the resulting cells.

The nucleotide sequence of the inserted portion containing the BO gene of the obtained plasmid vector was examined, and it was SEQ ID NO: 7.

The base sequence shown in SEQ ID NO: 7 is 1719 bp, corresponding to 572 amino acids. On the other hand, mature-type M. verrucaria -derived BO is composed of 534 amino acids (see SEQ ID NO: 1). Here, the 38-residue amino acid corresponding to this difference exists on the N-terminal side, and is a signal peptide responsible for secretion of the protein existing on the C-terminal side. This part is cleaved upon secretion after translation.

1-4: Insertion of AAA sequence Next, the plasmid vector prepared in 1-3 was partially modified so that the expression level of the recombinant protein was increased. Specifically, the 3 bases upstream (5′-side) from the start codon (ATG) were changed as shown in “Table 3”. The change of 3 bases was performed by the Quick-change Mutagenesis Kit (Stratagene) using the PCR primers shown in “Table 4”. The detailed experimental procedure followed the attached manual.

The base sequence was confirmed in the entire region of the BO gene including the changed part, and it was confirmed that it was changed as designed. Hereinafter, the plasmid vector after the sequence change is referred to as “pYES2 / CT-BO vector”.

(Example 2): Construction of secretory expression system of recombinant BO by S. cerevisiae 2-1: Transformation of S. cerevisiae with pYES2 / CT-BO vector.
Next, S. cerevisiae was transformed using the above pYES2 / CT-BO vector. For S. cerevisiae, a commercially available INVSc1 strain (Invitrogen) was used together with the pYES2 / CT vector. Here, S. cerevisiae was transformed by the lithium acetate method. The detailed experimental procedure was based on the manual attached to the pYES2 / CT vector. For selection of transformed yeast, SCGlu agar plate medium (see Table 5 for composition) was used.

2-2: Secretory expression of recombinant BO.
Colonies of transformants of S. cerevisiae with pYES2 / CT-BO vector were inoculated into 15 ml of SCGlu liquid medium, and cultured with shaking at 30 ° C. for 14 to 20 hours. The obtained bacterial cells were once precipitated by centrifugation (1500 × g, room temperature, 10 minutes).

Here, after discarding the SCGlu liquid medium, the obtained bacterial cells were added to 50 ml of SCGal medium (refer to “Table 6” for the composition) so that the turbidity (OD ₆₀₀ ) was about 0.5. This was subjected to shaking culture at 25 ° C. for 10 to 14 hours. After culturing, the cells were removed by centrifugation, and the remaining culture solution was concentrated to about 5 ml and dialyzed against 20 mM Na phosphate buffer (pH 7.4).

The purification of recombinant BO was performed by Ni-NTA affinity chromatography (His-trap HP (1 ml), Amersham Bioscience). The purification method followed the attached manual. The purified recombinant BO obtained was confirmed to have a purity of 100 by SDS-PAGE or the like. The yield of this was 0.36 mg when converted to 1 L culture.

(Example 3): Heat-resistant screening of recombinant BO by molecular evolution engineering technique Next, heat-resistant screening of recombinant BO by molecular evolution engineering technique was performed. Specifically, random mutation insertion using Error-prone PCR, creation of mutant BO gene library, transformation of S. cerevisiae using mutant gene library, and heat resistance using 96-well plate Screening was performed.

3-1: Insertion of random mutations using Error-prone PCR Random mutations were inserted by Error-prone PCR using the pYES2 / CT-BO vector as a template. The N-terminal PCR primer used here was designed to contain a unique BglII site (AGATCT) present 218 base pairs downstream from the start codon. Further, the C-terminal side was designed as follows so as to include an XbaI site (TCTAGA) (see “Table 7”).

Using these primers, Error-prone PCR was performed by GeneMorph PCR-Mutagenesis Kit (Stratagene). The reaction conditions were set with reference to the manual attached to the kit.

When the obtained PCR product was analyzed by agarose gel electrophoresis, a PCR fragment of about 1500 bp could be obtained. The mutation frequency calculated from the yield of the obtained PCR product was 1.5 sites per 1000 bp. Refer to the manual attached to the kit for the calculation method.

3-2: Preparation of mutant BO gene library The BO gene fragment randomly generated with mutations prepared in 3-1 above is prepared by the same method as shown in 1-3, pYES2 / CT-BO vector. Was incorporated into the BglII-XbaI site, and E. coliTO P10 strain was transformed. Here, a colony library of about 6600 transformants, that is, a plasmid library containing about 6600 mutant genes was obtained.

3-3: Transformation of S. cerevisiae with mutant BO gene library According to the method described in 3-2 above, S. cerevisiae INV Sc1 strain (Invitrogen) was transformed with the mutant gene library. A competent cell of S. cerevisiae INVSc1 was produced by the lithium acetate method. The obtained transformant library was subjected to heat-resistant screening using a 96-well plate.

3-4: Heat-resistant screening experiment using 96-well plate After dispensing 150 ml of SCGlu medium into a 96-well plate, one colony of the transformed yeast library prepared above was inoculated into each well. This was subjected to shaking culture at 27 ° C. for 20 to 23 hours. After culturing, the turbidity of each well was almost constant visually.

At this stage, the whole 96-well plate was centrifuged once (1500 × g, 20 ° C., 10 minutes) to once precipitate the cells. The SCGlu medium was completely removed so as not to disturb the cells precipitated on the bottom of each well. To this, 180 ml of SCGal sputum medium was dispensed and further shake-cultured at 27 ° C. for 8 hours. After this culture, centrifugation (1500 × g, 20 ° C., 10 minutes) was performed again to precipitate the cells. 100 ml of this supernatant was transferred to a new 96 well plate. When heat treatment was performed here, the sample solution on the 96-well plate was sealed with cellophane tape and then left in a dry oven at 80 ° C. for 15 minutes. After the heat treatment, it was rapidly cooled on an ice bath for 5 minutes and then allowed to stand at room temperature for 15 minutes. This was mixed with an equal amount of 20 mM ABTS solution (100 mM Tris-HCl pH 8.0). The state in which the solution in the well colored green with the reaction with ABTS was observed for 1 hour after the start of the reaction. Those having a strong color of ABTS compared to the wild type were picked up, and the corresponding cells were stored as a 20 glycerol stock at -80 ° C.

Figure 1 shows an example of heat resistance screening. FIG. 1 shows the state of coloration of ABTS 1 hour after the start of the reaction. All of the middle two columns (6th and 7th columns from the left) are wild-type recombinant BOs for comparison, and the 6th column is heat-treated in the same manner as the other wells. The seventh column is a comparison of the wild type recombinant BO without heat treatment.

Referring to FIG. 1, it can be seen that ABTS is colored more strongly than any of the wild type in the sixth row in the square-enclosed wells. In these wells, it is considered that mutant BO having improved thermostability compared to wild-type recombinant BO is expressed.

In this example, 50 heat-resistant screening shown in 3-4 was performed on a total of 4000 samples in a 96-well plate, and 26 transformed yeasts that were considered to express heat-resistant mutant BO were selected.

As a result of extracting a plasmid vector from the obtained 26 transformed yeasts and analyzing the nucleotide sequence of the BO gene region, it was found that the following 26 types of mutations were inserted into the BO gene. That is, a mutation in which the 49th glutamine from the N-terminus in the wild-type amino acid sequence is replaced with lysine (Q49K), a mutation in which the 72nd glutamine is replaced with glutamic acid (Q72E), and the valine in the 81st are replaced with leucine. Mutation (V81L), mutation in which 103rd alanine is replaced with proline (A103P), mutation in which 121th tyrosine is replaced with serine (Y121S), mutation in which 147th arginine is replaced with proline (R147P), 185th Mutation in which alanine was replaced with serine (A185S), mutation in which 210th proline was replaced with leucine (P210L), mutation in which 225th phenylalanine was replaced with valine (F225V), mutation in which 258th glycine was replaced with valine (G258V) Mutation in which 264th alanine is replaced with valine (A264V), 270th tyrosine is asthma Mutation substituted with aspartic acid (Y270D) Mutation where 299th serine was replaced with asparagine (S299N) Mutation where 322nd aspartic acid was replaced with asparagine (D322N) Mutation where 335th asparagine was replaced with serine ( N335S) 356th arginine substitution with leucine (R356L), 359th proline substitution with serine (P359S), 370th aspartic acid substitution with tyrosine (D370Y), 371th valine Is a mutation in which the alanine is replaced with alanine (V371A), a mutation in which the 381st valine is replaced with leucine (V381L), a mutation in which the 418th alanine is replaced with threonine (A418T), a mutation in which the 423rd proline is replaced with leucine (P423L ) Arginine at 437 was replaced with histidine (R437H), and methionine at 468 was Bali Substituted mutated to (M468V), 476 th leucine is substituted with proline mutation (L476P), 513 th valine mutation was substituted with leucine (V513L) was confirmed.

(Example 4): Mass expression of thermostable mutants by Pichia methanolica In the following, in order to express a large amount of 26 thermostable mutant candidates discovered by thermostable screening, a new yeast Pichia methanolica (hereinafter, We constructed a secretory expression system for recombinant BO using P. methanolica, and tried to express wild-type and thermostable mutant candidates in large quantities.

4-1: Preparation of pMETaB-BO vector and transformation of P. methanolica using the vector First, an expression vector for use in an expression system of P. methanolica was prepared. Since the pMETaB vector (Invitrogen) used here contains a secretion signal: α factor derived from S. cerevisiae, a gene corresponding to mature BO was inserted downstream thereof. Amplification of the mature BO gene region by PCR was performed using the pYES2 / CT-BO vector as a template and the primers shown in “Table 8” below.

The obtained 1500 bp amplified fragment was digested with restriction enzymes EcoRI and SpeI, and then ligated with the pMETaB vector digested with the same enzyme. During this ligation reaction, the same treatment as that shown in 1-3 was performed on the reaction product. Regarding the prepared pMETaB vector containing the BO gene region (hereinafter referred to as pMETaB-BO vector), the base sequence of the inserted BO gene portion was confirmed. In the case of mutant BO, the mutation was inserted into the pMETaB-BO vector prepared here by QuickChange Mutagenesis Kits (Invitrogen). All subsequent operations were performed in the same manner regardless of the wild type or mutant.

In addition to the pMETaB-BO vectors of 26 wild-type mutants and 26 heat-resistant mutant candidates as described above, a multi-mutant pMETaB-BO vector combining 2 or 3 or 4 of 26 heat-resistant mutant candidates is also available. A similar preparation was made and the nucleotide sequence was confirmed.

P. methanolica was transformed with all of the prepared pMETaB-BO vectors. PMAD11 strain (Invitrogen) was used for P. methanolica. Transformation was performed according to the method described in the manual attached to the pMETaB vector. Selection of transformed yeast was carried out using MD agar plate medium (see “Table 9” for composition). The competency of this reaction was ˜10 / 1 μg DNA, which almost coincided with the value described in the manual.

4-2: Large-scale expression of recombinant BO by P. methanolica The transformed yeast colonies on MD medium obtained 5-7 days after transformation were 3 mL of BMDY medium (see Table 10 for composition). Cultured overnight. A part of the obtained culture broth was again developed on an MD agar plate medium. The white purified colony obtained 2 to 3 days later was used for mass expression of the next item.

Next, it shifted to the mass expression work of recombinant BO by P. methanolica. Purified colonies of transformed yeast were inoculated into 50 mL of BMDY liquid medium and cultured overnight at 30 ° C. with shaking. At this time, the OD ₆₀₀ was 2-5. Here, the obtained bacterial cells were once precipitated by centrifugation (1500 × g, room temperature, 10 minutes), and after removing the BMDY liquid medium, only the bacterial cells were treated with the BMMY liquid medium (see “Table 11” for the composition). ) Suspended in 50-100 ml. This was cultured with shaking at 27 ° C. for 24 hours. Thereafter, methanol was added at a final concentration of 0.5% and further cultured for 24 hours under the same conditions. After performing this for up to 96 hours, the cells were removed by centrifugation, and the remaining culture solution was concentrated to about 5 to 10 ml and dialyzed against 50 mM Tris-HCl buffer (pH 7.6).

4-3. Purification of recombinant BO Subsequently, the recombinant BO was purified by anion exchange chromatography. The crude liquid containing recombinant BO prepared in the previous step was purified using an anion exchange column (HiTrap Q HP; bed volume: 5 ml; GE Healthcare Bioscience). Purification conditions were carried out with reference to a previous report (Biochemistry, 38, 3034-3042 (1999)).

Next, the recombinant BO was purified by hydrophobic chromatography. The column used for the hydrophobic chromatography is a Toyopearl Butyl-650M column (100 ml; 22 mm x 20 cm; Tosoh). Purification conditions were carried out with reference to a previous report (Biochemistry, 44, 7004-7012 (2005)). The UV-vis spectrum of recombinant BO (A264V) obtained after purification is shown in FIG.

The spectrum pattern of A264V shown in FIG. 2 completely matched that of the recombinant BO by P. pastris in the previous report (Protein Expression Purif., 41, 77 -83 (2005)).

The final yield of mass culture with P. methanolica was 11.7 mg / 1L maximum.

4-4: Evaluation of heat resistance Next, the heat resistance of recombinant BO by P. methanolica and commercially available BO (Amanoenzyme) was evaluated. The heat resistance was evaluated by comparing the residual activity after heat treatment. Here, “residual activity after heat treatment” (residual activity) may be referred to as “enzyme activity remaining rate” or “enzyme activity maintenance rate”. When the enzyme is subjected to a predetermined heat treatment It is a value representing how the activity changes before and after that. That is, the enzyme activity measurement is performed under the same conditions before and after the heat treatment, and the percentage value indicates how much the activity value after the heat treatment is present before the treatment. The condition of the “heat treatment” as a reference in the present invention is a static treatment at 60 ° C. for 1 hour or 65 ° C. for 30 minutes in a buffer solution, and the ratio of the enzyme activity values before and after this heat treatment is expressed as a percentage. .

The BO activity was measured using ABTS as a substrate, and the change in absorbance at 730 nm accompanying the progress of the reaction (derived from an increase in the reaction product of ABTS) was followed. The measurement conditions were as shown in “Table 12”. The BO concentration was adjusted so that the change in absorbance at 730 nm was about 0.01 to 0.2 per minute when measuring the activity. The reaction was started by adding an enzyme solution (5 to 20 μL) to a phosphate buffer solution (2980 to 2995 μL) containing ABTS.

A total of 26 heat-resistant BO mutant candidates expressed in P. methanolica (Q49K, Q72E, V81L, A103P, Y121S, R147P, SA185S, P210L, F225V, G258V, A264V, Y270D, S299N, D322N, N335S, P359S, D370Y, V371A, V381L, A418T, P423L, R437H, M468V, L476P, V513L) and multiple mutants combining these two or three or four were subjected to heat resistance experiments. For heat treatment of each enzyme solution, 150 ml of enzyme solution (100 mM potassium phosphate buffer (バ ッ フ ァ ー pH 6.0)) dispensed into a 500 ml tube in an ice bath was quickly transferred onto a heat block set at 60 ° C. and left for 1 hour. After that, the method of quickly returning to the ice bath again was adopted. The results of the heat resistance confirmation experiment are summarized in “Table 13”.

The denaturation temperature Tm of the 55 heat-resistant BO mutant candidates for which heat resistance was evaluated was measured by differential scanning microcalorimetry (hereinafter abbreviated as DSC: Different scanning calorimetry). DSC used VP-DSC of MicroCal. The enzyme solution was 2.0 to 2.5 mg / ml, and the temperature was raised at a rate of 60 ° C. per hour. The results are shown in “Table 13” together with the results of the heat resistance confirmation experiment.

In Table 13, several triple mutants or quadruple mutants having a residual enzyme activity rate exceeding 100% were observed. Then, the heat treatment temperature was raised to 65 ° C., and a similar heat resistance confirmation experiment was performed. The results are summarized in “Table 14”. In Table 14, the heat treatment time was 30 minutes.

As summarized in “Table 13” and “Table 14”, in the wild-type and commercially available BO, a remarkable decrease in the residual enzyme activity rate was observed by heat treatment. On the other hand, it was confirmed that many of the thermostable BO mutant candidates maintain high enzyme activity even by heat treatment and exhibit excellent heat resistance.

Furthermore, as shown in Table 14, mutant F225V / D322N / M468VM, variant F225V / D370Y / L476P, variant A264V / A418T / L476P, variant A264V / R437H / L476P and variant A103P / A264V / Y270D In the 5 thermostable BO mutants of / L476P, the residual enzyme activity rate was increased by heat treatment at 65 ° C. for 30 minutes. This reveals that these five mutants have outstanding characteristics that the enzyme activity is increased by heat treatment in addition to heat resistance.

(Example 5): Immobilization of thermostabilized BO mutant to electrode and evaluation of electrode performance In this example, one of the five thermostabilized BO mutants whose enzyme activity was increased by heat treatment (mutant A103P / A264V / Y270D / L476P) was immobilized together with a mediator (K ₃ [Fe (CN) ₆ ]) to prepare an electrode. Using the rotating electrode device, the catalyst current value of the produced electrode was measured, and performance evaluation as a fuel cell electrode was performed. The thermostabilized BO mutant was immobilized by two methods, the method according to the present invention and the conventional method, and the performance of the electrodes obtained by each method was compared. In addition, the commercially available BO was similarly fixed by two methods, and the performance of the obtained electrodes was compared.

5-1: Preparation of electrode (1) Electrode 1 (commercially available BO, conventional immobilization method)
A commercial product (Amanoenzyme) BO solution (buffer solution: 46.5 mM sodium phosphate solution) 80 μl, and 1-cm square carbon felt (CF2) stacked, poly-L-lysine (PLL) ) 80 μl of 2 wt% solution and 80 μl of 200 mM solution of K ₃ [Fe (CN) ₆ ] (hereinafter abbreviated as “FeCN”) were immersed. Commercially available BO was prepared by using a lyophilized product (enzyme activity 2.5 U / mg) in a 50 mg / ml solution (see the manufacturer's attachment for the definition of enzyme activity).

The CF2 soaked with the solution was dried in a dryer at 30 ° C. for 2 hours to evaporate water. After drying, a CF2 cage cut into a size of 6 mm in diameter was physically fixed to the tip of a 6 mm PFC electrode (carbon part is 3 mm in diameter) using a nylon net and an O-ring to obtain an electrode 1.

(2) Electrode 2 (commercial product BO, immobilization method according to the present invention)
In the same manner as in the electrode 1, CF2 soaked with each solution was dried with a dryer at 30 ° C. for 1 hour, and further dried at 60 ° C. for 1 hour to evaporate water. After drying, CF2 was cut out and fixed to the tip of the PFC electrode to obtain electrode 2.

(3) Electrode 3 (heat-resistant BO mutant / conventional immobilization method)
80 μl of mutant BO solution, 80 μl of immobilizing agent poly-L-lysine (PLL) 2 wt% solution, and 80 μl of FeCN 200 mM solution were immersed in a stack of two 1 cm square carbon felts (CF2). . The mutant BO solution was prepared and used so that the absorbance at 600 nm was comparable to that of the commercially available BO solution.

The CF2 soaked with the solution was dried in a dryer at 30 ° C. for 2 hours to evaporate water. After drying, the CF2 cage was cut out and fixed to the tip of the PFC electrode to obtain an electrode 3.

(4) Electrode 4 (heat-resistant BO mutant / immobilization method according to the present invention)
In the same manner as in the electrode 3, CF2 soaked with each solution was dried with a dryer at 30 ° C. for 1 hour, and further dried at 60 ° C. for 1 hour to evaporate water. After drying, CF2 was cut out and fixed to the tip of the PFC electrode to obtain an electrode 4.

5-2: Measurement of catalyst current value For electrodes 1 to 4, cyclic voltammetry (CV) was measured using a rotating electrode device. The CV measurement was performed by scanning the potential in the potential region from the initial potential of 0.6 V (vs. Ag | AgCl) to −0.35 V (vs. Ag | AgCl) at a speed of 10 mV / s. As the buffer solution, 46.5 mM sodium phosphate buffer solution (2 ml) was used, and pure oxygen was bubbled through this to obtain an oxygen saturated solution. The rotation speed of the electrode was 1000 rpm.

FIG. 3 shows a voltammogram obtained with the electrode 1. In the figure, the dotted line shows a voltammogram measured under argon, and the solid line shows a voltammogram measured under oxygen saturation. The difference between the current value under argon where the catalytic reaction of BO does not proceed and the current value under oxygen saturation where the catalytic reaction proceeds is defined as the catalytic current value, and the steady-state current at -0.2 V (vs.Ag | AgCl) The difference between the values was used as the catalyst current value for electrode performance evaluation.

The catalyst current values (mA / cm ² ) obtained with the electrodes 1 to 4 are shown in “Table 15”.

The catalyst current value of electrode 1 (commercial product BO, conventional immobilization method) was 6.60 ± 0.57 mA / cm ² . On the other hand, in the electrode 3 in which the commercial product BO was fixed by the fixing method according to the present invention, the catalyst current value was 2.06 ± 0.33 mA / cm ² . The catalyst current ratio of the electrode 2 to the electrode 1 is 44.8%. When a commercially available BO having low heat resistance is used, the electrode performance may be significantly reduced by setting the immobilization temperature to 60 ° C. confirmed.

The catalyst current value of electrode 3 (heat-resistant BO mutant / conventional immobilization method) was 3.81 ± 0.22 mA / cm ² . On the other hand, in the electrode 4 in which the heat-resistant BO mutant was immobilized by the immobilization method according to the present invention, the catalyst current value was 4.56 ± 0.46 mA / cm ² , and the catalyst current value was about 119% compared to the electrode 3. Increased to. This is because when the thermostabilized BO mutant is used, the enzyme activity of BO is increased by setting the immobilization temperature to 60 ° C., which makes it possible to produce an electrode having superior performance compared to the conventional immobilization method. It shows that it is obtained.

The fuel cell according to the present invention, the fuel cell electrode and the production method thereof, and the enzyme immobilization method to the fuel cell electrode include, for example, an electronic device, a moving body, a power device, a construction machine, a machine tool, a power generation system, It can be widely used in various devices, devices, and systems that require electric power, such as cogeneration systems.

Claims (13)

1. A method for immobilizing an enzyme on an electrode used in a fuel cell,
   As the enzyme, a mutant enzyme in which at least one amino acid residue is deleted, substituted, added or inserted in the wild-type amino acid sequence, and having a property that activity can be increased by heat treatment An enzyme immobilization method for immobilizing an enzyme in a temperature range in which the activity can be increased.
2. The enzyme immobilization method according to claim 1, wherein the electrode is a positive electrode, and the mutant enzyme is a mutant bilirubin oxidase.
3. The enzyme immobilization method according to claim 2, wherein the wild type amino acid sequence is the amino acid sequence of bilirubin oxidase derived from the incomplete filamentous fungus yro Myrothercium verrucari 配 列 shown in SEQ ID NO: 1.
4. As the mutant bilirubin oxidase, in the wild type amino acid sequence, 225th phenylalanine from the N-terminus is replaced with valine (F225V), 322rd aspartic acid is replaced with asparagine (D322N), and 468th methionine is replaced with valine. The method for immobilizing an enzyme according to claim 3, wherein the mutant bilirubin oxidase represented by SEQ ID NO: 2 (M468V) is used.
5. As the mutant bilirubin oxidase, in the wild type amino acid sequence, 225th phenylalanine from the N-terminal is replaced with valine (F225V), 370th aspartic acid is replaced with tyrosine (D370Y), and 476th leucine is replaced with proline. The method for immobilizing an enzyme according to claim 3, wherein the mutant bilirubin oxidase represented by SEQ ID NO: 3 (L476P) is used.
6. As the mutant bilirubin oxidase, in the wild type amino acid sequence, the 264th alanine from the N-terminus is replaced with valine (A264V), the 418th alanine is replaced with threonine (A418T), and the 476th leucine is replaced with proline ( The method for immobilizing an enzyme according to claim 3, wherein the mutant bilirubin oxidase represented by SEQ ID NO: 4 is used.
7. As the mutant bilirubin oxidase, in the wild-type amino acid sequence, the 264th alanine from the N-terminus is replaced with valine (A264V), the 437th arginine is replaced with histidine (R437H), and the 476th leucine is replaced with proline ( 4. The enzyme immobilization method according to claim 3, wherein the mutant bilirubin oxidase represented by SEQ ID NO: 5 is used.
8. As the mutant bilirubin oxidase, in the wild type amino acid sequence, the 103rd alanine from the N-terminus is replaced with proline (A103P), the 264th alanine from the N-terminus is replaced with valine (A264V), and the 270th tyrosine is asparagine. The enzyme immobilization method according to claim 3, wherein the mutant bilirubin oxidase represented by SEQ ID NO: 6 in which the acid is substituted (Y270D) and the 476th leucine is substituted with proline (L476P) is used.
9. The enzyme immobilization method according to any one of claims 4 to 8, wherein the temperature range is from 20 ° C to 65 ° C.
10. A method of manufacturing a fuel cell having a structure in which electrodes face each other via a proton conductor,
    A mutant enzyme having at least one amino acid residue deleted, substituted, added or inserted in the wild-type amino acid sequence, and having a property that the activity can be increased by heat treatment; A method for producing a fuel cell, comprising a step of fixing to the electrode in a temperature range in which the temperature can be increased.
11. A fuel cell having a structure in which electrodes face each other via a proton conductor,
    A mutant enzyme in which at least one or more amino acid residues are deleted, substituted, added or inserted in the wild-type amino acid sequence and has a characteristic that the activity can be increased by heat treatment is A fuel cell fixed to the electrode in a temperature range in which the temperature can be increased.
12. A method of manufacturing an electrode used in a fuel cell,
    A mutant enzyme having at least one amino acid residue deleted, substituted, added or inserted in the wild-type amino acid sequence, and having a property that the activity can be increased by heat treatment; A method for producing an electrode for a fuel cell, comprising a step of fixing to the electrode in a temperature range in which the temperature can be increased.
13. An electrode used in a fuel cell,
    A mutant enzyme in which at least one or more amino acid residues are deleted, substituted, added or inserted in the wild-type amino acid sequence and has a characteristic that the activity can be increased by heat treatment is A fuel cell electrode fixed to the electrode in a temperature range in which the temperature can be increased.
    

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