WO2010061822A1 - Procédé pour immobiliser une enzyme sur une électrode pour pile à combustible, pile à combustible, procédé pour fabriquer une pile à combustible, électrode pour pile à combustible, et procédé pour fabriquer une électrode pour pile à combustible - Google Patents

Procédé pour immobiliser une enzyme sur une électrode pour pile à combustible, pile à combustible, procédé pour fabriquer une pile à combustible, électrode pour pile à combustible, et procédé pour fabriquer une électrode pour pile à combustible Download PDF

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WO2010061822A1
WO2010061822A1 PCT/JP2009/069803 JP2009069803W WO2010061822A1 WO 2010061822 A1 WO2010061822 A1 WO 2010061822A1 JP 2009069803 W JP2009069803 W JP 2009069803W WO 2010061822 A1 WO2010061822 A1 WO 2010061822A1
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enzyme
electrode
mutant
fuel cell
replaced
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PCT/JP2009/069803
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English (en)
Japanese (ja)
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英之 汲田
貴晶 中川
秀樹 酒井
正也 角田
裕一 戸木田
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ソニー株式会社
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Priority to US13/130,475 priority Critical patent/US20110229776A1/en
Publication of WO2010061822A1 publication Critical patent/WO2010061822A1/fr

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • C12Y103/03005—Bilirubin oxidase (1.3.3.5)
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/88—Processes of manufacture
    • H01M4/8825—Methods for deposition of the catalytic active composition
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/90—Selection of catalytic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
    • Y02P70/50—Manufacturing or production processes characterised by the final manufactured product

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • saccharides such as glucose
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 immobilization method is provided.
  • 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.
  • the 225th phenylalanine from the N-terminal is replaced with valine (F225V)
  • the 322nd aspartic acid is replaced with asparagine (D322N)
  • the 468th methionine is replaced with valine.
  • 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.
  • 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
  • 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.
  • “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.
  • an enzyme 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.
  • 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.
  • FIG. 10 is a diagram showing an example of a voltammogram (electrode 1) measured in Example 5.
  • 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.
  • a monosaccharide such as glucose
  • an oxidase that promotes and decomposes the oxidation of the monosaccharide
  • a coenzyme oxidase which returns a coenzyme reduced by an oxidase to an oxidant is used.
  • NAD-dependent glucose dehydrogenase such as glucose dehydrogenase (GDH) is used as the oxidase
  • NADH oxidoreductase such as diaphorase is used as the coenzyme oxidase.
  • polysaccharides 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.
  • 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. .
  • degrading enzymes examples include amylase, glucosidase, dextrinase, sucrase, lactase, and cellulase.
  • 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.
  • 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.
  • 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.
  • 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)).
  • PLL poly-L-lysine
  • PAAcNa sodium polyacrylate
  • other polyanions or salts thereof can be used.
  • 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.
  • 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.
  • 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.
  • the 225th phenylalanine from the N-terminal is replaced with valine (F225V)
  • the 322nd aspartic acid is replaced with asparagine (D322N)
  • the 468th methionine is replaced with valine (M468V).
  • 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)
  • the 476th leucine is replaced with proline (L476P).
  • 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.
  • 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.
  • 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.
  • 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.
  • a material having high porosity and high conductivity is preferably used.
  • a metal material (metal or alloy), a carbon material with a strong skeleton, or the like can be used.
  • the porous material 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.
  • a resin material for example, a sponge-like 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 ⁇ m.
  • the material for covering the surface of the skeleton needs to be conductive and stable at the assumed operating potential.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m to 1 mm, and more generally Specifically, it is 1 to 600 ⁇ m.
  • 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 is 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.
  • VK3 2-methyl-1,4-naphthoquinone
  • ACNQ 2-amino-3-carboxy-1,4-naphthoquinone
  • ANQ 2-amino-1,4- Naphthoquinone
  • potassium hexacyanoferrate or the like can be used for the positive electrode, if necessary.
  • polysaccharide 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.
  • amylose and amylopectin are components contained in starch, and starch is a mixture of amylose and amylopectin.
  • glucoamylase 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 ⁇ -glucan such as starch to produce glucose
  • glucose dehydrogenase is an oxidase that oxidizes ⁇ -D-glucose to D-glucono- ⁇ -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 (HE
  • Buffer substances in general, as long as a pK a of 6 to 9, may be used What.
  • 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.
  • 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%).
  • PDA potato dextrose agar
  • 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).
  • mRNA Messenger RNA
  • total RNA mixture of mRNA, ribosomal RNA, and transfer RNA
  • 100 ⁇ g (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.
  • 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.
  • mature-type M. verrucaria -derived BO is composed of 534 amino acids (see SEQ ID NO: 1).
  • 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.
  • 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.
  • 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.
  • S. cerevisiae was transformed using the above pYES2 / CT-BO vector.
  • a commercially available INVSc1 strain Invitrogen
  • S. cerevisiae was transformed by the lithium acetate method.
  • the detailed experimental procedure was based on the manual attached to the pYES2 / CT vector.
  • SCGlu agar plate medium see Table 5 for composition
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • thermostable mutants by Pichia methanolica
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g DNA, which almost coincided with the value described in the manual.
  • 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).
  • 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 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 ⁇ L) to a phosphate buffer solution (2980 to 2995 ⁇ L) 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.
  • 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.
  • thermostable BO mutant candidates maintain high enzyme activity even by heat treatment and exhibit excellent heat resistance.
  • 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.
  • thermostabilized BO mutant Immobilization of thermostabilized BO mutant to electrode and evaluation of electrode performance
  • 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.
  • a mediator K 3 [Fe (CN) 6 ]
  • 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.
  • the commercially available BO was similarly fixed by two methods, and the performance of the obtained electrodes was compared.
  • Electrode 1 (commercially available BO, conventional immobilization method) A commercial product (Amanoenzyme) BO solution (buffer solution: 46.5 mM sodium phosphate solution) 80 ⁇ l, and 1-cm square carbon felt (CF2) stacked, poly-L-lysine (PLL) ) 80 ⁇ l of 2 wt% solution and 80 ⁇ l 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.
  • 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.
  • Electrode 3 heat-resistant BO mutant / conventional immobilization method 80 ⁇ l of mutant BO solution, 80 ⁇ l of immobilizing agent poly-L-lysine (PLL) 2 wt% solution, and 80 ⁇ l 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.
  • Electrode 4 heat-resistant BO mutant / immobilization method according to the present invention
  • 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.
  • FIG. 3 shows a voltammogram obtained with the electrode 1.
  • the dotted line shows a voltammogram measured under argon
  • 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
  • the catalyst current value of electrode 1 (commercial product BO, conventional immobilization method) was 6.60 ⁇ 0.57 mA / cm 2 .
  • 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%.
  • 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 .
  • 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.

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Abstract

La présente invention concerne un procédé d’immobilisation d’enzyme, une pile à combustible et une électrode pour la pile à combustible qui utilisent le procédé d’immobilisation d’enzyme, et un procédé pour fabriquer la pile à combustible et l’électrode. Le procédé d’immobilisation d’enzyme prévient la réduction d’activité enzymatique lorsque l’enzyme est immobilisée sur l’électrode, de manière à rendre possible l’obtention d’une valeur de courant de catalyseur élevée. Dans le procédé pour immobiliser une enzyme sur l’électrode utilisée dans la pile à combustible, un variant d’enzyme avec au moins un résidu d’acide aminé étant délété, substitué, ajouté, ou inséré dans une séquence d’acides aminés de type sauvage est utilisé en tant qu’enzyme, et le variant d’enzyme augmente d’activité par traitement thermique. L’immobilisation est effectuée dans une plage de température qui rend possible l’augmentation de l’activité du variant d’enzyme.
PCT/JP2009/069803 2007-12-04 2009-11-24 Procédé pour immobiliser une enzyme sur une électrode pour pile à combustible, pile à combustible, procédé pour fabriquer une pile à combustible, électrode pour pile à combustible, et procédé pour fabriquer une électrode pour pile à combustible WO2010061822A1 (fr)

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CN102884180A (zh) * 2010-05-13 2013-01-16 索尼公司 重组胆红素氧化酶及其生产方法
EP3892723A4 (fr) * 2018-12-05 2022-08-31 Ozeki Corporation Procédé d'amélioration de l'activité de la bilirubine oxydase et produit de bilirubine oxydase

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CN102884180A (zh) * 2010-05-13 2013-01-16 索尼公司 重组胆红素氧化酶及其生产方法
FR2975704A1 (fr) * 2011-05-24 2012-11-30 Centre Nat Rech Scient Bilirubine oxydase de magnaporthe oryzae et ses applications
EP3892723A4 (fr) * 2018-12-05 2022-08-31 Ozeki Corporation Procédé d'amélioration de l'activité de la bilirubine oxydase et produit de bilirubine oxydase

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