Allergen inactivation method and allergen inactivation device

Abstract

This method of inactivating an allergen comprises: an inactivation step (S104) for using a reduced redox protein to reduce and thereby inactivate an allergen that is present in a reaction system; and a reduction step (S103) for donating electrons from an electrode that has been connected to an external power source outside the reaction system to an oxidized redox protein generated by oxidation of the reduced redox protein in the inactivation step, thereby reducing the oxidized redox protein to reduced redox protein.

Description

[Technical Field]

[0001] The present disclosure relates to a method and an apparatus for inactivating allergens, which inactivate allergens by reducing disulfide bonds in allergen proteins. [Background technology]

[0002] The number of allergy sufferers is increasing year by year, especially in developed countries, and has become a serious social problem. Proteins with disulfide bonds are known to be allergy-inducing components (hereinafter also referred to as allergens). Disulfide bonds are very strong bonds, so proteins with such bonds are difficult to dissolve in their three-dimensional structure. Therefore, proteins with disulfide bonds are difficult to digest in digestive organs such as the stomach. Therefore, proteins with disulfide bonds are said to have a high possibility of causing allergies (in other words, to be highly allergenic).

[0003] For example, Patent Document 1 describes a method for attenuating the allergenicity of an allergen by using thioredoxin, a low-molecular-weight redox protein, to cleave the disulfide bond of the allergen. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2001-520027 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the method described in Patent Document 1 requires the addition of an excess amount of thioredoxin relative to the amount of allergens in a sample in order to cleave the disulfide bonds of all allergens contained in the sample. Therefore, it is difficult to say that the method described in Patent Document 1 efficiently inactivates allergens contained in a sample.

[0006] Therefore, the present disclosure provides an allergen deactivation method and an allergen deactivation device that can efficiently deactivate allergens. [Means for solving the problem]

[0007] A method for inactivating an allergen according to one embodiment of the present disclosure includes an inactivation step in which the allergen present in a reaction system is inactivated by reducing it with a reduced redox protein, and a reduction step in which the allergen is reduced to the reduced redox protein by donating electrons from an electrode connected to an external power source outside the reaction system to the oxidized redox protein produced by oxidizing the reduced redox protein in the first step.

[0008] Furthermore, an allergen deactivation device according to one aspect of the present disclosure includes an electrode for donating electrons to a redox protein that inactivates allergens by reducing them when a voltage is applied, a power source that applies a voltage to the electrode, and a control unit that controls the voltage application by the power source. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide an allergen deactivation method and an allergen deactivation device that can efficiently deactivate allergens. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram showing an example of the configuration of an allergen deactivation device according to the first embodiment. [Figure 2]FIG. 2 is a block diagram showing an example of the functional configuration of the allergen deactivation apparatus according to the first embodiment. [Figure 3A] FIG. 3A is a first schematic diagram showing components contained in a sample solution and electron transfer reactions between these components. [Figure 3B] FIG. 3B is a second schematic diagram showing components contained in a sample solution and electron transfer reactions between these components. [Figure 3C] FIG. 3C is a diagram illustrating an example of application of the fusion protein. [Figure 4] FIG. 4 is a flowchart showing an example of the operation of the allergen deactivation device according to the first embodiment. [Figure 5] FIG. 5 is a diagram showing an example of the configuration of the allergen deactivation device according to the second embodiment. [Figure 6] FIG. 6 is a schematic cross-sectional view of the working electrode taken along line VI-VI of FIG. [Figure 7] FIG. 7 shows electrophoretic images of Comparative Example 1 and Example 1 after SDS-PAGE. [Figure 8] FIG. 8 is a graph showing the decomposition rates of allergenic proteins after treatment with digestive enzymes in Comparative Example 1 and Example 1. [Figure 9] FIG. 9 shows electrophoretic images of Comparative Example 2 and Example 2 after SDS-PAGE. [Figure 10] FIG. 10 is a graph showing the decomposition rates of allergenic proteins after treatment with digestive enzymes in Comparative Example 2 and Example 2. [Figure 11] FIG. 11 shows electrophoretic images of Examples 3 and 4 after SDS-PAGE. [Figure 12] FIG. 12 is a graph showing the decomposition rates of allergenic proteins after treatment with digestive enzymes in Examples 3 and 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] (Findings that led to this disclosure) In recent years, the number of allergy sufferers has increased, becoming a serious social problem. As mentioned in the Background Art section, allergens are components that cause allergies. For example, most food allergens are proteins (also called allergenic proteins) contained in food. Proteins have a three-dimensional structure in which amino acids are linked in a chain and folded into a spiral or sheet shape. Food allergy symptoms are caused, for example, by IgE antibodies that bind to specific food allergens binding to a portion of the protein's three-dimensional structure that consists of a specific amino acid sequence. Therefore, the allergenicity of the allergen can be reduced by reducing the binding of IgE antibodies to the specific food allergen. Methods for reducing the allergenicity of allergens include, for example, denaturing the protein by adding heat, acid, or enzymes. When a protein denatures, its three-dimensional structure is disassembled and / or its amino acid sequence is cleaved. This results in a change in the portion of the protein that binds to the IgE antibody. However, disulfide bonds are very strong and are difficult to cleave even with heat, acid, and enzymes (e.g., digestive enzymes in the stomach). Therefore, proteins with disulfide bonds are difficult to dissolve in their three-dimensional structure. Therefore, if a protein with disulfide bonds has a disulfide bond in the portion that binds to IgE antibodies, the portion that binds to IgE antibodies is likely to be retained, and it is said that the protein has a high possibility of causing allergies (i.e., is highly allergenic).

[0012] Therefore, as one of the techniques for attenuating allergens, a technique for efficiently cleaving disulfide bonds in allergens is desired. For example, Patent Document 1 discloses a method for attenuating the allergenicity of allergens by cleaving disulfide bonds in allergenic proteins using thioredoxin, a low-molecular-weight redox protein.

[0013] However, in the method described in Patent Document 1, thioredoxin loses its reducing power when it cleaves the disulfide bonds of allergens. Specifically, thioredoxin (also called reduced thioredoxin) is oxidized to reduce disulfide bonds to thiol groups, thereby cleaving the disulfide bonds. At this time, reduced thioredoxin is transformed into oxidized thioredoxin (also called inactive thioredoxin), and loses its reducing power to reduce disulfide bonds. Therefore, the method described in Patent Document 1 requires the addition of an excess amount of thioredoxin to the sample in order to cleave the disulfide bonds of all allergens contained in the sample. Therefore, it is difficult to say that the method described in Patent Document 1 efficiently reduces allergens (i.e., efficiently inactivates allergens).

[0014] Furthermore, for example, according to the method described in Patent Document 1, when the sample is a food product, adding an excessive amount of thioredoxin to the food product leaves a large amount of unreacted thioredoxin remaining in the food product, which may alter other components in the food product. Therefore, applying the method described in Patent Document 1 to food products is inappropriate from the viewpoints of quality and consumer safety. Furthermore, Patent Document 1 does not state or suggest that inactive thioredoxin can be activated and used continuously.

[0015] As a result of extensive research aimed at solving the above problems, the present inventors have discovered a method for repeatedly activating a redox protein that has lost its reducing power by reducing allergens, thereby enabling efficient reduction of allergens in a sample using a smaller amount of the redox protein than is necessary to reduce all of the allergens in the sample.

[0016] Therefore, according to the present disclosure, it is possible to provide an allergen deactivation method and an allergen deactivation device that can efficiently deactivate allergens.

[0017] (One aspect of the present disclosure) An outline of one aspect of the present disclosure is as follows.

[0018] A method for inactivating an allergen according to one aspect of the present disclosure includes an inactivation step in which the allergen present in a reaction system is inactivated by reducing it with a reduced redox protein, and a reduction step in which the allergen is reduced to the reduced redox protein by donating electrons to the oxidized redox protein produced by oxidizing the reduced redox protein in the inactivation step from an electrode connected to an external power source outside the reaction system.

[0019] This allows the oxidized redox protein to be reduced to the reduced redox protein, so that the redox protein that has lost its activity can be activated and reused for inactivating allergens. Therefore, the allergen in the reaction system can be reduced using a small amount of redox protein relative to the amount of allergen. Therefore, this method for inactivating allergens allows the allergen to be inactivated efficiently.

[0020] In the allergen inactivation method according to one aspect of the present disclosure, in the reduction step, electrons may be donated from the electrode to an oxidoreductase, and electrons may be donated from the oxidized oxidoreductase to the oxidized oxidized-redox protein.

[0021] This allows the speed of electron transfer and the amount of energy donated from the electrode to the oxidized redox protein to be adjusted depending on the combination of the redox enzyme and the oxidized redox protein used, thereby improving the efficiency of the electron transfer reaction between the electrode and the redox protein.

[0022] In the allergen inactivation method according to one aspect of the present disclosure, in the reduction step, electrons may be donated from the electrode to a redox molecule, electrons may be donated from the redox molecule to an oxidized reduction enzyme, and electrons may be donated from the oxidized reduction enzyme to the oxidized redox protein.

[0023] This allows the rate of electron transfer and the amount of energy donated from the electrode to the oxidized redox protein to be adjusted depending on the combination of the redox molecule, redox enzyme, and oxidized redox protein used, thereby improving the efficiency of the electron transfer reaction between the electrode and the redox protein.

[0024] In the allergen inactivation method according to one aspect of the present disclosure, in the reduction step, electrons may be donated from the electrode to an electron mediator, electrons may be donated from the electron mediator to a redox molecule, electrons may be donated from the redox molecule to an oxidized reduction enzyme, and electrons may be donated from the oxidized reduction protein to the oxidized form of the redox protein.

[0025] This allows the transfer rate of electrons and the amount of energy donated from the electrode to the oxidized redox protein to be adjusted depending on the combination of the electron mediator, redox molecule, redox enzyme, and oxidized redox protein used, thereby improving the efficiency of the electron donor reaction between the electrode and the redox protein.

[0026] In the method for inactivating an allergen according to one aspect of the present disclosure, the allergen may have a disulfide bond.

[0027] As a result, the disulfide bonds of the allergen are reduced to thiol groups by the reduced redox protein, resulting in cleavage of the disulfide bonds of the allergen. Therefore, according to the method for inactivating allergens, the disulfide bonds of the allergens can be cleaved.

[0028] In the method for inactivating an allergen according to one aspect of the present disclosure, the redox protein may be any one of thioredoxin, glutathione, a protein having at least one thioredoxin-like domain, and a protein having at least one glutathione-like motif.

[0029] As a result, the redox protein has a cysteine-derived thiol group and can reduce disulfide bonds, so that the allergen can be inactivated by reducing the disulfide bonds of the allergen according to the method for inactivating an allergen.

[0030] In the method for inactivating an allergen according to one aspect of the present disclosure, the oxidoreductase may be either (i) an enzyme that catalyzes the reduction of oxidized thioredoxin, including NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase, or (ii) an enzyme that catalyzes the reduction of oxidized glutathione, including glutathione reductase.

[0031] Thus, when the redox protein is thioredoxin or glutathione, the redox enzyme can efficiently reduce the oxidized redox protein generated by reducing the disulfide bond to the reduced redox protein. Therefore, according to the method for inactivating an allergen, a redox protein that has lost its reducing power can be activated (i.e., reduced) with a small amount of the redox protein, thereby efficiently inactivating the allergen.

[0032] In the method for inactivating an allergen according to one aspect of the present disclosure, the redox molecule may be either nicotinamide adenine dinucleotide phosphate or ferredoxin.

[0033] This allows the redox molecule to efficiently donate electrons to the redox enzyme, thereby efficiently reducing the redox enzyme. Therefore, the redox enzyme can efficiently reduce the oxidized redox protein. Therefore, according to the method for inactivating allergens, a small amount of the redox protein can efficiently activate (i.e., reduce) the redox protein that has lost its reducing power, thereby efficiently inactivating the allergen.

[0034] In the method for inactivating an allergen according to one aspect of the present disclosure, the electron mediator may be a compound having a bipyridine skeleton.

[0035] As a result, since the electron mediator has multiple nitrogen-containing heterocycles, the multiple contributions of nitrogen contained in the nitrogen-containing heterocycles allow the electrons required for the reduction of the redox protein to be efficiently donated to the redox molecule. Therefore, according to the method for inactivating an allergen, electrons donated from the electrode via the electron mediator can be efficiently donated to the oxidized redox protein. As a result, the oxidized redox protein is efficiently activated (i.e., reduced), allowing the allergen to be efficiently inactivated with a small amount of redox protein. Therefore, according to the method for inactivating an allergen, the oxidized redox protein can be efficiently reduced to the reduced redox protein with a small amount of redox protein, allowing the allergen to be efficiently inactivated.

[0036] In the method for inactivating an allergen according to one aspect of the present disclosure, the reaction temperature in the reaction system may be 4°C or higher and lower than 60°C.

[0037] As a result, the reduced redox protein can reduce allergens in an environment of 4° C. or higher and lower than 60° C. Therefore, according to the method for inactivating allergens, allergens can be inactivated in an environment of 4° C. or higher and lower than 60° C.

[0038] In the allergen inactivation method according to one aspect of the present disclosure, the voltage applied to the electrodes by the external power supply may be −1.0 V or more and 0 V or less.

[0039] As a result, according to the allergen inactivation method, the voltage applied to the electrodes by the external power source is - 1.0 Since the applied voltage is adjusted to be in the range of V or more and 0 V or less, the efficiency of the electron donor / acceptor reaction can be adjusted depending on the components contained in the reaction system.

[0040] Furthermore, an allergen deactivation device according to one aspect of the present disclosure includes: An apparatus used in the above-mentioned method for inactivating allergens, The device comprises an electrode for donating electrons to a redox protein that inactivates allergens by reducing them when a voltage is applied, a power source for applying voltage to the electrode, and a control unit for controlling the voltage application by the power source.

[0041] As a result, the allergen deactivation device can reduce the oxidized form of the redox protein (so-called oxidized redox protein) generated by oxidizing the allergen through reduction to the reduced form of the redox protein (so-called reduced redox protein) by donating electrons from the electrode. Therefore, the allergen deactivation device can activate the redox protein that has lost its activity due to the redox reaction with the allergen and reuse it for reducing the allergen. Therefore, the allergen deactivation device can reduce the allergen using a small amount of redox protein relative to the amount of allergen, thereby enabling efficient inactivation of the allergen.

[0042] For example, in the allergen deactivation device according to one aspect of the present disclosure, the redox protein may be immobilized on the electrode.

[0043] This eliminates the need to add redox proteins to samples containing allergens in order to inactivate them. Therefore, the allergen deactivation device can easily reduce allergens, thereby efficiently inactivating them. Furthermore, the allergen deactivation device can prevent redox proteins from contaminating samples containing allergens.

[0044] For example, in the allergen deactivation device according to one aspect of the present disclosure, an oxidoreductase that donates electrons to the oxidative reduction protein may be immobilized on the electrode.

[0045] This eliminates the need to add an oxidoreductase to a sample containing an allergen in order to inactivate the allergen. Therefore, the allergen deactivation device can more easily reduce the allergen, thereby efficiently inactivating the allergen. Furthermore, the allergen deactivation device can prevent the sample containing the allergen from being contaminated with an oxidoreductase.

[0046] For example, in the allergen deactivation device according to one aspect of the present disclosure, a redox molecule that donates electrons to the redox enzyme may be immobilized on the electrode.

[0047] This eliminates the need to add additional redox molecules to a sample containing allergens in order to inactivate the allergens. Therefore, the allergen deactivation device can more easily reduce allergens, thereby efficiently inactivating them. Furthermore, the allergen deactivation device can prevent the sample containing allergens from being contaminated with additional redox molecules.

[0048] For example, in the allergen deactivation device according to one aspect of the present disclosure, an electron mediator that donates electrons to the redox molecule may be immobilized on the electrode.

[0049] This eliminates the need to add an electron mediator to a sample containing an allergen in order to inactivate the allergen. Therefore, the allergen deactivation device can more easily reduce the allergen, thereby efficiently inactivating the allergen. Furthermore, the allergen deactivation device can prevent the allergen-containing sample from being contaminated with an electron mediator.

[0050] For example, in the allergen deactivation device according to one aspect of the present disclosure, an electron mediator that mediates electron transfer between the electrode and the redox protein may be immobilized on the electrode.

[0051] This improves the efficiency of the electron transfer reaction between the electrode and the redox protein, allowing the allergen deactivation device to efficiently reduce allergens with a small amount of redox protein, thereby enabling the allergen deactivation device to efficiently inactivate allergens.

[0052] These comprehensive or specific aspects may be realized as a system, a method, an apparatus, an integrated circuit, a computer program, or a recording medium such as a computer-readable CD-ROM, or may be realized as any combination of a system, a method, an apparatus, an integrated circuit, a computer program, and a recording medium.

[0053] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0054] The embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement and connection configurations, steps, and step sequences shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, among the components in the following embodiments, components that are not described in the independent claims that represent the highest concepts are described as optional components. Furthermore, the drawings are not necessarily strict illustrations. In the drawings, substantially identical components are designated by the same reference numerals, and redundant descriptions may be omitted or simplified.

[0055] In addition, in each drawing, the explanation will be made by appropriately using the mutually orthogonal X-axis, Y-axis, and Z-axis directions. In particular, the positive side of the Z-axis direction may be referred to as the upper side, and the negative side as the lower side.

[0056] Furthermore, in this disclosure, terms indicating the relationship between elements, such as parallel and perpendicular, terms indicating the shape of elements, such as rectangle, and numerical values ​​do not only represent the strict meaning, but also include a substantially equivalent range, for example, a difference of about a few percent.

[0057] Also, in the drawings of this disclosure, dashed lines represent what is not visible from the surface and boundaries of areas.

[0058] (Embodiment 1) The first embodiment will be specifically described below with reference to FIGS. 1 to 4. FIG.

[0059] [Allergen deactivation device] [1. Overview] First, an overview of the allergen deactivation apparatus according to the first embodiment will be described with reference to Fig. 1. Fig. 1 is a diagram showing an example of the configuration of an allergen deactivation apparatus 100a according to the first embodiment.

[0060] The allergen deactivation device 100a includes: This is an apparatus used in the allergen inactivation method described below, This device continuously inactivates allergens by donating electrons to redox proteins that inactivate allergens by reducing them, thereby repeatedly activating the redox proteins that have lost their reducing power. The allergen deactivation device 100a can adjust the transfer speed and amount of energy of electrons donated from the electrode (working electrode 1a) to the redox protein by controlling the voltage applied to the electrode (working electrode 1a) by the power supply 20.

[0061] As mentioned above, allergens are components that induce allergies. Allergens have a site (specific binding site) that specifically binds to antibodies (e.g., IgE antibodies) of people with allergic diseases. Inactivating an allergen means weakening the allergenicity of the allergen, for example, by changing the structure (e.g., amino acid sequence) of the specific binding site of the allergen, making it difficult for IgE antibodies to recognize the specific binding site of the allergen.

[0062] For example, the allergen to be inactivated in embodiment 1 has a disulfide bond. An allergen having a disulfide bond is, for example, an allergenic protein. As described above, a disulfide bond is a very strong bond and is difficult to cleave even by heat, acid, and enzymes (for example, digestive enzymes in the stomach). Therefore, allergens having a disulfide bond are likely to cause allergies. When an allergen has a disulfide bond, reducing the allergen means reducing the disulfide bond of the allergen. Details of the reduction of allergens will be described later.

[0063] [2. Configuration] Next, the configuration of the allergen deactivation apparatus 100a according to the first embodiment will be described with reference to Figures 1 and 2. Figure 2 is a block diagram showing an example of the functional configuration of the allergen deactivation apparatus 100a according to the first embodiment.

[0064] The allergen deactivation device 100a according to the first embodiment includes an electrode (working electrode 1a) for donating electrons to a redox protein that inactivates allergens by reducing them when a voltage is applied, a power supply 20 that applies a voltage to the electrode (working electrode 1a), and a control unit 30 that controls the voltage application of the power supply 20. The electrode that donates electrons to the redox protein (hereinafter simply referred to as the working electrode 1a) is one component of the voltage application unit 10a.

[0065] [Voltage application section] The voltage application unit 10a donates electrons from an electrode (working electrode 1a) to the redox protein. The voltage application unit 10a is, for example, a three-electrode cell including a working electrode 1a, a reference electrode 2, a counter electrode 3, a cell 4, a lid 5, terminals 6a, 6b, and 6c, and leads 7a, 7b, and 7c. The voltage application unit 10a may also be, for example, a two-electrode cell including a working electrode 1a and a counter electrode 3.

[0066] The working electrode 1a is an electrode that sensitively responds electrochemically to trace components in the sample solution 9a on its surface. The counter electrode 3 is an electrode that sets a potential difference between the working electrode 1a and the counter electrode 3. The working electrode 1a and the counter electrode 3 are made of conductive materials. Examples of conductive materials include carbon materials, conductive polymer materials, semiconductors, and metals. Examples of carbon materials include carbon nanotubes, Ketjen Black (registered trademark), glassy carbon, graphene, fullerene, carbon fiber, carbon fabric, and carbon aerogel. Examples of conductive polymer materials include polyaniline, polyacetylene, polypyrrole, poly(3,4-ethylenedioxythiophene), poly(p-phenylenevinylene), polythiophene, and poly(p-phenylene sulfide). Examples of semiconductors include silicone, germanium, indium tin oxide (ITO), titanium oxide, copper oxide, and silver oxide. Furthermore, the metal may be, for example, gold, platinum, silver, titanium, aluminum, tungsten, copper, iron, or palladium. Here, the working electrode 1a is, for example, a glassy carbon electrode, and the counter electrode 3 is a platinum electrode. The conductive substance is not particularly limited as long as it is not decomposed by its own oxidation reaction.

[0067] The reference electrode 2 is an electrode that does not react with the components in the sample solution 9a and maintains a constant potential, and is used to control the potential difference between the working electrode 1a and the reference electrode 2 to a constant value using a power supply 20. Here, the reference electrode 2 is a silver / silver chloride electrode.

[0068] The cell 4 is a holding unit for holding a sample solution 9a containing an allergen. The sample solution 9a contains at least an allergen and a redox protein that reduces the allergen. In addition to the allergen and the redox protein, the sample solution 9a may also contain an oxidoreductase that reduces the redox protein. Furthermore, the sample solution 9a may also contain an allergen, a redox protein, and a redox enzyme, as well as a redox molecule that reduces the redox enzyme. Furthermore, the sample solution 9a may also contain an electron mediator involved in electron transfer between the electrode and the redox protein, in addition to the allergen, the redox protein, the oxidoreductase, and the redox molecule. In the following, an example will be described in which the sample solution 9a contains an allergen, a redox protein, a redox enzyme, a redox molecule, and an electron mediator. Note that reducing an allergen refers to reducing the disulfide bond of the allergen.

[0069] Here, each component contained in the sample solution 9a will be described with reference to FIG. 3A. FIG. 3A shows the components contained in the sample solution 9a and the electrons (e in the figure) between those components. - ) is a first schematic diagram showing an electron donor reaction. An electron donor reaction is a reaction involving the donor and donor of electrons, and is a so-called oxidation-reduction reaction. Each component in the reaction system is reduced when it receives an electron, and oxidized when it donates an electron. Therefore, each component has two forms: an oxidized form (ox) and a reduced form (red).

[0070] For example, as shown in Figure 3A, allergens (target molecules in the figure) ox ) has a disulfide bond (-SS-). Examples of allergenic proteins having disulfide bonds (hereinafter simply referred to as allergenic proteins) include allergens derived from foods such as beans, wheat, milk, seafood, eggs, and rice, allergens derived from environments such as pollen, animals, nematodes, mold, mildew, and mites, and allergens derived from animals such as animal hair and dander.

[0071] Allergens (target molecules) ox) disulfide bonds form the reduced form of redox proteins (redox proteins red ) is reduced to a thiol group by donating an electron. ox Above the figure, a schematic representation of the disulfide bond-containing portion (a) of the allergen before reduction is shown. red Below the diagram, a schematic representation of the portion (b) of the allergen after reduction corresponding to (a) above is shown. The circles in (a) and (b) represent amino acids, and the hatched circles represent amino acid sequences recognized by IgE antibodies. As shown in (a) and (b), when the disulfide bonds of the allergen are reduced, the loop portion of the amino acid sequence is released, resulting in the insertion of a different amino acid sequence in the middle of the amino acid sequence recognized by IgE antibodies. This increases the likelihood that the IgE antibody will not be able to recognize the specific binding site. Therefore, the allergenicity of the allergen is weakened.

[0072] Although the above (a) and (b) have been described using loop portions as examples, this is not limiting. In the three-dimensional structure of a protein, functional sites are formed when amino acid residues located far apart in the amino acid sequence of the protein's peptide chain come closer to each other. Therefore, when disulfide bonds are reduced, the connections between the secondary structures of the protein that were connected by the disulfide bonds are severed, and the functional sites formed by the connections between the secondary structures are no longer maintained. More specifically, disulfide bonds connect the secondary structures of a protein to each other, strengthening the three-dimensional structure of the protein. Therefore, when disulfide bonds are cleaved, the connections between the secondary structures are severed, increasing the degree of freedom (fluctuation) of the three-dimensional structure of the allergenic protein. As a result, the functional sites (e.g., three-dimensional conformational epitopes) of the allergenic protein are less likely to be maintained, increasing the likelihood that IgE antibodies will be unable to recognize the specific binding sites of the allergenic protein.

[0073] In addition, reduction of disulfide bonds increases the degree of freedom (fluctuation) of the three-dimensional structure of allergenic proteins, making it easier for digestive enzymes to act on the cleavage sites in the peptide chains of the allergenic proteins, making the allergenic proteins more susceptible to digestion by digestive enzymes.

[0074] In addition, in the present disclosure, as shown in FIG. 3B, the oxidoreductase can transfer electrons without the intervention of a redox molecule. FIG. 3B shows components contained in the sample solution 9a and electrons (e in the figure) between those components. - ) is a second schematic diagram showing the donor / acceptor reaction. The mechanism is thought to be that when (i) the shape of the binding site of the redox molecule with the redox enzyme is similar to the shape of the binding site of the electron mediator, and (ii) the redox potentials of the redox molecule and the electron mediator are equivalent, the redox enzyme can donate and accept electrons without the intervention of the redox molecule. Therefore, the redox enzyme is not particularly limited as long as it is an enzyme that reduces the redox protein, but it is preferable to use it in combination with an electron mediator and redox enzyme that satisfy the above (i) and (ii).

[0075] A redox protein is a protein, polypeptide, or oligopeptide of any size or structure. A redox protein inactivates an allergen by reducing it. For example, a redox protein cleaves the disulfide bond of an allergenic protein by reducing the disulfide bond to a thiol group. As a result, as described above, the increased conformational flexibility (fluctuation) of the allergenic protein due to the disulfide bond not only makes it difficult for IgE antibodies to identify the specific binding site of the allergenic protein, but also makes it easier for digestive enzymes to act on the cleavage site, thereby inactivating the allergenicity of the allergen. Examples of redox proteins that reduce disulfide bonds include thioredoxin, glutathione, proteins having at least one thioredoxin-like domain, and proteins having at least one glutathione-like motif. These redox proteins have thiol groups. For example, thioredoxin is a small redox protein with an active site motif consisting of the amino acid sequence Trp (tryptophan)-Cys (cysteine)-Gly (glycine)-Pro (proline)-Cys (cysteine). Thioredoxin can exist in two forms, reduced and oxidized, depending on the redox state of the two Cys thiol groups in the active site. Glutathione is a tripeptide consisting of Glu (glutamic acid)-Cys (cysteine)-Gly (glycine). Glutathione reduces disulfide bonds in allergenic proteins to thiol groups using the reducing power of the Cys thiol group. Reduced glutathione is a tripeptide consisting of the above three amino acids. Oxidized glutathione is a molecule formed by two molecules of reduced glutathione linked by a disulfide bond.

[0076] Furthermore, a protein having at least one thioredoxin-like domain is, for example, a protein having at least one thioredoxin-like domain containing a Cys-AAc1-AAc2-Cys active site. AAc1 and AAc2 may be any amino acid residues other than cysteine ​​residues. Furthermore, the number of amino acid residues between the two Cys residues in the active site is not limited to two (AAc1 and AAc2) and may be, for example, three or four. Furthermore, a thioredoxin-like domain may contain at least one Cys-AAc1-AAc2-Cys active site. For example, a thioredoxin-like domain may contain a Cys-AAc1-AAc2-Cys active site and a Cys-AAc1'-AAc2'-Cys active site, or a Cys-AAc1-AAc2-Cys-AAc1'-AAc2'-Cys active site. AAc1 and AAc1' may be the same or different amino acid residues, and AAc2 and AAc2' may be the same or different amino acid residues.

[0077] Furthermore, a protein having at least one glutathione-like motif is, for example, a protein having at least one AAc3-Cys-AAc4 active site. That is, the AAc3-Cys-AAc4 active site is a motif similar to the chemical properties of glutathione (so-called glutathione-like motif). AAc3 may be the same acidic amino acid as Glu in glutathione, and AAc4 may be the same neutral amino acid as Gly in glutathione. For example, AAc3 may be Glu, γ-Glu, Asp (aspartic acid), β-Asp, GluGly, γ-GluGly, AspGly, or β-AspGly. Furthermore, AAc4 may be, for example, Gly, Phg (phenylglycine), Ala (alanine), β-Ala, or Phe (phenylalanine).

[0078] The oxidoreductase is an enzyme that catalyzes the oxidation-reduction reaction of the redox protein. The oxidoreductase donates electrons to the redox protein. More specifically, the oxidoreductase donates electrons donated from the working electrode 1a to the oxidized redox protein. The oxidoreductase may donate electrons from the working electrode 1a via at least one of an electron mediator and a redox protein. The oxidoreductase is, for example, either (i) an enzyme that catalyzes the reduction of oxidized thioredoxin, including NADPH (nicotinamide adenine dinucleotide phosphate)-thioredoxin reductase or ferredoxin-thioredoxin reductase, or (ii) an enzyme that catalyzes the reduction of oxidized glutathione, including glutathione reductase.

[0079] For example, when the redox protein is thioredoxin, the oxidoreductase is an enzyme that catalyzes the reduction of oxidized thioredoxin, including NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase. The enzyme that catalyzes the reduction of oxidized thioredoxin is, for example, a polypeptide or protein having thioredoxin reduction activity. The enzyme may be, for example, NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase, or a mutant enzyme in which a portion of the amino acid sequence of NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase is mutated. Furthermore, the enzyme may be a metalloenzyme containing a metal atom such as iron, chromium, manganese, magnesium, calcium, cobalt, molybdenum, zinc, copper, or nickel in the active site of NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase. Alternatively, the enzyme may be a hybrid enzyme consisting of a fusion protein in which NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase is fused to thioredoxin via a linker peptide. In this case, the fusion protein receives electrons donated to the electron mediator from the electrode and then donates the electrons to the allergen.

[0080] The linker peptide is a linker peptide for fusing the above-mentioned thioredoxin reductase (e.g., NADPH-thioredoxin reductase or ferredoxin-thioredoxin reductase) with thioredoxin to create a fusion protein. The linker peptide fuses thioredoxin to thioredoxin reductase so that the redox-active disulfide of thioredoxin can interact with the redox-active disulfide of thioredoxin reductase.

[0081] Furthermore, for example, when the redox protein is glutathione, the oxidoreductase is an enzyme that catalyzes the reduction of oxidized glutathione, including glutathione reductase. The enzyme that catalyzes the reduction of oxidized glutathione is, for example, a polypeptide or protein having glutathione reducing activity. The enzyme may be, for example, a riboflavin-dependent glutathione reductase such as FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide), or an NADPH-dependent glutathione reductase.

[0082] FIG. 3C is a diagram illustrating an example of the application of a fusion protein. FIG. 3C shows an example in which the fusion protein is applied to the inactivation of an allergen. When the fusion protein is applied to the inactivation of an allergen, the fusion protein receives electrons donated to the mediator from the electrode and donates the electrons to the allergen. Then, the disulfide bond of the allergen (e.g., FIG. 3C (a)) is reduced to a thiol group (e.g., FIG. 3C (b)) by receiving electrons from the active fusion protein. As a result, the allergen is inactivated.

[0083] The redox molecule is a molecule that reduces the redox enzyme. The redox molecule reduces the redox enzyme by donating electrons to the redox enzyme. More specifically, the redox molecule reduces the redox enzyme by donating electrons donated from the working electrode 1a to the redox enzyme. The redox molecule may donate electrons from the working electrode 1a via an electron mediator. The redox molecule is, for example, either nicotinamide adenine dinucleotide phosphate (NADPH) or ferredoxin. The redox molecule may also be NADH (nicotinamide adenine dinucleotide).

[0084] The electron mediator is a redox substance that mediates electron transfer between the electrode and the redox protein. The electron mediator donates electrons to the redox molecule. More specifically, the electron mediator donates electrons received from the working electrode 1a to the redox molecule. The electron mediator may donate electrons directly to the oxidized redox protein. The electron mediator is not particularly limited as long as it is a substance that enables electron transfer between the electrode and the redox protein. The electron mediator may be selected depending on the redox potential of the target molecule to which the electron mediator donates electrons. The electron mediator may be, for example, a compound having a bipyridine skeleton, a compound having a quinone skeleton, or a compound having a phenylenediamine skeleton. These compounds may be used alone or in combination of two or more.

[0085] The compound having a bipyridine skeleton may be, for example, a compound having a 2,2'-bipyridine skeleton, a compound having a 2,4'-bipyridine skeleton, or a compound having a 4,4'-bipyridine skeleton, or may be a derivative thereof (e.g., a 4,4'-bipyridinium derivative). The compound having a bipyridine skeleton may be a bipyridine compound having a substituent on the bipyridine skeleton (so-called bipyridine derivative), or may be a bipyridine compound without a substituent. Examples of the substituent include hydrogen, halogen, a hydroxyl group, a nitro group, a carboxyl group, a carbonyl group, an amino group, an amide group, a sulfonic acid group, or an alkyl group, an aryl group, a heteroaromatic alkyl group, or a phenyl group substituted therewith. In addition, two adjacent substituents may form an aromatic ring. The same applies to the compounds having a quinone skeleton and the compounds having a phenylenediamine skeleton described below. When the electron mediator is a compound having a bipyridine skeleton, examples of the electron mediator include 1,1'-dimethyl-4,4'-bipyridinium (methyl viologen), 1-methyl-1'-carboxylmethyl-4,4'-bipyridinium, 1,1'-dicarboxymethyl-4,4'-bipyridinium, 1-methyl-1'-aminoethyl-4,4'-bipyridinium, 1,1'-diaminoethyl-4,4'-bipyridinium, 1-methyl-1'-ethyl-4,4'-bipyridinium, and 1-methyl-1'-propyl-4,4'-bipyridinium. The 4,4'-bipyridinium may be 1-methyl-1'-butyl-4,4'-bipyridinium, 1-methyl-1'-pentylhexyl-4,4'-bipyridinium, 1-methyl-1'-hexyl-4,4'-bipyridinium, 1-methyl-1'-heptyl-4,4'-bipyridinium, 1-methyl-1'-octyl-4,4'-bipyridinium, 1-methyl-1'-nonyl-4,4'-bipyridinium, or 1-methyl-1'-decyl-4,4'-bipyridinium, or may be a compound in which the methyl group at position 1 of these compounds is substituted with an ethyl group.

[0086] The compound having a quinone skeleton may be, for example, a compound having a benzoquinone skeleton, a compound having a naphthoquinone skeleton, a compound having an anthraquinone skeleton, or a derivative thereof. The compound having a quinone skeleton may or may not have a substituent. Since the substituent has been described above, a description thereof will be omitted here. When the electron mediator is a compound having a quinone skeleton, the electron mediator may be, for example, methylbenzoquinone, dimethylbenzoquinone (e.g., 2,5-dimethyl-1,4-benzoquinone, 2,3-dimethyl-1,4-benzoquinone, and 2,6-dimethyl-1,4-benzoquinone), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone, 2,3,5, Examples of the electron mediator include 6-tetrachloro-1,4-benzoquinone (chloranil), ubiquinone (CoQ), pyrroloquinoline quinone (PQQ), 1,2-naphthoquinone-4-sulfonic acid, 2-methyl-1,4-naphthoquinone (vitamin K3), 2-hydroxy-1,4-naphthoquinone, 1,2-dihydroxyanthraquinone (alizarin), 1,2,4-trihydroxyanthraquinone (purpurin), and 9,10-phenanthrenequinone. The electron mediator may also be, for example, a benzenediol in which the ketone group of the benzoquinone skeleton is substituted with a hydroxyl group. More specifically, it may be hydroquinone (1,4-benzenediol) in which the ketone group of 1,4-benzoquinone is substituted with a hydroxyl group, or resorcinol (1,3-benzenediol) in which the ketone group of 1,3-benzoquinone is substituted with a hydroxyl group.

[0087] The compound having a phenylenediamine skeleton may or may not have a substituent. When the electron mediator is a compound having a phenylenediamine skeleton, the electron mediator may be, for example, p-phenylenediamine, 2,3,5,6-tetramethyl-1,4-phenylenediamine, N,N-dimethyl-p-phenylenediamine, N,N'-diphenyl-p-phenylenediamine (DPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), or the like.

[0088] Referring again to FIG. 1, the lid 5 is provided with terminals 6a, 6b, and 6c that electrically connect the working electrode 1a, the reference electrode 2, and the counter electrode 3 to the power supply 20, respectively. Leads extend from each terminal, and the terminals electrode The working electrode 1a is connected to a terminal 6a via a lead 7a, the reference electrode 2 is connected to a terminal 6b via a lead 7b, and the counter electrode 3 is connected to a terminal 6c via a lead 7c.

[0089] [power supply] The power supply 20 applies a voltage to the electrode (working electrode 1a). More specifically, the power supply 20 applies a voltage between the working electrode 1a and the counter electrode 3 of the voltage application unit 10a in accordance with a control signal output from the control unit 30, and controls the potential difference between the working electrode 1a and the reference electrode 2 to a predetermined value. For example, the power supply 20 may apply a voltage to the working electrode 1a so that the voltage applied to the working electrode 1a is between −1.0 V and 0 V, with the reference electrode 2 being the reference (0 V). In this case, the reference electrode 2 is, for example, a silver / silver chloride electrode.

[0090] As shown in FIG. 2, the power supply 20 includes, for example, an acquisition unit 21, an information processing unit 22, and a voltage control unit .

[0091] The acquiring unit 21 acquires a control signal output from the control unit 30. The acquiring unit 21 may also acquire measurement data such as the potential of each electrode in the voltage applying unit 10a and the value of the current flowing through the sample solution 9a.

[0092] The information processing unit 22 processes the information acquired by the acquiring unit 21. For example, when the information processing unit 22 acquires a control signal from the acquiring unit 21, it outputs the acquired control signal to the voltage control unit 23. When the voltage control unit 23 starts applying a voltage to each electrode of the voltage application unit 10a, the information processing unit 22 acquires measurement data, such as the potential of each electrode in the voltage application unit 10a and the value of the current flowing through the sample solution 9a, acquired from the acquiring unit 21, and derives a voltage to be applied to the working electrode 1a based on the acquired data so as to maintain the potential difference between the working electrode 1a and the reference electrode 2 at a predetermined value. Then, the information processing unit 22 outputs a control signal to the voltage control unit 23 to control the voltage of the working electrode 1a using the derived voltage.

[0093] Based on the control signal output from the information processing unit 22, the voltage control unit 23 applies a voltage to each electrode of the voltage application unit 10a.

[0094] Although FIG. 1 shows an example in which the power supply 20 and the control unit 30 are separate entities, the power supply 20 may include the control unit 30.

[0095] [Control Unit] The control unit 30 performs information processing to control the application of voltage from the power supply 20. The control unit 30 is realized by, for example, a processor, a microcomputer, or a dedicated circuit. In FIG. 1, the control unit 30 is shown as a computer device.

[0096] The control unit 30 includes, for example, an acquisition unit 31, an information processing unit 32, a storage unit 33, and an output unit .

[0097] The acquisition unit 31 acquires, for example, information relating to an instruction input by a user (hereinafter, instruction information), and outputs the acquired instruction information to the information processing unit 32.

[0098] The information processing unit 32 derives conditions for applying a voltage to each electrode of the voltage application unit 10a (also referred to as voltage application conditions) based on, for example, the instruction information acquired by the acquisition unit 31. The instruction information may be, for example, the type of allergen, the amount of sample solution 9a, the processing completion time, the completion time, or the degree of inactivation (reduction) (for example, a percentage or a five-level display).

[0099] Furthermore, the information processing unit 32 may output to the output unit 34 a control signal that controls voltage application under conditions derived based on the instruction information, or may output to the output unit 34 a control signal that controls voltage application under voltage application conditions preset by the user.

[0100] The output unit 34 outputs the control signal obtained from the information processing unit 32 to the power supply 20 .

[0101] The storage unit 33 stores data such as instruction information acquired by the acquisition unit 31, computer programs executed by the control unit 30 (for example, application programs for controlling the power supply 20), and the like.

[0102] [3. Operation] Next, the operation of the allergen deactivation apparatus 100a according to the first embodiment will be specifically described with reference to Figures 1 to 4. Figure 4 is a flowchart showing an example of the operation of the allergen deactivation apparatus 100a according to the first embodiment.

[0103] First, a preparation step (not shown) before operating the allergen deactivation device 100a will be described. For example, the preparation step may be performed by a user. In the preparation step, first, a sample solution 9a is prepared. The user introduces a sample containing an allergen into the cell 4 of the voltage application unit 10a. Next, a redox protein, a redox enzyme, a redox molecule, and an electron mediator are added to the sample in the cell 4 to prepare the sample solution 9a. At this time, the allergens present in the sample solution 9a are inactivated by being reduced by the reduced redox protein. Note that the added redox protein, redox enzyme, redox molecule, and electron mediator may each be in a reduced form, or may be in a mixed state of oxidized and reduced forms.

[0104] Next, the user inserts and sets the electrodes into the sample solution 9a. The electrodes are a working electrode 1a, a reference electrode 2, and a counter electrode 3. The working electrode 1a is connected to a lead 7a extending from a terminal 6a arranged on the lid 5, the reference electrode 2 is connected to a lead 7b extending from a terminal 6b arranged on the lid 5, and the counter electrode 3 is connected to a lead 7c extending from a terminal 6c arranged on the lid 5.

[0105] Next, the user inputs information regarding instructions (so-called instruction information) such as the type of allergen, the amount of sample solution 9a, the processing completion time, the completion time, or the degree of inactivation (reduction) into the allergen deactivation device 100a.

[0106] In the above preparation step, the user prepares sample solution 9a by adding only reduced or a mixture of reduced and oxidized redox proteins, redox enzymes, redox molecules, and electron mediators to a sample containing allergens, but oxidized redox proteins, oxidized redox enzymes, oxidized redox molecules, and oxidized electron mediators may also be added. This allows allergens present in sample solution 9a to be inactivated after voltage application begins in step S102, thereby reducing variation in inactivation efficiency.

[0107] In the preparation step described above, the sample solution 9a is prepared by introducing a sample containing an allergen into the cell 4, but a sample solution 9a that has been prepared in advance may also be introduced into the cell 4.

[0108] Next, the operation of the allergen deactivation device 100a will be described. When instruction information is input by the user, the control unit 30 sets conditions for applying voltage to each electrode of the voltage application unit 10a (step S101). In setting the conditions, the control unit 30 derives voltage application conditions based on the input instruction information, and outputs a control signal to the power source 20 to control the voltage application of the power source 20 under the derived voltage application conditions. Note that in step S101, the user may select a program number associated with the voltage application conditions, and the control unit 30 may acquire the program number and set the voltage application conditions.

[0109] Next, upon receiving a control signal from the control unit 30, the power supply 20 starts applying a voltage to the electrodes in accordance with the control signal (step S102). For example, the power supply 20 applies a voltage between the working electrode 1a and the counter electrode 3 of the voltage application unit 10a, and controls the potential difference between the working electrode 1a and the reference electrode 2 to a predetermined value (for example, a value in the range of −1.0 V to 0 V). That is, the power supply 20 applies a voltage to the working electrode 1a so that the voltage applied to the working electrode 1a is −1.0 V to 0 V, with the reference electrode 2 set as a reference (0 V). In this case, the reference electrode 2 is, for example, a silver / silver chloride electrode. As a result, electrons are donated from the working electrode 1a to the oxidized redox protein in the sample solution 9a. As a result, the oxidized redox protein is reduced to a reduced redox protein (step S103). Next, the reduced redox protein reduces and inactivates the allergen in the sample solution 9a (step S104). Steps S103 and S104 are repeated while a voltage is being applied to the electrodes from the power supply 20. In step S103, the redox enzyme, redox molecule, and electron mediator in the sample solution 9a are also reduced from their oxidized forms to their reduced forms.

[0110] Next, the control unit 30 determines whether the process under the set conditions has been completed (step S105). The set conditions are, for example, the duration (time) of voltage application or the number of times voltage is applied (e.g., pulse voltage). If the control unit 30 determines that the process under the set conditions has not been completed (No in step S105), it causes the power source 20 to continue applying voltage (step S106). Then, steps S103 and S104 are repeated until the next determination (step S105) is made. On the other hand, if the control unit 30 determines that the process under the set conditions has been completed (Yes in step S105), it causes the power source 20 to end the voltage application (step S107). This completes the inactivation of allergens in the sample solution 9a.

[0111] Although the above preparation step has been described as being performed by a user, it may also be performed by the allergen deactivation device 100a. In this case, the allergen deactivation device 100a may further include an introduction unit (not shown), a collection unit (not shown), an inlet (not shown), and an outlet (not shown). For example, the introduction unit may introduce an allergen-containing sample, an oxidized-reduction protein, an oxidized-reduction enzyme, an oxidized-reduction molecule, and an electron mediator into the cell 4 through an introduction port provided in the cell 4. Furthermore, for example, the collection unit may collect the sample solution 9a in which the allergen has been inactivated, from an outlet provided in the cell 4 to the outside of the cell 4.

[0112] (Embodiment 2) Next, the second embodiment will be specifically described with reference to Fig. 5 and Fig. 6. In the second embodiment, differences from the first embodiment will be mainly described. Note that the description of the same content as the first embodiment will be simplified or omitted.

[0113] [Allergen deactivation device] [1. Overview] First, an overview of the allergen deactivation apparatus according to the second embodiment will be described with reference to Fig. 5. Fig. 5 is a diagram showing an example of the configuration of an allergen deactivation apparatus 100b according to the second embodiment.

[0114] The allergen deactivation device 100b differs from the first embodiment in that a redox protein that reduces allergens is immobilized on an electrode (here, the working electrode 1b), eliminating the need to prepare a sample solution. The allergen deactivation device 100b donates electrons to the redox protein that inactivates allergens present in a sample by reducing them, thereby repeatedly activating the redox protein that has lost its electrode reducing power, thereby continuously inactivating allergens. In other words, the allergen deactivation device 100b can directly inactivate allergens in a sample. This allows the allergen deactivation device 100b according to the second embodiment to inactivate allergens more simply. Furthermore, the allergen deactivation device 100b can prevent redox proteins and the like from contaminating a sample containing allergens.

[0115] [2. Configuration] Next, the configuration of the allergen deactivation device 100b according to the second embodiment will be described with reference to FIG.

[0116] 5, the allergen deactivation device 100b according to the second embodiment includes a voltage application unit 10b, a power supply 20, and a control unit 30. The allergen deactivation device 100b differs from the device according to the first embodiment in that it includes a working electrode 1b on the surface of which a redox protein is immobilized.

[0117] [Working electrode] The working electrode 1b is an electrode for donating electrons to the redox protein upon application of a voltage. The redox protein is immobilized on the working electrode 1b. Furthermore, in addition to the redox protein, an oxidoreductase that donates electrons to the redox protein may be immobilized on the working electrode 1b, or an oxidoreductase and a redox molecule that supplies electrons to the oxidoreductase may be immobilized on the working electrode 1b, or an oxidoreductase, a redox molecule, and an electron mediator that donates electrons to the redox molecule may be immobilized on the working electrode 1b. Furthermore, an electron mediator may be immobilized on the working electrode 1b in addition to the redox protein. Here, an example will be described in which an oxidoreductase protein, an oxidoreductase, a redox molecule, and an electron mediator are immobilized on the working electrode 1b.

[0118] Fig. 6 is a schematic cross-sectional view of the working electrode 1b shown in Fig. 5 taken along line VI-VI. As shown in Fig. 6, the working electrode 1b includes a base electrode 18, a redox protein 14 immobilized on the base electrode 18, a redox enzyme 15, a redox molecule 16, and an electron mediator 17. The base electrode 18 includes a substrate 11 made of an electrode material, a conductive polymer 12, and conductive particles 13. The redox protein 14, the redox enzyme 15, the redox molecule 16, and the electron mediator 17 (hereinafter also referred to as the redox protein 14) have been described above, and therefore will not be described here.

[0119] The substrate 11 is made of, for example, a porous electrode material. From the viewpoints of strength, rigidity, and lightness, the conductive material is, for example, carbon fiber, and may be, for example, polyacrylonitrile (PAN)-based carbon fiber, pitch-based carbon fiber, or rayon-based carbon fiber. Among these, PAN-based carbon fiber may be used from the viewpoint of the mechanical strength of the porous electrode material. The PAN-based carbon fiber may be, for example, a short-fiber randomly oriented mat formed from Torayca (registered trademark) yarn. The carbon fiber may be one type of carbon fiber or a plurality of different types of carbon fibers. Furthermore, known fibers such as glass fiber, aramid fiber, polyethylene terephthalate fiber, vinylon fiber, polyester fiber, amide fiber, or ceramic fiber may be used in combination with the carbon fiber.

[0120] The conductive polymer 12 is not particularly limited as long as it is a polymeric material having conductivity, and may be, for example, polyacetylene, polythiophene, polyfluorene, polyethylene vinylene, polyphenylene vinylene, polypyrrole, polyaniline, etc. The conductive polymer 12 may contain a dopant.

[0121] The conductive particles 13 are not particularly limited as long as they behave as good electrical conductors. For example, the conductive particles 13 may be conductive polymer particles such as polyacetylene particles, polyaniline particles, polypyrrole particles, polythiophene particles, polyisothianaphthene particles, and polyethylenedioxythiophene particles, carbon particles, carbon fiber particles, or metal particles. Among these, the conductive particles 13 may be carbon particles or metal particles because they exhibit high conductivity and stability.

[0122] Examples of carbon particles include carbon black, expanded graphite, flake graphite, graphite powder, graphite particles, graphene sheets, milled carbon fibers, carbon nanotubes, and carbon nanofibers including vapor grown carbon fibers (VGCF: registered trademark). Among these, carbon black and milled carbon fibers are preferred because they exhibit high conductivity and are inexpensive. Examples of carbon black include furnace black, acetylene black, thermal black, channel black, and Ketjen Black (registered trademark).

[0123] The metal particles are not particularly limited, but when carbon fibers are used as reinforcing fibers, they may be particles of platinum, gold, silver, copper, tin, nickel, titanium, cobalt, zinc, iron, chromium, or aluminum, particles of alloys containing these metals as the main component, tin oxide, indium oxide, or indium tin oxide (ITO), in order to prevent corrosion due to the potential difference with the carbon fibers.

[0124] The shape of the conductive particles 13 is not particularly limited, and may be spherical, non-spherical, or porous. From the viewpoint of forming conductive bridges between carbon fiber layers, it is preferable that the conductive particles 13 have a large aspect ratio.

[0125] The method for immobilizing the redox protein 14, etc. on the surface of the electrode is not particularly limited, and examples thereof include a method of chemically immobilizing the redox protein 14, etc. on the electrode, a method of indirectly immobilizing the redox protein 14, etc. on the electrode using a conductive polymer or a crosslinking agent, or a method of immobilizing the redox protein 14, etc. on the electrode via a monolayer-forming molecule. In the example of Fig. 6, the redox protein 14, etc. is immobilized on the electrode using a conductive polymer.

[0126] [3. Operation] The operation of the allergen deactivation device 100b according to the second embodiment is almost the same as that of the allergen deactivation device 100a according to the first embodiment shown in FIG. 4, and therefore a flow chart of an example of the operation will be omitted. The second embodiment differs from the operation of the allergen deactivation device 100a according to the first embodiment in that it reduces an oxidized redox protein immobilized on the working electrode 1b (specifically, step S103). Furthermore, the preparation step does not require a sample solution preparation step. More specifically, the allergen deactivation device 100b has been found to be a method in which an oxidized redox protein (e.g., oxidized thioredoxin) is repeatedly reduced to a reduced redox protein (e.g., reduced thioredoxin). This has enabled efficient inactivation of allergens using a smaller amount of redox protein than is required to inactivate all allergens.

[0127] [Modification of the second embodiment] In the second embodiment, an example in which at least a redox protein is immobilized on the working electrode 1b has been described. However, a redox protein does not have to be immobilized on the working electrode 1b. For example, it is sufficient that an electron mediator that mediates electron transfer between the working electrode 1b and the redox protein is immobilized on the working electrode. In this case, the sample solution contains an allergen and a redox protein. This improves the electron transfer efficiency compared to when the redox protein receives electrons directly from the working electrode, thereby improving the allergen inactivation efficiency. [Example]

[0128] The allergen inactivation method of the present disclosure will be specifically explained in the following examples, but the present disclosure is not limited to the following examples in any way.

[0129] In the following examples, the decomposition rate (digestibility) of allergens was examined with and without application of voltage to a sample solution containing the allergen in the following cases (1) and (2). In the following examples, an allergenic protein having a disulfide bond (hereinafter simply referred to as the allergenic protein) was used as the allergen.

[0130] (1) When redox proteins are not immobilized on the electrode surface When the redox protein is not immobilized on the electrode surface, the redox protein does not directly receive electrons from the electrode by interfacial electron transfer, but receives electrons indirectly from the electrode via an electron mediator, a redox molecule, or a redox enzyme, etc. Hereinafter, this is referred to as reduction of the redox protein by indirect electron transfer.

[0131] In this case, the redox protein is contained in a sample solution (hereinafter referred to as sample solution I) together with the allergen.

[0132] [Comparative Example 1] In Comparative Example 1, sample solution I was left standing overnight at low temperature without applying a voltage, and then a digestive enzyme was added and the solution was incubated under predetermined conditions.

[0133] (Preparation of sample solution I) Sample solution I was prepared by dissolving an allergen (target molecule), an oxidized redox protein (so-called an inactive redox protein), an oxidized redox enzyme that reduces the oxidized redox protein, a redox molecule that activates the oxidized redox enzyme, and an electron mediator that donates electrons to the oxidized redox molecule in phosphate-buffered saline (PBS) at pH 7.4. The allergen is an allergenic protein having a disulfide bond. The oxidized redox enzyme and redox protein used in Comparative Examples 1 and 2 and Examples 1 and 2 were NADPH-thioredoxin reductase and thioredoxin, respectively, and were prepared based on the description in Patent Document 1. NADPH was used as the redox molecule, and methyl viologen was used as the electron mediator.

[0134] (Inactivation of allergens) After preparing sample solution I, sample solution I was left standing overnight at a low temperature (for example, the temperature inside a refrigerator). No voltage was applied to sample solution I.

[0135] (Breakdown of allergens by digestive enzymes) After allowing sample solution I to stand overnight, digestive enzymes were added to sample solution I and the mixture was incubated at 37°C for 2 hours. Subsequently, the incubated sample solution I and molecular weight markers were subjected to electrophoresis using standard SDS-PAGE (Sodium dodecyl sulfate-Polyaclamide gel electrophoresis). The gel after electrophoresis was stained, and the intensity of the allergenic protein band was quantified by image analysis. Electrophoretic images are shown in Figure 7. Figure 7 shows electrophoretic images of Comparative Example 1 and Example 1 after SDS-PAGE. (a) of Figure 7 is an electrophoretic image of the molecular weight marker. (b) of Figure 7 is an electrophoretic image of Comparative Example 1, in which the allergenic protein band was observed at the position enclosed by the dashed line. Although not shown in Figure 7, a sample solution containing only the allergen (hereinafter referred to as the control sample solution) was subjected to SDS-PAGE electrophoresis as an indicator of 100% residual rate.

[0136] (Calculation of allergen decomposition rate) The values ​​representing the intensity of the bands of the allergenic proteins in the control sample solution and sample solution I of Comparative Example 1 were calculated. Then, the value for the control sample solution was set as 100% allergenic protein residual rate, and the allergenic protein residual rate for Comparative Example 1 was calculated by proportional calculation. As a result, the allergenic protein residual rate for Comparative Example 1 was 100%. Therefore, it was confirmed that the allergenic protein in sample solution I of Comparative Example 1 was not decomposed by the digestive enzyme (i.e., the decomposition rate was 0%). The calculation results of the decomposition rate are shown in Figure 8. Figure 8 is a graph showing the decomposition rates of allergenic proteins after treatment with digestive enzymes in Comparative Example 1 and Example 1.

[0137] [Example 1] In Example 1, a predetermined voltage was applied to sample solution I at low temperature overnight, and then a digestive enzyme was added and the solution was incubated under predetermined conditions. Sample solution I was prepared in the same manner as in Comparative Example 1.

[0138] (Inactivation of allergens) In Example 1, a predetermined voltage was applied to sample solution I overnight at low temperature using a three-electrode voltage application cell (e.g., voltage application unit 10a in FIG. 1) and a potentiostat (e.g., power supply 20 in FIG. 1). Of the three electrodes, a glassy carbon electrode was used as working electrode 1a, and an Ag / AgCl electrode was used as reference electrode 2. The predetermined voltage applied to sample solution I was controlled by the potentiostat so that the potential of working electrode 1a relative to reference electrode 2 was the reduction potential of the electron mediator.

[0139] (Breakdown of allergens by digestive enzymes) Subsequently, sample solution I after voltage application was collected, and a digestive enzyme was added to the collected sample solution I, followed by incubation at 37°C for 2 hours. Sample solution I after incubation and a molecular weight marker were subjected to SDS-PAGE electrophoresis in the same manner as in Comparative Example 1. When the gel after electrophoresis was stained, no bands of allergenic proteins were observed. The electrophoretic image is shown in Figure 7. (c) of Figure 7 is the electrophoretic image of Example 1, in which no bands of allergenic proteins were observed at the position surrounded by the dashed line.

[0140] (Calculation of allergen decomposition rate) As in Comparative Example 1, a numerical value representing the intensity of the band of the allergenic protein in Sample Solution I of Example 1 was calculated. Then, the numerical value representing the intensity of the band in Comparative Example 1 was set as 100% of the residual rate of the allergenic protein, and the residual rate of the allergenic protein in Example 1 was calculated by proportional calculation. As a result, in Example 1, the residual rate of the allergenic protein in Sample Solution I was 0%.

[0141] Next, the decomposition rate of the allergenic protein in Example 1 was calculated by subtracting the residual rate in Example 1 (0%) from the residual rate in Comparative Example 1 (100%). The calculation results of the decomposition rate are shown in Figure 8. As shown in Figure 8, in Example 1, the decomposition rate of the allergenic protein in Sample Solution I was 100%.

[0142] (2) Immobilization of redox proteins on the electrode surface When the redox protein is immobilized on the electrode surface, the redox protein is reduced by indirect electron transfer, as in (1) above.

[0143] In this case, the redox protein is not contained in the sample solution (hereinafter referred to as sample solution II), and sample solution II contains only the allergen as the target molecule.

[0144] Comparative Example 2 In Comparative Example 2, similarly to Comparative Example 1, sample solution II was left to stand overnight at low temperature without applying a voltage, and then a digestive enzyme was added and the solution was incubated under predetermined conditions.

[0145] (Preparation of sample solution) Sample solution II was prepared by dissolving the target molecule, an allergen, in PBS at pH 7.4. The allergen is an allergenic protein containing disulfide bonds.

[0146] (Production of electrodes with immobilized redox proteins) 1. Creating the base electrode substrate 60 mg of conductive carbon black (e.g., Ketjen Black) was ground using an agate pestle. 1800 μL of N-methyl-2-pyrrolidone (NMP) solution was gradually added to the ground conductive carbon black while mixing. 260 μL of a 10% (w / v) NMP solution of poly(4-vinylpyridine) (PVP) as a dispersing aid was gradually added while mixing. 1800 μL of NMP solution was then gradually added to the ground conductive carbon black while mixing. The mixture was transferred to a 25 ml container, and the conductive carbon black was dispersed in the NMP solution by sonication to obtain a carbon slurry. A 1 cm diameter carbon fiber mat (Torayca Mat) was impregnated with the resulting carbon slurry to obtain a prepreg. The prepreg was dried at 90°C for 3 hours to obtain a base electrode substrate.

[0147] 2. Immobilization of redox proteins A redox protein solution containing a redox protein, a redox enzyme, a redox molecule, and an electron mediator was prepared. Next, 25 μl of this redox protein solution was mixed with 4.1 μl of a 20% (w / v) poly-L-lysine solution, 4.4 μl of a 2.5% glutaraldehyde solution, 7.2 μl of a 10 mM Tris-HCl buffer, 1.5 μl of a 50 mg / ml BSA (bovine serum albumin) solution, and 12.8 μl of distilled water to prepare an immobilization solution. After impregnating a base electrode substrate with this immobilization solution, the base electrode substrate was allowed to stand at 4°C for at least 8 hours and dried. This resulted in an electrode (working electrode 1b in Figure 5) in which a redox protein, a redox enzyme, a redox molecule, and an electron mediator were immobilized on the surface of the base electrode substrate. Note that the redox protein immobilized on the electrode was an oxidized (inactive) redox protein.

[0148] (Inactivation of allergens and decomposition of allergens by digestive enzymes) The same procedure as in Comparative Example 1 was carried out, except that sample solution II was used instead of sample solution I. The results of electrophoresis are shown in Figure 9. Figure 9 shows electrophoretic images of Comparative Example 2 and Example 2 after SDS-PAGE. (a) of Figure 9 is an electrophoretic image of molecular weight markers. (d) of Figure 9 is an electrophoretic image of Comparative Example 2, in which a band of allergenic protein was observed at the position surrounded by the dashed line. Although not shown in Figure 9, a sample solution containing only allergens (hereinafter referred to as control sample solution) was subjected to SDS-PAGE electrophoresis as an indicator of 100% residual rate.

[0149] (Calculation of allergen decomposition rate) As in Comparative Example 1, values ​​indicating the intensity of the bands of the allergenic proteins in the control sample solution and sample solution II of Comparative Example 2 were calculated. Then, the value for the control sample solution was set to 100% allergenic protein residual rate, and the allergenic protein residual rate for Comparative Example 2 was calculated by proportional calculation. As a result, the allergenic protein residual rate for Comparative Example 2 was 100%. Therefore, it was confirmed that the allergenic protein in sample solution I) in Comparative Example 2 was not decomposed by the digestive enzyme (i.e., the decomposition rate was 0%). The calculation results of the decomposition rate are shown in Figure 10. Figure 10 is a graph showing the decomposition rate of the allergenic protein after treatment with digestive enzymes.

[0150] [Example 2] Example 2 was carried out in the same manner as Example 1, except that sample solution II was used instead of sample solution I, and an electrode (working electrode 1b in Figure 5) on which a redox protein or the like was immobilized was used.

[0151] (Inactivation of allergens) In Example 2, a three-electrode voltage application cell (e.g., voltage application unit 10b in FIG. 5) and a potentiostat (e.g., power supply 20 in FIG. 5) were used to apply a predetermined voltage to sample solution II at low temperature overnight. Of the three electrodes, the working electrode 1b was an electrode on which the above-mentioned redox protein or the like was immobilized, and the reference electrode was an Ag / AgCl electrode. The predetermined voltage applied to sample solution II was controlled by the potentiostat so that the potential of working electrode 1b relative to reference electrode 2 was the reduction potential of the electron mediator.

[0152] (Breakdown of allergens by digestive enzymes) Subsequently, sample solution II after voltage application was collected, and sample solution II was subjected to enzyme treatment with a digestive enzyme and electrophoresis by SDS-PAGE in the same manner as in Example 1. The electrophoretic images are shown in Figure 9. (e) of Figure 9 is the electrophoretic image of Example 2, in which a band of the allergenic protein was observed at the position surrounded by the dashed line.

[0153] (Calculation of allergen decomposition rate) As in Comparative Example 2, a numerical value representing the intensity of the band of the allergenic protein in sample solution II of Example 2 was calculated. Then, the numerical value representing the intensity of the band in Comparative Example 2 was set as 100% of the residual rate of the allergenic protein, and the residual rate of the allergenic protein in Example 2 was calculated by proportional calculation. As a result, in Example 2, the residual rate of the allergenic protein in sample solution II was 64%.

[0154] Next, the decomposition rate of the allergenic protein in Example 2 was calculated by subtracting the residual rate in Example 2 (64%) from the residual rate in Comparative Example 2 (100%). The calculation results of the decomposition rate are shown in Figure 10. As shown in Figure 10, in Example 2, the decomposition rate of the allergenic protein in sample solution II was 36%.

[0155] (Consideration) The results of Comparative Examples 1 and 2 suggest that, unless a voltage is applied to the sample solution, the oxidized redox protein is not reduced to the reduced redox protein. In other words, unless a voltage is applied to the sample solution, the disulfide bonds of the allergenic protein (hereinafter referred to as "allergen") are not reduced. Therefore, it is thought that the allergen was not decomposed by the digestive enzyme because the sample solution was subjected to enzymatic treatment with the digestive enzyme in a state in which the disulfide bonds of the allergen were not reduced (i.e., not cleaved).

[0156] On the other hand, the results of Examples 1 and 2 suggest that application of a voltage to the sample solution reduces the oxidized redox protein to a reduced redox protein. In other words, application of a voltage to the sample solution suggests that the oxidized redox protein is repeatedly reduced to a reduced redox protein through indirect electron transfer from the electrode. Furthermore, the higher digestion rates by digestive enzymes in Examples 1 and 2 compared to Comparative Examples 1 and 2 suggest that application of a voltage to the sample solution repeatedly reduces the oxidized redox protein through the above mechanism, and the reduced redox protein may continuously cleave the disulfide bonds of the allergen. Therefore, according to the allergen inactivation method of the present disclosure, application of a voltage to the sample solution is thought to donate electrons from the electrode to the oxidized redox protein, reducing the oxidized redox protein to a reduced redox protein. As a result, according to the allergen inactivation method of the present disclosure, the oxidized redox protein in the sample solution is repeatedly reduced to the reduced redox protein, thereby enabling efficient inactivation of allergens present in the sample solution.

[0157] Furthermore, based on the results of Examples 1 and 2, even when inactivation treatment was performed under the same voltage application conditions, the decomposition rate in Example 2 was lower, likely because the amount of redox protein immobilized on the electrode in Example 2 was smaller than the amount of redox protein added to sample solution I in Example 1. Furthermore, when the redox protein is immobilized on the electrode, electron transfer occurs near the electrode interface, making it difficult for allergens to be reduced unless they approach the electrode interface. Although no data are shown, an investigation of the stirring conditions for the sample solution revealed that constant stirring reduced the decomposition rate of allergens, indicating the need to consider the balance between inactivation of allergens near the electrode interface and reduction of the oxidized redox protein.

[0158] Furthermore, the improvement in the decomposition rate of allergenic proteins by digestive enzymes is thought to be due to the reduction of disulfide bonds in allergenic proteins in the sample solution by reduced redox proteins, making it easier for digestive enzymes to act on the allergenic proteins. More specifically, the improvement in the decomposition rate of allergenic proteins by digestive enzymes is thought to be due to the increased degree of freedom (i.e., fluctuation) of the three-dimensional structure of the allergenic proteins, which is caused by the reduction and cleavage of disulfide bonds connecting the secondary structures of the allergenic proteins.

[0159] Furthermore, as the three-dimensional conformation of allergenic proteins becomes more unstable, it becomes more difficult for IgE antibodies to identify the specific binding site of the allergenic protein, which is thought to weaken the allergenicity of the allergenic protein.

[0160] Therefore, according to the allergen inactivation method of the present disclosure, by applying a voltage to a sample solution, the redox protein can be repeatedly activated (i.e., reduced), and it is believed that the disulfide bonds of the allergen can be efficiently reduced with a small amount of the redox protein. Furthermore, according to the allergen inactivation method of the present disclosure, the disulfide bonds of the allergen can be efficiently reduced (cleaved), which increases the fluctuation in the three-dimensional structure of the allergenic protein, and therefore not only reduces the allergenicity of the allergenic protein but also improves its decomposition by digestive enzymes. Therefore, it is believed that the allergen inactivation method of the present disclosure can efficiently inactivate allergens.

[0161] (3) When the number of components in the sample solution is changed without fixing the electrode Next, allergen inactivation was performed by changing the number of components contained in the sample solution.

[0162] [Example 3] Example 3 was performed in the same manner as Example 1, except that sample solution III containing an electron mediator, an oxidoreductase, and an oxidoreductase protein was used instead of sample solution I. The oxidoreductase and oxidoreductase protein used in Example 3 were ferredoxin-thioredoxin reductase and thioredoxin, respectively, and were prepared according to the method described in Non-Patent Document 1 (Keisuke Yoshida et al., "Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts," Biochemical Journal, Portland Press, 2017, Vol. 474 (Pt. 8), pp. 1347-1360). The electrophoresis results are shown in Figure 11. (a) and (d) of Figure 11 show molecular weight markers, and (e) of Figure 11 shows a control sample (untreated). As shown in (b) of Figure 11, in Example 3, the residual rate of allergenic protein in sample solution III was 55%.

[0163] Next, the decomposition rate of the allergenic protein in Example 3 was calculated. The calculation results of the decomposition rate are shown in Figure 12. As shown in Figure 12(b), in Example 3, the decomposition rate of the allergenic protein in sample solution III was 45%.

[0164] [Example 4] Example 4 was carried out in the same manner as Example 1, except that sample solution IV containing an electron mediator and a fusion protein of an oxidoreductase and an oxidoreductase protein was used instead of sample solution I. A fusion protein of thioredoxin reductase and thioredoxin was prepared and used as the fusion protein. The results of electrophoresis are shown in Figure 11. As shown in Figure 11(c), the residual rate of the allergenic protein in sample solution IV was 42%.

[0165] Next, the decomposition rate of the allergenic protein in Example 4 was calculated. The calculation results of the decomposition rate are shown in Figure 12. As shown in Figure 12(c), in Example 4, the decomposition rate of the allergenic protein in sample solution IV was 58%.

[0166] (Consideration) The results of Examples 3 and 4 show that the residual rate of allergenic proteins was reduced to approximately 50% whether the sample solution contained three molecules other than the redox molecule (electron mediator, redox enzyme, and redox protein) or two molecules (electron mediator and redox enzyme-redox protein complex). This confirmed that the allergen inactivation effect was achieved regardless of the number of components involved in electron transfer.

[0167] The allergen deactivation method and allergen deactivation device according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications that a person skilled in the art could conceive of to the embodiments and other forms constructed by combining some of the components of the embodiments are also included within the scope of the present disclosure.

[0168] In the above embodiment, an example has been described in which allergens are efficiently inactivated using electrical energy by controlling the voltage application, but this is not limiting. For example, in embodiment 2, the sample 9b may be stirred to increase the reactivity between the redox protein immobilized on the working electrode 1b and the allergens in the sample 9b. That is, the control unit 30 of the allergen deactivation device 100b may derive stirring conditions such as the stirring speed, stirring time, and stirring interval in addition to the voltage application conditions, and output control signals related to voltage control and stirring control. In this case, the allergen deactivation device 100b may include a stirring unit that stirs the sample 9b in the cell 4 of the voltage application unit 10b. The stirring unit may include a stirring blade detachably attached to the lid 5, a motor that rotates the stirring blade, and a control unit that controls the movement of the motor. [Industrial Applicability]

[0169] According to the present disclosure, it is possible to provide an allergen inactivation method that can efficiently inactivate allergens derived from foods, drugs, pollen, animals, etc., and an apparatus that carries out the method. [Explanation of symbols]

[0170] 1a, 1b working electrode 2 Reference pole 3. Opposite 4 cells 5 Lid 6a, 6b, 6c terminals 7a, 7b, 7c leads 9a Sample solution 9b Sample 10a, 10b Voltage application section 11 Circuit Board 12 Conductive polymers 13 Conductive particles 14 Redox Proteins 15 Oxidoreductase 16 Redox Molecules 17 Electron Mediator 18 Base Electrode 20 Power supply 21 Acquisition Department 22 Information Processing Department 23 Voltage control section 24 Output section 30 Control Unit 31 Acquisition Department 32 Information Processing Department 33 Storage section 34 Output section 100a, 100b Allergen deactivation device

Claims

1. an inactivation step in which allergens present in the reaction system are inactivated by reduction using a reduced redox protein; a reduction step of reducing the oxidized redox protein to the reduced redox protein by donating electrons from an electrode connected to an external power source outside the reaction system to the oxidized redox protein produced by oxidation of the reduced redox protein in the inactivation step. Methods for inactivating allergens.

2. In the reduction step, electrons are donated from the electrode to the oxidoreductase, and electrons are donated from the oxidized oxidoreductase to the oxidized oxidized-form redox protein. The method for inactivating an allergen according to claim 1.

3. In the reduction step, electrons are donated from the electrode to the redox molecule, electrons are donated from the redox molecule to the redox enzyme, and electrons are donated from the redox enzyme to the oxidized redox protein. The method for inactivating an allergen according to claim 1.

4. In the reduction step, electrons are donated from the electrode to the electron mediator, electrons are donated from the electron mediator to the redox molecule, electrons are donated from the redox molecule to the oxidoreductase, and electrons are donated from the oxidoreductase to the oxidized redox protein. The method for inactivating an allergen according to claim 1.

5. The allergen has a disulfide bond. A method for inactivating an allergen according to any one of claims 1 to 4.

6. The redox protein is any one of thioredoxin, glutathione, a protein having at least one thioredoxin-like domain, and a protein having at least one glutathione-like motif. A method for inactivating an allergen according to any one of claims 1 to 5.

7. The oxidoreductase is either (i) an enzyme that catalyzes the reduction of oxidized thioredoxin, including NADPH (nicotinamide adenine dinucleotide phosphate)-thioredoxin reductase or ferredoxin-thioredoxin reductase, or (ii) an enzyme that catalyzes the reduction of oxidized glutathione, including glutathione reductase. The method for inactivating an allergen according to any one of claims 2 to 4.

8. The redox molecule is either nicotinamide adenine dinucleotide phosphate or ferredoxin. A method for inactivating an allergen according to claim 3 or 4.

9. the electron mediator is a compound having a bipyridine skeleton; The method for inactivating an allergen according to claim 4.

10. The reaction temperature in the reaction system is 4°C or higher and lower than 60°C. A method for inactivating an allergen according to any one of claims 1 to 9.

11. the voltage applied to the electrodes by the external power supply is −1.0 V or more and 0 V or less; A method for inactivating an allergen according to any one of claims 1 to 10.

12. An apparatus used in the method for inactivating an allergen according to any one of claims 1 to 11, an electrode for donating electrons to a redox protein that inactivates allergens by reducing them when a voltage is applied; a power source that applies a voltage to the electrodes; a control unit that controls the application of voltage to the power source; Equipped with Allergen deactivation device.

13. The redox protein is immobilized on the electrode. The allergen deactivation device according to claim 12.

14. Furthermore, an oxidoreductase that donates electrons to the oxidative reduction protein is immobilized on the electrode. The allergen deactivation device according to claim 12.

15. Furthermore, a redox molecule that donates electrons to the redox enzyme is immobilized on the electrode. The allergen deactivation device according to claim 14.

16. Furthermore, an electron mediator that donates electrons to the redox molecule is immobilized on the electrode. The allergen deactivation device according to claim 15.

17. an electron mediator that mediates electron transfer between the electrode and the redox protein is immobilized on the electrode; The allergen deactivation device according to claim 12 or 13.

Images