Reaction apparatus and method for producing product

Abstract

The present disclosure provides a technique that is advantageous from the viewpoint of suppressing the denaturation of an enzyme while continuously activating a coenzyme. This reaction apparatus (100) is provided with an enzyme (9), a working electrode (1), and a voltage application device (20). The enzyme (9) exhibits the activity thereof by the action of a coenzyme. The working electrode (1) electrochemically activates the coenzyme. The voltage application device (20) applies a voltage to the working electrode (1) to cause the transfer of electrons between the coenzyme and the working electrode (1). The enzyme (9) oxidizes or reduces a substrate through an enzymatic reaction in which the enzyme (9) is involved. In the reaction apparatus (100), when a voltage is applied to the working electrode (1), a non-contact state between the enzyme (9) and the working electrode (1) is maintained, and a contact state between the coenzyme and the working electrode (1) is maintained.

Description

Reactor and method for producing product

[0001] The present disclosure relates to a reactor and a method for producing a product.

[0002] Enzymes have traditionally been known as catalysts for chemical reactions in living organisms, acting only on specific reactant substrates and exhibiting catalytic activity in the production of specific products.

[0003] An example of an enzymatic reaction is an enzymatic reaction using glucose dehydrogenase. This enzymatic reaction requires a substrate, glucose, and a coenzyme, nicotinamide adenine dinucleotide (NAD). In this enzymatic reaction, the substrate is converted into gluconolactone, and the coenzyme is converted into reduced nicotinamide adenine dinucleotide (NADH).

[0004] For example, Patent Document 1 describes an electrochemical reaction device that contains glucose dehydrogenase and NAD and includes a first tank in which a working electrode is placed, and a second tank in which a counter electrode is placed. In this electrochemical reaction device, glucose dehydrogenase functions as a catalyst, and NAD participates as a coenzyme to cause an enzyme reaction. In addition, when a voltage is applied between the working electrode and the counter electrode, NADH produced by the enzyme reaction is oxidized to produce oxidized nicotinamide adenine dinucleotide (NADH). + ) is generated.

[0005] Patent Document 2 describes a method for inactivating allergens. This method includes an inactivation step and a reduction step. The inactivation step is a step in which allergens present in a reaction system are inactivated by reduction with a reduced redox protein. The reduction step is a step in which an oxidized redox protein is reduced to a reduced redox protein. In the inactivation step, the reduced redox protein is oxidized to generate an oxidized redox protein. The oxidized redox protein is reduced to a reduced redox protein by donating electrons to the redox protein from an electrode connected to an external power source.

[0006] International Publication No. 2023 / 074453 International Publication No. 2021 / 261511

[0007] The techniques described in the above patent documents need to be reconsidered from the viewpoint of continuously activating a coenzyme while suppressing enzyme denaturation. Therefore, the present disclosure provides a technique that is advantageous from the viewpoint of continuously activating a coenzyme while suppressing enzyme denaturation.

[0008] The present disclosure provides a reaction device comprising: a working electrode that electrochemically activates a coenzyme; and a voltage application device that applies a voltage to the working electrode to cause electrons to be exchanged between the coenzyme and the working electrode, wherein a substrate is oxidized or reduced by an enzymatic reaction involving an enzyme whose activity is expressed by the coenzyme, and wherein, when the voltage is applied to the working electrode, a non-contact state is maintained between the enzyme and the working electrode, and a contact state is maintained between the coenzyme and the working electrode.

[0009] The reaction apparatus of the present disclosure is advantageous from the viewpoint of continuously activating the coenzyme while suppressing the denaturation of the enzyme.

[0010] FIG. 1 is a schematic diagram of an example of a reaction apparatus according to an embodiment. FIG. 2 is a functional block diagram of an example of a reaction apparatus according to an embodiment. FIG. 3A is a schematic diagram of an example of an enzyme reaction mechanism. FIG. 3B is a schematic diagram of another example of an enzyme reaction mechanism. FIG. 4 is a graph showing the relationship between the current value of the working electrode and the voltage application time during product production. FIG. 5 is a flowchart showing an example of a method for producing a product according to an embodiment. FIG. 6 is a graph showing the results of enzyme activity evaluation in Example 1. FIG. 7 is a graph showing the results of enzyme activity evaluation in Comparative Example 1. FIG. 8 is a polyacrylamide gel electrophoresis (SDS-PAGE) image of samples according to Example 1, Comparative Example 2, and Reference Example 3. FIG. 9 is a graph showing the change in fructose concentration in Example 1 and Comparative Example 1.

[0011] (Findings that Form the Basis of the Present Disclosure) As described in Patent Document 1, the coenzyme can be regenerated by applying a voltage between the working electrode and the counter electrode, and the coenzyme can be repeatedly used for the enzymatic reaction. On the other hand, according to the investigations of the present inventors, it has been newly found that when an enzyme comes into contact with the working electrode to which a voltage is applied, the enzyme is denatured, and there is a possibility that the enzyme will no longer exhibit the desired catalytic action.

[0012] Therefore, the present inventors have conducted extensive research into the configuration of a reaction device that is advantageous from the viewpoint of continuously activating a coenzyme while suppressing enzyme denaturation. As a result, it has been newly discovered that by keeping the working electrode and the enzyme in a predetermined state when a voltage is applied to the working electrode, it is possible to continuously activate a coenzyme while suppressing enzyme denaturation. Based on this new finding, the present inventors have completed the reaction device of the present disclosure. In this disclosure, "continuously" means "repeatedly."

[0013] (Embodiments of the Present Disclosure) Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, the arrangement and connection of the components, steps, and the order of steps shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, each drawing is not necessarily an exact illustration. In each drawing, substantially identical configurations are assigned the same reference numerals, and redundant explanations may be omitted or simplified. Furthermore, below, terms indicating the relationship between elements, such as "parallel" and "perpendicular," terms indicating the shape of elements, such as "rectangular," and numerical ranges do not only indicate the strict meaning, but also include substantially equivalent ranges, for example, differences of a few percent. These embodiments shown as comprehensive or specific examples may be realized using a system, an apparatus, an integrated circuit, a computer program, or a computer-readable recording medium such as a CD-ROM, or may be realized using any combination of a system, a method, an integrated circuit, a computer program, and a recording medium.

[0014] (Embodiment) Hereinafter, an embodiment will be specifically described with reference to FIGS. 1 to 5. FIG.

[0015] FIG. 1 is a schematic diagram illustrating an example of a reaction apparatus according to an embodiment. As shown in FIG. 1, the reaction apparatus 100 includes a working electrode 1 and a voltage application device 20. The working electrode 1 is an electrode for electrochemically activating a coenzyme present in a liquid supplied to the reaction apparatus 100. The voltage application device 20 applies a voltage to the working electrode 1, causing electrons to be exchanged between the coenzyme and the working electrode 1. This electrochemically activates the coenzyme. The reaction apparatus 100 oxidizes or reduces a substrate through an enzymatic reaction involving an enzyme 9 whose activity is expressed by the coenzyme. In the reaction apparatus 100, when a voltage is applied to the working electrode 1, a non-contact state between the enzyme 9 and the working electrode 1 is maintained, and a contact state between the coenzyme and the working electrode 1 is maintained.

[0016] The reaction device 100 can provide a method for producing a product, which includes, for example, the following (I) and (II): (I) Applying a voltage to the working electrode 1 causes electron transfer between the coenzyme and the working electrode 1, thereby electrochemically activating the coenzyme; (II) The coenzyme activates the enzyme 9, and an enzyme reaction involving the enzyme 9 oxidizes or reduces the substrate to produce a product.

[0017] The coenzyme is present in the liquid supplied to the reaction device 100, and therefore is maintained in contact with the working electrode 1. In the reaction device 100, the coenzyme that has lost its activity due to the enzymatic reaction involving the enzyme 9 is activated by applying a voltage to the working electrode 1 by the voltage application device 20. This allows the coenzyme to be continuously activated. Furthermore, since a non-contact state between the enzyme 9 and the working electrode 1 is maintained when a voltage is applied to the working electrode 1, it is possible to prevent the enzyme 9 from coming into contact with the working electrode 1 to which a voltage is applied and causing the enzyme 9 to denature. As a result, the enzyme 9 can exhibit the desired catalytic activity for a long period of time.

[0018] In the reaction device 100, for example, a liquid containing a coenzyme and a substrate is supplied so as to come into contact with the working electrode 1. In addition to the coenzyme and the substrate, this liquid may contain a solvent such as water, a buffer such as Good's buffer, a salt such as sodium chloride, or a pH adjuster such as hydrochloric acid.

[0019] Enzyme 9 is not limited to a specific enzyme as long as its activity is expressed by a coenzyme and a substrate is oxidized or reduced by an enzymatic reaction involving enzyme 9. Enzyme 9 may be, for example, an enzyme that catalyzes an enzymatic reaction involving sugars such as fructose and glucose as substrates. Enzyme 9 may also be an enzyme that catalyzes an enzymatic reaction involving proteins such as ovomucoid contained in egg white and casein contained in dairy products as substrates.

[0020] FIG. 3A is a schematic diagram showing an example of the mechanism of an enzyme reaction. As shown in FIG. 3A, the enzyme 9 is activated by, for example, a reduced coenzyme, and a product is produced from the substrate by the enzyme reaction. In this case, the enzyme 9 reduces the substrate. As the enzyme reaction progresses, the reduced coenzyme is oxidized and converted into an oxidized coenzyme. The oxidized coenzyme receives electrons from the working electrode 1, and the reduced coenzyme is regenerated. Therefore, as long as the substrate is present, the enzyme reaction occurs continuously. For example, if the substrate is fructose and the enzyme 9 is mannitol dehydrogenase, mannitol can be produced from fructose by this mechanism. The fructose is, for example, D-fructose.

[0021] FIG. 3B is a diagram schematically illustrating another example of the mechanism of an enzyme reaction. As shown in FIG. 3B, the enzyme 9 is activated by, for example, an oxidized coenzyme, and a product is produced from the substrate by the enzyme reaction. In this case, the enzyme 9 oxidizes the substrate. As the enzyme reaction progresses, the oxidized coenzyme is reduced and converted into a reduced coenzyme. The reduced coenzyme donates electrons to the working electrode 1, and the oxidized coenzyme is regenerated. Therefore, the enzyme reaction occurs continuously. For example, when the substrate is D-glucose and the enzyme 9 is glucose dehydrogenase, D-glucono-1,5-lactone can be produced from D-glucose by this mechanism.

[0022] For example, a voltage is applied to the working electrode 1 for a predetermined period of time. For example, the period during which the voltage is applied to the working electrode 1 is adjusted depending on the initial concentration of the substrate in the liquid supplied to the reaction device 100. For example, the period during which the voltage is applied to the working electrode 1 becomes longer as the amount of liquid supplied to the reaction device 100 increases or the initial concentration of the substrate in this liquid increases.

[0023] In the reaction device 100, for example, a voltage may be applied to the working electrode 1 until the current value at the working electrode 1 becomes equal to or less than a predetermined value Ie. FIG. 4 is a graph showing the relationship between the current value at the working electrode 1 and the voltage application time during product production. As shown in FIG. 4, the current value at the working electrode 1 decreases as the voltage application time increases. When products are continuously produced from the substrate and the concentration of the substrate contained in the liquid supplied to the reaction device 100 decreases, the enzyme reaction becomes less likely to occur and the activation of the coenzyme also becomes less likely. Therefore, the amount of electron transfer between the working electrode 1 and the coenzyme decreases, and the current value at the working electrode 1 decreases. In other words, a small current value at the working electrode 1 suggests that the concentration of the substrate in the liquid supplied to the reaction device 100 has decreased.

[0024] The coenzyme is not limited to a specific coenzyme as long as it can exert the activity of enzyme 9. The coenzyme can be, for example, nicotinamide adenine dinucleotide (NADH or NAD + In this case, for example, as mannitol is produced from fructose by mannitol dehydrogenase, reduced NADH is oxidized, and oxidized NAD + In addition, for example, when D-glucono-1,5-lactone is produced from D-glucose by glucose dehydrogenase, oxidized NAD + is reduced to produce reduced NADH. The coenzyme is nicotinamide adenine dinucleotide phosphate (NADPH or NADP) + ) may also be used.

[0025] The reaction device 100 includes, for example, an enzyme 9. The enzyme 9 can be arranged in the reaction device 100 so that its activity is expressed by a coenzyme.

[0026] 1 , the reaction device 100 further includes, for example, a carrier 9c. The carrier 9c has an enzyme 9 immobilized thereon. In other words, the enzyme 9 is supported on the carrier 9c. With this configuration, it is easier to design a system for maintaining a non-contact state between the enzyme 9 and the working electrode 1 when a voltage is applied to the working electrode 1, compared to when the enzyme 9 is present alone.

[0027] The carrier 9c is not limited to a specific carrier as long as it can immobilize the enzyme 9. The carrier 9c is, for example, particulate. In this case, the surface area of ​​the enzyme 9 is likely to be large. The carrier 9c contains, for example, a carbon material, an organic polymer, a metal material, a gel, beads, porous glass, an inorganic oxide, or a polysaccharide. In this case, the activity of the enzyme 9 is likely to be maintained at a high level. An example of an organic polymer is an ion exchange resin. The carrier 9c may be flat, rod-shaped, or fibrous.

[0028] As shown in FIG. 1 , the reaction device 100 further includes, for example, a membrane 9m. The membrane 9m allows the coenzyme and substrate to pass through, while preventing the enzyme 9 from passing through. In the reaction device 100, the enzyme 9 is disposed in a space separated from the working electrode 1 by the membrane 9m. With this configuration, the coenzyme and substrate contained in the liquid supplied to the reaction device 100 pass through the membrane 9m and are introduced into the space separated from the working electrode 1, where an enzymatic reaction involving the enzyme 9 occurs. In addition, the enzyme 9 can be prevented from being introduced outside the space separated from the working electrode 1. Therefore, a non-contact state between the enzyme 9 and the working electrode 1 is maintained when a voltage is applied to the working electrode 1.

[0029] The membrane 9m is not limited to a specific membrane 9m as long as it allows the coenzyme and substrate to pass through and prevents the enzyme 9 from passing through. The membrane 9m is, for example, an electrically insulating membrane. In this case, the enzyme 9 is less susceptible to the effect of voltage application to the working electrode 1. The membrane 9m may be, for example, a woven fabric, a nonwoven fabric, or a net.

[0030] The membrane 9m is, for example, in the form of a bag, and the enzyme 9 is contained inside this bag. With this configuration, a non-contact state between the enzyme 9 and the working electrode 1 can be maintained with a simple configuration when a voltage is applied to the working electrode 1. The bag may be composed of only the membrane 9m, or a part of the bag may be composed of a material other than the membrane 9m. The membrane 9m may not form a bag, and may be fixed to a wall surface in contact with a space in the reaction device 100 to which a liquid containing a coenzyme and a substrate is supplied.

[0031] 1 , the reaction apparatus 100 further includes, for example, a reference electrode 2, a cell 4, a lid 5, terminals 6a, 6b, and 6c, leads 7a, 7b, and 7c, a stirrer 8, a control device 30, and a stirrer 40. The working electrode 1, the reference electrode 2, the counter electrode 3, the cell 4, the lid 5, the terminals 6a, 6b, and 6c, and the leads 7a, 7b, and 7c constitute an electrochemical reactor 10, which is a three-electrode cell. The electrochemical reactor 10 may also be configured as a two-electrode cell that includes the working electrode 1 and the counter electrode 3 and omits the reference electrode 2.

[0032] The cell 4 has, for example, a first space 4a and a second space 4b therein. A working electrode 1 and a reference electrode 2 are disposed in the first space 4a, and a counter electrode 3 is disposed in the second space 4b. The cell 4 has, for example, a connection portion 4c. The connection portion 4c connects the first space 4a and the second space 4b, and a separator 4s is disposed in the connection portion 4c. The separator 4s separates the space on the first space 4a side of the connection portion 4c from the space on the second space 4b side.

[0033] An enzyme 9 is disposed in the first space 4a. A liquid containing a coenzyme and a substrate can be supplied to the first space 4a. This allows the liquid to come into contact with the working electrode 1. An electrolyte solution such as a phosphate buffer solution can be accommodated in the second space 4b. This allows the electrolyte solution to come into contact with the counter electrode 3.

[0034] The separator 4s has, for example, ion conductivity and blocks the permeation of some of the components contained in the liquid supplied to the first space 4a and the electrolyte solution contained in the second space 4b. For example, the separator 4s blocks the permeation of the substrate and coenzyme contained in the liquid supplied to the first space 4a. The separator 4s has, for example, proton conductivity. The separator 4s includes, for example, a polymer having a perfluoro side chain containing a sulfonic acid group.

[0035] The surfaces of the working electrode 1 and the counter electrode 3 include, for example, a conductive material. The conductive material is, for example, a material that does not decompose when the conductive material is oxidized. Examples of conductive materials include carbon materials, conductive polymers, semiconductor materials, and metal materials. 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 polymers include polyaniline, polyacetylene, polypyrrole, poly(3,4-ethylenedioxythiophene), poly(p-phenylene vinylene), polythiophene, and poly(p-phenylene sulfide). Examples of semiconductor materials include silicon, germanium, indium tin oxide (ITO), titanium oxide, copper oxide, and silver oxide. Examples of metal materials include gold, platinum, silver, titanium, aluminum, tungsten, copper, iron, and palladium. The working electrode 1 may be a glassy carbon electrode, and the counter electrode 3 may be a platinum electrode.

[0036] The surface area of ​​the counter electrode 3 is, for example, larger than the surface area of ​​the working electrode 1 .

[0037] In the reaction device 100, the working electrode 1 may function as a cathode electrode and the counter electrode 3 may function as an anode electrode, or the working electrode 1 may function as an anode electrode and the counter electrode 3 may function as a cathode electrode.

[0038] The reference electrode 2 does not react with components contained in the liquid supplied to the first space 4 a and is maintained at a specific potential. The reference electrode 2 is, for example, a silver / silver chloride electrode. The reference electrode 2 can maintain a constant potential difference between the working electrode 1 and the counter electrode 3.

[0039] In the electrochemical reactor 10, the cell 4 is closed by, for example, a lid 5. Terminals 6a, 6b, and 6c are attached to the lid 5. A lead 7a extends from the terminal 6a and electrically connects the terminal 6a to the working electrode 1. A lead 7b extends from the terminal 6b and electrically connects the terminal 6b to the reference electrode 2. A lead 7c extends from the terminal 6c and electrically connects the terminal 6c to the counter electrode 3. The terminals 6a, 6b, and 6c are electrically connected to a voltage application device 20.

[0040] The voltage application device 20 applies a voltage between the working electrode 1 and the counter electrode 3 in accordance with, for example, a control signal output from the control device 30, and adjusts the potential difference between the working electrode 1 and the reference electrode 2 to a predetermined value. When the working electrode 1 functions as a cathode electrode, the voltage application device 20 applies a voltage to the working electrode 1 so that the potential at the working electrode 1 is between −1.0 V and 0 V, for example, with the potential of the reference electrode 2 set as a reference potential (0 V).

[0041] The control device 30 performs information processing to control the application of voltage by the voltage application device 20 and the movement of the motor (not shown) of the agitation device 40. The control device 30 includes, for example, a memory that stores a program for controlling the application of voltage by the voltage application device 20 and the movement of the motor of the agitation device 40, and an arithmetic device such as a processor that reads out the program and performs predetermined calculations.

[0042] In the stirring device 40, the operation of the motor is controlled in accordance with the control signal output from the control device 30. This adjusts the rotation speed and rotation time of the stirring bars 8 arranged in the first space 4 a and the second space 4 b, respectively.

[0043] Fig. 2 is a functional block diagram showing the reaction device 100. As shown in Fig. 2, the voltage application device 20 includes, for example, an acquisition unit 21, an information processing unit 22, and a voltage control unit 23.

[0044] The acquiring unit 21 acquires the 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 electrochemical reactor 10a and the value of the current flowing through the liquid inside the cell 4.

[0045] The information processing unit 22 processes the information acquired by the acquisition unit 21. For example, when the information processing unit 22 acquires a control signal from the acquisition 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 electrochemical reactor 10, the information processing unit 22 acquires measurement data acquired from the acquisition unit 21, such as the potential of each electrode of the electrochemical reactor 10 and the value of the current flowing through the liquid inside the cell 4. Based on the acquired data, the information processing unit 22 calculates a voltage to be applied to the working electrode 1 so as to maintain the potential difference between the working electrode 1 and the reference electrode 2 at a predetermined value. Then, it outputs a control signal to the voltage control unit 23 to adjust the voltage of the working electrode 1 to the calculated voltage.

[0046] The voltage control unit 23 applies a voltage to each electrode of the electrochemical reactor 10 based on the control signal output from the information processing unit 22 .

[0047] As shown in FIG. 1, the voltage application device 20 and the control device 30 may be separate entities, or the voltage application device 20 and the control device 30 may be integrally configured.

[0048] The control device 30 includes, for example, an acquisition unit 31, an information processing unit 32, a storage unit 33, and an output unit 34.

[0049] The acquisition unit 31 acquires, for example, instruction information, which is information relating to an instruction input by a user, and outputs the acquired instruction information to the information processing unit 32 .

[0050] The information processing unit 32 determines the conditions for applying a voltage to each electrode of the electrochemical reactor 10, for example, based on the instruction information acquired by the acquisition unit 31. The instruction information includes, for example, the type of enzyme 9, the amount of liquid supplied to the first space 4a, the completion time of the enzyme reaction, the completion time of the enzyme reaction, etc. The instruction information may include the ratio of the target concentration of the substrate to the initial concentration of the substrate in the liquid supplied to the first space 4a, or information corresponding to that ratio.

[0051] The information processing unit 32 may output to the output unit 34 a control signal that controls voltage application under conditions determined 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.

[0052] The output unit 34 outputs the control signal obtained from the information processing unit 32 to the voltage application device 20 .

[0053] 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 voltage application device 20), and the like.

[0054] 5 is a flowchart illustrating an example of a method for producing a product. For example, a liquid containing a substrate and a coenzyme is supplied to the first space 4a, and an electrolyte solution is accommodated in the second space 4b. Next, in step S101, instruction information is input to the reaction device 100. The instruction information includes, for example, the amount of the liquid containing the substrate and the coenzyme, the time required for the enzyme reaction to be completed, the time at which the enzyme reaction is completed, the ratio of the target concentration of the substrate to the initial concentration of the substrate in the liquid supplied to the first space 4a, or information corresponding to that ratio.

[0055] Next, in step S102, a condition for terminating the production of the product is set based on the instruction information. This condition is, for example, that a predetermined time has elapsed since the start of voltage application or that the current at the working electrode 1 has become equal to or less than a predetermined value Ie. In step S102, conditions related to a target value for the potential of the working electrode 1, a target value for the voltage between the working electrode 1 and the counter electrode 3, and the rotation speed of the stirring device 40 can be set.

[0056] Next, in step S103, voltage application is started. For example, the control device 30 generates a control signal in accordance with a target value of the potential of the working electrode 1 and a target value of the voltage between the working electrode 1 and the counter electrode 3. The voltage application device 20 applies a voltage between the working electrode 1 and the counter electrode 3 in accordance with the control signal acquired from the control device 30.

[0057] Next, in step S104, the coenzyme is activated, an enzymatic reaction involving the enzyme 9 occurs, and a product is produced from the substrate.

[0058] Next, the process proceeds to step S105, where it is determined whether the conditions for terminating the production of the product, which were set in step S102, are met. If the result of the determination in step S105 is negative, the process proceeds to step S107, where the application of voltage between the working electrode 1 and the counter electrode 3 is continued, and steps S104 and S105 are repeated.

[0059] If the determination in step S105 is positive, the process proceeds to step S106, where the voltage application by the voltage application device 20 is terminated. Thereafter, the liquid containing the product is collected from the first space 4a, and the series of processes is completed.

[0060] (Other Embodiments) The reaction apparatus and the method for producing a product have been described based on the embodiments. However, 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 can make to the embodiments and other embodiments formed by combining some of the components of the embodiments are included in the scope of the present disclosure.

[0061] (Additional Note) From the above description, the following techniques are disclosed.

[0062] (Technology 1) A reaction device comprising: a working electrode that electrochemically activates a coenzyme; and a voltage application device that applies a voltage to the working electrode to cause electrons to be exchanged between the coenzyme and the working electrode, wherein a substrate is oxidized or reduced by an enzymatic reaction involving an enzyme whose activity is expressed by the coenzyme, and wherein, when the voltage is applied to the working electrode, a non-contact state between the enzyme and the working electrode is maintained, and a contact state between the coenzyme and the working electrode is maintained.

[0063] (Technology 2) The reaction device according to Technology 1, further comprising the enzyme.

[0064] (Technology 3) The reaction device according to Technology 2, further comprising a carrier on which the enzyme is immobilized.

[0065] (Technology 4) The reaction device according to Technology 2 or 3, further comprising a membrane that allows permeation of the coenzyme and the substrate and blocks permeation of the enzyme, wherein the enzyme is disposed in a space separated from the working electrode by the membrane.

[0066] (Technology 5) The reaction device according to Technology 4, wherein the membrane forms a bag, and the enzyme is contained inside the bag.

[0067] (Technology 6) A method for producing a product, comprising: electrochemically activating the coenzyme by applying a voltage to a working electrode to cause electron transfer between the coenzyme and the working electrode; and causing an enzyme to become active by the coenzyme, and oxidizing or reducing a substrate through an enzymatic reaction involving the enzyme to produce a product, wherein a non-contact state between the enzyme and the working electrode is maintained when the voltage is applied to the working electrode.

[0068] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0069] Example 1 An apparatus corresponding to the reactor 100 shown in FIG. 1 was prepared. A gold film was formed on a rectangular glass plate having a short side length of 10 mm and a long side length of 20 mm in plan view, and platinum was plated on it. A working electrode was thus obtained. A platinum wire with a wire diameter of 0.5 mm and a length of 23 cm was wound into a coil and used as a counter electrode. A silver / silver chloride (Ag / AgCl) electrode was used as a reference electrode. The surface area of ​​the counter electrode was larger than that of the working electrode. A potentiostat OctoStat 30 provided by Ivium Technologies BV was used as a voltage application device. The working electrode, reference electrode, and counter electrode were electrically connected to the working electrode terminal, reference electrode terminal, and counter electrode terminal of the potentiostat, respectively, to form a three-electrode cell. An ion-exchange membrane, Nafion 117 provided by MTI Corporation, was used as a membrane separating the space inside the three-electrode cell where the working electrode and reference electrode were located from the space inside the three-electrode cell where the counter electrode was located. Nafion is a registered trademark. 20 mL of 2-morpholinoethanesulfonic acid (MES) buffer solution was placed in the space inside the three-electrode cell where the counter electrode was placed. The pH of the MES buffer solution was 5.5.

[0070] 20 mL of a fructose-containing solution obtained by dissolving fructose and NADH in a separately prepared MES buffer solution was placed in the space inside the three-electrode cell where the working electrode was located. D(+)-fructose provided by Fujifilm Wako Pure Chemical Industries, Ltd. was used as the fructose. β-nicotinamide adenine dinucleotide, reduced disodium salt provided by Sigma-Aldrich was used as the NADH. The fructose concentration in the fructose-containing solution was 355 mM. The NADH concentration in the fructose-containing solution was 5.6 mM.

[0071] Enzyme-loaded particles obtained by loading mannitol dehydrogenase onto particles of DuPont's ion-exchange resin Duolite A568 were placed in a nonwoven bag. In this state, the enzyme-loaded particles were immersed in a fructose-containing solution contained in the space inside the three-electrode cell where the working electrode was located. Duolite is a registered trademark. Fructose and NADH were permeable through the nonwoven fabric, but the enzyme-loaded particles were impermeable. Therefore, the mannitol dehydrogenase was positioned so as not to come into contact with the working electrode. Mannitol dehydrogenase (MDH) derived from Leuconostoc mesenteroides, provided by Sigma-Aldrich, was used as the mannitol dehydrogenase.

[0072] In the three-electrode cell, a predetermined voltage was applied to the working electrode at a temperature of 20° C. to 25° C. for 16 hours while stirring the fructose-containing liquid with a magnetic stirrer, and a treatment liquid according to Example 1 was obtained.

[0073] <Comparative Example 1> A treatment solution according to Comparative Example 1 was obtained in the same manner as in Example 1, except that MDH was dispersed in a fructose-containing solution instead of immersing the enzyme-attached particles in a nonwoven fabric bag in the fructose-containing solution. In this case, it is believed that MDH came into contact with the working electrode during stirring with the magnetic stirrer.

[0074] (Evaluation of Enzyme Activity) The enzymatic activity of MDH can be evaluated by quantifying the amount of NADH consumed in a solution containing MDH, NADH, and fructose. The amounts of NADH consumed within 10 minutes after adding 1 mM NADH and 1 mM fructose to each of the treatment solutions of Example 1 and Comparative Example 1 contained in a three-electrode cell were determined. The amount of NADH consumed in the treatment solution was determined by measuring the absorbance of the sample at a wavelength of 340 nm, and determining whether the molar extinction coefficient of NADH was 6.2 × 10. 3 The results are shown in Figures 6 and 7.

[0075] FIG. 6 is a graph showing the results of evaluating the enzyme activity in Example 1. FIG. 7 is a graph showing the results of evaluating the enzyme activity in Comparative Example 1. In FIGS. 6 and 7, the vertical axis represents the concentration of NADH. In FIG. 6, (a1) shows the concentration of NADH 10 minutes after adding 1 mM NADH and 1 mM fructose to a treatment solution according to Reference Example 1 obtained in the same manner as in Example 1, except that MDH was inactivated by heat denaturation. (b1) shows the concentration of NADH 10 minutes after adding 1 mM NADH and 1 mM fructose to a fructose-containing solution contained in the triode cell in Example 1 before voltage application. (c1) shows the concentration of NADH 10 minutes after adding 1 mM NADH and 1 mM fructose to a treatment solution according to Example 1 contained in the triode cell. In Figure 7, (d1) shows the NADH concentration 10 minutes after adding 1 mM NADH and 1 mM fructose to the treatment solution of Reference Example 2, which was obtained in the same manner as Comparative Example 1, except that MDH was inactivated by heat denaturation. (e1) shows the NADH concentration 10 minutes after adding 1 mM NADH and 1 mM fructose to the fructose-containing solution contained in the triode cell of Comparative Example 1 before voltage application. (f1) shows the NADH concentration 10 minutes after adding 1 mM NADH and 1 mM fructose to the treatment solution of Comparative Example 1 contained in the triode cell. (g1) shows the NADH concentration 60 minutes after adding 1 mM NADH and 1 mM fructose to the treatment solution of Comparative Example 1 contained in the triode cell. 6 (b1) and (c1) show that the NADH added to the treatment solution of Example 1 was almost completely consumed within 10 minutes, similar to the NADH added to the fructose-containing solution before voltage application in Example 1. On the other hand, FIG. 7 (f1) and (g1) show that the NADH in the treatment solution of Comparative Example 1 was hardly consumed 10 minutes and 60 minutes after addition. FIG. 7 shows that the activity of the enzyme can decrease when the enzyme comes into contact with the electrode to which a voltage is applied, as in Comparative Example 1.On the other hand, it can be seen from FIG. 6 that if the electrode to which a voltage is applied and the enzyme are kept out of contact as in Example 1, the decrease in enzyme activity can be suppressed.

[0076] (Evaluation of Enzyme Activity) In Example 1, a sample solution containing the enzyme after applying a predetermined voltage to the working electrode for 16 hours was prepared as the sample solution of Example 1. In Comparative Example 1, a sample solution containing the enzyme after applying a predetermined voltage to the working electrode for 16 hours was prepared as the sample solution of Comparative Example 1. In Example 1, a sample solution containing the enzyme before applying a predetermined voltage to the working electrode was prepared as the sample solution of Reference Example 3. These sample solutions were subjected to SDS-PAGE. FIG. 8 shows SDS-PAGE images of the treatment solutions of Example 1, Comparative Example 2, and Reference Example 3. In FIG. 8, (a2) is an SDS-PAGE image of a molecular weight marker, (b2) is an SDS-PAGE image of the sample solution of Reference Example 3, (c2) is an SDS-PAGE image of the sample solution of Comparative Example 1, and (d2) is an SDS-PAGE image of the sample solution of Example 1. Tetramers and dimers were detected in the SDS-PAGE image of the sample solution of Reference Example 3. In the SDS-PAGE image of the sample solution according to Comparative Example 1, mainly monomers were detected. On the other hand, in the SDS-PAGE image of the sample solution according to Example 1, tetramers and dimers were detected. (c2) suggests that in Comparative Example 1, the higher-order structure of the enzyme is destroyed by contact between the electrode to which a voltage is applied and the enzyme. On the other hand, (d2) suggests that in Example 1, the higher-order structure of the enzyme is maintained by maintaining a non-contact state between the working electrode to which a voltage is applied and the enzyme. The reason why the activity of the enzyme is maintained by applying a voltage to the working electrode for 16 hours in Example 1 is thought to be because the higher-order structure of the enzyme is maintained.

[0077] (Changes in Fructose Concentration) The fructose concentrations in the treatment solutions of Example 1 and Comparative Example 2 were measured using a Fructose Colorimetric / Fluorometric Assay Kit provided by Sigma-Aldrich. The results are shown in FIG. 9. FIG. 9 is a graph showing changes in fructose concentration in Example 1 and Comparative Example 1. In FIG. 9, the vertical axis represents fructose concentration. In FIG. 9, (a3) ​​represents the initial fructose concentration in the fructose-containing solutions of Example 1 and Comparative Example 1. (b3) represents the fructose concentration in the treatment solution of Comparative Example 1. (c3) represents the fructose concentration in the treatment solution of Example 1. The fructose concentration in the treatment solution of Example 1 was 319 mM compared to the initial concentration (355 mM), and it is understood that 10% of the fructose, on a substance mass basis, was changed by the enzymatic reaction. On the other hand, the fructose concentration in the treatment solution of Comparative Example 1 was 351 mM, and it is understood that about 1% of the fructose was converted by the enzyme reaction on a substance basis. According to Example 1, the destruction of the higher-order structure of the enzyme is suppressed by maintaining a non-contact state between the working electrode to which a voltage is applied and the enzyme, and it is thought that the catalytic action of the MDH, which maintains its activity, promotes the production of mannitol from fructose.

[0078] The reactor of the present disclosure can be used as a reactor for generating electrochemical reactions for enzymatic reactions such as converting fructose to mannitol.

[0079] 1 Working electrode 9 Enzyme 9c Carrier 9m Membrane 20 Voltage application device 100 Reaction device

Claims

1. An anode that electrochemically activates a coenzyme, and a voltage application device that applies a voltage to the anode to cause electron transfer between the coenzyme and the anode, wherein a substrate is oxidized or reduced by an enzymatic reaction involving an enzyme whose activity is expressed by the coenzyme, and when the voltage is applied to the anode, a non-contact state between the enzyme and the anode is maintained, and a contact state between the coenzyme and the anode is maintained. Reaction device.

2. The reaction device according to claim 1, further comprising the enzyme.

3. The reaction device according to claim 2, further comprising a carrier on which the enzyme is immobilized.

4. The reaction device according to claim 2, further comprising a membrane that allows the coenzyme and the substrate to permeate and blocks the permeation of the enzyme, wherein the enzyme is disposed in a space separated from the anode by the membrane.

5. The reaction device according to claim 4, wherein the membrane forms a bag, and the enzyme is accommodated inside the bag.

6. Electrochemically activating the coenzyme by causing electron transfer between the coenzyme and the anode by applying a voltage to the anode, expressing the activity of the enzyme by the coenzyme, and oxidizing or reducing the substrate by the enzymatic reaction involving the enzyme to produce a product, wherein when the voltage is applied to the anode, a non-contact state between the enzyme and the anode is maintained, and a contact state between the coenzyme and the anode is maintained. Method for producing a product.

Images