Reduction method of target molecule, reduction device and electrode

Abstract

This method for reducing a target molecule comprises: a first step for reducing a disulfide bond in a target molecule present in a reaction system by means of thioredoxin, the state of which changes between an oxidized form and a reduced form and which is in a reduced form; and a second step in which the state of the oxidized thioredoxin is changed to a reduced state by supplying electrons from an electrode connected to an external power source outside the reaction system to the thioredoxin which is oxidized by the disulfide bond in the first step and which changes to the oxidized state, thereby changing the state of the oxidized thioredoxin to the reduced state. The thioredoxin is immobilized on the surface of the electrode directly or via a linker, and the first step and the second step are carried out under conditions in which the reaction system is alkaline.

Description

Technical Field

[0001] The present disclosure relates to a method for reducing a target molecule, a device for reducing a disulfide bond, and an electrode. Background Art

[0002] It is known that by immobilizing an enzyme on an electrode, the transfer of electrons between the electrode and the enzyme can be utilized for various purposes. For example, Patent Document 1 discloses an enzyme electrode in which a carrier, an enzyme, and a mediator (also referred to as a mediator) are immobilized on a conductive member.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2006-058289 Summary of the invention

[0006] Problems to be solved by the invention

[0007] The enzyme electrode described in Patent Document 1 can be used as a sensor that observes electron transfer from the enzyme to the electrode and measures a current value.

[0008] However, in order to reduce the target molecule by reducing and cutting (in other words, cleaving) the disulfide bond possessed by the target molecule using electrons supplied from the electrode, an appropriate reduction method is required.

[0009] Therefore, an object of the present disclosure is to provide a more suitable reduction method and the like from the viewpoint of reducing a target molecule such as by cleaving a disulfide bond possessed by the target molecule.

[0010] Means for solving problems

[0011] A method for reducing a target molecule according to one embodiment of the present disclosure comprises the following steps: a first step of reducing disulfide bonds in a target molecule present in a reaction system by using thioredoxin which changes its state between an oxidized form and a reduced form and is in the reduced form; and a second step of reducing the thioredoxin which is oxidized by the disulfide bonds in the first step and changes its state to the oxidized form by supplying electrons from an electrode connected to an external power source outside the reaction system, thereby changing the state of the oxidized thioredoxin to the reduced form, wherein the thioredoxin is immobilized on the surface of the electrode directly or via a linker, and the first step and the second step are performed under alkaline conditions in the reaction system.

[0012] In addition, a reduction device involved in one embodiment of the present disclosure is a reduction device that reduces disulfide bonds in a target molecule present in a reaction system, and comprises: a unit that separates the above-mentioned reaction system from the outside, and an electrode connected to an external power source outside the above-mentioned reaction system, wherein a thioredoxin whose state changes between an oxidized form and a reduced form is fixed directly or via a linker on the surface of the above-mentioned electrode.

[0013] In addition, one embodiment of the present invention involves an electrode used as a reduction device for reducing disulfide bonds in a target molecule present in a reaction system, the electrode is connected to an external power source outside the reaction system, and a thioredoxin whose state changes between an oxidized form and a reduced form is fixed directly or via a linker on the surface of the electrode.

[0014] Effects of the Invention

[0015] According to the present disclosure, a more suitable reduction method and the like can be provided from the viewpoint of reducing a target molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing an example of the configuration of the disulfide bond cleaving device according to the present embodiment.

[0017] Figure 2 yes Figure 1 Cross-sectional view at line II-II.

[0018] Figure 3A This is a diagram schematically showing thioredoxin immobilized on an electrode.

[0019] Figure 3B is another diagram schematically showing thioredoxin immobilized on an electrode.

[0020] Figure 4 This is a flow chart showing an example of a method for cleaving a disulfide bond.

[0021] Figure 5 FIG. 1 is a first diagram for explaining the embodiment.

[0022] Figure 6 FIG. 2 is a second figure for explaining the embodiment.

[0023] Figure 7 FIG. 3 is a diagram for explaining the embodiment.

[0024] Figure 8 FIG. 4 is used to illustrate the embodiment.

[0025] Fig. 9 FIG5 is a fifth figure for explaining the embodiment.

[0026] Fig.10FIG6 is a sixth figure for explaining the embodiment. DETAILED DESCRIPTION

[0027] (Reaching this public view)

[0028] In order to reduce the target molecule by reducing the disulfide bond of the target molecule using the electrons supplied from the electrode and cutting it, it becomes necessary to have a molecular species that can accept the electrons supplied from the electrode and use the electrons as reducing force for the reduction of the target molecule. As such a molecular species, in one of the above examples, it is known that a combination of enzymes and proteins such as thioredoxin reductase and thioredoxin can be used in the past. Thioredoxin can change between the two states of oxidation and reduction. Moreover, the reduced thioredoxin has the function of cutting the disulfide bond of the target molecule. The thioredoxin after the disulfide bond is cut is oxidized in exchange for the cutting of the disulfide bond, and changes into the oxidized thioredoxin.

[0029] Thioredoxin reductase has the function of reducing oxidized thioredoxin using electrons to change it into reduced thioredoxin. Therefore, if electrons from an electrode can be supplied to thioredoxin reductase, oxidized thioredoxin can be changed into reduced thioredoxin. That is, the following cycle can be operated: the disulfide bond possessed by the target molecule is cut, the thioredoxin that has become oxidized is changed into a reduced form, and the disulfide bond possessed by the target molecule is cut again. However, since thioredoxin reductase is an enzyme, the optimal pH has been determined, and if it is out of a certain pH range, the function of changing thioredoxin into a reduced form becomes difficult to function. More specifically, it is known that the function of thioredoxin reductase decreases under alkaline conditions.

[0030] Furthermore, it is known that thioredoxin reductase has difficulty in directly accepting electrons from an electrode and requires the presence of an electron transporter generally referred to as an electron mediator (or simply referred to as a mediator).

[0031] On the other hand, there is a use of thioredoxin's function of cleaving disulfide bonds to cleave disulfide bonds of allergens (substances causing allergic reactions) contained in food. If the disulfide bonds of the allergens can be cleaved, the three-dimensional structure of the allergen changes or becomes easy to change, so it is possible that it becomes less likely to cause allergic reactions. That is, the development of technology for producing foods that are less likely to cause allergic reactions despite containing allergens by cleaving disulfide bonds is being promoted. Representative allergens include prolamins, albumin, and β-lactoglobulin, but are not limited to these. For all allergens, especially allergens with disulfide bonds near the molecular surface, it can be effectively applied to the above-mentioned situations.

[0032] The food production process may include a step of pretreating food materials under alkaline conditions. If disulfide bonds can be cleaved in this step, the process can be simplified and the food production cost can be reduced.

[0033] However, as described above, in the system using thioredoxin reductase, it is difficult to use it under alkaline conditions because the function of thioredoxin reductase is reduced. Therefore, in the present disclosure, from the perspective of reducing the target molecule such as cleaving the disulfide bond possessed by the target molecule as one example described above, a method for reducing the target molecule that can be used even if the reaction condition is alkaline is provided.

[0034] (One aspect of the present disclosure)

[0035] An outline of one aspect of the present disclosure is as follows.

[0036] The first embodiment of the present disclosure relates to a method for reducing a target molecule, comprising the following steps: a first step of reducing disulfide bonds in a target molecule present in a reaction system by using thioredoxin which changes its state between an oxidized form and a reduced form and is in the reduced form; and a second step of reducing the thioredoxin which is oxidized by the disulfide bonds in the first step and changes its state to the oxidized form by supplying electrons from an electrode connected to an external power source outside the reaction system, thereby changing the state of the oxidized thioredoxin to the reduced form, wherein the thioredoxin is immobilized on the surface of the electrode directly or via a linker, and the first step and the second step are performed under the condition that the reaction system is alkaline.

[0037] According to such a method for reducing a target molecule, by fixing thioredoxin on an electrode directly or via a linker, it is possible to supply electrons from the electrode. The reduced thioredoxin changes to an oxidized form each time the target molecule is reduced, and then changes to a reduced form by the electrons supplied from the electrode. Thus, as long as the electrons are continuously supplied from the electrode, thioredoxin can repeatedly reduce the target molecule. In addition, by fixing thioredoxin on an electrode, thioredoxin becomes less likely to remain in the reaction system, and can effectively function in food processing applications, etc. Furthermore, when thioredoxin is fixed on an electrode, the three-dimensional structure of thioredoxin changes, and the disulfide bond can be cut under alkaline conditions and the state of the oxidized thioredoxin can be changed to a reduced form. Therefore, from the perspective of cutting the disulfide bond possessed by the target molecule, a method for cutting the disulfide bond that can be used even if the reaction conditions are alkaline conditions can be achieved.

[0038] Furthermore, for example, the target molecule reduction method according to the second embodiment may be: in the target molecule reduction method according to the first embodiment, the first step and the second step are performed under a pH condition of the reaction system being greater than pH 8.5.

[0039] This makes it possible to cleave the disulfide bond and change the state of oxidized thioredoxin to a reduced form under alkaline conditions of a reaction system having a pH higher than pH 8.5.

[0040] Furthermore, for example, the method for reducing a target molecule according to the third embodiment may be such that, in the method for reducing a target molecule according to the first embodiment or the second embodiment, the linker comprises an alkyl chain, and the alkyl chain has 1 to 14 carbon atoms.

[0041] Thus, by fixing thioredoxin to the electrode via a linker including an alkyl chain having 1 to 14 carbon atoms, electrons can be donated from the electrode.

[0042] In addition, for example, the target molecule reduction method according to the fourth aspect may be: in the target molecule reduction method according to any one of the first to third aspects, the electrode comprises gold, platinum, glassy carbon, or ITO.

[0043] Thus, the disulfide bonds can be repeatedly cleaved using an electrode containing gold, platinum, glassy carbon or ITO and to which thioredoxin is immobilized.

[0044] Furthermore, for example, the method for reducing a target molecule according to the fifth aspect may be the method for reducing a target molecule according to any one of the first to fourth aspects, wherein the target molecule is at least one of prolamin, albumin, and β-lactoglobulin.

[0045] Thus, the disulfide bonds contained in at least any one of prolamin, albumin, and β-lactoglobulin can be repeatedly cleaved using the electrode to which thioredoxin is immobilized.

[0046] In addition, for example, the reduction method of the target molecule involved in Scheme 6 may also be: in the reduction method of the target molecule described in any one of Schemes 1 to 5, no electron transfer body for transferring electrons to and from the thioredoxin is fixed on the above-mentioned electrode, and the above-mentioned second step supplies electrons from the above-mentioned electrode to the above-mentioned oxidized thioredoxin without via the above-mentioned electron transfer body.

[0047] Thus, when electrons are supplied to thioredoxin, electrons can be supplied to thioredoxin from the electrode without passing through an electron carrier. At this time, if thioredoxin is fixed to the electrode directly or via a relatively short linker having a carbon number of 1 or more and 14 or less, near the electrode, the supply efficiency of electrons can be less likely to be reduced even without passing through an electron carrier. Furthermore, since it is not dependent on the reaction speed of the electron carrier accepting electrons and the reaction speed of the electron carrier releasing electrons, but is only controlled by the reaction speed of thioredoxin itself accepting electrons, it also has the advantage of being easy to improve the supply efficiency of electrons. In addition, since the number of molecular species fixed on the electrode is reduced, it is easier to design the electrode, and the manufacturing quality of the electrode can be more stabilized.

[0048] In addition, the reduction device involved in the seventh scheme of the present disclosure is a reduction device for reducing disulfide bonds in a target molecule present in a reaction system, and comprises: a unit that separates the above-mentioned reaction system from the outside, and an electrode connected to an external power source outside the above-mentioned reaction system, wherein a thioredoxin whose state changes between an oxidized form and a reduced form is fixed directly or via a linker on the surface of the above-mentioned electrode.

[0049] Such a reduction device can realize a reduction device that performs the method for reducing a target molecule described in any one of the first to sixth embodiments. Therefore, the disulfide bond cleaving device exhibits the same effects as the disulfide bond cleaving method described in any one of the first to sixth embodiments.

[0050] In addition, the electrode involved in the 8th scheme of the present disclosure is an electrode for a reduction device that reduces disulfide bonds in a target molecule present in a reaction system, wherein the above-mentioned electrode is connected to an external power source outside the above-mentioned reaction system, and thioredoxin whose state changes between an oxidized form and a reduced form is fixed on the surface of the above-mentioned electrode directly or via a linker.

[0051] Such an electrode can be used in a reduction device for performing the target molecule reduction method described in any one of the first to sixth aspects. Therefore, the electrode exerts the same effect as the target molecule reduction method described in any one of the first to sixth aspects.

[0052] It should be noted that these general or specific solutions can be implemented through systems, methods, devices, integrated circuits, computer programs or computer-readable CD-ROMs and other recording media, or can be implemented through any combination of systems, methods, devices, integrated circuits, computer programs and recording media.

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

[0054] It should be noted that the embodiments described below are all general or specific examples. The numerical values, shapes, materials, constituent elements, configuration positions of constituent elements and connection forms, steps, order of steps, etc. shown in the following embodiments are examples, and their main purpose is not to limit the claims. In addition, among the constituent elements in the following embodiments, the constituent elements not recorded in the independent claims representing the highest concept are described as optional constituent elements. In addition, the figures are not necessarily strictly illustrated. In each figure, the same symbol is marked for substantially the same structure, and repeated descriptions are sometimes omitted or simplified.

[0055] In the drawings, the X-axis direction, Y-axis direction, and Z-axis direction that are orthogonal to each other are appropriately used for description. In particular, the positive side of the Z-axis direction may be described as the upper side and the negative side may be described as the lower side.

[0056] In the present disclosure, terms such as parallel and perpendicular indicating the relationship between elements, terms such as rectangle indicating the shape of elements, and numerical values ​​do not have strict meanings but also include substantially equivalent ranges, for example, differences of several percent.

[0057] In addition, in the drawings of the present disclosure, dotted lines indicate boundaries of parts and regions that cannot be seen from the surface.

[0058] (Implementation Method)

[0059] Below, refer to Figure 1 to Figure 4 The implementation manner will be described in detail.

[0060] [Disulfide bond cleavage device]

[0061] [1. Summary]

[0062] First, as an example of a reduction device for a target molecule in an embodiment, refer to Figure 1 The outline of the disulfide bond cleaving device (hereinafter, sometimes simply referred to as a cleaving device) is described below. Figure 1 This is a diagram showing an example of the configuration of the disulfide bond cleaving device according to the present embodiment.

[0063] The cutting device 100 transfers electrons between the electrode and the thioredoxin by applying a voltage to the electrode. For example, the cutting device 100 supplies electrons from the electrode to the thioredoxin fixed on the surface of the electrode. As a result, electron transfer occurs between the electrode and the target molecule in the sample, so that the disulfide bond of the target molecule is reduced and cut. For example, the cutting device 100 applies a voltage to the electrode when the sample (e.g., sample solution) containing the target molecule is in a non-flowing state, so that electron transfer occurs between the thioredoxin fixed on the electrode and the target molecule in the sample, thereby reducing the target molecule. Moreover, by switching the liquid from a non-flowing state to a flowing state, the reduced target molecule is diffused in the liquid. By repeatedly switching the flowing state of the liquid in this way, the target molecule can be efficiently reduced with the entire liquid. At this time, the reaction system, i.e., the liquid containing the sample, becomes an alkaline condition. It should be noted that the alkaline condition refers to a condition in a pH range higher than neutral. Generally, neutral refers to a pH range with a specified range (e.g., pH6 to pH8) centered on pH7. Therefore, the alkaline condition in this embodiment refers to a pH range higher than pH8.

[0064] It should be noted that the non-flowing state of a liquid refers to, for example, a state in which the liquid is not stirred or vibrated (ie, is not subjected to external forces such as shear force or vibration) and no shaking or other movement is observed on the liquid surface.

[0065] [2. Composition]

[0066] Next, refer to Figure 1 to Figure 3B The configuration of the cutting device 100 in the embodiment will be described.

[0067] like Figure 1 As shown in the figure, the cutting device 100 comprises: a stirring section 40 for stirring a sample 9 containing a target molecule to make it flow; an electrode (cathode electrode 1) fixed with thioredoxin for reducing the target molecule by transferring electrons between the target molecule and the target molecule; a power supply 20 for applying a voltage to the electrode; and a control section 30 for controlling the power supply 20 and the stirring section 40.

[0068] The voltage applying unit 10 is, for example, a three-electrode unit including a cathode electrode 1 (also referred to as a working electrode), a reference electrode 2, a counter electrode 3, a unit 4, a cover 5, terminals 6a, 6b, 6c, and leads 7a, 7b, 7c. It should be noted that the voltage applying unit 10 may also be, for example, a two-electrode unit including a working electrode (cathode electrode 1) and a counter electrode 3.

[0069] The cathode electrode 1 and the counter electrode 3 are made of a conductive material. The conductive material may be, for example, a carbon material, a conductive polymer material, a semiconductor, or a metal.

[0070] First, for the cathode electrode 1, refer to Figure 2 , Figure 3A and Figure 3B Provide explanation. Figure 2 yes Figure 1 Cross-sectional view at line II-II. Figure 3A and Figure 3B This is a diagram schematically showing thioredoxin immobilized on an electrode.

[0071] The cathode electrode 1 is an electrode to which thioredoxin is fixed. The cathode electrode 1 includes, for example, a glass substrate 11, a titanium vapor-deposited layer 12 vapor-deposited on the glass substrate 11, a cathode substrate 13 formed on the titanium vapor-deposited layer 12, and a reaction layer 14 containing thioredoxin fixed on the cathode substrate 13.

[0072] For example, a conductive substrate such as gold, platinum, glassy carbon, or ITO (Indium Tin Oxide) may be used as the cathode substrate 13. The thickness of the cathode substrate 13 is not particularly limited.

[0073] No electron mediator is fixed to the cathode substrate 13. The electron mediator is a substance that transfers electrons between the electrode and thioredoxin fixed to the electrode, and examples thereof include viologen, quinone, and indophenol.

[0074] Thioredoxin immobilized on the surface of the electrode (so-called cathode substrate 13) is a protein that reduces the target molecule. Figure 3A As shown in , it is fixed to the cathode substrate 13 via a chain-like linker. The linker, for example, includes an alkyl chain having 1 to 14 carbon atoms. Alternatively, thioredoxin is Figure 3B As shown in , it is directly fixed to the surface of the electrode without a linker. It should be noted that the so-called "without a linker" means that no other molecular species intervene between the surface of the electrode and thioredoxin. However, the case where a part of the main chain or side chain of thioredoxin and a part involved in immobilization acts like a linker is also included in the case of not passing through a linker.

[0075] The reference electrode 2 is an electrode that maintains a certain potential without reacting with the components in the sample 9, and is used to control the potential difference between the cathode electrode 1 and the reference electrode 2 to be constant by the power supply 20. The reference electrode 2 is, for example, a silver / silver chloride electrode. The counter electrode 3 is, for example, a platinum electrode. In the present embodiment, the unit 4 separates the reaction system from the outside. The unit 4 connects the first space in which the cathode electrode 1 and the reference electrode 2 are arranged and the second space in which the counter electrode 3 is arranged via the connecting portion 4a. In the connecting portion 4a, there is a diaphragm 4b, and some components in the sample 9 in the first space and the sample 9a in the second space do not move relative to each other.

[0076] The power supply 20 applies a voltage between the cathode electrode 1 and the counter electrode 3 of the voltage applying unit 10 according to a control signal output from the control unit 30 , and controls the potential between the cathode electrode 1 and the reference electrode 2 to a predetermined value.

[0077] The control unit 30 performs information processing for applying voltage to the power source 20 and controlling the operation of a motor (not shown) of the stirring unit 40. The control unit 30 is realized by, for example, a processor, a microcomputer, or a dedicated circuit.

[0078] The stirring section 40 controls the operation of the motor according to the control signal output from the control section 30 , thereby controlling the rotation speed and rotation time of the stirring element 8 provided in the voltage applying section 10 .

[0079] In the method for producing a thioredoxin-immobilized electrode, a linker for fixing thioredoxin is fixed to an electrode. The linker is dissolved in a solution of ethanol:acetonitrile=1:1, and the electrode is immersed for example for more than 1 hour and allowed to stand for immobilization. In order to enable electrons to move from the electrode to thioredoxin, the linker is preferably an alkyl chain with a carbon number of 14 or less. For example, the linker is an alkyl chain with a carbon number of 1 or more and 14 or less. In particular, the linker has a carboxyl group or an amino group at one end and a thiol group at the other end.

[0080] Next, the linker fixed on the electrode is bound to thioredoxin. At this time, for example, the amino group of thioredoxin and the carboxyl group of the linker are bound by an amine coupling reaction. Thus, thioredoxin is fixed to the electrode via the linker.

[0081] It should be noted that when thioredoxin is directly immobilized on the electrode, the linker immobilization step is omitted.

[0082] [3. Method for reducing the target molecule (method for cleaving disulfide bonds)]

[0083] Next, as an example of a method for reducing a target molecule, refer to Figure 4 The method for cleaving a disulfide bond is described. Figure 4 This is a flow chart showing an example of a method for cleaving a disulfide bond.

[0084] The method for cutting the disulfide bond of the target molecule is to reduce the target molecule contained in the sample 9 by using a voltage applying unit 10 of a three-electrode system having a thioredoxin fixed electrode as a cathode electrode 1, an anode electrode as a counter electrode 3, and a reference electrode 2, thereby cutting the disulfide bond (S101). Step S101 is an example of the first step. Step S101 is performed under alkaline conditions. The surface area of ​​the anode electrode is, for example, sufficiently larger than that of the cathode electrode 1. As a result of cutting the disulfide bond, thioredoxin is oxidized and changes to an oxidized form. The sample 9 is an aqueous solution containing the target molecule.

[0085] Next, the thioredoxin converted to the oxidized form is reduced again to convert it to the reduced form (S102). Step S102 is an example of the second step. Step S102 is performed under alkaline conditions. Then, by repeatedly performing steps S101 and S102, the disulfide bonds of the target molecule can be repeatedly cut continuously. At this time, the voltage applied to the sample 9 can also be controlled in such a way that the potential of the cathode electrode 1 relative to the reference electrode 2 becomes the oxidation potential of thioredoxin.

[0086] [Example 1: Verification of the number of carbon atoms in the alkyl chain contained in the linker]

[0087] [1-1. Thioredoxin-immobilized electrode 1]

[0088] Hereinafter, the method for cleaving disulfide bonds of a target molecule using the thioredoxin-immobilized electrode of the present disclosure will be specifically described with reference to examples, but the present disclosure is not limited to the following examples at all.

[0089] In the following examples, β-lactoglobulin was used as the target molecule.

[0090] The target molecule was dissolved in phosphate buffered saline (PBS) at pH 7.4 and adjusted to 3.3 mg / ml.

[0091] The thioredoxin-immobilized electrode was prepared by adding a solution prepared by dissolving thioredoxin in phosphate buffered saline (PBS) at pH 7.4 onto a gold substrate and allowing the solution to stand to allow thioredoxin to adsorb onto the substrate.

[0092] [1-2. Thioredoxin-immobilized electrode 2]

[0093] The above-mentioned thioredoxin-immobilized electrode 1 is an electrode without a linker, i.e., an electrode directly adsorbing thioredoxin is prepared. In contrast, the thioredoxin-immobilized electrode 2 of this example is different in that thioredoxin is immobilized on a substrate via a linker (carboxyalkanethiol) having 5 carbon atoms. Therefore, in this example, first, a linker solution having 5 carbon atoms is prepared, and a self-assembled monomolecular film (SAM) of the linker is formed on a substrate. Then, the amino group of thioredoxin and the carboxyl group of the linker are bonded by an amine coupling reaction.

[0094] [1-3. Thioredoxin-immobilized electrode 3]

[0095] In the above-mentioned thioredoxin-immobilized electrode 2, the carbon number of the alkyl chain contained in the linker is 5. In contrast, in the thioredoxin-immobilized electrode 3 of this example, thioredoxin is immobilized on the substrate via a linker (carboxyalkanethiol) having 10 carbon atoms. Therefore, in this example, first, a linker solution having 10 carbon atoms is prepared, and a self-assembled monomolecular film (SAM) of the linker is formed on the substrate. Then, the amino group of thioredoxin and the carboxyl group of the linker are bonded by an amine coupling reaction.

[0096] [1-4. Thioredoxin-immobilized electrode 4 (Comparative example)]

[0097] In the above-mentioned thioredoxin-immobilized electrode 2, the carbon number of the alkyl chain contained in the linker is 5. In contrast, in the thioredoxin-immobilized electrode 4 of this example, thioredoxin is immobilized on the substrate via a linker (carboxyalkanethiol) having 15 carbon atoms. Therefore, in this example, first, a linker solution having 15 carbon atoms is prepared, and a self-assembled monomolecular film (SAM) of the linker is formed on the substrate. Then, the amino group of thioredoxin and the carboxyl group of the linker are bonded by an amine coupling reaction.

[0098] [1-5. Verification results]

[0099] exist Figure 1 The target molecule solution is introduced into the unit 4 described in the description as samples 9 and 9a, and each electrode is set. The electrode uses a three-electrode system in which the prepared thioredoxin fixed electrode is used as the working electrode, the Pt (platinum) electrode is used as the counter electrode, and the Ag / AgCl (silver / silver chloride) electrode is used as the reference electrode. Next, a stirrer 8 is placed in the target molecule solution, and the stirrer 8 is rotated at a specified rotation speed (rpm) while applying a specified voltage described later to the target molecule solution. It should be noted that the reaction system uses a phosphate buffered saline (PBS) with a pH of 7.4 as an initial condition, but it is confirmed that the pH rises to about 11 due to the hydroxyl species generated until the end of the reaction, and it is confirmed that the disulfide bond is cut under this condition. That is, the so-called reaction system is alkaline as long as it becomes alkaline (pH range higher than pH 8.0) during at least a part of the period from the start time of the reaction to the end time of the reaction. In particular, in the disulfide bond cleavage method of the present disclosure, a predetermined potential is applied, so if it is a normal aqueous solution system, the virtual pH rises slowly, and it is advantageous that the disulfide bond can be cleaved under alkaline conditions.

[0100] In order to confirm the cleavage of the disulfide bonds of the target molecule, the sample after voltage application was mixed with digestive enzymes and reacted at 37°C for 30 minutes, and then analyzed by SDS-PAGE. The sample before voltage application was similarly analyzed, and the relative amount of disulfide bond cleavage was calculated by image analysis when the sample was set as 100%.

[0101] here, Figure 5 : is a graph showing the relationship between the applied potential and the relative amount of disulfide bond cleavage in Example 1. Figure 5 In FIG. 1 , the horizontal axis shows the potential applied between the working electrode and the counter electrode, and the vertical axis shows the relative amount of disulfide bonds cut at each applied potential. Figure 5 As shown in , it is shown that the cleavage of disulfide bonds occurs rapidly from an applied potential of -0.8 V, with a peak at -1.0 V, and the relative amount of cleavage of disulfide bonds decreases at potentials lower than that. In the following examples, the cleavage of disulfide bonds is performed at an applied potential of -1.0 V.

[0102] The verification results of Example 1 are shown in Figure 6 middle. Figure 6 is a graph showing the verification results of Example 1. Figure 6 In the graph, the horizontal axis shows each thioredoxin-immobilized electrode species, and the vertical axis shows the relative amount of disulfide bonds that are cut in each thioredoxin-immobilized electrode species. Figure 6 As shown in , about 80% of the disulfide bonds were cut for the thioredoxin-immobilized electrode 1 (adsorption) to which thioredoxin was directly adsorbed. Also, about 90% of the disulfide bonds were cut for the thioredoxin-immobilized electrode 2 (C=5) to which thioredoxin was immobilized via a linker having 5 carbon atoms. Also, about 80% of the disulfide bonds were cut for the thioredoxin-immobilized electrode 3 (C=10) to which thioredoxin was immobilized via a linker having 10 carbon atoms. In contrast, less than 10% of the disulfide bonds were cut for the thioredoxin-immobilized electrode 4 (C=15) to which thioredoxin was immobilized via a linker having 15 carbon atoms.

[0103] From this result, it can be seen that the highest cleavage amount was shown in the thioredoxin-immobilized electrode 2. It is presumed that in the case of the electrode to which thioredoxin is immobilized by direct adsorption, there is no degree of freedom in the orientation direction of thioredoxin, etc., and thus the efficiency of cleavage of disulfide bonds is reduced, while in the case of the electrode to which thioredoxin is immobilized via a linker having 10 carbon atoms, the efficiency of electron transfer from the electrode surface to thioredoxin is reduced, and thus the efficiency of cleavage of disulfide bonds is reduced.

[0104] [Example 2: Verification of the appropriate pH range in the reaction system]

[0105] Next, the appropriate pH conditions of the reaction system were verified using the thioredoxin-immobilized electrode 2. Here, the case where disulfide bond cleavage is performed in a system that can maintain a pH of about 10 and the case where disulfide bond cleavage is performed in a system that can maintain a pH of about 8 or less are compared for explanation. Figure 7 This is a graph showing the change over time in the relative amount of disulfide bond cleavage when disulfide bonds are cleaved in a system capable of maintaining pH 10. Figure 8 This is a graph showing the change over time in the relative amount of disulfide bond cleavage when disulfide bonds are cleaved in a system that can maintain a pH of 8 or less. Figure 7 and Figure 8 In the graph, the black circle symbol and the solid line represent the relative amount of disulfide bond cleavage, and the open circle symbol and the dotted line represent the pH of the reaction system.

[0106] like Figure 7 and Figure 8 As shown in , in a system capable of maintaining a pH of about 8 or less, the disulfide bond cleavage reaction did not proceed except for the first 2 hours, whereas in a system capable of maintaining a pH of about 10, the disulfide bond cleavage reaction proceeded throughout the reaction period.

[0107] and then, Fig. 9 This is a graph showing the change over time in the relative amount of disulfide bond cleavage when disulfide bonds are cleaved under conditions where the pH changes over time from about 7.5 to about 10.5. Fig. 9 In the figure, the black circle symbol and the solid line represent the relative amount of disulfide bond cleavage, and the hollow circle symbol and the dotted line represent the pH of the reaction system. Fig.10 The relationship between the pH of the reaction system and the relative amount of disulfide bond cleavage is organized into a graph with the reaction time condition fixed.

[0108] like Fig. 9 and Fig.10 As shown in , it is shown that the disulfide bonds are cleaved rapidly from the time when the pH exceeds 8.5. These results show that the thioredoxin-immobilized electrode prepared in this example preferably cleaves the disulfide bonds under the pH condition of pH greater than 8.5.

[0109] The above is a description of the disulfide bond cleavage method, disulfide bond cleavage device and electrode according to the present disclosure based on the embodiments, but the present disclosure is not limited to these embodiments. As long as it does not depart from the gist of the present disclosure, various modifications that can be thought of by a person skilled in the art are applied to the technical solutions of the embodiments, and other embodiments constructed by combining some of the constituent elements in the embodiments are also included in the scope of the present disclosure.

[0110] Industrial Applicability

[0111] According to the present disclosure, it is useful in the reduction of a target molecule.

[0112] Explanation of symbols

[0113] 1 cathode electrode

[0114] 2 Reference electrode

[0115] 3 pairs of electrodes

[0116] Unit 4

[0117] 4a Connection

[0118] 4b Diaphragm

[0119] 5 cover

[0120] 6a terminal

[0121] 6b terminal

[0122] 6c terminal

[0123] 7a lead

[0124] 7b lead

[0125] 7c lead

[0126] 8 Stirring bar

[0127] 9. Sample 9a

[0128] 10 voltage applying unit

[0129] 11Glass Substrate

[0130] 12 Titanium Evaporated Layer

[0131] 13 Cathode substrate

[0132] 14 Reaction layer

[0133] 20 Power Supply

[0134] 30 Control Department

[0135] 40 Mixing unit

[0136] 100 Cutting device

Claims

1. A method for reducing a target molecule, comprising the following steps: A first step of reducing disulfide bonds in a target molecule in a reaction system by using thioredoxin that changes state between an oxidized form and a reduced form and is in the reduced form; and a second step of reducing the thioredoxin that has been oxidized by the disulfide bonds and changed into the oxidized form in the first step by supplying electrons from an electrode connected to an external power source outside the reaction system, thereby changing the state of the oxidized thioredoxin into the reduced form; in, The thioredoxin is fixed to the surface of the electrode directly or via a linker, The first step and the second step are performed under the condition that the reaction system is alkaline.

2. The method for reducing a target molecule according to claim 1, wherein: The first step and the second step are performed under the pH condition of the reaction system being higher than pH 8.

5.

3. The method for reducing a target molecule according to claim 1, wherein: The linker comprises an alkyl chain, The alkyl chain has 1 or more and 14 or less carbon atoms.

4. The method for reducing a target molecule according to claim 1, wherein: The electrodes include gold, platinum, glassy carbon or ITO.

5. The method for reducing a target molecule according to claim 1, wherein: The target molecule is at least any one of prolamin, albumin and β-lactoglobulin.

6. The method for reducing a target molecule according to any one of claims 1 to 5, wherein An electron mediator that transfers electrons to and from the thioredoxin is not immobilized on the electrode. The second step includes donating electrons from the electrode to the oxidized thioredoxin without via the electron mediator.

7. A reduction device for reducing disulfide bonds in a target molecule present in a reaction system, wherein: The reduction device comprises: a unit for isolating the reaction system from the outside, and an electrode connected to an external power source outside the reaction system, On the surface of the electrode, thioredoxin, which changes state between an oxidized form and a reduced form, is immobilized directly or via a linker.

8. An electrode for use in a reduction device for reducing disulfide bonds in a target molecule present in a reaction system, wherein: The electrode is connected to an external power source outside the reaction system, Furthermore, thioredoxin, which changes state between an oxidized form and a reduced form, is immobilized directly or via a linker on the surface of the electrode.

Images