JP2014160024A - Enzyme modified electrode, manufacturing method of enzyme modified electrode, electrochemical reaction device using the same, and manufacturing method of chemical substance using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme modified electrode capable of reproducing coenzyme by a simple method and easily purifying a specified substance, and to provide a novel substance measurement sensor capable of performing a quantitative measurement of a target substance at high sensitivity.SOLUTION: An enzyme modified electrode is formed by fixing enzyme, coenzyme, and an electron transport mediator which mediates an electron transport between a conductor and the coenzyme on the conductor by covalent bonding respectively through a high molecular compound having an amino group and a high molecular compound having a carboxy group. The content per unit area of the electron transport mediator is 10 nmol/cmor higher. An oxidation-reduction reaction of a chemical substance is executed using the enzyme modified electrode.

Description

  The present invention relates to an enzyme-modified electrode, a method for producing the enzyme-modified electrode, an electrochemical reaction device using the same, and a method for producing a chemical substance using the electrochemical reaction device. The present invention relates to a method for producing a chemical substance having optical activity by a chemical reaction apparatus.

  Chemical substances having various optical activities are used as raw materials for pharmaceuticals and agricultural chemicals. Enzymes are highly suitable biocatalysts for the synthesis of optically active chemical substances because they catalyze redox reactions stereospecifically. Several important optically active pharmaceutical intermediates are produced using enzymatic reactions. A redox reaction catalyzed by an enzyme requires a chemical substance called a coenzyme that directly transfers electrons and hydrogen ions to and from a substrate. In order to maintain the reaction, an amount of coenzyme corresponding to the base mass is necessary. However, since the coenzyme is generally expensive, it is disadvantageous in cost when producing a large amount of the target substance. In order to solve this problem, the entire reaction is designed so that the coenzyme regeneration reaction proceeds simultaneously with the target reaction (see, for example, Patent Document 1 and Patent Document 2).

  That is, a coenzyme oxidized (reduced) by reduction (oxidation) of the target substance is regenerated to its original state by being used for the oxidation (reduction) reaction by another oxidoreductase incorporated in the same reaction system. Is done. In the case of an enzyme reduction reaction, a coenzyme regeneration reaction uses a reaction in which glucose is oxidized with glucose oxidase to produce glucuronic acid, or a reaction in which formic acid is oxidized with formate dehydrogenase to formaldehyde. In these reactions, the oxidized coenzyme receives electrons and protons from glucose or formic acid and is reduced to a reduced coenzyme. However, this method has the disadvantages that the reaction system becomes complicated and the cost increases because it is necessary to introduce the enzyme and substrate required for coenzyme regeneration in addition to the enzyme required for the target reaction. In addition, there remains a problem that the cost is increased because the instability of enzyme activity and the inability to reuse the enzyme activity. Furthermore, in the system after the completion of the reaction, the enzyme used for the target reaction, the coenzyme, the enzyme used for the coenzyme regeneration, the substrate, and the products produced by the coenzyme regeneration reaction are mixed in addition to the target substance. It is also disadvantageous in terms of purification of the target substance.

  On the other hand, there is a method in which an enzyme gene that catalyzes a target reaction and an enzyme gene required for a coenzyme regeneration reaction are incorporated into a microbial cell and a target substance is produced by a reaction using the microbial cell (for example, Patent Document 3). reference.). This method is more advantageous than the above enzyme reaction from the viewpoint of purification of the target substance, but the coenzyme regeneration system substrate and product are not mixed with the target substance in the reaction system. In addition, although it is a general issue of bacterial cell reaction, it is necessary to suppress the problem of cell membrane permeability of the reaction substrate and target product and the generation of by-products caused by undesired reactions due to enzymes originally present in the bacterial cell. There is a problem.

  Also disclosed is a method of regenerating a coenzyme by an indirect electrochemical reaction using a chemical substance that catalyzes the transfer of electrons between the electrode and the coenzyme in the production of a compound by an oxidation-reduction reaction using an enzyme. (For example, refer to Patent Document 4). In this case, the oxidized coenzyme produced by the target reaction is regenerated to the original reduced coenzyme by receiving electrons from the electrode via the catalyst. Therefore, according to this method, the enzyme and the substrate required for coenzyme regeneration are not required, so that the reaction system is simplified. However, in the method disclosed here, since the enzyme used for the target reaction, the coenzyme, and the catalyst for coenzyme regeneration are mixed in the reaction solution, there remains a problem in terms of purification of the product.

  In Patent Document 5, for the purpose of coenzyme regeneration, a polymer layer having an amino group is formed on a carbon plate by electrolytic polymerization, and an electron transfer mediator is fixed via the amino group, and the electron transfer mediator is fixed. An electrode is further disclosed in which a coenzyme and / or an oxidoreductase for regenerating the coenzyme is immobilized on the formed electrode. Since these electrodes are exclusively electrodes for the coenzyme regeneration reaction, when synthesizing a chemical substance having optical activity, an enzyme that catalyzes the target reaction must be separately added to the reaction system. For this reason, this technique still has problems regarding reuse of the target enzyme, stability of activity, and purification of the target substance.

Japanese Patent No. 4426437 JP 2002-233395 A Japanese Patent Application Laid-Open No. 2004-1559587 Special table 2003-533208 gazette Japanese Patent Laid-Open No. 06-153904

  Chemical synthesis reactions using enzymes are very useful because they are substrate-specific, site-specific, and stereospecific. However, coenzymes are expensive, so they are expensive when producing large quantities of chemicals. Disadvantageous. In order to solve this problem, a method of combining a target reaction and a coenzyme regeneration reaction and a method using a microorganism incorporating a target enzyme gene and a gene that catalyzes the coenzyme regeneration reaction have been developed. These methods are economical because the target reaction can be performed while regenerating the coenzyme, but the substrate necessary for the coenzyme regeneration reaction is necessary, the purification of the target substance becomes complicated, and the by-product is There are problems such as generating. Therefore, it is desired to establish a method for regenerating the coenzyme by a simple method and easily purifying the target substance.

  The present inventors have intensively studied to solve the above problems. As a result, an enzyme-modified electrode in which an enzyme necessary for producing a target chemical substance, a coenzyme corresponding to the enzyme, an enzyme necessary for coenzyme regeneration, and an electron transfer mediator are fixed to a conductor, By using an enzyme-modified electrode containing a certain amount or more of an electron transfer mediator, it becomes possible to produce an electrode that can catalyze a highly selective reaction with a low overvoltage. In addition, effective use of enzymes, coenzymes, electron transfer mediators, and electrodes The present invention has been completed by finding out that leakage to the outside can be prevented and that the electrode can be used continuously and for a long time.

That is, the present invention has the following configuration.
[1] A polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, an enzyme 1, an enzyme 2, and an enzyme-modified electrode comprising a conductor, wherein the enzyme 1 is the electron transfer An enzyme that catalyzes electron transfer between a mediator and the coenzyme, wherein the enzyme 2 is an enzyme or enzyme group that catalyzes a target reaction, and the coenzyme is a coenzyme for the enzyme 2, The polymer compound having the amino group to which the electron transfer mediator is fixed by a covalent bond covers at least a part of the surface of the conductor to form a first coating layer, and has the carboxy group A polymer compound coats at least a part of the surface of the first coating layer to form a second coating layer, and the polymer compound having a carboxy group includes Enzyme 1, wherein the coenzyme and the enzyme 2 is fixed by a covalent bond, the content per unit area of the electron transfer mediator is 10 nmol / cm ² or more, the enzyme modified electrode.
[2] At least one of the enzyme 1 and the enzyme 2 is covalently fixed on the surface of the first coating layer or in the layer of the second coating layer, [1] The enzyme-modified electrode according to 1.
[3] The enzyme-modified electrode according to [1] or [2], wherein the enzyme 2 is an enzyme that catalyzes a redox reaction.
[4] The polymer compound having an amino group is polyallylamine, the polymer compound having a carboxy group is polyacrylic acid, the electron transfer mediator is pyrroloquinoline quinone, and the coenzyme is Nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, the enzyme 1 is diaphorase, the enzyme 2 is an enzyme containing at least one of formate dehydrogenase, alcohol dehydrogenase or aldehyde dehydrogenase, The enzyme-modified electrode according to any one of [1] to [3], wherein the conductor is graphite felt.
[5] A method for producing an enzyme-modified electrode in which a polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, enzyme 1 and enzyme 2 are immobilized on a conductor, The enzyme 1 is an enzyme that catalyzes electron transfer between the electron transfer mediator and the coenzyme, the enzyme 2 is an enzyme or a group of enzymes that catalyzes a target reaction, and the coenzyme is the enzyme 2 The conductor is immersed in a solution obtained by mixing the polymer compound having the amino group in a reaction solution of the electron transfer mediator and the condensing agent, and is applied to at least a part of the surface of the conductor. Forming a first coating layer, and then dipping in a solution containing the polymer compound having a carboxy group to form a second coating on at least a part of the surface of the first coating layer Forming, said the second coating layer, wherein the enzyme 1, comprising the step of sequentially securing the coenzyme and the enzyme 2, the manufacturing method of the enzyme modified electrode.
[6] A method for producing an enzyme-modified electrode in which a polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, enzyme 1 and enzyme 2 are immobilized on a conductor, The enzyme 1 is an enzyme that catalyzes electron transfer between the electron transfer mediator and the coenzyme, the enzyme 2 is an enzyme or a group of enzymes that catalyzes a target reaction, and the coenzyme is the enzyme 2 The conductor is immersed in a solution obtained by mixing the polymer compound having the amino group in a reaction solution of the electron transfer mediator and the condensing agent, and is applied to at least a part of the surface of the conductor. A step of forming a first coating layer, a step of fixing the enzyme 1 to the first coating layer, before the enzyme 1 is fixed in a solution containing the polymer compound having a carboxy group A step of immersing the conductor on which the first coating layer is formed to form a second coating layer on at least a part of the surface of the first coating layer; and the coenzyme in the second coating layer And a method for producing an enzyme-modified electrode, comprising the step of sequentially fixing the enzyme 2.
[7] The method for producing an enzyme-modified electrode according to [5] or [6], wherein the enzyme 2 modified with the coenzyme is fixed to the second coating layer.
[8] An electrochemical reaction apparatus for continuously or discontinuously performing an electroenzymatic reaction using an oxidoreductase as a catalyst, and at least one of the above-described [1] to [4] An electrochemical reaction device comprising a pair of electrodes that are enzyme-modified electrodes according to claim 1, a solvent containing an electrolyte, and a substrate.
[9] A production method for producing a chemical substance by an oxidation reaction or a reduction reaction using the electrochemical reaction device according to [8].

  By using the enzyme-modified electrode of the present invention, the reaction can be carried out with a simple reaction system and at a low cost as compared with the case where a chemical substance is produced using a conventional enzymatic method. Can be made to react more quickly and with higher current efficiency. Moreover, since all the components related to the reaction are fixed to the electrode, the target substance from the reaction system can be easily purified. In particular, the asymmetric reduction reaction of ketones using a biocatalyst can be performed with a simple reaction system and at low cost.

  According to the enzyme-modified electrode of the present invention, the chemical substance is oxidized or reduced by the catalytic action of the enzyme and the electrons supplied from the power source. Since this reaction is an enzyme reaction, it proceeds under mild conditions, and since all substances necessary for the target reaction and the coenzyme regeneration reaction are immobilized on the electrode, the current efficiency is high. That is, most of the input electrons are consumed in a reaction that generates a product by oxidizing or reducing a chemical substance through regeneration of the coenzyme.

  According to the enzyme-modified electrode of the present invention and the electrochemical reaction apparatus using the same, the corresponding aldehydes or alcohols from the carboxylic acids and the corresponding alcohols from the aldehydes and / or ketones are efficient under mild conditions. Can be manufactured automatically. Moreover, the corresponding aldehydes or carboxylic acids can be efficiently produced from the alcohols, and the corresponding carboxylic acids can be efficiently produced from the aldehydes under mild conditions. Furthermore, the alcohols and / or aldehydes produced by this method can be produced in a state having stereospecificity because they use enzymes as catalysts. Similarly, according to the enzyme-modified electrode of the present invention and the electrochemical reaction device using the same, not only methanol but also formaldehyde or formic acid can be produced by reducing carbon dioxide, and formamide or formic acid can be obtained by oxidizing methanol. Alternatively, carbon dioxide, formaldehyde can be oxidized to formic acid or carbon dioxide, and formic acid can be oxidized to produce carbon dioxide.

It is a figure which shows the measurement result by the cyclic voltammetry of the pyrroloquinoline quinone modification graphite felt electrode 1 of the comparative example 1. It is a figure which shows the measurement result by the cyclic voltammetry of the pyrroloquinoline quinone modification graphite felt electrode 2 of Example 1. FIG. It is a figure which shows the measurement result of the gas chromatography and the high performance liquid chromatography in methanol production using the electrode of Examples 2, 3 and Comparative Example 2. It is a figure which shows the measurement result by the cyclic voltammetry of the electrode of Example 3 in various substrate concentration. It is a figure which shows the measurement result of the gas chromatography in a methanol production using the electrode of Example 3, 4 and a high performance liquid chromatography. It is a figure which shows the measurement result of the gas chromatography in a methanol production using the electrode of the comparative example 3, and a high performance liquid chromatography. 6 is a diagram showing measurement results of gas chromatography and high performance liquid chromatography in methanol production using the electrode of Example 5. FIG. It is a figure which shows the measurement result of the gas chromatography in a methanol production using the electrode of Example 6, and a high performance liquid chromatography.

(Enzyme modified electrode)
In the enzyme-modified electrode of the present invention, a polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, an enzyme 1, and an enzyme 2 are fixed to a conductor. The polymer compound having an amino group and the polymer compound having a carboxy group are charged polymer compounds. An electron transfer mediator is a compound having a function of mediating electron transfer between a conductor and a coenzyme. Enzyme 1 is an enzyme that catalyzes electron transfer between an electron transfer mediator and a coenzyme. Enzyme 2 is an enzyme or enzyme group that catalyzes the target reaction. The conductor is a felt made of a fibrous conductive material, and a polymer compound having an amino group covers at least a part of the surface of the conductor to form a first coating layer, An electron transfer mediator is fixed on one coating layer by a covalent bond, and a polymer compound having a carboxy group covers at least a part of the surfaces of the electron transfer mediator and the first coating layer, Two coating layers are formed, and a coenzyme and an enzyme are immobilized on the second coating layer by a covalent bond.

In the enzyme-modified electrode of the present invention, the content per unit area of the electron transfer mediator is 10 nmol / cm ² or more. Content per unit area of the electron transfer mediator in the enzyme modified electrode is preferably 10~50nmol / cm ^2, more preferably 15~30nmol / cm ^2. If the content of the electron transfer mediator is 20 nmol / cm ² or more, it is preferable because electron transfer proceeds reversibly.

  In addition, the enzyme-modified electrode of the present invention has a reduction potential and a potential when it is oxidized again after the reduction reaction (hereinafter referred to as “reoxidation potential”) in terms of electrode reaction behavior when measured by cyclic voltammetry (CV). It is preferable that it is 0.04-0.2V. The difference between the reduction potential and the reoxidation potential represents the magnitude of the resistance of the enzyme-modified electrode. It can be seen that the smaller the difference, the faster the electron transfer rate. If the difference between the reduction potential and the reoxidation potential is 0.2 V or less, it is preferable because electron transfer proceeds quasi-reversibly, and if it is 0.059 V or less, it is more preferable because electron transfer proceeds reversibly. And it is preferable for the difference between the reduction potential and the reoxidation potential to be 0.04 V or more, since this indicates that the multi-layer structure is fixed. The difference between the reduction potential and the reoxidation potential is more preferably 0.04 to 0.1 V, further preferably 0.05 to 0.07 V, and most preferably 0.05 to 0.059 V.

(conductor)
The conductor used in the present invention is a felt made of a conductive material. The conductor may be any material as long as it is capable of transferring electrons by being electrically connected to an external circuit when the enzyme-modified electrode is used. Examples thereof include glassy carbon. In particular, from the viewpoint of the size of the specific surface area, a felt made of graphite (hereinafter sometimes referred to as graphite felt) is preferable.

(Enzymes and coenzymes)
The enzyme 1 used in the present invention is an enzyme that catalyzes the redox reaction between the electron transfer mediator and the coenzyme, the enzyme 2 is an enzyme that catalyzes the redox reaction, and the coenzyme performs the redox reaction. Any substance may be used as long as it corresponds to the enzyme 2 to be catalyzed, and can be appropriately selected depending on the reaction system of the chemical substance to be produced. For example, enzyme 1 can include diaphorase and the like, enzyme 2 can include enzymes classified in enzyme number (EC) group 1 such as formate dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase and the like, and nicotinamide as a coenzyme Adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, thiamine diphosphate, vitamin B6, pantothenic acid, biotin, folic acid, vitamin B12, pyrroloquinoline quinone, topaquinone, tryptophan-triple Examples include coenzymes such as tofil quinone, lysine tyrosyl quinone, cystenyl-tryptophan quinone and the like, and these can be used in appropriate combination depending on the intended compound to be produced.

(Electronic transfer mediator)
The electron transfer mediator used in the present invention may be any as long as it mediates electron transfer between the conductor and the coenzyme. For example, ferrocene, potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, phenazine Examples include methosulfate, p-benzoquinone, 2,6-dichlorophenolindophenol, methylene blue, potassium β-naphthoquinone-4-sulfonate, phenazine etsulfate, vitamin K, viologen, pyrroloquinoline quinone, and the like.

(Polymer compound having amino group, polymer compound having carboxy group)
Examples of the polymer compound having an amino group and the polymer compound having a carboxy group include polyallylamine and polyacrylic acid. When a polymer compound having no charge is used as the polymer compound, the electron transfer mediator, enzyme, and coenzyme cannot be covalently immobilized to the polymer compound having an amino group and the polymer compound having a carboxy group. . Therefore, it is necessary to use a polymer compound having a charge as the polymer compound having an amino group and the polymer compound having a carboxy group. In this case, as the polymer compound having a carboxy group, a polymer compound having the same charge as the polymer compound having an amino group in water, or a polymer compound having a charge paired with the polymer compound having an amino group in water is used. Can be used. Compared to the case where a polymer compound having the same charge as a polymer compound having an amino group in water is used as the polymer compound having a carboxy group, the polymer having a charge paired with the polymer compound having an amino group in water It is preferable to use a compound because the polymer compound having a carboxy group is stably coated on the surface of the polymer compound having an amino group by electrostatic interaction between the two polymer compounds.

  In order to covalently bond an enzyme, a coenzyme and an electron transfer mediator to a reactive functional group of a polymer compound having an amino group or a polymer compound having a carboxy group, for example, the amino group and the carboxy group are dehydrated. It is preferable to use a condensing agent for condensation (for example, water-soluble carbodiimide).

  The method of immobilizing the enzyme, the coenzyme and the electron transfer mediator to the conductor via a covalently bonded polymer compound having an amino group or a polymer compound having a carboxy group is not particularly limited. The body is immersed in a solution containing an enzyme, a coenzyme, an electron transfer mediator, a polymer compound having the above functional group, and a condensing agent, or the solution is applied to a conductor, sprayed, and then dried. Thus, a conductor coated with a polymer compound in which an enzyme, a coenzyme and a conductor are covalently bonded can be obtained. The above-mentioned enzyme, coenzyme and electron transfer mediator may be fixed in one step, or divided into two or more steps, for example, an electron transfer mediator is attached to a conductor via a polymer compound having an amino group. After fixing, enzyme 1, coenzyme and enzyme 2 may be fixed on the coated polymer compound having amino group, but the electron transfer mediator and enzyme 1 are fixed through the polymer compound having amino group. After that, it is more preferable to coat the electrode on which the electron transfer mediator and the enzyme 1 are fixed with a polymer compound having a carboxy group, and then fix the coenzyme and the enzyme 2 sequentially or simultaneously.

(Production of enzyme-modified electrode)
The production of the enzyme-modified electrode is not particularly limited, but can be performed, for example, as follows. For example, a pyrroloquinoline quinone (electron transfer mediator) and a methanol aqueous solution obtained by reacting the pyrroloquinoline quinone with a condensing agent (for example, water-soluble carbodiimide) for dehydration condensation of an amino group and a carboxyl group have an amino group. A graphite felt coated with polyallylamine covalently bonded to pyrroloquinolinequinone can be obtained by immersing graphite felt in an aqueous methanol solution mixed with a molecular compound (for example, polyallylamine) and then drying under reduced pressure. Next, an aqueous methanol solution containing, for example, diaphorase (enzyme 1) and a condensing agent (for example, water-soluble carbodiimide) for dehydration condensation of an amino group and a carboxyl group is coated with polyallylamine in which the pyrroloquinoline quinone is covalently bonded. The graphite felt covered with the polyallylamine in which the pyrroloquinoline quinone and the diaphorase are covalently bonded can be obtained by immersing the graphite felt and drying under reduced pressure. Further, after immersing the graphite felt coated with polyallylamine in which the pyrroloquinoline quinone and diaphorase are covalently bonded to an aqueous methanol solution containing a polymer compound having a carboxy group (for example, polyacrylic acid), polyacrylic acid is dried under reduced pressure. Thus, it is possible to obtain a graphite felt coated with polyallylamine in which pyrroloquinoline quinone and diaphorase are covalently bonded. Furthermore, polyallylamine in which pyrroloquinoline quinone coated with polyacrylic acid and diaphorase are covalently bonded to an aqueous methanol solution containing alcohol dehydrogenase (enzyme 2), nicotinamide adenine dinucleotide (coenzyme), and water-soluble carbodiimide. After immersing the coated graphite felt and drying under reduced pressure, the graphite felt coated with a polymer compound in which pyrroloquinoline quinone, diaphorase, alcohol dehydrogenase, and nicotinamide adenine dinucleotide are covalently fixed is used. An enzyme-modified electrode can be produced.

  In the present invention, in the above preparation method, polyacrylic acid is bonded after diaphorase is covalently bonded to the amino group of polyallylamine which has not been reacted with pyrroloquinoline quinone of graphite felt coated with polyallylamine covalently bonded to pyrroloquinoline quinone. The graphite felt coated with polyallylamine covalently bonded with pyrroloquinolinequinone is immersed in an aqueous methanol solution containing a polymer compound having a carboxy group (for example, polyacrylic acid) and dried under reduced pressure. After obtaining graphite felt coated with polyallylamine covalently bonded with pyrroloquinoline quinone coated with diaphorase, a condensing agent (for example, water-soluble carbodiimide) for dehydration condensation of amino group and carboxyl group Immersed in an aqueous methanol solution, and then dried under reduced pressure, pyrroloquinoline quinone and diaphorase coated with polyacrylic acid may be obtained graphite felt coated with polyallylamine covalently bound.

  In the above production method, a polyacrylic acid is added to an aqueous methanol solution containing alcohol dehydrogenase, nicotinamide adenine dinucleotide, and water-soluble carbodiimide, that is, an aqueous solution in which enzyme 2 and coenzyme are first activated with a condensing agent. A graphite felt coated with polyallylamine covalently bonded to pyrroloquinoline quinone and diaphorase was immersed in the coating, but first, pyrroloquinoline quinone and diaphorase coated with polyacrylic acid in an aqueous solution containing a coenzyme and a condensing agent. After immersing the graphite felt coated with polyallylamine covalently bound to, it may be immersed in an aqueous solution containing enzyme 2 and a condensing agent.

(Electrochemical reactor)
The production of the chemical substance using the enzyme-modified electrode can be performed with an electrochemical reaction device having the following configuration. The electrochemical reaction apparatus includes at least a stabilized power source connected to a power source, the enzyme-modified electrode respectively connected to the stabilized power source, a pair of electrodes, a reference electrode, a tube for ventilating nitrogen gas, etc., exhaust And a water tank with a lid having a water jacket for temperature control and a water jacket for temperature control. Here, the three electrodes are installed so as to be immersed in the solution when the solution is added, and the tip of the tube on the side where nitrogen gas or the like is discharged is below the surface of the solution when the solution is added. So that it is installed in the aquarium. Further, the inside of the water tank is blocked from the outside air by closing the lid. The power source to which the stabilized power source is connected may be a general household outlet, but may also be a natural energy-derived power source such as a fuel cell, solar power generation, hydroelectric power generation, wind power generation, or geothermal power generation.

  When a chemical substance reduction reaction is performed in the electrochemical reaction device having the above-described configuration, after filling a water tank with a chemical substance serving as a substrate and an electrolytic solution, for example, a phosphate buffer solution, a stabilized power source is used between the reference electrode and the enzyme-modified electrode. Is kept at −0.6 ± 0.1V. Moreover, the temperature of the electrolyte in the water tank can be maintained at an appropriate temperature by flowing a temperature-controlled solvent through the water jacket. During energization, the chemical substance serving as a substrate is reduced by receiving electrons and hydrogen ions from the coenzyme by the action of the enzyme at the interface between the solution and the electrode. The coenzyme that is oxidized by passing electrons and hydrogen ions to the chemical substance that is the substrate receives electrons and hydrogen ions from pyrroloquinoline quinone by the action of diaphorase, and becomes a reduced coenzyme again. Furthermore, pyrroloquinoline quinone, which has been oxidized by passing electrons and hydrogen ions to the oxidized coenzyme, has electrons derived from the power source and hydrogen ions present in the electrolyte via the conductors that make up the enzyme-modified electrode. And becomes reduced pyrroloquinoline quinone again. Through the above series of reactions, the chemical substance serving as the substrate is reduced by the catalytic action of the enzyme, and the electrons and hydrogen ions consumed in this reaction are supplied from the power source and the electrolyte, respectively. In the case of an oxidation reaction, the above series of reactions proceeds in the reverse direction.

(Enzyme modified electrode for producing L-phenylalaninol)
Hereinafter, the case where racemic phenylalanine is reduced to produce L-phenylalaninol will be described as an example.
In that case, the first coating layer is coated with polyallylamine in which pyrroloquinoline quinone, which is an electron transfer mediator, and diaphorase are covalently bonded, and then, as the second coating layer, alcohol dehydrogenase, a coenzyme of the alcohol dehydrogenase, is coated. An enzyme-modified electrode that is a graphite felt coated with polyacrylic acid covalently bonded to nicotinamide adenine dinucleotide can be used.

  The alcohol dehydrogenase used here is not limited to its origin, so long as it has an activity defined by EC 1.1.1.1, EC 1.1.1.2, or EC 1.1.1.71. The diaphorase may be an enzyme having an activity defined by EC1.8.1.4 regardless of its origin.

  Further, the nicotinamide adenine dinucleotide used here may be oxidized or reduced, regardless of its origin, may be its free acid or salt, and may be fixed in any state. The sodium salt is more preferred. Furthermore, the pyrroloquinoline quinone used here may be oxidized or reduced, regardless of its origin, may be its free acid or salt, and may be fixed in any state. Acid is more preferred.

(Method for producing L-phenylalaninol)
Production of L-phenylalaninol by reduction of racemic phenylalanine using the enzyme-modified electrode can be carried out in an electrochemical reaction apparatus having the following constitution. The electrochemical reaction apparatus includes a stabilized water source connected to a power source, the enzyme-modified electrode connected to the stabilized power source, a pair of electrodes, a reference electrode, and a water tank with a lid having a water jacket for temperature control. Composed. Here, the three electrodes are installed so as to be immersed in the solution when the solution is added. Further, the inside of the water tank is blocked from the outside air by closing the lid. The power source to which the stabilized power source is connected may be a general household outlet, but may also be a natural energy-derived power source such as a fuel cell, solar power generation, hydroelectric power generation, wind power generation, or geothermal power generation.

  When producing L-phenylalaninol by reduction of racemic phenylalanine, the water tank is filled with an electrolyte, for example, racemic phenylalanine containing a phosphate buffer, and nitrogen gas is added to the racemic phenylalanine containing the electrolyte. It is preferable to keep the potential between the reference electrode and the enzyme-modified electrode at -0.5 ± 0.1 V with a stabilized power supply after removing oxygen present in the solution by aeration. Moreover, the temperature of the electrolyte in the water tank can be maintained at an appropriate temperature by flowing a temperature-controlled solvent through the water jacket. During energization, only L-phenylalanine receives electrons and hydrogen ions from nicotinamide adenine dinucleotide and is reduced to L-phenylalaninol by the action of alcohol dehydrogenase at the interface between the solution and the electrode. By the way, nicotinamide adenine dinucleotide that has been oxidized by passing electrons and hydrogen ions to L-phenylalanine receives electrons and hydrogen ions from pyrroloquinoline quinone by the action of diaphorase, and becomes reduced nicotinamide adenine dinucleotide again. . In addition, pyrroloquinoline quinone, which has been oxidized by passing electrons and hydrogen ions to oxidized nicotinamide adenine dinucleotide, is present in the electrolyte and the electrons derived from the power source via the conductor constituting the enzyme-modified electrode. It receives hydrogen ions and becomes reduced pyrroloquinoline quinone again.

  Through the above series of reactions, L-phenylalanine is reduced to L-phenylalaninol by the catalytic action of the enzyme, and electrons and hydrogen ions consumed in this reaction are supplied from a power source and an electrolyte, respectively. This reduction reaction proceeds unless the supply of electrons from the power source is interrupted or until there is no L-phenylalanine in the electrolyte, and L-phenylalaninol accumulates in the electrolyte. Moreover, it is possible to produce | generate L-phenylalaninol continuously by continuing supplying L-phenylalanine.

  The present invention will be described in detail by the following examples, but the present invention is not limited to these examples.

  The definitions of the abbreviations of the present invention are as follows.

GF: graphite felt (specific surface area: 0.7 m ² / g), PAA: polyallylamine, PAAc: polyacrylic acid, PQQ: pyrroloquinoline quinone, NADH: nicotinamide adenine dinucleotide, FDH: formate dehydrogenase, ADH: alcohol dehydrogenase , Dp: diaphorase, WSC: water-soluble carbodiimide, SCE: saturated calomel electrode, Ag / AgCl: silver / silver chloride electrode, polyacrylic acid (weight average molecular weight 1,400,000 manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyallylamine (weight average molecular weight 70,000 made by Nittobo)

[Measurement by cyclic voltammetry (CV)]
An “electrochemical QCM analyzer (ALS model 420A)” manufactured by BBS was used.
Measurement conditions were as follows: an electrode to be evaluated as a working electrode, a platinum wire as a counter electrode, SCE as a reference electrode, or Ag / AgCl in 10 ml of 0.1 M phosphate buffer (pH 7). After removing nitrogen gas for 10 minutes to remove oxygen, the voltage was continuously changed within a certain range with respect to the reference electrode at a sweep rate of 50 mV / sec. Recording was performed using a Y recorder (WX1200).

[Measurement by gas chromatography]
“Mass spectrometer JMS-MS700V” manufactured by JEOL Ltd. was used.
Measurement conditions were: InertCap 1 0.25 mm inner diameter, 30 m long glass capillary column packed with dimethylpolysiloxane, column temperature, 100 ° C., sample inlet temperature 300 ° C., detector temperature 250 ° C., argon gas 3.0 kg / cm ^2. (294.2 KPa). For all of methanol, formic acid, formaldehyde, amino acids, and 2-amino alcohols, a standard material sample having a qualitative and known concentration was prepared based on the retention time with the standard material, and a calibration curve was drawn for quantification.

[Measurement by high performance liquid chromatography]
“UV-2075”, “PU-2080”, and “CO-2065” manufactured by JASCO Corporation were used.
The measurement conditions are for formic acid and formaldehyde, an ODS-120 stainless steel column (made by Tosoh Corporation) with an inner diameter of 4.6 mm and a length of 15 cm, and a mobile phase of 0.9 mass% phosphoric acid aqueous solution (water distilled twice) ), Flow rate 0.5 mL / min. Column temperature 30 ° C. For amino acids and 2-amino alcohols, CHIROBIOTIC T stainless steel column (manufactured by Sigma-Aldrich) having an inner diameter of 4.6 mm and a length of 25 cm, the mobile phase was methanol containing 15 mM ammonium formate, and the flow rate was 1 mL / min. Formic acid, formaldehyde, amino acids, and 2-aminoalcohols were each qualitatively prepared with a standard substance sample having a known concentration based on the retention time with the standard substance, and a calibration curve was drawn for quantification.

  The water used in the present invention is purified water having a conductivity of 18 MΩcm made by a pure water production apparatus (Elix-UV3, 60 L tank, Milli-Q Academic, ASM) manufactured by Nihon Millipore.

Comparative Example 1
(Preparation of PQQ-modified GF electrode 1)
In 5 ml of a 60 (v / v)% methanol aqueous solution, 100 μl of PAA (manufactured by Nitto Boseki Co., Ltd., weight average molecular weight 70,000), PQQ 5 μmol and WSC 6 μmol were dissolved. GF (5 cm × 2 cm × 0.5 cm) was immersed in this solution at room temperature for 72 hours to be reacted. After completion of the reaction, GF was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. The electrode thus obtained was designated as PQQ-modified GF electrode 1.

Example 1
(Preparation of PQQ-modified GF electrode 2)
5 μmol of PQQ was dissolved in 5 ml of 60 (v / v)% methanol aqueous solution, 6 μmol of WSC was added and mixed for 2 minutes, and then 100 μl of PAA was added. GF (5 cm × 2 cm × 0.5 cm) was immersed in this solution at room temperature for 72 hours to be reacted. After completion of the reaction, GF was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. The electrode thus obtained was designated as PQQ-modified GF electrode 2.

Test example 1
(Evaluation by CV of PQQ-modified GF electrodes with different production methods)
The electrode reaction behavior of the PQQ modified GF electrode 1 and the PQQ modified GF electrode 2 was evaluated by CV. PQQ-modified GF electrode 1 (1 cm × 1 cm × 0.5 cm) or PQQ-modified GF electrode 2 (1 cm × 1 cm × 0.5 cm) as working electrode in 10 ml of 0.1 M phosphate buffer (pH 7), platinum as counter electrode As the wire and reference electrode, SCE was immersed in the case of the PQQ-modified GF electrode 1, and Ag / AgCl was immersed in the case of the PQQ-modified GF electrode 2. After removing nitrogen gas for 10 minutes to remove oxygen, the PQQ-modified GF electrode 1 is set to −1.0 V to 0.4 V with respect to the SCE, and the PQQ-modified GF electrode 2 is set to Ag / AgCl. On the other hand, the voltage was continuously changed between -1.0 V and 0.6 V at a sweep rate of 50 mV / sec. The result of the PQQ modified GF electrode 1 is shown in FIG. 1, and the result of the PQQ modified GF electrode 2 is shown in FIG.

A significant increase in peak current was confirmed in the PQQ-modified GF electrode 2 than in the PQQ-modified GF electrode 1. Moreover, the reduction potential of the PQQ-modified GF electrode 1 was −0.3 V with respect to SCE, the reoxidation potential was −0.1 V with respect to SCE, and the difference between them was 0.2 V. On the other hand, the reduction potential of the PQQ-modified GF electrode 2 was −0.15V with respect to Ag / AgCl, the reoxidation potential was −0.09V with respect to Ag / AgCl, and the difference between them was 0.06V. It was. Since the difference between the reduction potential and the reoxidation potential of the PQQ modified GF electrode 2 is smaller than the difference between the reduction potential and the reoxidation potential of the PQQ modified GF electrode 1, the electron transfer rate is higher for the PQQ modified GF electrode 2 than for the PQQ modified GF electrode 2. It became clear that it was faster than the electrode 1. Further, the difference between the reduction potential and the reoxidation potential when reversible electron transfer is performed is generally said to be 0.059 V, and reversible electron transfer is performed at the PQQ-modified GF electrode 2. It became clear that A fixed amount of PQQ than the area of the reduction wave was calculated to be 20 nmol / cm ² in PQQ-modified GF electrode 1 at 0.2 nmol ^/ cm 2, PQQ modified GF electrode 2. From the above results, the PQQ-modified GF electrode 2 has a fixed amount of PQQ increased by 100 times compared to the PQQ-modified GF electrode 1, and as a result, the performance has been remarkably improved. Here, the usefulness of the PQQ-modified GF electrode prepared in Example 1 was shown.

Comparative Example 2
(Preparation of enzyme-immobilized PQQ-modified GF electrode 1)
PQQ-modified GF electrode prepared in Comparative Example 1 in 5 ml of 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000) 1 (5 cm × 2 cm × 0.5 cm) was soaked at room temperature for 2 hours and allowed to stand, and then dried under reduced pressure to prepare a PQQ-modified GF electrode 1 coated with PAAc. This electrode was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 5 mM NADH and 6 μmol WSC for 72 hours at room temperature. After completion of the reaction, GF was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 1 covered with the NAAc-immobilized PAAc was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 20 μM Dp and 6 μmol WSC at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 1 coated with PAAc on which Dp and NADH are fixed is immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of ADH and 6 μmol of WSC at room temperature for 72 hours. It was. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 1.

Example 2
(Preparation of enzyme-immobilized PQQ-modified GF electrode 2)
PQQ-modified GF electrode prepared in Example 1 in 5 ml of 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000) 2 (5 cm × 2 cm × 0.5 cm) was soaked at room temperature for 2 hours and allowed to stand, and then dried under reduced pressure to prepare a PQQ-modified GF electrode 2 coated with PAAc. This electrode was immersed in 5 ml of a 60 (v / v)% aqueous methanol solution containing 20 μM Dp and WSC 6 μmol for 72 hours at room temperature. After completion of the reaction, GF was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 coated with PAAc to which Dp was immobilized was immersed in 5 ml of a 60 (v / v)% aqueous methanol solution containing 5 mM NADH and 6 μmol of WSC at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 coated with PAAc on which NADH and Dp are fixed is immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of ADH and WSC 6 μmol at room temperature for 72 hours. It was. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 2.

Example 3
(Preparation of enzyme-immobilized PQQ-modified GF electrode 3)
Dp20 μM and WSC 6 μmol were dissolved in 5 ml of 60 (v / v)% methanol aqueous solution and mixed for 2 minutes, and then the PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) prepared in Example 1 was immersed for 72 hours at room temperature. I let you. After completion of the reaction, GF was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, a PQQ-modified GF electrode in which Dp is fixed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000). 2 (5 cm × 2 cm × 0.5 cm) was reacted at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, after dissolving NADH 5 mM and WSC 6 μmol in 60 ml of 60 (v / v)% methanol aqueous solution, PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) fixed with PAAc-coated Dp was soaked at room temperature for 72 hours. Reacted. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, 2.5 enzyme units of ADH and 6 μmol of WSC were dissolved in 5 ml of 60 (v / v)% methanol aqueous solution and mixed for 2 minutes, and then PQQ-modified GF electrode 2 on which Dp coated with PAAc on which NADH was immobilized was immobilized. (5 cm × 2 cm × 0.5 cm) was immersed and reacted at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 3.

Test example 2
(Effect of electric potential on electrolytic reaction using enzyme-immobilized PQQ-modified GF electrode)
The electrode prepared in Example 2 (1 cm × 1 cm × 0.5 cm) as a working electrode and a platinum plate (2 cm as a counter electrode) in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM D, L-phenylalanine × 2 cm × 0.1 cm), and Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then −0.50 V, or −0.55 V, or −0.60 V, or −0 with respect to Ag / AgCl under a nitrogen gas atmosphere. An electric potential was applied so as to be .65V or −0.70V, and the current was applied. A part of the reaction solution was withdrawn at regular intervals, and the product (L-phenylalaninol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. Table 1 shows the results.

  From the results shown in Table 1, it was found that L-phenylalanine was selectively reduced to produce L-phenylalaninol when any set potential was applied. D-phenylalanine was recovered unreacted. Although the electrolysis time is shortened as the set potential is increased, the recovery rate of D-phenylalanine is deteriorated and the current efficiency is lowered. However, L-phenylalaninol may be produced in a yield of almost 100% ee. did it.

Test example 3
(Comparison of methanol productivity by electrolytic reduction of formic acid using enzyme-immobilized PQQ-modified GF electrodes 1 to 3)
An electrode (1 cm × 1 cm × 0.5 cm) prepared in Example 2, 3 or Comparative Example 2 as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM formic acid, and a platinum plate as a counter electrode (2 cm × 2 cm × 0.1 cm), Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was extracted at regular intervals, and the product (methanol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The results are shown in FIG.

  After the start of the reaction, the enzyme-immobilized PQQ-modified GF electrode 1 (Comparative Example 2) is 7 hours, and the enzyme-immobilized PQQ-modified GF electrode 2 (Example 2) is 45 minutes. In Example 3), the reaction was completed in 40 minutes. In addition, the production amounts per unit time and unit capacity were 0.05, 0.4, and 0.5 g / L / h, respectively, and the enzyme-immobilized PQQ modified electrode 3 showed the highest productivity among the comparisons.

Test example 4
(Effect of substrate concentration on electron transfer of enzyme-immobilized PQQ-modified GF electrode)
The electrode (1 cm × 1 cm × 0.5 cm) prepared in Example 3 as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM, 20 mM, 30 mM, or 40 mM formic acid, counter electrode As a platinum plate (2 cm × 2 cm × 0.1 cm) and Ag / AgCl as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then the voltage was continuously changed at a sweep rate of 50 mV / sec between -1.0 V and 0.6 V with respect to Ag / AgCl. It was. The results are shown in FIG.

  As the formic acid concentration increased, a large reduction current flowed, but 40 mM formic acid did not cause a significant increase in reduction current compared to 30 mM formic acid. An increase in the reduction current indicates an increase in the reaction rate, and it has been clarified that the reaction rate becomes the fastest at a formic acid concentration of 30 mM under the electrolysis conditions used here.

Example 4
(Preparation of enzyme-immobilized PQQ-modified GF electrode 4)
Dp20 μM and WSC 6 μmol were dissolved in 5 ml of 60 (v / v)% methanol aqueous solution and mixed for 2 minutes, and then the PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) prepared in Example 1 was immersed for 72 hours at room temperature. I let you. After completion of the reaction, GF was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, a PQQ-modified GF electrode in which Dp is fixed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000). 2 (5 cm × 2 cm × 0.5 cm) was reacted at room temperature for 2 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, the PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) on which Dp coated with PAAc was fixed was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 6 μmol of WSC for 5 minutes, and then 25 μmol of NADH. Was added and allowed to react for 72 hours at room temperature. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, 6 μmol of WSC was added to 5 ml of a 60 (v / v)% aqueous methanol solution containing 2.5 enzyme units of ADH and stirred for 2 minutes. The solution was immersed in the PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) on which Dp was coated with PAAc on which NADH was immobilized, and reacted at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 4.

Test Example 5
(Comparison of methanol productivity by electroreduction of formic acid using enzyme-immobilized PQQ-modified GF electrodes 3 and 4)
The electrode produced in Example 3 or 4 (1 cm × 1 cm × 0.5 cm) as a working electrode and a platinum plate (2 cm × 2 cm) as a counter electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 30 mM formic acid × 0.1 cm), Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was extracted at regular intervals, and the product (methanol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The results are shown in FIG.

  After the start of the reaction, the reaction was completed in about 2.5 hours with the enzyme-immobilized PQQ-modified GF electrode 3 (Example 3) and about 1 hour with the enzyme-immobilized PQQ-modified GF electrode 4 (Example 4). The production amounts per unit time and unit volume were 0.72 and 1.8 g / L / h, respectively, and the enzyme-immobilized PQQ modified electrode 4 showed higher productivity than the enzyme-immobilized PQQ modified electrode 3.

  From the results of Test Example 3 and Test Example 5, it was shown that the method for producing the enzyme-immobilized PQQ-modified GF electrode shown in Example 4 was the most excellent.

Test Example 6
(Reusability of enzyme-immobilized PQQ-modified GF electrode 3)
The electrode prepared in Example 3 (1 cm × 1 cm × 0.5 cm) as a working electrode and a platinum plate (2 cm × 2 cm × 0) as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM formic acid. 0.1 cm), Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was extracted at regular intervals, and the product (methanol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The same operation was repeated 5 times using the same electrode. Table 2 shows the results.

  From the results of Table 2, as the electrode is repeatedly used, the electrolysis time and the amount of energization increase and the current efficiency decreases, so that the enzyme, coenzyme, and electron transfer mediator fixed to GF may drop off, Alternatively, the enzyme may be deactivated, but it was found that methanol can be obtained in a yield of 100% even when the enzyme-immobilized PQQ-modified GF electrode 3 is repeatedly used.

Test Example 7
(Reusability of enzyme-immobilized PQQ-modified GF electrode 4)
The electrode prepared in Example 4 (1 cm × 1 cm × 0.5 cm) as a working electrode and a platinum plate (2 cm × 2 cm × 0) as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 30 mM formic acid. 0.1 cm), Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was extracted at regular intervals, and the product (methanol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The same operation was repeated 5 times using the same electrode. Table 3 shows the results.

  From the results shown in Table 3, almost no change was observed in electrolysis time, energization amount, and current efficiency in the repeated operation of 5 times. Therefore, the enzyme-immobilized PQQ-modified GF electrode 4 can obtain methanol with a yield of 100% even when used repeatedly, and the enzyme, coenzyme, and electron transfer mediator immobilized on GF are stably held. I found out.

Test Example 8
(Production of L-2-amino alcohol and D-amino acid from D, L-amino acid using enzyme-immobilized PQQ-modified GF electrode)
The electrode prepared in Example 2 (1 cm × 1 cm × 0.5 cm) as a working electrode and a platinum plate (2 cm × 2 cm × 0) as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM amino acid. 0.1 cm), Ag / AgCl was immersed as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was withdrawn at regular intervals, and the product was qualitatively and quantitatively analyzed by high performance liquid chromatography. The above operations are performed for phenylalanine, alanine, and valine. From D, L-phenylalanine to L-phenylalaninol and D-phenylalanine, from D, L-alanine to L-alaninol and D-alanine, and from D, L-valine to L -Valinol and D-valine were produced respectively. The results are shown in Table 4.

  From the results in Table 4, the corresponding 2-aminoalcohol and D-amino acid were obtained from any of the amino acids used with an enantiomeric excess of 99% ee or more. Therefore, the enzyme-immobilized PQQ-modified GF electrode using ADH can be used not only to produce methanol by electroreducing formic acid but also to produce 2-amino alcohol from L-amino acid. It has been shown. Furthermore, the electrode was shown to be L-form specific as a reaction substrate.

Test Example 9
(Comparison of electrocatalytic reduction reaction of D, L-phenylalanine using enzyme-immobilized PQQ-modified GF electrodes 1 to 4)
The electrode prepared in Comparative Example 2, Example 2, Example 3 or Example 4 (1 cm × 1 cm) as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM D, L-phenylalanine × 0.5 cm), a platinum plate (2 cm × 2 cm × 0.1 cm) as a counter electrode, and Ag / AgCl as a reference electrode. Nitrogen gas was bubbled for 10 minutes to remove dissolved oxygen in the solution, and then a potential was applied to the Ag / AgCl at −0.65 V in a nitrogen gas atmosphere to energize. A part of the reaction solution was withdrawn at regular intervals, and the product was qualitatively and quantitatively analyzed by high performance liquid chromatography. L-phenylalaninol and D-phenylalanine were respectively produced from D, L-phenylalanine. The results are shown in Table 5.

  From the results of Table 5, the yield was 98% or more for all, and the enantiomeric excess was 99% ee or more for all. With the four types of electrodes used, the reaction proceeded the fastest with the electrode prepared in Example 4. The relationship between each electrode shown here and the electrolysis time is consistent with the results of the electrolysis reaction of formic acid shown in Test Example 3 and Test Example 5, and in order to improve the performance of the enzyme-modified electrode, the PQQ fixation amount is increased. It was confirmed that fixing Dp to the PAA layer and adding NADH after activating PAAc with WSC were important.

Comparative Example 3
(Preparation of enzyme-immobilized PQQ-modified GF electrode for carbon dioxide reduction 1)
PQQ-modified GF electrode prepared in Comparative Example 1 in 5 ml of 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000) 1 (5 cm × 2 cm × 0.5 cm) was soaked at room temperature for 2 hours and allowed to stand, and then dried under reduced pressure to prepare a PQQ-modified GF electrode 1 coated with PAAc. This electrode was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 5 mM NADH and 6 μmol WSC for 72 hours at room temperature. After completion of the reaction, GF was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 1 covered with the NAAc-immobilized PAAc was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 20 μM Dp and 6 μmol WSC at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 1 coated with PAAc on which Dp and NADH are fixed is immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of ADH and 6 μmol of WSC at room temperature for 72 hours. It was. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 1 coated with PAAc on which ADH, Dp, and NADH were immobilized was immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of FDH and WSC 6 μmol at room temperature for 72 hours. Reacted. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 1 for carbon dioxide reduction.

Example 5
(Production of enzyme-immobilized PQQ-modified GF electrode for carbon dioxide reduction 2)
PQQ-modified GF electrode prepared in Example 1 in 5 ml of 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000) 2 (5 cm × 2 cm × 0.5 cm) was soaked at room temperature for 2 hours and allowed to stand, and then dried under reduced pressure to prepare a PQQ-modified GF electrode 2 coated with PAAc. This electrode was immersed in 5 ml of a 60 (v / v)% aqueous methanol solution containing 20 μM Dp and WSC 6 μmol for 72 hours at room temperature. After completion of the reaction, GF was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 coated with PAAc to which Dp was immobilized was immersed in 5 ml of a 60 (v / v)% aqueous methanol solution containing 5 mM NADH and 6 μmol of WSC at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 coated with PAAc on which NADH and Dp are fixed is immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of ADH and WSC 6 μmol at room temperature for 72 hours. It was. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 coated with PAAc to which ADH, NADH, and Dp are immobilized is immersed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of FDH and WSC 6 μmol at room temperature for 72 hours. Reacted. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 2 for carbon dioxide reduction.

Example 6
(Production of enzyme-immobilized PQQ-modified GF electrode for carbon dioxide reduction 3)
Dp20 μM and WSC 6 μmol were dissolved in 5 ml of 60 (v / v)% methanol aqueous solution and mixed for 2 minutes, and then the PQQ-modified GF electrode 2 (5 cm × 2 cm × 0.5 cm) prepared in Example 1 was immersed for 72 hours at room temperature. I let you. After completion of the reaction, GF was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, a PQQ-modified GF electrode in which Dp is fixed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 0.1 (w / v)% PAAc (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 1,400,000). 2 (5 cm × 2 cm × 0.5 cm) was reacted at room temperature for 2 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, followed by rinsing three times with 100 ml of 30 (v / v)% aqueous methanol solution, and then dried under reduced pressure. Next, the PQQ-modified GF electrode 2 on which Dp coated with PAAc was fixed was immersed in 5 ml of a 60 (v / v)% aqueous methanol solution containing 5 mM NADH and 6 μmol of WSC at room temperature for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 on which Dp is covered with the PAAc on which NADH is fixed is placed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of ADH and 6 μmol of WSC at room temperature for 72 hours. Immerse and react. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. Next, the PQQ-modified GF electrode 2 on which Dp is coated with PAAc on which ADH and NADH are fixed is placed in 5 ml of a 60 (v / v)% methanol aqueous solution containing 2.5 enzyme units of FDH and WSC 6 μmol at room temperature. The reaction was immersed for 72 hours. After completion of the reaction, the reaction mixture was dried under reduced pressure, rinsed three times with a 30 (v / v)% aqueous methanol solution and dried under reduced pressure. The electrode thus obtained was designated as an enzyme-immobilized PQQ-modified GF electrode 3 for carbon dioxide reduction.

Test Example 10
(Production comparison of methanol from carbon dioxide using enzyme-immobilized PQQ-modified GF electrodes 1 to 3 for carbon dioxide reduction)
An electrode (2 cm × 1 cm × 0.5 cm) prepared in Example 5, Example 6 or Comparative Example 3 as a working electrode in 10 ml of 0.1 M phosphate buffer (pH 7) containing 0.3 M sodium bicarbonate. ), A platinum plate as a counter electrode, and Ag / AgCl as a reference electrode. Carbon dioxide gas was bubbled through the phosphate buffer solution for 5 minutes, and then a potential was applied to −0.65 V with respect to Ag / AgCl. A part of the reaction solution was extracted at regular intervals, and the product (methanol) was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The results are shown in FIGS.

  As shown in FIG. 6, the reaction is completed in 12 hours with the enzyme-immobilized PQQ-modified GF electrode 1 for carbon dioxide reduction after the start of the reaction. As shown in Fig. 8, the reaction was completed in 8 hours, and the reaction was completed in 6 hours in the enzyme-immobilized PQQ-modified GF electrode 3 for reducing carbon dioxide, as shown in Fig. 8. Further, the production amounts per unit time and unit capacity are 0.007, 0.011, and 0.015 g / L / h, respectively, and the carbon dioxide reducing enzyme immobilized PQQ-modified GF electrode 2 is immobilized with the carbon dioxide reducing enzyme. Productivity 1.6 times higher than PQQ-modified GF electrode 1, and the enzyme-immobilized PQQ-modified GF electrode 3 for carbon dioxide reduction is 1.4 times higher than the enzyme-immobilized PQQ-modified GF electrode 2 for carbon dioxide reduction Productivity was shown. From this result, it was shown that the method for producing the enzyme-immobilized PQQ-modified GF electrode for carbon dioxide reduction shown in Example 6 was the most excellent. The difference between the enzyme-immobilized PQQ-modified GF electrode 1 for carbon dioxide reduction and the enzyme-immobilized PQQ-modified GF electrode 2 for carbon dioxide reduction differs only in the amount of PQQ immobilized, and the enzyme-immobilized PQQ-modified GF electrode 2 for carbon dioxide reduction. The carbon dioxide reducing enzyme-immobilized PQQ-modified GF electrode 3 is different only in the order of Dp fixation. As described above, the result that the amount of PQQ fixation and the order of Dp fixation affect the performance of the enzyme-modified electrode supports the result shown in Test Example 3.

Test Example 11
(Methanol production from formic acid by flow-type electrolytic layer)
HX-201 manufactured by Hokuto Denko Co., Ltd. was used for the flow type electrolytic layer. The electrode (diameter 0.8 cm × 5 cm) produced in Example 2 processed into a cylindrical shape as a working electrode, a platinum wire (diameter 0.3 mm × 300 mm) as a counter electrode, and Ag / AgCl as a reference electrode were used. After removing dissolved oxygen by bubbling nitrogen gas through 50 ml of 0.1 M phosphate buffer (pH 7) containing 10 mM formic acid and 0.3 M sodium hydrogen carbonate for 10 minutes, it becomes −0.5 V against Ag / AgCl. Thus, the liquid was passed through the flow-type electrolytic layer to which a potential was applied at a constant flow rate. The effluent was collected, and the product was qualitatively and quantitatively analyzed by gas chromatography and high performance liquid chromatography. The conversion rate of formic acid to methanol is the ratio of formic acid charged into the flow-type electrolytic layer to methanol, and is calculated from the formula of formic acid in the effluent / amount of formic acid before charging × 100. The The results are shown in Table 6.

  From the results of Table 6, the conversion rate of formic acid into methanol was 100% at a flow rate of 0.05 to 0.20 mL / min. However, the current efficiency decreased as the flow rate increased, and the conversion rate was 40% at a flow rate of 1.0 mL / min. The electrolytic reaction by the flow type electrolytic layer is greatly influenced by the performance, size and flow rate of the electrode. Further, the conversion rate can be made 100% even at a high flow rate by using a circulation type.

Claims (9)

A polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, an enzyme 1, an enzyme 2, and an enzyme-modified electrode comprising a conductor,
The enzyme 1 is an enzyme that catalyzes electron transfer between the electron transfer mediator and the coenzyme,
The enzyme 2 is an enzyme or enzyme group that catalyzes a target reaction,
The coenzyme is a coenzyme for the enzyme 2;
The polymer compound having the amino group to which the electron transfer mediator is fixed by a covalent bond covers at least a part of the surface of the conductor to form a first coating layer;
The polymer compound having a carboxy group coats at least part of the surface of the first coating layer to form a second coating layer;
The enzyme 1, the coenzyme and the enzyme 2 are fixed to the polymer compound having a carboxy group by a covalent bond,
The content per unit area of the electron transfer mediator is 10 nmol / cm ² or more,
Enzyme modified electrode.   The enzyme according to claim 1, wherein at least one of the enzyme 1 and the enzyme 2 is covalently immobilized on the surface of the first coating layer or in the layer of the second coating layer. Modified electrode.   The enzyme-modified electrode according to claim 1 or 2, wherein the enzyme 2 is an enzyme that catalyzes a redox reaction. The polymer compound having an amino group is polyallylamine,
The polymer compound having a carboxy group is polyacrylic acid,
The electron transfer mediator is pyrroloquinoline quinone;
The coenzyme is nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate;
The enzyme 1 is diaphorase,
The enzyme 2 is an enzyme containing at least one of formate dehydrogenase, alcohol dehydrogenase or aldehyde dehydrogenase,
The enzyme-modified electrode according to any one of claims 1 to 3, wherein the conductor is graphite felt. A method for producing an enzyme-modified electrode comprising a conductor having a polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, enzyme 1 and enzyme 2,
The enzyme 1 is an enzyme that catalyzes electron transfer between the electron transfer mediator and the coenzyme,
The enzyme 2 is an enzyme or enzyme group that catalyzes a target reaction,
The coenzyme is a coenzyme for the enzyme 2;
The conductor is immersed in a solution in which the polymer compound having the amino group is mixed in a reaction solution of the electron transfer mediator and a condensing agent, and a first coating layer is formed on at least a part of the surface of the conductor. Forming step,
Next, a step of immersing in a solution containing the polymer compound having a carboxy group to form a second coating layer on at least a part of the surface of the first coating layer;
Including sequentially fixing the enzyme 1, the coenzyme and the enzyme 2 to the second coating layer,
A method for producing an enzyme-modified electrode. A method for producing an enzyme-modified electrode comprising a conductor having a polymer compound having an amino group, a polymer compound having a carboxy group, an electron transfer mediator, a coenzyme, enzyme 1 and enzyme 2,
The enzyme 1 is an enzyme that catalyzes electron transfer between the electron transfer mediator and the coenzyme,
The enzyme 2 is an enzyme or enzyme group that catalyzes a target reaction,
The coenzyme is a coenzyme for the enzyme 2;
The conductor is immersed in a solution in which the polymer compound having the amino group is mixed in a reaction solution of the electron transfer mediator and a condensing agent, and a first coating layer is formed on at least a part of the surface of the conductor. Forming step,
Immobilizing the enzyme 1 on the first coating layer;
A conductor containing the first coating layer on which the enzyme 1 is fixed is immersed in a solution containing the polymer compound having a carboxy group, and is applied to at least a part of the surface of the first coating layer. Forming a second coating layer;
Including sequentially fixing the coenzyme and the enzyme 2 to the second coating layer,
A method for producing an enzyme-modified electrode.   The method for producing an enzyme-modified electrode according to claim 5 or 6, wherein the enzyme 2 modified with the coenzyme is fixed to the second coating layer.   An electrochemical reaction device for continuously or discontinuously carrying out an electroenzymatic reaction using an oxidoreductase as a catalyst, and at least one of the reaction vessels according to any one of claims 1 to 4, An electrochemical reaction device comprising a pair of electrodes that are enzyme-modified electrodes, a solvent containing an electrolyte, and a substrate.   A production method for producing a chemical substance by an oxidation reaction or a reduction reaction using the electrochemical reaction device according to claim 8.

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