KR20090023541A - Electrochemical method for cofactor regeneration - Google Patents

Abstract

A method for electrochemically regenerating a coenzyme used for a biosensor or an enzymatic biofuel cell is provided to simply regenerate the coenzyme at low costs. A method for electrochemically regenerating a coenzyme includes oxidization of the coenzyme by directly delivering electron to a metal oxide electrode or reduction of the coenzyme by directly accepting the electron from the metal oxide electrode. The metal of the metal oxide electrode shows the second to fourteenth group and the third to sixth period among the periodic table of the chemical elements. The metal of the metal oxide electrode is selected from the group consisting of tin, iridium, titanium, chrome, copper, manganese, iron, tungsten, nickel, zinc, niobium, magnesium, aluminium, ruthenium, lead and their mixtures.

Description

Electrochemical Regeneration of Coenzyme {ELECTROCHEMICAL METHOD FOR COFACTOR REGENERATION}

The present invention relates to a method for regenerating a coenzyme using an electrochemical method, an enzymatic redox method using the same, and an enzyme biosensor and an enzyme fuel cell. More specifically, the coenzyme may be directly delivered or delivered to a metal oxide electrode. It is oxidized or reduced, and is characterized in that no electron transfer medium is used.

Redox enzymes are widely used in the synthesis of high value-added fine chemicals, pharmaceuticals, food additives or herbicides. Such oxidoreductases require coenzymes such as nicotinamide adenine dinucleotides (including reduced forms and phosphates. NAD (H), NADP (H)) and the like. Coenzymes are low molecular weight substances and are essential for enzymatic reactions. In particular, pyridine-based coenzymes are widely used. In the reaction using oxidoreductase, it is necessary to supply a constant coenzyme to produce a product, but the problem is that the cost is very high.

In order to solve this problem, many researchers are conducting researches to regenerate coenzymes. Such methods include enzyme-based methods, photochemical methods, and electrochemical methods.

Enzyme-based method is to use a separate enzyme to regenerate coenzyme. In order to use this method, it is necessary to add a substrate that can react with the enzyme required to regenerate the coenzyme. Have. The most widely used enzyme for the regeneration of coenzyme using enzyme is formate dehydrogenase, which generates carbon dioxide as a by-product. The use of this enzyme has the advantage of low price of formic acid used as a substrate and easy separation of the product.However, the use of enzyme increases the overall process cost, and the activity of the enzyme decreases due to changes in the surrounding environment. There is a problem of lowering the efficiency.

The photochemical method uses a photosensitizer or a semiconductor to convert light energy into chemical energy, and the reaction mechanism is very complicated and stays conceptually.

The electrochemical method is a method of using an electrode as an electron acceptor or electron donor that deprives or supplies electrons necessary for redox of coenzyme. This method is economical because it does not require a separate enzyme for regenerating the coenzyme and uses low-cost electricity. However, electron transfer media should be used because direct electron transfer between the coenzyme and the electrode is difficult. That is, in the reduction reaction of the coenzyme, the electron transfer medium receives electrons from the electrode and is reduced, and the electrons transferred from the reduced electron transfer medium to the coenzyme are oxidized to reduce the crude enzyme. Examples of widely used electron transfer mediators include viologen-based dyes, ferrocene derivatives, and quinone-based materials. These electron transfer mediators sometimes not only cause the problem of inhibiting the activity of enzymes, but also have the trouble of immobilizing not only enzymes and coenzymes but also electron transfer mediators in order to perform a continuous reaction.

The present invention has been made to solve the problems of the prior art as described above, an object of the present invention is to provide a new electrochemical coenzyme regeneration method that does not use a conventional electron transfer medium.

Another object of the present invention is to provide an enzymatic redox method using the coenzyme regenerated by the electrochemical coenzyme regeneration method.

Another object of the present invention is to provide an enzyme biosensor or an enzyme fuel cell, wherein the coenzyme used is regenerated by the electrochemical coenzyme regeneration method.

Electrochemical coenzyme regeneration method of the present invention to achieve the object as described above,

The coenzyme is oxidized by directly transferring electrons to the metal oxide electrode,

The coenzyme is reduced by receiving electrons directly from the metal oxide electrode.

It is characterized by.

In addition, the metal of the metal oxide is a group 2 to 14 of the periodic table, it is preferable that the third to sixth cycle.

In addition, the metal of the metal oxide is preferably selected from the group consisting of tin, iridium, titanium, chromium, copper, manganese, iron, tungsten, nickel, zinc, niobium, magnesium, aluminum, ruthenium, lead, and mixtures thereof.

In addition, the coenzyme is nicotinamide adenine dinucleotide (NAD ⁺ ), reduced nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate. ⁺ ), Reduced nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), flavin adenine dinucleotide (FAD ⁺ ), and reduced flavin adenine dinucleotide, is selected from the group consisting of a reduced form. FADH ₂₎ are preferred.

In addition, the metal oxide electrode

Anodizing the metal, and

Annealing the anodized metal

It may be prepared by a manufacturing method comprising a.

In addition, the metal is preferably tin.

In addition, the metal oxide electrode

Coating a metal on the glass electrode, and

Heat-treating the coated metal

It may be prepared by a manufacturing method comprising a.

In addition, the metal is preferably titanium.

In addition, the metal oxide electrode may be prepared by mixing 100 parts by weight of the metal oxide, 2 to 25 parts by weight of carbon black and 2 to 25 parts by weight of the binder.

In addition, the binder is polytetrafluoroethylene, poly (vinylidene fluoride), poly (vinylidene chloride), poly (acrylic ether sulfone), poly (ether ether ketone), and Nafion ^® (a sulfonated tetrafluoroethylene air It is preferable to select from the group consisting of).

On the other hand, the enzymatic redox method of the present invention is characterized in that it comprises an oxidation step or a reduction step of the coenzyme regenerated by the electrochemical coenzyme regeneration method.

In addition, the enzyme of the enzymatic redox method may be dehydrogenase (dehydrogenase), reductase (reductase), or oxidase (oxygenase).

In addition, the enzyme of the enzymatic redox method is alcohol dehydrogenase (Lcohol dehydrogenase), L-leucine dehydrogenase (L-leucine dehydrogenase), L-alanine dehydrogenase (L-alanine dehydrogenase), L-phenylalanine dehydrogenase (L) -phenylalanine dehydrogenase, D-lactate dehydrogenase, R-hydroxy isocarproate dehydrogenase, L-lactate dehydrogenase, carnitine dehydrogenase Carnitine dehydrogenase, dihydrofolate reductase, glycerol dehydrogenase, menthone reductase, xylose reductase, glucose dehydrogenase , Aldose reductase, 12a-hydroxysteroid dehydrogenase, 3a-hydroxysteroid dehydrogenase (3a- hydroxysteroid dehydrogenase, styrene monooxygenase, carbonyl reductase, glutamate dehydrogenase, formate dehydrogenase, glucose-6-p-dehydrogenase 6-p-dehydrogenase, morphine dehydrogenase, morphinone reductase, or cyclohexanone monooxygenase.

On the other hand, the enzyme biosensor of the present invention is characterized in that the coenzyme is regenerated by the electrochemical coenzyme regeneration method.

On the other hand, the enzyme fuel cell of the present invention is characterized in that the coenzyme is regenerated by the electrochemical coenzyme regeneration method.

The coenzyme regeneration method of the present invention can simply regenerate the coenzyme at low cost, and can be applied to various enzyme reactions. Accordingly, the present invention has an advantage that can be widely applied to various fields such as biosensors, biofuel cells as well as electrochemical bioreactors.

The present invention is characterized in that the coenzyme is oxidized by directly transferring electrons to the metal oxide electrode, or the coenzyme is regenerated by reducing the coenzyme by receiving electrons directly from the metal oxide electrode, thereby eliminating the need for a separate electron transfer medium. .

The method for producing such a metal oxide electrode is not limited as long as it functions as an electrode, for example, a method of anodizing and subsequently annealing a metal, coating and heat-treating a metal on a glass electrode, and mixing the metal oxide with carbon black and the like The method of bonding with a binder, etc. are mentioned. Of course, the electrode manufacturing method is not limited thereto.

Specifically, using a metal tin plate having a purity of 99.9% or more as a positive electrode, an aluminum plate as a negative electrode, and an aqueous 10 to 1000 mM oxalic acid solution as an electrolyte, supplying a voltage between 5 and 15 V, and anodizing for 1 to 10 minutes. After the reaction, black tin oxide (SnO) is obtained, and the white tin oxide (SnO ₂ ) obtained by annealing at 400 to 650 ° C. for 2 to 5 hours is used as an electrode of the present invention.

Alternatively, a titanium dioxide electrode may be prepared by applying a mixture of an aqueous polyethylene glycol solution and an aqueous titanium dioxide solution to a fluorinated tin oxide glass electrode and then heat-treating the same at 100 to 500 ° C. for 20 minutes to 2 hours.

In addition, it is also possible to produce an electrode by mixing an oxide such as iridium with carbon black, adding and mixing a binder and fixing it to a mold.

The binder is not limited to the surface of the synthetic resin that can bond the metal oxide and carbon black to each other, but polytetrafluoroethylene, poly (vinylidene fluoride), poly (vinylidene chloride), poly (acrylic ether sulfone), poly (ether ether ketone), and Nafion ^® is preferably selected from the group consisting of (a sulfonated tetrafluoroethylene copolymer).

As described above, the metal oxide electrode used in the present invention is not limited to the preparation method as long as it can directly exchange electrons with the coenzyme.

Regeneration of the coenzyme is carried out using the obtained metal oxide electrode. As a preferred example, the metal oxide electrode is a working electrode, the platinum electrode is a counter electrode, and the Ag / AgCl electrode is a reference electrode. In the electrochemical reactor used as a reference electrode), the coenzyme regeneration reaction can be performed by a known method. In this reaction, the coenzyme can be reduced by directly transferring electrons generated from the metal oxide electrode to the coenzyme without using a separate electron transfer medium.

A redox enzyme reaction is carried out using the coenzyme regenerated by the above method. In a preferred embodiment, the metal oxide electrode is used in an electrochemical reactor using a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. Enzymatic redox method can be performed using the regenerated coenzyme.

The electrochemical coenzyme regeneration method and the enzymatic redox method using the regenerated coenzyme of the present invention described above can be applied to various fields such as bioreactors. In particular, in systems involving redox reactions by enzymes such as biosensors and fuel cells, the coenzyme required for the enzyme reaction is more preferably regenerated by the electrochemical coenzyme regeneration method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to Examples.

Example

Example  1-1. Fabrication of Tin Oxide Electrode

Anodization was carried out using a metal tin plate having a purity of 99.9% or more as an anode. An aqueous 500 mM oxalic acid solution was used as an electrolyte, and an aluminum plate having a purity of 99.9% or more was used as a cathode. In order to anodize, a voltage of 8 V was supplied and the reaction time was 2 minutes. As a result, black tin oxide (SnO) was obtained. Annealing was performed for 3 hours at 500 ° C. to obtain white tin oxide (SnO ₂ ).

Example  1-2. Electrochemical Characterization of Tin Oxide

In order to analyze the electrochemical properties of tin oxide, cyclic voltammetry was performed using potentiostat. The tin oxide was analyzed in phosphate buffer solution of pH 7.5, 100 mM using working electrode, platinum electrode as counter electrode and Ag / AgCl electrode as reference electrode. The scan range was -0.2 to -1.2 V and the scan speed was 20 mV / Sec. As a result of drawing the graph from the experimental results, it was confirmed that the tin oxide has a redox potential. As shown in FIG. 2, it was confirmed that the oxidation potential was around -0.5 V and the reduction potential was near -0.9V. It was confirmed that the manufactured tin oxide electrode can be used to regenerate coenzyme without adding an electron transfer medium because tin oxide on the surface of the electrode functions as an electron transfer medium.

Example  1-3. Regeneration of Coenzyme Using Tin Oxide Electrodes

The experiment was carried out using an electrochemical reactor for coenzyme regeneration. The reaction progress is schematically shown in FIG. The tin oxide prepared in Example 1-1 was reacted using a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. The size of the working electrode was 2 × 2.5 cm, and the externally supplied voltage was -0.5 V for oxidation and -0.95 V for reduction. The initial concentration of the coenzyme (NADH or NAD ⁺ ) was 0.5 mM, which was reacted in a phosphate buffer solution of pH 7.5, 100 mM in a volume of 20 ml. Concentration analysis of the coenzyme was performed using a UV spectrophotometer.

3 and 4 are graphs showing the redox reaction of the coenzyme with time when the tin oxide according to the present invention is used as a working electrode. Figure 3 shows a tendency to decrease the concentration of NAD (P) H because the coenzyme NAD (P) H is oxidized, Figure 4 shows a tendency to increase the concentration because the coenzyme NAD ⁺ is reduced and converted to NADH Is showing.

Example  1-4. Using coenzyme regenerated with tin oxide electrode Enzymatic  Redox Method

The actual redox enzyme reaction was examined using the coenzyme regeneration method described above. The tin oxide produced from Example 1 was reacted using a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. The size of the working electrode was 2 × 2.5 cm, and the external voltage applied to the oxidation reaction was -0.5 V. Alcohol dehydrogenase was used as redox enzyme and 0.5 mM NADP ⁺ was used as coenzyme. The material used as a substrate was 2-propanol, and the initial concentration was 10 mM. It was reacted in a volume of 20 ml at pH 7.5, 100 mM phosphate buffer solution, 40 ^° C. The concentration of reactants and products was analyzed by gas chromatography.

5 is a graph showing the conversion rate with time in the enzyme reaction using the tin oxide according to the present invention as a working electrode. It gave it in the initial reaction NADP ⁺ NADPH is the reduced form is reduced by the enzyme reaction is re-played to NADP ⁺ by the oxidation reaction at the tin oxide electrode showed a greater than 90% conversion.

Example  2-1. Fabrication of Titanium Dioxide Electrode

Fluorine doped tin oxide (FTO) glass electrode is mixed with 20 g of polyethylene glycol (PEG) and 1 g / 3 ml of titanium dioxide (TiO ₂ ) in the same amount and applied on FTO glass. The titanium dioxide coated FTO glass was heat-treated at a temperature of 300 ° C. for 1 hour to pyrolyze the PEG, leaving only titanium dioxide on the FTO glass electrode. (The thickness of titanium dioxide can be adjusted by the amount applied on the glass.)

Example  2-2. Electrochemical Characterization of Titanium Dioxide

In order to analyze the electrochemical properties of titanium dioxide, a cyclic voltammetry experiment was conducted using electrostatic potential. Titanium dioxide was analyzed in phosphate buffer solution of pH 7.8, 100 mM, using working electrode, platinum electrode as counter electrode and Ag / AgCl electrode as reference electrode. The scan range was +0.4 to -1.4 V and the scan speed was 20 mV / sec. As a result of drawing the graph from the experimental results, it was confirmed that the titanium dioxide has a redox potential near -0.7 V as shown in FIG. Titanium dioxide electrode produced through this result was confirmed that the titanium dioxide on the surface of the electrode can be used to regenerate the coenzyme without the addition of an electron transfer medium because the role of the electron transfer medium.

Example  2-3. Using Coenzyme Regenerated by Titanium Dioxide Electrode Enzymatic  Redox Method

In the actual redox enzyme reaction, the coenzyme regeneration method described above was used. The reaction was performed using titanium dioxide produced in Example 2-1 using a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. The working electrode was 2.5 × 5 cm in size, and the external voltage for the oxidation reaction was -0.7 V. Alcohol dehydrogenase was used as redox enzyme and 0.5 mM NADPH was used as coenzyme. The material used as a substrate was 2-propanol, and the initial concentration was 5 mM. This was reacted in a volume of 20 ml at pH 7.8, 100 mM phosphate buffer solution, 40 ^° C. The concentration of reactants and products was analyzed by gas chromatography.

Figure 7 is a graph showing the conversion rate with time in the enzyme reaction using titanium dioxide as the working electrode. The NADPH oxidized in the initial reaction is oxidized and the oxidized form of NADP ⁺ is used for the enzymatic reaction, and the reduced form of NADPH produced after the enzymatic reaction is oxidized on the titanium dioxide electrode and regenerated to NADP ⁺ , without the electron transfer mediator. Coenzyme regeneration was performed and enzyme reaction was well progressed.

Example  3-1. Fabrication of Metal Oxide Electrodes

10 mg of the metal oxide of Table 1 was mixed with 1 mg of carbon black and 1 mg of polytetrafluoroethylene, and then fixed to a stainless steel frame to prepare a metal oxide electrode used in the present invention.

Metal oxide Oxidation potential (V) Reduction potential (V) ZnO -0.228, 0.109, 0.249 - Cu ₂ O 0.388 -0.516 Fe ₂ O ₃ -0.002 -0.716 NiO - -0.105 Mn ₃ O ₄ -0.113 - Cr ₂ O ₃ 0.239 -0.213 RuO ₂ - -0.442 IrO ₂ -0.208 -

Example  3-2. Electrochemical Characterization of Metal Oxides

In order to analyze the electrochemical characteristics of metal oxides, cyclic voltammetry was conducted using electrostatic potential. The metal oxide electrode of Example 3-1 was analyzed in a phosphate buffer solution of pH 7.0, 100 mM using the working electrode, the platinum electrode as the counter electrode, and the Ag / AgCl electrode as the reference electrode. The redox potential of the metal oxide is shown in Table 1 above. It was confirmed that the prepared metal oxide electrode can be used to regenerate the coenzyme without the addition of the electron transfer medium because the metal oxide on the electrode surface acts as an electron transfer medium.

Example  3-3. Regeneration of Coenzyme Using Metal Oxide Electrode

The coenzyme regeneration experiment was performed using an electrochemical reactor. The reaction was performed using the metal oxide electrode prepared in Example 3-1 as the working electrode, the platinum electrode as the counter electrode, and the Ag / AgCl electrode as the reference electrode. The size of the working electrode is 0.56 cm ² , the voltage supplied from the outside is shown in FIG. 8. The initial concentration of the coenzyme (NADH or NAD ⁺ ) was 0.5 mM, which was reacted in a phosphate buffer solution of pH 7.0, 50 ° C., 100 mM in a volume of 5 ml. Concentration analysis of coenzyme was carried out using a UV spectrophotometer. 8 is a graph showing the oxidation reaction of the coenzyme with time when the metal oxide electrode according to the present invention is used as a working electrode, and shows a tendency to increase NAD ⁺ because the coenzyme NADH is oxidized.

1 is a schematic diagram of a redox reaction of a coenzyme using an electrode.

2 is a graph showing a cyclic voltage current curve of tin oxide.

3 is a graph showing the oxidation reaction of NAD (P) H using a tin oxide electrode.

4 is a graph showing a reduction reaction of NAD ⁺ using a tin oxide electrode.

5 is a graph showing the conversion of 2-propanol by the reaction of alcohol dehydrogenase when using a tin oxide electrode.

6 is a graph showing a cyclic voltage current curve of titanium dioxide.

7 is a graph showing the conversion of 2-propanol by alcohol dehydrogenase reaction when using a titanium dioxide electrode.

8 is a graph showing the oxidation reaction of NADH in the electrode made of a mixture of metal oxide and carbon black.

Claims (14)

The coenzyme is oxidized by directly transferring electrons to the metal oxide electrode, The coenzyme is reduced by receiving electrons directly from the metal oxide electrode. Electrochemical coenzyme regeneration method characterized in that. The method of claim 1, The metal of the metal oxide is a group 2 to 14 of the periodic table, the electrochemical coenzyme regeneration method, characterized in that the third to sixth cycle. The method of claim 1, The metal of the metal oxide is electrochemically selected from the group consisting of tin, iridium, titanium, chromium, copper, manganese, iron, tungsten, nickel, zinc, niobium, magnesium, aluminum, ruthenium, lead, and mixtures thereof. Coenzyme Regeneration Method. The method of claim 1, The coenzyme is nicotinamide adenine dinucleotide (NAD ⁺ ), reduced nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate.NADP ⁺ ), Reduced nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), flavin adenine dinucleotide (FAD ⁺ ), and reduced flavin adenine dinucleotide, FADH ₂ ) electrochemical coenzyme regeneration method, characterized in that selected from the group consisting of. The method of claim 1, wherein the metal oxide electrode Anodizing the metal, and Annealing the anodized metal Electrochemical coenzyme regeneration method characterized in that prepared by the production method comprising a. The method of claim 5, wherein Electrochemical coenzyme regeneration method, characterized in that the metal is tin. The method of claim 1, wherein the metal oxide electrode Coating a metal on the glass electrode, and Heat-treating the coated metal Electrochemical coenzyme regeneration method characterized in that prepared by the production method comprising a. The method of claim 7, wherein The metal is an electrochemical coenzyme regeneration method, characterized in that titanium. The method of claim 1, wherein the metal oxide electrode An electrochemical coenzyme regeneration method comprising a mixture of 100 parts by weight of a metal oxide, 2 to 25 parts by weight of carbon black and 2 to 25 parts by weight of a binder. The method of claim 9, The binder is an ethylene, poly (vinylidene fluoride) as polytetrafluoroethylene, poly (vinylidene chloride), poly (acrylic ether sulfone), poly (ether ether ketone), and Nafion ^® (a sulfonated tetrafluoroethylene copolymer) Electrochemical coenzyme regeneration method, characterized in that selected from the group consisting of. Enzymatic redox method comprising the step of oxidizing or reducing the coenzyme regenerated by the electrochemical coenzyme regeneration method of any one of claims 1 to 10. The method of claim 11, wherein The enzyme of the enzymatic redox method is an enzymatic redox method, characterized in that the dehydrogenase (dehydrogenase), reductase (reductase), or oxidase (oxygenase). Enzyme biosensor characterized in that the coenzyme is regenerated by the electrochemical coenzyme regeneration method of any one of claims 1 to 10. Enzyme fuel cell, characterized in that the coenzyme is regenerated by the electrochemical coenzyme regeneration method of any one of claims 1 to 10.

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