Description

ELECTROCHEMICAL METHOD FOR COFACTOR REGENERATION

Technical Field

[1] The present invention relates to a method for regenerating a cof actor using an electrochemical method, a method for performing enzymatic oxidation-reduction using the method, an enzymatic biosensor, and an enzymatic fuel cell. More particularly, the present invention is characterized in that a cofactor is oxidized or reduced as a result of direct transfer of electron to or from a metal oxide electrode, and an electron transferring mediator is not used. Background Art

[2] Oxidoreductases are being extensively used in the synthesis of fine chemical products, pharmaceutical products, food additives, herbicides and the like, which have high added- values in an industrial viewpoint. In order for these oxidoreductases to be activated, they need cofactors such as nicotinamide adenine dinucleotide (including the reduced forms and the phosphate, NAD(H), NADP(H)). Cofactors are low molecular weight materials which are essential in enzymatic reactions, and in particular, pyridine nucleotide cofactors are being widely used. In a reaction utilizing an oxidoreductase, a continuous supply of cofactors is required to produce the products, but there arises a problem that large expenses are required.

[3] In order to solve such problems, researchers are extensively conducting research to regenerate cofactors, and such methods include methods of using an enzyme, photochemical methods, electrochemical methods, and the like.

[4] The method of using an enzyme is a method of using a special enzyme for regenerating a cofactor, and use of this method faces problems in that a substrate capable of reacting with the enzyme necessary for the regeneration of the cofactor must be supplied, and thereby side products are generated. The enzyme most widely used in the method for regenerating cofactors using an enzyme, is formate dehydrogenase which generates carbon dioxide as a side product. In the case of using this enzyme, there are advantages that the price of formic acid that is used as the substrate is inexpensive, and separation of the product is easy. However, the use of an enzyme leads to problems that the overall process costs increase, and the activity of the enzyme is deteriorated along with the changes in the surrounding environment, thus the process efficiency being lowered.

[5] The photochemical method is a method of using a photosensitizer or a semiconductor to convert light energy to chemical energy, the method remaining only conceptual

because of its very complicated reaction mechanism.

[6] The electrochemical method is a method of using an electrode as an electron receptor or an electron donor which withdraws or supplies electrons necessary for the oxidation-reduction of cof actors. Since this method does not require any additional enzymes to regenerate cofactors, and makes use of inexpensive electricity, the method may be said to be an economically efficient method. However, the method needs to use an electron transfer mediator, because direct electron transfer between cofactors and the electrode is difficult. That is, in the reduction reaction of a cof actor, the electron transfer mediator is reduced by having electron transferred thereto from the electrode, and the electrons carried by the reduced electron transfer mediator are transferred to the cofactor, so that the electron transfer mediator itself is oxidized while the cofactor is reduced. Examples of widely used electron transfer mediators include viologen dyes, ferrocene derivatives, quinine substances, and the like. These electron transfer mediators often cause a problem of inhibiting the activity of enzymes, and also, in order to perform a reaction in the continuous mode, the process requires cumbersome fixation of the enzyme and the cofactor as well as the electron transfer mediator. Disclosure of Invention Technical Problem

[7] The present invention was designed to solve the problems of the prior art as described above, and it is an object of the present invention to provide a new electrochemical method for cofactor regeneration, which method does not utilize conventional electron transfer mediators.

[8] It is another object of the present invention to provide an enzymatic method for oxidation-reduction, which utilizes a cofactor that has been regenerated by the electrochemical method for cofactor regeneration.

[9] It is still another object of the present invention to provide an enzymatic biosensor or an enzymatic fuel cell, characterized in that the cofactor used therein is regenerated by the electrochemical method for cofactor regeneration. Technical Solution

[10] The electrochemical method for cofactor regeneration of the present invention is characterized in that, in order to achieve the object as described above, electrons are directly transferred to a metal oxide electrode to oxidize the cofactor, or electrons are directly transferred from the metal oxide electrode to reduce the cofactor.

[11] The metal of the metal oxide may belong to Group 2 to Group 14, and Period 3 to

Period 6, in the Periodic Table of Elements.

[12] 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.

[13] The cofactor is preferably selected from the group consisting of nicotinamide adenine dinucleotide (NAD ⁺ ), the reduced form of nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD ⁺ ), and the reduced form of flavin adenine dinucleotide (FADH ).

[14] Furthermore, the metal oxide electrode can be produced by a method for production thereof comprising:

[15] anodizing a metal, and

[16] annealing the anodized metal.

[17] The above-mentioned metal is preferably tin.

[18] The metal oxide electrode can be produced by a method for production thereof comprising:

[19] coating a glass electrode with a metal, and

[20] thermally treating the coated metal.

[21] The above-mentioned metal is preferably titanium.

[22] The metal oxide electrode can be produced by mixing 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.

[23] The binder is preferably selected from the group consisting of polytetrafluo- roethylene, poly(vinylidene fluoride), poly(vinylidene chloride), poly(acryl ether sulfone), poly (ether ether ketone), and Nafion® (sulfonated tetrafluoroethylene copolymer).

[24] Meanwhile, the enzymatic method for oxidation-reduction of the present invention is characterized by comprising oxidizing or reducing a cofactor which has been regenerated by the electrochemical method for cofactor regeneration.

[25] The enzyme used in the enzymatic method for oxidation-reduction may be a dehydrogenase, a reductase, or an oxygenase.

[26] The enzyme used in the enzymatic method for oxidation-reduction may be an alcohol dehydrogenase, a L-leucine dehydrogenase, a L-alanine dehydrogenase, a L- phenylalanine dehydrogenase, a D-lactate dehydrogenase, a R-hydroxyisocaproate dehydrogenase, a L-lactate dehydrogenase, a carnitine dehydrogenase, a dihydrofolate reductase, a glycerol dehydrogenase, a menthone reductase, a xylose reductase, a glucose dehydrogenase, an aldose reductase, a 12a-hydroxysteroid dehydrogenase, a 3 a-hydroxy steroid dehydrogenase, a styrene monooxygenase, a carbonyl reductase, a glutamate dehydrogenase, a formate dehydrogenase, a glucose-6-p-dehydrogenase, a morphine dehydrogenase, a morphinone reductase, or a cyclohexanone monooxygenase.

[27] Meanwhile, the enzymatic biosensor of the present invention is characterized in that

the cofactor is regenerated by the electrochemical method for cofactor regeneration. [28] The enzymatic fuel cell of the present invention is characterized in that the cofactor is regenerated by the electrochemical method for cofactor regeneration.

Advantageous Effects

[29] The method for cofactor regeneration of the present invention is capable of simply regenerating a cofactor at low costs, and can be applied to various enzymatic reactions. Therefore, the present invention has an advantage that the invention can be widely applied to various fields such as electrochemical bioreactors, as well as biosensors and biofuel cells. Brief Description of the Drawings

[30] Fig. 1 is a schematic diagram showing the process of an oxidation-reduction reaction of a cofactor using an electrode.

[31] Fig. 2 is a graph showing a cyclic voltammogram of tin oxide.

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

[33] Fig. 4 is a graph showing the reduction reaction NAD ⁺ using a tin oxide electrode.

[34] Fig. 5 is a graph showing the conversion of 2-propanol through the reaction of an alcohol dehydrogenase in the case of using a tin oxide electrode.

[35] Fig. 6 is a graph showing a cyclic voltammogram of titanium dioxide.

[36] Fig. 7 is a graph showing the conversion of 2-propanol through the reaction of an alcohol dehydrogenase in the case of using a titanium dioxide electrode.

[37] Fig. 8 is a graph showing the oxidation reaction of NADH in an electrode produced with a mixture of a metal oxide and carbon black. Best Mode for Carrying Out the Invention

[38] The most prominent feature of the present invention is that a cofactor is regenerated as electrons are directly transferred to a metal oxide electrode to oxidize the cofactor, or electrons are directly transferred from the metal oxide electrode to reduce the cofactor, and thereby there is no need for any additional electron transfer mediator.

[39] The method for producing such a metal oxide electrode is not limited as long as the electrode functions as an electrode, and there may be mentioned, for example, a method of anodizing and subsequently annealing a metal; a method of coating a glass electrode with a metal, and thermally treating the metal; a method of mixing a metal oxide with carbon black or the like, and binding the mixture with a binder; and the like. As a matter of course, the method for producing an electrode is not limited to these.

[40] Specifically, an anodization reaction is performed for 1 to 10 minutes, by using a metal tin plate having a purity of 99.9% or higher as an anode, an aluminum plate as a cathode, and a 10 to 1000 mM aqueous solution of oxalic acid as an electrolyte, and

applying a voltage between 5 and 15 V, to thus obtain black tin oxide (SnO), and then the black tin oxide is subjected to annealing at 400 to 65O ⁰ C for 2 to 5 hours, to thus obtain white tin oxide (SnO ). This resulting white tin oxide is used as the electrode of the present invention.

[41] Alternatively, a titanium dioxide electrode can also be produced by applying a mixture of an aqueous polyethylene glycol solution and an aqueous titanium dioxide solution onto a fluorinated tin oxide glass electrode, and then thermally treating the resulting electrode at 100 to 500 ⁰ C for 20 minutes to 2 hours.

[42] In addition, an electrode can also be produced by mixing an oxide of iridium or the like with carbon black, adding and mixing a binder to the mixture, and fixing the resulting mixture in a frame.

[43] The binder is not limited as long as it is a synthetic resin capable of binding a metal oxide and carbon black, but it is preferably selected from the group consisting of poly- tetrafluoroethylene, poly(vinylidene fluoride), poly(vinylidene chloride), poly(acryl ether sulfone), poly(ether ether ketone), and Nafion® (sulfonated tetrafluoroethylene copolymer).

[44] As such, the metal oxide electrode used in the present invention is not limited in the method of production thereof, as long as the electrode is capable of performing direct donation and reception of electrons with a cofactor.

[45] A cofactor regeneration reaction is carried out using a metal oxide electrode thus obtained, and as a preferred example, the cofactor regeneration reaction can be carried out by a known method in an electrochemical reactor which utilizes the aforementioned metal oxide electrode as a working electrode, a platinum electrode as a counter electrode, and an Ag/ AgCl electrode as a reference electrode. In this reaction, electrons generated in the metal oxide electrode can be directly transferred to the cofactor to reduce the cofactor, even without using any additional electron transfer mediator.

[46] An enzymatic oxidation-reduction is carried out using a cofactor regenerated by the above-described method, and as a preferred example, the enzymatic method for oxidation-reduction can be carried out using a cofactor regenerated in an electrochemical reactor which utilizes the aforementioned metal oxide electrode as a working electrode, a platinum electrode as a counter electrode, and an Ag/AgCl electrode as a reference electrode.

[47] The electrochemical method for cofactor regeneration of the present invention described above, and the enzymatic method for oxidation-reduction using a cofactor regenerated by the same method, can be applied to various fields such as bioreactors. In particular, in the case of a system accompanied by an oxidation-reduction reaction involving an enzyme, such as a biosensor or a fuel cell, it is more preferable that the

cofactor required in the enzymatic reaction be regenerated by the electrochemical method for cofactor regeneration of the present invention.

[48] Hereinafter, the present invention will be described in more detail by way of

Examples, but the scope of the invention is not intended to be limited to the Examples. Mode for the Invention

[49] Example 1-1. Production of tin oxide electrode

[50] An anodization reaction was performed using a metal tin plate having a purity of

99.9% or higher as an anode. A 500 mM aqueous solution of oxalic acid was used as an electrolyte, and an aluminum plate having a purity of 99.9% or higher was used as a cathode. A voltage of 8 V was applied to carry out the anodization, and the reaction time was set to 2 minutes. As a result, black tin oxide (SnO) could be obtained. This was subjected to annealing at 500 ⁰ C for 3 hours, to thus obtain white tin oxide (SnO ).

[51] Example 1-2. Analysis of electrochemical properties of tin oxide

[52] To analyze the electrochemical properties of tin oxide, a cyclic voltammetry experiment was performed using a potentiostat. The analysis was performed using tin oxide as a working electrode, a platinum electrode as a counter electrode, and an Ag/ AgCl electrode as a reference electrode, in a 100 mM phosphate buffered solution at pH 7.5. The scanning range was from -0.2 to -1.2 V, and the scanning speed was 20 mV/sec. A graph was plotted from the experimental results, and as a result, it could be verified that tin oxide has oxidation and reduction potentials. As shown in Fig. 2, it was confirmed that tin oxide has an oxidation potential near -0.5 V, and a reduction potential near -0.9 V. From this, it was confirmed that since the tin oxide electrode thus produced can be used in the regeneration of a cofactor, even though an electron transfer mediator is not additionally added, because the tin oxide on the electrode surface plays a role as an electron transfer mediator.

[53] Example 1-3. Regeneration of cofactor using tin oxide electrode

[54] An experiment for cofactor regeneration was performed using an electrochemical reactor. The process procedure of the reaction is schematically shown in Fig. 1. The reaction was carried out using the tin oxide produced in the Example 1-1 above as 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 2x2.5 cm, and the voltage applied from an external source was -0.5 V during the oxidation reaction, and -0.95 V during the reduction reaction. The initial concentration of the cofactor (NADH or NAD ⁺ ) was 0.5 mM, and this cofactor was used in the reaction in a volume of 20 ml, in a 100 mM phosphate buffered solution at pH 7.5. The analysis of the cofactor concentration was carried out using a UV spectrophotometer.

[55] Fig. 3 and Fig. 4 are graphs showing the oxidation-reduction reaction of the cofactor

over time, in the case of using the tin oxide according to the present invention as a working electrode. Fig. 3 shows a tendency of a decrease in the concentration of the cofactor NAD(P)H, because NAD(P)H is oxidized, while Fig. 4 shows a tendency of an increase in the concentration, because the cofactor NAD ⁺ is reduced and converted to NADH.

[56] Example 1-4. Enzymatic method for oxidation-reduction using cofactor regenerated with tin oxide electrode

[57] The method for cofactor regeneration described above was used for an actual enzymatic oxidation-reduction reaction. The reaction was carried out using the tin oxide produced in Example 1 as 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 2x2.5 cm, and the voltage applied from an external source for the oxidation reaction was -0.5 V. An alcohol dehydrogenase was used as the oxi- doreductase, and 0.5 mM NADP ⁺ was used as the cofactor. The substance used as the substrate was 2-propanol, and the initial concentration was 10 mM. This system was used in the reaction in a volume of 20 ml, in a 100 mM phosphate buffered solution at pH 7.5, at 4O ⁰ C. An analysis of the concentrations of the reactant and the product was performed using gas chromatography.

[58] Fig. 5 is a graph showing the conversion rate over time in an enzymatic reaction using the tin oxide according to the present invention as a working electrode. NADP ⁺ introduced in the initial reaction was reduced by the enzymatic reaction, and the reduced form, NADPH, underwent an oxidation reaction at the tin oxide electrode to be regenerated again to NADP ⁺ , so that a conversion rate of 90% or higher was exhibited.

[59] Example 2-1. Production of titanium dioxide electrode

[60] A 20 mM aqueous solution of polyethylene glycol (PEG) and an aqueous solution of titanium dioxide (TiO ) at 1 g/3 ml were mixed in equal amounts, and the mixture was applied on a fluorine-doped tin oxide (FTO) glass electrode. The FTO glass coated with titanium dioxide was thermally treated at a temperature of 300 ⁰ C for 1 hour to thermally degrade and remove PEG, and only titanium dioxide was allowed to remain on the FTO glass electrode (at this time, the thickness of titanium dioxide can be controlled by the amount applied on the glass).

[61] Example 2-2. Analysis of electrochemical properties of titanium dioxide

[62] To analyze the electrochemical properties of titanium dioxide, a cyclic voltammetry experiment was performed using a potentiostat. The analysis was performed using titanium dioxide as a working electrode, a platinum electrode as a counter electrode, and an Ag/ AgCl electrode as a reference electrode, in a 100 mM phosphate buffered solution at pH 7.8. The scanning range was from +0.4 to -1.4 V, and the scanning

speed was 20 mV/sec. A graph was plotted from the experimental results, and as a result, it could be verified that titanium dioxide has a redox potential near -0.7 V, as shown in Fig. 6. From this, it was confirmed that since the titanium dioxide electrode thus produced can be used in the regeneration of a cofactor, even though an electron transfer mediator is not additionally added, because the titanium dioxide on the electrode surface plays a role as an electron transfer mediator.

[63] Example 2-3. Enzymatic method for oxidation-reduction using cofactor regenerated with titanium dioxide electrode

[64] The method for cofactor regeneration specified above was used for an actual enzymatic oxidation-reduction reaction. The reaction was carried out using the titanium dioxide produced in Example 2-1 as 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 2.5x5 cm, and the voltage applied from an external source for the oxidation reaction was -0.7 V. An alcohol dehydrogenase was used as the oxi- doreductase, and 0.5 mM NADPH was used as the cofactor. The substance used as the substrate was 2-propanol, and the initial concentration was 5 mM. This system was used in the reaction in a volume of 20 ml, in a 100 mM phosphate buffered solution at pH 7.8, at 4O ⁰ C. An analysis of the concentrations of the reactant and the product was performed using gas chromatography.

[65] Fig. 7 is a graph showing the conversion rate over time in an enzymatic reaction using titanium dioxide as a working electrode. NADPH introduced in the initial reaction was oxidized so that the oxidized form, NADP ⁺ was used in the enzymatic reaction, and the reduced from, NADPH, generated after the enzymatic reaction underwent an oxidation reaction again at the titanium dioxide electrode to be regenerated into NADP ⁺ . Thus, cofactor regeneration was achieved without an electron transfer mediator, and the enzymatic reaction also proceeded smoothly.

[66] Example 3-1. Production of metal oxide electrode

[67] 10 mg of the metal oxides shown in the following Table 1 were respectively mixed with 1 mg of carbon black and 1 mg of polytetrafluoroethylene, and then the mixture was fixed in a stainless steel frame, to produce metal oxide electrodes used in the present invention.

[68] Table 1

[Table 1] [Table ]

[69] Example 3-2. Analysis of electrochemical properties of metal oxide

[70] To analyze the electrochemical properties of the metal oxides, a cyclic voltammetry experiment was performed using a potentiostat. The analysis was performed using the metal oxide electrodes of Example 3-1 as working electrodes, a platinum electrode as a counter electrode, and an Ag/ AgCl electrode as a reference electrode, in a 100 mM phosphate buffered solution at pH 7.0. The results of the experiment, that is, the redox potential of the metal oxides, are presented in Table 1 in the above. From this, it was confirmed that since the metal oxide electrodes thus produced can be used in the regeneration of a cofactor, even though an electron transfer mediator is not additionally added, because the metal oxides on the electrode surfaces play a role as an electron transfer mediator.

[71] Example 3-3. Regeneration of cofactor using metal oxide electrode

[72] An experiment of cofactor regeneration was carried out using an electrochemical reactor. The reaction was carried out using the metal oxide electrodes produced in Example 3-1 above as working electrodes, a platinum electrode as a counter electrode, and an Ag/ AgCl electrode as a reference electrode. The size of the working electrode

2 was 0.56 cm , and the voltage applied from an external source was as shown in Fig. 8. The initial concentration of the cofactor (NADH or NAD ⁺ ) was 0.5 mM, and this system was used in the reaction in a volume of 5 ml, in a 100 mM phosphate buffered solution at pH 7.0, at 5O ⁰ C. An analysis of the concentration of the cofactor was performed using a UV spectrophotometer. Fig. 8 is a graph showing the oxidation reaction of a cofactor over time in the case of using the metal oxide electrodes according to the present invention as working electrodes, and exhibits a tendency of an increase in NAD ⁺ , as a result of oxidation of the cofactor NADH.

[73]