KR101077622B1 - Method for Photochemical Regenerating Oxidoreductase Cofactor Using Xanthene Dyes - Google Patents

Abstract

The present invention relates to an optical regeneration method of an oxidoreductase cofactor using xanthene dye, and more particularly, to (i) an oxidative oxidoreductase cofactor, (ii) xanthene dye and (iii) sacrificial electron donor. The present invention relates to an optical regeneration method of an oxidoreductase cofactor comprising irradiating light to a regeneration solution containing an oxidoreductase cofactor.

When the oxidoreductase cofactor is regenerated using xanthene dyes according to the present invention, the oxidoreductase cofactor can be regenerated at a high speed, thereby increasing the efficiency of various biocatalytic reactions using the oxidoreductase enzyme. This can be useful.

Xanthene, oxidoreductase cofactor, NADH, FADH, eosin Y

Description

Method for Photochemical Regenerating Oxidoreductase Cofactor Using Xanthene Dyes}

The present invention relates to an optical regeneration method of an oxidoreductase cofactor using xanthene dye, and more particularly, to (i) an oxidative oxidoreductase cofactor, (ii) xanthene dye and (iii) sacrificial electron donor. The present invention relates to an optical regeneration method of an oxidoreductase cofactor comprising irradiating light to a regeneration solution containing an oxidoreductase cofactor.

The enzyme consists of a main enzyme (apoenzyme) and coenzyme (coenzyme) consisting of proteins, the coenzyme is divided into cofactors or prosthetic groups depending on whether the metal ion. Coenzyme, which we usually refer to, refers strictly to a cofactor, which temporarily accepts atoms or groups of atoms released from a substrate and delivers them to other substances. Typically, nicotinamide cofactor NAD, NADP and flavin cofactors FAD and FMN. The nicotinamide cofactors and flavin cofactors or their oxidized forms are used as co-factors essential for the redox biocatalysis carried out by many types of oxidoreductases (E. Siu et al ., Biotechnol. Prog. , 23: 293, 2007; WA van der Donk and H. Zhao, Curr. Opin. Biotechnol. 14: 421, 2003).

Such biocatalytic reactions are becoming increasingly important in laboratory organic synthesis and various industrial fields. In order to increase the efficiency of the biocatalyst reaction, cofactors for the continuous reaction of the enzymes need to be continuously regenerated, but unlike hydrolytic enzymes which are widely used in various ways, they are satisfactory in the use of redox enzymes. Cofactor recovery method has not been established, so it is not widely commercialized. Electrochemical regeneration has thus been considered an attractive alternative to the existing second enzyme / substrate regeneration (F. Hollmann and A. Schmid, Biocatal. Biotransform., 22:63, 2004). . However, electrochemical regeneration method had in ^{NAD (P) + NAD (P} ) reduced the disadvantage in the voltage condition is thermodynamically favored due to slow the electron transfer rate between the electrode and the NAD (P) ⁺ poor reproduction efficiency to H in.

To solve this problem, a method of transferring electrons between an electrode and NAD (P) ⁺ using a homogeneous redox mediator has been developed (H. Jaegfeldt et al ., Anal. Chem., 53: 1979). , 1981; J. Wang and J. Liu Anal. Chim. Acta , 284: 385, 1993; J. Wang et al. , Anal. Chim. Acta , 360: 171, 1998).

Specifically, methyl viologen has been used for indirect electrochemical regeneration with flavoase (Ferredoxin reductase (FDR) or Lipoamide Dehydrogenase (LipDH)) as an electron transfer medium for NAD (P) H. (Dicosimo et al. J Org Chem , 46: 4622, 1981), electron transfer of ruthenium (II) complex (hexamethylbenzene-2,2′-bipyridinechloro) ruthenium (II) to reduction of ketones with alcohols (Ogo S, Abura T, Watanabe Y, Organometallics 21: 2964, 2002; Yaw Kai Yan et al. J Biol Inorg Chem, 11: 483, 2006), a rhodium (III) complex Pentamethylcyclopentadienyl-2,2'-bipyridinechloro) rhodium (III): [Cp * Rh (bpy) H ₂ O] ²⁺ (hereinafter M _ox ) for electron transfer to NAD (P) ⁺ (R. Ruppert et al ., Tetrahedron Lett ., 28: 6583, 1987; F. Hollmann et al ., Angew. Chem ., 113: 190, 2001; Angew. Chem. Int. Ed. , 40: 169, 2001; K. Vuorilehto et al ., Bioelectroc hemistry, 65: 1, 2004), the rhodium III complex has been used for the regeneration of FADH ₂ (F. Hollmann et al. Journal of Molecular Catalysis B: Enzymatic 19-20: 167, 2003).

However, the electrochemical regeneration method has a disadvantage in that the productivity is limited because the yield is essentially dependent on the surface area of the electrode. The use of a large surface area electrode allows for higher reaction rates and higher productivity, but increases the cost of infrastructure.

On the other hand, photochemical cofactor regeneration using light is already used in the name of photosynthesis in nature. Natural photosynthesis absorbs light from the photosystem to obtain high energy electrons, reductively regenerates ATP (Adenosine Triphosphate) cofactors and NAD (Nicotinamide Adenosine Dinucleotide) cofactors and uses them as raw materials for glucose synthesis.

On the other hand, Eosin Y, a kind of xanthene dye, has been used as a sensitizer for dye-sensitized solar cells (DSSC) in the field of solar energy conversion research (T. Yoshida et al. , Adv. Mater. , 12: 1214, 2000; S.-S. Kim et al. , Sol.Energy Mater.Sol. Cells , 79: 495, 2003). Particularly in the visible region, Eosin Y is an excellent material with high quantum yield in hybrid DSSC devices.

Eosin Y has also been used in photochemical hydrogen production by immobilization with platinum catalysts on conductive substrates (R. Abe et al ., J. Photochem. Photobiol. A: Chem, 137: 63, 2000; Q. Li et al ., J. Phys. Chem. C , 111: 11494, 2007). Eosin Y's quantum efficiency for hydrogen production through visible light has been reported to be much higher than for other organic dyes such as merocyanine and coumarin. Although Eosin Y shows relatively high quantum efficiency, hydrogen production through other organic dyes is possible through the same principle as previously announced, and thus, existing xanthene dye groups such as Eosin Y, merocyanine, coumarin, N719, etc. The dyes used in dye-sensitized solar cells and hydrogen production are expected to function similar to Eosin Y.

However, no xanthene dye has ever been used to regenerate oxidoreductase cofactors, and no effects have been reported when used in combination with known media. In addition, conventionally known photochemical cofactor regeneration technology has a very low reaction rate in the visible region (I. Willner et al., J. Chem. Soc. Perkin Trans. 2: 559, 1990; Q. Shi et al. , J. Mol. Catal. B-Enzym 43:44, 2006).

Accordingly, the present inventors have made efforts to develop a high-efficiency photochemical oxidoreductase cofactor regeneration method by mimicking the cofactor regeneration that occurs through the photosynthesis of natural photosynthesis. When the xanthene dye was added to the regeneration solution to be contained, it was confirmed that the xanthene dye acts as a photocatalyst, so that the cofactor regeneration by light is achieved at a high speed, thereby completing the present invention.

An object of the present invention is to provide a high-efficiency photochemical oxidoreductase cofactor regeneration method that mimics the cofactor regeneration that occurs through the photosynthetic photosystem.

In order to achieve the above object, the present invention is to irradiate the oxidoreductase cofactor regeneration solution containing (i) oxidized oxidoreductase cofactor, (ii) xanthene dye and (iii) sacrificial electron donor An optical regeneration method of an oxidoreductase cofactor is provided.

The present invention is also directed to a oxidoreductase cofactor regeneration solution containing (i) an oxidative oxidoreductase cofactor, (ii) xanthene dye, (iii) sacrificial electron donor and (iv) a redox mediator. It provides an optical regeneration method of the oxidoreductase cofactor, characterized in that to irradiate.

The present invention also relates to an oxidoreductase reactor comprising an artificial light source and a quartz reactor (i) an oxidative oxidoreductase cofactor, (ii) Eosin Y, (iii) Triethanolamine (TEOA) and (iv) Provided is an oxidoreductase cofactor regeneration solution containing ruthenium II complex, and irradiated with light to provide an optical regeneration method of an oxidoreductase cofactor.

When the oxidoreductase cofactor is regenerated using xanthene dyes according to the present invention, the oxidoreductase cofactor can be regenerated at a high speed, thereby increasing the efficiency of various biocatalytic reactions using the oxidoreductase enzyme. This can be useful.

The present invention relates to an oxydori which is irradiated with a oxidoreductase cofactor regeneration solution containing (i) an oxidative oxidoreductase cofactor, (ii) xanthene dye and (iii) a sacrificial electron donor. A method for optical regeneration of ductase cofactors.

Oxidative oxidoreductase cofactors of the oxidative type that are subject to photochemical reduction of the present invention are nicotinamide cofactors NAD ⁺ (nicotinamide adenine dinucleotide), NADP ⁺ (nicotinamide adenine dinucleotide phosphate) or flavin cofactor FAD ⁺ (flavin adenine dinucleotide) ), FMN ⁺ (flavin mononucleotide), and preferably NAD ⁺ .

In the present invention, the sacrificial electron donor is a material that provides electrons to fill the electron pores generated by the transfer of electrons excited from the dye to the redox mediator, and from the viewpoint of those skilled in the art, triethanolamine (TEOA), Ethylenediaminetetraacetic acid (Ethylenediaminetetraacetic acid (EDTA), citric acid, formic acid, ascorbic acid, ascorbic acid, oxalic acid or water, etc. may be used, but preferably TEOA or EDTA). Can be.

The xanthene dye used in the present invention may be used in the system in which the redox mediator and the sacrificial electron donor are present, 1) a mediator for delivering electrons of high energy state to the redox mediator, and 2) the redox mediator. It acts as a double mediator by acting as a mediator to obtain electrons for delivery from the sacrificial electron donor.

From the viewpoint of those skilled in the art, the xanthene dye may be any dye capable of generating an electric current by excitation of electrons through light, but preferably includes fluorescein, and some hydrogen elements are used as a basic structure. Eosin Y, Eosin B, Erythrosine B, Phloxine B, Rose Bengal, Mulbromin Ethyl Eosin, Rhodamine B, Rhodamine 6G, and one or more selected from the group consisting of sulforhodamine B may be used, more preferably. May use Eosin Y (FIG. 1).

Although the addition amount of the said dye is not restrict | limited, Preferably, it is preferable to add in the density | concentration of 0.1 micrometer-100 micrometers, More preferably, it is good to add in the concentration of 1 micrometer-100 micrometers.

In the optical regeneration method of the oxidoreductase cofactor of the present invention, the oxidoreductase cofactor regeneration solution may further contain a redox mediator. The redox mediator may be any one or more selected from the group consisting of methyl viologen, ruthenium II complex, and rhodium III complex, and the redox mediator may be an oxidative derivative of oxidation type. It is used as a primary mediator for electron transfer to other cofactors.

Among the redox mediators, methyl viologen was used for indirect electrochemical regeneration with Flavoenzyme (Ferredoxin reductase (FDR) or Lipoamide Dehydrogenase (LipDH)) as an electron transfer mediator for NAD (P) H. (Dicosimo et al. J Org Chem , 46: 4622, 1981), a ruthenium (II) complex (hexamethylbenzene-2,2′-bipyridinechloro) ruthenium (II) is used for the reduction of ketones to alcohols. Ogo S et al., Organometallics, 21: 2964, 2002; Yaw Kai Yan et al. J Biol Inorg Chem, 11: 483, 2006), the rhodium (III) complex (Penta) Methylcyclopentadienyl-2,2'-bipyridinechloro) rhodium (III): [Cp * Rh (bpy) H ₂ O] ²⁺ (hereinafter M _ox ) is a medium for electron transfer to NAD (P) ⁺ (R. Ruppert, S. et al., Tetrahedron Lett. M 28: 6583, 1987; F. Hollmann et al. , Angew. Chem ., 113: 190, 2001; Angew. Chem. Int. Ed. 40: 169 , 2001; K. Vuorilehto et al. , Bioelectrochemistry , 65: 1, 2004) and It has been used as a medium for electron transfer to FAD ⁺ (F. Hollmann et al. Journal of Molecular Catalysis B : Enzymatic 19-20: 167, 2003). Thus, the redox mediator is used to improve the regeneration kinetics of oxidoreductase cofactors by delivering electrons and protons.

The ruthenium II complex may preferably be (hexamethylbenzene-2,2′-bipyridinechloro) ruthenium (II), and the rhodium III complex may be (pentamethylcyclopentadienyl-2,2′-bipyridine Preference is given to using chloro) rhodium (III).

However, in the present invention, the presence of the redox mediator is effective in regenerating the cofactor in the form having activity in the enzymatic reaction and speeding up the regeneration of the cofactor, even if the redox mediator does not exist, The presence of redox mediators does not limit the scope of the present invention because photochemical regeneration of cofactors is possible directly through dye sensitization.

Photochemical regeneration of the oxidoreductase cofactor of the present invention can be carried out in an oxidoreductase bioreactor. The reactor preferably comprises a system comprising a quartz reactor containing a reaction solution comprising a photosensitization dye, a redox mediator and a sacrificial electron donor, and a light source for irradiating light.

The light source used in the system may preferably be natural sunlight and may be an artificial tungsten-halogen lamp, xenon lamp or short wavelength laser device.

In the method of the present invention, the oxidoreductase cofactor containing oxidized oxidoreductase cofactor, xanthene dye, and sacrificial electron donor is irradiated with light and stirred with a light source, followed by stirring. Creates. The output of the light source is preferably 5W ~ 2000W. If it is lower than the output range, the light intensity may be weak, so that the catalytic function may not appear. At too high output, the biocatalytic reaction may not occur or unwanted reaction may occur due to the degeneration of enzymes and other substances by light and heat.

Optical regeneration method of the oxidoreductase cofactor of the present invention, as shown in Figure 2, the dye and the redox mediator to form a self-assembled complex (step a); Transferring electrons to the redox mediator by the dye absorbing light in the dye-redox mediator complex of step a to the redox mediator (step b); Repeating step b, in which the dye-redox mediator complex of step a is supplemented with electrons from the sacrificial electron donor (step c); And the redox mediator receiving the electrons and protons passes the electrons and protons to the oxidative oxidoreductase cofactor to form a reduced oxidoreductase cofactor (step d). In other words, the xanthene dye used in the present invention acts as a catalyst having activity against light in the presence of a redox mediator to participate in the transfer of electrons.

In one embodiment of the present invention, when rhodium complex (III) M is used as the redox mediator and Eosin Y is used as the xanthene dye, as shown in FIG. 3, Eosin Y and the redox mediator M are self-assembled complexes Eosin Y Forming M (step a) transferring electrons to the M electrons excited by Eosin Y by absorbing light in the Eosin Y-M complex of step a (step b); Repeating step b in which the Eosin Y-M complex of step a is supplemented with electrons from the sacrificial electron donor (step c); After receiving electrons and reducing M ( ₁ ), M _red1 receives protons and becomes M _red2 , and then transfers electrons and protons to oxidized oxidoreductase cofactors to form reduced oxidoreductase cofactors (step d).

In order to confirm the effect of the present invention, the conversion rate from NAD ⁺ to NADH in the absence or presence of Eosin Y, NADH was not generated at all in the absence of Eosin Y, but when Eosin Y is present It was confirmed that NADH is generated at a high speed. In addition, as the amount of Eosin Y increases, the amount of NADH produced increases, it was confirmed that the production rate increases (see Experimental Example 3).

In one aspect of the present invention, as a result of examining the conversion rate of α-ketoglutamic acid to glutamic acid in order to confirm the function of the regenerated NADH by the method of the present invention, it was confirmed that glutamic acid was produced by the regenerated NADH of the present invention. In addition, it was found that the production rate of the resulting glutamic acid was proportional to the concentration of the added dye (see Experimental Example 4).

From this, the xanthene dye used in the present invention acts as a catalyst that is activated by light in the presence of a sacrificial electron donor and is involved in the transfer of protons and electrons, thereby contributing to increasing the regeneration rate of the oxidoreductase cofactor. (See Experimental Example 5) . Therefore, the method of the present invention can increase the efficiency of various redox catalysis reactions using oxidoreductase enzymes, and is particularly effective under poor environments such as a high pH environment. It is also contemplated that the method of the present invention can be extended not only for the regeneration of oxidoreductase cofactors but also for chemical reactions of other substances.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In the following examples, redox mediators were added to regenerate oxidoreductase cofactors, but the redox mediators played a role in regenerating the cofactors in a form active for enzymatic reactions and increasing the rate of cofactor regeneration. It will be apparent to those skilled in the art that photochemical regeneration of direct cofactors via dye sensitization is possible even without the presence of redox mediators.

Example: Regeneration of NADH by Photochemical Method Using Rhodium III Complex and Xanthene Dye

Rhodium III complex (pentamethylcyclopentadienyl-2,2'-bipyridinechloro) rhodium (III) (hereinafter referred to as M; M = [Cp ^* ) as an organometallic medium for transferring electrons between NAD ⁺ and the electrode Rh (bpy) Cl] ⁺ ; Cp ^* = C ₅ Me ₅ , bpy = 2,2′-bipyridine). The rhodium III complex M was synthesized by K ㆆ lle and Gr ㅴ tzel's methods (F. Hollmann, B. Witholt, A. Schmid, J. Mol. Catal . B 19-20 , 167, 2002).

In brief, the rhodium precursor (RhCl ₃ ㅇ H ₂ O) was dissolved in methanol with 1 equivalent of hexamethyl dewarbenzene and refluxed for 24 hours, and the resulting red precipitate was filtered and filtered back into methanol. After melting, 2 equivalents of 2,2'-bipyridine was added to give a yellow solution. A precipitate of the final rhodium III complex M was obtained by using diethyl ether.

In order to regenerate NADH by the photochemical method, an artificial light source and a quartz reactor capable of irradiating light from the artificial tube source were manufactured. As an artificial light source, a tungsten-halogen lamp having a power of 400 W was used, and in particular, a filter for filtering light having a wavelength of 420 nm or less was used to irradiate light in the visible region. In addition, a filter capable of filtering light having wavelengths in the heat and infrared regions was constructed using cooling water to exclude possible results obtained from the heat of the light source. Light from the light source was irradiated onto the reaction solution contained in the quartz reactor.

Rhodium III complex M, a redox mediator for NADH production, was used at a concentration of 0.25 mM, and xanthene dyes used as catalysts that absorbed light and were active in redox reactions were used at concentrations of 5 μM, 10 μM or 50 μM. NAD ⁺ was used at a concentration of 1 mM or 0.2 mM.

Phosphate buffer (100 mM, pH 7.0) was used as an electrolyte. After the addition of TEOA, which is a sacrificial solution, to the buffer, M, xanthene dye and NAD ⁺ were added, stirred and dissolved, and then irradiated with light. , NADH was regenerated.

Experimental Example 1 Effect of Rhodium III Complex M on the Absorption / Fluorescence Spectra of Eosin Y

To spectroscopically investigate the intermolecular interactions of rhodium III complex M with dye Eosin Y, various concentrations of rhodium III complex M were present after dissolving Eosin Y in a phosphate buffer (100 mM, pH 7.0) at a concentration of 5 μM. The resulting absorbance and fluorescence spectra were measured.

As a result, as shown in a) of FIG. 4, the absorption spectrum of Eosin Y decreased as the concentration of M increased and the position of the peak point moved in the long wavelength direction. When Eosin Y and M coexist in a solution, the negatively charged carboxyl functional groups of Eosin Y are bound to each other by a mutually attracting interaction with the metal of M having a positive charge (Rh ²⁺ ), As a result, the charge of the carboxyl functional groups of Eosin Y is neutralized, causing a change in the absorption spectrum. In this case, since M does not have an absorbance between the inspection regions 440nm and 560nm, it was determined that this result is not a phenomenon in which the absorption spectra of the two materials simply overlap.

In addition, as a result of investigating the fluorescence spectrum of Eosin Y with the solution under the above conditions, as shown in b) of FIG. 4, the decrease in intensity and the shift of the peak point in the long wavelength direction were observed in the fluorescence spectrum similarly to the absorption spectrum. This supports the formation of a complex of Eosin YM. The Eosin Y fluorescence spectrum is attached to a material such as TiO ₂ and shifts in the long wavelength direction under the influence of the electronegativity and polarity of the metal ion. In addition, decreasing the intensity of the spectrum indicates that M prevented the radioactive decay of the excited electrons of the light absorbing Eosin Y. It has been reported that the excited electrons of Eosin Y are transferred to the surface of semiconductor metal oxide (ZrO ₂ , TiO ₂ , Al ₂ O ₃ ) nanoparticles, resulting in reduced fluorescence (S. Pelet et al ., J. Phys. Chem. B , 107: 3215, 2003).

Experimental Example 2: Rhodium III Complex M, NAD ⁺ And cyclic voltage-current diagram in the presence of Eosin Y

To investigate the kinetics of the presence of NAD ⁺ and Eosin Y in addition to the redox mediator Rhodium III complex M, Eosin Y in the presence of complex M alone, NAD ^{+ in} addition to complex M, and each of them Cyclic voltammograms were measured. Eosiun Y was dissolved in phosphate buffer (100 mM, pH 7.0) at 50 μM, complex M at 0.25 mM, and NAD ⁺ at 1 mM, and the test rate was measured at 100 mVs ⁻¹ .

As a result, as shown in Figure 5a, the reduction potential (reduction potential) at the cathodic peak current (E _pc ) of M was measured at about -0.7V with or without NAD ⁺ .

On the other hand, when Eosin Y was added thereto, a change in cyclic voltage-current diagram was observed (b of FIG. 4). The cathodic peak shown in b of FIG. 4 was mainly measured at -0.75 V shifted by 0.05 V in the negative direction at the M cathode current. This phenomenon, which can be interpreted as the M's cathodic transition, shows that when Eosin Y coexists in the same solution as the stabilizing agent, Eosin Y and M are simultaneously reduced through complex formation. it means.

From the Eosin Y-M complex following the cyclic voltammogram of M on the GC electrode, it can be seen that M of Eosin Y and M is more susceptible to reduction. The electrons will thus be transferred in the direction of Eosin Y to M.

Experimental Example 3 Measurement of NADH Regeneration Rate According to Eosin Y Concentration and pH

In order to examine the pH range in which the catalytic function of Eosin Y can be performed, NADH regeneration experiments were performed at various pH conditions.

As a result, as shown in a of FIG. 6, the total yield of NADH obtained after the visible light irradiation reaction for one hour was different depending on pH. Total NADH output increased until pH 8.0 was reached. This is because the pH is increased and the ratio of ionization of the carboxyl group of Eosin Y is increased, thereby facilitating complex formation with M. The pK _a of Eosin Y appears at 3.0 and 5.0, which is the protonation of hydroxyl and carboxyl groups, respectively. Therefore, below pH 5.0, the protonated carboxyl group interferes with the formation of the complex with M, thereby preventing electron transfer, thereby preventing NADH regeneration. On the other hand, NADH output decreased at pH 9.0, which is thought to be due to a decrease in catalytic activity of M at high pH.

In addition, in order to confirm the visible light activity of the Eosin YM complex, NADH regeneration was performed with or without light as a variable. As a result, as shown in b of FIG. 6, the reduction reaction of NAD ⁺ to NADH was not detected in the dark reaction for one hour, but the reduced NADH began to be detected immediately after irradiation with visible light (λ ?? 420 nm). The rate of photochemical reduction was proportional to the concentration of Eosin Y and at pH 7.0, 5 μM of Eosin Y yielded maximum yield in 30 minutes (FIG. 6B).

Experimental Example 4: Validation of biocatalytic reaction efficiency using regenerated NADH according to the present invention

In order to examine the efficiency of the present invention, NADH regenerated by the photochemical regeneration method of the present invention was applied to the biocatalytic reaction of alpha-ketoglutamic acid to L-glutamic acid by glutamate dehydrogenase.

For the biocatalytic reaction, 0.1M phosphate buffer solution (pH 8.0) was added to the reactor, 15% by weight of triethanolamine, 0.2 mM NAD +, 0. mM complex M, 10 mM alpha-ketoglutamic acid, 100 mM (NH ₄ ) ₂ SO ₄ , 40U glutamic acid dehydrogenase was added, and then, xanthene dye Eosin Y was added at different concentrations at 10 μM and 50 μM. The solution was irradiated with light and stirred to proceed the reaction for 40 minutes, and then the conversion rate (%) was measured.

As a result, as shown in FIG. 7, the conversion rate in the case of the experiment without the irradiation of light or without adding Eosin Y or M was 0%. The conversion rate was 57.9% for 10 μM Eosin Y and 81.4% for 50 μM Eosin Y, indicating that the conversion increased with the amount of Eosin Y added. This is because NADH regeneration occurs by Eosin Y and NADH regeneration rate is increased according to the concentration of Eosin Y. Accordingly, the rate of biocatalytic reaction using NADH as a cofactor is also increased. It was confirmed that the efficiency of the catalytic reaction could be increased.

Experimental Example 5: NADH regeneration efficiency according to the type of xanthene dye

To determine the activity of NADH regeneration of other xanthene dyes, including Eosin Y, in each experiment, 5 μM xanthene dyes and 250 μM M, 1 mM at pH 7.4, 0.1 M phosphate buffer, 15 wt% triethanolamine The reaction was carried out using NAD ⁺ . As shown in FIG. 8, most xanthene dyes, including Eosin Y, were shown to be effective in NADH formation. The reaction rate for NADH formation was Rose Bengal> Phloxine B> Erythrosine B> Eosin Y>Merbromin> Ethyl Eosin> Fluorescein.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 shows the chemical structure of 11 xanthene dyes.

Figure 2 shows the electrochemical conversion of rhodium III complex M.

3 shows a schematic diagram of photochemical cofactor regeneration using Eosin Y.

4 shows the effect of complex M on the absorption / fluorescence spectrum of Eosin Y obtained at pH 7.0.

5 shows a cyclic voltage-current diagram using the composite M and Eosin Y on Glassy Carbon.

Figure 6 shows the results of photochemical NADH regeneration experiments with pH and concentration of Eosin Y (-○-: 0μM, .-Δ-: 3μM,-□-: 1μM,-◇-: 5μM).

Figure 7 shows the effect of the presence of light and the concentration of Eosin Y on the L- glutamic acid conversion of alpha-ketoglutamic acid by photochemically regenerated NADH cofactor.

8 shows the results of photochemical NADH regeneration using 11 xanthene dyes.

Claims (10)

(i) an oxidized oxidoreductase cofactor selected from the group consisting of NAD ⁺ , NADP ⁺ , FAD ⁺ and FMN ⁺ ; (ii) xanthene dyes; And (iii) irradiating the regeneration solution of the oxidoreductase cofactor containing the sacrificial electron donor with light. delete According to claim 1, The xanthene dye is Fluorescein (Fluorescein), Eosin Y (Eosin Y), Eosin B (Eosin B), Erythrosine B (Phloxine B), From the group consisting of Rose Bengal, Merbromin, Ethyl Eosin, Rhodamine B, Rhodamine 6G, and Sulforhodamine B Optical regeneration method of oxidoreductase cofactor, characterized in that at least one dye selected. The method of claim 1, wherein the sacrificial electron donor is triethanolamine (TEOA), ethylenediaminetetraacetic acid (EDTA), citric acid, formic acid, ascorbic acid, oxalic acid (Oxalic acid), an alcohol and an optical regeneration method of oxidoreductase cofactors selected from the group consisting of water. The optical regeneration method of an oxidoreductase cofactor according to claim 1, wherein the light is selected from the group consisting of tungsten-halogen lamp light, xenon lamp light, short wavelength laser light and sunlight. The optical regeneration method of an oxidoreductase cofactor according to claim 1, which is performed in an oxidoreductase reactor including an artificial light source and a quartz reactor. The optical regeneration method of an oxidoreductase cofactor according to claim 1, wherein the xanthene dye is added at a concentration of 0.1 to 100 µM. The method of claim 1, wherein the oxidoreductase cofactor regeneration solution further comprises any one or more redox mediators selected from the group consisting of methylviologen, ruthenium II complex and rhodium III complex. Method for photochemical regeneration of oxidoreductase cofactor. 9. The method of claim 8 wherein the ruthenium II complex is (hexamethylbenzene-2,2'-bipyridinechloro) ruthenium (II). 9. The method of claim 8, wherein the rhodium III complex is (pentamethylcyclopentadienyl-2,2'-bipyridinechloro) rhodium (III).

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