KR101249410B1 - Photocatalytic production method of oxidoreductase cofactors using Pt Nanoparticles - Google Patents

Abstract

The present invention comprises the steps of: (a) adding an oxidative oxidase cofactor and platinum (Pt) nanoparticles to an oxidoreductase reactor; And (b) relates to a photochemical regeneration method of the oxidoreductase cofactor comprising the step of irradiating light to the reactor of step (a), redox reduction by photoreaction in the presence of platinum nanoparticles as redox mediator The present invention relates to a method of photochemical regeneration by converting to an enzyme cofactor, and may be very useful for increasing the efficiency of various biocatalytic reactions using oxidoreductase cofactors.

Description

Photocatalytic production method of oxidoreductase cofactors using Pt Nanoparticles}

The present invention comprises the steps of: (a) adding platinum (Pt) nanoparticles to the oxidative oxidoreductase cofactor; And (b) irradiating light to the mixture of step (a). The present invention relates to a photochemical regeneration method of an oxidoreductase cofactor, comprising: reducing oxidation by photoreaction in the presence of platinum nanoparticles as redox mediators. The present invention relates to a method for regenerating by converting to a reductase cofactor.

The present invention is the result of support from the SRC / ERC program of MOST / KOSEF (R11-2005-008-000-0) and the National Research Foundation of Korea Grant funded by the Korean Government (2009-0087304).

As fossil fuels are gradually depleted, various attempts have been made to find alternative energy sources. For example, attempts have been made to convert radiant electromagnetic energy into chemical energy in the form of reducing power, namely NADH, using photochemical systems.

Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP) are important coenzymes found in cells, and NADH, NADPH are reduced forms of NAD and NADDP, respectively.

NAD is used in glycolysis and TCA circuits in cell respiration, and the reducing potential stored in NADH is converted to ATP or used for anabolism through an electron transport system. NADPH provides reducing power and is used for assimilation such as fatty acid and nucleic acid synthesis. NADP acts as an important oxidant in the photosynthetic initial reaction, ie the photolysis of water, to form NADPH. NADPH provides reducing power to the Calvin cycle of photosynthesis.

FAD and FMN are a type of complement molecule of the flavin enzyme group, and are involved in hydrogen and electron transfer in the redox system in vivo. FAD is also called riboflavin adenine dinucleotide, is a nucleotide consisting of a compound of riboflavin, two phosphate groups and adenosine, and FMN is also called riboflavin phosphate, and corresponds to the phosphate ester of riboflavin. FAD and FMN are important materials for hydrogen and electron transfer in the redox system of the living body. It acts as a coenzyme of the flavin enzyme family and is involved in electron transfer from the substrate to the electron acceptor.

Natural photosynthesis occurs in the chloroplasts in the cells of green plants. In light reactions, O ₂ is produced, and in the dark, glucose is synthesized and water is produced. More specifically, the thylakoid membrane has a chlorophyll and an electron transport system, so that a light reaction occurs. The light reaction can be divided into two stages: water photolysis and photophosphorylation. Water photolysis process is electron (e ^-) to which the water is decomposed by the absorbed light energy to chlorophyll produce hydrogen ions and oxygen. The photophosphorylation process converts the light energy absorbed by chlorophyll into chemical energy to produce ATP. When light energy is absorbed by chlorophyll, chlorophyll is excited and releases electrons. Create During photophosphorylation, NAHPH ₂ is also produced, which is used for cancer reactions. The cancer reaction occurs in the chloroplast stroma and synthesizes glucose from carbon dioxide using ATP and NADPH ₂ produced in the light reaction.

Systems that mimic nature's photosynthesis usually contain three independent components: photosensitisers, electron relays, and catalysts.

NADH / NADPH is used as a cofactor in various biological reactions performed by many redox enzymes. Photocatalytic production of NADH / NADPH has been studied in the context of solar energy conversion and artificial photosynthesis. Cofactors used in stoichiometric amounts for biological reactions are expensive for widespread application and require the development of NADH and NADPH regeneration systems to efficiently perform solar energy capture and conversion. In addition, since visible light reaches 46% of total solar energy, efforts have been made to develop visible-propulsive systems.

As an example of a system comprising three components for the generation of NADH / NADPH light by visible light, a light-harvesting inorganic dye is used as a methyl viologen as an electron transfer / catalyst pair in the presence of an electron donor. viologen) and a system for coupling with dehydrogenase.

However, few attempts have been made for non-enzymatic photochemical regeneration of NADH / NADPH, and in the presence of Ru (bpy) ₃ ²⁺ as a photosensitizer, the mononuclear Rh complex is an electron transfer and hydride transfer catalyst. There is only an example used as. Co-fixing of photosensitisers, redox mediators, catalysts or enzymes has also been undertaken to bring together all the components necessary for more practical applications. For example, studies have shown that hydrogenase immobilized on photosensitizer CdS particles produces NADH. However, there is a problem that such integrated systems are usually less active than homogeneous systems.

In addition, although Korean Patent Publication No. 2010-0011160 discloses an electrochemical regeneration method of an oxidoreductase cofactor using metal nanoparticles, a primary redox mediator such as a ruthenium II complex is additionally required. Regeneration yield is not good enough.

The present invention is to solve the above problems, in order to provide a system for the efficient photochemical regeneration of oxidoreductase cofactor, by introducing a platinum (Pt) nanoparticles (Pt NanoParticle, PtNP) I can solve it.

The present invention provides a method of photochemically regenerating oxidoreductase cofactors by using platinum (Pt) nanoparticles having excellent photosensitizer, electron transfer and catalytic activity. When the platinum (Pt) nanoparticle solution of the present invention is irradiated with visible light in the presence of an electron donor, it was found that the oxidative oxidoreductase cofactor is converted into a reduced yield in excellent yield, thereby completing the present invention.

The present invention relates to a photochemical regeneration method of oxidoreductase cofactors using platinum nanoparticles and light irradiation, and is very useful for enhancing the efficiency of various biocatalytic reactions in which the platinum nanoparticles use oxidoreductase cofactors. Can be.

In particular, compared with the conventional electrochemical regeneration method, the photochemical regeneration method of the present invention showed an excellent NADH regeneration yield more than three times.

FIG. 1 (A) shows the results of measuring the production of NADH after irradiation with 0.2 mM NAD ⁺ , 0.4 M TEOA, 0.15 M phosphate buffer (pH = 7.0) at 25 ° C. for 12 hours [PtNP: 1 unit (92.5 μM Pt atoms); 1: 0.01 mM [Cp * Rh (bpy) (H ₂ O)] ²⁺ ; eosin: 0.05 mM].
FIG. 1B shows a unit of PtNP 0.2 mM NAD ⁺ , 0.4 M TEOA, 0.15 M phosphate buffer (pH = 7.0), light irradiation at 25 ° C. for 0, 3, 6, 9, 12 hours (λ> 400). nm), followed by UV-vis absorption spectrum measurement of NADH.
Figure 2 (A) is the result of measuring the photochemical NADH formation rate according to PtNP concentration in 0.2 Mm NAD ⁺ , 0.4 M TEOA, 0.15 M phosphate buffer (pH = 7.0) [PtNP, 1 unit: ,, 2 units: ●, 3 units: ▲, 4 units: ▼].
Figure 2 (B) shows the number of conversions for 0.2 Mm NAD ⁺ , 0.4 M TEOA, PtNP per hour for 12 hours. The conversion number was calculated assuming nanoparticles containing 2800 Pt atoms relative to the amount of PtNPs.
FIG. 3 shows the results of measuring the effect of pH on photoregeneration of NADH after 9 hours of light irradiation (λ> 400 nm) in PtNP 1 unit 0.2 mM NAD ⁺ , 0.4 M TEOA, 0.15 M phosphate buffer.
Figure 4 (A) is the result of measuring the degree of NADH production according to the concentration of TEOA in NAD ⁺ , 0.15 M phosphate buffer of 0.2 mM PtNP 1 unit.
4B is a result of measuring the NADH production rate according to the concentration of NAD ⁺ in PtNP 1 unit 0.2 mM NAD ⁺ , 0.4 M TEOA, 0.15 M phosphate buffer (pH = 7.0) [0.1 Mm: ■ , 0.2 mM: ▲, 0.3 mM: ●].
Figure 5 illustrates the electron transfer relationship between PtNP, NAD ⁺ and TEOA.
Figure 6 shows the results of absorption spectrum change, (A) is [1 unit of PtNP, 0.01 mM Cp * Rh (bpy) (H ₂ O)] ^{2 +} , 0.05 mM Eosin, 0.2 mM NAD ⁺ and 0.4 M TEOA], (B) is the result of [1 unit of PtNP, 0.01 mM Cp * Rh (bpy) (H ₂ O)] ²⁺ , 0.2 mM NAD ⁺ and 0.4 M TEOA].
FIG. 7 shows the results of fluorescence spectral measurement of 0.5 μM eosin after and without addition of 4 units of PtNP, with an excitation wavelength of 524 nm.
Fig. 8 shows the results of measuring changes in absorption spectrum when 0, 3, 6, 9, and 12 hours, (A) shows two units of PtNP, and (B) shows three units of PtNP (0.15M). 0.2 mM NAD ⁺ and 0.4 M TEOA conditions in phosphate buffer).
9 shows the effect of pH on the light regeneration of NADH regenerated in a system consisting of 1 unit PtNP, 0.2 mM NAD ⁺ , and 0.4 M TEOA in 0.15 M phosphate buffer after 0, 3, 6, 9, 12 hours of light irradiation. Is measured.
10 is a result of measuring the change in absorption spectrum in NADH regeneration according to the concentration of TEOA ((A) is 200 mM, (B) is 300 mM).
11 shows the results of measuring absorption spectrum change in NADH regeneration using Au nanoparticles.
12 shows the results of measuring absorption spectrum change in NADH regeneration using Pd nanoparticles.
Figure 13 is the result of measuring the degree of electrochemical NADH production using PtNP.
Fig. 14 shows the results of measuring absorption spectrum change in NADH regeneration according to EDTA 1 mM, an electron donor.
FIG. 15 shows the results of measuring absorption spectrum change in NADH regeneration according to triethylamine (Et ₃ N) 400 mM as an electron donor.
Fig. 16 shows the results of measuring absorption spectrum changes in NADH regeneration according to sodium formate (Na-formate) 100 mM, which is an electron donor.

Hereinafter, the present invention will be described in detail.

The present invention provides a photochemical regeneration method of an oxidoreductase cofactor comprising the following two steps.

Step (a): adding platinum (Pt) nanoparticles to an oxidative oxidoreductase cofactor

Step (b): irradiating the mixture of step (a) with light.

In the present invention, step (a) is the step of adding the platinum nanoparticles to the oxidoreductase cofactor of the oxidation type.

Oxidized oxidoreductase cofactors of the reduction type of the present invention are nicotinamide cofactor NAD ⁺ (nicotinamide adenine dinucleotide), NADP ⁺ (nicotinamide adenine dinucleotide phosphate) or flavin cofactor FAD ⁺ (flavin adenine dinucleotide), FMN ^It may be a ⁺ (flavin mononucleotide), preferably NAD ⁺ .

In the present invention, an electron donor may be further added to the oxidoreductase reactor of step (a), and triethanolamine is preferable as the electron donor.

In addition, in the present invention, the concentration of the platinum nanoparticles added to the oxidoreductase reactor is preferably added at a concentration of 1 unit or more (wherein 1 unit contains 92.5 μM of Pt atoms), more Specifically, it can be added at a concentration of 2 units to 4 units.

In the present invention, it is preferable to adjust the pH in step (a) to 7 or more. Preferably the pH can be adjusted to 7 to 10.

In the present invention, the light irradiated to the mixture of step (a) may be visible light.

In the present invention, in the case of step (a), it is preferable to be carried out in a buffer, and in particular, but not limited to, a phosphate buffer, Tris buffer, ammonium phosphate buffer or MES buffer may be used.

In the present invention, the method may further include removing air from the mixture before irradiating the mixture of step (a), for example, by removing the air through evacuation.

In the present invention, photoregeneration of oxidoreductase cofactors is possible regardless of whether the reaction conditions are aerobic or anaerobic conditions.

In the present invention, the platinum nanoparticles can act alone as a light absorption unit. Specifically, the platinum nanoparticles may act not only as an electron transfer / catalyst, but also as a photosensitizer, and may collect photoelectrons under visible light.

The present invention also relates to a reactor comprising an oxidative oxidoreductase cofactor and platinum nanoparticles; And a photochemical regeneration device of an oxidoreductase cofactor including a light irradiation device.

The present invention also relates to a method for reducing an enzyme using an oxidoreductase cofactor prepared by the above method. In order to confirm the function of the regenerated NADH by the method of the present invention, by examining the conversion rate of alpha-glutamate to L-glutamate, it was confirmed that L-glutamate is produced by the regenerated NADH of the present invention (See Example 8 below).

The convertible enzymes include, but are not limited to, glutamate, formate dehydrogenase, or glucose-6-phosphate dehydrogenase.

Acronym Definition:

NAD: Nicotinamide Adenine Dinucleotide

NADP: Nicotinamide Adenine Dinucleotide Phosphate

FAD: flavin adenine dinucleotide

FMN: flavin mononucleotide

PtNP: Platinum nanoparticles

TEOA: triethanolamine

Cp *: pentamethylcyclopentadienyl

bpy: 2,2'-bipyridine

Hereinafter, the specific configuration and operation of the present invention will be described through examples, but the scope of the present invention is not limited to the following examples.

[ Example ]

 [Reagent Preparation]

 (1) Water was purified with MilliQ purification system. All reagents were purchased from Aldrich and used for further purification.

(2) [Cp ^* Rh (bpy)] ²⁺ was prepared according to a known method.

Manufacturing example  One. Pt  Preparation of Nanoparticles

PtNP was prepared by reducing potassium tetrachloroplatinate (K ₂ PtCl ₄ ) with polyvinylpyrrolidone (MW = 10k).

More specifically, K ₂ PtCl ₄ The hydrothermal mixture (11.5 mM, 120 mL) was reacted with polypinylpyrrolidone (3 g) for 4 hours. The reaction was allowed to react by stirring at 90 to 100 ° C. for 4 hours, after which the solution appeared dark brown to black.

Here, one unit of PtNP solution is meant to contain 92.5 μM of Pt atoms.

Comparative Production Example  One. Au  Preparation of Nanoparticles

Au nanoparticles were prepared by reducing H ₂ AuCl ₄ with polyvinylpyrrolidone (MW = 10k).

More specifically, H ₂ AuCl ₄ (11.5 mM, 120 mL) was reacted with polypinylpyrrolidone (3 g) for 4 hours. The reaction was allowed to react by stirring at 90 to 100 ° C. for 4 hours, after which the solution appeared red.

Comparative Production Example  2. Pd  Preparation of Nanoparticles

Pd nanoparticles were prepared by reducing K ₂ PdCl ₄ with polyvinylpyrrolidone (MW = 10k).

More specifically, K ₂ PdCl ₄ (11.5 mM, 120 mL) was reacted with polypinylpyrrolidone (3 g) for 4 hours. The reaction was allowed to react by stirring at 90 to 100 ° C. for 4 hours, after which the solution appeared pale black.

Example  One. Pt  Observation of the size and distribution of nanoparticles

The size and distribution of Pt nanoparticles prepared according to the method described above were measured by TEM and dynamic light scattering instrument (Malvern, Zetasizer Nano ZS). The nanoparticles were 5 ± 2 nm in size and had a near circular shape.

Example  2. UV - vis  Spectrum and TEM  video

UV-vis spectra were measured with a Hewlett Packard 8458 spectrophotometer. Transmission electron microscopy (TEM) images were observed with a JEOL 2010FX electron microscope (operated at 200 kV). Transmission spectra were collected using a Perkin-Elmer LS55 fluorescence spectrophotometer.

UV-vis was carried out to measure the concentration of NADH at 340 nm, in the case of fluorescence image to show that the fluorescence of eosin is quenched by the platinum nanoparticles.

Example  3. NADH  Photochemical Regeneration Measurement

For photochemical regeneration of NADH, it was performed in 0.15 M phosphate buffer (1 mL, pH 5-9) in the presence of 1 unit of PtNP, 0.4 M TEOA and 0.2 mM NAD ⁺ at 25 ° C. Photoreaction was carried out in a 3 mL glass cuvette (vial) equipped with a magnetic stirrer and stopper.

A sample containing all the components (1 mL) was placed in a cuvette and reacted, and the degassed reaction was repeated by evacuation to prepare a degassed sample and subjected to Ar flushing. . A 330 W Xe lamp equipped with a 420 nm cutoff filter was used as the light source. The production of NADH was determined by measuring UV absorbance at 340 nm (ε = 6220 cm ⁻¹ M ⁻¹ ).

At room temperature, when NAD ⁺ was photochemically reduced under conditions of Eosin / PtNP / [Cp * Rh (bpy) (H ₂ O)] ²⁺ / TEOA in phosphate buffer, after 12 hours of irradiation with a 420 nm cutoff filter, NAD ^{It was found that +} was converted to NADH by about 40% (see FIG. 1A and FIG. 6A). The photochemical reaction occurred even in the absence of eosin, but a similar amount of NADH (36%) was obtained (see FIG. 6 (B)). The results show that PtNP can act as a light-absorbing unit alone.

On the other hand, there was a clear spectral change near 340 nm during photolysis even with [Cp * Rh (bpy) (H ₂ O)] ²⁺ and eosin removed, producing 59% NADH under the same conditions (see FIG. 1). The results indicate that the consumption of TEOA results in visible light-driven electron flow from PtNP to NAD ⁺ and that PtNP can act as a photosensitizer as well as an electron transfer / catalyst alone. You can see the point.

When both PtNP and TEOA were removed from the system, photosensitized formation of NADH was not observed. This should be noted that the fluorescence of eosin is quenched (quenched) by PtNP (see FIG. 7), but the energy transfer from excited eosin does not improve the yield of NADH.

To compare the photocatalytic activity of 1 unit of PtNP (1 unit of PtNP solution contains 92.5 μM of Pt atoms) and 0.01 mM of [Cp * Rh (bpy) (H ₂ O)] ²⁺ , NAD ⁺ reduction [Cp * Rh (bpy) ( H 2 O)] 2+ with the reaction, and [Cp * Rh (bpy) ( H 2 O)] seen performed under TEOA present and the reaction with the ^{2 +} / eosin for. The result was 4% in the former and 36% in the latter. In the case of photoexcited eosin, the energy was transferred to [Cp * Rh (bpy) (H ₂ O)] ²⁺ , but [Cp * Rh (bpy) (H ₂ O)] ²⁺ alone When used, the amount of NADH produced was negligibly small (see FIG. 1A). The results indicate that PtNP is more active as a photocatalyst system that converts light energy and generates NADH than [Cp * Rh (bpy) (H ₂ O)] ²⁺ / eosin.

In the case of Au nanoparticles prepared in Comparative Preparation Example 1 and Pd nanoparticles prepared in Comparative Preparation Example 2, only a slight change in the amount of NADH was observed under the same conditions (see FIGS. 11 and 12).

Molecular oxygen is known to readily react with chemically or electrochemically reduced methyl viologen or [Cp * Rh (bpy) (H ₂ O)] ²⁺ . In addition, the yield is sharply limited when oxygen is present, which appears to be due to the depolarization of Pt particles and the reoxidation of a reduced relay in hydrogen photoregeneration. To investigate the effect of O ₂ on photoexcited PtNP, the degree of NADH regeneration was measured under aerobic and anaerobic conditions with PtNP / TEOA. As a result, there was no significant difference in the rate of formation of NADH, indicating that the photoreaction was insensitive to oxygen, and that the activated surface of PtNP had a low affinity for O ₂ in the presence of NAD ⁺ and TEOA. In the case of the regeneration method of the present invention, it has been shown to exhibit excellent regeneration efficiency regardless of aerobic and anaerobic conditions.

Comparative example  One. NADH  Electrochemical Regeneration Measurement

Electrochemically -0.8 using 1 unit PtNP (92.5 μM) and 0.2 mM NAD ⁺ coenzyme, 0.1 mM Cp * Rh (bpy) (H ₂ O)] ²⁺ at the same concentration as used in Example 3 above. When reacted for 12 hours at V, the yield was 18% (see Fig. 13). This results in much lower than 59% NADH photochemical regeneration yield using the same concentration of PtNP and coenzyme.

Example  4. PtNP According to the concentration of NADH of Photogeneration rate  Measurement result

NADH photogeneration initial rate (initial rate) was shown to be dependent on the concentration of PtNP (see Figure 2 (A) and Figure 8). Increasing the amount of PtNP from 1 to 4 units increased the initial rate for NADH formation, and the final yield after irradiation up to 12 hours increased from 59% to 86%. The increase curve was linear at low concentrations of PtNP, but at high concentrations it appears to be due to a limiting substrate.

When the concentration of PtNP was 3 units or more, a constant yield was obtained, which means that the photochemical step became turnover-limiting. The maximum conversion rate was 86%, and the maximum conversion rate was shown when PtNP was 3 units. Using 1 unit of PtNP yielded a yield of 59%, corresponding to ˜300 turnover per hour, assuming 5 nm Pt nanoparticles contain 2800 Pt elements. As expected, the turnover number decreased with increasing PtNP concentration (see FIG. 2B).

Example  5. pH In accordance NADH Photogeneration  Measure

The pH conditions were adjusted to 5.6, 6.0, 7.0, 8.0 and 9.1, respectively, by adjusting the pH of the phosphate buffer. The degree of generation of NADH was measured using solutions having different pHs (see FIG. 3).

The NADH regeneration yield was maximum when the pH was 7.0, and a significant amount of NADH was produced even at pH 8.0 and 9.1 (see FIG. 9). It can be seen that NADH production is significantly inhibited at higher hydrogen concentrations. The decrease in the rate of NADH production under acidic pH conditions appears to be the result of protonation of TEOA which is converted to less efficient electron donors.

Example  6. TEOA Concentration of NADH  Impact on Regeneration

The result of the concentration-dependent experiment of TEOA is as shown in Fig. 4A.

NADH production also increased with increasing TEOA concentration in the presence of 1 unit of PtNP. When the concentration of TEOA was 0.2 to 0.4 M, NADH conversion was 36 to 55% after 9 hours (see FIG. 10). It can be seen that the oxidation step of TEOA is also a rate step.

Comparative example  2. other electronic Donor NADH  Impact on Regeneration

Ethylenediaminetetraacetic acid (EDTA), triethylamine (Et ₃ N) and sodium formate (Na-formate) were used instead of TEOA as electron donors, and the concentrations were 1, 400 and 100 mM, respectively. It was confirmed that the amount of NADH production after the reaction for 12 hours was very small (see FIGS. 14 to 16). Yields were about 3, 2 and 5%, respectively.

Example  7. NAD ⁺  According to concentration NADH  Play speed measurement

If the surface of the PtNP is required for the catalytic reaction, there will be no effect on the (regeneration) rate because the surface is saturated with the substrate when the concentration of NAD ⁺ increases above a certain level. Experimental results (regeneration) rate was found to be a constant value when 0.2 mM NAD ⁺ or more, and recalled the 0th order reaction (see Fig. 4 (B)).

Taken together all the experimental results, it can be seen that PtNP and TEOA are useful for visible-propulsive reduction of NAD ⁺ (see Fig. 5). The photoexcited PtNP can reduce or oxidatively quench (quench) NAD ⁺ and TEOA, respectively. Photo-propulsive PtNP can induce charge separation, excited electrons are transferred to NAD ⁺ for reduction, and valence band holes are reductively reclaimed by TEOA. As a result, it can be seen that PtNP can capture an incident photon under visible light.

Example  8. Created NADH Enzyme reaction

The reaction conditions (PtNP, NAD, TEOA, pH = 7) using 5 mM alpha-glutamate, 80 mM ammonium phosphate, 2 units glutamate dehydrogenase 9 After 85 hours of conversion, 85% conversion was obtained. The reaction product was analyzed using HPLC with L-glutamate (column was 150 mm ODS-3V in length, separated with 0.08% aqueous phosphoric acid solution and detected at 210 nm).

It is also possible to react with other enzymes such as formate dehydrogenase, glucose-6-phosphate dehydrogenase.

Claims (11)

(a) adding platinum (Pt) nanoparticles to a redox oxidase cofactor at a concentration of 1 unit to 4 units; And (b) photochemical regeneration method of the oxidoreductase cofactor comprising the step of light irradiation to the mixture of step (a):
However, 1 unit here means containing 92.5 micrometers Pt atom.
The method of claim 1,
The photochemical regeneration method of the oxidoreductase cofactor, wherein the oxidoreductase cofactor of the oxidized type in step (a) is any one selected from the group consisting of NAD ⁺ , NADP ⁺ , FAD ⁺ and FMN ⁺ .
The method of claim 1,
In the step (a), the oxidoreductase cofactor photochemical regeneration method, characterized in that further adding an electron donor (electron donor).
The method of claim 3,
The electron donor is triethanolamine, characterized in that the photochemical regeneration of the oxidoreductase cofactor.
delete delete The method of claim 1,
The photochemical regeneration method of the oxidoreductase cofactor, characterized in that to adjust the pH of step (a) to 7 or more.
The method of claim 1,
The photochemical regeneration method of the oxidoreductase cofactor, characterized in that the light of step (b) is visible light.
The method of claim 1,
The photochemical regeneration method of the oxidoreductase cofactor, characterized in that step (a) is carried out in a buffer.
delete delete

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