Photocatalytic artificial coenzyme regeneration system based on heterojunction photocatalyst and application thereof

Abstract

This invention discloses a photocatalytic artificial coenzyme regeneration system based on a heterojunction photocatalyst and its application. The heterojunction photocatalyst consists of four components: graphitic carbon nitride (g-C3N4), zinc acetate, N,N-dimethylformamide, and hexahydroxytriphenyl. g-C3N4 is first obtained from urea through high-temperature calcination. Then, it is gradually mixed with other components according to a specific procedure and dried. Finally, the dried material is calcined again at high temperature to obtain the photocatalyst g-C3N4 / Zn-MOF. An electron mediator and an electron sacrificial agent are then added to construct the photocatalytic artificial coenzyme regeneration system. The photocatalytic artificial coenzyme regeneration system constructed in this invention significantly improves the efficiency of photocatalytic artificial coenzyme regeneration and achieves higher efficiency than the natural coenzyme NAD+. + BANA, an artificial coenzyme with higher regeneration efficiency + The photocatalytic artificial coenzyme regeneration system was successfully coupled with an enzyme reaction.

Description

TECHNICAL FIELD

[0001] The application relates to a photocatalytic artificial coenzyme regeneration system based on a heterojunction photocatalyst and application thereof, and belongs to the technical field of photocatalytic artificial coenzyme regeneration. BACKGROUND

[0002] Oxidoreductases are an important part of enzymes, and the oxidation-reduction reactions (EC.1.X.X.X) catalyzed and regulated by the oxidoreductases play a very important role in the field of biocatalysis of chiral drugs and fine chemical synthesis. However, the key to the use of most oxidoreductases in cell-free biocatalysis is to use a certain amount of oxidoreduction equivalent, namely nicotinamide adenine dinucleotide (phosphate) NAD(P) / H. However, in the process of enzymatic reaction, the coenzyme is converted into the corresponding oxidized or reduced form while providing the oxidoreduction equivalent, resulting in the consumption of the coenzyme. In addition, due to the complicated regeneration process of the coenzyme in nature, the high cost and low stability of the natural coenzyme synthesis make it a major limiting factor in many applications for medium or even low-cost products. In order to solve this problem, it is necessary to reduce the production cost and improve the stability of the corresponding coenzyme.

[0003] The nicotinamide coenzyme biomimetic (NCBs) with simpler structure and higher stability is expected to further improve the stability of the coenzyme and reduce the production cost. The artificial coenzyme is modified from the natural coenzyme, however, most of the current researches are focused on how to regenerate the natural coenzyme NAD(P)H, and there are few reports on the regeneration of the artificial coenzyme. There are some reports on the regeneration of the natural coenzyme using semiconductor materials under light conditions, and the application of the photocatalytic natural coenzyme regeneration method can further reduce the cost of the coenzyme through the circulation of the coenzyme. In the process of photocatalytic coenzyme regeneration, the photoexcited electrons are transferred to the surface of the photocatalyst to reduce the oxidized coenzyme, but the recombination of the photoexcited electron-hole pairs limits the efficiency of the coenzyme regeneration. In addition, compared with the natural coenzyme, some artificial coenzymes have higher oxidation-reduction potential, and therefore, to realize the photocatalytic regeneration of the artificial coenzyme, a photocatalyst with more easily generated photoexcited electron-hole pairs and more difficult recombination is needed, so that the application tries to develop a photocatalytic system suitable for the regeneration of the artificial coenzyme. SUMMARY

[0004] To solve the above problems, in the application, a heterojunction photocatalyst g-C3N4 / Zn-MOF is synthesized by using the high separation ability of the photoexcited electron-hole pairs of the heterojunction photocatalyst, and an electron mediator and an electron sacrificial agent are added to construct a high-efficiency photocatalytic artificial coenzyme regeneration system. The system promotes the transfer of the photoexcited electrons by combining two semiconductors with different energy band structures to improve the efficiency of the artificial coenzyme regeneration, especially for the artificial coenzyme BANA +The light catalytic regeneration exhibits extremely excellent effect, and the regeneration efficiency is much higher than that of natural coenzyme NAD + .

[0005] The first object of the present application is to provide a light catalytic artificial coenzyme regeneration system, which comprises a heterojunction photocatalyst containing zinc-containing metal organic framework@graphitic carbon nitride, and the artificial coenzyme is selected from one of the structures shown below:

[0006]

[0007] Further, the light catalytic artificial coenzyme regeneration system further comprises a redox electron mediator and an electron sacrificial agent.

[0008] Further, the redox electron mediator includes but is not limited to Cp*Rh(bpy)H2O 2+ , etc.

[0009] Further, the electron sacrificial agent includes but is not limited to formic acid, formate, or tertiary amine (such as triethanolamine), etc.

[0010] Further, in the light catalytic artificial coenzyme regeneration system, the amount ratio of the heterojunction photocatalyst, the redox electron mediator and the electron sacrificial agent is 0.1-0.5 mg: 25-200 μM: 100-1000 mM.

[0011] Further, the preparation method of the zinc-containing metal organic framework@graphitic carbon nitride comprises the following steps:

[0012] S1, dispersing the graphitic carbon nitride and N,N-dimethylformamide in water, then adding zinc salt, stirring, heating to 60-80℃, adding hexahydroxytriphenyl, continuing to stir, and collecting the solid after the reaction is completed;

[0013] S2, drying the solid collected in S1, and calcining at 500-650℃ to obtain the zinc-containing metal organic framework@graphitic carbon nitride.

[0014] Further, the mass ratio of the graphitic carbon nitride, zinc salt, N,N-dimethylformamide and hexahydroxytriphenyl is 80-120: 5-15: 10-30: 5-15.

[0015] The second object of the present application is to provide the application of the light catalytic artificial coenzyme regeneration system in artificial coenzyme regeneration.

[0016] Further, the heterojunction photocatalyst, the redox electron mediator and the electron sacrificial agent are mixed, irradiated, and the reduction of the artificial coenzyme is carried out.

[0017] Furthermore, the wavelength of the light source is 420–500 nm, the power of the light source is 2–12 W, and the temperature is 25–37 °C.

[0018] A third objective of this invention is to provide the application of the aforementioned photocatalytic artificial coenzyme regeneration system in the synthesis of redox enzyme-catalyzed products.

[0019] The fourth objective of this invention is to provide a method for product synthesis using oxidoreductase catalysis, wherein the heterojunction photocatalyst, redox electron mediator, electron sacrificial agent, oxidoreductase, and substrate are mixed and reacted under light to synthesize the product.

[0020] Furthermore, the oxidoreductase includes, but is not limited to, succinyl oxalisase, equine liver alcohol dehydrogenase, glucose dehydrogenase, etc., and correspondingly, the substrate includes, but is not limited to, tea aroma ketone, trans-cinnamaldehyde, etc.

[0021] Furthermore, the sequence of the senescent enzyme is shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0022] Furthermore, the wavelength of the light source is 420–500 nm, the power of the light source is 2–12 W, and the temperature is 25–37 °C.

[0023] The beneficial effects of this invention are:

[0024] The photocatalytic artificial coenzyme regeneration system of the present invention, containing a heterojunction photocatalyst, assembles g-C3N4 with excellent photosensitivity and Zn-MOF with excellent electron conductivity, thereby improving the coenzyme regeneration efficiency. Furthermore, during the regeneration of the artificial coenzyme, an artificial coenzyme with superior regeneration efficiency compared to the natural coenzyme is used, and it is successfully coupled with the enzyme reaction, thus improving the conversion rate of the enzyme reaction. Attached Figure Description

[0025] Figure 1 Cold field emission scanning electron microscope image of the heterojunction photocatalyst g-C3N4 / Zn-MOF used.

[0026] Figure 2 The structural formulas of the artificial coenzyme and natural coenzyme used in Examples 1-3 of this invention are shown.

[0027] Figure 3 The photocatalytic artificial coenzyme regeneration system constructed in Example 1 of this invention is effective against the natural coenzyme NAD. + And a comparison of the regeneration efficiency of different artificial coenzymes with that of g-C3N4 as the photocatalyst.

[0028] Figure 4 The conversion rate results are for the photocatalytic artificial coenzyme regeneration system constructed in Example 2 of this invention coupled with the reduction of tea aroma ketone by the old yellow enzyme XenA.

[0029] Figure 5 Conversion rate results of the photocatalytic artificial coenzyme regeneration system constructed in Example 3 of the present application and the reaction coupling of the old yellow enzyme TsER reducing the theaspirone. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with the drawings and specific examples so that those skilled in the art can better understand the present application and implement it.

[0031] The scheme provided by the present application is as follows:

[0032] The present application designs a photocatalytic artificial coenzyme regeneration system for using a heterojunction photocatalyst, wherein the heterojunction photocatalyst is composed of four components of graphite carbon nitride g-C3N4, zinc acetate, N,N-dimethylformamide and hexahydrotriphenyl, and the mass ratio among the four is 80-120:5-15:10-30:5-15.

[0033] The photocatalytic artificial coenzyme regeneration system is constructed according to the following steps:

[0034] Step one, put urea into a ceramic crucible, wrap and seal the crucible with tin paper, and place it in a muffle furnace for calcination. The obtained light yellow powder is graphite carbon nitride g-C3N4.

[0035] Step two, take g-C3N4 and add it into water containing N,N-dimethylformamide, ultrasonically disperse the suspension, and place the dispersed suspension on a magnetic stirrer for slow stirring at room temperature. During the stirring process, add zinc acetate dihydrate, and after a period of time, increase the temperature to 60-80℃, add hexahydrotriphenyl, and continue to maintain slow stirring. After the stirring is completed, centrifuge and collect the suspension, wash it with anhydrous ethanol, and then dry it at 60-70℃.

[0036] Step three, place the obtained dry mixture into a ceramic crucible, wrap and seal the crucible with tin paper, and place it in a muffle furnace for calcination. The obtained granular light yellow powder is the heterojunction photocatalyst g-C3N4 / Zn-MOF.

[0037] Step four, add the heterojunction photocatalyst g-C3N4 / Zn-MOF, an electron mediator (Cp*Rh(bpy)H2O 2+ ), and an electron sacrificial agent (triethanolamine) into a photocatalytic reactor, and then add an oxidized artificial coenzyme to obtain the constructed photocatalytic artificial coenzyme regeneration system.

[0038] In step one, the process conditions for muffle furnace calcination are as follows: firing at 500-650℃ for 1-4 hours, and the temperature rising rate during the temperature rising process is 3-7℃ / min.

[0039] In step two, the process of ultrasonic dispersion is to place the suspension in an ultrasonic cleaner with a frequency of 30-50 kHz and ultrasonic treatment for 20-30 minutes. The stirring speed of the magnetic stirrer is in the range of 100-200 r / min.

[0040] In step three, the process conditions of muffle calcination are as follows: calcination at 350-450℃ for 1-4 hours, and the heating rate is 3-7℃ / min.

[0041] In step four, the parameters of the photocatalytic reactor are as follows: the wavelength of light is 420-500 nm, the power of light source is 2-12 W, the temperature is set to 25-37℃, the concentration of heterojunction photocatalyst g-C3N4 / Zn-MOF is 0.1-0.5 mg, the concentration of electron mediator (Cp*Rh(bpy)H2O 2+ ) is 25-200 μM, and the concentration of electron sacrificial agent (triethanolamine) is 100-1000 mM.

[0042] Example 1

[0043] Photocatalytic artificial coenzyme regeneration system for reducing different oxidation forms of artificial coenzyme + The steps are as follows:

[0044] (1) Put 10 g of urea into a ceramic crucible, wrap the crucible with tin paper to seal, and place it in a muffle furnace for calcination. Burn at 550℃ for two hours, and the heating rate during the heating process is 5℃ / min. A light yellow powder is obtained;

[0045] (2) Take 100 mg of the light yellow powder obtained in step (1) and add it to 5 mL of water containing 20 mg of N,N-dimethylformamide. Ultrasonically disperse the suspension in an ultrasonic cleaner with a frequency of 30 kHz for 20 min. Place the dispersed suspension on a magnetic stirrer and slowly stir at room temperature, with the stirring speed set to 120 r / min. Add 10 mg of zinc acetate dihydrate (or 10 mg of manganese chloride tetrahydrate, or 10 mg of copper sulfate pentahydrate) during stirring. After two hours, increase the temperature to 70℃, add 10 mg of hexahydroxytriphenyl, and continue to slowly stir for four hours. Centrifuge the suspension after stirring, wash it three times with anhydrous ethanol, and then dry it at 65℃;

[0046] (3) Put the obtained dry mixture into a ceramic crucible, wrap the crucible with tin paper to seal, and place it in a muffle furnace for calcination, and burn at 550°C for two hours, the heating rate during the heating process is 5°C / min, and the obtained granular light yellow powder is the heterojunction photocatalyst g-C3N4 / Zn-MOF (corresponding to g-C3N4 / Mn-MOF or g-C3N4 / Cu-MOF).

[0047] (4) 0.4 mg of the heterojunction photocatalyst g-C3N4 / Zn-MOF (g-C3N4 / Mn-MOF or g-C3N4 / Cu-MOF), 50 μM of the electron mediator (Cp*Rh(bpy)H2O 2+ ), 800 mM of the electron sacrificial agent (triethanolamine) were added to the photocatalytic reactor, and then 5 mM of the oxidized artificial coenzyme (including BANA + , BNA + , P2NA + and P3NA + ) and NAD + (structure formula see Figure 2 ) were added, the illumination wavelength was set to 460 nm, the illumination power was 10 W, the temperature was set to 30°C, and finally the reduction of the oxidized coenzyme was carried out, and the reaction time was 40 minutes.

[0048] Figure 3 The efficiency of the photocatalytic artificial coenzyme regeneration system was shown, and the artificial coenzyme BANA + had the highest conversion rate of 73.86% under the same reaction conditions, higher than the natural coenzyme NAD + 54.71%, and also higher than the yield of 48.32% of the experimental group with g-C3N4 as the photocatalyst under the same conditions.

[0049] Example 2

[0050] The photocatalytic artificial coenzyme regeneration system was coupled with the reduction of the old yellow enzyme XenA and the reaction of theamylase, and the steps of Example 2 and Example 1 were basically the same, and the only difference was that:

[0051] Step (4), 0.4 mg of the heterojunction photocatalyst g-C3N4 / Zn-MOF, 50 μM of the electron mediator (Cp*Rh(bpy)H2O 2 + ), 800 mM of the electron sacrificial agent (triethanolamine) were added to the photocatalytic reactor, and then 1 mM of the oxidized artificial coenzyme (including BANA + , BNA + , P2NA + and P3NA + ) and NAD +, 0.05 mg / mL XenA (sequence see SEQ ID NO. 1), 10 mM theaflavins, light wavelength set to 460 nm, light power is 10 W, temperature set to 30℃, finally the reduction of theaflavins, reaction time 90 minutes.

[0052] Figure 4 The conversion rate results of the constructed photocatalytic artificial coenzyme regeneration system coupled with the reduction of theaflavins by old yellow enzyme XenA are shown in Table 1, and the experimental group adding artificial coenzyme BANA + has the highest conversion rate under the same reaction conditions: more than 99% after 90 minutes of reaction, under the same reaction conditions, the conversion rate of the experimental group adding natural coenzyme NAD + is only 74.24%.

[0053] Example 3

[0054] The photocatalytic artificial coenzyme regeneration system is coupled with the reduction of theaflavins by old yellow enzyme TsER, and the steps of this embodiment 3 and embodiment 2 are basically the same, and the only difference is that:

[0055] The old yellow enzyme XenA in step (4) of embodiment 2 is replaced by old yellow enzyme TsER (sequence see SEQ ID NO. 2).

[0056] Figure 5 The conversion rate results of the constructed photocatalytic artificial coenzyme regeneration system coupled with the reduction of theaflavins by old yellow enzyme TsER are shown in Table 2, and the experimental group adding artificial coenzyme BANA + has the highest conversion rate under the same reaction conditions: 75.15% after 90 minutes of reaction, under the same reaction conditions, the conversion rate of the experimental group adding natural coenzyme NAD + is only 59.71%.

[0057] Obviously, the above examples are only examples for the purpose of clarity, and are not a limitation on the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A photocatalytic artificial coenzyme regeneration system characterized by comprising: The photocatalytic artificial coenzyme regeneration system comprises a heterojunction photocatalyst, a redox electron mediator and an electron sacrificial agent, the heterojunction photocatalyst contains zinc-containing metal organic framework@graphitic carbon nitride, and the artificial coenzyme is selected from one of the structures shown in the following: ； The redox mediator is [Cp*Rh(bpy)H2O] 2+ ; The electron sacrificial agent is selected from one or more of formic acid, formate, and tertiary amine; The preparation method of the zinc-containing metal organic framework@graphitic carbon nitride comprises the following steps: S1, dispersing the graphitic carbon nitride and N, N-dimethylformamide in water, then adding a zinc salt, stirring, heating to 60-80℃, adding hexahydroxytriphenyl, continuing to stir, and collecting the solid after the reaction is completed; S2, drying the solid collected in S1, and calcining at 500-650℃ to obtain the zinc-containing metal organic framework@graphitic carbon nitride.

2. The photocatalytic coenzyme regeneration system according to claim 1, wherein In the photocatalytic artificial coenzyme regeneration system, the amount ratio of the heterojunction photocatalyst, the redox electron mediator and the electron sacrificial agent is 0.1-0.5 mg: 25-200 μM: 100-1000 mM.

3. The photocatalytic artificial coenzyme regeneration system of claim 1 or 2 is applied in artificial coenzyme regeneration.

4. Use according to claim 3, characterized in that, The application is to mix the heterojunction photocatalyst, the redox electron mediator and the electron sacrificial agent, and to regenerate the artificial coenzyme under light irradiation.

5. Use according to claim 4, characterized in that, The wavelength during light irradiation is 420-500 nm, the light source power is 2-12 W, and the temperature is 25-37℃.

6. The photocatalytic artificial coenzyme regeneration system of claim 1 or 2 is applied in enzyme catalytic reaction.

7. A method for the synthesis of a product catalyzed by an oxidoreductase enzyme, characterized in that, The photocatalytic artificial coenzyme regeneration system of claim 1 or 2 is mixed with an oxidoreductase and a substrate, and the product is synthesized by reaction under light irradiation; wherein the oxidoreductase is old yellow enzyme, horse liver alcohol dehydrogenase or glucose dehydrogenase, and the substrate is theaspirone or trans-cinnamaldehyde.

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