Alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide artificial coenzymes, preparation method and application thereof

Abstract

The invention discloses an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes, as well as a preparation method and application thereof. First, a carbon-nitrogen precursor and an alkali metal chloride are ground in a mortar, deionized water is added after grinding, and the uniformly mixed material is transferred to a porcelain boat, covered with a ceramic cover, placed in a tube furnace, and slowly heated and calcined under an N2 atmosphere. After the calcination is completed, the obtained solid product is ground into powder and washed multiple times with deionized water until the pH value of the mixed solution reaches 7-8. The mixture is then centrifuged to obtain a solid, which is added to a beaker, and then deionized water is added for ultrasonic treatment. After the ultrasonic treatment is completed, the material is finally centrifuged and the obtained solid is dried to obtain the carbon-nitrogen catalyst to be prepared. The prepared carbon-nitrogen catalyst can effectively utilize visible light, which dominates the solar spectrum, and has excellent NAD(P)H and nicotinamide-based artificial coenzyme regeneration performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of material preparation, and in particular relates to an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes, as well as a preparation method and application thereof. Background Art

[0002] Nicotinamide cofactor (NAD(P) / NAD(P)H) is a universal proton / electron carrier that plays an important role in biological redox reactions in vivo and in vitro. For example, NAD(P) + NAD(P)H regulates cellular metabolism in vivo. Meanwhile, reactions catalyzed by oxidoreductases in vitro typically require redox cofactors such as NAD(P)H to provide electrons and protons. These cofactors are expensive and require continuous regeneration for practical use. Nicotinamide cofactor-dependent oxidoreductases are used in a wide range of industrial and biotechnological applications. In these enzyme catalysis, the nicotinamide cofactor plays a crucial role in electron and energy transfer. However, these cofactors exhibit poor stability to extreme conditions such as high temperature, organic solvents, and acidic / alkaline pH, limiting the potential of oxidoreductases in industrial applications. Therefore, the development of artificial cofactors and oxidoreductases that are favorable to artificial cofactors has become particularly important, as they facilitate the development of in vitro enzymatic synthesis systems in terms of availability and cost. Given that traditional enzyme-catalyzed NAD(P)H regeneration is limited to natural coenzymes and chemical energy, the development of catalysts that extend the reach of natural coenzymes to artificial coenzymes, as well as catalytic systems driven by renewable energy rather than chemical energy, has practical applications.

[0003] To this end, the present invention proposes an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes, as well as a preparation method and application thereof. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration using NAD(P)H and nicotinamide-based artificial coenzymes, as well as its preparation method and application. The catalyst has the characteristics of good catalytic effect, simple preparation method, wide raw material source, and safety and environmental protection.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention provides a method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes, comprising the following steps:

[0007] 1) Grind the carbon-nitrogen precursor and the corresponding alkali metal chloride in a mortar, add deionized water and mix until evenly mixed;

[0008] 2) Transfer the uniformly mixed materials from step 1) to a porcelain boat, cover it with a ceramic lid, place it in a tube furnace, and slowly heat and calcine it under a nitrogen atmosphere. After calcination, grind the resulting solid product into powder for later use;

[0009] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0010] 4) The solid obtained in step 3) is added to a beaker, and then deionized water is added for ultrasonic treatment. After the ultrasonic treatment, the material is centrifuged, and the obtained solid is dried and packaged to obtain the alkali metal cation-doped carbon nitride microsphere catalyst to be prepared.

[0011] Furthermore, in step 1), the carbon-nitrogen precursor is cyanamide, dicyandiamide or melamine, preferably melamine; and the alkali metal chloride is lithium chloride, sodium chloride, potassium chloride, rubidium chloride or cesium chloride, preferably cesium chloride.

[0012] Furthermore, in step 1), the ratio of the volume of deionized water to the mass of the carbon-nitrogen precursor and the mass of the metal chloride is 1:1-5:0.5-4, preferably 1:3:2, with the volume unit being mL and the mass unit being g.

[0013] Furthermore, in step 2), the heating rate is 1.5-2.5°C / h, preferably 2.2°C / h; the calcination temperature is 450-600°C, preferably 550°C; and the calcination time is 3-5 h, preferably 4 h.

[0014] Furthermore, in step 4), the ultrasonic time is 0-36 h, preferably 24 h, the drying temperature is 60-110 ° C, preferably 70 ° C, and the drying time is 10-26 h, preferably 24 h.

[0015] The invention provides an alkali metal cation-doped carbon nitride microsphere catalyst prepared by the method.

[0016] The present invention also proposes an application of the alkali metal cation-doped carbon nitride microsphere catalyst in photocatalytic regeneration of cofactors.

[0017] Further, the following steps are included:

[0018] First, add carbon nitride catalyst and triethanolamine into sodium phosphate buffer, perform ultrasonic dispersion treatment, and then add and oxidized coenzyme to obtain a mixed solution, which is then transferred to a photocatalytic reactor, stirred under sealed conditions, and nitrogen is introduced, and reacted under the irradiation of a xenon lamp light source to perform photocatalytic regeneration of the coenzyme.

[0019] Furthermore, the ratio of the mass of the carbon nitride catalyst to the volume of the sodium phosphate buffer is 1:0.5-2, preferably 1:1, with the mass unit being g and the volume unit being mL; the volume ratio of triethanolamine to the sodium phosphate buffer is 0.1-0.5:1, preferably 0.15:1, with the volume unit being mL; The molar ratio of oxidized coenzyme is 0.2:3.

[0020] Furthermore, the nitrogen flow rate is 1-30 mL / min, preferably 10 mL / min; the wavelength of the xenon lamp is 300-800 nm, preferably a visible light wavelength of 420-800 nm.

[0021] Principle of the present invention:

[0022] The present invention modifies the carbon-nitrogen material by alkali metal cations of different radii. The doped ions embedded in the modified carbonized carbon material and a large number of cyano and hydroxyl functional groups formed on the surface achieve the effect of regulating the intrinsic energy level structure of the material, so that the prepared carbon-nitrogen catalyst can effectively utilize the visible light that dominates the solar spectrum.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1) The present invention first grinds a carbon-nitrogen precursor and a corresponding alkali metal chloride in a mortar, adds deionized water after grinding, mixes evenly, and sets aside for use. The evenly mixed material is transferred to a porcelain boat, covered with a ceramic lid, placed in a tube furnace, and slowly heated and calcined under a N2 atmosphere. After the calcination is completed, the obtained solid product is ground into powder for use. The obtained powder is washed multiple times with deionized water to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8. The mixture is then centrifuged to obtain a solid, which is added to a beaker, and then deionized water is added for ultrasonic treatment. After the ultrasonic treatment is completed, the material is finally centrifuged, and the obtained solid is dried and packaged to obtain the carbon-nitrogen catalyst to be prepared. The prepared carbon-nitrogen catalyst can effectively utilize the visible light that dominates the solar spectrum, thereby having good NAD(P)H and nicotinamide artificial coenzyme regeneration performance;

[0025] 2) The present invention has the characteristics of simple method, wide source of raw materials, the prepared catalyst does not contain precious metals, good catalytic effect, and safety and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 11 is a SEM image of the carbon nitride microsphere catalyst prepared in Examples 1 to 12 of the present invention;

[0027] Figure 2 FT-IR images of carbon nitride microsphere catalysts prepared in Examples 1 to 8 of the present invention;

[0028] Figure 3 Zeta potential diagrams of carbon nitride microsphere catalysts prepared in Examples 1 to 12 of the present invention;

[0029] Figure 4 This is a comparison chart of the regenerated NADH performance of the carbon nitride microsphere catalysts prepared in Examples 1 to 8 of the present invention;

[0030] Figure 5 This is a comparison chart of the regenerated NADH performance of the carbon nitride microsphere catalysts prepared in Examples 9 to 12 of the present invention;

[0031] Figure 6 This is a comparison chart of the catalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes in Examples I-1 to I-6 of the present invention;

[0032] Figure 7 This is a time-reduced coenzyme regeneration yield comparison chart of Example I-7 of the present invention;

[0033] Figure 8 This is a time-reduced coenzyme regeneration yield comparison chart of Example I-8 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the described scope.

[0035] Example 1

[0036] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0037] 1) Weigh 12 g of cesium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0038] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0039] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0040] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 1.

[0041] Example 2

[0042] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0043] 1) Weigh 12 g of rubidium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0044] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0045] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0046] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 2.

[0047] Example 3

[0048] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0049] 1) Weigh 12 g of potassium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0050] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0051] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0052] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 3.

[0053] Example 4

[0054] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0055] 1) Weigh 12 g of sodium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0056] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0057] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0058] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 4.

[0059] Example 5

[0060] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0061] 1) Weigh 12 g of lithium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0062] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0063] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0064] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 5.

[0065] Example 6

[0066] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0067] 1) Weigh 3 g of melamine and grind it in a mortar. Add 2 mL of deionized water and mix thoroughly.

[0068] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0069] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0070] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 6.

[0071] Example 7

[0072] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0073] 1) Weigh 12 g of cesium chloride and 3 g of cyanamide, grind them in a mortar, and then add 2 mL of deionized water to mix until well combined.

[0074] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0075] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0076] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 7.

[0077] Example 8

[0078] A method for preparing a cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0079] 1) Weigh 12 g of cesium chloride and 3 g of dicyandiamide, grind them in a mortar, and then add 2 mL of deionized water to mix until evenly mixed.

[0080] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0081] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0082] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 8.

[0083] Example 9

[0084] A method for preparing a cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0085] 1) Weigh 12 g of cesium chloride and 3 g of melamine in a mortar and grind them. Add 4 mL of deionized water and mix thoroughly.

[0086] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0087] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0088] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 24 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a 70°C vacuum oven and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 9.

[0089] Example 10

[0090] 1) Weigh 12 g of cesium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0091] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0092] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0093] 4) Transfer the solid obtained in step 3) to a beaker, add 200 mL of deionized water, and sonicate for 0 h. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a vacuum oven at 100°C and dry it for 24 h. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 10.

[0094] Example 11

[0095] A method for preparing a cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0096] 1) Weigh 12 g of cesium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0097] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0098] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0099] 4) Transfer the solid from step 3) to a beaker, add 200 mL of deionized water, and sonicate for 12 hours. After completion, centrifuge to remove the precipitate. Finally, place the solid precipitate in a vacuum oven at 100°C and dry it for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 11.

[0100] Example 12

[0101] A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes comprises the following steps:

[0102] 1) Weigh 12 g of cesium chloride and 3 g of melamine in a mortar and grind them. Add 2 mL of deionized water and mix thoroughly.

[0103] 2) Transferring the uniformly ground and mixed materials from step 1) to a porcelain boat, covering it with a ceramic lid, and placing it in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 550°C at a heating rate of 2.2°C / h and calcined for 4 h. After calcination, the resulting solid product was ground into a powder for later use.

[0104] 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid;

[0105] 4) Transfer the solid from step 3) to a beaker, add 200 mL of deionized water, and sonicate for 36 hours. After completion, centrifuge the precipitate and finally dry it in a vacuum oven at 100°C for 24 hours. This yields the final alkali metal cation-doped carbon nitride microsphere catalyst 12.

[0106] The alkali metal cation-doped carbon nitride catalyst prepared above was tested using the following method:

[0107] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 60 mg NAD + The mixed solution was then transferred to a photocatalytic reactor, sealed and stirred, with nitrogen gas introduced at a rate of 10 mL / min for 30 minutes, and heated in a 37°C water bath. A 300-W xenon lamp light source, equipped with a visible light filter with a cutoff wavelength of 420 nm, was then used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of the catalytic coenzyme.

[0108] The reaction solution was sampled every 5 minutes to measure NADH concentration. 1 mL of the centrifuged supernatant was diluted with 2 mL of phosphate buffer and the absorbance was measured at 340 nm. The relationship between NADH concentration and absorbance was determined by comparing the absorbance with the standard curve.

[0109] The calculation formula is: .

[0110] from Figure 1 Scanning electron microscopy (SEM) images show that the morphologies of alkali metal cation-doped carbon nitride catalysts 1-12 exhibit irregular granular and flake structures. Alkali metal cations of varying radius lead to varying degrees of microspherical morphology. The addition of water and the duration of ultrasonication during catalyst grinding both influence the formation of microspheres.

[0111] Further from Figure 2 The Fourier transform infrared (FT-IR) spectrum shows that the graphite carbon nitride material modified with a larger ionic radius is the key factor for the alkali metal cation-doped carbon nitride microsphere catalyst to have a high coenzyme regeneration yield. Carbon nitride catalyst 6 is the original unmodified graphite carbon nitride, which does not contain a cyanide peak (2178 cm -1 ) and hydroxyl peak (3200 cm -1 -3600cm -1 , 1150cm-1 , 1000cm -1 ), the basic heptazine ring structure (1200cm -1 -1800cm -1 ), as the radius of the doped alkali metal cation increases, the catalyst presents a large number of cyano groups (2178 cm -1 ) and hydroxyl peak (3200 cm -1 -3600cm -1 , 1150cm -1 , 1000cm -‍1 ), indicating the introduction of a large number of these two types of functional groups. The heptazine ring structure is consistent with that of graphitic carbon nitride materials, indicating that the heptazine ring structure has not been destroyed. With the increase of sonication time, the amount of cyano and hydroxyl groups gradually increases. After sonication for more than 24 hours, the excessive content of cyano and hydroxyl groups leads to the destruction of the overall carbon-nitrogen structure, reducing the visible light response and the ability to regenerate the coenzyme. Therefore, after 24 hours of sonication, the alkali metal cation-doped carbon nitride microsphere catalyst 1, modified with Cs ions with the largest ionic radius, is the optimal catalyst.

[0112] From the zeta-potential diagram ( Figure 3 ) As can be seen, due to the incorporation of alkali metal cations into the carbon nitride matrix, when the ions are dispersed in solution, the surface cations tend to dissolve in water, forming colloidal particles with a negative zeta potential. The negative potential in solution is related to the radius of the doped alkali metal cation. A larger ionic radius results in a more negative zeta potential, enabling faster adsorption of reactants onto the catalyst surface and facilitating coenzyme regeneration. The optimal catalyst, 1, is the alkali metal cation-doped carbon nitride microsphere catalyst with the largest ionic radius, modified with cesium chloride, exhibiting the most negative zeta potential at -18.0 mV, demonstrating the best NADH regeneration performance.

[0113] Experimental study on photocatalytic regeneration of NADH by alkali metal cation-doped carbon nitride microspheres catalyst ( Figure 4 ) showed that as the radius of the doped alkali metal cation increased, the concentration of NADH regeneration increased significantly, and carbon-nitrogen catalyst 1 had the highest NADH yield. The concentration of NADH regeneration increased with the extension of ultrasonication time. After ultrasonication for more than 24 h, the NADH concentration decreased again. Therefore, 24 h of ultrasonication was the optimal time.

[0114] pass Figure 4 and Figure 5 As can be seen from the NADH production comparison chart, the catalysts prepared from cyanamide and dicyandiamide precursors can regenerate NADH, but the effect is not as good as that of melamine (compared to Figure 4Alkali metal chlorides such as lithium chloride, sodium chloride, potassium chloride, and rubidium chloride can all be used to regenerate NADH, but none are as effective as cesium chloride. The amount of water used in catalyst preparation, the duration of ultrasonic treatment, the number of washes, the drying temperature, and the drying time can all reduce the catalytic NADH concentration. Optimum yields were achieved using optimized conditions.

[0115] Comparison of application conditions of the catalyst of Example 1

[0116] Example I-1

[0117] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 60 mg NADP + The mixed solution was then transferred to a photocatalytic reactor, sealed and stirred, with nitrogen gas introduced at a rate of 10 mL / min for 30 minutes, and heated in a 37°C water bath. A 300 W xenon lamp light source, equipped with a visible light filter with a 420 nm cutoff wavelength, was used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of NADPH.

[0118] Example I-2

[0119] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 26 mg of nicotinamide-based artificial coenzyme NAD + A mixed solution of NADH-m1 (3 mM) was prepared. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-venting. The solution was then heated in a 37°C water bath. A 300 W xenon lamp light source, equipped with a visible light filter with a 420 nm cutoff wavelength, was used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of NADH-m1.

[0120] Example I-3

[0121] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 27 mg of nicotinamide-based artificial coenzyme NAD +A mixed solution of NADH-m2 (3 mM) was obtained. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-venting. The solution was then heated in a 37°C water bath. A 300 W xenon lamp light source was then used. A visible light filter with a cutoff wavelength of 420 nm was used to control the wavelength of the xenon lamp output to visible light greater than 420 nm. The light was then vertically irradiated into the photocatalytic reactor to initiate the photocatalytic regeneration of NADH-m2.

[0122] Example I-4

[0123] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 29 mg of nicotinamide-based artificial coenzyme NAD + A mixed solution of NADH-m3 (3 mM) was obtained. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-venting. The solution was then heated in a 37°C water bath. A 300 W xenon lamp light source was then used. A visible light filter with a cutoff wavelength of 420 nm was used to control the wavelength of the xenon lamp output to visible light greater than 420 nm. The light was then vertically irradiated into the photocatalytic reactor to initiate the photocatalytic regeneration of NADH-m3.

[0124] Example I-5

[0125] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed. Then, 2.6 mg (0.2 mM) and 30 mg of nicotinamide-based artificial coenzyme NAD + A mixed solution of NADH-m4 (3 mM) was obtained. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-aeration in a 37°C water bath. A 300 W xenon lamp light source, equipped with a visible light filter with a 420 nm cutoff wavelength, was then used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of NADH-m4.

[0126] Example I-6

[0127] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to sodium phosphate buffer and ultrasonically dispersed, and then 2.6 mg of (0.2 mM) and 32 mg of nicotinamide-based artificial coenzyme NAD +A mixed solution of NADH-m5 (3 mM) was prepared. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-venting. The solution was then heated in a 45°C water bath. A 300 W xenon lamp light source, equipped with a visible light filter with a 420 nm cutoff wavelength, was used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of NADH-m5.

[0128] Example I-7

[0129] The reaction was carried out at 37°C, and the reaction system consisted of glucose (100 mM), NAD(P) + The mixture is composed of nicotinamide or other artificial coenzymes (3 mM), glucose dehydrogenase (GDH), and 30 ml of sodium phosphate buffer. Transfer the mixed solution to a 500 mL photocatalytic reactor, add a magnetic stirrer, seal the reactor, and bubble nitrogen at a rate of 10 mL / min for 30 minutes. Heat in a 37°C water bath. Measure the absorbance using a UV-visible spectrophotometer.

[0130] Example I-8

[0131] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to the sodium phosphate buffer, and then 2.6 mg of (0.2 mM) and 60 mg of nicotinamide-based artificial coenzyme NAD + The mixed solution was then transferred to a photocatalytic reactor, sealed and stirred, with nitrogen gas introduced at a rate of 10 mL / min for 30 minutes, and heated in a 25°C water bath. A 300 W xenon lamp light source, equipped with a visible light filter with a cutoff wavelength of 420 nm, was then used to vertically illuminate the photocatalytic reactor to carry out the photocatalytic regeneration of NADH.

[0132] Example I-9

[0133] First, 30 mg of carbon nitride catalyst and 4.5 mL of triethanolamine were added to the sodium phosphate buffer, and then 2.6 mg (0.2 mM) and 60 mg, NAD +, (3 mM) to obtain a mixed solution. The mixed solution was then transferred to a photocatalytic reactor, stirred in a sealed container, and nitrogen was introduced at a rate of 10 mL / min for 30 minutes before pre-venting. The solution was then heated in a 45°C water bath. A 300 W xenon lamp light source was then used. A visible light filter with a cutoff wavelength of 420 nm was used to control the wavelength of the xenon lamp output to visible light greater than 420 nm. The light was then vertically irradiated into the photocatalytic reactor to initiate the photocatalytic regeneration of NADH.

[0134] pass Figure 6 As can be seen in Examples (I-1) through (I-6), NADPH and other nicotinamide-based artificial coenzymes can be photocatalytically regenerated to concentrations comparable to NADH. As alternatives to natural cofactors, synthetic nicotinamide cofactor analogs, due to their low cost and excellent chemical stability, even improve biocatalytic efficiency. These synthetic artificial coenzymes can operate in more complex reactions with a variety of other redox enzymes. The photocatalytic regeneration of artificial coenzymes will promote the low-cost use of redox biocatalysts in chemical production, thereby broadening the use of redox enzymes in industrial biocatalysis.

[0135] from Figure 7 It can be seen from Example 1-7 that when GDH is used as the regeneration enzyme, NAD + and NADP + The regeneration efficiency of GDH was slightly higher than that of photocatalysis. However, for the nicotinamide-based artificial coenzymes, the regeneration efficiency of GDH was negligible. In contrast, the photocatalytic method was able to regenerate all artificial coenzymes with considerable efficiency, demonstrating the advantages of this catalytic system in practical applications.

[0136] from Figure 8 It can be seen that in Example 1-8 and Example 1-9, the increase or decrease of temperature will reduce the regeneration concentration of NADH (comparison Figure 4 The catalyst 1 in the reaction mixture was used), so 37 °C was selected as the reaction temperature.

Claims

1. A method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes, characterized in that The steps include: 1) Grind the carbon-nitrogen precursor and the corresponding alkali metal chloride in a mortar, add deionized water and mix until evenly mixed; 2) Transfer the uniformly mixed materials from step 1) to a porcelain boat, cover it with a ceramic lid, place it in a tube furnace, and slowly heat and calcine it under a nitrogen atmosphere. After calcination, grind the resulting solid product into powder for later use; 3) washing the powder obtained in step 2) with deionized water several times to wash away residual water-soluble chloride salts until the pH value of the mixed solution is 7-8, and then centrifuging the mixture to obtain a solid; 4) adding the solid obtained in step 3) to a beaker, then adding deionized water for ultrasonic treatment. After the ultrasonic treatment, the material is centrifuged, and the obtained solid is dried and packaged to obtain the alkali metal cation-doped carbon nitride microsphere catalyst to be prepared; In step 1), the ratio of the volume of deionized water to the mass of the carbon-nitrogen precursor and the mass of the metal chloride is 1:1-5:0.5-4, with the volume unit being mL and the mass unit being g; In step 1), the carbon and nitrogen precursor is cyanamide, dicyandiamide or melamine; and the alkali metal chloride is lithium chloride, sodium chloride, potassium chloride, rubidium chloride or cesium chloride.

2. The method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes according to claim 1, characterized in that In step 2), the heating rate is 1.5-2.5°C / h; the calcination temperature is 450-600°C; and the calcination time is 3-5 h.

3. The method for preparing an alkali metal cation-doped carbon nitride microsphere catalyst for photocatalytic regeneration of NAD(P)H and nicotinamide-based artificial coenzymes according to claim 1, characterized in that In step 4), the ultrasonication time is 0-36 h, the drying temperature is 60-110° C., and the drying time is 10-26 h.

4. An alkali metal cation-doped carbon nitride microsphere catalyst prepared by the method according to any one of claims 1 to 3.

5. Use of the alkali metal cation-doped carbon nitride microsphere catalyst as claimed in claim 4 in photocatalytic regeneration of cofactors.

6. The use according to claim 5, characterized in that The steps include: First, add carbon nitride catalyst and triethanolamine into sodium phosphate buffer, perform ultrasonic dispersion treatment, and then add and oxidized coenzyme to obtain a mixed solution, which is then transferred to a photocatalytic reactor, stirred under sealed conditions, and nitrogen is introduced, and reacted under the irradiation of a xenon lamp light source to perform photocatalytic regeneration of the coenzyme.

7. The use according to claim 6, characterized in that The ratio of the mass of the carbon nitride catalyst to the volume of the sodium phosphate buffer is 1:0.5-2, with the mass unit being g and the volume unit being mL; the volume ratio of triethanolamine to the sodium phosphate buffer is 0.1-0.5:1, with the volume unit being mL; The molar ratio of oxidized coenzyme is 0.2:

3.

8. The use according to claim 7, characterized in that The nitrogen flow rate was 1-30 mL / min; the xenon lamp wavelength was 300-800 nm.

Images