A bifunctional photo-enzyme composite catalyst and a preparation method and application thereof

Abstract

This invention provides a bifunctional photo-enzyme composite catalyst, its preparation method, and its application, belonging to the fields of nanocomposite material preparation and clean energy technology. In this invention, the bifunctional photo-enzyme composite catalyst can utilize photogenerated holes to oxidize NADH to NAD under visible light. + NAD + Furthermore, it promotes the catalytic oxidation of ethanol by ethanol dehydrogenase, while photogenerated electrons converge on Pt and split water to produce hydrogen. The bifunctional photo-enzyme composite catalyst does not require the addition of any sacrificial agent, avoiding the waste of photogenerated holes and making full use of photogenerated charge carriers. The raw materials for the preparation of the bifunctional photo-enzyme composite catalyst are inexpensive and readily available, and the preparation process is simple and feasible, which has good practical value.

Description

Technical Field

[0001] This invention belongs to the field of nanocomposite material preparation and clean energy technology, specifically relating to a bifunctional photo-enzyme composite catalyst, its preparation method and application. Background Technology

[0002] Hydrogen has advantages such as abundant and inexpensive raw materials, non-toxicity, and high production capacity; however, naturally occurring hydrogen is extremely rare. Therefore, the use of photocatalytic technology to split water and produce hydrogen has attracted widespread attention from researchers. g-C3N4, as an organic semiconductor polymer, possesses a band gap width (2.7 eV) and suitable conduction and valence band positions, endowing it with oxidation and reduction capabilities, making it promising for broad applications in photocatalysis. Its photocatalytic hydrogen production principle is as follows: when the incident light energy (hν) is greater than the band gap of g-C3N4, electrons in the valence band jump to the conduction band, forming photogenerated electrons. Strongly oxidizing photogenerated holes are left in the valence band. These excited electron-hole pairs migrate to the surface of the g-C3N4 catalyst and participate in redox reactions. Specifically, photogenerated electrons migrate to H2O molecules or H+ adsorbed on the catalyst surface. + The photogenerated holes are transported and reduced, while the additional sacrificial agent consumes them. Although the addition of the sacrificial agent can promote the hydrogen evolution half-reaction, it also wastes the photogenerated holes, preventing them from being effectively utilized.

[0003] Enzyme-catalyzed reactions can synthesize a variety of value-added fine chemicals with high selectivity under mild conditions. Coenzymes are an indispensable component in these reactions; in oxidoreductase-catalyzed reactions, NAD(H) is often required as an oxidant or reductant. However, within cells, the NAD(P)H / NAD(P) ratio... + It can be reversibly converted through coupling with substrate metabolism, thereby maintaining intracellular redox balance in living cells. However, in in vitro enzyme-catalyzed reactions, an excess of NAD(P) must be added to maintain the reaction. The high price of NAD(P) reduces the economic viability of adding an external coenzyme, thus limiting its industrial application in enzyme catalysis. Currently, photocatalytic NAD(P)H regeneration, as a relatively novel regeneration method, is attracting increasing attention due to its controllable reaction and utilization of visible light. However, current photocatalytic coenzyme regeneration systems mainly focus on photogenerated electrons, where the photocatalytic material absorbs light energy and generates photogenerated electrons, which are then used as the driving force for NAD... + The C4 molecule undergoes hydrogenation reduction to obtain the reduced coenzyme 1,4-NADH. This process still requires the addition of a sacrificial agent to capture holes in order to promote the reaction, which also results in some energy waste.

[0004] Therefore, it is necessary to develop a method that can simultaneously utilize photogenerated holes to oxidize NADH and generate NAD.+ A bifunctional photo-enzyme composite catalyst that can also utilize photogenerated electrons to split water and produce hydrogen. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bifunctional photo-enzyme composite catalyst, its preparation method, and its applications. In this invention, the bifunctional photo-enzyme composite catalyst can utilize photogenerated holes to oxidize NADH to NAD under visible light. + NAD + Furthermore, it promotes the catalytic oxidation of ethanol by ethanol dehydrogenase, while photogenerated electrons converge on Pt and split water to produce hydrogen. The bifunctional photo-enzyme composite catalyst does not require the addition of any sacrificial agent, avoiding the waste of photogenerated holes and making full use of photogenerated charge carriers. The raw materials for the preparation of the bifunctional photo-enzyme composite catalyst are inexpensive and readily available, and the preparation process is simple and feasible, which has good practical value.

[0006] This invention first provides a bifunctional photo-enzyme composite catalyst, denoted as ADH / UIO-66 / CN, which is composed of ADH / UIO-66 nanoparticles loaded on a two-dimensional CN plane; the ADH / UIO-66 is alcohol dehydrogenase (ADH) immobilized in the mesoporous channels of UIO-66.

[0007] Preferably, the mass ratio of ADH / UIO-66 to CN in the bifunctional photo-enzyme composite catalyst is 1-4:10.

[0008] This invention also provides a method for preparing the above-mentioned bifunctional photo-enzyme composite catalyst, specifically including the following steps:

[0009] ADH was slowly added to the dispersion of UIO-66 to obtain a mixed system. The mixed system was immobilized and reacted at a constant temperature. After the reaction was completed, the mixture was centrifuged and washed to obtain ADH / UIO-66.

[0010] CN and ADH / UIO-66 were dispersed in PBS buffer and stirred to react. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the bifunctional photoenzyme composite catalyst.

[0011] Preferably, the UIO-66 is prepared by the following method: dissolving zirconium chloride and terephthalic acid in DMF to obtain a DMF solution containing zirconium chloride and a DMF solution containing terephthalic acid;

[0012] Then, bovine serum albumin and DMF solution containing zirconium chloride were added to the DMF solution containing terephthalic acid. After mixing evenly, the mixture was reacted at 80°C for 24 hours. After the reaction was completed, the mixture was centrifuged, washed, and dried. Then, it was calcined at 325°C for 2 hours to obtain mesoporous UIO-66.

[0013] Preferably, in the mixed system, the final concentration of UIO-66 is 0.2-1.2 mg / mL, and the final concentration of ADH is 0.1-0.5 mg / mL.

[0014] Preferably, the immobilization reaction is carried out at room temperature for 2–12 hours.

[0015] Preferably, the CN is prepared by the following method: after drying, urea is ground and heated from room temperature to 550°C at a heating rate of 2.5°C / min, and calcined for 4 hours. Then, the calcined product is washed with nitric acid overnight, filtered, washed, and dried to obtain the finished product.

[0016] Preferably, the pH of the PBS buffer is 7.4, and its formulation is 0.27 g / L potassium dihydrogen phosphate, 1.42 g / L disodium hydrogen phosphate, 8 g / L sodium chloride, and 0.2 g / L potassium chloride.

[0017] The stirring reaction was carried out at room temperature for 2 hours.

[0018] Preferably, in the bifunctional photoenzyme composite catalyst, the mass ratio of ADH / UIO-66 to CN is 1-4:10.

[0019] This invention also provides the application of the bifunctional photo-enzyme composite catalyst in the photocatalytic splitting of water to produce hydrogen while simultaneously regenerating coenzymes.

[0020] This invention also provides the application of the above-mentioned bifunctional photo-enzyme composite catalyst in the photocatalytic splitting of water to produce hydrogen while simultaneously catalyzing the production of acetaldehyde from ethanol.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) In this invention, UIO-66 is introduced as an enzyme carrier into the CN-based photo-enzyme coupling system. UIO-66 is used as an immobilization "container" for alcohol dehydrogenase (ADH), which confines the free ADH in the mesoporous channels of UIO-66. This not only improves the immobilization capacity and stability of alcohol dehydrogenase, but also enables the recycling of alcohol dehydrogenase.

[0023] (2) This invention utilizes photogenerated holes to oxidize NADH and generate NAD. + NAD + This process further promotes the catalytic oxidation of ethanol by alcohol dehydrogenase. Simultaneously, photogenerated electrons converge on Pt and split water to produce hydrogen. No sacrificial agent is required in this process, avoiding the waste of photogenerated holes and ensuring full utilization of photogenerated carriers.

[0024] (3) In the bifunctional photocatalyst-enzyme composite catalyst of the present invention, CN and ADH serve as the active centers for photocatalysis and enzyme catalysis, respectively, achieving the conversion of ethanol to acetaldehyde while simultaneously producing hydrogen through photocatalytic water splitting. The hydrogen production performance of the bifunctional photocatalyst-enzyme composite catalyst of the present invention is 3.84 times that of pure CN, exhibiting good stability and maintaining high photocatalytic activity even after four cycles. Furthermore, the acetaldehyde conversion rate of the bifunctional photocatalyst-enzyme composite catalyst of the present invention can reach 1.82 mmol / g / h. Therefore, the bifunctional photocatalyst-enzyme composite catalyst has great potential in the field of clean energy. Attached Figure Description

[0025] Figure 1 Transmission electron micrographs of CN(a) and ADH / UIO-66 / CN(b).

[0026] Figure 2 The plot shows the unit immobilization load of ADH / UIO-66 under different UIO-66 contents (a), ADH concentrations (b), and immobilization times (c).

[0027] Figure 3 The diagrams show the pH stability verification (a), temperature stability verification (b), storage stability verification (c), and cycling capacity verification (d) for free ADH and immobilized ADH.

[0028] Figure 4 XRD spectra of UIO-66, ADH / UIO-66, CN, and ADH / UIO-66 / CN.

[0029] Figure 5 Fourier transform infrared spectra of UIO-66, ADH / UIO-66, CN, and ADH / UIO-66 / CN.

[0030] Figure 6 The images show the laser confocal images of ADH / UIO-66(ac) and ADH / UIO-66 / CN(df), where a represents ADH / UIO-66 under bright field, b represents ADH / UIO-66 under fluorescent field, c represents ADH / UIO-66 under binding field, d represents ADH / UIO-66 / CN under bright field, e represents ADH / UIO-66 / CN under fluorescent field, and f represents ADH / UIO-66 / CN under binding field.

[0031] Figure 7 Thermogravimetric analysis curves for UIO-66, ADH / UIO-66, and ADH / UIO-66 / CN are shown.

[0032] Figure 8The bar chart (a) shows the hydrogen production and acetaldehyde production of ADH / UIO-66 / CN with different composite ratios under visible light, and the recycling effect of ADH / UIO-66 / CN-2 (AUC-2) (b). In Figure b, the four lines represent the results of four consecutive cycles of hydrogen production. Detailed Implementation

[0033] 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 thereto.

[0034] In the following embodiments, ADH / UIO-66 / CN-X (abbreviated as AUC-X) refers to the composite material in which the mass ratio of ADH / UIO to CN is X:10.

[0035] Example 1: Preparation of ADH / UIO-66 immobilized enzyme

[0036] (1) Preparation of carrier material UIO-66:

[0037] Weigh out 0.1630 g of zirconium chloride and 0.1163 g of terephthalic acid and dissolve them in 10 mL of DMF to obtain a DMF solution containing zirconium chloride and a DMF solution containing terephthalic acid.

[0038] Then, 2 mg of bovine serum albumin and DMF solution containing zirconium chloride were added to the DMF solution containing terephthalic acid. The mixed solution was reacted at 80 °C for 24 h. After the reaction was completed, the solution was centrifuged, washed, and dried. Then, it was calcined at 325 °C for 2 h to obtain mesoporous UIO-66.

[0039] (2) Immobilization of alcohol dehydrogenase (ADH):

[0040] 1 mg of mesoporous UIO-66 was dispersed in 700 μL of 10 mM PBS buffer (pH 7.4), and then 300 μL of 1 mg / mL alcohol dehydrogenase solution was added. The mixture was then stirred and immobilized at room temperature for 12 h. After immobilization, the enzyme was centrifuged and washed to obtain immobilized ADH / UIO-66, which was stored at 4 °C for later use.

[0041] Figure 1 The images show transmission electron microscopy (TEM) images of CN and ADH / UIO-66 / CN. As can be seen from the images, pure CN without ADH / UIO-66 loading has a smooth two-dimensional planar structure. After loading with ADH / UIO-66, it is clearly visible that ADH / UIO-66 nanoparticles of approximately 70 nm are loaded onto the two-dimensional plane of CN. Furthermore, the ADH / UIO-66 nanoparticles fix ADH within the mesoporous channels of UIO-66 through physical adsorption, which can be clearly seen using laser confocal microscopy.

[0042] Example 2: Optimization of the ADH / UIO-66 Immobilization Process

[0043] (1) Effect of UIO-66 content in carrier material on immobilization process:

[0044] 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg of UIO-66 were weighed and dispersed in 700 μL of 10 mM PBS buffer (pH 7.4). Then, 300 μL of 1 mg / mL alcohol dehydrogenase solution was added. The mixture was stirred and immobilized at room temperature for 12 h. After immobilization, the mixture was centrifuged and washed to obtain the immobilized enzyme ADH / UIO-66.

[0045] Figure 2 (a) is a graph showing the unit immobilization load of ADH / UIO-66 at different UIO-66 contents. As can be seen from the graph, the unit immobilization load of ADH on UIO-66 gradually increases with increasing UIO-66 content, reaching its maximum when the UIO-66 content reaches 1.0 mg. Further increasing the UIO-66 content decreases the unit immobilization load of immobilized alcohol dehydrogenase. This is because when the UIO-66 content reaches 1.0 mg, the alcohol dehydrogenase in the immobilized system participates in immobilization to almost its maximum extent.

[0046] (2) Effect of enzyme concentration on the immobilization process:

[0047] Weigh 1.0 mg UIO-66 and disperse it in 500 μL of PBS buffer with pH 7.4 and a concentration of 10 mM. Then add 100–500 μL of ethanol dehydrogenase solution with a concentration of 1 mg / mL and make up the total immobilization volume to 1 mL. Stir and immobilize at room temperature for 12 h. After immobilization, centrifuge and wash to obtain immobilized enzyme ADH / UIO-66.

[0048] Figure 2 (b) shows the unit immobilization capacity of ADH / UIO-66 at different ADH concentrations. As can be seen from the figure, the unit immobilization capacity of ADH / UIO-66 gradually increases with increasing enzyme concentration, reaching its maximum at a concentration of 0.3 mg / mL. Further increases in enzyme concentration do not significantly improve the immobilization capacity. This is because at a concentration of 0.3 mg / mL, all adsorption sites on UIO-66 are saturated, so even with continued increases in enzyme concentration, excess alcohol dehydrogenase cannot be immobilized.

[0049] (3) The effect of immobilization time on the immobilization process:

[0050] Weigh 1.0 mg UIO-66 and disperse it in 700 μL of PBS buffer with pH 7.4 and a concentration of 10 mM. Then add 300 μL of ethanol dehydrogenase solution with a concentration of 1 mg / mL. Stir and immobilize at room temperature for 2–12 h. After immobilization, centrifuge and wash to obtain immobilized enzyme ADH / UIO-66.

[0051] Figure 2 (c) This graph shows the unit immobilization load of ADH / UIO-66 at different immobilization times. As can be seen from the graph, the immobilization load of ADH on the UIO-66 carrier material increases with time, and the rate of increase in immobilization load becomes increasingly rapid. After 8 hours, the rate of increase slows down significantly. Although the immobilization load still increases after 8 hours, the increase is not very significant. This indicates that the immobilization of ADH in UIO-66 is completed after 8 hours.

[0052] In summary, the optimized immobilization conditions of ADH on UIO-66 are: UIO-66 content of 1.0 mg, alcohol dehydrogenase concentration of 0.3 mg / mL, and immobilization time of 8 h.

[0053] Example 3: Study on the enzymatic properties of ADH / UIO-66

[0054] (1) pH stability:

[0055] In this embodiment, the effect of pH environment on the enzyme activity of ADH / UIO-66 was investigated by preparing PBS buffer with a pH value of 5.0–9.0. ADH / UIO-66 and free ADH were placed in PBS buffer for 1 hour, and their enzyme activities were measured. The relative enzyme activities were obtained based on the optimal enzyme activity. The results are as follows: Figure 3 As shown in a.

[0056] Figure 3 (a) is a pH stability verification graph for free ADH and immobilized ADH. The graph shows that both free ADH and ADH / UIO-66 exhibit good stability at pH values ​​greater than 7.0, but enzyme activity decreases when the pH value is less than 7.0. Furthermore, after standing for 1 hour under different pH conditions, the enzyme activity of ADH / UIO-66 is higher than that of free ADH, indicating that ADH / UIO-66 also possesses good pH stability even under acidic conditions.

[0057] (2) Temperature stability:

[0058] Free ADH and ADH / UIO-66 were placed at 40, 45, 50, 55, and 60°C for 1 hour respectively, and their remaining enzyme activity was tested. The relative enzyme activity was obtained based on the optimal enzyme activity. The results are as follows: Figure 3 As shown in b.

[0059] Figure 3 (b) is a temperature stability verification graph for free ADH and immobilized ADH. The graph shows that free ADH retained 72.49% of its enzyme activity after standing at 40℃ for 1 h; however, the enzyme activity dropped sharply at 50℃ and was completely inactivated at 60℃. In contrast, ADH / UIO-66 retained 18.61% activity at 60℃. Furthermore, due to the spatial confinement effect of the pores of UIO-66 on ADH, the activity of ADH / UIO-66 was higher than that of free ADH at the same temperature.

[0060] (3) Storage stability:

[0061] The prepared ADH / UIO-66 and free ADH were stored at 4°C. Enzyme activity was measured every 3 days. Based on the optimal enzyme activity, relative enzyme activity was obtained to investigate the storage stability of the immobilized and free enzymes. The test results are as follows: Figure 3 As shown in c.

[0062] Figure 3 (c) This is a storage stability verification graph for free ADH and immobilized ADH. The graph shows that the activity of free ADH decreased significantly, and after 9 days, half of its activity was lost. In contrast, ADH / UIO-66 retained approximately 69.08% of its initial activity after standing at 4°C for 30 days. This demonstrates that ADH / UIO-66 exhibits good storage stability and significant potential for industrial applications.

[0063] (4) Reusability of ADH / UIO-66:

[0064] The reusability of ADH / UIO-66 was verified by designing multiple consecutive reaction batches and detecting the activity of ADH / UIO-66. The specific process is as follows:

[0065] Mix 850 μL of PBS buffer, 0.01 mg ADH / UIO-66, and 100 μL of ethanol (4 mM) thoroughly. Then add 50 μL of NAD (coenzyme) solution (10 mM) and react at 37 °C for 5 min. After the reaction, centrifuge and retain the supernatant. Repeat the above operation 8 times to obtain 8 supernatants, and measure the absorbance of NADH at 340 nm. Based on the optimal enzyme activity, obtain the relative enzyme activity data. The test results are as follows: Figure 3 As shown in d.

[0066] Figure 3(d) shows the cycling performance verification graphs for free ADH and immobilized ADH. The graph indicates that the residual activity of ADH / UIO-66 reaches 76.58% after 5 cycles and remains at 61.58% after 8 cycles. This phenomenon may be due to two reasons: firstly, the small size of ADH / UIO-66 may lead to its loss during centrifugation; secondly, it may be due to the deformation and inactivation of some ADH / UIO-66 during its reaction with the substrate.

[0067] In summary, immobilizing ADH within the pores of UIO-66 can effectively improve the stability, cycling capacity, and other enzymatic properties of the immobilized enzyme, which is beneficial for the industrial application of ADH.

[0068] Example 4: Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-1

[0069] (1) The preparation method of CN is the same as that in Example 4;

[0070] (2) Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-1:

[0071] Weigh 10 mg CN and sonicate it in 5 mL of phosphate buffer. Then add 1 mg ADH / UIO-66 and stir magnetically for 2 h. After the reaction is complete, centrifuge, wash with distilled water, freeze dry overnight to obtain the product ADH / UIO-66 / CN-1.

[0072] Example 5: Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-2

[0073] (1) The preparation method of CN is the same as that in Example 4;

[0074] (2) Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-2:

[0075] Weigh 10 mg CN and sonicate it in 5 mL of phosphate buffer. Then add 2 mg ADH / UIO-66 and stir magnetically for 2 h. After the reaction is complete, centrifuge, wash with distilled water, freeze dry overnight to obtain the product ADH / UIO-66 / CN-2.

[0076] Example 6: Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-3

[0077] (1) Preparation of CN:

[0078] Urea was dried in an oven at 80°C for 24 hours, ground, and placed in a crucible. The crucible was then heated in a muffle furnace from room temperature to 550°C at a heating rate of 2.5°C / min for 4 hours. The calcined product was then removed and acid-washed overnight in 1 mol / L nitric acid, filtered, and washed 5–8 times with distilled water until neutral. It was then dried overnight in a vacuum drying oven. 1 g of the dried product was weighed into a porcelain boat and heated in a muffle furnace from room temperature to 500°C at a heating rate of 5°C / min for 4 hours to obtain CN.

[0079] (2) Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-3

[0080] 10 mg of CN was weighed and ultrasonically dispersed in 5 mL of phosphate buffer. Then, 3 mg of ADH / UIO-66 was added and the mixture was magnetically stirred for 2 h. After the reaction was completed, the mixture was centrifuged, washed with distilled water, and freeze-dried overnight to obtain the bifunctional photoenzyme composite catalyst, denoted as ADH / UIO-66 / CN-3.

[0081] Figure 4 The XRD patterns of UIO-66, ADH / UIO-66, CN, and ADH / UIO-66 / CN are shown. The figures reveal distinct characteristic peaks in UIO-66 at 7.17, 8.16, 11.93, 17.71, 25.63, and 33.12°, corresponding to the (111), (200), (022), (400), (442), and (137) crystal planes, respectively, consistent with literature reports. The characteristic peaks in the XDR pattern of ADH / UIO-66 largely overlap with those of UIO-66, indicating that the addition of ADH did not affect the basic framework of the support material. Meanwhile, in the XRD spectrum of ADH / UIO-66 / CN, not only were the characteristic peaks of UIO-66 observed, but new peaks (12.78° and 27.33°) also appeared, which correspond to the (100) and (002) crystal planes of g-C3N4, indicating the successful preparation of ADH / UIO-66 / CN.

[0082] To further verify the phase composition of all samples, Fourier transform infrared (FT-IR) was applied. Figure 5 As shown, UIO-66 is at 1707cm -1 The characteristic peak at 1557 cm⁻¹ is the stretching vibration of the carbon-oxygen double bond (C=O) in the carboxyl group. -1 The point is the stretching vibration of C=C in the benzene ring, 1408 cm⁻¹. -1 The point is the stretching vibration of O=CO, 748cm. -1 The vibrations at these locations are Zr-O-Zr stretching vibrations. When the ADH is fixed on the UIO-66 carrier material, the FT-IR values ​​at 657, 1103, and 1240 cm⁻¹ are... -1New peaks appear at these locations, corresponding to the out-of-plane bending vibration absorption of -NH2 and the stretching vibration absorption of CN in the enzyme molecule, respectively. Additionally, peaks appear at 3500-3100 cm⁻¹. -1 The broad peak at 1200-1600 cm⁻¹ corresponds to the vibrational absorption of NH. ADH / UIO-66 / CN shows this peak at 1200-1600 cm⁻¹. -1 A series of diffraction peaks are observed within the range of g-C3N4, which is caused by g-C3N4. Among them, the peaks are located in the 1540-1635 cm⁻¹ range. -1 A series of spectral bands at 1239-1460 cm⁻¹ originate from the stretching mode of CN, while at 1239-1460 cm⁻¹... -1 The peaks at these locations belong to the aromatic CN heterocycle mode. Additionally, peaks at 3462 and 748 cm⁻¹ also appear. -1 The observation of NH vibrational absorption in the enzyme molecule and Zr-O-Zr stretching vibration in UIO-66 further demonstrates the successful preparation of ADH / UIO-66 / CN.

[0083] Figure 6 These are laser confocal images of ADH / UIO-66(ac) and ADH / UIO-66 / CN(df). Laser confocal microscopy (CLSM) was used to observe whether ADH was successfully immobilized on the UIO-66 carrier material. The fluorescence field from the CLSM images (…) Figure 6 b) The presence of FITC-labeled ADH is clearly visible, emitting green fluorescence. Combined with bright-field images ( Figure 6 a) and fluorescence field images clearly show that ADH was successfully immobilized on UIO-66. Furthermore, by observing the fluorescence field images of ADH / UIO-66 / CN ( Figure 6 e) It was found that ADH / UIO was uniformly distributed on g-C3N4.

[0084] Figure 7 The figures show the thermogravimetric analysis (TGA) curves for UIO-66, ADH / UIO-66, and ADH / UIO-66 / CN. As shown, the mass loss of UIO-66 is mainly concentrated in the 500-600℃ range, corresponding to the thermal decomposition of the ligands in the MOF. In addition, the loss before 120℃ is due to the evaporation of water molecules. Compared to UIO-66, ADH / UIO-66 exhibits an additional mass loss in the 234-500℃ range, caused by the thermal decomposition of ADH. Furthermore, ADH / UIO-66 / CN also shows an additional mass loss after 600℃, caused by the thermal decomposition of g-C3N4. The TGA results further confirm the successful preparation of ADH / UIO-66 / CN.

[0085] Example 7: Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-4

[0086] (1) The preparation method of CN is the same as that in Example 4;

[0087] (2) Preparation of bifunctional photoenzyme composite catalyst ADH / UIO-66 / CN-4:

[0088] Weigh 10 mg CN and sonicate it in 5 mL of phosphate buffer. Then add 4 mg ADH / UIO-66 and stir magnetically for 2 h. After the reaction is complete, centrifuge, wash with distilled water, freeze dry overnight to obtain the product ADH / UIO-66 / CN-4.

[0089] Example 8: Performance Testing of ADH / UIO-66 / CN Photo-Enzyme Composite Catalyst

[0090] The performance of the ADH / UIO-66 / CN photo-enzyme composite catalyst was tested using a 300W xenon lamp as the light source and a 420nm filter. The specific procedures are as follows:

[0091] (1) Disperse 10 mg of sample in 20 mL of deionized water and stir magnetically for 10 min. After the dispersion is uniform, add 0.3 mL of chloroplatinic acid solution with a concentration of 1 mg / mL, 0.4 mL of anhydrous ethanol and 0.0284 g of reduced coenzyme I disodium salt (β-NADH).

[0092] (2) Pour the mixture into the reactor, exhaust with argon gas for 20 minutes, turn on the light to irradiate the reaction, and calculate the corresponding hydrogen content and acetaldehyde content by analyzing the peak area obtained by gas chromatography and liquid chromatography.

[0093] Figure 8 The bar chart (a) shows the hydrogen production and acetaldehyde yield of ADH / UIO-66 / CN with different composite ratios under visible light, and the cycling performance of ADH / UIO-66 / CN-2 (AUC-2). It can be seen that the hydrogen production performance of AUC-2 is 3.84 times that of pure g-C3N4, and it exhibits good stability, maintaining high photocatalytic activity even after four cycles. Furthermore, the acetaldehyde conversion rate of AUC-2 can reach 1.82 mmol / g / h. This indicates that the bifunctional ADH / UIO-66 / CN photo-enzyme composite photocatalyst has great potential in the field of clean energy.

[0094] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a bifunctional photo-enzyme composite catalyst, characterized in that, include: ADH was slowly added to the dispersion of UIO-66 to obtain a mixed system. The mixed system was immobilized at a constant temperature. After the reaction was completed, the mixture was centrifuged and washed to obtain ADH / UIO-66. In the mixed system, the final concentration of UIO-66 was 0.2-1.2 mg / mL and the final concentration of ADH was 0.1-0.5 mg / mL. CN and ADH / UIO-66 were dispersed in PBS buffer and stirred to react. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the bifunctional photoenzyme composite catalyst. The mass ratio of ADH / UIO-66 to CN was 1-4:

10. The CN was prepared by the following method: after drying, urea was ground and heated from room temperature to 550°C at a heating rate of 2.5°C / min, and calcined for 4 h. The calcined product was then washed with nitric acid overnight, filtered, washed, and dried to obtain the finished product.

2. The method for preparing the bifunctional photo-enzyme composite catalyst according to claim 1, characterized in that, The immobilization reaction was carried out at room temperature for 2–12 h.

3. The method for preparing the bifunctional photo-enzyme composite catalyst according to claim 1, characterized in that, The pH of the PBS buffer is 7.

4.

4. The method for preparing the bifunctional photo-enzyme composite catalyst according to claim 1, characterized in that, The stirring reaction was carried out at room temperature for 2 hours.

5. The bifunctional photoenzyme composite catalyst prepared by the preparation method according to any one of claims 1-4, characterized in that, The bifunctional photo-enzyme composite catalyst is designated as ADH / UIO-66 / CN; the bifunctional photo-enzyme composite catalyst is composed of ADH / UIO-66 nanoparticles loaded on a two-dimensional CN plane; the ADH / UIO-66 is an alcohol dehydrogenase ADH immobilized in the mesoporous channels of UIO-66.

6. The bifunctional photo-enzyme composite catalyst according to claim 5, characterized in that, The mass ratio of ADH / UIO-66 to CN in the bifunctional photo-enzyme composite catalyst is 1-4:

10.

7. The application of the bifunctional photo-enzyme composite catalyst prepared by the method of any one of claims 1 to 4, or the bifunctional photo-enzyme composite catalyst of any one of claims 5 to 6, in the photocatalytic splitting of water to produce hydrogen while simultaneously regenerating coenzymes.

8. The application of the bifunctional photo-enzyme composite catalyst prepared by the method of any one of claims 1 to 4, or the bifunctional photo-enzyme composite catalyst of any one of claims 5 to 6, in the photocatalytic splitting of water to produce hydrogen while simultaneously catalyzing the production of acetaldehyde from ethanol.

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