A method for constructing an electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system

Abstract

This invention discloses a method for constructing an electrocatalytic and immobilized enzyme cascade catalytic inert C-H bond hydroxylation reaction system. The method first uses cobalt nitrate hexahydrate and melamine as precursors to prepare a Co-N@CNTs composite catalyst by pyrolysis under an argon atmosphere. This catalyst is then drop-coated onto a carbon felt surface for in-situ cathodic electrocatalytic generation of hydrogen peroxide. Next, using trialdehyde-resorcinol and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine as ligands, 1,4-dioxane and mesitylene as solvents, and glacial acetic acid as a catalyst, a Schiff base reaction is performed to prepare TpTa COF. This COF is then used as a carrier to immobilize AaeUPO, achieving substrate enrichment and enzyme molecule protection. This cascade system combines electrochemical in-situ hydrogen peroxide production with immobilized enzyme catalysis, efficiently driving the inert C-H bond hydroxylation reaction while effectively inhibiting the damage of high-concentration hydrogen peroxide to the active sites of AaeUPO.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, specifically to a method for catalyzing the hydroxylation of inert CH bonds using a combination of electrocatalysis and immobilized enzymes. Background Technology

[0002] Alcohols are important structural units constituting fine chemicals, fragrances, and bioactive molecules. The selective hydroxylation of CH bonds to synthesize alcohols not only provides a crucial pathway for the high-value transformation of basic chemical raw materials but also opens up new directions for the green synthesis of fine chemicals, pharmaceutical intermediates, and biofuels, holding significant importance in the chemical industry. In modern chemistry, introducing functional groups into the inert framework of organic molecules is a considerable challenge. Traditional chemical methods suffer from poor selectivity, numerous byproducts, and high energy consumption, making it difficult to achieve a balance between selectivity and reactivity. Biocatalysis has proven to be an effective tool for overcoming these obstacles. Enzyme catalysts, with their significant substrate specificity, chemoselectivity, regioselectivity, and enantioselectivity, provide new synthetic strategies for oxygen functionalization reactions, greatly simplifying synthetic pathways and significantly reducing the energy consumption of many chemical reactions.

[0003] Nonspecific peroxygenases (UPOs, EC 1.11.2.1) are a class of heme oxidases derived from fungi. In the field of biocatalysis, UPOs exhibit excellent catalytic activity and broad substrate specificity in the selective hydroxylation of CH bonds. They are independent of NAD(P)H, require no additional cofactors or complex electron transport systems, and utilize only simple H2O2 as an oxidant to achieve the hydroxylation of CH bonds in aliphatic, aromatic, and heterocyclic compounds under mild conditions. However, high concentrations of H2O2 can easily cause partial oxidative degradation of the heme active sites, leading to enzyme inactivation. Constructing a stable and efficient in-situ H2O2 generation system and maintaining the H2O2 concentration in the reaction system to balance enzyme reactivity and oxidative inactivation are key factors in maximizing the catalytic activity of UPOs. Electrochemical methods are an environmentally friendly, efficient, and sustainable ideal strategy for in-situ H2O2 generation, requiring no additional chemical reagents and avoiding the accumulation of byproducts in the reaction mixture. Furthermore, the poor stability of free enzymes, their difficulty in recovery and reuse, and the generally poor water solubility of organic substrates limit the contact between UPO and substrates. Designing and selecting suitable immobilized enzyme carriers to improve enzyme stability and substrate enrichment, thereby enhancing the catalytic efficiency of UPO, is a key issue in the catalytic application of UPO. These problems severely hinder its application in various reactions involving the catalytic hydroxylation of inert CH bonds. Therefore, developing reaction systems capable of continuously and efficiently catalyzing the hydroxylation of inert CH bonds is of great significance to the development of the field of organic chemical synthesis. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, this invention provides a method for the highly efficient catalytic hydroxylation of inert CH bonds through electrochemical in-situ generation of H₂O₂ coupled with immobilized AaeUPO. First, a Co-N@CNTs composite catalyst is prepared by pyrolysis in Ar₂ using Co(NO₃)₂·6H₂O and melamine as precursors. The Co-N@CNTs are then drop-coated onto a carbon felt surface for in-situ cathodic electrocatalytic generation of H₂O₂. Next, COF is selected as the carrier for the immobilized enzyme, protecting the enzyme molecule while simultaneously enriching the substrate to increase the local substrate concentration near the enzyme molecule, thereby improving catalytic efficiency. The immobilized AaeUPO is then combined with the electrochemical in-situ generation of H₂O₂ to achieve the hydroxylation of inert CH bonds.

[0005] To address the aforementioned technical problems, this invention proposes a method for constructing an electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system. Using Co(NO3)2·6H2O and melamine as precursors, a composite catalyst Co-N@CNTs is prepared by pyrolysis in Ar2. The Co-N@CNTs are then drop-coated onto a carbon felt surface for in-situ cathodic electrocatalysis to generate H2O2. A covalent organic framework material TpTaCOF is used as the carrier for the immobilized enzyme AaeUPO, protecting the enzyme molecule while simultaneously enriching the substrate to increase the local substrate concentration near the enzyme molecule, thereby improving catalytic efficiency. The covalent organic framework material TpTaCOF... COF was prepared via a Schiff base reaction using trialdehyde-resorcinol and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine as ligands, 1,4-dioxane and mesitylene as solvents, and glacial acetic acid as a catalyst. By combining immobilized enzymes with electrochemical in-situ H₂O₂ generation, highly efficient conversion of the AaeUPO-catalyzed CH bond hydroxylation reaction was achieved, driven by in-situ hydrogen peroxide production, while effectively inhibiting the damage of high-concentration hydrogen peroxide to the active sites of AaeUPO. The specific steps are as follows:

[0006] Step 1: Preparation of two-electron oxygen reduction cathode material: Co(NO3)2·6H2O, melamine, and ethanol were mixed and continuously ground to form a uniform powder, wherein the molar volume ratio of Co(NO3)2·6H2O, melamine, and ethanol was 3-4 mmol: 60-80 mmol: 2-3 mL; the powder was placed in a crucible and calcined in Ar2, then washed with 0.5-0.7 M H2SO4 solution at 60-90 ℃ for 2-6 h, then centrifuged and freeze-dried to obtain the catalyst, denoted as Co-N@CNTs; Co-N@CNTs, Nafion solution, and isopropanol aqueous solution were mixed and ultrasonically treated to form a uniform catalyst dispersant; an appropriate amount of the catalyst dispersant was drop-coated onto the surface of carbon felt and dried at room temperature to serve as the two-electron oxygen reduction cathode material;

[0007] Step 2, Synthesis of covalent organic framework material: Trialdehyde resorcinol and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were placed in a Pyrex tube, and 1,4-dioxane and mesitylene were added to dissolve them. 6 M glacial acetic acid was added dropwise, and the solution was subjected to freezing-vacuuming-thawing cycles several times after sonication. Then, the solution was placed in an oil bath at 100-120 °C for 24-72 h. After centrifugation, washing and drying, the covalent organic framework material was obtained, denoted as TpTaCOF.

[0008] Step 3: Synthesis of covalently organic framework immobilized enzyme: Add an appropriate amount of TpTa COF obtained in step 2 to a 0.25%-3.0% glutaraldehyde solution and stir at 10-35 °C for 1-5 h. Then centrifuge, wash, and dry. Add the glutaraldehyde-activated TpTa COF to phosphate buffer and sonicate to disperse evenly. Then add a certain concentration of AaeUPO crude enzyme solution and shake in a constant temperature shaker for a certain time. Then centrifuge, wash, and freeze-dry to obtain the covalently organic framework immobilized enzyme, denoted as AaeUPO@TpTa COF.

[0009] Step 4: Mix the AaeUPO@TpTa COF obtained in Step 3, phosphate buffer, acetonitrile, and substrate to obtain a mixed solution;

[0010] Step 5: In an oxygen-saturated H-type electrolytic cell consisting of a cathode chamber, a proton exchange membrane, and an anode chamber, a platinum sheet electrode is used as the anode, an Ag / AgCl electrode as the reference electrode, the two-electron oxygen reduction cathode material prepared in Step 1 is used as the cathode, and the mixed solution prepared in Step 4 is used as the reaction solution. An external voltage of -0.6V is applied to construct an electroenzyme cascade catalytic CH bond hydroxylation reaction system. The substrate is an aromatic alkane substrate, which is efficiently converted into the corresponding chiral alcohol product in the reaction system.

[0011] Furthermore, in the method for constructing the reaction system described in this invention, wherein:

[0012] In step 1, the pyrolysis calcination treatment in Ar2 is carried out in a muffle furnace, with a heating rate of 2-5 °C / min; the calcination temperature is 600-800 °C; and the calcination time is 1-5 h. The Nafion solution has a volume concentration of 5%, and the isopropanol aqueous solution has a C3H8O:H2O ratio of 1:1. The mass-to-volume ratio of Co-N@CNTs, Nafion solution, and isopropanol aqueous solution is 1-10 mg: 20-50 μL: 1-2 ml.

[0013] In step 2, the molar volume ratio of trialdehyde phloroglucinol, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane, mesitylene, and glacial acetic acid is 0.1-0.2 mmol: 0.15-0.3 mmol: 1.5-3 ml: 3-6 ml: 0.3-0.6 ml.

[0014] In step 3, the concentration of the crude AaeUPO enzyme solution is 50-100 U / mL, preferably 90 U / mL; the mass-to-volume ratio of the glutaraldehyde-activated TpTa COF to the crude AaeUPO enzyme solution is 5-10 mg / mL, preferably 10 mg / mL.

[0015] In step 4, the mass-to-volume ratio of AaeUPO@TpTa COF, phosphate buffer, and acetonitrile in the mixed solution is 60-100 mg:9 mL:1 mL, preferably 90 mg:9 mL:1 mL; the substrate concentration is 5-10 mmol / L, preferably 10 mmol / L.

[0016] In this invention, the aromatic alkane substrate is any one of 4-ethylbenzoic acid, ethylbenzene, n-propylbenzene, o-chloroethylbenzene, 1-chloro-3-ethylbenzene, 1-chloro-4-ethylbenzene, 2-ethyltoluene, 3-ethyltoluene, 4-ethyltoluene, o-bromoethylbenzene, 1-bromo-3-ethylbenzene, p-bromoethylbenzene, 1-ethyl-2-fluorobenzene, 1-ethyl-3-fluorobenzene, and p-fluoroethylbenzene.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) The two-electron oxygen reduction cathode material synthesized in this invention contains a two-electron oxygen reduction electrocatalyst, which successfully realizes the in-situ production of H2O2 to drive the reaction of AaeUPO catalyzing the hydroxylation of inert CH bonds, and effectively inhibits the enzyme inactivation caused by the partial oxidation and degradation of heme active sites due to high concentration of H2O2.

[0019] (2) In this invention, covalent organic frameworks (COFs) were used as immobilized enzyme carriers to synthesize covalent organic framework immobilized enzymes, which improved the stability and reusability of the enzymes and successfully solved the problems of poor stability, difficulty in recovery and reuse of free enzymes. By using COFs materials to enrich substrates and increase the local substrate concentration near the enzyme molecules, the problem of poor water solubility of organic substrates, which leads to limited contact between UPO and substrates, was successfully solved, thus improving catalytic efficiency.

[0020] (3) The electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system constructed in this invention is used to catalyze the conversion of aromatic alkane substrates into corresponding chiral alcohol products. For example, it is used to catalyze the conversion of 4-ethylbenzoic acid (EBA) into 4-hydroxybenzoic acid (HEBA). After 16 hours of reaction, the yield reaches over 99%, which is 1.7 times that of the free enzyme system. After repeated use 6 times, AaeUPO@TpTa COF still retains about 56% of its initial activity, and this reaction system also has good catalytic activity and selectivity for the hydroxylation reactions of other substrates.

[0021] (4) The reaction system constructed in this invention achieves the hydroxylation of inert CH bonds under very mild and green conditions, which is an environmentally friendly and sustainable method for the hydroxylation of inert CH bonds. Attached Figure Description

[0022] Figure 1 A schematic diagram of the reaction route for constructing the reaction system of this invention;

[0023] Figure 2 Scanning electron microscope image of Co-N@CNTs material;

[0024] Figure 3 X-ray diffraction pattern of Co-N@CNTs material;

[0025] Figure 4 Fine Co2p spectrum of Co-N@CNTs material;

[0026] Figure 5 The images are SEM and TEM images of TpTa COF and AaeUPO@TpTa COF, where (a) and (b) are SEM and TEM images of TpTa COF, and (c) and (d) are SEM and TEM images of AaeUPO@TpTa COF.

[0027] Figure 6 Fourier transform infrared spectra of TpTa COF, AaeUPO, and AaeUPO@TpTa COF;

[0028] Figure 7 X-ray diffraction (XRD) patterns of TpTa COF and AaeUPO@TpTa COF powders;

[0029] Figure 8 Thermogravimetric analysis (TGA) plots of TpTa COF and AaeUPO@TpTa COF;

[0030] Figure 9 N2 adsorption-desorption isotherms for TpTa COF and AaeUPO@TpTa COF;

[0031] Figure 10 CLSM plot of AaeUPO@TpTa COF;

[0032] Figure 11 This is a diagram showing the effect of reusing AaeUPO@TpTa COF. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the following embodiments are not intended to limit the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments and comparative examples are conventional methods; the raw materials or test materials used are typical products purchased from the market, unless otherwise specified. In the following embodiments and comparative examples, the quantitative experiments were all repeated three times, and the results were averaged.

[0034] Example 1:

[0035] The preparation process for a cathode material with two-electron oxygen reduction properties is as follows:

[0036] 1) Co(NO3)2·6H2O (3.5 mmol) and melamine (70 mmol) were mixed, and 2 mL of ethanol was added. The mixture was continuously ground in agate slurry to form a uniform light green powder. The resulting mixture was placed in a crucible and heated to 800 °C at a heating rate of 3 °C / min under an Ar2 atmosphere and held for 3.5 h. The product was washed with 0.5 M H2SO4 solution at 90 °C for 4 h, and finally collected by centrifugation (12000 r / min). The product was then freeze-dried to obtain Co-N@CNTs. Its morphology is as follows: Figure 2 As shown, melamine pyrolysis forms many carbon nanotube structures with diameters ranging from 50 to 200 nm.

[0037] X-ray diffraction analysis of Co-N@CNTs, such as Figure 3 The Co-N@CNTs catalyst shown exhibits a diffraction peak at 26.4°, which belongs to the (002) plane of CNTs, indicating the presence of graphitic carbon in the catalyst. Comparison with standard cards reveals diffraction peaks consistent with metallic Co, with peaks at 44.2°, 51.5°, and 75.6° attributed to the (111), (200), and (220) planes of metallic Co, respectively, confirming the successful synthesis of the catalyst.

[0038] like Figure 4 As shown, in the fine Co2p spectrum of Co-N@CNTs, the main peaks at 780.7 eV (2p3 / 2) and 796.4 eV (2p1 / 2) are attributed to Co-N.

[0039] 2) Mix 5 mg of the Co-N@CNTs catalyst prepared in step 1) with an isopropanol aqueous solution (C3H8O:H2O = 1:1) containing 50 μL of Nafion solution (5% vol%) and sonicate for 30 min to form a uniform catalyst dispersion. Then, drop-coat 200 μL of the catalyst dispersion onto a carbon felt (1.5 cm × 2 cm), dry at room temperature, and use it as the working electrode.

[0040] Example 2:

[0041] Determination of hydrogen peroxide yield under different applied voltages:

[0042] Hydrogen peroxide production was determined in a three-electrode sealed H-type electrolyzer containing O2-saturated PBS (100 mM, pH=5). The electrolyzer consisted of a cathode chamber, a proton exchange membrane, and an anode chamber. The working electrode obtained in Example 1 was placed in the cathode chamber, the Ag / AgCl electrode was used as the reference electrode, and the platinum sheet electrode was placed in the anode chamber as the counter electrode.

[0043] Chronoamperometry (it) tests were performed under different voltages, and the H2O2 concentration in the cathode cell was measured after 2 hours.

[0044] The H2O2 concentration in the cathode cell was measured six times sequentially by applying -0.8 V (vs. Ag / AgCl), -0.6 V (vs. Ag / AgCl), -0.4 V (vs. Ag / AgCl), -0.2 V (vs. Ag / AgCl), 0 V (vs. Ag / AgCl), and 0.2 V (vs. Ag / AgCl). Samples (samples 1A, 2A, 3A, 4A, 5A, and 6A) were taken. The H2O2 concentration was determined by iodometric titration followed by ultraviolet spectrophotometry. 400 μL of the reaction solution was taken from the electrolytic cell, and 300 μL of 0.5 M potassium hydrogen phthalate (C8H5KO4) and 300 μL of potassium iodide aqueous solution (0.4 M KI, 0.05 M NaOH, 10...) were added. -4 (Mammonium molybdate) was mixed thoroughly. I was measured at 351 nm using a UV spectrophotometer. 3− The absorbance value is used for the quantitative determination of H2O2.

[0045] The yields of hydrogen peroxide synthesized in situ under different applied voltages for the above six samples are shown in Table 1 below.

[0046] Table 1

[0047]

[0048] In Table 1, the H2O2 yield was calculated based on the H2O2 standard curve: Y = 0.0135X + 0.1251; where Y is the absorbance value measured by a UV spectrophotometer at 351 nm; and X is the H2O2 concentration in μM. In this invention, the applied voltage is the key variable controlling the in-situ generation rate of hydrogen peroxide, and the generation efficiency varies significantly under different voltage conditions. Based on the comprehensive performance evaluation of the electroenzyme cascade catalytic system, the optimal applied voltage that balances hydrogen peroxide generation efficiency and enzyme catalytic activity can be effectively screened.

[0049] Example 3:

[0050] The preparation process for TpTa COF and immobilization of AaeUPO is as follows:

[0051] 1) Synthesis of TpTa COF: TpTa COF was synthesized using a Schiff base reaction. 0.1 mmol Tp and 0.15 mmol Ta were placed in a 10 mL Pyrex tube, along with 1,4-dioxane (1.5 mL) and mesitylene (3 mL). The mixture was then sonicated for 10 min to ensure uniform dispersion. Subsequently, 0.3 mL of glacial acetic acid (6 M) was added to the system, and the solution was subjected to three cycles of freezing-vacuuming-thawing (0.2 MPa). The mixture was then placed in an oil bath at 120 °C for 48 h. The resulting orange-yellow powder was filtered and washed three times with tetrahydrofuran (THF) and dichloromethane (DCM) to remove unreacted monomers. The final product was dried under vacuum at 60 °C to obtain TpTa COF.

[0052] 2) Synthesis of AaeUPO@TpTa COF: 100 mg of TpTa COF obtained in step 1) was added to 10 mL of 1.0 wt% glutaraldehyde solution and stirred at 25 °C for 4.0 h. The glutaraldehyde-functionalized TpTa COF was washed three times with ultrapure water and dried overnight in a vacuum oven at 60 °C. AaeUPO was immobilized using a covalent bonding method. 10 mg of glutaraldehyde-activated TpTa COF was added to 2 mL of PBS buffer solution (100 mM, pH = 4) and ultrasonically dispersed. Then, 0.6 mg / mL of crude AaeUPO enzyme solution was added, and the mixture was shaken at 200 r / min for 1.5 h in a constant temperature shaker at 25 °C. AaeUPO@TpTa COF was separated by centrifugation and washed three times with PBS buffer solution (100 mM, pH = 4) to remove free AaeUPO. The AaeUPO@TpTa COF was stored at 4 °C for later use.

[0053] Figure 5The images show SEM and TEM images of TpTa COF and AaeUPO@TpTa COF, where (a) and (b) are SEM and TEM images of TpTa COF, and (c) and (d) are SEM and TEM images of AaeUPO@TpTa COF. The morphology and internal structure of TpTa COF and AaeUPO@TpTa COF were observed using scanning electron microscopy and transmission electron microscopy, respectively. Figure 6 The TpTa COF shown is a network structure formed by interconnected strips. The morphology of AaeUPO@TpTa COF is similar to that of TpTa COF, indicating that the structure of TpTa COF was not damaged by enzyme loading.

[0054] The functional groups of TpTa COF, AaeUPO, and AaeUPO@TpTa COF were characterized using FT-IR, such as... Figure 7 As shown, compared with TpTa COF, AaeUPO@TpTa COF at 1100 cm⁻¹ -1 and 3251 cm -1 New characteristic peaks appeared, which correspond to the tensile vibrations of CN and NH, respectively, and are consistent with the characteristic peaks of free AaeUPO, proving the successful fixation of AaeUPO.

[0055] X-ray diffraction analysis was performed on TpTa COF and AaeUPO@TpTa COF, such as... Figure 8 As shown, four distinct diffraction peaks were observed in TpTa COF at 2θ = 5.6°, 10.06°, 14.82°, and 25.43°, which belong to the (100), (110), (200), and (001) crystal planes, respectively, proving that the prepared TpTa COF has high crystallinity. The characteristic peaks of AaeUPO@TpTa COF after enzyme loading were not significantly different from those of TpTa COF, indicating that the crystal structure of TpTa COF was not destroyed after enzyme immobilization.

[0056] The thermal stability of TpTa COF and AaeUPO@TpTa COF was characterized using thermogravimetric analysis, such as... Figure 9 As shown, the first stage of weight loss is below 150℃, with only about 3% mass loss, due to the evaporation of residual solvent. The second stage of weight loss is in the range of 150~500℃. Compared with pure TpTa COF, AaeUPO@TpTa COF has a greater weight loss because, in addition to the decomposition of some COF organic ligands, the enzyme protein also decomposes within this temperature range. The third stage of weight loss is in the range of approximately 500~800℃, mainly due to the degradation of COF.

[0057] The changes in specific surface area and pore size of the COF material before and after immobilization were analyzed by N2 adsorption-desorption characterization. The results are as follows: Figure 10 As shown, the specific surface areas of TpTa COF and AaeUPO@TpTa COF are 689.5 m². 2 / g and 382.2 m 2 / g, the specific surface area of ​​TpTaCOF decreases after enzyme loading because the enzyme molecules occupy part of the space.

[0058] To further demonstrate the successful immobilization of AaeUPO, AaeUPO@TpTa COF was characterized by laser scanning confocal microscopy (CLSM). Rhodamine B-stained AaeUPO was covalently immobilized on glutaraldehyde-activated TpTa COF as a support. The results are as follows: Figure 11 As shown, the COF surface exhibits uniform red fluorescence, confirming that the enzyme molecules are stably anchored on the carrier surface, indicating the successful immobilization of AaeUPO.

[0059] Example 4:

[0060] Using the electrode prepared in Example 1 as the working electrode and the AaeUPO@TpTa COF prepared in Example 3, a reaction system for the electrocatalytic and immobilized enzyme cascade catalytic hydroxylation of inert CH bonds was constructed. The specific operation procedure is as follows:

[0061] The electroenzyme cascade catalytic hydroxylation of 4-ethylbenzoic acid was carried out in an oxygen-saturated H-type electrolytic cell consisting of a cathode chamber, a proton exchange membrane, and an anode chamber. Figure 1 As shown, a platinum sheet electrode was used as the anode, and an Ag / AgCl electrode was used as the reference electrode. A carbon felt coated with Co-N@CNTs catalyst was used as the cathode in the cathode bath. 10 mL of deionized water was added to the anode chamber, and 90 mg of AaeUPO@TpTa COF (prepared in Example 8), 10 mM 4-ethylbenzoic acid, 1 mL of acetonitrile as a co-solvent, and 9 mL of phosphate buffer (100 mM, pH = 5) were added to the cathode chamber. Simultaneously, O2 was bubbled (100 mL / min), and the reaction was carried out at an applied voltage of -0.2 V (vs. Ag / AgCl) for 2 h. The reaction solution was centrifuged, diluted with ACN, and filtered through a 0.22 μm organic filter to remove insoluble impurities. The product, 4-hydroxybenzoic acid, was detected by liquid chromatography.

[0062] The liquid chromatography detection conditions were as follows: The concentrations of EBA and HEBA were determined using an HPLC-20AD XR HP high-performance liquid chromatography system (Shimadzu Corporation, Japan). Column: C18-RP; Detection temperature: 25°C; Flow rate: 0.5 mL / min; Mobile phase: Gradient elution using a mixture of 0.1% trifluoroacetic acid solution (Solution A) and 0.095% trifluoroacetic acid acetonitrile solution (Solution B), with the volume fraction of Solution B changing over time as follows: 0 min: 35%, to 7 min: 80%, to 9 min: 35%, to 10 min: 35%. EBA and HEBA were detected using a UV-Vis detector (237 nm).

[0063] The above process was repeated with applied voltages of −0.2 V (vs. Ag / AgCl), −0.3 V (vs. Ag / AgCl), −0.4 V (vs. Ag / AgCl), −0.5 V (vs. Ag / AgCl), −0.6 V (vs. Ag / AgCl), −0.7 V (vs. Ag / AgCl), and −0.8 V (vs. Ag / AgCl). The yields of EBA to HEBA converted from EBA to HEBA by the electroenzyme cascade for the seven samples (1B, 2B, 3B, 4B, 5B, 6B, and 7B) are shown in Table 2.

[0064] Table 2

[0065]

[0066] As can be seen from the data in Table 2, the yield of HEBA is not directly positively correlated with the yield of H2O2. In samples 1B, 2B, 3B, and 4B, the low yield of H2O2 makes it impossible to ensure the supply of H2O2, while in samples 6B and 7B, the high yield of H2O2 may lead to the destruction of the active center of AaeUPO, which will also cause a decrease in the yield of HEBA.

[0067] Comparative Example

[0068] The preparation procedures and conditions were basically the same as those for sample 5B, except that AaeUPO@TpTa COF was replaced with free AaeUPO enzyme solution. Electrocatalysis and immobilized enzyme cascade catalysis of inert CH bonds were performed using the sample from the comparative example. Tests showed that the yield of 4-hydroxybenzoic acid was only 56.7% within 16 hours.

[0069] Example 5:

[0070] Following the reaction conditions of sample 5B, the prepared AaeUPO@TpTa COF was cycled 6 times, and its performance was evaluated by calculating the relative activity based on the enzyme activity measured at the time of addition. Figure 11The results show that the enzyme activity can still be retained by 56% after being reused 6 times, indicating that the catalyst has good reusability.

[0071] Comparing the test results of sample 5B and the comparative sample, the yield of 4-hydroxybenzoic acid in the free enzyme of the comparative sample was only 56.7% within 16 h, while the yield of 4-hydroxybenzoic acid in sample 5B, which uses AaeUPO@TpTa COF to generate H2O2 in situ via electrochemical binding, reached 99.1% within 16 h. The AaeUPO@TpTa COF system of this invention is 1.7 times that of the free enzyme AaeUPO system. This significant improvement is attributed to: (1) AaeUPO@TpTa COF has higher stability than free AaeUPO; (2) TpTa COF enrichment increases the substrate concentration near the enzyme molecule, shortens the substrate mass transfer distance, and improves catalytic efficiency.

[0072] Example 6:

[0073] Table 3 shows the yields of products synthesized from different substrates under an applied voltage of −0.6 V (vs. Ag / AgCl) and following the procedure of Example 4.

[0074] Table 3

[0075]

[0076] In summary, the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system constructed in this invention uses Co-N@CNTs with two-electron oxygen reduction capability as the working electrode for in-situ hydrogen peroxide production. This not only achieves efficient conversion of the in-situ hydrogen peroxide-driven AaeUPO-catalyzed inert CH bond hydroxylation reaction but also effectively inhibits the damage of high-concentration hydrogen peroxide to the AaeUPO active site. By covalently immobilizing AaeUPO on GA-activated COF material TpTaCOF, the resulting immobilized enzyme AaeUPO@TpTaCOF exhibits high stability and excellent reusability.

[0077] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments described above. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above embodiments only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application.

Claims

1. A method for constructing an electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system, characterized in that, A composite catalyst, Co-N@CNTs, was prepared by pyrolysis in Ar2 using Co(NO3)2·6H2O and melamine as precursors. Co-N@CNTs were then drop-coated onto a carbon felt surface for in-situ cathodic electrocatalytic generation of H2O2. A covalent organic framework material, TpTa COF, was used as a carrier for the immobilized enzyme AaeUPO, protecting the enzyme molecule and simultaneously enriching the substrate to increase the local substrate concentration near the enzyme molecule, thereby improving catalytic efficiency. The TpTa COF was prepared via a Schiff base reaction using trialdehyde-resorcinol and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine as ligands, 1,4-dioxane and mesitylene as solvents, and glacial acetic acid as a catalyst. Combining the immobilized enzyme with in-situ electrochemical H2O2 generation achieved highly efficient conversion of the AaeUPO-catalyzed CH bond hydroxylation reaction driven by in-situ hydrogen peroxide production, while effectively inhibiting the damage of high-concentration hydrogen peroxide to the active sites of AaeUPO.

2. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, The specific steps are as follows: Step 1: Preparation of two-electron oxygen reduction cathode material: Co(NO3)2·6H2O, melamine, and ethanol were mixed and continuously ground to form a uniform powder, wherein the molar volume ratio of Co(NO3)2·6H2O, melamine, and ethanol was 3-4 mmol: 60-80 mmol: 2-3 mL. The powder was placed in a crucible and subjected to pyrolysis calcination in Ar2. After washing with 0.5-0.7 M H2SO4 solution at 60-90 °C for 2-6 h, the powder was centrifuged and freeze-dried to obtain the composite catalyst, denoted as Co-N@CNTs. Co-N@CNTs, Nafion solution, and isopropanol aqueous solution were mixed and then subjected to ultrasonic treatment to form a uniform catalyst dispersant. Take an appropriate amount of catalyst dispersant and drop it onto the surface of carbon felt. After drying at room temperature, it is used as a two-electron oxygen reduction cathode material. Step 2, Synthesis of covalent organic framework materials: Trialdehyde phloroglucinol and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were placed in a Pyrex tube, and 1,4-dioxane and mesitylene were added to dissolve them. 6 M glacial acetic acid was added dropwise, and the solution was sonicated and then subjected to several cycles of freezing-vacuuming-thawing. The solution was then placed in an oil bath at 100-120 °C for 24-72 h, followed by centrifugation, washing, and drying to obtain a covalent organic framework material, denoted as TpTaCOF. Step 3: Synthesis of covalently organic framework-immobilized enzymes: An appropriate amount of TpTa COF obtained in step 2 was added to a 0.25%-3.0% glutaraldehyde solution and stirred at 10-35 °C for 1-5 h. Then, the mixture was centrifuged, washed, and dried. The glutaraldehyde-activated TpTa COF was added to phosphate buffer and ultrasonically dispersed. A certain concentration of crude AaeUPO enzyme solution was then added and the mixture was shaken in a constant temperature shaker for a certain period of time. Finally, the mixture was centrifuged, washed, and freeze-dried to obtain a covalently immobilized organic framework enzyme, denoted as AaeUPO@TpTa COF. Step 4: Mix the AaeUPO@TpTa COF obtained in Step 3, phosphate buffer, acetonitrile, and substrate to obtain a mixed solution; Step 5: In an H-type electrolytic cell saturated with oxygen and consisting of a cathode chamber, a proton exchange membrane, and an anode chamber, a platinum sheet electrode is used as the anode, an Ag / AgCl electrode is used as the reference electrode, the two-electron oxygen reduction cathode material prepared in step 1 is used as the cathode, and the mixed solution prepared in step 4 is used as the reaction solution. The applied voltage is -0.6V, thereby constructing an electroenzyme cascade catalytic CH bond hydroxylation reaction system. The substrate is an aromatic alkane substrate, which is efficiently converted into the corresponding chiral alcohol product in the reaction system.

3. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, In step 1, the pyrolysis calcination treatment in Ar2 is carried out in a muffle furnace, with a heating rate of 2-5 ℃ / min, a calcination temperature of 600-800 ℃, and a calcination time of 1-5 h.

4. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, In step 1, the volume concentration of the Nafion solution is 5%, the ratio of C3H8O to H2O in the isopropanol aqueous solution is 1:1, and the mass-volume ratio of Co-N@CNTs, Nafion solution, and isopropanol aqueous solution is 1-10 mg: 20-50 μL: 1-2 ml.

5. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, In step 2, the molar volume ratio of trialdehyde phloroglucinol, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane, mesitylene, and glacial acetic acid is 0.1-0.2 mmol: 0.15-0.3 mmol: 1.5-3 ml: 3-6 ml: 0.3-0.6 ml.

6. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, In step 3, the concentration of the crude AaeUPO enzyme solution is 50-100 U / mL, and the mass-to-volume ratio of the glutaraldehyde-activated TpTa COF to the crude AaeUPO enzyme solution is 5-10 mg / mL.

7. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 6, characterized in that, The concentration of the crude AaeUPO enzyme solution was 90 U / mL, and the mass-to-volume ratio of the glutaraldehyde-activated TpTa COF to the crude AaeUPO enzyme solution was 10 mg / mL.

8. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, In step 4, the mass-to-volume ratio of AaeUPO@TpTa COF, phosphate buffer, and acetonitrile in the mixed solution is 60-100 mg: 9 mL: 1 mL, and the substrate concentration is 5-10 mmol / L.

9. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 8, characterized in that, In the mixed solution, the mass-to-volume ratio of AaeUPO@TpTa COF, phosphate buffer, and acetonitrile was 90 mg: 9 mL: 1 mL, and the substrate concentration was 10 mmol / L.

10. The method for constructing the electrocatalytic and immobilized enzyme cascade catalytic inert CH bond hydroxylation reaction system according to claim 1, characterized in that, The aromatic alkane substrate is any one of 4-ethylbenzoic acid, ethylbenzene, n-propylbenzene, o-chloroethylbenzene, 1-chloro-3-ethylbenzene, 1-chloro-4-ethylbenzene, 2-ethyltoluene, 3-ethyltoluene, 4-ethyltoluene, o-bromoethylbenzene, 1-bromo-3-ethylbenzene, p-bromoethylbenzene, 1-ethyl-2-fluorobenzene, 1-ethyl-3-fluorobenzene, and p-fluoroethylbenzene.

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