A bimetallic artificial enzyme based on 4,4'-biphenylalanine and copper cobalt, and a preparation method and application thereof

Abstract

This invention discloses a bimetallic artificial enzyme based on 4,4′-biphenylalanine and copper-cobalt, its preparation method, and its applications. The artificial enzyme is formed by the self-assembly of 4,4′-biphenylalanine (Bip), cobalt ions, and copper ions through coordination and π-π stacking, denoted as Bip / (Co+Cu). In preparation, Bip is dissolved in an aqueous NaOH solution, and aqueous solutions of cobalt and copper salts are added. The mixture is then allowed to self-assemble under static conditions, followed by centrifugation, washing, and vacuum drying to obtain the product. The artificial enzyme obtained by this invention possesses both organophosphorus hydrolase-like and nitroreductase-like activities. It can catalyze the hydrolysis of organophosphorus compounds such as methyl parathion and can catalyze the reduction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride. This artificial enzyme exhibits good thermal stability, ionic strength tolerance, organic solvent tolerance, and reusability, making it suitable for the environmental remediation of organophosphorus pesticides and nitroaromatic pollutants.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a bimetallic artificial enzyme that possesses both organophosphorus hydrolase-like and nitroreductase-like activities, formed by the self-assembly of 4,4′-biphenylalanine with transition metal cobalt and copper ions through coordination. It belongs to the field of artificial enzyme preparation and catalytic degradation technology of environmental pollutants. Specifically, it is a bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt, along with its preparation method and application. Background Technology

[0002] Enzymes are a class of biocatalysts that can efficiently catalyze chemical reactions under mild conditions (Nature 2012, 485, 185-194), and are widely used in medicine, food processing, biomanufacturing, and environmental remediation. However, natural enzymes typically rely on complex protein structures to maintain their catalytic activity and are easily inactivated in complex environments such as high temperatures, strong acids and bases, organic solvents, and high salt concentrations. Furthermore, the expression, isolation, and purification of natural enzymes are costly, and they are difficult to recover and reuse in their free state. These drawbacks limit their large-scale application in industrial and environmental remediation fields. Therefore, developing artificial enzyme materials with enzyme-like catalytic activity, high stability, simple preparation, and easy recyclability is of great significance.

[0003] Organophosphorus compounds are widely found in pesticides, flame retardants, plasticizers, and some industrial chemicals (FEMSmicrobiology reviews, 2006, 30(3), 428-471). Among them, organophosphorus pesticides such as methyl parathion have become important environmental pollutants due to their large usage, high toxicity, and high environmental mobility. Organophosphorus hydrolases can catalyze the cleavage of phosphoester bonds in organophosphorus compounds, thereby reducing the toxicity of pollutants, and are important biocatalytic templates for the removal of organophosphorus pollutants (Frontiers in bioengineering and biotechnology, 2019, 7, 289). However, natural organophosphorus hydrolases also suffer from problems such as high cost, insufficient stability, and poor adaptability to complex environments (Environmental Science and Pollution Research, 2016, 23(9), 8200-8218). The active sites of natural organophosphorus hydrolases usually contain bimetallic centers, where metal ions can participate in substrate polarization, water molecule activation, and transition state stabilization. Therefore, constructing artificial organophosphorus hydrolase mimics with bimetallic synergistic catalytic centers is an effective way to achieve stable degradation of organophosphorus pollutants.

[0004] Furthermore, 4-nitrophenol is a common nitroaromatic pollutant and an important intermediate product of the hydrolysis of some organophosphorus pesticides containing p-nitrophenyl groups (Chemosphere, 2017, 172, 52-71). 4-Nitrophenol exhibits high water solubility, toxicity, and environmental persistence risks, while its catalytic reduction product, 4-aminophenol, has relatively low toxicity and can serve as an important intermediate in pharmaceuticals, dyes, and fine chemicals (Journal of Photochemistry and Photobiology B: Biology, 2019, 196, 111502). Therefore, reducing 4-nitrophenol to 4-aminophenol has both pollutant detoxification and resource utilization value.

[0005] Currently, metal-organic frameworks (Journal of the American Chemical Society, 2018, 140(17), 5678-5681), metal oxides (Theranostics, 2020, 10(2), 687-706), nanomaterials (Chemical Society Reviews, 2019, 48(4), 1004-1076), and peptide self-assembly materials (Journal of Materials Chemistry B, 2016, 4(26), 4605-4611) have all been used to construct enzyme-mimicking systems. However, some materials suffer from problems such as complex preparation methods, non-uniform active sites, insufficient substrate affinity, unsatisfactory cycling stability, or limited environmental adaptability. Amino acids and their derivatives have good biocompatibility and well-defined coordination functional groups, which can coordinate with metal ions through amino, carboxyl, and side chain structures, and form supramolecular structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic interactions, and π-π stacking. Among them, 4,4′-biphenylalanine possesses a rigid biphenyl skeleton, which can enhance the ability to build aromatic stacking and hydrophobic microenvironments (CCS Chemistry, 2022, 4(8), 2816-2828), thus facilitating substrate enrichment and the formation of ordered coordination structures. Therefore, constructing artificial enzymes with multifunctional catalytic activity based on 4,4′-biphenylalanine and cobalt and copper bimetallic ions has promising research and application prospects. Summary of the Invention

[0006] The purpose of this invention is to address the problems of poor stability, high preparation cost, and difficulty in reusing natural organophosphorus hydrolases, as well as the complexity of existing enzyme preparation processes and insufficient multifunctional catalytic performance. This invention provides a bimetallic artificial enzyme that uses 4,4′-biphenylalanine as an organic ligand and cobalt and copper ions as bimetallic active centers, achieving self-assembly through coordination and π-π stacking. This artificial enzyme possesses both organophosphorus hydrolase-like and nitroreductase-like activities, and can be used for the hydrolytic degradation of organophosphorus pollutants such as methyl parathion, and for the reduction of 4-nitrophenol to 4-aminophenol. The preparation method of this invention is simple, the reaction conditions are mild, the raw materials are readily available, the materials have good stability, and it is easy to recover and recycle.

[0007] The technical solution of this invention is summarized as follows:

[0008] A bimetallic artificial enzyme based on 4,4′-biphenylalanine and copper-cobalt is disclosed. The bimetallic artificial enzyme is self-assembled from 4,4′-biphenylalanine, cobalt ions, and copper ions through coordination interactions. 4,4′-biphenylalanine coordinates with cobalt and copper ions through its amino and carboxyl groups, and forms a supramolecular aggregate through π-π stacking between the biphenyl backbones. The bimetallic artificial enzyme is a bulk supramolecular aggregate with a mesoporous structure. The bimetallic artificial enzyme exhibits both organophosphorus hydrolase-like and nitroreductase-like activities.

[0009] The method for preparing the bimetallic artificial enzyme based on 4,4′-biphenylalanine and copper-cobalt of the present invention includes the following steps:

[0010] 1) Dissolve 4,4′-biphenylalanine in an aqueous sodium hydroxide solution to obtain an alkaline Bip aqueous solution; prepare an aqueous solution of cobalt salt and an aqueous solution of copper salt, mix the aqueous solutions of cobalt salt and copper salt and add them dropwise to the alkaline Bip aqueous solution to dissolve, stirring during the dropwise addition, and then allow it to stand and self-assemble to obtain a bimetallic artificial enzyme suspension.

[0011] 2) Centrifuge the bimetallic artificial enzyme suspension obtained in step 1), discard the supernatant, collect the precipitate, wash with water and vacuum dry to obtain the bimetallic artificial enzyme solid.

[0012] The water used to prepare the NaOH and transition metal salt solutions is at least deionized water; the concentration of the sodium hydroxide aqueous solution is 20-50 mM; the concentration of 4,4′-biphenylalanine in the Bip alkaline aqueous solution is 5-20 mM; the concentration of the cobalt salt aqueous solution is 15-100 mM; and the concentration of the copper salt aqueous solution is 15-100 mM.

[0013] The dissolution of Bip is carried out using ultrasonic-assisted dispersion and constant temperature water bath heating at 60-80 ℃; the static self-assembly is carried out at a temperature of 40-70 ℃ for 6-24 h.

[0014] The centrifugation conditions are 9000-12000 rpm for 15-30 min; the temperature of the vacuum drying oven is set to 40-80 ℃, and the drying time is 24-72 h, until the precipitate containing a small amount of moisture is dried to powder Bip / (Co+Cu).

[0015] The Bip, Co 2+ With Cu 2+ The molar ratio is 6:2-4:2-4.

[0016] The application of the bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt in the hydrolytic degradation of organophosphorus compounds.

[0017] The organophosphorus compounds include one or more of methyl parathion, p-nitrophenyl phosphate, and bis(p-nitrophenyl) phosphate.

[0018] The application of the bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt in the reduction and transformation of nitro aromatic compounds.

[0019] The nitroaromatic compound is 4-nitrophenol, and the reduction is converted into the reduction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride.

[0020] This invention uses 4,4′-biphenylalanine and Co 2+ Cu 2+ A bimetallic coordination assembly was constructed. The Bip molecule coordinates with metal ions via amino and carboxyl groups, while π-π stacking interactions between the biphenyl skeletons promote the formation of an ordered supramolecular structure. The resulting material exhibits uniform distribution of Co and Cu elements. 2+ Cu 2+ The molar ratio of Bip to Bip is approximately 1:1:2; the material exhibits a bulk supramolecular aggregate morphology and a mesoporous structure, with a BET specific surface area of ​​21.15 m². 2 ·g -1 With an average pore size of approximately 8.31–19.26 nm, it can provide a spatial basis for the diffusion of organophosphorus substrates and the release of products.

[0021] In this invention, Bip / (Co+Cu) prepared at a molar ratio of 6:3:3 is used as a representative sample. Its catalytic hydrolysis of methyl parathion follows the Michaelis-Menten kinetics, with a Km of 0.41 mM and a Vmax of 4.3 μM·min. -1 The catalytic efficiency is 0.2097 (g·L). -1 ) -1 ·min -1This indicates that it has good substrate affinity and catalytic efficiency for methyl parathion. Bip / (Co+Cu) retained 89.3% catalytic activity after incubation at 70 °C for 30 min, its activity remained relatively stable under 600 mM NaCl conditions, maintained 89% activity in 20% dimethyl sulfoxide, and 81.3% activity in 60% ethanol. After six cycles, it still retained 80.1% catalytic activity, demonstrating the high stability of the artificial enzyme Nα-CDH-Zn.

[0022] The Bip / (Co+Cu) of this invention can also catalyze the reduction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride. Compared with control systems such as single Bip component, single metal assembly, and metal ion mixture, Bip / (Co+Cu) can simultaneously exhibit higher organophosphorus hydrolase-like activity and nitroreductase-like activity, indicating that the bimetallic coordination environment constructed by the Bip ligand is conducive to the formation of an artificial enzyme system with dual catalytic performance.

[0023] Compared with existing technologies, the advantages of this invention are:

[0024] 1) This invention uses 4,4′-biphenylalanine, cobalt salt and copper salt as raw materials to obtain bimetallic artificial enzymes through coordination self-assembly. The preparation conditions are mild, the operation is simple and the raw materials are readily available.

[0025] 2) The prepared artificial enzyme has both organophosphorus hydrolase-like activity and nitro reductase-like activity, and can be used for the treatment of organophosphorus pesticides and nitro aromatic pollutants.

[0026] 3) The prepared artificial enzyme has good thermal stability, ionic strength tolerance, organic solvent tolerance, stability in actual water samples and reusability, and is suitable for catalytic transformation of pollutants under complex environmental conditions. Attached Figure Description

[0027] Figure 1 The image shows a scanning electron microscope image and elemental distribution map of Bip / (Co+Cu) in Example 4.

[0028] Figure 2 This is a diagram showing the relative activity of different component assemblies in Example 5 for catalyzing the hydrolysis of methyl parathion.

[0029] Figure 3 The reaction process and kinetic curves of the Bip / (Co+Cu) catalytic methyl hydrolysis of thiophosphorus in Example 6 are shown.

[0030] Figure 4 The graph shows the relative catalytic activity of Bip / (Co+Cu) in Example 7 under different temperatures, ionic strengths, DMSO contents, and ethanol contents.

[0031] Figure 5 The graph shows the relative catalytic activity of Bip / (Co+Cu) in Example 8 after storage in Tris-HCl buffer, tap water, and lake water.

[0032] Figure 6 This is a graph showing the relative catalytic activity of Bip / (Co+Cu) before and after repeated use in Example 9.

[0033] Figure 7 This is a graph showing the relative catalytic activity of Bip / (Co+Cu) hydrolysis of different organophosphorus substrates in Example 10.

[0034] Figure 8 This is a graph showing the relative catalytic activity of Bip / (Co+Cu) for the reduction of 4-nitrophenol in Example 11.

[0035] Figure 9 The graph shows the effect of different buffer solutions on the catalytic reduction activity of 4-nitrophenol by Bip / (Co+Cu) in Example 12.

[0036] Figure 10 This is a graph showing the optimized concentration of the reducing agent in the Bip / (Co+Cu) catalytic reduction of 4-nitrophenol in Example 13.

[0037] Figure 11 This is a graph showing the relative activity of Bip / (Co+Cu) in catalyzing the reduction of 4-nitrophenol after treatment with different DMSO concentrations, ethanol concentrations, and temperatures in Example 14.

[0038] Figure 12 The reusability of the Bip / (Co+Cu) catalytic reduction of 4-nitrophenol in Example 15 is demonstrated. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, but the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention in any way.

[0040] A bimetallic artificial enzyme based on 4,4′-biphenylalanine and copper-cobalt is formed by the self-assembly of 4,4′-biphenylalanine, cobalt ions and copper ions through coordination, denoted as Bip / (Co+Cu), where Bip is 4,4′-biphenylalanine, and Co and Cu represent cobalt ions and copper ions, respectively.

[0041] The present invention provides a method for preparing a bimetallic artificial enzyme based on 4,4′-biphenylalanine, comprising the following steps:

[0042] 1) Dissolve 4,4′-biphenylalanine in NaOH aqueous solution to obtain Bip alkaline aqueous solution; prepare aqueous solutions of cobalt and copper chloride salts; mix the cobalt and copper chloride salt solutions and add them dropwise to the Bip alkaline aqueous solution while stirring magnetically during the addition; let stand at 40-70 ℃ for 6-24 h to complete self-assembly;

[0043] 2) Centrifuge at 9000-12000 rpm for 15-30 min, discard the supernatant and collect the precipitate. Wash the precipitate with deionized water 3-5 times. Dry the precipitate obtained from the last centrifugation under vacuum to obtain the bimetallic artificial enzyme Bip / (Co+Cu) solid.

[0044] In step 1), the water used to prepare the NaOH and transition metal salt solutions is deionized water; the concentration of the NaOH aqueous solution is 20-50 mM; the concentration of Bip in the Bip alkaline aqueous solution is 5-20 mM; the concentration of the cobalt chloride aqueous solution is 15-100 mM; and the concentration of the copper chloride aqueous solution is 15-100 mM.

[0045] In step 1), ultrasonic-assisted dispersion and 60-80 ℃ constant temperature water bath heating are used during the dissolution of Bip.

[0046] In step 1), Bip and Co 2+ With Cu 2+ The molar ratio is 6:2-4:2-4, preferably 6:3:3.

[0047] In step 2), the temperature of the vacuum drying oven is set to 40-80 ℃, and the drying time is 24-72 h, until the precipitate containing a small amount of moisture is dried to powder Bip / (Co+Cu).

[0048] The bimetallic artificial enzyme of the present invention can be used for the hydrolytic degradation of organophosphorus compounds, including one or more of methyl parathion, p-nitrophenyl phosphate and bis(p-nitrophenyl) phosphate.

[0049] The bimetallic artificial enzyme of the present invention can also be used for the reductive conversion of nitroaromatic compounds, including 4-nitrophenol, wherein the reductive conversion is the conversion of 4-nitrophenol to 4-aminophenol in the presence of a reducing agent.

[0050] Example 1: Synthesis of a bimetallic artificial enzyme with a molar ratio of Bip, CoCl2 and CuCl2 of 6:3:3.

[0051] 1) First, prepare an alkaline solution for dissolving the ligand. Weigh 868.6 mg of 4,4′-biphenylalanine (Bip) and dissolve it in 240 mL of 20 mM NaOH solution. Disperse with ultrasonic assistance and heat in a 70 °C constant temperature water bath until Bip is completely dissolved, yielding a homogeneous 15 mM alkaline aqueous solution of Bip. Simultaneously, prepare 15 mM CoCl2 and 15 mM CuCl2 solutions using deionized water. Under room temperature conditions, according to the Bip, Co... 2+ With Cu 2+ The molar ratio of the two solutions was 6:3:3. The prepared CoCl2 and CuCl2 solutions were mixed and slowly added dropwise to a Bip alkaline aqueous solution. With the addition of metal ions, a blue flocculent precipitate formed in the system, indicating that a coordination assembly reaction had occurred. After the metal ion solution had been completely added, the reaction system was placed in a 60 °C constant temperature water bath and allowed to stand for 6 h.

[0052] 2) After the reaction was completed, the product precipitate was collected by centrifugation at 10,000 rpm for 20 min using a high-speed centrifuge. The supernatant was discarded, and the product was resuspended in deionized water and washed. The washing was repeated 3 times to remove unreacted metal ions and impurities. The washed sample was then dried in a vacuum drying oven at 60 ℃ for 24 h. The resulting material was named Bip / (Co+Cu). Finally, the dried sample was sealed and stored for later use.

[0053] Example 2: Synthesis of a bimetallic artificial enzyme with a molar ratio of Bip, CoCl2 and CuCl2 of 6:4:2.

[0054] 1) First, prepare an alkaline solution for dissolving the ligand. Weigh 1158.1 mg of 4,4′-biphenylalanine (Bip) and dissolve it in 240 mL of 50 mM NaOH solution. Disperse with ultrasonic assistance and heat in an 80 °C constant temperature water bath until Bip is completely dissolved, yielding a homogeneous 20 mM alkaline aqueous solution of Bip. Simultaneously, prepare 100 mM CoCl2 solution and 50 mM CuCl2 solution using deionized water. Under room temperature conditions, according to the Bip, Co... 2+ With Cu 2+ The molar ratio of the two solutions was 6:4:2. The prepared CoCl2 and CuCl2 solutions were mixed and slowly added dropwise to a Bip alkaline aqueous solution. With the addition of metal ions, a blue flocculent precipitate formed in the system, indicating that a coordination assembly reaction had occurred. After the metal ion solution had been completely added, the reaction system was placed in a 40 °C constant temperature water bath and allowed to stand for 24 h.

[0055] 2) After the reaction was completed, the product precipitate was collected by centrifugation at 12,000 rpm for 15 min using a high-speed centrifuge. The supernatant was discarded, and the product was resuspended in deionized water and washed. The washing was repeated 5 times to remove unreacted metal ions and impurities. The washed sample was then dried in a vacuum drying oven at 40 °C for 72 h. The resulting material was named Bip / (Co+Cu). Finally, the dried sample was sealed and stored for later use.

[0056] Example 3: Synthesis of a bimetallic artificial enzyme with a molar ratio of Bip, CoCl2 and CuCl2 of 6:2:4.

[0057] 1) First, prepare an alkaline solution for dissolving the ligand. Weigh 289.5 mg of 4,4′-biphenylalanine (Bip) and dissolve it in 240 mL of 20 mM NaOH solution. Disperse with ultrasonic assistance and heat in a 60 °C constant temperature water bath until Bip is completely dissolved, yielding a homogeneous 5 mM alkaline aqueous solution of Bip. Simultaneously, prepare 50 mM CoCl2 and 100 mM CuCl2 solutions using deionized water. Under room temperature conditions, according to the Bip, Co... 2+ With Cu 2+ The molar ratio of the two solutions was 6:2:4. The prepared CoCl2 and CuCl2 solutions were mixed and slowly added dropwise to a Bip alkaline aqueous solution. With the addition of metal ions, a blue flocculent precipitate formed in the system, indicating that a coordination assembly reaction had occurred. After the metal ion solution had been completely added, the reaction system was placed in a 70 °C constant temperature water bath and allowed to stand for 15 h.

[0058] 2) After the reaction was completed, the product precipitate was collected by centrifugation at 9000 rpm for 30 min using a high-speed centrifuge. The supernatant was discarded, and the product was resuspended in deionized water and washed. The washing was repeated 4 times to remove unreacted metal ions and impurities. The washed sample was then dried in a vacuum drying oven at 80 ℃ for 48 h. The obtained material was named Bip / (Co+Cu). Finally, the dried sample was sealed and stored for later use.

[0059] Example 4: Morphological and structural characterization of Bip / (Co+Cu)

[0060] The morphology of the Bip / (Co+Cu) prepared in Example 1 was observed using scanning electron microscopy, and its elemental composition and distribution were analyzed using EDS. The experimental results are as follows: Figure 1 As shown, where Figure 1 (a) is a scanning electron microscope image of Bip / (Co+Cu), showing its bulk supramolecular aggregate structure of varying sizes, indicating that 4,4′-biphenylalanine plays a role in the formation of Co+Cu. 2+ and Cu 2+Stable solid assemblies were formed through coordination and π-π stacking of the biphenyl skeleton. Figure 1 (b) shows the elemental distribution of N, Co and Cu. All elements can be detected in the assembly and are distributed relatively evenly, indicating that Co and Cu were successfully introduced into the Bip assembly and formed a uniformly distributed bimetallic coordination structure, providing a structural basis for subsequent organophosphorus hydrolysis and nitro reduction bifunctional catalysis.

[0061] Example 5: Determination of the organophosphorus hydrolysis activity of Bip / (Co+Cu)

[0062] The organophosphorus hydrolase-like activity of Bip / (Co+Cu) was determined using methyl parathion (MP) as a model substrate. A 2.5 mM MP substrate solution was prepared using methanol, and the Bip / (Co+Cu) prepared in Example 1 was prepared into a 1 mg / mL dispersion using 30 mM Tris-HCl buffer (pH 9.0). In a 1 mL reaction system, 100 μL of MP substrate solution, 100 μL of catalyst dispersion, and 800 μL of Tris-HCl buffer (30 mM, pH 9.0) were added sequentially, mixed, and reacted at 25 °C for 3 min. After the reaction, the mixture was centrifuged at 12000 rpm, and the supernatant was measured at 405 nm using a microplate reader. The catalytic activity was evaluated based on the characteristic absorption of 4-nitrophenol at 405 nm.

[0063] To eliminate the non-specific catalytic effects of other components, a blank system without catalyst was set up, as well as systems with Bip / Cu, Bip / Co, pure Bip, and Cu catalysts added, respectively. 2+ Co 2+ Co 2+ With Cu 2+ The activity of mixed ions, Bip / Co, and Bip / Cu physical mixtures was determined under the same conditions. The experimental results are as follows: Figure 2 As shown, Bip / (Co+Cu) exhibits the highest MP hydrolysis activity, indicating that its catalytic ability mainly comes from the reaction of Bip and Co. 2+ Cu 2+ The bimetallic supramolecular structure formed by co-assembly.

[0064] Example 6: Determination of kinetic parameters for Bip / (Co+Cu) catalytic MP hydrolysis

[0065] To analyze the kinetics of Bip / (Co+Cu)-catalyzed MP hydrolysis, the reaction progress was measured over 15 min using Bip / (Co+Cu) at a concentration of 0.1 mg / mL (prepared in Example 1), MP substrate at a concentration of 0.25 mM, 30 mM Tris-HCl buffer (pH 9.0), and at 25 °C. The results are as follows: Figure 3 As shown in (a), a good linear relationship was observed in the first 3 minutes of the reaction, so 3 minutes was selected as the standard reaction time for subsequent activity comparison.

[0066] Using the Bip / (Co+Cu) catalyst prepared in Example 1 as a catalyst, the initial reaction rate was determined in the MP concentration range of 0-0.70 mM. The catalyst concentration was fixed at 0.05 mg / mL, and the reaction buffer was 30 mM Tris-HCl (pH 9.0). The kinetic parameters were fitted using the Michaelis-Menten model. The experimental results are as follows: Figure 3 As shown in (b), the Bip / (Co+Cu)-catalyzed MP hydrolysis follows the Michaelis-Menten kinetics, with a Vmax of 4.3 μM·min. -1 The Km value is 0.41 mM, and the catalytic efficiency is 0.2097 (g·L⁻¹). -1 ) -1 ·min -1 This indicates that Bip / (Co+Cu) has good substrate affinity and catalytic efficiency for MP.

[0067] Example 7: Determination of the catalytic stability of Bip / (Co+Cu)

[0068] To investigate the catalytic stability of Bip / (Co+Cu) under different environmental conditions, the effects of temperature, ionic strength, and organic solvent on the MP hydrolysis activity of Bip / (Co+Cu) prepared in Example 1 were studied. In the temperature stability experiment, Bip / (Co+Cu) was incubated at 0, 10, 25, 40, 50, 60, and 70 °C for 30 min, respectively. After returning to room temperature, the residual activity was measured according to the method in Example 4, and the relative activity was calculated with the activity measured at 25 °C as 100%. The experimental results are as follows: Figure 4 As shown in (a), Bip / (Co+Cu) retains 89.3% of its catalytic activity after incubation at 70 °C, and retains more than 90% of its activity after incubation at other temperatures, indicating that the artificial enzyme has good thermal stability.

[0069] In the ionic strength experiment, 0, 100, 200, 300, 400, 500, and 600 mM NaCl were added to the 30 mM Tris-HCl buffer in the reaction system, respectively. The relative activity was calculated with the system activity without added NaCl as 100%. The results are as follows: Figure 4 As shown in (b), changes in ionic strength have virtually no effect on the catalytic activity of Bip / (Co+Cu), indicating that it has good ionic strength tolerance.

[0070] In the organic solvent tolerance test, 0%, 10%, 20%, 30%, 40%, 50%, and 60% (v / v) of DMSO or ethanol were added to the reaction system, and the relative activity was calculated with the activity in the organic solvent-free system as 100%. The results are as follows: Figure 4 As shown in (c) and (d), when the DMSO content is 20%, Bip / (Co+Cu) still retains 89% of its activity; when the ethanol concentration is 60%, Bip / (Co+Cu) still retains 81.3% of its activity, indicating that this bimetallic artificial enzyme has good organic solvent tolerance.

[0071] Example 8: Long-term stability determination of Bip / (Co+Cu) in actual water samples

[0072] To evaluate the stability of Bip / (Co+Cu) in a real aquatic environment, the Bip / (Co+Cu) catalyst prepared in Example 1 was prepared into a 1 mg / mL dispersion using Tris-HCl buffer, laboratory tap water, and filtered lake water, respectively. The systems were incubated at 4 °C for 7 days. Every 24 hours, 100 μL samples were taken, centrifuged at 12000 rpm, the supernatant was discarded, and the precipitate was washed with deionized water. Residual catalytic activity was then measured according to the method described in Example 4, and the relative activity was calculated with the initial activity measured on day 0 as 100%. The experimental results are as follows: Figure 5 As shown, Bip / (Co+Cu) retained nearly 90% of its activity after 7 days in Tris-HCl buffer, and still retained about 65% of its activity after 7 days of incubation in a complex lake environment, indicating that it has good resistance to environmental interference and long-term stability.

[0073] Example 9: Reusability determination of Bip / (Co+Cu)

[0074] The hydrolytic activity of Bip / (Co+Cu) prepared in Example 1 on MP was determined according to the method described in Example 4. After each reaction, the Bip / (Co+Cu) precipitate in the reaction system was separated by centrifugation and washed three times with deionized water to remove residual substrate and product. The washed catalyst was then added back to a fresh reaction system for the next round of catalytic reaction. The above operation was repeated, and the changes in catalytic activity after each cycle were recorded. The experimental results are as follows: Figure 6 As shown, Bip / (Co+Cu) retains 80.1% of its catalytic activity after 6 cycles, indicating that it has good reusability.

[0075] Example 10: Catalytic universality of Bip / (Co+Cu) for different organophosphorus substrates

[0076] To investigate the catalytic universality of Bip / (Co+Cu) for different organophosphorus substrates, methyl parathion (MP), p-nitrophenyl phosphate (PNPP), and bis(p-nitrophenyl) phosphate (BNPP) were selected as model substrates. The hydrolytic activity of Bip / (Co+Cu) prepared in Example 1 was determined according to the method described in Example 5, and the relative activity was calculated with the degradation activity of Bip / (Co+Cu) for MP as 100%. The experimental results are as follows: Figure 7 As shown, Bip / (Co+Cu) exhibits high hydrolytic activity for both MP and PNPP, but relatively low activity for BNPP with larger molecular sizes. This result indicates that Bip / (Co+Cu) possesses a certain degree of substrate size selectivity; its mesoporous structure promotes the diffusion and access of small-sized substrates to metal active sites, while larger substrates face steric hindrance when entering the channels and approaching the active sites.

[0077] Example 11: Determination of the nitro reduction catalytic activity of Bip / (Co+Cu)

[0078] The nitroreductase-like activity of Bip / (Co+Cu) was determined using 4-nitrophenol as a substrate and NaBH4 as a reducing agent. The Bip / (Co+Cu) prepared in Example 1 was dispersed in Tris-HCl buffer (30 mM, pH 9.0) to prepare a 1 mg / mL dispersion. A reaction system was constructed with a final concentration of 50 μM for 4-nitrophenol and 20 mM for NaBH4. After thorough mixing, the mixture was reacted at 25 °C for 4 min. After the reaction, the reaction solution was centrifuged at 13000 rpm for 5 min. 200 μL of the supernatant was added to a colorimetric system consisting of 200 μL NaOH solution (0.5%, w / w) and 200 μL phenol solution (5%, w / w). The mixture was thoroughly mixed and incubated at 60 °C for 30 min. The mixture was then cooled to 25 °C, and the absorbance at 630 nm was measured to characterize the amount of 4-aminophenol generated.

[0079] The catalytic activity of the original Bip / (Co+Cu) assembly was defined as 100%, and the relative activities of the Bip / (Co+Cu), Bip / Co, and Bip / Cu assemblies were compared. The experimental results are as follows: Figure 8 As shown, the single Co assembly exhibits low activity, while the single Cu assembly shows activity close to that of the bimetallic assembly, but still lower than Bip / (Co+Cu). This indicates that the Co / Cu ratio is beneficial for maintaining high catalytic activity for the reduction of 4-nitrophenol, where Cu... 2+ Possibly in BH4 - It plays a more important role in activation, electron transfer, or substrate activation.

[0080] Example 12: Effect of different buffer systems on the Bip / (Co+Cu) catalytic activity for the reduction of 4-nitrophenol.

[0081] Using the method described in Example 11, HEPES, Tris-HCl, and PB (all with pH 8) were selected as reaction buffers. The reduction reaction of 4-nitrophenol catalyzed by Bip / (Co+Cu) prepared in Example 1 was carried out under the same conditions, and the amount of 4-aminophenol produced was determined by colorimetric method. The relative activities of Bip / (Co+Cu) in the Tris-HCl buffer were calculated as 100% for the other two buffers.

[0082] Experimental results are as follows Figure 9 As shown, the relative activity of Bip / (Co+Cu) was highest in HEPES buffer, approximately 157%; the activity in Tris-HCl buffer was defined as 100%; and the relative activity in PB buffer was approximately 47%. These results indicate that the type of buffer plays a crucial role in regulating the catalytic performance of Bip / (Co+Cu) in the reduction of 4-nitrophenol, with HEPES being more favorable for the 4-nitrophenol reduction reaction.

[0083] Example 13: Effect of NaBH4 concentration on the reductive activity of Bip / (Co+Cu) nitro group.

[0084] The Bip / (Co+Cu) prepared in Example 1 was dispersed in Tris-HCl buffer to prepare a 1 mg / mL dispersion. Using 4-nitrophenol as the substrate, with a final concentration of 50 μM, NaBH4 was added at final concentrations of 10, 20, 30, 40, 50, and 60 mM to construct reaction systems under different reducing agent concentrations. After thorough mixing of all components, the reaction was carried out at 25 °C for 4 min. After the reaction, the reaction solution was centrifuged at 13000 rpm for 5 min, and 200 μL of the supernatant was added to the colorimetric system. The absorbance at 630 nm was measured according to the method described in Example 11. The experimental results are as follows: Figure 10 As shown, the NaBH4 concentration affects the efficiency of the Bip / (Co+Cu) catalytic reduction of 4-nitrophenol to 4-aminophenol. Optimizing the NaBH4 concentration can further improve the reduction reaction efficiency.

[0085] Example 14: Determination of the environmental stability of Bip / (Co+Cu) catalytic reduction of 4-nitrophenol.

[0086] To evaluate the stability of Bip / (Co+Cu) in catalyzing the reduction of 4-nitrophenol under different environmental conditions, the effects of DMSO concentration, ethanol concentration, and temperature treatment on the catalytic activity of the Bip / (Co+Cu) prepared in Example 1 were investigated. In the DMSO stability experiment, the catalytic activity of Bip / (Co+Cu) without DMSO treatment was defined as 100%. The experimental results are as follows: Figure 11 As shown in (a), when the volume fraction of DMSO is 10%, 20%, 30% and 40%, the relative activities of Bip / (Co+Cu) are approximately 95%, 90%, 89% and 84%, respectively, indicating that it has good DMSO tolerance.

[0087] In the ethanol stability experiment, the catalytic activity of Bip / (Co+Cu) without ethanol treatment was defined as 100%. The experimental results are as follows: Figure 11 As shown in (b), when the volume fraction of ethanol is 10%, 20%, 30% and 40%, the relative activities of Bip / (Co+Cu) are approximately 96%, 91%, 87% and 82%, respectively, indicating that it has good tolerance to ethanol.

[0088] In the temperature stability experiment, Bip / (Co+Cu) was incubated at 0, 10, 25, 40, 50, 60, and 70 °C for 30 min, respectively. After returning to room temperature, the residual activity was measured according to the method described in Example 11, and the catalytic activity after treatment at 25 °C was defined as 100%. The experimental results are as follows: Figure 11As shown in (c), Bip / (Co+Cu) retains approximately 95% and 97% of its relative activity after treatment at 0 ℃ and 10 ℃, respectively. After treatment at 40 ℃, its activity is close to the initial level. After treatment at 50 ℃ and 60 ℃, it retains approximately 95% and 89% of its initial activity, respectively. After treatment at 70 ℃, it still retains approximately 84% of its reduction catalytic activity, indicating that it has good thermal stability in the reduction reaction of 4-nitrophenol.

[0089] Example 15: Determination of the reusability of Bip / (Co+Cu)-catalyzed reduction of 4-nitrophenol.

[0090] To evaluate the reusability of Bip / (Co+Cu) in the reduction reaction of 4-nitrophenol, the catalytic activity of the Bip / (Co+Cu) prepared in Example 1 after multiple uses was determined using the method described in Example 11. After each reaction, the Bip / (Co+Cu) precipitate in the reaction system was separated and washed three times with deionized water to remove residual substrate and product. The washed catalyst was then reused in the next round of catalytic reaction. The above operation was repeated, and the changes in catalytic activity after each cycle were recorded.

[0091] Experimental results are as follows Figure 12 As shown, with the catalytic activity of the first cycle defined as 100%, the relative activity of Bip / (Co+Cu) remained at approximately 95% and 92% after the second and third cycles, respectively; after the fourth and fifth cycles, the relative activities were approximately 87% and 82%, respectively; and after six consecutive cycles, it still maintained approximately 77% of the initial catalytic activity. These results indicate that Bip / (Co+Cu) possesses good structural stability and reusability in the NaBH4-mediated reduction of 4-nitrophenol.

[0092] Example 16: Preparation and catalytic activity comparison of Bip with different single metal ion assemblies.

[0093] To further illustrate the roles of copper and cobalt ions in the bimetallic artificial enzyme of this invention, the same coordination self-assembly method as in Example 1 was used, with 4,4′-biphenylalanine as the organic ligand, and copper chloride, cobalt chloride, zinc chloride, manganese chloride, and ferric chloride as metal salts, respectively, to assemble a single-metallic artificial enzyme with Bip. Specifically, Bip was dissolved in NaOH aqueous solution, and Cu... 2+ Co 2+ Zn 2+ Mn 2+ and Fe 3+The corresponding metal salt aqueous solution was slowly added dropwise to the Bip alkaline aqueous solution, and the molar ratio of Bip to metal ions was controlled at 2:1. After stirring, standing at 60 °C for 6 h for self-assembly, centrifugation, washing and vacuum drying, Bip / Cu, Bip / Co, Bip / Zn, Bip / Mn and Bip / Fe single metal assemblies were obtained respectively.

[0094] The above-mentioned single metal assemblies were dispersed in 30 mM Tris-HCl buffer (pH 9.0) and their organophosphorus hydrolase-like activities were determined using methyl parathion as a substrate, according to the method described in Example 5. At the same time, their nitroreductase-like activities were determined using 4-nitrophenol as a substrate and NaBH4 as a reducing agent, according to the method described in Example 11.

[0095] Experimental results show that both Bip / Cu and Bip / Co exhibit significant enzyme-like catalytic activity, far exceeding that of the Bip / Zn, Bip / Mn, and Bip / Fe assemblies, which showed only low activity or were close to the blank system. This indicates that in the Bip-metal coordination self-assembly system, Cu… 2+ and Co 2+ This is more conducive to the formation of effective catalytic sites. The synergistic assembly of the two as bimetallic centers can significantly improve the catalytic performance of artificial enzymes, thus providing experimental basis for the selection of Cu / Co bimetallic system to construct Bip / (Co+Cu) in this invention.

[0096] The technical solutions disclosed and proposed in this invention can be implemented by those skilled in the art by appropriately modifying the conditions and routes, etc., based on the content of this document. Although the methods and preparation techniques of this invention have been described through preferred embodiments, those skilled in the art can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit, and scope of this invention to achieve the final preparation technique. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the spirit, scope, and content of this invention.

Claims

1. A bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt, characterized in that, The bimetallic artificial enzyme is self-assembled from 4,4′-biphenylalanine, cobalt ions, and copper ions through coordination interactions. 4,4′-biphenylalanine coordinates with cobalt and copper ions via amino and carboxyl groups, and forms a supramolecular aggregate through π-π stacking between the biphenyl backbones. The bimetallic artificial enzyme is a bulk supramolecular aggregate with a mesoporous structure. The bimetallic artificial enzyme possesses both organophosphorus hydrolase-like and nitroreductase-like activities.

2. A method for preparing a bimetallic artificial enzyme based on 4,4′-biphenylalanine and copper-cobalt as described in claim 1, characterized in that, Includes the following steps: 1) Dissolve 4,4′-biphenylalanine in an aqueous sodium hydroxide solution to obtain an alkaline Bip aqueous solution; prepare an aqueous solution of cobalt salt and an aqueous solution of copper salt, mix the aqueous solutions of cobalt salt and copper salt and add them dropwise to the alkaline Bip aqueous solution to dissolve, stirring during the dropwise addition, and then allow it to stand and self-assemble to obtain a bimetallic artificial enzyme suspension. 2) Centrifuge the bimetallic artificial enzyme suspension obtained in step 1), discard the supernatant, collect the precipitate, wash with water and vacuum dry to obtain the bimetallic artificial enzyme solid.

3. The preparation method according to claim 2, characterized in that, The water used to prepare the NaOH and transition metal salt solutions is at least deionized water; the concentration of the sodium hydroxide aqueous solution is 20-50 mM; the concentration of 4,4′-biphenylalanine in the Bip alkaline aqueous solution is 5-20 mM; the concentration of the cobalt salt aqueous solution is 15-100 mM; and the concentration of the copper salt aqueous solution is 15-100 mM.

4. The preparation method according to claim 2, characterized in that, The dissolution of Bip is carried out using ultrasonic-assisted dispersion and constant temperature water bath heating at 60-80 ℃; the static self-assembly is performed at a temperature of 40-70 ℃ for 6-24 h.

5. The preparation method according to claim 2, characterized in that, Centrifugation conditions: 9000-12000 rpm for 15-30 min; vacuum drying oven temperature setting: 40-80 ℃; drying time: 24-72 h, until the precipitate containing a small amount of moisture is dried to powder Bip / (Co+Cu).

6. The preparation method according to claim 2, characterized in that, Bip, Co 2+ with Cu 2+ in a molar ratio of 6:2-4:2-4.

7. The application of the bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt as described in claim 1 in the hydrolytic degradation of organophosphorus compounds.

8. The application according to claim 7, characterized in that, The organophosphorus compounds include one or more of methyl parathion, p-nitrophenyl phosphate, and bis(p-nitrophenyl) phosphate.

9. The application of the bimetallic artificial enzyme constructed based on 4,4′-biphenylalanine and copper-cobalt as described in claim 1 in the reductive conversion of nitroaromatic compounds.

10. The application according to claim 9, characterized in that, The nitroaromatic compound is 4-nitrophenol, and the reduction is converted into the reduction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride.

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