Preparation method and application of cross-linked enzyme aggregate biocatalytic membrane

Abstract

The application discloses a preparation method of a cross-linked enzyme aggregate biological catalytic membrane, and mainly comprises the following steps: taking a polyacrylonitrile ultrafiltration membrane as a base film, loading polydopamine modified UiO-66-NH2 nanoparticles to the surface of the polyacrylonitrile ultrafiltration membrane as an intermediate layer, the adsorption characteristics of the intermediate layer help to enrich micro-pollutants and promote enzyme catalysis, and loading cross-linked enzyme aggregates on the treated polyacrylonitrile ultrafiltration membrane by a pressure-assisted self-assembly technology, so as to obtain the cross-linked enzyme aggregate biological catalytic membrane. The preparation method is simple in operation and easy to implement. The cross-linked enzyme aggregate technology is used to combine separation, purification and immobilization together, one-step purification and immobilization of the enzyme are realized, and the immobilization amount is increased. The cross-linked enzyme aggregate biological catalytic membrane is used for removing micro-pollutants in water, separating dyes and salt ions in wastewater or removing methyl parathion in water, and the removal rate is more than 90%, so that the cross-linked enzyme aggregate biological catalytic membrane has good effects.

Description

Technical Field

[0001] This invention relates to the preparation of a high-efficiency biocatalytic membrane based on cross-linked aggregate technology, the immobilization of enzymes, and membrane separation applications; specifically, it relates to the preparation and application of a cross-linked enzyme aggregate (CLEAs) biocatalytic membrane. Background Technology

[0002] With rapid industrialization and urbanization, as well as a dramatic increase in medical and agricultural activities, it is expected that more micropollutants will exist in water bodies in the near future. Many micropollutants remain and accumulate in water bodies, seriously endangering the ecological environment and human health. Biocatalytic membranes use membranes as enzyme immobilization carriers, combining the advantages of membrane separation and enzyme catalysis. They utilize membranes to retain pollutants and then degrade them through enzymes. Simultaneously, they can also retain large molecules formed by the polymerization of small molecule pollutants after degradation, thus enhancing retention and achieving highly efficient removal of micropollutants from water. Therefore, they have extremely high scientific research and application value.

[0003] Compared to traditional methods such as adsorption, biodegradation, chemical oxidation, and membrane separation, combining separation membranes with biological enzymes to prepare biocatalytic membranes can achieve highly efficient removal of micropollutants, providing a new approach for the gentle and green treatment of wastewater. This allows for continuous operation, improves enzyme stability, and promotes enzyme reuse. Laccase is a copper-rich redox enzyme that utilizes the unique redox center of copper ions, using oxygen as an electron acceptor to degrade various substrates while simultaneously reducing molecular oxygen to water, catalyzing a cycle without the need for additional substances. However, the volatility and poor reusability of free laccase during use limit its industrial application. To achieve integrated membrane separation and enzyme catalysis, enabling continuous operation, improving enzyme stability, and promoting enzyme reuse, the development of a biocatalytic membrane capable of efficiently removing pollutants and thus achieving gentle and green wastewater treatment is urgently needed. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, this invention proposes a method for removing micropollutants and separating salt contaminants from water using cross-linked enzyme aggregates (CLEAs) biocatalytic membranes, achieving efficient, mild, and environmentally friendly removal of micropollutants from water. Cross-linked laccase aggregates (Lac-CLEAs) are immobilized onto a base membrane using pressure-assisted self-assembly technology. Polydopamine (PDA)-modified UiO-66-NH2 (UiO-66-NH2@PDA) is loaded onto the base membrane surface as an intermediate layer. This method is simple to operate and easy to implement. The cross-linked enzyme aggregate technology combines separation, purification, and immobilization, achieving one-step purification and immobilization of the enzyme and increasing the immobilization capacity. Pre-loading UiO-66-NH2@PDA onto the HPAN base membrane as an intermediate layer compensates for the interface defects of Lac-CLEAs while stabilizing them. The adsorption properties of UiO-66-NH2@PDA also contribute to the enrichment of micropollutants, resulting in excellent removal of micropollutants and separation of salt contaminants from water. The biocatalytic membrane prepared by this method is used in research on the removal of micropollutants and salt staining in water, thus providing a new strategy for the application of biocatalytic membranes.

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing a cross-linked enzyme aggregate biocatalytic membrane, comprising: using a polyacrylonitrile ultrafiltration membrane as the base membrane, loading polydopamine-modified UiO-66-NH2 nanoparticles onto the surface of the polyacrylonitrile ultrafiltration membrane as an intermediate layer, and loading cross-linked enzyme aggregates onto the treated polyacrylonitrile ultrafiltration membrane using pressure-assisted self-assembly technology, thereby obtaining a cross-linked enzyme aggregate biocatalytic membrane.

[0006] Furthermore, in the method for preparing the cross-linked enzyme aggregate biocatalytic membrane of the present invention, wherein:

[0007] The cross-linked enzyme aggregates are cross-linked laccase aggregates or cross-linked triglyceride phospholipase aggregates, abbreviated as Lac-CLEAs and PTE-CLEAs, respectively. The prepared cross-linked enzyme aggregate biocatalytic membranes are cross-linked laccase aggregate biocatalytic membranes or cross-linked triglyceride phospholipase aggregate biocatalytic membranes, abbreviated as Lac-CLEAs / UiO-66-NH2@PDA and PTE-CLEAs / UiO-66-NH2@PDA, respectively.

[0008] The preparation method of the UiO-66-NH2 nanoparticles is as follows: a certain mass of NH2-BDC and ZrCl4 are weighed and dissolved in an appropriate amount of DMF to obtain a mixed solution A. In solution A, the mass of NH2-BDC is 310 mg and the mass of ZrCl4 is 400 mg, measured in 100 mL of DMF. Then, CH3COOH and H2O are added to solution A to obtain solution B. In solution B, the volume ratio of DMF, CH3COOH and H2O is 2000:60:1. Solution B is ultrasonically treated to make the solution homogeneous. Then, it is reacted at 100℃ for 24 h, followed by washing with DMF 3 times and water 3 times, and centrifuged for 10 min to obtain the product, which is the UiO-66-NH2 nanoparticles.

[0009] The specific steps for modifying the UiO-66-NH2 nanoparticles with polydopamine are as follows: A certain amount of UiO-66-NH2 nanoparticles and an appropriate amount of dopamine hydrochloride are dissolved in an appropriate amount of Tris-HCl buffer solution, wherein the concentration of the Tris-HCl buffer solution is 50 mmol / L and the pH is 8.5; the mass of the UiO-66-NH2 nanoparticles is 100 mg and the mass of the dopamine hydrochloride is 200 mg, measured in 100 mL of Tris-HCl buffer solution; the mixture is magnetically stirred at room temperature until homogeneous, then washed three times with water, and centrifuged for 10 min. The resulting product is the polydopamine-modified UiO-66-NH2 nanoparticles, abbreviated as UiO-66-NH2@PDA.

[0010] The method for synthesizing the cross-linked laccase aggregates is as follows: Under ice bath conditions, a certain amount of laccase and bovine serum albumin are added to an appropriate amount of PBS buffer. The concentration of the PBS buffer is 50 mmol / L, and the pH is 7.0, to obtain a mixed solution C. A PBS buffer containing ammonium sulfate is prepared, wherein the concentration of the PBS buffer is 50 mmol / L, the pH is 7.0, and the mass-to-volume ratio of ammonium sulfate is 0.5 g / L. The mixed solution C is added to the PBS buffer containing ammonium sulfate at a volume ratio of 1:3 to obtain a mixed solution D. In mixed solution D, the mass-to-volume concentration of laccase is (0.25–1.25) mg / mL, and the mass-to-volume concentration of bovine serum albumin is (0–0.05) mg / mL, and is not equal to 0. The mixture is allowed to stand for 3 hours. Subsequently, an appropriate amount of glutaraldehyde with a concentration of 37.5 mmol / L is added under magnetic stirring, wherein the volume ratio of glutaraldehyde to mixed solution C is 3:100. Cross-linking is carried out for 1 hour, and the resulting solution is the solution of the cross-linked laccase aggregates.

[0011] The method for synthesizing the cross-linked triphospholipase aggregate is basically the same as the method for synthesizing the cross-linked laccase aggregate described above, except that the laccase is replaced with triphospholipase. The mass-volume concentration of triphospholipase in the reaction system is (0.25-0.75) mg / mL, and the final solution obtained is the solution of the cross-linked triphospholipase aggregate.

[0012] Polyacrylonitrile ultrafiltration membranes were soaked in NaOH solution at 50℃ for 1 hour and washed three times with ultrapure water to obtain hydrolyzed PAN membranes, which were then used after soaking in ultrapure water. The hydrolyzed PAN membranes were fixed in an ultrafiltration cup with the active side facing upwards. A dispersion of polydopamine-modified UiO-66-NH2 nanoparticles was poured into the ultrafiltration cup, submerging the PAN membrane. The dry mass ratio of UiO-66-NH2 nanoparticles to the base membrane area was (0~0.125) mg / cm². 2 The nitrogen pressure was adjusted to allow the droplets to fully permeate and flow out, followed by washing three times with ultrapure water. This process loaded polydopamine-modified UiO-66-NH2 nanoparticles onto the surface of the polyacrylonitrile ultrafiltration membrane, which is referred to as the UiO-66-NH2@PDA-modified membrane. A solution of cross-linking enzyme aggregates was poured into an ultrafiltration cup to immerse the UiO-66-NH2@PDA-modified membrane, and the nitrogen pressure was adjusted to allow the droplets to slowly drip down until all droplets flowed out completely. The resulting membrane was washed three times with ultrapure water, yielding the cross-linking enzyme aggregate biocatalytic membrane. This membrane was then stored in ultrapure water at 4°C. Depending on the solution of cross-linking enzyme aggregates used, the final product is either a cross-linked laccase aggregate biocatalytic membrane or a cross-linked phosphotriesterase aggregate biocatalytic membrane.

[0013] The cross-linked laccase aggregate biocatalytic membrane prepared according to the present invention is used to remove micropollutants from water. The micropollutants include one or more of bisphenol A, phenol, hydroquinone, 2,4-dichlorophenol, tetracycline hydrochloride, and doxycycline hydrochloride. The removal rate of bisphenol A can be as high as 95%, the removal rate of phenol can be as high as 70%, the removal rate of 2,4-dichlorophenol can be as high as 80%, the removal rate of tetracycline hydrochloride can be as high as 85%, and the removal rate of doxycycline hydrochloride can be as high as 90%.

[0014] The biocatalytic membrane prepared by this invention utilizes its pore size sieving and electrostatic repulsion to achieve salt / dye separation, thereby removing dyes from wastewater. The dyes include one or more of Rhodamine B, crystal violet, Chrome Black T, Acid Fuchs Red, Malachite Green, and Congo Red; the rejection rates for Rhodamine B, crystal violet, Chrome Black T, Acid Fuchs Red, and Malachite Green can all reach up to 90%, and the rejection rate for Congo Red can reach up to 85%.

[0015] The cross-linked triphospholipase aggregate biocatalytic membrane prepared in this invention was used to remove methyl paraoxon from water, with a removal rate of 75% to 90%.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] (1) In this invention, UiO-66-NH2@PDA is preloaded on the base film as an intermediate layer to compensate for the interface defects of Lac-CLEAs and stabilize Lac-CLEAs. The adsorption characteristics of UiO-66-NH2@PDA also help to enrich micro pollutants, thereby increasing the local concentration of micro pollutants and promoting the removal of micro pollutants.

[0018] (2) The preparation method of the present invention uses a simple cross-linked enzyme aggregate technology to immobilize laccase and phosphotriesterase. This technology combines separation, purification and immobilization, realizes one-step purification and immobilization of enzymes and increases the amount of immobilization. The process is simple, enhances the stability of enzymes, has high enzyme activity per unit volume, and makes biological enzymes easier to apply to industrial production.

[0019] (3) In this invention, hydrolyzed polyacrylonitrile (HPAN) base membrane is selected as the immobilization carrier to fix CLEAs on the membrane. This can solve the viscosity problem of CLEAs, separate the catalytic coupling, improve the catalytic efficiency, and facilitate the reuse of CLEAs.

[0020] (4) The materials and reagents used in this invention for immobilized enzymes and membrane separation are all conventional bulk products and are inexpensive.

[0021] (5) The method described in this invention is used for water treatment, specifically for the removal of micropollutants and the separation of salt stains in water. The Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane achieves a BPA removal rate of up to 97.13%, while the removal rates for malachite green (BG4) and crystal violet (CV) dyes are 97.78% and 94.52%, respectively. The retention rates for the four common salt ions are all below 15%. The PTE-CLEAs / UiO-66-NH2@PDA biocatalytic membrane achieves a MPO removal rate of 94.04%. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation and use of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane in Example 1;

[0023] Figure 2Scanning electron microscope images of the HPAN-based membrane, the UiO-66-NH2@PDA membrane, and the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane, where (a, d) are the HPAN-based membrane, (b, e) are the UiO-66-NH2@PDA membrane, and (c, f) are the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0024] Figure 3(a) shows the infrared spectra of the HPAN-based membrane, the UiO-66-NH2@PDA membrane, and the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0025] Figure 3(b) shows the XRD patterns of the HPAN-based membrane, the UiO-66-NH2@PDA membrane, and the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0026] Figure 4 Laser confocal morphology of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0027] Figure 5(a) shows the removal rate of BPA of different concentrations by the Lac-CLEAs / UiO-66-NH2@PDA membrane and the BPA concentration in the permeate.

[0028] Figure 5(b) PWP and BPA removal rates of Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membranes obtained with different laccase concentrations;

[0029] Figure 5(c) PWP and BPA removal rates of Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membranes obtained with different BSA concentrations;

[0030] Figure 5(d) Removal rates of different substrates by Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0031] Figure 6(a) shows the dye retention performance of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0032] Figure 6(b) shows the salt retention performance of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0033] Figure 7(a) shows the storage stability test results of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane;

[0034] Figure 7(b) shows the stability test results of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane during repeated use;

[0035] Figure 8 The graph shows the antifouling stability test results of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane.

[0036] Figure 9 The PWP and MPO removal rates of PTE-CLEAs / UiO-66-NH2@PDA biocatalytic membranes obtained for different PTE concentrations. Detailed Implementation

[0037] The present invention proposes a design for preparing a cross-linked enzyme aggregate (CLEAs) biocatalytic membrane and applying it to the removal of micropollutants and salt staining in water. The design involves immobilizing cross-linked enzyme aggregates (CLEAs), specifically cross-linked laccase aggregates (Lac-CLEAs) or phosphotriesterase aggregates (PTE-CLEAs), onto a base membrane using pressure-assisted self-assembly technology. To compensate for the interfacial defects of the disordered cross-linked enzyme aggregate layer, polydopamine (PDA)-modified UiO-66-NH2 (UiO-66-NH2@PDA) is first loaded onto the base membrane surface as an intermediate layer. This compensates for the interfacial defects of the disordered cross-linked enzyme aggregate layer. Simultaneously, the adhesive surface facilitates the adhesion of the cross-linked enzyme aggregates. Furthermore, the adsorption properties of UiO-66-NH2@PDA help enrich micropollutants, promote enzyme catalysis, enable continuous operation, improve enzyme stability, and promote enzyme reuse. The Lac-CLEAs / UiO-66-NH2@PDA (cross-linked laccase aggregate biocatalytic membrane) prepared in this invention is used for salt / pollutant separation and exhibits excellent salt / pollutant separation performance, thereby achieving efficient removal of micro-pollutants. The PTE-CLEAs / UiO-66-NH2@PDA (cross-linked triphospholipase aggregate biocatalytic membrane) prepared in this invention can effectively remove organophosphorus pesticides (MPO) from water.

[0038] In this invention, the relevant testing methods include: Pure water permeate flux is tested using a dead-end filtration system with pressure provided by nitrogen (N2). Dye rejection performance is tested by filtering dyes of different molecular weights and charge properties. Salt ion rejection performance is tested by filtration, and conductivity at different concentrations is measured at room temperature using a conductivity meter. BPA removal rate is tested using a dead-end filtration system with pressure provided by nitrogen (N2), and the peak area corresponding to different concentrations of BPA is determined using high-performance liquid chromatography (HPLC) to establish concentration relationships. MPO removal rate is tested using a dead-end filtration system with pressure provided by nitrogen (N2), and the absorbance is measured using a UV-Vis spectrophotometer to calculate the corresponding permeate concentration.

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the following embodiments are by no means intended to limit the present invention.

[0040] Example 1: Preparation of a cross-linked laccase aggregate biocatalytic membrane (Lac-CLEAs / UiO-66-NH2@PDA) as follows Figure 1 As shown, prepare according to the following steps:

[0041] Step 1) Preparation of UiO-66-NH2 nanoparticles: The specific process is as follows: Weigh 0.1242 g of NH2-BDC and 0.1598 g of ZrCl4, dissolve them in 40 mL of N,N-dimethylformamide (DMF), add 1.2 mL of CH3COOH and 20 μL of H2O, and sonicate for a period of time to make the solution homogeneous. The system is reacted at 100 °C for 24 h, then washed 3 times with DMF and 3 times with water. After centrifugation at 10000 rpm for 10 min, the product obtained is UiO-66-NH2 nanoparticles.

[0042] Step 2) Synthesis of the intermediate layer UiO-66-NH2@PDA: The specific process is as follows: Weigh 20 mg of UiO-66-NH2 nanoparticles prepared in step 1) and 40 mg of dopamine hydrochloride, dissolve them in 20 mL of Tris-HCl (50 mmol / L, pH = 8.5) buffer, stir magnetically at room temperature for a certain period of time, and after stirring evenly, wash with water 3 times, and centrifuge at 10000 rpm for 10 min. The obtained product is polydopamine-modified UiO-66-NH2 nanoparticles, abbreviated as UiO-66-NH2@PDA.

[0043] Step 3) Synthesis of Lac-CLEAs, the specific process is as follows:

[0044] 7.5 mg of ammonium sulfate was added to 15 mL of PBS (50 mmol / L, pH = 7.0) buffer to obtain PBS buffer containing ammonium sulfate, denoted as Mixed Solution 1. Under ice bath conditions, 15 mg of Lac and 0.6 mg of BSA were added to 5 mL of PBS (50 mmol / L, pH = 7.0) buffer, and the resulting solution was denoted as Mixed Solution 2. Under ice bath conditions, Mixed Solution 2 was added to Mixed Solution 2. In this system, the mass-volume concentration of Lac was 0.75 mg / mL and the mass-volume concentration of BSA was 0.03 mg / mL. The mixture was allowed to stand for 3 h. Then, 150 μL of glutaraldehyde (37.5 mmol / L) was added while magnetically stirring, and crosslinking was carried out for 1 h. The resulting product was Lac-CLEAs.

[0045] Step 4) Lac-CLEAs are used immediately after preparation. The mixed solution containing Lac-CLEAs is loaded onto the UiO-66-NH2@PDA pre-modified membrane by pressure filtration, thereby obtaining a cross-linked enzyme aggregate biocatalytic membrane. The specific process is as follows:

[0046] The dry mass of UiO-66-NH2 nanoparticles to the base film area ratio is 0.075 mg / cm². 2 To prepare a dispersion of UiO-66-NH2@PDA nanoparticles, 2.34 mg of UiO-66-NH2@PDA nanoparticles obtained in step 2) were dispersed in 20 mL of ultrapure water to obtain a dispersion for later use.

[0047] The commercial PAN ultrafiltration membrane was soaked in NaOH solution at 50℃ for 1 hour and washed three times with ultrapure water to obtain hydrolyzed PAN (HPAN) membrane, which was then soaked in ultrapure water for use. The HPAN membrane was fixed in an ultrafiltration cup with the active side facing up. The above dispersion was poured into the ultrafiltration cup to immerse the HPAN membrane. The N2 pressure was adjusted to allow the droplets to completely permeate and flow out. Then, it was washed three times with ultrapure water. Thus, UiO-66-NH2 nanoparticles modified with polydopamine were loaded onto the surface of the polyacrylonitrile ultrafiltration membrane. This membrane is referred to as the UiO-66-NH2@PDA modified membrane.

[0048] The Lac-CLEAs mixed solution synthesized in step 3) above was poured into an ultrafiltration cup to immerse the membrane modified with UiO-66-NH2@PDA. The N2 pressure was adjusted to allow the droplets to drip slowly until all the droplets flowed out completely. The resulting membrane was washed three times with ultrapure water. The resulting membrane is a cross-linked laccase aggregate biocatalytic membrane, denoted as Lac-CLEAs / UiO-66-NH2@PDA. The membrane was then stored in ultrapure water at 4°C.

[0049] Example 2: Performance of Lac-CLEAs / UiO-66-NH2@PDA

[0050] 1) Observe the surface and cross-sectional morphology. The surface and cross-sectional morphology of the prepared HPAN-based membrane, UiO-66-NH2@PDA membrane, and Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane were observed using scanning electron microscopy. Figure 2 (a, b) It can be seen that compared with the smooth and flat original HPAN base film, the surface of the UiO-66-NH2@PDA film has undergone a visual change. The UiO-66-NH2@PDA nanoparticles are clearly visible and relatively uniformly distributed. Due to the three-dimensional structure of the UiO-66-NH2 regular octahedron, the surface is slightly rougher and has a large number of pores compared with the base film. Figure 2 (d) shows the cross-sectional morphology of the base membrane when the loading of UiO-66-NH2@PDA on the membrane is 0.075 mg / cm³. 2 At that time, from the cross-section Figure 2 (e) It was found that the thickness of the UiO-66-NH2@PDA interlayer was approximately 1.72 μm. After loading Lac-CLEAs, from Figure 2(c) It can be seen that it successfully adheres to the membrane surface, as shown in the cross-section. Figure 2 (f) It can be seen that the thickness of the Lac-CLEAs layer is about 156 nm, and the thickness of the entire separation layer is 1.876 μm.

[0051] 2) Surface chemical characterization. The surface chemical properties of the HPAN base film, UiO-66-NH2@PDA, Lac-CLEAs / UiO-66-NH2@PDA biocatalytic film, and Lac-CLEAs were characterized by Fourier transform infrared spectroscopy (FT-IR). The crystal structure of the HPAN base film, UiO-66-NH2@PDA film, and Lac-CLEAs / UiO-66-NH2@PDA biocatalytic film was characterized by X-ray diffraction (XRD).

[0052] As shown in Figure 3(a), the HPAN base film at 2240 and 2938 cm⁻¹ -1 Absorption peaks belonging to the -OH functional groups in C≡N and -COOH appeared at 770 cm⁻¹. After being loaded with UiO-66-NH₂@PDA, the peaks were observed at 770 cm⁻¹. -1 A new absorption peak at 3200 cm⁻¹ appeared, attributed to the Zr-O bond in UiO-66-NH₂@PDA. -1 The nearby absorption band corresponds to the -OH group of UiO-66-NH2@PDA. After loading Lac-CLEAs, the absorption band is at 1653 cm⁻¹. -1 and 1392cm -1 The absorption peaks at θ belong to the C=O stretching vibration and CN stretching vibration of the amide bond in Lac-CLEAs. XRD characterization was performed on the surface crystal structure of the HPAN-based membrane, UiO-66-NH2@PDA membrane, and Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane. As shown in Figure 3(b), all three membranes exhibited characteristic peaks of amorphous polyacrylonitrile polymer at 2θ = 17.65°, 22.74°, and 26.04°. The UiO-66-NH2@PDA membrane showed a characteristic peak at 7.81° belonging to the UiO-66-NH2(111) crystal plane, but the peak intensity was relatively low. This may be due to the encapsulation of UiO-66-NH2 by PDA and the relatively low loading of UiO-66-NH2@PDA. After loading with Lac-CLEAs, the characteristic peaks of the UiO-66-NH2(111) crystal plane were masked, and the XRD pattern was similar to that of the HPAN-based membrane.

[0053] Example 3: Enzyme distribution on the surface of Lac-CLEAs / UiO-66-NH2@PDA

[0054] The distribution of laccase on the Lac-CLEAs / UiO-66-NH2@PDA composite membrane was characterized using CLSM. Figure 4 As shown, laccase molecules stained with Rhodamine B exhibit red fluorescence after excitation at a specific wavelength. The laccase is relatively uniformly distributed on the membrane surface, basically completely covering the membrane surface, proving the successful loading of Lac-CLEAs on the membrane surface.

[0055] Example 4: Testing of BPA and different substrate removal by Lac-CLEAs / UiO-66-NH2@PDA and optimization of Lac concentration.

[0056] The removal rate of BPA by the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane prepared in Example 1 and the BPA concentration in the permeate were tested. The BPA concentration ranged from 2 to 100 mg / L. As shown in Figure 5(a), the BPA removal rate first increased and then gradually decreased. When the concentration was below 10 mg / L, the BPA removal rate gradually increased, while the BPA concentration in the permeate remained basically unchanged, consistently below 1 mg / L. This is because the higher the substrate concentration, the faster the catalytic reaction rate of laccase, thus maintaining a low BPA concentration in the permeate. With further increases in substrate concentration, a BPA removal rate of over 80% was achieved. Even when the substrate concentration reached 100 mg / L, the biocatalytic membrane could still achieve a BPA removal rate of over 70%, and the removal rate could be further improved by extending the reaction time, demonstrating that the biocatalytic membrane has a good applicability range for substrate concentrations.

[0057] The effects of different Lac concentrations on the water flux and BPA removal rate of the Lac-CLEAs / UiO-66-NH2@PDA membrane were tested. Lac-CLEAs / UiO-66-NH2@PDA membranes with different Lac concentrations were prepared according to the same process as in Example 1, except that in step 3), the mass-to-volume ratio of Lac in the reaction system was controlled at 0.25 mg / L, 0.50 mg / L, 0.75 mg / L, 1.0 mg / L, and 1.25 mg / L, respectively. The test results for multiple samples are shown in Figure 5(b). With increasing Lac concentration, the membrane structure became relatively more compact, and the water flux decreased from 114.21 LMH / bar to 52.74 LMH / bar. When the Lac concentration increased to 0.75 mg / mL, the BPA removal rate reached 97.13%. When the laccase concentration exceeded 0.75 mg / mL, the BPA removal rate still increased slightly. In this invention, the concentration of laccase is limited to 0.25–1.25 mg / mL, and the BPA removal rate is above 70%.

[0058] The effects of different BSA concentrations on the water flux and BPA removal rate of the Lac-CLEAs-UiO-66-NH2@PDA membrane were tested. Lac-CLEAs / UiO-66-NH2@PDA membranes with different BSA concentrations were prepared according to the same process as in Example 1, except that in step 3), the BSA mass-to-volume ratio in the reaction system was controlled at 0.001 mg / L, 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, and 0.05 mg / L, respectively. The results of multiple samples are shown in Figure 5(c). With increasing BSA concentration, the membrane became denser, which was conducive to improving laccase activity, while the water flux decreased and the BPA removal rate gradually increased. However, when the BSA concentration exceeded 0.03 mg / mL, the BPA removal rate decreased. This may be because excessive BSA concentration encapsulates too much laccase, affecting its active site and leading to insufficient cross-linking of enzyme molecules, making them prone to leakage. Further research was conducted using a BSA concentration of 0.03 mg / mL. In summary, when the UiO-66-NH2@PDA loading was 0.075 mg / mL... 2 When the laccase concentration was 0.75 mg / mL and the BSA concentration was 0.03 mg / mL, the BPA removal rate reached a maximum of 97.13%, and the water flux was 63.54 LMH / bar.

[0059] To broaden the application range of the catalytic membrane, in addition to BPA, as shown in Figure 5(d), the removal rates of five other substrates, phenol (10 mg / L), hydroquinone (HQ, 10 mg / L), 2,4-dichlorophenol (2,4-DCP, 10 mg / L), tetracycline hydrochloride (TC, 20 mg / L), and doxycycline hydrochloride (DC, 50 mg / L), prepared in Example 1 were also tested. These rates were 73.24%, 81.48%, 93.32%, 89.51%, and 91.40%, respectively. The removal rates were higher because laccase exhibited higher catalytic efficiency for BPA and 2,4-dichlorophenol, while phenol and hydroquinone showed relatively higher resistance to laccase oxidation, resulting in lower removal rates. Therefore, the prepared catalytic membrane has certain substrate applicability.

[0060] Example 5: Testing the dye and salt rejection performance of Lac-CLEAs / UiO-66-NH2@PDA

[0061] The retention efficiency of the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane for various dyes with different molecular weights and charges was investigated. These dyes included Rhodamine B (RhB, +, Mw = 479.01), Crystal Violet (CV, +, Mw = 407.98), Chrome Black T (CBT, -, Mw = 461.38), Acid Fuchs Red (AF, -, Mw = 585.54), Congo Red (CR, -, Mw = 696.66), and Malachite Green (BG4, +, Mw = 927.02). The main mechanisms were pore size sieving and electrostatic repulsion. Figure 6(a) shows the retention rate of malachite green (BG4) dye by the membrane prepared in Example 1, which was 97.78%. This was mainly due to the pore size sieving effect, which was effective against RhB, CV, CBT, AF, and CR. The retention rates of the five dyes were 90.16%, 94.52%, 93.97%, 91.18%, and 88.54%, respectively. Based on the pore size sieving and electrostatic repulsion mechanism, the Lac-CLEAs / PDA@UiO-66-NH2 biocatalytic membrane has the functions of adsorption and retention of dyes, thus achieving a high dye retention rate.

[0062] The rejection rates of four salt ions by the Lac-CLEAs / UiO-66-NH2@PDA biocatalytic membrane were tested. Figure 6(b) shows that the membrane prepared in Example 1 exhibits better permeation performance for salt ions, with rejection rates of MgSO4 > Na2SO4 > MgCl2 > NaCl. This is because the negative charge on the membrane surface has a greater repulsive effect on divalent salt ions than on monovalent salt ions. The rejection rates for MgSO4, Na2SO4, MgCl2, and NaCl were 14.35%, 12.9%, 8.06%, and 5.7%, respectively. The membrane exhibits high rejection rates for dyes and high permeation rates for salts, indicating that it simultaneously possesses salt / dye separation capabilities.

[0063] Example 6: Storage stability and reusability of Lac-CLEAs / UiO-66-NH2@PDA

[0064] The storage stability of the biocatalytic membrane was tested, and the enzyme activity on the membrane was tested at the same time interval. The results are shown in Figure 7(a). During the 10-day test, the enzyme activity on the membrane was still 70% of the initial enzyme activity, indicating that the cross-linked laccase aggregates were effectively fixed on the membrane. The reason for the decrease in enzyme activity may be due to the leakage of enzyme molecules during storage and the inactivation of the enzyme molecules themselves.

[0065] The prepared Lac-CLEAs / UiO-66-NH2@PDA membrane was tested for reusability. The membrane was washed three times with ultrapure water after each cycle. Figure 7(b) shows that after 6 cycles, the membrane maintained a BPA removal rate of 78.21%, indicating good reusability. With increasing cycle number, the BPA removal efficiency decreased. This may be due to the accumulation of BPA oxidation products on the membrane surface, affecting the contact between the enzyme and BPA, or it may be due to the shedding of Lac-CLEAs during reuse and washing.

[0066] Example 7: Antifouling stability of Lac-CLEAs / UiO-66-NH2@PDA

[0067] Membrane fouling significantly impacts membrane lifespan and separation efficiency during operation. Due to hydrogen bonding or electrostatic interactions, contaminants typically adhere to the membrane surface, leading to filter cake formation, clogging pores, and reducing permeate flux. Although cleaning removes most contaminants from the membrane due to external forces, some stubborn contaminants remain, causing irreversible fouling. Using BSA as a model contaminant, a steady-state filtration experiment was conducted with 6 cycles and 6 hours of cleaning. The membrane's antifouling ability was evaluated by examining the flux recovery rate (FRR). Figure 8 As shown, irreversible fouling caused a slight decrease in the FRR of the Lac-CLEAs / UiO-66-NH2@PDA membrane with increasing cycle number. After 6 cycles, the FRR of the membrane remained above 80% and gradually stabilized, indicating that the membrane has good antifouling stability and can maintain good performance during operation. This may be because the membrane surface is negatively charged, which has a certain repulsive effect on BSA, so the BSA adhering to the membrane surface is easily removed by washing.

[0068] Example 8: Preparation of PTE-CLEAs / UiO-66-NH2@PDA membrane

[0069] The preparation method is basically the same as in Example 1, except that in step 3), laccase (Lac) is replaced with phosphotriethesterase (PTE). The steps include: step 1) preparing UiO-66-NH2 nanoparticles, step 2) synthesizing the intermediate layer UiO-66-NH2@PDA, step 3) synthesizing PTE-CLEAs, and step 4) loading the mixed solution containing Lac-CLEAs onto the UiO-66-NH2@PDA pre-modified membrane by pressure filtration, finally obtaining the PTE-CLEAs / UiO-66-NH2@PDA membrane.

[0070] Example 9: Optimization of PTE concentration during the preparation of PTE-CLEAs / UiO-66-NH2@PDA membrane.

[0071] Following the preparation method of Example 8, multiple sample membranes with different PTE concentrations were prepared in the reaction system during the synthesis of PTE-CLEAs in step 3). The PTE concentrations were 0.15 mg / mL, 0.30 mg / mL, 0.45 mg / mL, 0.60 mg / mL, and 0.75 mg / mL. The effects of different PTE concentrations on the water flux and MPO removal rate of the PTE-CLEAs / UiO-66-NH2@PDA membrane were tested, and the results are as follows: Figure 9 As shown, in this invention, the PTE mass-volume concentration in the reaction system of step 3) is limited to 0.25-0.75 mg / mL. When the PTE concentration is 0.60 mg / mL, the MPO removal rate reaches 94.04%, and the water flux of the membrane is 76.65 LMH / bar.

[0072] 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 specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many changes under the guidance of the present invention without departing from the spirit of the present invention, and these changes are all within the protection scope of the present invention.

Claims

1. A method for the preparation of a cross-linked enzyme aggregate biocatalytic membrane, characterized in that, Using a polyacrylonitrile ultrafiltration membrane as the base membrane, polydopamine-modified UiO-66-NH2 nanoparticles are loaded onto the surface of the polyacrylonitrile ultrafiltration membrane as an intermediate layer. Crosslinking enzyme aggregates are loaded onto the treated polyacrylonitrile ultrafiltration membrane using pressure-assisted self-assembly technology, thereby obtaining a crosslinking enzyme aggregate biocatalytic membrane. The cross-linked enzyme aggregate is a cross-linked laccase aggregate or a cross-linked triphospholipase aggregate; The cross-linked laccase aggregate biocatalytic membrane prepared by loading the cross-linked laccase aggregates onto a treated polyacrylonitrile ultrafiltration membrane is a cross-linked laccase aggregate biocatalytic membrane; The cross-linked phospholipase aggregate biocatalytic membrane prepared by loading the cross-linked phospholipase aggregate onto a treated polyacrylonitrile ultrafiltration membrane is a cross-linked phospholipase aggregate biocatalytic membrane.

2. The method for preparing a cross-linked enzyme aggregate biocatalytic membrane according to claim 1, characterized in that, The preparation method of the UiO-66-NH2 nanoparticles is as follows: a certain mass of NH2-BDC and ZrCl4 are weighed and dissolved in an appropriate amount of DMF to obtain mixed solution A. In mixed solution A, the mass of NH2-BDC is 310 mg and the mass of ZrCl4 is 400 mg, measured in 100 mL of DMF. Then, CH3COOH and H2O are added to mixed solution A to obtain mixed solution B. In mixed solution B, the volume ratio of DMF, CH3COOH and H2O is 2000:60:

1. Mixed solution B is ultrasonically treated to make the solution homogeneous. Then, it is reacted at 100 °C for 24 h, followed by washing with DMF 3 times and water 3 times, centrifuged for 10 min to obtain the product, which is UiO-66-NH2 nanoparticles.

3. The method of preparing a cross-linked enzyme aggregate biocatalytic membrane according to claim 1, wherein, The specific steps for modifying the UiO-66-NH2 nanoparticles with polydopamine are as follows: A certain amount of UiO-66-NH2 nanoparticles and an appropriate amount of dopamine hydrochloride are dissolved in an appropriate amount of Tris-HCl buffer solution, wherein the concentration of the Tris-HCl buffer solution is 50 mmol / L and the pH is 8.5; the mass of the UiO-66-NH2 nanoparticles is 100 mg and the mass of the dopamine hydrochloride is 200 mg, measured in 100 mL of Tris-HCl buffer solution; the mixture is magnetically stirred at room temperature until homogeneous, then washed three times with water, and centrifuged for 10 min. The resulting product is the polydopamine-modified UiO-66-NH2 nanoparticles, abbreviated as UiO-66-NH2@PDA.

4. The method for preparing a cross-linked enzyme aggregate biocatalytic membrane according to claim 1, characterized by, The method for synthesizing the cross-linked laccase aggregates is as follows: Under ice bath conditions, a certain amount of laccase and bovine serum albumin are added to an appropriate amount of PBS buffer. The concentration of the PBS buffer is 50 mmol / L, and the pH is 7.0, to obtain a mixed solution C. A PBS buffer containing ammonium sulfate is prepared, wherein the concentration of the PBS buffer is 50 mmol / L, the pH is 7.0, and the mass-to-volume ratio of ammonium sulfate is 0.5 g / L. The mixed solution C is added to the PBS buffer containing ammonium sulfate at a volume ratio of 1:3 to obtain a mixed solution D. In mixed solution D, the mass-to-volume concentration of laccase is (0.25~1.25) mg / mL, and the mass-to-volume concentration of bovine serum albumin is (0~0.05) mg / mL, and not equal to 0. The mixture is allowed to stand for 3 hours. Subsequently, an appropriate amount of glutaraldehyde with a concentration of 37.5 mmol / L is added under magnetic stirring, wherein the volume ratio of glutaraldehyde to mixed solution C is 3:

100. Cross-linking is carried out for 1 hour, and the resulting solution is the solution of the cross-linked laccase aggregates.

5. The method for preparing the cross-linked enzyme aggregate biocatalytic membrane according to claim 1, characterized in that, The method for synthesizing the cross-linked phospholipase aggregates is as follows: Under ice bath conditions, a certain amount of phospholipase and bovine serum albumin are added to an appropriate PBS buffer with a concentration of 50 mmol / L and pH=7.0 to obtain a mixed solution C; a PBS buffer containing ammonium sulfate is prepared, wherein the concentration of the PBS buffer is 50 mmol / L, pH=7.0, and the mass-to-volume ratio of ammonium sulfate is 0.5 g / L; the mixed solution C is added to the PBS buffer containing ammonium sulfate at a volume ratio of 1:3 to obtain a mixed solution D, wherein the mass-to-volume concentration of phospholipase in mixed solution D is (0.25~0.75) mg / mL, and the mass-to-volume concentration of bovine serum albumin is (0~0.05) mg / mL, and is not equal to 0; the mixture is allowed to stand for 3 h; then, an appropriate amount of glutaraldehyde with a concentration of 37.5 mmol / L is added under magnetic stirring, wherein the volume ratio of glutaraldehyde to mixed solution C is 3:100, and cross-linking is performed for 1 h. The resulting solution is the solution of the cross-linked phospholipase aggregates.

6. The method for preparing the cross-linked enzyme aggregate biocatalytic membrane according to claim 1, characterized in that, Polyacrylonitrile ultrafiltration membranes were soaked in NaOH solution at 50 °C for 1 h and washed three times with ultrapure water to obtain hydrolyzed PAN membranes, which were then used after soaking in ultrapure water. The hydrolyzed PAN membranes were fixed in an ultrafiltration cup with the active side facing upwards. A dispersion of polydopamine-modified UiO-66-NH2 nanoparticles was poured into the ultrafiltration cup, submerging the PAN membrane. The dry mass ratio of UiO-66-NH2 nanoparticles to the base membrane area was (0~0.125) mg / cm². 2 Adjust the nitrogen pressure to allow the droplets to fully permeate and flow out, then wash with ultrapure water three times. Thus, the polydopamine-modified UiO-66-NH2 nanoparticles are loaded onto the surface of the polyacrylonitrile ultrafiltration membrane, which is referred to as the UiO-66-NH2@PDA modified membrane. The solution of cross-linking enzyme aggregates was poured into an ultrafiltration cup to immerse the membrane modified with UiO-66-NH2@PDA. The nitrogen pressure was adjusted to allow the droplets to drip slowly until all the droplets flowed out completely. The resulting membrane was washed three times with ultrapure water to obtain the cross-linking enzyme aggregate biocatalytic membrane. The membrane was then soaked in ultrapure water and stored at 4°C.

7. Use of a cross-linked enzyme aggregate biocatalytic membrane produced according to the production method of claim 6, characterized in that, The cross-linked enzyme aggregate biocatalytic membrane is a cross-linked laccase aggregate biocatalytic membrane; The cross-linked laccase aggregate biocatalytic membrane is used to remove micropollutants from water; the micropollutants include one or more of bisphenol A, phenol, hydroquinone, 2,4-dichlorophenol, tetracycline hydrochloride and doxycycline hydrochloride. Salt / dye separation is achieved by utilizing the pore size sieving and electrostatic repulsion of the cross-linked laccase aggregate biocatalytic membrane.

8. Use of a cross-linked enzyme aggregate biocatalytic membrane produced according to the production method of claim 6, characterized in that, The cross-linked enzyme aggregate biocatalytic membrane is a cross-linked phosphotriacylase aggregate biocatalytic membrane, and the cross-linked phosphotriacylase aggregate biocatalytic membrane is used to remove methyl parathion in water, and the removal rate is above 75%.

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