Hybrid hydrogel immobilized double-enzyme cascade reactor as well as preparation method and application thereof

Abstract

The invention relates to a hybrid hydrogel immobilized double-enzyme cascade reactor as well as a preparation method and application thereof. A biomimetic mineralization method is adopted for preparing a metal organic framework compound embedded with combined enzyme, the metal organic framework compound is dispersed in a water-soluble monomer, combined enzyme cascade reaction enzyme is utilized for catalyzing monomer polymerization, in-situ immobilization without damage to double enzymes is achieved while a hybrid hydrogel network structure is formed, and the immobilized double-enzyme cascade reactor is obtained. The hybrid hydrogel immobilized double-enzyme cascade reactor prepared by the invention is high in enzyme immobilization rate, good in enzyme stability, controllable in double-enzyme immobilization proportion, simple to operate, mild in condition and easy to implement; the prepared hybrid hydrogel immobilized double-enzyme cascade reactor has adsorption and degradation functions, phenolic compounds in water can be effectively enriched, the local concentration of the phenolic compounds near immobilized double enzymes is improved, the efficiency of catalytic degradation of the phenolic compounds through double-enzyme cascade reaction is improved, and the service life of the reactor is prolonged. The method can be applied to efficient treatment of wastewater containing phenolic compounds.

Description

Technical Field

[0001] The invention relates to the technical field of immobilized enzyme engineering, and in particular to a hybrid hydrogel-immobilized dual-enzyme cascade reactor and a preparation method and application thereof. Background Art

[0002] Multi-enzyme cascade reaction refers to a process in which multiple enzymes participate in step-by-step catalysis in a coordinated manner. The product (or intermediate) formed by the catalysis of the previous enzyme can continue to participate in the next enzyme catalytic reaction as a substrate of the subsequent enzyme. The intermediates of the multi-enzyme cascade reaction are transported between the active sites of different enzymes, which can maintain a high local concentration of the intermediates and reduce the diffusion loss during the material transport process, thereby achieving a specific, dedicated and efficient catalytic reaction. Inspired by this, people restrict or locate multiple enzymes with different functions on the surface or inside of a specific carrier through physical, chemical or biological methods, and construct an immobilized multi-enzyme cascade reactor based on the efficient synergistic catalytic mechanism of the multi-enzyme cascade reaction. It combines the synergistic efficiency of the multi-enzyme cascade reaction with the excellent stability and reusability of the immobilized enzyme, and can combine the catalytic properties of different enzymes to increase the concentration of reactants around the enzyme, thereby significantly improving the enzyme catalytic efficiency. Among them, glucose oxidase and horseradish peroxidase are often used to construct immobilized dual enzyme cascade reactors because of their simple raw materials and stable cascade reactions under various conditions.

[0003] Suitable immobilization carriers and immobilization processes are the basis for constructing high-activity and high-synergy dual-enzyme cascade reactors. Metal-organic frameworks (MOFs) are composed of metal cation clusters coordinated with organic ligands with a three-dimensional network structure, and have high crystallinity and porosity. Among various MOF materials, ZIF-8 has important application prospects in enzyme immobilization such as encapsulation and pore adsorption due to its mild synthesis conditions, good hydrothermal stability, large specific surface area and adjustable pore size, and has become a commonly used new carrier material for enzyme immobilization. However, most ZIF materials will gradually degrade in an acidic environment and lose their porous structure, resulting in the leakage of the immobilized enzyme, and the ZIF-8 immobilized enzyme in powder form is difficult to separate and reuse during use.

[0004] Polymer hydrogels are considered to be ideal carrier materials for immobilized enzymes due to their excellent enzyme compatibility, stimulus responsiveness, and flexible structure. Hydrogel-immobilized enzymes can provide a hydrophilic microenvironment for the immobilized enzymes inside to maintain the activity of the enzymes, and can regulate the activity of the immobilized enzymes by changing their hydrophilicity or volume through external stimuli. However, the chemical cross-linking method used in the traditional preparation process of hydrogel-immobilized enzymes will destroy the spatial structure of the enzymes, thereby reducing the activity of the enzymes.

[0005] Therefore, it is necessary to develop a new hybrid hydrogel-immobilized enzyme cascade reactor that can immobilize multiple enzymes in situ and retain the activity of multiple enzymes. Summary of the invention

[0006] The present invention aims to provide a hybrid hydrogel immobilized dual enzyme cascade reactor and a preparation method and application thereof, wherein the cascade reactor comprises a reaction core and a hydrogel layer on the surface of the reaction core; the reaction core comprises a combined enzyme wrapped in ZIF-8; the hydrogel layer comprises a hybrid hydrogel; the hybrid hydrogel is immobilized by GOx in O 2 In the presence of 2 O 2 , the generated H 2 O 2 The ACAC is transferred to the HRP-catalyzed substrate to generate ACAC free radicals, thereby initiating free radical copolymerization of the monomer and the cross-linker to form a hybrid hydrogel three-dimensional cross-linked network that wraps the ZIF-8 complex.

[0007] In a first aspect, the present invention provides a hybrid hydrogel-immobilized dual-enzyme cascade reactor, wherein the hybrid hydrogel-immobilized dual-enzyme cascade reactor comprises a reaction core and a hybrid hydrogel layer on the surface of the reaction core;

[0008] The reaction core includes an immobilized dual enzyme ZIF-8 complex;

[0009] The immobilized dual-enzyme ZIF-8 complex comprises ZIF-8 and a combined enzyme encapsulated in ZIF-8.

[0010] Optionally, the enzyme combination includes any one of glucose oxidase / horseradish peroxidase and pyranose oxidase / horseradish peroxidase.

[0011] Optionally, the hybrid hydrogel layer comprises a monomeric free radical polymer.

[0012] Optionally, the monomer includes a water-soluble monomer; the water-soluble monomer includes any one of N,N-dimethylacrylamide, polyethylene glycol methacrylate, polyethylene glycol acrylate, and hydroxyethyl acrylate.

[0013] In a second aspect, the present invention provides a method for preparing a hybrid hydrogel immobilized dual enzyme cascade reactor, comprising the following steps:

[0014] Adding the combined enzyme and 2-methylimidazole to a zinc nitrate solution, stirring for 2-4 hours, centrifuging, washing, and drying to obtain an immobilized dual-enzyme ZIF-8 complex;

[0015] The molar ratio of zinc ion to 2-methylimidazole in the zinc nitrate is 1:4; the mass ratio of the two enzymes in the combined enzyme is (1-2): (1-3);

[0016] In water, under the catalysis of the combined enzyme and the action of a crosslinking agent, the immobilized dual-enzyme ZIF-8 complex polymerizes with the monomer and substrate to obtain a hybrid hydrogel immobilized dual-enzyme cascade reactor; the substrate includes glucose and acetylacetone.

[0017] Optionally, the formation of the hybrid hydrogel comprises the following steps:

[0018] Glucose oxidase oxidizes glucose to produce H 2 O 2 , and then H 2 O 2 Transferred to HRP, so that HRP catalyzes ACAC to produce ACAC free radicals; ACAC free radicals undergo free radical copolymerization with water-soluble monomers and cross-linking agents to form a hybrid hydrogel three-dimensional cross-linked network structure.

[0019] Optionally, the cross-linking agent includes any one of modified silica nanoparticles and N,N-methylenebisacrylamide.

[0020] Optionally, the mass ratio of the immobilized dual-enzyme ZIF-8 complex to the monomer is 1:(10-100).

[0021] Optionally, the mass of the cross-linking agent is 1%-4% of the mass of the monomer.

[0022] Optionally, the concentration of glucose in water is 5-20 mmol / L.

[0023] Optionally, the concentration of acetylacetone in water is 0.1-1 mmol / L.

[0024] Optionally, the conditions under which the enzyme combination is catalyzed include: a temperature of 30°C.

[0025] In a third aspect, the present invention provides an application of a hybrid hydrogel immobilized dual enzyme cascade reactor in wastewater treatment, wherein the application includes degrading phenolic compounds in the wastewater.

[0026] Optionally, the phenolic compound includes at least one of phenol, bisphenol A, 4-chlorophenol, ferulic acid, and caffeic acid.

[0027] Optionally, the hybrid hydrogel-immobilized dual-enzyme cascade reactor has good reusability; after being reused 5 times, the activity of the cascade reactor relative to the first use drops to 20%.

[0028] The beneficial effects of the present invention include:

[0029] (1) The preparation method of the cascade reactor provided by the present invention can achieve efficient immobilization of enzymes with controllable enzyme ratios by embedding dual enzymes in ZIF-8 through a biomimetic mineralization method, and can effectively retain the dual enzyme activities; and the outer layer of the hybrid hydrogel of the present invention is constructed in situ by enzyme-catalyzed polymerization of water-soluble monomers through the cascade reaction of the immobilized dual enzymes themselves, without requiring harsh reaction conditions and external reaction reagents, and simultaneously realizes the construction of the hybrid hydrogel network and the in situ damage-free embedding of the immobilized dual enzymes during the enzyme-catalyzed polymerization process, thereby avoiding the problem that conventional free radical polymerization reaction systems are prone to changes in enzyme configurations or even reduced activity, and the method is simple, the conditions are mild, and it is easy to implement;

[0030] (2) The hybrid hydrogel provided by the present invention uses ZIF-8 and KH570-SiO 2 Nanoparticles improve the cross-linking density and network structure of hybrid hydrogels, effectively enhance the mechanical strength of hybrid hydrogels, and enhance the structural stability of ZIF-8 embedded therein, thereby solving the problem of easy degradation in acidic solutions leading to leakage of immobilized enzymes. At the same time, they provide a hydrophilic microenvironment for the immobilized dual enzymes, thereby enhancing the activity of the dual enzyme cascade reaction.

[0031] (3) The hybrid hydrogel immobilized dual enzyme cascade reactor provided by the present invention can effectively enrich phenolic compounds in water, thereby increasing the local concentration of phenolic compounds near the immobilized dual enzymes, and improving the efficiency of the dual enzyme cascade reaction in catalyzing the degradation of phenolic compounds. It is highly efficient and environmentally friendly, has a good degradation effect on phenolic compounds, and its products are non-toxic or low-toxic, which conforms to the concept of green chemistry and can be applied to the efficient treatment of wastewater containing phenolic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A reaction mechanism diagram for preparing a hybrid hydrogel-immobilized dual enzyme cascade reactor for preparing the catalytic monomer polymerization of the present invention;

[0033] Figure 2 is a scanning electron microscope image of GOx / HRP@ZIF-8 prepared in Preparation Example 1;

[0034] Figure 3 This is a scanning electron microscopy image of the GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor prepared in Example 1;

[0035] Figure 4 It is a comparison chart of the cascade reaction activity of different GOx / HRP@ZIF-8 in the preparation examples;

[0036] Figure 5 is the compressive stress-strain curve of GOx / HRP@ZIF-8@PDMAA hybrid hydrogel;

[0037] Figure 6 The UV absorption spectra (a) and concentration-absorbance working curves (b) of 4-chlorophenol at different concentrations;

[0038] Figure 7 The concentration-absorbance working curves of phenol (a), bisphenol A (b), ferulic acid (c) and caffeic acid (d);

[0039] Figure 8 The degradation rate-time curve of 4-chlorophenol degradation in GOX / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor;

[0040] Fig. 9 is the effect of pH on the degradation of 4-chlorophenol, phenol and bisphenol A by GOX / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor;

[0041] Fig.10 is the effect of pH on the degradation of ferulic acid and caffeic acid in a GOX / HRP@ZIF-8@PDMAA hybrid hydrogel-immobilized dual enzyme cascade reactor;

[0042] Fig.11 is the degradation amount-time curve of bisphenol A, 4-chlorophenol and phenol in the GOX / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor;

[0043] Fig.12 is the degradation amount-time curve of ferulic acid and caffeic acid in GOX / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor;

[0044] Fig.13 This is a verification of the degradation and reuse effect of the GOX / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor. DETAILED DESCRIPTION

[0045] The present invention will be further described through the following embodiments in conjunction with the accompanying drawings.

[0046] In a first aspect, an embodiment of the present invention provides a hybrid hydrogel-immobilized dual-enzyme cascade reactor, wherein the hybrid hydrogel-immobilized dual-enzyme cascade reactor comprises a reaction core and a hybrid hydrogel layer on the surface of the reaction core;

[0047] The reaction core includes an immobilized dual enzyme ZIF-8 complex;

[0048] The immobilized dual-enzyme ZIF-8 complex comprises ZIF-8 and a combined enzyme encapsulated in ZIF-8.

[0049] In some embodiments, the enzyme combination includes any one of glucose oxidase / horseradish peroxidase and pyranose oxidase / horseradish peroxidase.

[0050] In some embodiments, the hybrid hydrogel layer includes a monomeric free radical polymer.

[0051] In some embodiments, the monomer includes a water-soluble monomer; the water-soluble monomer includes any one of N,N-dimethylacrylamide, polyethylene glycol methacrylate, polyethylene glycol acrylate, and hydroxyethyl acrylate.

[0052] In a second aspect, an embodiment of the present invention provides a method for preparing a hybrid hydrogel immobilized dual enzyme cascade reactor, comprising the following steps:

[0053] Adding the combined enzyme and 2-methylimidazole to a zinc nitrate solution, stirring for 2-4 hours, centrifuging, washing, and drying to obtain an immobilized dual-enzyme ZIF-8 complex;

[0054] The molar ratio of zinc ion to 2-methylimidazole in the zinc nitrate is 1:4; the mass ratio of the two enzymes in the combined enzyme is (1-2): (1-3);

[0055] In water, under the catalysis of the combined enzyme and the action of a crosslinking agent, the immobilized dual-enzyme ZIF-8 complex polymerizes with the monomer and substrate to obtain a hybrid hydrogel immobilized dual-enzyme cascade reactor; the substrate includes glucose and acetylacetone.

[0056] In some embodiments, the formation of the hybrid hydrogel comprises the following steps:

[0057] Glucose oxidase oxidizes glucose to produce H 2 O 2 , and then H 2 O 2 Transferred to HRP, so that HRP catalyzes ACAC to produce ACAC free radicals; ACAC free radicals undergo free radical copolymerization with water-soluble monomers and cross-linking agents to form a hybrid hydrogel three-dimensional cross-linked network structure.

[0058] In some embodiments, the cross-linking agent includes any one of modified silica nanoparticles and N,N-methylenebisacrylamide.

[0059] In some embodiments, the mass ratio of the immobilized dual-enzyme ZIF-8 complex to the monomer is 1:(10-100).

[0060] In some embodiments, the mass of the cross-linking agent is 1%-4% of the mass of the monomer.

[0061] In some embodiments, the concentration of glucose in water is 5-20 mmol / L.

[0062] In some embodiments, the concentration of acetylacetone in water is 0.1-1 mmol / L.

[0063] In some embodiments, the conditions under which the enzyme combination is catalyzed include: a temperature of 30°C.

[0064] In a third aspect, an embodiment of the present invention provides an application of a hybrid hydrogel-immobilized dual-enzyme cascade reactor in wastewater treatment, wherein the application includes degrading phenolic compounds in wastewater.

[0065] In some embodiments, the phenolic compound includes at least one of phenol, bisphenol A, 4-chlorophenol, ferulic acid, and caffeic acid.

[0066] In some embodiments, the hybrid hydrogel-immobilized dual-enzyme cascade reactor has good reusability; after the cascade reactor is reused 5 times, the activity of the cascade reactor decreases to 20% relative to the first use.

[0067] In the embodiment of the present invention, glucose oxidase is labeled as GOx, horseradish peroxidase is labeled as HRP, pyranose oxidase is labeled as P2Ox, acetylacetone is labeled as ACAC, and acetylacetone free radical is labeled as ACAC. · , γ-methacryloxypropyltrimethoxysilane functionalized silica nanoparticles were labeled as KH570-SiO 2 , N,N-methylenebisacrylamide was labeled as MBA, N,N-dimethylacrylamide was labeled as DMAA, polyethylene glycol methacrylate was labeled as PEGMA, polyethylene glycol acrylate was labeled as PEGA, hydroxyethyl acrylate was labeled as HEMA, ZIF-8 immobilized with glucose oxidase / horseradish peroxidase was labeled as GOx / HRP@ZIF-8, and ZIF-8 immobilized with pyranose oxidase / horseradish peroxidase was labeled as P2Ox / HRP@ZIF-8.

[0068] Preparation Example 1

[0069] Preparation Example 1 of the present invention provides a method for preparing GOx / HRP@ZIF-8 by a biomimetic mineralization method, comprising the following steps:

[0070] Dissolve GOx and HRP in ultrapure water to obtain a combined enzyme aqueous solution, wherein the concentration of GOx is 2 mg / L and the concentration of HRP is 3 mg / mL;

[0071] Take 1 mL of the combined enzyme aqueous solution and add it to 2 mL of 0.125 mol / L Zn(NO 3 ) 2 6H 2O aqueous solution, and after uniform mixing, a mixed system 1 is obtained, in which the mass ratio of GOx:HRP is 2:3;

[0072] Add 2 mL of 0.5 mol / L 2-methylimidazole aqueous solution to the mixed system 1, stir vigorously for 2 h, centrifuge at 6000 rpm for 15 min, collect the supernatant 1, add deionized water to the precipitate, wash and centrifuge, take the supernatant 2, repeat the washing, take the supernatant 3, and combine the supernatants obtained three times;

[0073] The obtained precipitate was freeze-dried to obtain a light brown powder, namely GOx / HRP@ZIF-8.

[0074] Preparation Example 2

[0075] Preparation Example 2 of the present invention provides a method for preparing GOx / HRP@ZIF-8 by a biomimetic mineralization method, which is different from Preparation Example 1 in that the concentrations of GOx and HRP in the combined enzyme aqueous solution are both 2 mg / mL, the mass ratio of GOx:HRP in the mixed system is 1:1, and the other steps and conditions are the same as in Preparation Example 1 to obtain GOx / HRP@ZIF-8.

[0076] Preparation Example 3

[0077] Preparation Example 3 of the present invention provides a method for preparing GOx / HRP@ZIF-8 by a biomimetic mineralization method, which is different from Preparation Example 1 in that the GOx concentration in the combined enzyme aqueous solution is 2 mg / mL, the HRP concentration is 4 mg / mL, the mass ratio of GOx:HRP in the mixed system is 1:2, and the other steps and conditions are the same as in Preparation Example 1 to obtain GOx / HRP@ZIF-8.

[0078] Preparation Example 4

[0079] Preparation Example 4 of the present invention provides a method for preparing GOx / HRP@ZIF-8 by a biomimetic mineralization method, which is different from Preparation Example 1 in that the GOx concentration in the combined enzyme aqueous solution is 4 mg / mL, the HRP concentration is 2 mg / mL, the mass ratio of GOx:HRP in the mixed system is 2:1, and the other steps and conditions are the same as in Preparation Example 1 to obtain GOx / HRP@ZIF-8.

[0080] Preparation Example 5

[0081] Preparation Example 5 of the present invention provides a method for preparing P2Ox / HRP@ZIF-8 by a bionic mineralization method, which is different from Preparation Example 1 in that GOx is replaced by P2Ox, and the other steps and conditions are the same as those in Preparation Example 1 to obtain P2Ox / HRP@ZIF-8.

[0082] Example 1

[0083] Example 1 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual-enzyme cascade reactor, comprising the following steps:

[0084] Take 1.5 g DMAA, dissolve it in 7 mL ultrapure water, add 0.12 g GOx / HRP@ZIF-8 prepared in Preparation Example 1, 0.015 g glucose, 280 μL ACAC, 0.04 g KH570-SiO 2 , stirring at 300 rpm for 30 min;

[0085] 1 mL was placed in a polytetrafluoroethylene round mold and polymerized in a constant temperature drying oven at 30°C for 1-4 h to prepare a GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor.

[0086] Example 2

[0087] Example 2 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual-enzyme cascade reactor, which is different from Example 1 in that GOx / HRP@ZIF-8 is replaced with P2Ox / HRP@ZIF-8 prepared in Preparation Example 5 to obtain a P2Ox / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual-enzyme cascade reactor.

[0088] Example 3

[0089] Example 3 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual enzyme cascade reactor, which is different from Example 1 in that KH570-SiO 2 By replacing it with MBA and keeping the other step conditions the same, a GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor was prepared, and GOx / HRP@ZIF-8@PDMAA did not contain silica particles.

[0090] Example 4

[0091] Example 4 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual-enzyme cascade reactor, which is different from Example 1 in that DMAA is replaced by PEGMA, and other steps and conditions remain the same to obtain a GOx / HRP@ZIF-8@PPEGMA hybrid hydrogel immobilized dual-enzyme cascade reactor.

[0092] Example 5

[0093] Example 5 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual-enzyme cascade reactor, which is different from Example 1 in that DMAA is replaced by PEGA, and other steps and conditions remain consistent to obtain a GOx / HRP@ZIF-8@PPEGA hybrid hydrogel immobilized dual-enzyme cascade reactor.

[0094] Example 6

[0095] Example 6 of the present invention provides a method for preparing a hybrid hydrogel immobilized dual-enzyme cascade reactor, which is different from Example 1 in that DMAA is replaced by HEMA, and other steps and conditions remain the same to obtain a GOx / HRP@ZIF-8@PHEMA hybrid hydrogel immobilized dual-enzyme cascade reactor.

[0096] Performance Verification

[0097] 1. Morphological determination of GOx / HRP@ZIF-8 and GOx / HRP@ZIF-8@PDMAA

[0098] The morphology of GOx / HRP@ZIF-8 prepared in Preparation Example 1 and GOx / HRP@ZIF-8@PDMAA prepared in Example 1 was characterized by SEM. Figure 2 , Figure 3 As shown;

[0099] See also Figure 3 The GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor prepared by the present invention has an obvious three-dimensional mesh structure, and GOx / HRP@ZIF-8 is embedded in the hydrogel pore wall.

[0100] 2. Activity determination of GOx / HRP@ZIF-8

[0101] Take the GOx / HRP@ZIF-8 of different mass ratios prepared in Preparation Examples 1-4, add 0.2mmol / L 2-azinobis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt solution and different concentrations (1, 2, 3, 4, 5mmol / L) of glucose solution; after mixing, measure the absorbance of the product after the dual enzyme cascade catalysis at 416nm. The results are as follows Figure 4 shown.

[0102] See also Figure 4 , when the mass ratio of GOx to HRP in GOx / HRP@ZIF-8 was 2:3, the activity of the dual-enzyme cascade reaction reached the optimal state.

[0103] 3. Determination of the immobilization rate of GOx / HRP@ZIF-8

[0104] Prepare different concentration gradients of bovine serum albumin (BSA) and prepare 0.1 mg / mL Coomassie brilliant blue solution;

[0105] Take 1 mL of BSA solution and 2 mL of Coomassie Brilliant Blue solution, mix them evenly, let them stand for 3 minutes, measure the maximum absorption peak of BSA at 585 nm using a UV-visible spectrophotometer, and make a concentration-absorbance BSA standard curve;

[0106] The combined supernatant in the preparation example was diluted to 500 mL;

[0107] 1 mL of the fixed solution was taken and mixed evenly with 2 mL of Coomassie Brilliant Blue solution and allowed to stand for 3 min. The absorbance at 585 nm was measured and substituted into the BSA standard curve, A=11.4775C+0.16103, and the GOx / HRP immobilization rate was calculated to be 69.77%.

[0108] 4. Compressive stress-strain curves of the hybrid hydrogel-immobilized dual-enzyme cascade reactor

[0109] The compressive stress-strain curve of GOx / HRP@ZIF-8@PDMAA was tested by an electronic universal testing machine. The cylindrical sample had a diameter of 10 mm, a height of 10 mm, a compression speed of 2 mm / min, and a maximum compressive strain of 70%. The results are shown in Figure 5 shown.

[0110] See also Figure 5 When the compression strain is 70%, the strength of GOx / HRP@ZIF-8@PDMAA hybrid hydrogel reaches 77KPa, and the hybrid hydrogel sample is still intact at this time, indicating that the hybrid hydrogel prepared by the present invention has good toughness, and the three-dimensional cross-linked network structure combines GOx / HRP@ZIF-8 and KH570-SiO 2 Nanoparticles provide a good mechanical basis for it.

[0111] 5. Concentration-absorbance working curve of phenolic compounds

[0112] PBS solutions of pH=6 with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L and 25 mg / L of 4-chlorophenol were prepared respectively, 3 parallel samples were set for each concentration, the total volume of each sample was 5 mL, and glucose was dissolved in each sample until the final molar concentration of glucose in the solution was 3 mmol / L;

[0113] Take 2.5mL of each sample solution and add 250μL of potassium ferrocyanide solution and 4-aminoantipyrine solution. Mix well and let stand for 3min. Measure the absorption spectrum of 400-700nm with UV-visible spectrophotometer. Take the absorbance value of the maximum absorption peak at 505nm. Draw the concentration-absorbance working curve with concentration as the horizontal axis and absorbance as the vertical axis. The results are shown in Figure 6 shown.

[0114] See also Figure 6 b. There is a linear relationship between the concentration of 4-chlorophenol and the corresponding absorbance value at a wavelength of 505 nm, and the working curve equation is A=0.0907C-0.0378. The concentration of 4-chlorophenol in the solution can be calculated from the absorbance value at 505 nm through the working curve.

[0115] In the same manner, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L and 25 mg / L PBS solutions of phenol or bisphenol A at pH 6, 10 mg / L, 20 mg / L, 25 mg / L, 30 mg / L and 50 mg / L PBS solutions of ferulic acid at pH 7, and 5 mg / L, 10 mg / L, 25 mg / L, 40 mg / L and 50 mg / L PBS solutions of caffeic acid at pH 7 were prepared, and the concentration-absorbance working curves of phenol, bisphenol A, ferulic acid and caffeic acid were obtained by the same test method. The results are shown in FIG. Figure 7 shown.

[0116] See also Figure 7 The working curve of phenol is A=0.1088C+0.1099, the working curve of bisphenol A is A=0.06138C+0.02951, the working curve of ferulic acid is A=0.03533C-0.1072, and the working curve of caffeic acid is A=0.01419C-0.01754.

[0117] 6. Degradation effect of 4-chlorophenol (4-CP) by hybrid hydrogel-immobilized dual enzyme cascade reactor

[0118] Prepare 4-chlorophenol PBS solutions of different concentrations, namely 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L, and set up 3 parallel samples for each concentration. Dissolve glucose in each sample until the molar concentration in the solution is 3 mmol / L, and the total volume of each sample is 5 mL.

[0119] Each sample was added with 0.1 g of GOx / HRP@ZIF-8@PDMAA hybrid hydrogel-immobilized dual enzyme cascade reactor and degraded at 25 °C for 300 min;

[0120] Take 2.5mL of degradation solution, add 250μL of potassium ferricyanide solution and 4-aminoantipyrine solution, mix well and let stand for 3min, measure the absorbance at 505nm until the absorbance remains stable, and calculate the effect of time on the degradation of 4-chlorophenol through the concentration-absorbance working curve. The results are as follows: Figure 8 shown.

[0121] See also Figure 8 The maximum degradation rate of 4-chlorophenol at four different concentrations was about 85%, and the time required to reach equilibrium was about 240 minutes.

[0122] 7. Effect of pH on the degradation efficiency of various phenolic compounds in a hybrid hydrogel-immobilized dual-enzyme cascade reactor

[0123] Prepare PBS solutions with a pH of 3-10 and a concentration of 20 mg / L 4-CP, phenol, bisphenol A, ferulic acid, and caffeic acid. Prepare 8 groups of each phenolic compound, and the total volume of each group is 5 mL. Dissolve glucose in the solution until the final molar concentration of glucose is 3 mmol / L.

[0124] 0.1 g of GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor prepared in Example 1 was added to each group and degraded at 25°C for 300 min; 2.5 mL of degradation solution was added to 250 μL of potassium ferrocyanide solution and 4-aminoantipyrine solution, mixed and allowed to stand for 3 min, and the absorbance at 505 nm was measured. The results are as follows: Figure 9-10 shown.

[0125] See also Fig. 9 At pH = 6, the biological activity of GOx / HRP reached its best. At this time, the GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor achieved the best effect in degrading phenol, bisphenol A and 4-chlorophenol, with the maximum degradation rates of 85%, 77% and 88%, respectively. When the pH of the system is too high, it will have an adverse effect on the degradation rate; therefore, the optimal pH for GOx / HRP@ZIF-8@PDMAA to degrade phenol, bisphenol A and 4-chlorophenol is 6.

[0126] See also Fig.10 When pH = 7, the GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor achieved the best effect in degrading ferulic acid and caffeic acid, with the maximum degradation rates being approximately 92% and 87%, respectively. Therefore, the optimal pH for GOx / HRP@ZIF-8@PDMAA to degrade ferulic acid and caffeic acid is 7.

[0127] 8. Degradation effect of hybrid hydrogel-immobilized dual enzyme cascade reactor on various phenolic compounds

[0128] Prepare PBS solutions of phenol, bisphenol A and 4-chlorophenol at pH 6 with a concentration of 20 mg / L, and PBS solutions of ferulic acid and caffeic acid at pH 7 with a concentration of 20 mg / L, and dissolve glucose in the solutions to a molar concentration of 3 mmol / L, respectively. The total volume of each sample solution is 5 mL;

[0129] 0.1 g GOx / HRP@ZIF-8@DMAA hybrid hydrogel immobilized dual enzyme cascade reactor was added and degraded at 25 °C for 300 min;

[0130] Take 2.5 mL of degradation solution, add 250 μL of potassium ferrocyanide solution and 4-aminoantipyrine solution, mix well and let stand for 3 minutes, measure the absorbance at 505 nm, and record it as A. t ,Depend on Figure 7 The residual concentration C of phenolic compounds at this time was calculated from the concentration-absorbance working curve of phenolic compounds shown in FIG. t ;

[0131] Degradation amount q t (unit: mg / g) is used to characterize the degradation performance of the hybrid hydrogel-immobilized dual enzyme cascade reactor on phenolic compounds. The formula is: q t =(C 0 -C t )*V / m;

[0132] Among them, C 0 represents the initial concentration of phenolic compounds, C t represents the residual concentration of phenolic compounds at time t, m is the dry weight of GOx / HRP@ZIF-8@DMAA hybrid hydrogel, and V is the volume of the entire system;

[0133] The measured data are shown in Table 1. Figure 11-12 shown.

[0134] Table 1 Degradation ability of GOx / HRP@ZIF-8@PDMAA on various phenolic compounds

[0135]

[0136] As shown in Table 1, the degradation capacity of the GOx / HRP@ZIF-8@PDMAA hybrid hydrogel immobilized dual enzyme cascade reactor for five phenolic compounds was higher than 0.81 mg / g, indicating that the hybrid hydrogel immobilized dual enzyme cascade reactor can efficiently degrade phenolic compounds.

[0137] See also Figure 11-12 Since the principle of phenol degradation by the hybrid hydrogel-immobilized dual-enzyme cascade reactor is to first adsorb the phenolic compounds and then achieve the purpose of degrading the phenolic compounds through the HRP / GOX cascade reaction embedded in the hydrogel, for the five phenolic compounds, the degradation rate is very high in the early stage. As time goes by, the rate gradually decreases. In the later stage, it mainly plays a stable degradation role and finally reaches the maximum degradation amount.

[0138] 9. Verification of the reuse effect of the hybrid hydrogel-immobilized dual-enzyme cascade reactor

[0139] In a 20 mg / L 4-chlorophenol PBS buffer, glucose was dissolved to a molar concentration of 3 mmol / L in the solution, and 0.1 g GOx / HRP@ZIF-8@PDMAA was added and degraded at room temperature until the degradation equilibrium time;

[0140] Take 2.5 mL of degradation solution, add 250 μL of potassium ferrocyanide solution and 4-aminoantipyrine solution, mix well and let stand for 3 min, and measure the absorbance at 505 nm;

[0141] After each absorbance measurement, the hybrid hydrogel was taken out and the surface water was dried with filter paper, then put into the same conditions for the degradation effect measurement, and the absorbance was measured again;

[0142] The absorbance of the first degradation was taken as the group with 100% activity to determine the reusability of the hybrid hydrogel-immobilized dual-enzyme cascade reactor. Fig.13 As shown, the measurement was repeated 5 times.

[0143] See also Fig.13 After each repeated use, the degradation rate of 4-chlorophenol gradually decreased. After 5 cycles, the relative activity of the cascade reactor decreased to 20%, indicating that the hybrid hydrogel-immobilized dual-enzyme cascade reactor has good reusability.

[0144] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A hybrid hydrogel immobilized dual enzyme cascade reactor, characterized in that: The hybrid hydrogel-immobilized dual-enzyme cascade reactor comprises a reaction core and a hybrid hydrogel layer on the surface of the reaction core; The reaction core includes an immobilized dual enzyme ZIF-8 complex; The immobilized dual-enzyme ZIF-8 complex comprises ZIF-8 and a combined enzyme encapsulated in ZIF-8.

2. The hybrid hydrogel immobilized dual enzyme cascade reactor according to claim 1, characterized in that: The combined enzyme includes any one of glucose oxidase / horseradish peroxidase and pyranose oxidase / horseradish peroxidase.

3. The hybrid hydrogel immobilized dual enzyme cascade reactor according to claim 1, characterized in that: The hybrid hydrogel layer includes a monomeric free radical polymer.

4. The hybrid hydrogel immobilized dual enzyme cascade reactor according to claim 3, characterized in that: The monomer includes a water-soluble monomer; the water-soluble monomer includes any one of N,N-dimethylacrylamide, polyethylene glycol methacrylate, polyethylene glycol acrylate, and hydroxyethyl acrylate.

5. A method for preparing a hybrid hydrogel immobilized dual enzyme cascade reactor as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: Adding the combined enzyme and 2-methylimidazole to a zinc nitrate solution, stirring for 2-4 hours, centrifuging, washing, and drying to obtain an immobilized dual-enzyme ZIF-8 complex; The molar ratio of zinc ion to 2-methylimidazole in the zinc nitrate is 1:4; the mass ratio of the two enzymes in the combined enzyme is (1-2): (1-3); In water, under the catalysis of the combined enzyme and the action of a crosslinking agent, the immobilized dual-enzyme ZIF-8 complex polymerizes with the monomer and substrate to obtain a hybrid hydrogel immobilized dual-enzyme cascade reactor; the substrate includes glucose and acetylacetone.

6. The preparation method according to claim 5, characterized in that: The cross-linking agent includes any one of modified silica nanoparticles and N,N-methylenebisacrylamide.

7. The preparation method according to claim 5, characterized in that: The mass ratio of the immobilized dual-enzyme ZIF-8 complex to the monomer in the monomer aqueous solution is 1:(10-100).

8. An application of the hybrid hydrogel immobilized dual enzyme cascade reactor according to any one of claims 1 to 4 in wastewater treatment, characterized in that: The application includes degrading phenolic compounds in the wastewater.

9. The use according to claim 8, characterized in that: The phenolic compound includes at least one of phenol, bisphenol A, 4-chlorophenol, ferulic acid and caffeic acid.

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