Core-shell material immobilized enzyme as well as preparation and application thereof

Abstract

The invention provides a core-shell material immobilized enzyme, a preparation method thereof and application of the core-shell material immobilized enzyme in catalyzing phenol hydroxylation to prepare benzenediol. The core-shell material immobilized enzyme comprises multifunctional magnetic nanoparticles modified by phase-change lysozyme which takes phase-change lysozyme as a shell and magnetic Fe3O4 nanoparticles as a core, and cross-linked aggregation enzyme connected to the multifunctional magnetic nanoparticles modified by the phase-change lysozyme, the cross-linked aggregation enzyme is formed by adding a cross-linking agent into horse radish peroxidase for cross-linking. The magnetic material is modified by the phase-change lysozyme, so that the surface of the magnetic material contains abundant functional groups and the surface of the material has pollution resistance, the loading capacity and the catalytic efficiency of enzyme are improved due to the formation of aggregating enzyme, and the stability of the enzyme is enhanced; the method for preparing benzenediol by catalyzing phenol hydroxylation through the core-shell material immobilized enzyme is a green synthesis process, and is environment-friendly and high in catalytic efficiency, the immobilized enzyme can be recycled, and the cost is saved.

Description

Technical Field

[0001] The invention relates to the field of catalyst materials, and in particular to a core-shell material immobilized enzyme and a preparation and application thereof. Background Art

[0002] Catechol (orthocatechol and hydroquinone) is an important intermediate in the pharmaceutical industry, used to prepare drugs such as berberine and isoproterenol. It is also an important basic organic chemical raw material, widely used in the production of dyes, light stabilizers, photosensitive materials, fragrances, preservatives, promoters, special inks, electroplating materials, and lacquer flame retardants. In addition, it is also a widely used astringent and antioxidant. In recent years, people's demand for catechol has continued to increase, and its application range has continued to expand.

[0003] At present, hydroquinone is mainly prepared by the bisphenol A method or the aniline oxidation method, and catechol is mainly prepared by hydrolysis of 2,4-disulfonic acid phenol or o-chlorophenol. The traditional synthesis route has many disadvantages, such as complex process, many by-products, difficult product separation, and severe corrosion of equipment. The research on the hydroxylation reaction of phenol has gradually developed in the direction of multiphase catalysts, but the catalytic performance is not easy to regenerate, the preparation steps are relatively cumbersome, the reaction conditions are relatively harsh, and it is difficult to control and easy to produce multiple by-products. Therefore, it is urgent to develop a green, efficient and simple process route for the synthesis of hydroquinone. In recent years, the method of producing hydroquinone by hydroxylation of phenol hydroperoxide under catalysis has the highlights of mild reaction conditions and simple process, and has gradually become a research hotspot.

[0004] Initially, Fenton's reagent was used as an efficient homogeneous catalyst in the hydroxylation of phenol, but the process had many by-products, low yield of hydroquinone and low selectivity. Later, the research on the hydroxylation of phenol gradually developed in the direction of heterogeneous catalysts. The catalysts currently studied mainly include composite metal oxides, heteropoly compounds and zeolite molecular sieve catalysts, among which iron-based catalysts show excellent catalytic performance in this catalytic reaction and are increasingly attracting the attention of researchers. However, its catalytic performance is not easy to regenerate, the preparation steps are relatively cumbersome, the reaction conditions are relatively harsh, and it is difficult to control and easily produce multiple by-products. Therefore, an environmentally friendly, simple and easy to prepare, and highly catalytically efficient immobilized enzyme is needed to catalyze the hydroxylation of phenol to prepare hydroquinone. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a core-shell material immobilized enzyme and its preparation and application, so as to solve the technical problems that the existing catalyst preparation steps are cumbersome, the reaction conditions are relatively harsh, and it is difficult to control and easily produce multiple by-products, and the catalytic performance is difficult to regenerate.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: According to one aspect of the present invention, a core-shell material immobilized enzyme is provided, comprising multifunctional magnetic nanoparticles modified with phase-change lysozyme and cross-linked aggregated enzymes connected to the multifunctional magnetic nanoparticles modified with phase-change lysozyme, wherein the multifunctional magnetic nanoparticles modified with phase-change lysozyme have phase-change lysozyme as a shell and magnetic Fe3O4 nanoparticles as a core, and the cross-linked aggregated enzymes are formed by cross-linking by adding a cross-linking agent to horseradish peroxidase.

[0007] In the above core-shell material immobilized enzyme, the cross-linking agent is 1,3,5-benzenetricarboxylic acid.

[0008] According to another aspect of the present invention, a method for preparing the above-mentioned core-shell material immobilized enzyme is provided, comprising the following steps: S1. preparing a lysozyme solution in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, and preparing a tri(2-carboxyethyl)phosphine solution in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, and mixing equal volumes of the two solutions to obtain a phase-change lysozyme solution; S2. adding Fe3O4 nanoparticles to the phase-change lysozyme solution, uniformly dispersing by ultrasonication, incubating, washing, and drying to obtain a multifunctional magnetic nanomaterial modified with phase-change lysozyme; S3. activating the carboxyl functional groups on the surface of the multifunctional magnetic nanomaterial modified with phase-change lysozyme to obtain an activated multifunctional magnetic nanomaterial modified with phase-change lysozyme; S4. precipitating horseradish peroxidase with a precipitant to obtain a precipitated enzyme, and adding a cross-linking agent to the precipitated enzyme to form a cross-linked aggregated enzyme; S5. adding the cross-linked aggregated enzyme to the activated phase-change lysozyme modified multifunctional magnetic nanomaterial to react to obtain a core-shell material immobilized enzyme.

[0009] In the preparation method of the core-shell material immobilized enzyme, in step S1, a lysozyme solution with a concentration of 1 mg / mL to 3 mg / mL is prepared in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution at pH=7.4, and a tris(2-carboxyethyl)phosphine solution with a concentration of 40 mM to 60 mM is prepared in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution at pH=5.

[0010] In the preparation method of the core-shell material immobilized enzyme, in step S2, the mixture is incubated in a constant temperature water bath at 37°C for 50-70 minutes. After the reaction is completed, the obtained material is washed alternately 5 to 7 times with ultrapure water and anhydrous ethanol under the action of a magnet, and then dried in a vacuum oven.

[0011] In the above-mentioned method for preparing the core-shell material immobilized enzyme, in steps S1 and S2, the mass ratio of tri(2-carboxyethyl)phosphine, lysozyme, and Fe3O4 nanoparticles is 1:15~20:15~20.

[0012] In the above-mentioned method for preparing the core-shell material immobilized enzyme, in step S3, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used to activate the carboxyl functional groups on the surface of the multifunctional magnetic nanomaterial modified with phase-change lysozyme, and the remaining 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are washed away with 2-morpholineethanesulfonic acid (MES) buffer at pH=6 to obtain an activated phase-change lysozyme modified multifunctional magnetic nanomaterial.

[0013] In the preparation method of the core-shell material immobilized enzyme, in step S4, the precipitant is saturated ammonium sulfate, and the cross-linking agent is 1,3,5-benzenetricarboxylic acid. Before adding the cross-linking agent to the precipitated enzyme, the carboxyl group on the cross-linking agent is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the activated cross-linking agent is added to the precipitated enzyme to react for 50-70 minutes to form a cross-linked aggregated enzyme.

[0014] In the above-mentioned method for preparing the core-shell material immobilized enzyme, in steps S4 and S5, the mass ratio of saturated ammonium sulfate, horseradish peroxidase (HRP), and activated phase-change lysozyme-modified multifunctional magnetic nanomaterial is 1:20-25:30-35.

[0015] According to yet another aspect of the present invention, there is provided use of the above core-shell material immobilized enzyme in catalyzing phenol hydroxylation to prepare hydroquinone.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The core-shell material immobilized enzyme is simple and easy to prepare, has mild reaction conditions and high efficiency, and can catalyze the hydroxylation of phenol to prepare diphenol without easily producing multiple by-products. In addition, the immobilized enzyme has stable performance and can be reused many times. It is also environmentally friendly, meets the requirements of modern green chemical industry, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0018] Figure 1 It is a schematic flow chart of the method for preparing the core-shell material immobilized enzyme of the present invention; Figure 2 This is a transmission electron microscope image of the core-shell material immobilized enzyme HRP@PTL@Fe3O4 DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0020] The core-shell material immobilized enzyme of the present invention comprises multifunctional magnetic nanoparticles modified by phase-change lysozyme and cross-linked aggregated enzymes connected to the multifunctional magnetic nanoparticles modified by phase-change lysozyme, wherein the multifunctional magnetic nanoparticles modified by phase-change lysozyme use phase-change lysozyme as a shell and magnetic Fe3O4 nanoparticles as a core, and the cross-linked aggregated enzymes are formed by adding a cross-linking agent (1,3,5-benzenetricarboxylic acid) to horseradish peroxidase for cross-linking. Due to the modification of the magnetic material by phase-change lysozyme, the surface contains rich functional groups and the surface of the material has anti-pollution property, and the formation of aggregated enzymes increases the enzyme loading and the enzyme catalytic efficiency, and enhances the enzyme stability.

[0021] like Figure 1 As shown, the method for preparing the core-shell material immobilized enzyme of the present invention comprises the following steps: Step 1. Preparation of phase-changing lysozyme (PTL)-modified multifunctional magnetic nanomaterials (PTL@Fe3O4 magnetic nanomaterials), including: S1. Prepare 1 mg / mL to 3 mg / mL lysozyme solution in 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution at pH = 7.4, prepare 40 mM to 60 mM tris(2-carboxyethyl)phosphine (TCEP) solution in HEPES buffer solution at pH = 5, take equal volumes of the two solutions and mix them to obtain a phase-change lysozyme solution; S2. Add Fe3O4 nanoparticles to the phase-change lysozyme solution to make it evenly dispersed by ultrasonication, and incubate it in a constant temperature water bath at 37°C for 50-70 minutes. After the reaction is completed, wash the obtained material alternately with ultrapure water and anhydrous ethanol for a total of 5 to 7 times under the action of a magnet, and dry it in a vacuum oven to obtain a multifunctional magnetic nanomaterial (PTL@Fe3O4 magnetic nanomaterial) modified with phase-change lysozyme (PTL).

[0022] In this step, the mass ratio of TCEP, lysozyme, and Fe3O4 nanoparticles is 1:15~20:15~20.

[0023] Step 2. Preparation of core-shell material immobilized enzyme HRP@PTL@Fe3O4, including: S3. Activate the carboxyl functional groups on the surface of the PTL@Fe3O4 magnetic nanomaterial prepared in step S1 using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and then wash away the remaining EDC and NHS with a pH=6 MES (2-morpholineethanesulfonic acid) buffer to obtain an activated phase-change lysozyme (PTL)-modified multifunctional magnetic nanomaterial for later use. Preferably, the concentration of EDC and NHS is 0.1M; S4. horseradish peroxidase (HRP) is precipitated with saturated ammonium sulfate as a precipitant to obtain a precipitated enzyme, wherein the volume ratio of horseradish peroxidase to the precipitant is 1:9, during which EDC and NHS are used to activate the carboxyl group on the cross-linking agent 1,3,5-benzenetricarboxylic acid, and the activated cross-linking agent is added to the precipitated enzyme for reaction for 50-70 minutes to form a cluster between the enzyme molecules to obtain a cross-linked aggregated enzyme (i.e., a clustered enzyme), preferably, the concentration of EDC and NHS is 0.1 M; S5. Add the cross-linked aggregated enzyme to the activated phase-change lysozyme (PTL) modified multifunctional magnetic nanomaterials and react at room temperature for 4-5 hours to obtain the core-shell material immobilized enzyme HRP@PTL@Fe3O4. The mass ratio of saturated ammonium sulfate, horseradish peroxidase (HRP), and activated phase-change lysozyme (PTL) modified multifunctional magnetic nanomaterials is 1:20-25:30-35.

[0024] The core-shell material immobilized enzyme of the present invention is used to catalyze the hydroxylation of phenol to prepare hydroquinone, comprising the following steps: adding the core-shell material immobilized enzyme HRP@PTL@Fe3O4 and 30% H2O2 to a phenol solution (preferably 9mM), the solvent used for the phenol solution is preferably methanol, and incubating at 37°C for 30-50min after oscillation and mixing; then separating the reaction solution with a magnet, adding a sodium sulfite solution to the supernatant to remove the remaining hydrogen peroxide, obtaining a hydroquinone product, analyzing its composition by gas chromatography, and calculating the phenol conversion rate by an area normalization method. Preferably, the final concentration ratio of HRP@PTL@Fe3O4, 30% H2O2, and phenol is 1:2:1, the concentration of the sodium sulfite solution is preferably 1mol / L, and the dosage is 3-5mL. Among them, the solid core-shell material immobilized enzyme HRP@PTL@Fe3O4 obtained by magnetic separation is rinsed and can be reused.

[0025] The core-shell material immobilized enzyme of the present invention has a "core@shell" structure, with phase-change lysozyme as the shell and magnetic nanoparticles as the core (the electron microscope image of which is shown in FIG. Figure 2As shown), horseradish peroxidase is then connected to the multifunctional magnetic nanoparticles modified with phase-change lysozyme by 1,3,5-benzenetricarboxylic acid to form a cross-linked aggregate enzyme to obtain the immobilized enzyme HRP@PTL@Fe3O4. The core-shell material immobilized enzyme is simple and easy to prepare, with mild reaction conditions and high efficiency. It catalyzes the hydroxylation of phenol to prepare diphenol, and is not easy to produce multiple by-products. The immobilized enzyme has stable performance and can be reused many times. It has high catalytic efficiency, and the immobilized enzyme can be recycled and reused, which saves costs. It is a green synthesis process, and it is also environmentally friendly, in line with the requirements of modern green chemical industry, and has good application prospects.

[0026] The present invention will be further described below with reference to the embodiments, but is not limited thereto.

[0027] Example 1 Step 1: Prepare 2 mg / mL lysozyme in 10 mM HEPES buffer solution at pH = 7.4, prepare 50 mM tris (2-carboxyethyl) phosphine (TCEP) in 10 mM HEPES buffer solution at pH = 5, take equal volumes of the two solutions and mix them to 20 mL, add 20 mg of Fe3O4 nanoparticles, make them evenly dispersed by ultrasonication, incubate them in a constant temperature water bath at 37 ° C for 1 hour, after the reaction is completed, wash the obtained material with ultrapure water and anhydrous ethanol alternately for a total of six times under the action of a magnet, dry it in a vacuum oven, and obtain PTL@Fe3O4 magnetic nanomaterials for use. Prepare 6 mg of the above-prepared PTL@Fe3O4 magnetic nanomaterials, activate the carboxyl functional groups on its surface with 0.1 M EDC and NHS, and then wash away the remaining EDC and NHS with MES buffer at pH = 6 to obtain the activated phase-change lysozyme (PTL) modified multifunctional magnetic nanomaterials. HRP was precipitated using saturated ammonium sulfate as a precipitant, with the volume ratio of enzyme to precipitant being 1:9. During this period, the carboxyl groups on the cross-linker 1,3,5-benzenetricarboxylic acid were activated with 0.1 M EDC and NHS. The activated cross-linker was added to the precipitated enzyme for reaction for 1 hour to form a cluster between the enzyme molecules. Finally, the clustered enzyme was added to the prepared activated magnetic nanomaterial for reaction for 5 hours to obtain the core-shell material immobilized enzyme HRP@PTL@Fe3O4.

[0028] Step 2: Add 0.1 mL of the HRP@PTL@Fe3O4 prepared above and 0.2 mL of 30% H2O2 to 9 mM and 100 mL of phenol-methanol solution, shake and mix, and incubate at 37°C for 50 min. Then separate the reaction solution with a magnet, add 3 mL of 1 M sodium sulfite solution to the supernatant to remove the remaining H2O2, and obtain the hydroquinone product. The composition is analyzed by gas chromatography, and the phenol conversion rate is calculated to be 72% by the area normalization method.

[0029] Embodiment 2, 3 The method was the same as that in Example 1, except that the incubation time in step 2 was changed to 30 min and 70 min, to obtain a hydroquinone product, analyze its composition by gas chromatography, and calculate the phenol conversion rate by the area normalization method.

[0030] Table 1 Effect of incubation time on product yield Example 4 The core-shell material immobilized enzyme HRP@PTL@Fe3O4 obtained by magnetic separation after the reaction in Example 1 was used, and after being washed alternately with ultrapure water and anhydrous ethanol for a total of 5 to 7 times, it was used again to catalyze the hydroxylation of phenol to prepare hydroquinone, and this was repeated 7 times. The reaction results are shown in Table 2, indicating that the immobilized enzyme of the present invention has stable performance and can be reused many times.

[0031] Table 2 Effect of the number of times the core-shell material immobilized enzyme HRP@PTL@Fe3O4 was used on the catalytic reaction of phenol hydroxylation to prepare diphenol The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify or improve the technical solutions recorded in the above embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein by equivalents; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A core-shell material immobilized enzyme, characterized in that: The invention comprises multifunctional magnetic nanoparticles modified with phase-change lysozyme and cross-linked aggregation enzymes connected to the multifunctional magnetic nanoparticles modified with phase-change lysozyme, wherein the multifunctional magnetic nanoparticles modified with phase-change lysozyme use phase-change lysozyme as a shell and magnetic Fe3O4 nanoparticles as a core, and the cross-linked aggregation enzymes are formed by adding a cross-linking agent to horseradish peroxidase for cross-linking.

2. The core-shell material immobilized enzyme according to claim 1, characterized in that: The cross-linking agent is 1,3,5-benzenetricarboxylic acid.

3. The method for preparing the core-shell material immobilized enzyme according to claim 1 or 2, characterized in that: The following steps are involved: S1. preparing a lysozyme solution in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, and preparing a tri(2-carboxyethyl)phosphine solution in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, mixing equal volumes of the two solutions to obtain a phase-change lysozyme solution; S2. Fe3O4 nanoparticles were added to the phase-change lysozyme solution, ultrasonically dispersed, incubated, washed, and dried to obtain a multifunctional magnetic nanomaterial modified with phase-change lysozyme; S3. activating the carboxyl functional groups on the surface of the phase-change lysozyme-modified multifunctional magnetic nanomaterial to obtain an activated phase-change lysozyme-modified multifunctional magnetic nanomaterial; S4. using a precipitant to precipitate horseradish peroxidase to obtain a precipitated enzyme, and adding a cross-linking agent to the precipitated enzyme to form a cross-linked aggregated enzyme; S5. Adding the cross-linked aggregation enzyme to the activated phase-change lysozyme-modified multifunctional magnetic nanomaterial to react, thereby obtaining a core-shell material-immobilized enzyme.

4. The method for preparing a core-shell material immobilized enzyme according to claim 3, characterized in that: In step S1, a lysozyme solution with a concentration of 1 mg / mL to 3 mg / mL is prepared in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution at pH=7.4, and a tris(2-carboxyethyl)phosphine solution with a concentration of 40 mM to 60 mM is prepared in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution at pH=5.

5. The method for preparing a core-shell material immobilized enzyme according to claim 3, wherein in step S2, the mixture is incubated in a constant temperature water bath at 37°C for 50-70 minutes. After the reaction is completed, the obtained material is washed alternately 5-7 times with ultrapure water and anhydrous ethanol under the action of a magnet, and then dried in a vacuum oven.

6. The method for preparing a core-shell material immobilized enzyme according to claim 3, wherein in steps S1 and S2, the mass ratio of tri(2-carboxyethyl)phosphine, lysozyme, and Fe3O4 nanoparticles is 1:15-20:15-20.

7. The method for preparing a core-shell material immobilized enzyme according to claim 3, wherein in step S3, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used to activate the carboxyl functional groups on the surface of the multifunctional magnetic nanomaterial modified with the phase-change lysozyme, and the remaining 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are washed away with a 2-morpholineethanesulfonic acid (MES) buffer at pH=6 to obtain the activated phase-change lysozyme modified multifunctional magnetic nanomaterial.

8. The method for preparing a core-shell material immobilized enzyme according to claim 3, wherein in step S4, the precipitant is saturated ammonium sulfate, and the cross-linking agent is 1,3,5-benzenetricarboxylic acid. Before adding the cross-linking agent to the precipitated enzyme, the carboxyl group on the cross-linking agent is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the activated cross-linking agent is added to the precipitated enzyme to react for 50-70 minutes to form the cross-linked aggregated enzyme.

9. The method for preparing a core-shell material immobilized enzyme according to claim 3, wherein in steps S4 and S5, the mass ratio of saturated ammonium sulfate, horseradish peroxidase, and activated phase-change lysozyme-modified multifunctional magnetic nanomaterial is 1:20-25:30-35.

10. Use of the core-shell material immobilized enzyme according to claim 1 or 2 in catalyzing the hydroxylation of phenol to prepare diphenol.

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