CN113073095B - Magnetic immobilized laccase with high enzyme activity, preparation method thereof and method for efficiently degrading dye - Google Patents

Magnetic immobilized laccase with high enzyme activity, preparation method thereof and method for efficiently degrading dye Download PDF

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CN113073095B
CN113073095B CN202110535494.3A CN202110535494A CN113073095B CN 113073095 B CN113073095 B CN 113073095B CN 202110535494 A CN202110535494 A CN 202110535494A CN 113073095 B CN113073095 B CN 113073095B
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laccase
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CN113073095A (en
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陈志明
朱庆鹏
汪春梅
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Anhui Polytechnic University
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    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
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    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
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Abstract

The invention provides a magnetic immobilized laccase with high enzyme activity, a preparation method thereof and a method for efficiently degrading dye, wherein a solvothermal method is adopted to synthesize magnetic ZnFe 2 O 4 And (3) the nano particles are functionalized by amino functional groups, and finally, the magnetic immobilized laccase with high activity, high stability and high recycling property is prepared by crosslinking by using a bifunctional reagent glutaraldehyde. The magnetic immobilized laccase prepared by the invention has the advantages of simple operation, economy, environmental protection, high atom utilization rate, green and no pollution; the carrier structure is designed, and the prepared magnetic ZnFe 2 O 4 The immobilized enzyme and the substrate show ultrahigh substrate affinity, so that the enzyme activity of the immobilized laccase is greatly improved; the magnetic immobilized laccase has continuous and stable catalytic capability, simple and convenient reusability and green sustainability, greatly reduces the production cost, and has wide application prospect in the fields of catalytic oxidation, water body treatment, environmental remediation and the like.

Description

Magnetic immobilized laccase with high enzyme activity, preparation method thereof and method for efficiently degrading dye
Technical Field
The invention belongs to the field of immobilized laccase, and particularly relates to a magnetic immobilized laccase with high enzyme activity, a preparation method thereof and a method for efficiently degrading dye.
Background
Laccase is a multi-copper oxidase, has a wider substrate range, can realize the catalytic oxidation of polyphenols, arylamines, thiols and partial natural macromolecules, has green reaction, has only water as a byproduct, and accords with the modern green chemistry concept. The free laccase has high efficiency and can rapidly complete the catalytic reaction, but the free laccase is difficult to separate from the water body for recycling, so that the cost is greatly increased. To solve the limitations of free laccase, one has physically or chemically linked the free enzyme to water-insoluble materials to prepare immobilized laccase. However, the immobilization of free enzymes on a carrier loses a great deal of enzyme activity, and how to prepare immobilized laccase with high enzyme activity is the important content of laccase research.
The magnetic nano particles are materials with good magnetic response capability, the enrichment and separation of products can be easily realized through an externally applied magnetic field, and the magnetic nano particles have good biocompatibility and wide application prospect in the aspect of immobilized laccase. Various magnetically immobilized enzymes such as Fe have been reported so far 3 O 4 Graphene, fe 3 O 4 @MOF、Fe 3 O 4 @SiO 2 (International Journal of Biological Macromolecules 138,1-12 (2019); bioresource Technology 317 (2020); chin.J.chem.30,2849-2860 (2012)), but laccase loses a large amount of enzyme activity during the immobilization process in the literature and cannot be popularized in large scale in industrial production.
Disclosure of Invention
The invention aims to provide the magnetic immobilized laccase with high enzyme activity and the preparation method thereof, and the synthesis method is simple, has mild conditions, and the prepared magnetic immobilized laccase has high enzyme activity, high recovery rate and good stability and can effectively play a role in an acid environment.
The invention also aims to provide a method for efficiently degrading the dye, which utilizes the magnetic immobilized laccase treatment with high enzyme activity.
The specific technical scheme of the invention is as follows:
a preparation method of a magnetic immobilized laccase with high enzyme activity comprises the following steps:
1) Preparation of aminated magnetic ZnFe 2 O 4 A nanoparticle;
2) Preparation of double-functional magnetic ZnFe 2 O 4 A nanoparticle;
3) Using dual-functional magnetic ZnFe 2 O 4 The nanoparticles immobilize laccase.
The preparation method of the step 1) comprises the following steps:
1-1) adding polyalcohol and polyethylene glycol into an iron source, a zinc source and sodium acetate, reacting at high temperature, washing, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) the magnetic ZnFe obtained in step 1-1) 2 O 4 Dispersing the nano particles in a solvent, adding a silane coupling agent, reacting at high temperature, washing, and separating to obtain the amino magnetic ZnFe 2 O 4 A nanoparticle;
further, in the step 1-1), the molar ratio of the iron source to the zinc source to the sodium acetate is 1: (0.5-1): (6-12);
the iron source in the step 1-1) is ferric chloride or ferric sulfate; the zinc source is zinc chloride, zinc nitrate or zinc sulfate; the polyol is ethylene glycol; the polyethylene glycol is PEG-200;
the concentration of the iron source in the step 1-1) is 0.03-0.1mol/L;
the volume ratio of the ethylene glycol to the polyethylene glycol is 40:1-2;
the high temperature reaction in the step 1-1) means that the reaction is carried out for 6-24 hours at 160-280 ℃;
further, in the step 1-2), the solvent is distilled water or deionized water;
the silane coupling agent in the step 1-2) is a silane coupling agent containing amino functional groups; preferably 3-aminopropyl triethoxysilane;
the magnetic ZnFe of step 1-2) 2 O 4 The mass ratio of the nano particles to the silane coupling agent is 1: (3-13).
The reaction at the high temperature in the step 1-2) means that the reaction is carried out for 20-28h at the temperature of 110-150 ℃;
the volume ratio of the solvent to the silane coupling agent in the step 1-2) is 20:1-2;
the step 2) is specifically as follows:
magnetic ZnFe to be aminated 2 O 4 Dispersing nano particles in a bifunctional reagent solution, culturing, washing and separating to obtain the bifunctional magnetic ZnFe 2 O 4 A nanoparticle;
the bifunctional reagent solution in the step 2) refers to an aqueous solution of which the solvent is a bifunctional reagent; the concentration is 2-3wt%;
bifunctional reagent in bifunctional reagent solution in step 2) and preparation of aminated magnetic ZnFe 2 O 4 Magnetic ZnFe for nanoparticles 2 O 4 The mass ratio of the nano particles is 1-10;
the difunctional reagent in the step 2) is a dialdehyde difunctional reagent; preferably glutaraldehyde;
the culture method in the step 2) comprises the following steps: stirring and culturing at 25-45deg.C for 2-7 hr;
the step 3) is specifically as follows: will double functional magnetic ZnFe 2 O 4 Dispersing the nano particles in a solvent, adding laccase solution, culturing, washing and separating to obtain magnetic ZnFe 2 O 4 The enzyme is immobilized.
Further, in the step 3), the solvent is distilled water or deionized water;
the laccase activity in the laccase solution in the step 3) is 8.8U/mL; the concentration of laccase solution is 0.07mg/ml;
the culture method in the step 3) comprises the following steps: stirring and culturing at 25-45deg.C for 2-6 hr;
laccase contained in laccase solution in step 3) and preparation of bifunctional magnetic ZnFe 2 O 4 Magnetic ZnFe for nanoparticles 2 O 4 The mass ratio of the nano particles is (0.002-0.012): 1.
The magnetic immobilized laccase with high enzyme activity is prepared by the method.
The invention provides a method for efficiently degrading dye, which comprises the following steps:
s1, dispersing magnetic immobilized laccase with high enzyme activity in a dye;
s2, adding ABTS into the reaction system, and carrying out oscillation reaction.
Further, the dye in the step S1 is triphenylmethane dye, azo dye or anthraquinone dye; the dye concentration is 5 mg/L-200 mg/L;
step S2, oscillating reaction at 600-900 rpm;
preferably, the triphenylmethane dye in the step S1 is bright green; the azo dye is reactive red; anthraquinone dye is brilliant blue;
the dye concentration in step S1 is as follows: 40mg/L bright green, 20mg/L active red and 100mg/L bright blue;
dual-functional magnetic ZnFe 2 O 4 The mass and dye volume ratio of the nano particle immobilized laccase is 1: (0.38-0.42) mg/mL.
Further, the ABTS in the step S2 is 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, and the concentration in the system is 0.025-0.05mM;
the oscillating reaction temperature in the step S2 is 30-70 ℃, preferably 50 ℃; the reaction time is 15min-90min.
More preferably, the magnetic immobilized laccase with high enzyme activity is used for efficiently degrading three dyes, and the specific process is as follows:
taking 10mg of magnetic immobilized laccase with high enzyme activity, adding 3.9mL of triphenylmethane dye solution with pH of 3.0, adding 100 mu L of 1mmol/L ABTS solution, placing in a 50 ℃ mixing instrument, uniformly mixing for reaction, and performing magnetic separation after reacting for 60 min.
Magnetic ZnFe 2 O 4 The immobilized enzyme can degrade azo dye effectively, and the specific process is as follows:
taking 10mg of magnetic immobilized laccase with high enzyme activity, adding 3.9mL of azo dye solution with pH of 3.0, adding 100 mu L of 1mmol/L ABTS solution, placing in a 50 ℃ mixing instrument for mixing and reacting uniformly, and performing magnetic separation after reacting for 15 min.
Magnetic ZnFe 2 O 4 The immobilized enzyme can degrade anthraquinone dye effectively, and the specific process is as follows:
taking 10mg of high-enzyme activity magnetic immobilized laccase, adding 3.9mL of anthraquinone dye solution with pH value of 3.0, adding 100 mu L of 1mmol/L ABTS solution, placing in a 50 ℃ mixing instrument, uniformly mixing for reaction, and performing magnetic separation after reacting for 60 min.
The technology not mentioned in the present invention refers to the prior art.
The invention firstly uses 3-aminopropyl triethoxy silane to make magnetic ZnFe 2 O 4 Nanoparticle functionalized modified amino (-NH) 2 ) Covalently binding magnetic ZnFe with glutaraldehyde as bifunctional reagent 2 O 4 Nanoparticle, finally covalently immobilizing laccase.
The invention adopts a solvothermal method to prepare the magnetic ZnFe in one step 2 O 4 Nanoparticles, then passAmino functional groups are modified on the surfaces of the magnetic nano particles in a heating mode to obtain the aminated magnetic ZnFe 2 O 4 And (3) nanoparticles. The aminated magnetic ZnFe prepared by the invention 2 O 4 The nano particles are hollow structures and have the particle size of about 100nm-200nm. The dosage of the silane coupling agent influences the activity of the follow-up immobilized enzyme, the excessive silane coupling agent leads to the steric hindrance of covalent bonding reaction, the laccase solid-borne quantity is reduced, and the activity is reduced.
The invention adopts a covalent fixing mode to prepare the magnetic ZnFe 2 O 4 Immobilized enzyme: first, aminated magnetic ZnFe 2 O 4 The nanoparticles are crosslinked with a bifunctional reagent and then immobilized with laccase after washing. The dosage and the concentration of the difunctional reagent play a decisive role in the enzyme activity of the immobilized enzyme, and glutaraldehyde is used as the difunctional reagent, so that the glutaraldehyde has a sterilization effect and the concentration of glutaraldehyde needs to be controlled; the magnetic ZnFe of the invention 2 O 4 Compared with the traditional magnetic Fe, the nanoparticle immobilized enzyme 3 O 4 The activity of the immobilized enzyme of the nano particles is improved by 15 to 25 percent.
Compared with the prior art, the invention has the following beneficial effects:
1. preparation of amino-modified magnetic ZnFe 2 O 4 The nanoparticle method is simple, safe and low in toxicity, and the raw material utilization rate is high;
2. magnetic ZnFe 2 O 4 The surface of the nanoparticle is rich in amino functional groups, can react with aldehyde groups to generate Schiff base, and can be directly used for covalently fixing laccase;
3. magnetic ZnFe with surface rich in hydroxyl groups prepared by solvothermal method 2 O 4 The hydrophilicity of the nano particles is enhanced, and the dispersity of the magnetic nano particles is improved.
4. Adopting water phase to connect silane coupling agent to make the silane coupling agent in magnetic ZnFe 2 O 4 The surface of the nanoparticle is coated in multiple layers, so that amino functional groups are enriched, and laccase immobilization capacity is increased.
5. Magnetic ZnFe 2 O 4 The nano particles are used for immobilizing laccase in a mild process, at normal temperature and normal pressure, so that the activity retention of enzyme protein is facilitated, and the immobilization process is carried outThe recovery rate of the middle enzyme activity can reach 80.1% -121.3%;
6. amino-modified magnetic ZnFe 2 O 4 The nano particles are used for covalently fixing laccase through a bifunctional reagent, the immobilization amount is 2-5mg/g, and the high enzyme activity expression of low-load enzymes is realized;
7. through laccase enzyme activity research and carrier structure design, the magnetic ZnFe is made 2 O 4 The immobilized enzyme has excellent affinity with the substrate, so that the immobilized enzyme has higher enzyme activity;
8. magnetic ZnFe 2 O 4 The immobilized enzyme is immobilized by covalent immobilization, so that the laccase and the magnetic carrier are in strong interaction of chemical bond linkage, and the immobilized enzyme has good stability, repeatability and environmental tolerance;
9. the magnetic ZnFe prepared by the invention 2 O 4 The immobilized enzyme improves the heat resistance of laccase to 122%, improves the acid resistance to 129%, improves the organic matter resistance to 164%, improves the metal ion resistance to 188%, improves the protein inhibitor resistance by more than 50 times, and is higher than the immobilized laccase prepared by the prior art.
10. Compared with the prior art, the magnetic ZnFe prepared by the invention 2 O 4 Immobilized enzyme degradation dye efficiency is increased to 550% and is achieved with less enzyme usage.
11. Magnetic ZnFe 2 O 4 The immobilized enzyme has good magnetic property, can be rapidly enriched and separated from a reaction system under an external magnetic field, reduces the production cost, and has wide application prospect in the aspects of environmental management, catalysis and the like.
Drawings
FIG. 1 shows the aminated magnetic ZnFe prepared in example 1 2 O 4 A nanoparticle transmission electron microscope detection diagram;
FIG. 2 is an amino magnetic ZnFe prepared in example 1 2 O 4 A nanoparticle X-ray powder diffraction pattern; in fig. 2, the abscissa is the diffraction angle 2θ (°), and the ordinate Intensity (a.u.); the diffraction peaks of the product are 18.3 degrees, 30.1 degrees, 35.5 degrees, 43.1 degrees, 53.6 degrees, 57.0 degrees, 62.7 degrees, and the diffraction peaks are compared with the standard spectrum,71.0 °, 74.2 °, 79.0 °, respectively corresponding to diffraction peaks (111), (220), (311), (400), (422), (511), (440), (620), (533), (444) of cubic phase zinc iron tetraoxide;
FIG. 3 shows the magnetic ZnFe powder prepared in example 1 2 O 4 A comparison graph of the thermostability of immobilized enzyme and free enzyme; in fig. 3, the abscissa represents Time (h), and the ordinate represents relative activity Relative activity (%); both immobilized enzyme and free enzyme are kept at 60℃for the same time (1-10 h);
FIG. 4 shows the magnetic ZnFe powder prepared in example 1 2 O 4 A fixed enzyme recycling effect diagram; in FIG. 4, the abscissa indicates the Number of cycles, and the ordinate indicates the relative activity Relative activity (%);
FIG. 5 shows the magnetic ZnFe powder prepared in example 1 2 O 4 The effect diagram of the immobilized enzyme to repeatedly degrade bright green, active red and bright blue dyes; in FIG. 5, the abscissa indicates the Number of cyclic Degradation times of cycles, and the ordinate indicates the Degradation rate Degradation (%);
FIG. 6 is an aminated magnetic ZnFe prepared in example 2 2 O 4 A nanoparticle transmission electron microscope detection diagram;
FIG. 7 is an amino magnetic ZnFe prepared in example 3 2 O 4 A nanoparticle transmission electron microscope detection diagram;
FIG. 8 is an amino magnetic ZnFe prepared in example 4 2 O 4 Nanoparticle transmission electron microscopy images.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1
A preparation method of a magnetic immobilized laccase with high enzyme activity comprises the following steps:
1) Preparation of aminated magnetic ZnFe 2 O 4 The nanoparticle operation process is as follows:
1-1) weighing 0.9g FeCl 3 ·6H 2 O，3.6g NaAc·3H 2 O，0.49g Zn(NO 3 ) 2 Placing the mixture into a polytetrafluoroethylene lining, adding 40mL of ethylene glycol and 1mL of PEG-200, uniformly stirring to obtain yellow emulsion, reacting the reaction kettle at 200 ℃ for 8 hours, and cooling to room temperature; washing the product with ethanol for 1 time, washing with distilled water for 2 times, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) magnetic ZnFe prepared by 1-1) 2 O 4 Dispersing the nano particles in 60mL of distilled water, and adding 3mL of 3-aminopropyl triethoxysilane; introducing shielding gas argon, heating to 120 ℃ and refluxing for 24 hours, washing with distilled water for 1 time, and separating to obtain the aminated magnetic ZnFe 2 O 4 And (3) nanoparticles. As can be seen from FIG. 1, the aminated magnetic ZnFe prepared in example 1 2 O 4 The particle size of the nano particles is about 120-150nm;
2) Aminated magnetic ZnFe 2 O 4 Preparation of difunctional modified magnetic ZnFe from nanoparticles 2 O 4 The operation process of the nanoparticle immobilized laccase is as follows:
2-1) the aminated magnetic ZnFe prepared in step 1-2) 2 O 4 The nanoparticles were dispersed into 50ml of 2% glutaraldehyde aqueous solution (w/w); stirring the mixture in a water bath at the temperature of 32 ℃ for backlight reaction for 4 hours, washing the mixture for 1 time by distilled water after the completion of the backlight reaction, and separating the mixture to obtain the magnetic ZnFe modified by the bifunctional reagent 2 O 4 A nanoparticle;
2-2) the difunctional modified magnetic ZnFe to be produced 2 O 4 Dispersing the nanoparticles into 50mL distilled water17mL of laccase solution is added, the laccase activity in the laccase solution is 8.8U/mL, and the concentration of the laccase solution is 0.07mg/mL; stirring the mixture in a water bath at the temperature of 32 ℃ for backlight reaction for 2 hours, washing the mixture once by distilled water, and separating the mixture to obtain the magnetic ZnFe 2 O 4 The nanoparticle is immobilized with an enzyme.
Determination of magnetic ZnFe 2 O 4 The operation steps of the immobilized enzyme catalytic activity are as follows:
1) 1mmol of guaiacol is weighed and dissolved in 1L of distilled water, 1mmol/L of guaiacol aqueous solution is prepared, and the guaiacol aqueous solution is placed in a refrigerator at 4 ℃ for standby;
2) Determination of magnetic ZnFe by the Bradford method 2 O 4 The amount of laccase immobilized by nano particles is measured by using an oxidized guaiacol method to measure magnetic ZnFe 2 O 4 Catalytic activity of nanoparticle immobilized laccase. Respectively placing the magnetic immobilized laccase (or the free enzyme) at 60 ℃ for preserving for the same time, then measuring the enzyme activity by using an oxidation guaiacol method, and researching the thermal stability of the immobilized laccase (or the free enzyme); the immobilized laccase is recovered from the reaction liquid, and the enzyme activity is measured by using an oxidized guaiacol method, so that the recycling condition of the magnetic immobilized enzyme is studied.
Determination of magnetic ZnFe 2 O 4 The operation steps of the immobilized enzyme for efficiently degrading the dye are as follows:
1) 10mg of magnetic ZnFe is taken 2 O 4 Immobilized enzyme, adding 3.9mL of different types of dyes (the dye concentration is respectively 40mg/L bright green, 20mg/L active red and 100mg/L bright blue degradation rate), adding 0.1mL of 1 mmol/LABSS solution, and carrying out oscillation reaction under the condition of 600rpm, testing the absorbance of each dye at a specific wavelength in different time intervals by an ultraviolet spectrophotometer, and researching the dynamics of degrading the dye by the immobilized enzyme;
2) 10mg of magnetic immobilized laccase with high enzyme activity is taken, 3.9mL of bright green solution of triphenylmethane dye with pH of 3.0 is added, 100 mu L of 1mmol/L ABTS solution is added, and the mixture is placed in a 50 ℃ mixing instrument for mixing reaction, and the mixture is subjected to oscillation reaction for 60min under the condition of 600rpm and then subjected to magnetic separation.
Magnetic ZnFe 2 O 4 The immobilized enzyme can degrade azo dye effectively, and the specific process is as follows:
taking 10mg of high-enzyme activity magnetic immobilized laccase, adding 3.9mL of azo dye active red solution with pH of 3.0, adding 100 mu L of 1mmol/L ABTS solution, placing in a 50 ℃ mixing instrument for mixing and reacting uniformly, oscillating at 600rpm for 15min, and performing magnetic separation.
Magnetic ZnFe 2 O 4 The immobilized enzyme can degrade anthraquinone dye effectively, and the specific process is as follows:
taking 10mg of high-enzyme activity magnetic immobilized laccase, adding 3.9mL of pH 3.0 anthraquinone dye brilliant blue solution, adding 100 mu L of 1mmol/L ABTS solution, placing in a 50 ℃ mixing instrument for mixing and reacting uniformly, oscillating at 600rpm for 60min, and performing magnetic separation.
After the degradation reaction, each time the immobilized laccase is recovered from the reaction liquid, the absorbance of each dye at a specific wavelength is tested by an ultraviolet spectrophotometer, and the repeatability of the degradation dye of the immobilized enzyme is studied. The results are shown in FIG. 5.
Example 1 detection results: detecting the in-magnetic ZnFe 2 O 4 The immobilization amount of the nano particles is 3.22mg/g; the magnetic ZnFe 2 O 4 The recovery rate of the enzyme activity of the immobilized enzyme is 94.9%; the magnetic ZnFe 2 O 4 The immobilized enzyme has a Miq constant of 0.54mM, approaching that of the free enzyme; the magnetic ZnFe 2 O 4 The enzyme activity of the immobilized enzyme is 224.4U/g; the magnetic ZnFe 2 O 4 After the immobilized enzyme is placed for 10 hours at 60 ℃, the enzyme activities of the immobilized enzyme can still be respectively maintained at 76.0 percent, compared with the free enzyme which is raised to 122 percent (as shown in figure 3); the magnetic ZnFe 2 O 4 After the immobilized enzyme is mixed with different pH buffers and placed for 2 hours at the temperature of 30 ℃, the enzyme activity of the immobilized enzyme in the pH 2.3 buffer can still be kept 93.3 percent, compared with the enzyme activity of the immobilized enzyme which is raised to 129 percent; the immobilized enzyme moves to the acid direction relative to the optimal pH of the free enzyme, which indicates that the immobilized enzyme can play an effective role in a more acid environment; the magnetic ZnFe 2 O 4 After the immobilized enzyme and different organic matters 1:1 (w/w) are mixed and placed for 2 hours at 30 ℃, 99.6% of relative activity of the immobilized enzyme in acetone is reserved, and compared with the free enzyme, the immobilized enzyme is raised to 164%; the magnetic ZnFe 2 O 4 Mixing immobilized enzyme and metal ion at 30deg.C for 2 hr, and immobilizing enzyme in Fe 3+ 83.7% relative activity retention in solution, up to 188% compared to free enzyme; the magnetic ZnFe 2 O 4 After the immobilized enzyme and the protein inhibitor are mixed and placed for 2 hours at 30 ℃, 77.2% of relative activity of the immobilized enzyme in ethylenediamine is still reserved, and the immobilized enzyme is improved by more than 50 times compared with free enzyme; the magnetic ZnFe 2 O 4 After 10 cycles of immobilized enzyme, there was still 69.8% retention of relative activity (see FIG. 4).
Detecting the magnetic ZnFe 2 O 4 The degradation rates of the immobilized laccase to 40mg/L bright green, 20mg/L active red and 100mg/L bright blue are respectively 51.3%, 85.4% and 59.5% after 5min, 15min and 60min, and tend to the degradation end point; the magnetic ZnFe 2 O 4 The degradation rate of the immobilized laccase to 40mg/L bright green, 20mg/L active red and 100mg/L bright blue is 95.0%, 85.3% and 86.1%, respectively, after 10 times of cyclic degradation, the magnetic ZnFe is obtained 2 O 4 The degradation rates of the immobilized laccase on bright green, active red and bright blue are 92.5%, 65.8% and 79.7% respectively (as shown in figure 5);
example 2
A preparation method of a magnetic immobilized laccase with high enzyme activity comprises the following steps:
1) Preparation of aminated magnetic ZnFe 2 O 4 The nanoparticle operation process is as follows:
1-1) weighing 0.9g FeCl 3 ·6H 2 O，3.6g NaAc·3H 2 O，0.49g Zn(NO 3 ) 2 Placing the mixture into a polytetrafluoroethylene lining, adding 40mL of ethylene glycol and 1mL of PEG-200, uniformly stirring to obtain yellow emulsion, reacting the reaction kettle at 240 ℃ for 8 hours, and cooling to room temperature; washing the product with ethanol for 1 time, washing with distilled water for 2 times, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) preparation of ZnFe from 1-1) 2 O 4 Dispersing the nano particles in 60mL of distilled water, and adding 3mL of 3-aminopropyl triethoxysilane; introducing shielding gas argon, heating to 120 ℃ and refluxing for 24 hours, washing with distilled water for 1 time, and separating to obtain the aminated magnetic ZnFe 2 O 4 And (3) nanoparticles. From FIG. 6, it can be seen that example 2 is preparedAminated magnetic ZnFe 2 O 4 The particle size of the nano particles is about 130-165nm;
2) Aminated magnetic ZnFe 2 O 4 Preparation of difunctional modified magnetic ZnFe from nanoparticles 2 O 4 The operation process of the nanoparticle immobilized laccase is as follows:
2-1) the aminated magnetic ZnFe to be prepared 2 O 4 Dispersing the nano particles into 50mL 2% glutaraldehyde aqueous solution (w/w), stirring in a water bath at 32 ℃ for backlight reaction for 2h, washing with distilled water for 1 time after finishing, and separating to obtain the magnetic ZnFe modified by the bifunctional reagent 2 O 4 A nanoparticle;
2-2) the difunctional modified magnetic ZnFe to be produced 2 O 4 Dispersing the nanoparticles into 50mL of distilled water, adding 17mL of laccase solution with laccase activity of 8.8U/mL, laccase solution concentration of 0.07mg/mL, stirring in a water bath at 32 ℃ for backlight reaction for 4h, washing once with distilled water, and separating to obtain magnetic ZnFe 2 O 4 The nanoparticle is immobilized with an enzyme.
Determination of magnetic ZnFe 2 O 4 The procedure for the immobilization of the catalytic activity of the enzyme was as in example 1:
determination of magnetic ZnFe 2 O 4 The procedure for the efficient degradation of the dye by immobilized enzymes is the same as in example 1:
detecting the laccase in magnetic ZnFe 2 O 4 The immobilization amount of the nano particles is 3.01mg/g; the magnetic ZnFe 2 O 4 The recovery rate of the enzyme activity of the immobilized enzyme is 95.8%; the magnetic ZnFe 2 O 4 The enzyme activity of the immobilized enzyme is 205.7U/g;
detecting the magnetic ZnFe 2 O 4 The degradation rate of immobilized enzyme to bright green is 90.1%; the magnetic ZnFe 2 O 4 The degradation rate of the immobilized enzyme to bright green after 10 times of cyclic use is 82.6 percent.
Example 3
A preparation method of a magnetic immobilized laccase with high enzyme activity comprises the following steps:
1) Preparation of aminated magnetic ZnFe 2 O 4 Nanoparticle manipulation processes such asThe following steps:
1-1) weighing 0.9g FeCl 3 ·6H 2 O，3.6g NaAc·3H 2 O，0.49g Zn(NO 3 ) 2 Placing the mixture into a polytetrafluoroethylene lining, adding 40mL of ethylene glycol and 1mL of PEG-200, uniformly stirring to obtain yellow emulsion, reacting the reaction kettle at 180 ℃ for 20h, and cooling to room temperature; washing the product with ethanol for 1 time, washing with distilled water for 2 times, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) preparation of ZnFe from 1-1) 2 O 4 Dispersing the nano particles in 60mL of distilled water, adding 3mL of 3-aminopropyl triethoxysilane, introducing argon, heating to 120 ℃ and refluxing for 24h, washing with distilled water for 1 time, and separating to obtain the aminated magnetic ZnFe 2 O 4 A nanoparticle; as can be seen from FIG. 7, the aminated magnetic ZnFe prepared in example 3 2 O 4 The particle size of the nano particles is about 115-135nm;
2) Aminated magnetic ZnFe 2 O 4 Preparation of difunctional modified magnetic ZnFe from nanoparticles 2 O 4 The operation process of the nanoparticle immobilized laccase is as follows:
2-1) the aminated magnetic ZnFe to be prepared 2 O 4 Dispersing the nano particles into 50mL 2% glutaraldehyde aqueous solution (w/w), stirring in water bath at 40 ℃ for 4h, washing with distilled water for 1 time after finishing, and separating to obtain the magnetic ZnFe modified by the bifunctional reagent 2 O 4 A nanoparticle;
2-2) the difunctional modified magnetic ZnFe to be produced 2 O 4 Dispersing the nanoparticles into 50mL of distilled water, adding 12mL of laccase solution with laccase activity of 8.8U/mL, laccase solution concentration of 0.07mg/mL, stirring in water bath at 40 ℃ for 2h, washing once with distilled water, and separating to obtain magnetic ZnFe 2 O 4 The nanoparticle is immobilized with an enzyme.
Determination of magnetic ZnFe 2 O 4 The procedure for the immobilization of the catalytic activity of the enzyme was as in example 1:
determination of magnetic ZnFe 2 O 4 The procedure for the efficient degradation of the dye by immobilized enzymes is the same as in example 1:
detecting the presence of the laccaseIn magnetic ZnFe 2 O 4 The immobilization amount of the nano particles is 2.51mg/g; the magnetic ZnFe 2 O 4 The recovery rate of the enzyme activity of the immobilized enzyme is 80.4%; the magnetic ZnFe 2 O 4 The enzyme activity of the immobilized enzyme is 174.6U/g;
detecting the magnetic ZnFe 2 O 4 The degradation rate of immobilized enzyme to bright green is 87.8%; the magnetic ZnFe 2 O 4 The degradation rate of the immobilized enzyme to bright green after the immobilized enzyme is recycled for 10 times is 79.6 percent.
Example 4
A preparation method of a magnetic immobilized laccase with high enzyme activity comprises the following steps:
1) Preparation of aminated magnetic ZnFe 2 O 4 The nanoparticle operation process is as follows:
1-1) weighing 0.9g FeCl 3 ·6H 2 O，3.6g NaAc·3H 2 O，0.49g Zn(NO 3 ) 2 Placing the mixture into a polytetrafluoroethylene lining, adding 40mL of ethylene glycol and 1mL of PEG-200, uniformly stirring to obtain yellow emulsion, reacting the reaction kettle at 260 ℃ for 6 hours, and cooling to room temperature; washing the product with ethanol for 1 time, washing with distilled water for 2 times, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) preparation of ZnFe from 1-1) 2 O 4 The nano particles are dispersed in 60mL of distilled water, 3mL of 3-aminopropyl triethoxysilane is added, and the magnetic ZnFe 2 O 4 The mass ratio of the nano particles to the 3-aminopropyl triethoxysilane is 1:10, introducing argon, heating to 120 ℃ and refluxing for 24 hours, washing with distilled water for 1 time, and separating to obtain the aminated magnetic ZnFe 2 O 4 And (3) nanoparticles. As can be seen from FIG. 8, the aminated magnetic ZnFe prepared in example 4 2 O 4 The particle size of the nanoparticles is about 125-150nm.
2) Aminated magnetic ZnFe 2 O 4 Preparation of difunctional modified magnetic ZnFe from nanoparticles 2 O 4 The operation process of the nanoparticle immobilized laccase is as follows:
2-1) the aminated magnetic ZnFe to be prepared 2 O 4 The nanoparticles were dispersed into 50mL of 4% glutaraldehyde aqueous solution (w/w)Stirring the mixture in a water bath at 30 ℃ for backlight reaction for 4 hours, washing the mixture with distilled water for 1 time after the completion of the backlight reaction, and separating the mixture to obtain the magnetic ZnFe modified by the bifunctional reagent 2 O 4 A nanoparticle;
2-2) the difunctional modified magnetic ZnFe to be produced 2 O 4 Dispersing the nanoparticles into 50mL of distilled water, adding 20mL of laccase solution with laccase activity of 8.8U/mL, laccase solution concentration of 0.07mg/mL, stirring in a water bath at 30 ℃ for backlight reaction for 2h, washing once with distilled water, and separating to obtain magnetic ZnFe 2 O 4 The nanoparticle is immobilized with an enzyme.
Determination of magnetic ZnFe 2 O 4 The procedure for the immobilization of the catalytic activity of the enzyme was as in example 1:
determination of magnetic ZnFe 2 O 4 The procedure for the efficient degradation of the dye by immobilized enzymes is the same as in example 1:
detecting the laccase in magnetic ZnFe 2 O 4 The immobilization amount of the nano particles is 4.23mg/g; the magnetic ZnFe 2 O 4 The recovery rate of the enzyme activity of the immobilized enzyme is 119.6%; the magnetic ZnFe 2 O 4 The enzyme activity of the immobilized enzyme is 287.3U/g;
detecting the magnetic ZnFe 2 O 4 The degradation rate of immobilized enzyme to bright green is 99.2%; the magnetic ZnFe 2 O 4 The degradation rate of the immobilized enzyme to bright green after the immobilized enzyme is recycled for 10 times is 94.1 percent respectively.
The invention firstly adopts a solvothermal method to synthesize magnetic ZnFe 2 O 4 And (3) the nano particles are functionalized by amino functional groups, and finally, the magnetic immobilized laccase with high activity, high stability and high recycling property is prepared by crosslinking by using a bifunctional reagent glutaraldehyde. The magnetic immobilized laccase prepared by the invention has the advantages of simple operation, economy, environmental protection, high atom utilization rate, green and no pollution; the carrier structure is designed, and the prepared magnetic ZnFe 2 O 4 The immobilized enzyme and the substrate show ultrahigh substrate affinity, so that the enzyme activity of the immobilized laccase is greatly improved; the magnetic immobilized laccase has continuous and stable catalytic capability, simple and convenient reusability and green sustainability, and greatly reduces the productionThe production cost is high, so that the catalyst has wide application prospect in the fields of catalytic oxidation, water body treatment, environmental remediation and the like.

Claims (6)

1. The preparation method of the magnetic immobilized laccase with high enzyme activity is characterized by comprising the following steps of:
1) Preparation of aminated magnetic ZnFe 2 O 4 A nanoparticle;
2) Preparation of double-functional magnetic ZnFe 2 O 4 A nanoparticle;
3) Using dual-functional magnetic ZnFe 2 O 4 Fixing laccase by nano particles;
step 1) the aminated magnetic ZnFe 2 O 4 The preparation method of the nanoparticle comprises the following steps:
1-1) adding polyalcohol and polyethylene glycol into an iron source, a zinc source and sodium acetate, reacting at high temperature, washing, and separating to obtain magnetic ZnFe 2 O 4 A nanoparticle;
1-2) the magnetic ZnFe obtained in step 1-1) 2 O 4 Dispersing the nano particles in a solvent, adding a silane coupling agent, reacting at high temperature, washing, and separating to obtain the amino magnetic ZnFe 2 O 4 A nanoparticle;
the magnetic ZnFe of step 1-2) 2 O 4 The mass ratio of the nano particles to the silane coupling agent is 1:3-13;
the silane coupling agent in the step 1-2) is 3-aminopropyl triethoxysilane;
the step 2) is specifically as follows: magnetic ZnFe to be aminated 2 O 4 Dispersing nano particles in a bifunctional reagent solution, culturing, washing and separating to obtain the bifunctional magnetic ZnFe 2 O 4 A nanoparticle; bifunctional reagent in bifunctional reagent solution and preparation of aminated magnetic ZnFe 2 O 4 Magnetic ZnFe for nanoparticles 2 O 4 Nanoparticles 1-10:1; the bifunctional reagent is glutaraldehyde.
2. The method according to claim 1, wherein the molar ratio of the iron source to the zinc source to sodium acetate in step 1-1) is 1: 0.5-1:6-12.
3. The process according to claim 1 or 2, wherein the high temperature reaction in step 1-1) is a reaction between 6 and 24h at 160 ℃ and 280 ℃.
4. The preparation method according to claim 1, wherein the step 3) specifically comprises: will double functional magnetic ZnFe 2 O 4 Dispersing the nano particles in a solvent, adding laccase solution, culturing, washing and separating to obtain magnetic ZnFe 2 O 4 Immobilizing enzymes; laccase contained in laccase solution in step 3) and preparation of bifunctional magnetic ZnFe 2 O 4 Magnetic ZnFe for nanoparticles 2 O 4 The mass ratio of the nano particles is 0.002-0.012:1.
5. A magnetically immobilized laccase with high enzymatic activity, which is prepared by the preparation method of any one of claims 1 to 4.
6. A method for efficiently degrading dye, which is characterized in that the method for preparing the magnetic immobilized laccase with high enzyme activity by using the preparation method of any one of claims 1-4 comprises the following steps:
s1, dispersing magnetic immobilized laccase with high enzyme activity in a dye;
s2, adding ABTS into the reaction system, and carrying out oscillation reaction.
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