Amino acid magnetic modified biochar immobilized laccase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of environmental pollution treatment, in particular to amino acid magnetic modified biochar immobilized laccase as well as a preparation method and application thereof. Comprising the steps of preparation of magnetic modified biochar, preparation of amino acid modified biochar and preparation of amino acid magnetic modified biochar immobilized laccase. The preparation method comprises the following steps: firstly, preparing magnetic modified biochar (MBC) through a coprecipitation method, then loading an amino functional group on the surface of the MBC, carrying out amino acid modification by utilizing arginine, and immobilizing laccase by utilizing a glutaraldehyde cross-linking agent on the modified biochar, so as to finally obtain the amino acid modified magnetic biochar immobilized laccase. The invention further provides application of the amino acid modified magnetic biochar immobilized laccase to removal of chlorpyrifos (CP) in a water environment and a soil environment, the amino acid modified magnetic biochar immobilized laccase has high adsorption capacity and degradation capacity, can effectively remove CP in a water body and soil, has the benefits of being safe, efficient and free of pollution, and is suitable for industrial production. The method can be widely applied to the pollution remediation field.

Description

Technical Field

[0001] The invention relates to the technical field of environmental pollution control, and in particular to an amino acid magnetic modified biochar immobilized laccase, a preparation method and an application thereof. Background Art

[0002] The problems caused by the large-scale use of pesticides to the water and soil environment are becoming increasingly prominent. Chlorpyrifos (CP) is one of the most widely used organophosphorus pesticides. CP is the fourth largest non-systemic broad-spectrum insecticide with high volatility, second only to thiosulfan, acephate and monocrotophos. However, only about 0.1% of most pesticides work on target species, and about 90-99% of the chemicals remain in the environment due to their stubbornness and persistence. Therefore, CP will spread widely through the atmosphere and rivers to pollute the environment. The use of CP has caused the residue in groundwater to exceed the regulatory guidelines, and the residual concentration of CP can reach 3.27-9.31 μg / L.

[0003] The existing technologies for remediating CP pollution in the environment are mainly advanced oxidation process technology and plant microbial remediation technology. However, advanced oxidation process technology is mainly applicable to the remediation of CP pollution in wastewater; plant microbial remediation is limited by environmental factors for soil application, and has the disadvantages of long remediation cycle and strong specificity. Therefore, for CP pollution in the soil environment, it is necessary to develop efficient and environmentally friendly functional materials to treat soil CP pollution.

[0004] Laccase is a multi-copper oxidase. Laccase can be combined with natural or synthetic oxidizing mediators to form a laccase-mediator system (LMS), which has broad application prospects in the degradation of pollutants such as phenols, pesticides, dyes, and food processing. However, laccase also has some defects, mainly because it is easily affected by the external environment and loses its activity. For example, under extreme temperature or pH conditions, it is easy to denature, resulting in inactivation of laccase. The low stability of free laccase in harsh environments, non-recyclability, and high production costs limit the large-scale application of laccase in the environment. Immobilized enzyme technology refers to combining enzymes with solid materials in a specific way. This technology not only helps the recovery and repeated use of enzymes, but also improves the efficiency and stability of the reaction. After being immobilized, the specificity of laccase to the substrate will not change, which can improve the stability of laccase to pH and temperature changes, making it easy to transport and store.

[0005] Biochar is a black, carbon-rich and porous solid material that can be produced through the thermochemical conversion of agricultural waste biomass. Therefore, it has the function of waste recycling and environmental protection. Biochar has a loose porous structure, rich functional groups on the surface, excellent ion exchange capacity, large specific surface area and significant adsorption capacity, and is both hydrophobic and stable. These advantages make it widely used in many fields such as fixed carriers, soil remediation, greenhouse gas emission reduction, electrochemical materials, adsorbents and catalysts.

[0006] Therefore, there is an urgent need for a method to improve the immobilization effect of laccase and improve the removal efficiency of CP in pesticide environmental pollution. Summary of the invention

[0007] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an immobilized laccase with low cost, simple preparation method, environmental friendliness, good repair effect and magnetic recovery, and its preparation method and application, so as to improve the removal efficiency of CP pollutants in the environment.

[0008] To achieve the above objectives, the present invention provides the following technical solution: an amino acid magnetically modified biochar immobilized laccase, which is obtained by loading amino functional groups on the surface of the magnetically modified biochar to bind to the laccase and immobilizing the laccase.

[0009] The preparation method of amino acid magnetic modified biochar immobilized laccase comprises the following steps: (1) Preparation of magnetically modified biochar: Add biomass powder to Fe 3+ and Fe 2+ A solution of 100 μm was prepared by adding an alkali source thereto, and after standing for 1 day to settle, the supernatant was removed, and after drying, the solution was placed in a tube furnace for pyrolysis under a N2 atmosphere to obtain magnetically modified biochar; (2) Preparation of amino acid modified biochar: The magnetically modified biochar is added to a solution containing amino acids, mixed, shaken, centrifuged, washed with distilled water and then dried to obtain unactivated amino acid-modified biochar; The unactivated amino acid-modified biochar was added to a buffer solution of pH=5, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added to the solution, and the solution was shaken at room temperature, filtered, washed, and dried to obtain the activated amino acid-modified biochar; (3) Preparation of laccase immobilized on amino acid magnetically modified biochar: The activated amino acid-modified biochar is mixed with a buffer solution containing laccase, and after adding a glutaraldehyde cross-linking agent, an oscillation reaction is performed to obtain the amino acid-modified magnetic biochar immobilized laccase.

[0010] Furthermore, in (1), Fe 3+ and Fe 2+ Derived from ferric chloride and ferrous sulfate, Fe 3+ with Fe 2+ The added molar ratio is 2:1, the mass ratio of biomass powder to iron ions is 1:1, and the alkali source is sodium hydroxide.

[0011] Furthermore, the specific steps of (1) include mixing 10 g corn stalk powder, 0.02 mol Fe 3+ and 0.01 mol Fe 2+ Dispersed in 300 mL of pure water, stirred and then added with NaOH to adjust the pH to 10-13, allowed to stand for one day, the supernatant removed, the precipitate washed with pure water to pH = 7, then dried at 120 °C, placed in a tubular furnace in a N2 atmosphere, the pyrolysis temperature was gradually increased to 600 °C at a heating rate of 5 °C / min, kept at a constant temperature for 2 h, and fired to obtain magnetically modified biochar.

[0012] Furthermore, in (2), 0.01 mol of amino acid is added per gram of magnetically modified biochar, the amino acid is arginine, and the mass ratio of unactivated amino acid-modified biochar, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2:1:2.

[0013] Furthermore, the specific steps of (2) include mixing 0.3 g of magnetically modified biochar with 0.05 M, 60 mL of arginine, placing the mixture in an oscillating box for 24 hours, centrifuging the mixture at 3000 revolutions per minute for 15 minutes, washing the MBC with distilled water to remove excess acid, repeating the washing process three times to ensure complete removal of organic compounds, and then drying in a hot oven at 60° C. for 24 hours to obtain unactivated amino acid-modified biochar; 0.1 g of unactivated amino acid-modified biochar was added to a buffer solution of pH 5, 0.05 g of N-hydroxysuccinimide and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added to the solution, and the solution was shaken at room temperature for 4 h, filtered, washed, and dried to obtain activated amino acid-modified biochar.

[0014] Furthermore, in (3), the mass ratio of amino acid modified biochar to laccase is 3:1-1.25, the concentration of amino acid modified biochar in the buffer is 1 mg / mL, and the volume fraction of glutaraldehyde is 0.25%-8%; The buffer solution is a citric acid phosphate buffer solution prepared with citric acid and disodium hydrogen phosphate, with a pH of 3 to 8; the temperature of the oscillation box is 15 to 55°C, and the fixed time is 4 to 6 hours.

[0015] Furthermore, the specific steps of (3) include taking 60 mg of amino acid modified biochar and placing it in a conical flask, adding 60 mL of citric acid phosphate buffer solution with a pH of 5, adding 20 mg of laccase, placing the mixture in a 200 rpm shaking box and maintaining it in dark room temperature conditions, shaking for 1 hour, adding 4% glutaraldehyde solution as a cross-linking agent to immobilize laccase, and then shaking in the dark for 2 hours, filtering and washing the precipitate three times with citric acid phosphate buffer solution with a pH of 5, and naturally air-drying to obtain amino acid magnetic modified biochar immobilized laccase.

[0016] The invention discloses an application of laccase immobilized on biochar with magnetic amino acid modification, wherein the laccase immobilized on biochar with magnetic amino acid modification is used for removing CP pollution in the environment.

[0017] Furthermore, amino acid magnetically modified biochar immobilized laccase accounting for 0.5% of the soil mass was added to a soil environment with a CP concentration of 20 mg / kg, and 1-hydroxybenzotriazole was added to form a laccase-mediator system to remove CP. The soil was ground and passed through a 60-mesh sieve, and then acetonitrile was used to extract pesticides in the soil.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The amino acid-modified magnetic biochar-immobilized laccase composite material of the present invention has high adsorption and degradation capacity for CP, has the benefits of safety, high efficiency and no pollution, and can be widely used in the field of pollution remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the synthesis of an embodiment of the present application; Figure 2 The immobilized laccase activity under different conditions in the examples of this application (a) The effect of pH on the fixation effect (b) Effect of glutaraldehyde addition on fixation effect (c) Effect of laccase addition on fixation effect (d) The effect of cross-linking time on fixation effect; Figure 3 This is the response surface regression model of laccase immobilized by amino acid magnetically modified biochar in the embodiment of the present application; Figure 4 Characterization diagram of the embodiment of this application (a) BC scanning electron microscopy characterization, (b) BC energy spectrum point scanning element scanning, (c) MBC scanning electron microscopy characterization, (d) MBC energy spectrum point scanning element scanning, (e) SEM characterization of MBC@LC. (f) MBC@LC energy spectrum point scanning element scanning, (g) Scanning electron microscopy characterization of NH2-MBC@LC. (h) NH2-MBC@LC energy spectrum point scanning element scanning; Figure 5 Fourier transform infrared spectroscopy analysis of laccase in the present application example; Figure 6 Fourier infrared spectroscopy analysis of BC, MBC, MBC@LC, and NH2-MBC@LC in the examples of this application; Figure 7 This is a nitrogen adsorption and desorption curve diagram of the embodiment of the present application (a) Nitrogen adsorption-desorption curve of BC, (b) MBC nitrogen adsorption and desorption curves, (c) Nitrogen adsorption and desorption curves of MBC@LC. (d) Nitrogen adsorption and desorption curves of NH2-MBC@LC; Figure 8 This is a comparison of the adsorption and desorption of the three materials MBC, MBC@LC, and NH2-MBC@LC in the examples of this application; Fig. 9 Comparison of hysteresis curves of BC and MBC in the embodiment of the present application; Fig.10 This is the XPS analysis diagram of the C element in the embodiment of this application (a) is the C element XPS analysis of the BC treatment group. (b) is the C element XPS analysis of the MBC treatment group. (c) is the C element XPS analysis of the MBC@LC treatment group. (d) C element XPS analysis of NH2-MBC@LC treated group; Fig.11 This is the XPS analysis diagram of the O element in the embodiment of the present application (a) is the XPS analysis of O element in the BC treatment group. (b) is the O element XPS analysis of the MBC treatment group. (c) XPS analysis of O element in MBC@LC treatment group. (d) XPS analysis of O element in the NH2-MBC@LC treatment group; Fig.12 This is the N element XPS analysis diagram of the embodiment of this application (a) is the N element XPS analysis of the BC treatment group. (b) N element analysis of the MBC treatment group. (c) N element analysis of the MBC@LC treatment group. (d) N element analysis of the NH2-MBC@LC treatment group; Fig.13 This is the XPS analysis diagram of the S element in the embodiment of the present application (a) is the S element XPS analysis of the BC treatment group. (b) is the S element XPS analysis of the MBC treatment group. (c) S element analysis of the MBC@LC treatment group. (d) S element analysis of the NH2-MBC@LC treatment group; Fig.14 This is the XPS analysis diagram of Fe element in the embodiment of this application (a) is the XPS analysis of Fe element in the MBC treatment group. (b) XPS analysis of Fe element in the MBC@LC treatment group. (c) XPS analysis of Fe element in the NH2-MBC@LC treatment group; Fig.15 The stability of LC, MBC@LC, and NH2-MBC@LC in different pH environments in the examples of this application; Fig.16 The stability of LC, MBC@LC, and NH2-MBC@LC in different temperature environments in the examples of this application; Fig.17 The reusability of LC, MBC@LC, and NH2-MBC@LC in the embodiments of this application; Fig.18 For the examples of this application, MBC and NH2-MBC are fitted for the adsorption kinetics of pseudo-second-order kinetics; Fig.19 This is a comparison of the degradation effects of different laccase mediator systems on CP in the examples of this application; Fig. 20 The degradation kinetics of CP by different treatment groups in the examples of this application; Fig.21 This is the degradation effect of different treatment groups in the examples of this application on CP in non-sterile soil; Fig. 22 This is the degradation effect of different treatment groups in the examples of this application on CP in sterilized soil; Fig.23 Schematic diagram of the degradation mechanism of CP by the NH2-MBC@LC composite material according to the embodiment of the present application; Fig.24 This is a comparison chart of MBC@LC and NH2-MBC@LC in the embodiments of the present application. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] The laccase in this application is sourced from Beijing Solebow Company, with the product number: L8540 (Aspergillus laccase).

[0022] Embodiment 1: The invention discloses an amino acid magnetically modified biochar immobilized laccase. The amino acid magnetically modified biochar immobilized laccase is obtained by loading an amino functional group on the surface of the magnetically modified biochar to combine the amino functional group with the laccase and immobilize the laccase.

[0023] Preparation method of amino acid magnetic modified biochar immobilized laccase, Figure 1 is a synthetic schematic diagram, (1) Preparation of magnetically modified biochar Corn stalk pretreatment: Grind the corn stalks until they can pass through a 10-mesh sieve, wash off impurities on the surface of the stalk powder with clean water, and then dry it in a 60°C oven.

[0024] After adding 10g corn stalk powder to 300mL ultrapure water, add ferric chloride and ferrous sulfate solids, 0.02mol and 0.01mol respectively, and stir evenly after all are dissolved. Use a rubber-tipped dropper to add 1M sodium hydroxide while stirring to adjust the pH to about 10-12, and let it settle for 12h. Remove the supernatant, wash the precipitate with pure water to pH=7, then dry it at 120℃, place it in a tubular furnace in a N2 atmosphere, and heat it at a rate of 5℃ / min. When it is heated to 400℃, it is retained for 30min, and then gradually raised to 600℃, and kept at a constant temperature for 2h to sinter magnetic modified biochar (MBC).

[0025] (2) Preparation of amino acid magnetically modified biochar 0.3 g of magnetically modified biochar was mixed with 0.05 M, 60 mL of arginine. The mixture was shaken in an oscillating box at 200 rpm for 24 hours, and then centrifuged at 3000 rpm for 15 minutes. The MBC was washed with distilled water to remove excess acid. The washing process was repeated three times to ensure complete removal of organic compounds. The MBC was then dried in a hot oven at 60 ° C for 24 hours to obtain unactivated biochar rich in amino acid modification.

[0026] The unactivated biochar was added to a citric acid phosphate buffer solution with a pH of 5, and N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added to the solution in a ratio of 1:2. Specifically, 0.1 g of unactivated biochar was added with 0.1 g of EDC and 0.05 g of NHS. The mixture was shaken at 200 rpm for 4 h at room temperature, filtered, washed, and dried at 60 °C to obtain activated amino acid magnetic modified biochar (NH2-MBC).

[0027] (3) Preparation of laccase immobilized on amino acid magnetically modified biochar Adsorption cross-linking method was used to prepare the MBC@LC composite material. In a 100 mL conical flask, 60 mg of magnetically modified biochar was suspended in 60 mL of citric acid phosphate buffer solution with different pH values ​​(the pH value was controlled at 5), 20 mg of laccase was added, and the mixture was placed in an oscillation box at a shaking rate of 200 rpm and a constant temperature of 25°C in the dark. After oscillation for 1 hour, 4% GA solution was added for cross-linking laccase, and then protected from light for 4 hours, then filtered, and the solid residue was washed 3 times with the same buffer solution, and the MBC@LC composite material was obtained after natural air drying. The enzyme activity of the obtained material was 166.24 U / g. Figure 2 The enzymatic activity of immobilized laccase under different conditions (a) The effect of pH on the fixation effect (b) The effect of glutaraldehyde addition on the fixation effect (c) The effect of laccase addition on the fixation effect (d) The effect of cross-linking time on the fixation effect; Figure 3 Response surface regression model of immobilized laccase.

[0028] 1. Evaluation of immobilized laccase activity: Laccase can oxidize 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) to produce ABTS + Free radicals show a blue-green color in the solution, so the ABTS method is used to determine the activity of laccase. The specific steps are: prepare 0.5mmol / L ABTS solution in advance, take 2.5mL pH5 citric acid phosphate buffer solution, add 0.4mL ABTS solution, take 0.1mL of free laccase solution or 10mg of immobilized laccase, so that the final reaction system reaches 3mL, and use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 420nm to reflect the activity of free enzyme and immobilized enzyme. The calculation formula of enzyme activity is as follows: Free laccase activity (U / L) =

[0029] Immobilized laccase activity (U / mg) =

[0030] Abs is the change in absorbance, df is the dilution factor, V is the volume of the reaction system (L), and cf is the conversion factor from molar to micromolar concentration 106 , ε is the molar extinction coefficient ε = 36,000 L / mol·cm, t is the reaction time, v is the volume of the laccase solution (L), and M is the mass of the immobilized laccase biochar (mg).

[0031] Relative enzyme activity (%)

[0032] U1 represents the enzyme activity of free laccase or immobilized laccase (U / L, U / mg), and U2 represents the maximum enzyme activity of free laccase or immobilized laccase in batch experiments.

[0033] Determination of laccase fixation: The protein concentration was determined by the BCA method using an ELISA reader (the amount of initial free laccase protein - the amount of protein in the supernatant = the amount of laccase fixed by biochar). 0.8 mL of protein standard preparation solution was added to a tube of protein standard (20 mg BSA) and fully dissolved to prepare a 25 mg / mL protein standard solution. Take an appropriate amount of 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL. According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. In a 96-well plate, standard lines with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL were configured, and 200 μL of BCA working solution was added to each well and placed at 37°C for 20-30 minutes. The absorbance at A562 wavelength was measured using an enzyme-labeled instrument, and the protein concentration of the sample was calculated based on the standard curve and the sample volume used.

[0034] The first-order linear equation is obtained: y=17524x+0.2507, and the correlation coefficient R 2 =0.999. Where y is the absorbance at 562nm and x is the protein concentration. Using this first-order equation, the concentration of laccase in the solution can be calculated from the absorbance of the solution. Under optimal conditions, the immobilization amount of laccase is about 80%.

[0035] The above technical solution has the characteristics of simple operation, little environmental pollution, good laccase fixation effect, etc.

[0036] 2. Characterization of laccase immobilized on amino acid magnetically modified biochar: Free laccase, BC, MBC, MBC@LC and NH2-MBC@LC materials were characterized using different instruments and methods.

[0037] Figure 4 In order to analyze the surface morphology of BC, MBC, MBC@LC and NH2-MBC@LC materials using scanning electron microscopy (SEM), and observe the effects of modification and enzyme loading on the surface morphology and structure of biochar, Figure 4 As can be seen in Figure a, the biochar presents a tubular structure, indicating that the biochar retains part of the original structure of the corn stalk after preparation, giving it a larger specific surface area and pore size. Figure 4 From a and c, we can see that Figure 4 Many irregular particles are added to the surface of biochar in middle c, proving that Fe3O4 is successfully loaded on the surface of biochar after magnetic modification. Figure 4 In c and e, you can see Figure 4 Many small aggregates appeared on the surface of e, proving that the adsorption cross-linking method successfully loaded laccase. Figure 4 Comparison between e and g shows that amino acid modification makes the biochar surface smoother. By analyzing the surface elements of each material through energy spectrum scanning (EDS), it was found that the Fe content on the biochar surface increased significantly after magnetic modification, further proving that Fe3O4 was successfully loaded on the biochar surface.

[0038] Brunauer-Emmett-Teller (BET) nitrogen adsorption-desorption curves were used to measure the specific surface area, pore volume and pore size distribution of BC, MBC, MBC@LC and NH2-MBC@LC materials. N2 was used as the adsorption gas, the degassing time was 8h and the degassing temperature was 200℃. Figure 7 It can be seen that the adsorption curves of different materials conform to the type I isotherm, which is usually manifested as a rapid increase in adsorption at a lower relative pressure, and a saturation value of adsorption after reaching a certain relative pressure. This isotherm usually reflects the micropore filling phenomenon on the microporous adsorbent. Among the different materials, it can be seen that MBC has the best adsorption effect, followed by the NH2-MBC@LC treatment group, the MBC@LC treatment group, and the worst is the BC treatment group, indicating that the specific surface area of ​​the four different materials is MBC>NH2-MBC@LC>MBC@LC>BC. It can be concluded that magnetically modified biochar can significantly increase the specific surface area of ​​the original biochar, but because laccase will occupy the pores on the surface of biochar, the specific surface area of ​​the magnetic biochar loaded with laccase will decrease, proving that laccase is successfully loaded on biochar, and further amino acid modification can load new functional groups on the surface of biochar, thereby increasing the specific surface area compared to magnetically modified biochar. Figure 8 Comparison of adsorption and desorption of three materials: MBC, MBC@LC, and NH2-MBC@LC.

[0039] Fig.10 (a) is the C element XPS analysis of the BC treatment group, (b) is the C element XPS analysis of the MBC treatment group, (c) is the C element XPS analysis of the MBC@LC treatment group, and (d) is the C element XPS analysis of the NH2-MBC@LC treatment group.

[0040] Fig.11(a) is the O element XPS analysis of the BC treatment group, (b) is the O element XPS analysis of the MBC treatment group, (c) is the O element XPS analysis of the MBC@LC treatment group, and (d) is the O element XPS analysis of the NH2-MBC@LC treatment group.

[0041] Fig.12 (a) is the N element XPS analysis of the BC treatment group, (b) is the N element analysis of the MBC treatment group, (c) is the N element analysis of the MBC@LC treatment group, and (d) is the N element analysis of the NH2-MBC@LC treatment group.

[0042] Fig.13 (a) is the S element XPS analysis of the BC treatment group, (b) is the S element XPS analysis of the MBC treatment group, (c) is the S element analysis of the MBC@LC treatment group, and (d) is the S element analysis of the NH2-MBC@LC treatment group. Fig.14 (a) is the Fe element XPS analysis of the MBC treatment group, (b) is the Fe element XPS analysis of the MBC@LC treatment group, and (c) is the Fe element XPS analysis of the NH2-MBC@LC treatment group.

[0043] Fig.24 This is a comparison chart of MBC@LC and NH2-MBC@LC.

[0044] Fourier transform infrared (FTIR) spectroscopy was used to characterize the surface functional groups of free laccase, BC, MBC, MBC@LC, and NH2-MBC@LC. Data were recorded at 400–4000 cm -1 In the wavelength range, Figure 5 3200-3600 cm -1 In the range of 1650-1700 cm, a distinct broad absorption peak appeared, and its characteristic absorption was related to the vibration of hydroxyl (-OH) or amino (-NH). This indicates that there are a certain number of hydroxyl groups in the laccase molecule, which may be related to its catalytic activity and enzyme stability. -1 In the region of 1100-1300 cm, a strong absorption peak was observed, which is usually attributed to the carbonyl (C=O) vibration of the carbonyl group. The presence of this peak suggests the presence of a possible peptide bond or other carbonyl-containing functional group, which is particularly important for the structure-function relationship of enzymes. In addition, in the region of 1100-1300 cm -1 In the range of 1000 cm-1, a series of absorption peaks appear again, which are usually related to the stretching vibration of CO and CN. Finally, at 1000 cm-1 -1 In the following area, the spectrum shows several weak absorption peaks, which may be related to some more complex molecular structure information, such as the characteristics of peptide chains or cross-linked structures.

[0045] exist Figure 6BC, MBC, MBC@LC, and NH2-MBC@LC are at 3200-3600 cm -1 In the range of 1600-1650 cm -1 The absorption peaks of C=C appear in the range of 1380 and 1450 cm -1 There are two -CH3 absorption peaks; MBC@LC and NH2-MBC@LC at 2910-2930 cm -1 Absorption peaks appear in the range of 1125 to 1135 cm-1, which may be related to the stretching vibration of the CH bonds of -CH3 and -CH2; BC and MBC are at 1125 to 1135 cm-1 -1 The absorption peaks at 870 to 875 cm -1 The absorption peaks around 621 to 626 cm indicate that the hydrogen atoms on the aromatic ring are replaced by other atoms or groups. -1 The absorption peaks around 1014 cm-1 may be related to the in-plane bending vibration of CC=O; the absorption peaks of MBC@LC and NH2-MBC@LC at 1014 cm-1 -1 The absorption peak may be related to the stretching vibration of CO bond. The stretching vibration of Fe-O shows two main peaks, one at 540-650 cm -1 , and the other at 400-450 cm -1 interval.

[0046] Magnetic measurements were performed using a vibrating sample magnetometer (VSM). Fig. 9 It can be seen that compared with the original biochar, the magnetic modified biochar significantly increases the magnetism of the biochar. The magnetic modified biochar sample has no hysteresis loop in the magnetization curve, indicating that after the external magnetic field is removed, the internal magnetic field strength of the material drops to zero, revealing superparamagnetic behavior, which means that the coercive force (Hc) is zero. Superparamagnetism means that when there is no external magnetic field, the magnetic moment of the magnetic particles is randomly oriented, resulting in no magnetism on a macro scale; under the action of an external magnetic field, the magnetic moments will be arranged uniformly, showing magnetism. The magnetic property of the magnetic modified biochar is 18.13emu / g, proving that the magnetic modified biochar has been successfully prepared.

[0047] X-ray photoelectron spectrometer (XPS) was used to determine the molecular structure and atomic valence state of the surface of BC, MBC, MBC@LC, and NH2-MBC@LC materials. XPS analysis of different materials showed that there were three peaks containing C functional groups at binding energies of 284.8, 286.6, and 288.4 eV, representing CC, COC, and OC=O functional groups, respectively. Among them, CC and COC functional groups changed most significantly. Among them, the NH2-MBC@LC treatment group had the highest content of OC=O functional groups (11.82%), indicating that amino acid-modified biochar can increase the oxygen-containing functional groups of biochar, which is beneficial to the removal of pollutants.

[0048] Different materials contain O functional groups with two peaks at binding energies of 530.5 and 532.3 eV, representing CO and COH functional groups, respectively. The magnetically modified biochar changes more significantly than other materials, with a new peak at 536.8, which may be due to the appearance of alcohol and phenol functional groups on the biochar surface during the magnetic modification process.

[0049] Different materials containing N functional groups have two peaks at binding energies of 399 and 400.8 eV, representing pyridinic nitrogen (Phrrolic-N) and graphitic nitrogen (Graphitic-N), respectively. Among them, MBC@LC and NH2-MBC@LC materials have higher graphitic nitrogen contents. Graphitic nitrogen has high thermal stability and is not easy to change at high temperatures, indicating that magnetically modified biochar has better stability after immobilizing laccase.

[0050] There are two peaks at 164.5 and 169.5 eV for the S functional groups in different materials, representing R-SH and metal sulfate, respectively. It can be seen that the immobilization of laccase can significantly increase the content of R-SH functional groups. Since the thiol functional group is easily oxidized, it has strong reducibility. Weakly oxidizing substances can react with thiol to form disulfide, among which disulfide bonds can play an important role in maintaining the spatial structure of proteins. Thiol also has catalytic ability and can effectively remove pollutants, thus confirming that NH2-MBC@LC has a good removal ability for pollutants.

[0051] There are four peaks at binding energies of 711, 713, 724.5, and 733.4 eV for Fe-containing functional groups in different materials, representing divalent and trivalent iron-containing functional groups, respectively, proving that Fe3O4 is successfully loaded on the biochar surface after magnetic modification.

[0052] The present invention also explores the stability and reusability of the material under different environments. The pH value, temperature and reusability of free laccase and immobilized laccase were studied with relative activity as the evaluation index, where the maximum activity was 100%. For pH stability, 10 mg of free laccase and 10 mg of immobilized laccase were added to a centrifuge tube containing 30 mL of citric acid phosphate buffer (pH range 3-8). Then, the residual activity of free laccase and immobilized laccase was measured. For thermal stability, the process was similar to pH stability, except that the centrifuge tube was immersed in a water bath with different temperature gradients (ranging from 15°C to 55°C) to achieve the optimal temperature. For reusability, the enzyme activity of free laccase and immobilized laccase was determined by the ABTS method ten times in a row.

[0053] from Fig.15 As can be seen in the figure, the highest enzyme activity of laccase under different conditions was selected as the standard to calculate the relative enzyme activity of laccase at different pH. It can be observed that the activity of free laccase reached the highest level at pH 5. At pH 4, the relative activity was slightly reduced to about 90%. Thereafter, the enzyme activity decreased at pH 3 and pH 6. Under the conditions of pH 7 and pH 8, the activity of laccase decreased significantly, both below 70%. These results show that laccase exhibits higher activity in a weakly acidic environment, especially under the condition of pH 5, where its activity is extremely significant. This may be related to the conformational stability of the enzyme and the charge state of its active site. In a lower or higher pH environment, the enzyme may denature or lose functionality, thereby affecting the catalytic ability of the enzyme. The activity of immobilized laccase will lose some of its activity during the fixation process. After amino acid modification, the change of immobilized laccase affected by pH is reduced. In general, the activity of amino acid modified biochar is higher than that of unmodified materials at different pH. The phenomenon is more significant under high pH conditions. Under high pH conditions, the relative activity of amino acid modified biochar is about 20% to 30% higher than that of unmodified materials. Under pH 6, 7, and 8, the activity of laccase in NH2-MBC@LC material is higher than that of free laccase. The possible reasons are as follows: amino acid modification can increase the polar functional groups on the surface of biochar, thereby improving the interaction with laccase, such as hydrogen bonds and ionic bonds, which helps to improve the immobilization efficiency and activity of the enzyme; amino acid modification may form a thin film on the surface of biochar, which may regulate the local pH value around the enzyme, so that the enzyme can remain in an environment close to its optimal pH even when the external pH changes, thereby maintaining a high activity; the modified biochar may have a higher specific surface area and more active sites, which helps to increase the adsorption amount of the enzyme, thereby increasing the total amount and activity of the immobilized enzyme.

[0054] exist Fig.16In the experiment, thermal stability can be used to explore the temperature tolerance of free laccase and immobilized laccase. The free laccase and immobilized laccase have the highest enzyme activity at 35°C. The enzyme activity of free laccase and immobilized laccase shows the same trend under temperature change, that is, as the temperature increases, the enzyme activity increases first and then decreases, indicating that the increase in temperature within the suitable range of laccase will increase the activity of laccase and accelerate the reaction rate of laccase and substrate. As the temperature continues to rise, the protein structure will be destroyed, resulting in a decrease in enzyme activity. The trend of the reduction of the activity of immobilized laccase at high temperature is significantly lower than that of free laccase. The laccase activity of NH2-MBC@LC composite materials is higher than that of free laccase at 45°C and 55°C, indicating that immobilization can improve the stability of laccase at high temperature.

[0055] exist Fig.17 In the figure, it can be seen that the immobilized laccase can significantly improve the stability of laccase in terms of reusability. In addition, it can be seen that amino acid-modified biochar can improve the reusability of materials compared with non-amino acid-modified materials. The reason is that amino acid modification can improve the fixation ability of laccase and increase the diversity of functional groups on the surface of biochar. The introduced amino acids may provide additional hydrogen bonds or electrostatic effects for biochar, making the interaction between laccase and biochar stronger. This enhanced binding can reduce the shedding of enzymes during the reaction and improve the stability of repeated use; and the addition of amino acids can improve the microenvironment on the surface of biochar and reduce the inactivation of enzymes; because amino acids themselves have good biocompatibility, the modified biochar may be more suitable for long-term use in the biocatalytic process, thus improving the enzyme activity of laccase under repeated use.

[0056] 2. Adsorption kinetics of CP by biochar The present invention investigates the adsorption kinetics of CP by biochar materials. 10 mg MBC and NH2-MBC were weighed in 50 mL centrifuge tubes respectively, and 30 mL of a 20 mg / L pesticide solution was added. The temperature was set to 25 °C, the oscillation frequency was 150 rpm, and the oscillation time was set to 2 min, 4 min, 6 min, 8 min, 10 min, 20 min, 30 min, 1 h, 2 h, and 3 h respectively. The supernatant was taken to determine the concentration of the pesticide. Three parallels were used in each group, and the adsorption kinetics curve was drawn according to the adsorption amount. Fig.18 It can be seen that for the adsorption rate of CP, NH2-MBC>MBC material.

[0057] An application of amino acid magnetic modified biochar immobilized laccase, the amino acid modified magnetic biochar immobilized laccase composite material has a strong adsorption capacity, rich functional groups on the surface, and a large specific surface area. The composite material also has a strong ability to remove pollutants, and the laccase contained on the surface of the material can oxidize and reduce CP adsorbed by the composite material under the condition of the participation of a mediator.

[0058] Prepare 1g / L pesticide stock solution in advance, mix the pesticide and biochar in the soil for 5 minutes, and prepare 20mg / kg contaminated soil. The design of the treatment group (CP, CP+LC, CP+BC, CP+MBC, CP+MBC@LC, CP+MBC@LC +HBT, CP+NH2-MBC@LC, CP+NH2-MBC@LC+HBT) is divided into sterilized soil and non-sterilized soil for each treatment, and cultured in a dark environment. Degradation kinetics: Samples were taken at 1d, 3d, 7d, 14d, and 21d to measure the pesticide content. The extraction of chlorpyrifos in the soil was carried out by the QuEChERS method. Through the previous spiked determination, the recovery rate of the pesticide was above 90%, which proved that this method can be used to extract CP in the soil and detect the residual concentration in high-performance liquid chromatography.

[0059] Determination of CP: The mobile phase of CP was methanol: water = 90:10, the flow rate was 1.0 mL / min, the column temperature was 30 °C, the detection wavelength was 300 nm, the injection volume was 10 μL, the CP standard substance was dissolved in acetonitrile to obtain a 1 g / L mother solution, and chlorpyrifos solutions with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 mg / L were prepared. The absorbance was determined by high performance liquid chromatography, and the fitting equation was y = 6.123x + 0.2485, where y is the absorbance and x is the CP concentration. Correlation coefficient R 2 =0.9992, which shows that the concentration and absorbance have a good linear relationship.

[0060] The degradation rate of CP is the ratio of the degraded concentration to the concentration before the reaction, expressed as a percentage. The calculation formula is as follows: Degradation rate (%) =

[0061] The addition amount of NH2-MBC@LC composite material was 0.5% of the soil mass. The selected laccase mediators were 1-hydroxybenzotriazole, syringaldehyde, and 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS). The optimal laccase mediator was 1-hydroxybenzotriazole, and the concentration of the mediator was 3mmol / L.

[0062] (1) Selection of mediator At 25 °C and pH 5, 10 mg of LC was added, the CP concentration was 20 mg / L, ABTS, HBT, and syringaldehyde (3 mmol / L) were selected as mediators, and the degradation experiment was carried out in a brown headspace bottle and a dark environment. Samples were taken to determine the CP concentration within 1, 3, 6, 12, 24, and 48 h of culture.

[0063] Fig.19The variation trend of CP removal rate over time is reflected in the figure. The degradation rate of CP increases with time. The degradation rate of CP increases rapidly before 6 hours. The degradation rate of chlorpyrifos reaches about 80% after adding free laccase, laccase-HBT mediator, and laccase-syringaldehyde mediator at 6 hours. Then the degradation rate growth slows down gradually and eventually tends to be flat. This is because at the beginning of the reaction, the concentration of chlorpyrifos is high, the interaction between laccase and substrate is frequent, and the degradation rate increases rapidly. As the substrate is gradually degraded, the concentration of the substrate decreases, resulting in a decrease in the chance of enzyme binding to the substrate, thereby reducing the degradation rate. Moreover, with long-term reaction, products that are harmful to the activity of laccase and changes in the acid-base environment may be produced, causing the degradation rate to gradually tend to be flat.

[0064] The laccase-mediator system (LMS) formed by laccase and mediator can degrade non-phenolic substances. In order to verify the role of mediators in laccase degradation of CP, three mediators (ABTS, HBT, syringaldehyde) were designed to verify the promoting effect of mediators on degradation. At the final reaction time of 2d, the degradation rates of CP in different treatment groups were as follows: laccase-HBT mediator > laccase-syringaldehyde mediator > free laccase > laccase-ABTS mediator. The degradation rate of CP by laccase-HBT mediator was significantly higher than that of other treatment groups. Therefore, the selected laccase mediator was HBT.

[0065] (2) Degradation kinetics At 25 °C and pH 5, 10 mg of LC, MBC@LC, and NH2-MBC@LC were added respectively, the CP concentration was 20 mg / L, and HBT (3 mmol / L) was selected as the mediator. The degradation experiment was carried out in a brown headspace bottle and a dark environment. Samples were taken within 1, 3, 6, 12, 24, and 48 h of culture to determine the CP concentration.

[0066] from Fig. 20 It can be concluded that the degradation rates of CP by different materials are compared as follows: NH2-MBC@LC+HBT>MBC@LC+HBT>NH2-MBC@LC>LC+HBT>MBC@LC>LC. The results show that amino acid-modified biochar can effectively improve the degradation rate of chlorpyrifos by materials. Among them, the laccase-HBT mediator system composed of laccase and HBT has a higher degradation effect on CP than that of modified biochar. This is because in the presence of the mediator HBT, the addition of the mediator expands the substrate range of laccase, and the redox potential of laccase is improved, thereby being able to more effectively catalyze the oxidation of organic pollutants such as chlorpyrifos.

[0067] Secondly, amino acid-modified biochar increases the surface functionalization of the material by introducing amino functional groups, thereby improving the adsorption capacity of organic pollutants such as chlorpyrifos. This modification can increase the surface polarity of biochar and promote interaction with pollutants, thereby improving the adsorption efficiency. Amino acid modification may provide a more suitable microenvironment for laccase, thereby increasing the activity of immobilized laccase. Laccase can more effectively catalyze the oxidation of chlorpyrifos in a suitable microenvironment and promote its degradation. There may be a synergistic effect between amino acid-modified biochar and laccase, making the modified biochar not only an adsorbent, but also a carrier of the enzyme, improving the overall degradation efficiency.

[0068] (3) Removal of CP in soil environment Field soil was collected in Junan County, Linyi. After removing impurities, the soil samples were divided into two parts. One of them was sterilized. 40g of soil was weighed and placed in a glass bottle. 1g / L CP mother solution was added to prepare simulated contaminated soil with a CP concentration of 20mg / kg. LC, BC, MBC, MBC@LC, NH2-MBC@LC, MBC@LC and HBT, NH2-MBC@LC and HBT were added to account for 0.5% of the mass of the soil to prepare different treatment groups. The soil moisture content was maintained at 60%. Samples were taken at 1d, 3d, 7d, 14d, and 21d. After grinding the soil through a 60-mesh sieve, 10g of soil was weighed and added to a 50mL centrifuge tube, and acetonitrile (10 mL) was added. After being placed in an oscillating box and oscillated at 200rpm for 6h, NaCl (2g) and anhydrous magnesium sulfate (4g) were added to the soil sample. Then the sample was oscillated for 30min and centrifuged at 4000rpm for 5min. 1 mL of the acetonitrile supernatant of the soil sample was filtered through a 0.22 μm filter membrane and then subjected to HPLC analysis.

[0069] Fig.21 , 22It can be seen from the two figures that the degradation rate of CP in soil by NH2-MBC@LC was the highest during the reaction time of 21 days, reaching about 70% in non-sterilized soil. The removal effect of chlorpyrifos in all treatment groups was NH2-MBC@LC+HBT>MBC@LC+HBT>NH2-MBC@LC>MBC@LC> MBC>LC>BC, and it reached about 60% in sterilized soil. The removal effect of chlorpyrifos in all treatment groups was NH2MBC@LC+HBT>NH2MBC@LC>MBC@LC+HBT>MBC@LC>MBC>LC>BC. The experimental results show that NH2-MBC@LC can effectively remove CP in soil, NH2-MBC can improve the stability and activity of LC in soil, and the porous structure of NH2-MBC provides more surface area, enabling LC to have better contact with CP in soil, thereby improving the degradation efficiency. The laccase-mediator system composed of laccase and mediator substance HBT can further promote the removal of CP, which is about 9% higher than the group without mediator treatment. This is because HBT is a commonly used redox mediator that can play a role in the LC catalytic reaction. It improves the catalytic efficiency of LC by promoting electron transfer, making the CP degradation process more efficient. The removal rate of CP by MBC is about 6% higher than that of BC. This is because magnetic materials are introduced into MBC during the preparation process. These materials can not only increase the surface area and porosity of BC, but also may bring stronger adsorption characteristics. Moreover, magnetic modification enables the iron-containing functional groups on the surface of BC to promote CP degradation.

[0070] (4) Degradation mechanism of chlorpyrifos, Fig.23 Schematic diagram of the degradation mechanism of CP by NH2-MBC@LC composite material.

[0071] In order to explore the degradation mechanism of chlorpyrifos by immobilized laccase functional materials, the degradation products of chlorpyrifos in the NH2-MBC@LC treated group with HBT mediator at different time periods were determined by HPLC-MS / MS. It was found that chlorpyrifos was first converted into methyl chlorpyrifos, chlorpyrifos-oxy and o-diethyl o-hydrogen thiophosphate by the cleavage of CC bond, P=S double bond and CO bond, respectively. Methyl chlorpyrifos was converted into 3,5,6-trichloro-2-pyridinol (TCP) by the cleavage of PO bond. Chlorpyrifos-oxy subsequently had two degradation pathways. The first one was the cleavage of OP bond to convert it into 3,5,6-trichloro-2-pyridinol (TCP), and the second one was the cleavage of CO bond to generate P=O double bond, similar to the detachment of functional groups on benzene ring to generate diethyl phosphate. Studies have shown that TCP is the most toxic of the byproducts of chlorpyrifos. Therefore, exploring the degradation of TCP is a key indicator of the remediation of chlorpyrifos pollution. At the final reaction time of 21 days, a high concentration of 2-chloro-6-methoxypyridine byproduct was found, indicating that TCP further undergoes alkylation and dechlorination to generate 2-chloro-6-methoxypyridine, which is the key to the complete detoxification of chlorpyrifos. In the subsequent degradation reaction, diethyl phosphate further sheds its methyl group to generate dimethyl phosphate, and continues to degrade to generate CO2 and H2O under the action of NH2-MBC@LC.

[0072] Table 1 CP degradation related byproducts

[0073] This application adopts Fe 2+ and Fe 3+ Magnetic modified biochar was prepared by co-precipitation method under alkaline environment. On the basis of magnetic modified biochar, amino functional groups were loaded on the surface of biochar for amino acid modification. The introduction of amino acids can significantly improve the catalytic performance, adsorption performance and environmental friendliness of biochar. The development of amino acid modified biochar has the following significant advantages: (1) Enhanced adsorption of cations. (2) Improved water solubility and dispersibility of biochar. (3) Improved catalytic activity of biochar. Studies have shown that the surface active sites of biochar are significantly increased by amino acid modification, thereby effectively promoting its catalytic degradation of organic pollutants.

[0074] Embodiment 2: The difference between this embodiment and Example 1 is that the citric acid phosphate buffer solution is changed to 3, 4, 6, 7, and 8, and the rest is exactly the same as Example 1, and MBC@LC is obtained. The enzyme activities are 117.8U / g, 131.2U / g, 130.2U / g, 91.2U / g, and 95.6U / g, respectively.

[0075] Embodiment 3: The difference between this embodiment and Example 1 is that the proportion of the added glutaraldehyde solution is changed to 0.25%, 0.5%, 1%, 2%, and 8%, and the rest is exactly the same as Example 1, and MBC@LC is obtained. The enzyme activities are 115.37U / g, 129.33U / g, 152.4U / g, 161.6U / g, and 137.0U / g, respectively.

[0076] Embodiment 4: The difference between this embodiment and embodiment 1 is that the amount of laccase added is changed to 5, 10, 15, and 25 mg, and the rest is exactly the same as embodiment 1, and MBC@LC is obtained. The enzyme activities are 83.12 U / g, 109.4 U / g, 157.6 U / g, and 170.7 U / g, respectively.

[0077] Embodiment 5: The difference between this embodiment and Example 1 is that the cross-linking oscillation time is changed to 2, 3, 6, and 8 hours, and the rest is exactly the same as Example 1, and MBC@LC is obtained. The enzyme activities are 170.9 U / g, 185.1 U / g, 144.7 U / g, and 115.2 U / g, respectively.

[0078] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An amino acid magnetically modified biochar immobilized laccase, characterized in that: By loading amino functional groups on the surface of magnetically modified biochar, making it combine with laccase, and immobilizing laccase, amino acid-modified magnetic biochar-immobilized laccase is obtained.

2. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 1, characterized in that the steps include: (1) Preparation of magnetically modified biochar: Add biomass powder to Fe 3+ and Fe 2+ A solution of 100 μm was prepared by adding an alkali source thereto, and after standing for 1 day to settle, the supernatant was removed, and after drying, the solution was placed in a tube furnace for pyrolysis under a N2 atmosphere to obtain magnetically modified biochar; (2) Preparation of amino acid modified biochar: The magnetically modified biochar is added to a solution containing amino acids, mixed, shaken, centrifuged, washed with distilled water and then dried to obtain unactivated amino acid-modified biochar; The unactivated amino acid-modified biochar was added to a buffer solution of pH=5, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added to the solution, and the solution was shaken at room temperature, filtered, washed, and dried to obtain the activated amino acid-modified biochar; (3) Preparation of laccase immobilized on amino acid magnetically modified biochar: The activated amino acid-modified biochar is mixed with a buffer solution containing laccase, and after adding a glutaraldehyde cross-linking agent, an oscillation reaction is performed to obtain the amino acid-modified magnetic biochar immobilized laccase.

3. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: In (1), Fe 3+ and Fe 2+ Derived from ferric chloride and ferrous sulfate, Fe 3+ with Fe 2+ The added molar ratio is 2:1, the mass ratio of biomass powder to iron ions is 1:1, and the alkali source is sodium hydroxide.

4. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: The specific steps of (1) include mixing 10 g corn stalk powder, 0.02 mol Fe 3+ and 0.01 mol Fe 2+ Dispersed in 300 mL pure water, stirred and then added with NaOH to adjust the pH to 10-13, allowed to stand for one day, the supernatant removed, the precipitate washed with pure water to pH = 7, then dried at 120 °C, placed in a tubular furnace in a N2 atmosphere, the pyrolysis temperature was gradually increased to 600 °C at a heating rate of 5 °C / min, kept at a constant temperature for 2 h, and fired to obtain magnetically modified biochar.

5. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: In the above (2), 0.01 mol of amino acid is added per gram of magnetically modified biochar, the amino acid is arginine, and the mass ratio of unactivated amino acid-modified biochar, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2:1:

2.

6. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: The specific steps of (2) include mixing 0.3 g of magnetically modified biochar with 0.05 M, 60 mL of arginine, placing the mixture in an oscillating box for 24 hours, centrifuging the mixture at a speed of 3000 revolutions per minute for 15 minutes, washing with distilled water to remove excess acid, repeating the washing process three times to ensure complete removal of organic compounds, and then drying in a hot oven at 60° C. for 24 hours to obtain unactivated amino acid-modified biochar; 0.1 g of unactivated amino acid-modified biochar was added to a buffer solution of pH 5, 0.05 g of N-hydroxysuccinimide and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added to the solution, and the solution was shaken at room temperature for 4 h, filtered, washed, and dried to obtain activated amino acid-modified biochar.

7. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: In (3), the mass ratio of amino acid modified biochar to laccase is 3:1-1.25, the concentration of amino acid modified biochar in the buffer is 1 mg / mL, and the volume fraction of glutaraldehyde is 0.25%-8%; The buffer solution is a citric acid phosphate buffer solution prepared with citric acid and disodium hydrogen phosphate, with a pH of 3 to 8; the temperature of the oscillation box is 15 to 55°C, and the fixed time is 4 to 6 hours.

8. The method for preparing amino acid magnetically modified biochar immobilized laccase according to claim 2, characterized in that: The specific steps of (3) include taking 60 mg of amino acid modified biochar and placing it in a conical flask, adding 60 mL of citric acid phosphate buffer solution with a pH of 5, adding 20 mg of laccase, placing the mixture in an oscillating box at 200 rpm and maintaining it in the dark at room temperature, after oscillating for 1 hour, adding 4% glutaraldehyde solution as a cross-linking agent to immobilize laccase, and then oscillating in the dark for 2 hours, filtering and washing the precipitate three times with citric acid phosphate buffer solution with a pH of 5, and obtaining amino acid magnetic modified biochar immobilized laccase after natural air drying.

9. An application of amino acid magnetically modified biochar immobilized laccase according to claim 1, characterized in that: Laccase immobilized on amino acid magnetically modified biochar for the removal of chlorpyrifos pollution in the environment.

10. The use of amino acid magnetic modified biochar immobilized laccase according to claim 9, characterized in that: In a soil environment with a chlorpyrifos concentration of 20 mg / kg, 0.5% of the soil mass of amino acid magnetically modified biochar immobilized laccase was added, and 1-hydroxybenzotriazole was added to form a laccase-mediator system to remove chlorpyrifos. The soil was ground and passed through a 60-mesh sieve, and the pesticides in the soil were extracted using acetonitrile.

Images