Amino acid magnetic modified biochar immobilized laccase and its preparation method and application

Abstract

The present invention relates to the technical field of environmental pollution control, specifically to an amino acid magnetically modified biochar-immobilized laccase, its preparation method, and its application. This method comprises the preparation of magnetically modified biochar, the preparation of amino acid-modified biochar, and the preparation of amino acid magnetically modified biochar-immobilized laccase. First, magnetically modified biochar (MBC) is prepared by a coprecipitation method. Then, amino functional groups are loaded onto the surface of the MBC, and amino acids are modified using arginine. Laccase is immobilized on the modified biochar using a glutaraldehyde crosslinker, resulting in the amino acid-modified magnetic biochar-immobilized laccase. The present invention also provides the application of the amino acid-modified magnetic biochar-immobilized laccase for the removal of chlorpyrifos (CP) in water and soil environments. The amino acid-modified magnetic biochar-immobilized laccase has high adsorption and degradation capabilities, effectively removing CP from water and soil. It is safe, efficient, and pollution-free, and can be widely used in pollution remediation.

Description

Technical Field

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

[0002] The extensive use of pesticides is causing increasingly serious environmental problems in water and soil. Chlorpyrifos (CP) is one of the most widely used organophosphorus pesticides. CP is a highly volatile, non-systemic, broad-spectrum insecticide, ranking fourth after endosulfan, acephate, and monocrotophos. However, for most pesticides, only approximately 0.1% is effective against target species, while approximately 90-99% of the chemical remains in the environment due to its stubbornness and persistence. Consequently, CP can be widely disseminated through the atmosphere and rivers, contaminating the environment. The use of CP has resulted in residues in groundwater exceeding regulatory guidelines, with residual concentrations reaching 3.27-9.31 μg / L.

[0003] Existing technologies for remediating CP pollution in the environment primarily rely on advanced oxidation processes and phyto-microbial remediation. However, advanced oxidation processes are primarily suitable for remediating CP pollution in wastewater, while phyto-microbial remediation in soil is limited by environmental factors and suffers from drawbacks such as long remediation cycles and high specificity. Therefore, the development of highly efficient and environmentally friendly functional materials is needed to address CP pollution in soil environments.

[0004] Laccase is a multi-copper oxidase that can be combined with natural or synthetic oxidizing mediators to form a laccase-mediator system (LMS). This system has broad application prospects in the degradation of pollutants such as phenols, pesticides, and dyes, as well as in food processing. However, laccases also have some drawbacks, primarily being susceptible to environmental influences, leading to loss of activity. For example, extreme temperature and pH conditions can lead to denaturation and inactivation of laccases. The low stability of free laccase in harsh environments, its non-recyclability, and high production costs limit its large-scale application in environmental applications. Immobilized enzyme technology involves binding the enzyme to a solid material in a specific manner. This technique not only facilitates enzyme recovery and reuse, but also improves reaction efficiency and stability. Immobilized laccase maintains its substrate specificity, enhances its stability to pH and temperature fluctuations, and facilitates transport and storage.

[0005] Biochar is a black, carbon-rich, porous solid material produced through the thermochemical conversion of agricultural waste biomass. Therefore, it has the potential to recycle waste and protect the environment. Biochar has a loose, porous structure, abundant surface functional groups, excellent ion exchange capacity, a large specific surface area, and significant adsorption capacity, along with both hydrophobicity and stability. These advantages have led to its widespread application in a variety of fields, including as a stationary carrier, 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 enhance 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, as well as its preparation method and application, 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 so that it combines with the laccase and immobilizes the laccase.

[0009] The preparation method of amino acid magnetic modified biochar immobilized laccase comprises the following steps:

[0010] (1) Preparation of magnetically modified biochar:

[0011] Add biomass powder to the Fe 3+ and Fe 2+ Add an alkali source to the solution, let it stand for 1 day to settle, remove the supernatant, dry it, and then place it in a tube furnace for pyrolysis under N2 atmosphere to obtain magnetic modified biochar;

[0012] (2) Preparation of amino acid modified biochar:

[0013] 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;

[0014] 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.

[0015] (3) Preparation of laccase immobilized on amino acid magnetically modified biochar:

[0016] The activated amino acid-modified biochar was mixed with a buffer solution containing laccase, and after adding glutaraldehyde cross-linking agent, the mixture was shaken to obtain amino acid-modified magnetic biochar immobilized laccase.

[0017] Furthermore, in (1), Fe 3+ and Fe 2+ Derived from ferric chloride and ferrous sulfate, Fe 3+ with Fe 2+ The addition molar ratio is 2:1, the mass ratio of biomass powder to iron ions is 1:1, and the alkali source is sodium hydroxide.

[0018] Furthermore, the specific steps of (1) include: mixing 10g corn straw powder, 0.02 mol Fe 3+ and 0.01 mol Fe 2+ Disperse in 300 mL pure water, stir and add NaOH to adjust the pH to 10-13, let it stand for one day, remove the supernatant, wash the precipitate with pure water to pH=7, then dry at 120°C, place in a tubular furnace under N2 atmosphere, and pyrolyze at a heating rate of 5°C / min to 600°C, keep constant temperature for 2h, and sinter to obtain magnetic modified biochar.

[0019] 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.

[0020] 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 the biochar in a hot oven at 60°C for 24 hours to obtain unactivated amino acid-modified biochar;

[0021] 0.1 g of unactivated amino acid-modified biochar was added to a buffer solution with a pH of 5, 0.05 g of N-hydroxysuccinimide and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added to the solution, and the mixture was shaken at room temperature for 4 h. The activated amino acid-modified biochar was then filtered, washed, and dried.

[0022] 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 solution is 1 mg / mL, and the volume fraction of glutaraldehyde is 0.25%~8%;

[0023] 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.

[0024] 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 the dark at room temperature, shaking it for 1 hour, adding 4% glutaraldehyde solution as a cross-linking agent to immobilize the laccase, and then shaking it 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 it to obtain amino acid magnetic modified biochar immobilized laccase.

[0025] The invention discloses an application of amino acid magnetically modified biochar immobilized laccase, wherein the amino acid magnetically modified biochar immobilized laccase is used for removing CP pollution in the environment.

[0026] 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.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The amino acid-modified magnetic biochar-immobilized laccase composite material of the present invention has high adsorption and degradation capabilities for CP, is safe, efficient, and pollution-free, and can be widely used in the field of pollution remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the synthesis of the embodiments of the present application;

[0030] Figure 2 The immobilized laccase activity under different conditions in the examples of this application

[0031] (a) The effect of pH on the fixation effect

[0032] (b) Effect of glutaraldehyde addition on fixation effect

[0033] (c) Effect of laccase addition on fixation effect

[0034] (d) The effect of cross-linking time on the fixation effect;

[0035] Figure 3 This is the response surface regression model of laccase immobilized on amino acid magnetically modified biochar in the embodiment of the present application;

[0036] Figure 4 Characterization diagram of the embodiment of this application

[0037] (a) BC scanning electron microscopy characterization,

[0038] (b) BC energy spectrum point scan element scan,

[0039] (c) MBC scanning electron microscopy characterization,

[0040] (d) MBC energy spectrum point scan element scan,

[0041] (e) Scanning electron microscopy characterization of MBC@LC.

[0042] (f) MBC@LC energy spectrum point scanning element scanning,

[0043] (g) Scanning electron microscopy characterization of NH2-MBC@LC,

[0044] (h) NH2-MBC@LC energy spectrum point scanning element scanning;

[0045] Figure 5 Fourier transform infrared spectroscopy analysis of laccase in the examples of this application;

[0046] Figure 6 Fourier infrared spectroscopy analysis of BC, MBC, MBC@LC, and NH2-MBC@LC in the examples of this application;

[0047] Figure 7 This is a nitrogen adsorption and desorption curve diagram of the embodiment of this application

[0048] (a) Nitrogen adsorption and desorption curves of BC,

[0049] (b) MBC nitrogen adsorption and desorption curves,

[0050] (c) Nitrogen adsorption and desorption curves of MBC@LC.

[0051] (d) Nitrogen adsorption and desorption curves of NH2-MBC@LC;

[0052] 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;

[0053] Figure 9 Comparison of BC and MBC hysteresis curves in the embodiment of this application;

[0054] Figure 10 This is the C element XPS analysis diagram of the embodiment of this application

[0055] (a) is the C element XPS analysis of the BC treatment group,

[0056] (b) is the C element XPS analysis of the MBC treatment group.

[0057] (c) is the C element XPS analysis of the MBC@LC treatment group.

[0058] (d) C element XPS analysis of the NH2-MBC@LC treated group;

[0059] Figure 11 This is the XPS analysis diagram of the O element in the embodiment of this application

[0060] (a) is the XPS analysis of O element in the BC treatment group.

[0061] (b) is the O element XPS analysis of the MBC treatment group.

[0062] (c) is the O element XPS analysis of the MBC@LC treatment group.

[0063] (d) XPS analysis of O element in the NH2-MBC@LC treatment group;

[0064] Figure 12 This is the N element XPS analysis diagram of the embodiment of this application

[0065] (a) is the N element XPS analysis of the BC treatment group,

[0066] (b) N element analysis of the MBC treatment group,

[0067] (c) N element analysis of the MBC@LC treatment group.

[0068] (d) N element analysis of the NH2-MBC@LC treatment group;

[0069] Figure 13 This is the XPS analysis diagram of the S element in the embodiment of this application

[0070] (a) is the S element XPS analysis of the BC treatment group,

[0071] (b) is the S element XPS analysis of the MBC treatment group.

[0072] (c) S element analysis of the MBC@LC treatment group.

[0073] (d) S element analysis of the NH2-MBC@LC treatment group;

[0074] Figure 14 This is the XPS analysis diagram of Fe element in the embodiment of this application

[0075] (a) is the XPS analysis of Fe element in the MBC treatment group.

[0076] (b) XPS analysis of Fe element in the MBC@LC treatment group.

[0077] (c) XPS analysis of Fe element in the NH2-MBC@LC treatment group;

[0078] Figure 15 The stability of LC, MBC@LC, and NH2-MBC@LC in different pH environments.

[0079] Figure 16 The stability of LC, MBC@LC, and NH2-MBC@LC in different temperature environments in the examples of this application;

[0080] Figure 17 The reusability of LC, MBC@LC, and NH2-MBC@LC in the examples of this application;

[0081] Figure 18 For the examples of this application, MBC and NH2-MBC were fitted with pseudo-second-order adsorption kinetics;

[0082] Figure 19 Comparison of the degradation effects of different laccase mediator systems on CP in the examples of this application;

[0083] Figure 20 This is the degradation kinetics of CP by different treatment groups in the examples of this application;

[0084] Figure 21 This is the degradation effect of different treatment groups in the examples of this application on CP in non-sterile soil;

[0085] Figure 22 This is the degradation effect of different treatment groups in the examples of this application on CP in sterilized soil;

[0086] Figure 23 Schematic diagram of the degradation mechanism of CP by the NH2-MBC@LC composite material according to the embodiment of the present application;

[0087] Figure 24 This is a comparison chart of MBC@LC and NH2-MBC@LC in the examples of this application. DETAILED DESCRIPTION

[0088] 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.

[0089] The laccase in this application is sourced from Beijing Solebaugh Company, with the product number: L8540 (Aspergillus laccase).

[0090] Example 1:

[0091] The invention discloses an amino acid magnetically modified biochar immobilized laccase. The amino acid-modified magnetic biochar immobilized laccase is obtained by loading amino functional groups on the surface of the magnetically modified biochar to combine with the laccase and immobilize the laccase.

[0092] Preparation method of amino acid magnetic modified biochar immobilized laccase, Figure 1 A schematic diagram of the synthesis.

[0093] (1) Preparation of magnetically modified biochar

[0094] Corn straw pretreatment: Grind the corn straw until it passes through a 10-mesh sieve, wash the surface impurities of the straw powder with clean water, and then dry it in a 60°C oven.

[0095] To 10g of corn straw powder, add 300mL of ultrapure water, followed by 0.02mol of ferric chloride and 0.01mol of ferrous sulfate solids, respectively. After complete dissolution and stirring, add 1M sodium hydroxide using a rubber-tipped pipette while stirring to adjust the pH to around 10-12. The mixture was allowed to settle for 12 hours. The supernatant was removed, and the precipitate was washed with pure water to a pH of 7. The mixture was then dried at 120°C and pyrolyzed in a tube furnace under a nitrogen atmosphere at a heating rate of 5°C / min. The temperature was raised to 400°C, held for 30 minutes, then gradually increased to 600°C and held at this temperature for 2 hours to produce magnetically modified biochar (MBC).

[0096] (2) Preparation of amino acid magnetically modified biochar

[0097] 0.3 g of magnetically modified biochar was mixed with 60 mL of 0.05 M arginine. The mixture was shaken in an incubator 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. This washing process was repeated three times to ensure complete removal of organic compounds. The MBC was then dried in a 60°C oven for 24 hours to obtain unactivated biochar rich in amino acid modification.

[0098] 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 at room temperature for 4 h, filtered, washed, and dried at 60 °C to obtain activated amino acid magnetic modified biochar (NH2-MBC).

[0099] (3) Preparation of laccase immobilized on amino acid magnetically modified biochar

[0100] The MBC@LC composite was prepared using an adsorption cross-linking method. In a 100-mL conical flask, 60 mg of magnetically modified biochar was suspended in 60 mL of citric acid-phosphate buffer solutions of varying pH values ​​(controlled at pH 5). 20 mg of laccase was added, and the mixture was placed in a shaker at 200 rpm in the dark at 25°C. After 1 hour of shaking, a 4% GA solution was added to cross-link the laccase. The mixture was protected from light for another 4 hours, then filtered, washed three times with the same buffer, and air-dried to obtain the MBC@LC composite. The resulting material had an enzyme activity of 166.24 U / g. Figure 2 The enzymatic activity of immobilized laccase under different conditions (a) The effect of pH on the immobilization effect (b) The effect of glutaraldehyde addition on the immobilization effect (c) The effect of laccase addition on the immobilization effect (d) The effect of cross-linking time on the immobilization effect; Figure 3 is the response surface regression model of immobilized laccase.

[0101] 1. Evaluation of immobilized laccase activity:

[0102] Laccase can oxidize 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) to produce ABTS + Free radicals appear blue-green in solution, so the ABTS method is used to determine laccase activity. The specific steps are: prepare 0.5 mmol / L ABTS solution in advance, take 2.5 mL of pH 5 citrate phosphate buffer solution, add 0.4 mL of ABTS solution, take 0.1 mL of free laccase solution or 10 mg of immobilized laccase, and make the final reaction system reach 3 mL. Use a UV spectrophotometer to measure the absorbance at a wavelength of 420 nm to reflect the activity of free enzyme and immobilized enzyme. The enzyme activity is calculated as follows:

[0103] Free laccase activity (U / L) =

[0104] Immobilized laccase activity (U / mg) =

[0105] 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. 6 , ε is the molar extinction coefficient ε = 36,000 L / mol·cm, t is the reaction time, v is the volume of laccase solution (L), and M is the mass of immobilized laccase biochar (mg).

[0106] Relative enzyme activity (%)

[0107] U1 represents the enzymatic activity of free laccase or immobilized laccase (U / L, U / mg), and U2 represents the maximum enzymatic activity of free laccase or immobilized laccase in batch experiments.

[0108] Determination of laccase immobilization: Protein concentration was determined using the BCA assay using a microplate reader (initial free laccase protein amount minus supernatant protein amount = biochar immobilization of laccase). 0.8 mL of protein standard solution was added to a tube of protein standard (20 mg BSA) and fully dissolved to prepare a 25 mg / mL protein standard solution. An appropriate amount of the 25 mg / mL protein standard was diluted to a final concentration of 0.5 mg / mL. Based on the number of samples, an appropriate amount of BCA working solution was prepared by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mixing thoroughly. Standard markers with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL were prepared in a 96-well plate. 200 μL of BCA working solution was added to each well and incubated at 37°C for 20-30 minutes. The absorbance at wavelength A562 was measured using a microplate reader, and the protein concentration of the sample was calculated based on the standard curve and the sample volume used.

[0109] 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 562 nm, and x is the protein concentration. Using this first-order equation, the laccase concentration in the solution can be calculated from the absorbance. Under optimal conditions, the immobilization level of laccase is approximately 80%.

[0110] The above technical solution has the advantages of simple operation, little environmental pollution, good laccase fixation effect, etc.

[0111] 2. Characterization of laccase immobilized on amino acid magnetically modified biochar:

[0112] Free laccase, BC, MBC, MBC@LC and NH2-MBC@LC materials were characterized using different instruments and methods.

[0113] 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 to 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 4Many irregular particles were added to the surface of the biochar in the middle c, proving that the magnetic modification successfully loaded Fe3O4 on the biochar surface. Figure 4 You can see in c and e Figure 4 Many small aggregates appeared on the surface of the middle e, proving that the adsorption cross-linking method was successful in loading laccase. Figure 4 Comparison of images e and g shows that amino acid modification resulted in a smoother biochar surface. Analysis of the surface elements of each material using energy-dispersive spectroscopy (EDS) revealed a significant increase in the Fe content on the biochar surface after magnetic modification, further demonstrating the successful loading of Fe₃O₄ on the biochar surface.

[0114] 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 8 h, and the degassing temperature was 200°C. Figure 7 The adsorption curves of the different materials conform to a Type I isotherm, typically characterized by a rapid increase in adsorption at low relative pressures and a saturation adsorption value after reaching a certain relative pressure. This isotherm typically reflects the micropore filling phenomenon of the microporous adsorbent. Among the different materials, MBC exhibits the best adsorption effect, followed by the NH2-MBC@LC treatment group, then the MBC@LC treatment group, and the worst is the BC treatment group. This indicates that the specific surface areas of the four different materials follow the order: MBC > NH2-MBC@LC > MBC@LC > BC. It can be concluded that magnetically modified biochar significantly increases the specific surface area of ​​the original biochar. However, because laccase occupies the pores on the biochar surface, the specific surface area of ​​the laccase-loaded magnetic biochar decreases, demonstrating that laccase is successfully loaded onto the biochar. Further amino acid modification can load new functional groups on the biochar surface, 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.

[0115] Figure 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.

[0116] Figure 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.

[0117] Figure 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.

[0118] Figure 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.

[0119] Figure 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.

[0120] Figure 24 A comparison chart of MBC@LC and NH2-MBC@LC.

[0121] The surface functional groups of free laccase, BC, MBC, MBC@LC, and NH2-MBC@LC were characterized by Fourier transform infrared (FTIR) spectroscopy. The 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 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, a series of absorption peaks appear again, which are usually related to the stretching vibration of CO and CN. Finally, at 1000 cm -1 In the following area, the spectrum shows several weak absorption peaks, which may involve some more complex molecular structure information, such as the characteristics of peptide chains or cross-linking structures.

[0122] exist Figure 6 BC, MBC, MBC@LC, and NH2-MBC@LC are at 3200-3600 cm -1 In the range of 1600-1650 cm -1The 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, which may be related to the stretching vibration of the CH bonds of -CH3 and -CH2; BC, MBC are at 1125 to 1135 cm -1 The absorption peaks at 870 to 875 cm-1 may indicate that the sample contains primary alcohol structures. -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 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 The absorption peak may be related to the stretching vibration of the CO bond. The stretching vibration of Fe-O is manifested as two main peaks, one at 540-650 cm -1 , and the other at 400-450 cm -1 interval.

[0123] Magnetic measurements were performed using a vibrating sample magnetometer (VSM). Figure 9 As can be seen in the figure, the magnetically modified biochar significantly increases its magnetic properties compared to the original biochar. The absence of a hysteresis loop in the magnetization curve of the magnetically modified biochar sample indicates that the internal magnetic field strength of the material drops to zero after the external magnetic field is removed, revealing superparamagnetic behavior, which means that the coercive force (Hc) is zero. Superparamagnetism refers to the fact that in the absence of an external magnetic field, the magnetic moments of magnetic particles are randomly oriented, resulting in a lack of macroscopic magnetism. However, under the influence of an external magnetic field, the magnetic moments align uniformly, exhibiting magnetism. The magnetic properties of the magnetically modified biochar are 18.13 emu / g, confirming the successful preparation of the magnetically modified biochar.

[0124] X-ray photoelectron spectroscopy (XPS) was used to determine the molecular structure and atomic valence states of the surfaces of BC, MBC, MBC@LC, and NH2-MBC@LC materials. XPS analysis of the different materials revealed three peaks containing carbon functional groups at binding energies of 284.8, 286.6, and 288.4 eV, representing CC, COC, and OC=O functional groups, respectively. CC and COC functional groups showed the most significant changes. The NH2-MBC@LC treatment group had the highest OC=O functional group content (11.82%), indicating that amino acid-modified biochar can increase the amount of oxygen-containing functional groups in biochar, thereby facilitating pollutant removal.

[0125] 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. Among them, the magnetically modified biochar changes more significantly than other materials, with a new peak appearing 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.

[0126] Different materials contain N functional groups with 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 content. Because graphitic nitrogen has high thermal stability and is not easy to change at high temperatures, it shows that the magnetically modified biochar has better stability after immobilizing laccase.

[0127] Different materials containing S functional groups exhibit two peaks at binding energies of 164.5 and 169.5 eV, representing R-SH and metal sulfate, respectively. Immobilization of laccase significantly increases the content of R-SH functional groups. Because thiol functional groups are highly susceptible to oxidation and therefore possess strong reducing properties, weakly oxidizing substances can react with thiols to form disulfides, where disulfide bonds play a crucial role in maintaining the spatial structure of proteins. Thiols also possess catalytic activity, effectively removing pollutants. This demonstrates the excellent pollutant removal capabilities of NH2-MBC@LC.

[0128] There are four peaks at binding energies of 711, 713, 724.5, and 733.4 eV for Fe 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.

[0129] The present invention also investigated the stability and reusability of the material under different environments. Using relative activity as an evaluation metric, the pH, temperature, and reusability of free and immobilized laccases were studied, with maximum activity being 100%. For pH stability, 10 mg of free and 10 mg of immobilized laccase were added to a centrifuge tube containing 30 mL of citrate-phosphate buffer (pH range 3-8). The residual activity of the free and immobilized laccases was then measured. For thermal stability, the procedure was similar to that for pH stability, except that the centrifuge tubes were immersed in a water bath with a temperature gradient (ranging from 15°C to 55°C) to achieve the optimal temperature. For reusability, the enzymatic activity of free and immobilized laccases was measured ten times using the ABTS assay.

[0130] from Figure 15As can be seen in the figure, the relative activity of laccase at different pH levels was calculated by selecting the highest activity under different conditions as the standard. It can be observed that free laccase activity reached its highest level at pH 5. At pH 4, the relative activity decreased slightly, reaching approximately 90%. Thereafter, the enzyme activity decreased at pH 3 and pH 6. At pH 7 and pH 8, the laccase activity decreased significantly, both below 70%. These results indicate that laccase exhibits high activity in weakly acidic environments, particularly at pH 5, where its activity is particularly pronounced. This may be related to the conformational stability of the enzyme and the charge state of its active site. Lower or higher pH levels may lead to enzyme denaturation or functional loss, thus affecting the enzyme's catalytic ability. Immobilized laccase activity loses some activity during the immobilization process. After amino acid modification, the effect of pH on immobilized laccase is reduced. Overall, the amino acid-modified biochar exhibits higher activity than the unmodified material at all pH levels, a phenomenon more pronounced at high pH, ​​where the relative activity of the amino acid-modified biochar is approximately 20% to 30% higher than that of the unmodified material. The NH2-MBC@LC material exhibits higher laccase activity than free laccase at pH 6, 7, and 8. This is likely due to three factors: amino acid modification increases the number of polar functional groups on the biochar surface, thereby enhancing interactions with the laccase, such as hydrogen and ionic bonds, which contributes to improved enzyme immobilization efficiency and activity; amino acid modification may form a thin film on the biochar surface, which may regulate the local pH around the enzyme, keeping the enzyme near its optimal pH even under external pH fluctuations, thereby maintaining high activity; and the modified biochar may have a higher specific surface area and more active sites, which may increase the amount of enzyme adsorbed, thereby increasing the total amount and activity of the immobilized enzyme.

[0131] exist Figure 16 Thermal stability can be used to explore the temperature tolerance of free and immobilized laccases. Both free and immobilized laccases showed the highest activity at 35°C. The activity of both showed the same trend under temperature changes: with increasing temperature, the activity first increased and then decreased. This indicates that increasing the temperature within the laccase's optimal range increases laccase activity and accelerates the reaction between laccase and substrate. Further increases in temperature disrupt the protein structure, leading to decreased activity. The activity of immobilized laccase decreased significantly less at high temperatures than that of free laccase. The NH2-MBC@LC composite material had higher laccase activity than free laccase at 45°C and 55°C, indicating that immobilization can improve the high-temperature stability of laccase.

[0132] exist Figure 17In the results, it can be seen that 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 to 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 to biochar, making the interaction between laccase and biochar stronger. This strengthened binding can reduce the shedding of enzyme during the reaction and improve the stability of repeated use. In addition, the addition of amino acids can improve the microenvironment on the surface of biochar and thus reduce enzyme inactivation. Since amino acids themselves have good biocompatibility, the modified biochar may be more suitable for long-term use in biocatalysis processes, thereby improving the enzymatic activity of laccase under repeated use.

[0133] 2. Adsorption kinetics of CP by biochar

[0134] The present invention investigates the adsorption kinetics of CP by biochar materials. 10 mg of 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. Figure 18 It can be seen that for the adsorption rate of CP, NH2-MBC>MBC material.

[0135] An application of laccase immobilized on amino acid magnetically modified biochar. The amino acid-modified magnetic biochar-immobilized laccase composite material has strong adsorption capacity, rich surface functional groups, and a large specific surface area. The composite material also has strong pollutant removal capabilities. The laccase contained on the material surface can oxidize and reduce CP adsorbed by the composite material under the conditions of mediator participation.

[0136] A 1g / L pesticide stock solution was prepared in advance. The pesticide and biochar were mixed in soil for 5 minutes to create a 20mg / kg contaminated soil. Treatment groups (CP, CP+LC, CP+BC, CP+MBC, CP+MBC@LC, CP+MBC@LC +HBT, CP+NH2-MBC@LC, CP+NH2-MBC@LC+HBT) were designed. Each treatment consisted of sterilized and non-sterilized soil, and incubated in the dark. Degradation kinetics: Pesticide content was measured at 1, 3, 7, 14, and 21 days. Chlorpyrifos was extracted from soil using the QuEChERS method. Preliminary spiked assays demonstrated pesticide recovery rates exceeding 90%, demonstrating the suitability of this method for extracting CP from soil. Residual concentrations were determined by high-performance liquid chromatography.

[0137] Determination of CP: The CP mobile phase 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, and the injection volume was 10 μL. The CP standard substance was dissolved in acetonitrile to obtain a 1 g / L mother solution. Chlorpyrifos solutions with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 mg / L were prepared. The absorbance was determined by HPLC. The fitting equation was y = 6.123x + 0.2485, where y is the absorbance and x is the CP concentration. The correlation coefficient R 2 =0.9992, which shows that the concentration and absorbance have a good linear relationship.

[0138] 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:

[0139] Degradation rate (%) =

[0140] The addition amount of NH2-MBC@LC composite material was 0.5% of the soil mass, and the selected laccase mediator substances were 1-hydroxybenzotriazole, syringaldehyde, and 2,2'-hydrazino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS). The optimal laccase mediator substance was 1-hydroxybenzotriazole, and the concentration of the mediator substance was 3mmol / L.

[0141] (1) Selection of mediator

[0142] At a temperature of 25°C and a pH of 5, 10 mg of LC was added, the CP concentration was 20 mg / L, and ABTS, HBT, and syringaldehyde (3 mmol / L) were selected as mediators. The degradation experiment was carried out in a brown headspace bottle in a dark environment. Samples were taken to determine the CP concentration within 1, 3, 6, 12, 24, and 48 hours of culture.

[0143] Figure 19The figure shows the temporal trend of CP removal. The degradation rate of CP increases continuously with time, with rapid growth before 6 hours. The degradation rate of chlorpyrifos reaches approximately 80% after 6 hours of addition of free laccase, laccase-HBT mediator, and laccase-syringaldehyde mediator. The degradation rate then slows, eventually leveling off. This is because the chlorpyrifos concentration is high at the beginning of the reaction, leading to frequent interactions between laccase and substrate and a rapid increase in the degradation rate. As the substrate is gradually degraded, its concentration decreases, reducing the opportunities for enzyme-substrate binding and thus slowing the degradation rate. Furthermore, with prolonged reaction times, the degradation rate gradually flattens due to the production of products that are detrimental to laccase activity and changes in the acid-base environment.

[0144] The laccase-mediator system (LMS) formed by laccase and a mediator can degrade non-phenolic compounds. To verify the role of mediators in laccase degradation of CP, three mediators (ABTS, HBT, and syringaldehyde) were designed to verify their promoting effect on degradation. At a final reaction time of 2 days, the degradation rates of CP among the different treatment groups ranked in the order: laccase-HBT mediator > laccase-syringaldehyde mediator > free laccase > laccase-ABTS mediator. The laccase-HBT mediator had a significantly higher degradation rate of CP than the other treatment groups. Therefore, HBT was selected as the laccase mediator.

[0145] (2) Degradation kinetics

[0146] 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 in a dark environment. Samples were taken within 1, 3, 6, 12, 24, and 48 hours of culture to determine the CP concentration.

[0147] from Figure 20 It can be concluded that the degradation rate of CP by different materials is 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 the 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, which can more effectively catalyze the oxidation of organic pollutants such as chlorpyrifos.

[0148] Secondly, amino acid-modified biochar increases surface functionality by introducing amino groups, thereby improving its adsorption capacity for organic pollutants such as chlorpyrifos. This modification can increase the surface polarity of the biochar, promoting interaction with pollutants and thus improving adsorption efficiency. Amino acid modification may provide a more suitable microenvironment for laccase, thereby enhancing the activity of immobilized laccase. In this suitable microenvironment, laccase can more effectively catalyze the oxidation of chlorpyrifos and promote its degradation. A synergistic effect may exist between amino acid-modified biochar and laccase, allowing the modified biochar to serve not only as an adsorbent but also as an enzyme carrier, improving overall degradation efficiency.

[0149] (3) Removal of CP in soil environment

[0150] Field soil was collected in Junan County, Linyi. After impurities were removed, the soil sample was divided into two portions. One portion was sterilized, and 40 g of soil was weighed and placed in a glass vial. 1 g / L CP stock solution was added to prepare simulated contaminated soil containing 20 mg / kg CP. LC, BC, MBC, MBC@LC, NH2-MBC@LC, MBC@LC and HBT, and NH2-MBC@LC and HBT were added to the soil at 0.5% soil mass to prepare different treatment groups. The soil moisture content was maintained at 60%. Samples were collected on day 1, 3, 7, 14, and 21. After grinding through a 60-mesh sieve, 10 g of soil was weighed and placed in a 50 mL centrifuge tube. Acetonitrile (10 mL) was added. After shaking in an incubator at 200 rpm for 6 h, NaCl (2 g) and anhydrous magnesium sulfate (4 g) were added to the soil sample. The sample was shaken for 30 min and then centrifuged at 4000 rpm for 5 min. 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.

[0151] Figure 21 、 22As can be seen from the two figures, 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 by all treatment groups was NH2-MBC@LC+HBT>MBC@LC+HBT>NH2-MBC@LC>MBC@LC> MBC>LC>BC, and reached about 60% in sterilized soil. The removal effect of chlorpyrifos by 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 degradation efficiency. The laccase-mediator system, formed by laccase and the mediator HBT, further promoted CP removal, reaching approximately 9% higher levels than the untreated group. This is because HBT, a commonly used redox mediator, plays a role in LC-catalyzed reactions. By promoting electron transfer, it enhances the catalytic efficiency of LC, making CP degradation more efficient. MBC exhibited approximately 6% higher CP removal than BC. This is due to the inclusion of magnetic materials during the preparation of MBC. These materials not only increase the surface area and porosity of BC but also potentially enhance adsorption properties. Furthermore, the magnetic modification allows the BC surface to be loaded with iron-containing functional groups, which promote CP degradation.

[0152] (4) Degradation mechanism of chlorpyrifos, Figure 23 Schematic diagram of the degradation mechanism of CP by NH2-MBC@LC composite material.

[0153] In order to explore the degradation mechanism of chlorpyrifos by immobilized laccase functional material, the degradation products of chlorpyrifos in the NH2-MBC@LC treatment group with HBT mediator at different time periods were determined by high performance liquid chromatography-mass spectrometry. It was found that chlorpyrifos was first converted into methyl chlorpyrifos, chlorpyrifos-oxy and o-diethyl o-hydrothiophosphate 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 was the cleavage of OP bond to convert it into 3,5,6-trichloro-2-pyridinol (TCP), and the second was the cleavage of CO bond to form P=O double bond, similar to the shedding of functional groups on the benzene ring to form diethyl phosphate. Studies have shown that TCP is the most toxic of chlorpyrifos byproducts. Therefore, investigating the degradation of TCP is a key indicator of chlorpyrifos contamination remediation. At the final 21-day reaction time, a high concentration of 2-chloro-6-methoxypyridine was observed as a byproduct, indicating that TCP undergoes further alkylation and dechlorination to form 2-chloro-6-methoxypyridine, which is key to the complete detoxification of chlorpyrifos. In subsequent degradation reactions, diethyl phosphate further sheds its methyl group to form dimethyl phosphate, which continues to degrade under the action of NH2-MBC@LC to produce CO2 and H2O.

[0154] Table 1 CP degradation-related byproducts

[0155]

[0156] This application adopts Fe 2+ and Fe 3+ Magnetic modified biochar was prepared by co-precipitation under alkaline conditions. 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 amino acid modification significantly increases the surface active sites of biochar, thereby effectively promoting its catalytic degradation of organic pollutants.

[0157] Example 2:

[0158] This example differs from Example 1 in that the citrate phosphate buffer solution was changed to 3, 4, 6, 7, and 8. The remainder of the solution was identical to Example 1, yielding MBC@LC with enzyme activities of 117.8 U / g, 131.2 U / g, 130.2 U / g, 91.2 U / g, and 95.6 U / g, respectively.

[0159] Example 3:

[0160] This example differs from Example 1 in that the proportions of glutaraldehyde solution added were changed to 0.25%, 0.5%, 1%, 2%, and 8%. The remaining reactions were identical to those in Example 1, yielding MBC@LC with enzyme activities of 115.37 U / g, 129.33 U / g, 152.4 U / g, 161.6 U / g, and 137.0 U / g, respectively.

[0161] Example 4:

[0162] This example differs from Example 1 in that the laccase addition amount was changed to 5, 10, 15, and 25 mg, and the rest was identical to Example 1, yielding MBC@LC with enzyme activities of 83.12 U / g, 109.4 U / g, 157.6 U / g, and 170.7 U / g, respectively.

[0163] Example 5:

[0164] This example differs from Example 1 in that the crosslinking oscillation time was changed to 2, 3, 6, and 8 h. The remainder of the reaction was identical to Example 1, yielding MBC@LC with enzyme activities of 170.9 U / g, 185.1 U / g, 144.7 U / g, and 115.2 U / g, respectively.

[0165] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing laccase immobilized on amino acid magnetically modified biochar, characterized in that: By loading amino functional groups on the surface of magnetically modified biochar, making it bind to laccase and immobilizing laccase, amino acid-modified magnetic biochar-immobilized laccase is obtained. The steps include: (1) Preparation of magnetically modified biochar: 10g corn straw powder, 0.02 mol Fe 3+ and 0.01 mol Fe 2+ The mixture was dispersed in 300 mL of pure water, stirred, and then adjusted to pH 10-13 by adding NaOH. The mixture was allowed to stand for one day, the supernatant was removed, and the precipitate was washed with pure water to pH 7. The mixture was then dried at 120°C and placed in a tube furnace under a nitrogen atmosphere. The pyrolysis temperature was gradually increased to 600°C at a heating rate of 5°C / min and kept constant for 2 hours. The magnetically modified biochar was then fired. (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 was mixed with a buffer solution containing laccase, and after adding glutaraldehyde as a cross-linking agent, the mixture was shaken to obtain amino acid-modified magnetic biochar-immobilized laccase. 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; 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 solution 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 3-8; the shaking box temperature is 15-55°C, and the fixed time is 4-6 hours; 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 and shaking for 24 hours, then 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 at 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 mixture was shaken at room temperature for 4 h, filtered, washed, and dried to obtain activated amino acid-modified biochar. 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 a 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 the dark at room temperature, shaking it for 1 hour, adding 4% glutaraldehyde solution as a cross-linking agent to immobilize the laccase, and then shaking it in the dark for 2 hours, filtering and washing the precipitate three times with a citric acid phosphate buffer solution with a pH of 5, and air-drying it naturally to obtain the amino acid magnetic modified biochar immobilized laccase; In (1), Fe 3+ and Fe 2+ Derived from ferric chloride and ferrous sulfate.

2. Application of an amino acid magnetic modified biochar immobilized laccase, amino acid magnetic modified biochar immobilized laccase Prepared according to the method of claim 1, characterized in that: Amino acid magnetic modified biochar immobilized laccase for the removal of chlorpyrifos pollution in the environment; In a soil environment with a chlorpyrifos concentration of 20 mg / kg, laccase immobilized on amino acid magnetically modified biochar accounting for 0.5% of the soil mass was added, and then 1-hydroxybenzotriazole was added to form a laccase-mediator system to remove chlorpyrifos.

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