Biochar immobilized laccase method for efficiently degrading crop continuous cropping phenolic acid autotoxic substances

Abstract

The invention relates to a method for efficiently degrading phenolic acid autotoxic substances in continuous cropping of crops by using biochar immobilized laccase. Pyrolyzing the tobacco stem and straw powder in a muffle furnace to obtain tobacco stem and straw biochar BC; the preparation method comprises the following steps: soaking BC and potassium carbonate in a mass ratio of 1: 4 for 2-4 hours, drying, pyrolyzing in a muffle furnace after drying, air-cooling to room temperature, washing with deionized water, drying and sieving to obtain potassium carbonate modified tobacco stem and straw biochar CBC; dispersing CBC in an HNO3 solution, mixing for 3-5 hours at the speed of 300rpm and the temperature of 30 DEG C, washing with deionized water until the pH value is 7, drying overnight, and storing at room temperature to obtain functionalized CBC; placing the functionalized CBC in a 50mM phosphate buffer solution for 20-30 hours, adding 1.0% w / v glutaraldehyde with the same volume as the functionalized CBC, oscillating at room temperature, washing with redundant glutaraldehyde deionized water, and drying overnight to obtain glutaraldehyde-containing CBC; and carrying out enzyme immobilization on the glutaraldehyde-containing CBC and a 5-10U / mL laccase solution according to a solid-to-liquid ratio of 1g: 10mL at 25-30 DEG C to obtain the immobilized laccase. Autotoxic substances are effectively degraded, the yield and quality of peppers are improved, and ecological restoration of soil is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of laccase immobilization, and in particular to a biochar immobilization laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession. Background Art

[0002] Pepper is one of the important vegetable industries in my country. However, the limited arable land area and regional climatic conditions have caused pepper to have continuous cropping problems. Studies have shown that the main reason for the formation of continuous cropping problems is the accumulation of self-toxic substances secreted by pepper roots. Phthalic acid (PA) is a common phenolic acid organic compound and a type of self-toxic substance secreted by pepper roots. The long-term accumulation of phthalic acid in the soil will destroy the biodiversity and ecological functions of the soil, have an adverse effect on the root structure, and inhibit the growth and development of plants. Affected by this, the leaves of plants turn yellow, grow slowly, or even die, which in turn affects the yield. Therefore, it is crucial to find a suitable treatment method for the removal of PA.

[0003] The use of enzymes as catalysts is widely used in the study of pollutant removal due to their low toxicity, greenness, mild reaction conditions and high selectivity. Laccase is a type of multi-copper oxidase widely found in organisms such as fungi, plants and insects, which can directly catalyze the oxidation of various compounds and reduce oxygen to water. Laccase catalyzes the oxidation of various difficult-to-degrade environmental pollutants, such as phenols, lignin, aromatic amines, carboxylic acids, pesticides, dyes and other toxic compounds. Laccase has attracted attention for its ability to catalyze the oxidation of various compounds and is a biocatalyst with application potential and development prospects. However, the instability and non-reusability of free laccase have become the main limiting factors for practical applications.

[0004] Enzyme immobilization has become an effective solution to solve the instability and non-reusability of enzymes. Currently, the commonly used immobilization methods include adsorption, embedding, covalent bonding and cross-linking. The cross-linking method uses a cross-linking agent to make the carrier and the enzyme more firmly bonded, and the enzyme is not easy to leak from the carrier, thereby improving the degradation of pollutants. At present, there are many materials used as immobilization carriers. However, their preparation methods are complicated, the immobilization process is complicated and the cost is high, which is not suitable for widespread application in soil. Biochar is a carbon-rich solid obtained by high-temperature pyrolysis of biomass. It has abundant raw material sources, low cost, high porosity, high surface area, surface functional groups and environmental friendliness, showing great degradation potential in pollutant treatment. At the same time, biochar can improve soil properties, soil microbial community structure and allelopathic substances to inhibit self-toxicity. Therefore, biochar is used as an enzyme immobilization material to degrade PA. However, in the preparation process of biochar, factors such as pyrolysis temperature, time and atmosphere will significantly affect its physical and chemical properties, thereby affecting the immobilization effect of laccase and the subsequent degradation ability of PA. In addition, environmental factors in the soil such as humidity, temperature, pH, and other coexisting substances will affect the performance of biochar-immobilized laccase composites and need to be comprehensively considered during research and application. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a biochar immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping, which has high stability, can effectively degrade self-toxic substances, improve pepper yield and quality, and promote soil ecological restoration.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] A biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping comprises the following steps:

[0008] S1, placing tobacco straw powder in a sealed crucible in a muffle furnace for pyrolysis to obtain tobacco straw biochar BC;

[0009] S2, soaking tobacco straw biochar and potassium carbonate in a mass ratio of 1:4 for 2-4 hours, drying, sealing the dried biochar crucible and pyrolyzing it in a muffle furnace, then air-cooling to room temperature, washing with deionized water, drying, sieving, and obtaining potassium carbonate-modified tobacco straw biochar CBC;

[0010] S3, dispersing the potassium carbonate modified tobacco straw biochar CBC in HNO3 solution, mixing at 300rpm and 30℃ for 3-5h to obtain a functionalized biochar suspension, washing with deionized water to a pH of 7, drying overnight, and storing at room temperature to obtain functionalized CBC;

[0011] S4, placing the functionalized CBC in step S3 in 50 mM phosphate buffer for 20-30 h, adding the same volume of 1.0% w / v glutaraldehyde, shaking at room temperature, washing the excess glutaraldehyde with deionized water, drying overnight, and storing to obtain the glutaraldehyde-containing CBC;

[0012] S5. Mix the glutaraldehyde-containing CBC in step S4 with 5-10 U / mL laccase Lac solution at a solid-liquid ratio of 1 g:10 mL at 25-30° C. to immobilize the enzyme, thereby obtaining immobilized laccase LC-CBC.

[0013] Moreover, the pyrolysis temperature in the muffle furnace in step S1 is 300-600° C., and the pyrolysis time is 2-4 hours.

[0014] Moreover, the pyrolysis temperature of the muffle furnace in step S2 is 400-700° C., the pyrolysis time is 2-4 hours, and the pyrolysis rate is 5° C. / min.

[0015] Furthermore, in step S4, the mixture was shaken at 300 rpm at room temperature for 10-15 h.

[0016] Also, the temperature for overnight drying is 25-45°C.

[0017] Moreover, the concentration of the HNO3 solution is 75-95%.

[0018] A method for degrading phthalic acid by using biochar-immobilized laccase, comprising the following steps:

[0019] Immobilized laccase is added to a 20 mg / L phthalic acid solution, and the solution is oscillated at an optimum temperature in a constant temperature oscillator. The sample is filtered through a 0.22 μ filter membrane, and the phthalic acid concentration of the obtained sample is determined by a high performance liquid chromatograph. The reaction pH is 4.0-6.0, the temperature is 25-35° C., the initial phthalic acid concentration is 20 mg / L, the immobilized laccase dosage is 1-4 U / mL, and the reaction time is 10-12 h. The immobilized laccase is the immobilized laccase LC-CBC prepared by the above method.

[0020] A method for removing phthalic acid and other similar phenolic acid substances in soil, comprising the steps of:

[0021] Immobilized laccase is added to the soil, the pH of the soil is 3.0-7.0, the temperature is 15-45°C, the dosage of the immobilized laccase is 10-50U / mL, the degradation time is 10-12h, and the immobilized laccase is the immobilized laccase LC-CBC prepared by the above method.

[0022] A method for improving the germination rate of peppers in soil, comprising the following steps:

[0023] Immobilized laccase is added to the pepper soil solution containing phthalic acid, the pH of the solution is controlled to be 4-8, the temperature is 20-40°C, and the reaction is carried out for 10-12 hours. The immobilized laccase is the immobilized laccase LC-CBC prepared by the above method.

[0024] A soil conditioner for improving the germination rate of pepper in soil, comprising the following steps:

[0025] The immobilized laccase is mixed evenly with an inert filler and a binder in a mass ratio of 30-60:30-50:10-20, and the mixture is made into a granular or powdery soil conditioner product by extrusion and granulation, wherein the immobilized laccase is the immobilized laccase LC-CBC prepared by the above method;

[0026] The inert filler is at least one of vermiculite, perlite, ceramsite or rock wool; and the binder is at least one of starch, bentonite or sodium carboxymethyl cellulose.

[0027] The advantages and positive effects of the present invention are:

[0028] 1. By comparing the tobacco stem biochar modified by potassium carbonate, the tobacco stem biochar modified by potassium hydroxide, and the tobacco stem biochar modified by potassium citrate, the present invention finds that the combination of biochar and potassium carbonate shows better advantages. The experiments confirm that the surface areas of CBC, KBC, and NBC after different alkali etching are 644.95, 243.36, and 482.24 m 2 / g. The present invention selects tobacco straw biochar as an immobilization carrier, fixes laccase to the biochar by glutaraldehyde cross-linking, investigates the effects of different modification methods on the loading amount and activity of laccase, and determines the enzyme stability by exploring the pH value, thermal stability, storage and operation stability, and repeatability of laccase (Lac) and potassium carbonate-modified biochar-immobilized laccase (LC-CBC), and concludes that LC-CBC has better stability.

[0029] 2. The present invention uses different alkali-etched tobacco stem biochar as a carrier for immobilized laccase, and determines the optimal immobilized laccase carrier by loading amount and enzyme activity. Under optimized conditions, the laccase loading amount reaches 177.32U / g. The immobilized laccase has good tolerance to high pH and high temperature, and has good thermal stability and storage stability. The immobilized enzyme can be reused for 7 reaction cycles while retaining 51% of its activity. Phthalic acid is treated with laccase-immobilized biochar. Phthalic acid degradation was confirmed by HPLC and GC-MS analysis. The high adsorption potential of biochar and the efficient catalytic effect of laccase make the removal rate of phthalic acid reach 100%. At the same time, the degraded phthalic acid is more conducive to the growth and germination of peppers. Therefore, biochar can be used as a low-cost carrier for immobilized laccase and used to remove phthalic acid and other similar phenolic acid substances in the soil.

[0030] 3. The present invention uses treated phthalic acid, untreated phthalic acid and control samples to culture pepper seeds, and it is found that phthalic acid has an inhibitory effect on the germination of pepper seeds, but the inhibitory effect on seed germination is reduced after phthalic acid is treated with potassium carbonate modified biochar immobilized laccase. Taking distilled water as a control, the untreated phthalic acid reduces the germination rate of pepper seeds by 18% compared with the CK group, while the germination rate of the treated phthalic acid is not significantly different from the control treatment, indicating that phthalic acid after potassium carbonate modified biochar treatment can reduce the inhibition of pepper seed germination, which is beneficial to the germination of pepper seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a desorption isotherm model diagram of the tobacco straw biochar, potassium carbonate activated biochar, potassium hydroxide activated biochar, and potassium citrate activated biochar of the present invention.

[0032] Figure 2 This is the pore size distribution diagram of the tobacco straw biochar, potassium carbonate activated biochar, potassium hydroxide activated biochar, and potassium citrate activated biochar of the present invention.

[0033] Figure 3 This is a diagram showing the laccase loading and enzyme activity on biochar prepared by different alkali treatment methods in the present invention.

[0034] Figure 4 It is a Fourier transform infrared spectra of the potassium carbonate activated biochar, laccase and potassium carbonate modified biochar immobilized laccase of the present invention.

[0035] Figure 5 Scanning electron microscope images of laccase immobilized on tobacco straw biochar, potassium carbonate activated biochar, and potassium carbonate modified biochar of the present invention.

[0036] Figure 6This is a diagram showing the element distribution of the potassium carbonate activated biochar of the present invention.

[0037] Figure 7 This is a diagram showing the element distribution of laccase immobilized on potassium carbonate-modified biochar of the present invention.

[0038] Figure 8 This is the distribution diagram of carbon, oxygen and nitrogen elements of the potassium carbonate activated biochar of the present invention.

[0039] Fig. 9 This is the distribution diagram of carbon, oxygen and nitrogen elements of laccase immobilized on potassium carbonate modified biochar of the present invention.

[0040] Fig.10 This is a graph showing the effect of pH on the stability of laccase in free laccase and laccase immobilized in potassium carbonate-modified biochar.

[0041] Fig.11 This is a graph showing the effect of temperature on the stability of laccase in free laccase and laccase immobilized in potassium carbonate-modified biochar.

[0042] Fig.12 This is a graph showing the effect of storage time on the stability of laccase in free laccase and laccase immobilized in potassium carbonate-modified biochar.

[0043] Fig.13 This is a graph showing the effect of pH on the degradation efficiency of phthalic acid by laccase, tobacco straw biochar-immobilized laccase and potassium carbonate-modified biochar-immobilized laccase of the present invention.

[0044] Fig.14 This is a graph showing the effect of temperature on the degradation efficiency of phthalic acid by laccase, tobacco straw biochar-immobilized laccase and potassium carbonate-modified biochar-immobilized laccase of the present invention.

[0045] Fig.15 This is a graph showing the effect of phthalic acid on the degradation efficiency of phthalic acid by laccase, tobacco straw biochar-immobilized laccase and potassium carbonate-modified biochar-immobilized laccase of the present invention.

[0046] Fig.16 This is a graph showing the effects of the dosage of laccase, tobacco straw biochar-immobilized laccase on the degradation efficiency of phthalic acid by laccase and potassium carbonate-modified biochar-immobilized laccase.

[0047] Fig.17 This is a graph showing the effect of time on the degradation efficiency of phthalic acid by free laccase, tobacco straw biochar-immobilized laccase and potassium carbonate-modified biochar-immobilized laccase.

[0048] Fig.18 This is an influence diagram of the degradation efficiency of different phenolic acid substances by the laccase of the present invention, the laccase immobilized on tobacco straw biochar and the laccase immobilized on potassium carbonate modified biochar.

[0049] Fig.19 This is a diagram showing the reusability of the potassium carbonate-modified biochar-immobilized laccase in the degradation of polycyclic aromatic hydrocarbons and the oxidation of 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) of the present invention.

[0050] Fig. 20 This is a graph showing the effects of distilled water, phthalic acid, and degraded phthalic acid on pepper seed germination.

[0051] Fig.21 This is a diagram showing the degradation mechanism of phthalic acid by laccase immobilized on potassium carbonate-modified biochar of the present invention.

[0052] Fig. 22 This is an analysis of the degradation products of phthalic acid and the acute toxicity of phthalic acid by laccase immobilized on potassium carbonate-modified biochar of the present invention.

[0053] Fig.23 This is a graph showing the degradation products of phthalic acid and the chronic toxicity of phthalic acid by laccase immobilized on potassium carbonate-modified biochar of the present invention. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0055] Material preparation: Tobacco stalk biomass was collected locally in Yunnan. Laccase (Lac) (from Trametes versicolor); potassium hydroxide (KOH 99.99%), potassium citrate (C6H5K3O7 98%), potassium carbonate (K2CO3 99%), potassium dihydrogen phosphate (KH2PO4 99.5%), sodium dihydrogen phosphate (NaH2PO4 99%), glutaraldehyde (GA 50% H2O), Coomassie brilliant blue G250, 2,2'-azobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), phthalic acid (PA 99.5%), cinnamic acid (TC 99%), coumaric acid (CA 97%) and p-hydroxybenzoic acid (HBA 99.5%) were purchased from Shanghai MacLean Biochemical Co., Ltd. Ferulic acid (FA 99%) and disodium hydrogen phosphate (Na2HPO4 99.99%) were purchased from Aladdin Biochemical Technology. Methanol (HPLC) was purchased from Merck, Germany, nitric acid (HNO3 85%) was purchased from Sinopharm Chemical Reagent Co., Ltd., and the water used in the experiment was deionized water (Merck Millipore Milli-Q EQ 7008).

[0056] Example 1

[0057] A method for immobilizing laccase on biochar for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping. This embodiment provides a method for immobilizing laccase on CBC, comprising the following steps:

[0058] S1, placing tobacco straw powder in a muffle furnace at 500°C for 2h to pyrolyze to obtain tobacco straw biochar BC;

[0059] S2, soaking tobacco straw biochar and potassium carbonate in a mass ratio of 1:4 for 2 h, drying in an oven at 110 ° C, sealing the dried biochar in a crucible and pyrolyzing it at 600 ° C in a muffle furnace for 2 h, with a heating rate of 5 ° C / min, and then air-cooling to room temperature, washing the prepared biochar with deionized water several times to remove ash and alkali, and then drying at 105 ° C and sieving to obtain CBC;

[0060] S3, 20 g CBC was dispersed in 400 mL 85% HNO3, mixed at 300 rpm and 30°C for 4 h to obtain functionalized biochar, washed with deionized water to pH 7, dried at 80°C overnight, and stored at room temperature to obtain functionalized CBC;

[0061] S4, placing the functionalized CBC in step S3 in 50 mL 50 mM phosphate (PBS) buffer (pH 7.0) for 24 h, then adding the same volume of 1.0% w / v GA to the solution, shaking at room temperature (25° C.) for 12 h, washing the excess GA with deionized water, drying at 80° C. overnight, and storing to obtain CBC containing glutaraldehyde;

[0062] S5, mixing the glutaraldehyde-containing CBC in step S4 with Lac at a solid-liquid ratio of 1 g:10 mL at 25° C. (300 rpm) to immobilize the enzyme to obtain LC-CBC;

[0063] S6. Incubate the mixture in step S5 for 3 h, separate the enzyme-loaded biochar LC-CBC, wash it twice with buffer, and measure the enzyme activity.

[0064] Example 2

[0065] A method for immobilizing laccase on biochar for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping. This embodiment provides a method for immobilizing laccase on KBC, comprising the following steps:

[0066] S1, placing tobacco straw powder in a muffle furnace at 500°C for 2h to pyrolyze to obtain tobacco straw biochar BC;

[0067] S2, soaking tobacco straw biochar and potassium hydroxide in a mass ratio of 1:4 for 2 h, drying in an oven at 110 ° C, sealing the dried biochar in a crucible and pyrolyzing it at 600 ° C in a muffle furnace for 2 h, with a heating rate of 5 ° C / min, and then air-cooling to room temperature, washing the prepared biochar with deionized water several times to remove ash and alkali, and then drying at 105 ° C and sieving to obtain KBC;

[0068] S3, 20 g KBC was dispersed in 400 mL 85% HNO3, mixed at 300 rpm and 30 °C for 4 h to obtain a functionalized biochar suspension, washed with deionized water to a pH of 7, dried at 80 °C overnight, and stored at room temperature to obtain functionalized CBC;

[0069] S4, placing the functionalized KBC in step S3 in 50 mL 50 mM phosphate (PBS) buffer (pH 7.0) for 24 h, then adding the same volume of 1.0% w / v GA to the solution, shaking at room temperature (25° C.), washing the excess GA with deionized water, drying at 80° C. overnight, and storing to obtain glutaraldehyde-containing CBC;

[0070] S5, mixing the glutaraldehyde-containing KBC and Lac in step S4 at a solid-liquid ratio of 1 g:10 mL at 25° C. (300 rpm) to immobilize the enzyme to obtain LC-KBC;

[0071] S6. Incubate the mixture in step S5 for 3 h, separate the enzyme-loaded biochar LC-KBC, wash it twice with buffer, and measure the enzyme activity.

[0072] Example 3

[0073] A method for immobilizing laccase on biochar for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping is provided in this embodiment. The method comprises the following steps:

[0074] S1, placing tobacco straw powder in a muffle furnace at 500°C for 2h to pyrolyze to obtain tobacco straw biochar BC;

[0075] S2, soaking tobacco straw biochar and potassium citrate in a mass ratio of 1:4 for 2 h, drying in an oven at 110 ° C, sealing the dried biochar in a crucible and pyrolyzing it at 600 ° C in a muffle furnace for 2 h, with a heating rate of 5 ° C / min, and then air-cooling to room temperature, washing the prepared biochar with deionized water several times to remove ash and alkali, and then drying at 105 ° C and sieving to obtain NBC;

[0076] S3, 20 g NBC was dispersed in 400 mL 85% HNO3, mixed at 300 rpm and 30 °C for 4 h to obtain a functionalized biochar suspension, washed with deionized water to a pH of 7, dried at 80 °C overnight, and stored at room temperature to obtain a functionalized CBC;

[0077] S4, placing the NBC in step S3 in 50 mL 50 mM phosphate (PBS) buffer (pH 7.0) for 24 h, then adding the same volume of 1.0% w / v GA to the solution, shaking at room temperature (25°C), washing the excess GA with deionized water, drying at 80°C overnight, and storing to obtain CBC containing glutaraldehyde;

[0078] S5, mixing the NBC and Lac in step S4 at a solid-liquid ratio of 1 g:10 mL at 25° C. (300 rpm) to immobilize the enzyme to obtain LC-NBC;

[0079] S6. Incubate the mixture in step S5 for 3 h, separate the enzyme-loaded biochar LC-NBC, wash it twice with buffer, and measure the enzyme activity.

[0080] Example 4

[0081] A method for immobilizing laccase on biochar for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping is provided in this embodiment, and the method comprises the following steps:

[0082] S1, placing tobacco straw powder in a muffle furnace at 500°C for 2h to pyrolyze to obtain tobacco straw biochar BC;

[0083] S2, mixing BC and Lac in step S1 at a solid-liquid ratio of 1 g:10 mL at 25°C (300 rpm) for enzyme immobilization to obtain LC-BC;

[0084] S3. Incubate the mixture in step S2 for 3 h, separate the enzyme-loaded biochar LC-BC, wash it twice with buffer, and measure the enzyme activity.

[0085] Performance tests and results for the above embodiments

[0086] 1. Enzyme activity and enzyme loading

[0087] The controlled variable method was used to determine the enzyme activity and loading amount of the four immobilized laccases prepared to obtain the optimal biochar-immobilized laccase material.

[0088] Lac activity is defined as the amount of Lac required to oxidize 1 μmol ABTS per unit time. The Lac sample to be tested and ABTS (0.3 mL) were added to a PBS buffer solution at pH 4 for 20 minutes. Then, ABTS oxidation was measured by measuring the absorbance change at a wavelength of 420 nm using a UV-visible spectrophotometer (TU-1810D, China), and the enzyme activity of Lac was calculated using equation (1). The enzyme activity of immobilized Lac was calculated using equation (2):

[0089]

[0090] Where ΔA represents the absorbance change; Df represents the dilution factor; V represents the reaction volume mL; ε represents the molar extinction coefficient (3.6×10 4 M -1 cm -1 ); T represents the reaction time in min; v represents the amount of enzyme in mL and m represents the amount of biochar fixed by laccase in g.

[0091] In addition, the highest measured laccase activity was taken as 100%, and the remaining laccase activities were compared with it to obtain the relative activity calculated using equation (3):

[0092]

[0093] In the formula, A represents enzyme activity U / mL or U / g and A max Represents the maximum enzyme activity measured in the experiment.

[0094] The total protein content in the supernatant before and after the reaction was determined by the Coomassie brilliant blue method. The enzyme concentration was measured at 595 nm by a UV-visible spectrophotometer (TU-1810D, China), and the enzyme load was calculated using equation (4).

[0095]

[0096] Where C0 represents the initial concentration of the enzyme in mg / mL; C1 represents the laccase concentration in the supernatant after immobilization in mg / mL; V represents the volume of the solution in mL; and m represents the total mass of the material in mg.

[0097] 2. Stability Assessment

[0098] The pH stability was tested by immersing free and immobilized laccase in PBS buffer with different pH (i.e., 3, 4, 5, 6, 7, and 8) at 30°C for 2 h. The storage stability was evaluated by maintaining free and immobilized laccase at 4°C and 25°C for 30 days, and the enzyme activity was measured every 5 days. The reusability of immobilized laccase was studied for up to 7 reaction cycles using ABTS as substrate. The enzyme activity is expressed as relative activity.

[0099] 3. PA degradation experiment

[0100] Prepare several portions of 3mL of 20mg / L PA solution, add appropriate amount of immobilized laccase and equal concentration of Lac. Effects of different reaction pH (from 5.0 to 8.0), different reaction temperature (from 25 to 55℃), different initial PA concentration (from 20 to 70mg / L), different enzyme dosage (from 1 to 10U / mL) and different reaction time (1-24h) on PA degradation efficiency. Constant temperature oscillator was oscillated at 30℃. In each independent experiment, one parameter was changed while the other parameters remained unchanged. All samples were performed in triplicate. The degradation of PA by immobilized laccase was repeated 7 times. At the end of each degradation, the supernatant was aspirated, and the immobilized laccase was rinsed twice with PBS (20mM, pH 6.0) to remove residual substrate, and then PA solution was added for the next cycle. Prepare several 3 mL 20 mg / L FA, TC, CA and HBA solutions, add appropriate amount of immobilized laccase and equal concentration of Lac, oscillate at 30°C in a constant temperature oscillator, and aspirate the supernatant at the end of degradation. All samples were prepared in triplicate. The samples were filtered with a filter membrane and the concentration was determined by high performance liquid chromatography (HPLC-Agilent 1260, USA).

[0101] IV. Biological Tests

[0102] Select plump pepper seeds and immerse them in 3% sodium hypochlorite solution for disinfection for 5 minutes, and rinse them with distilled water 5 times. Three layers of qualitative filter paper were laid on the culture dish, and 5mL of PA solution, PA solution after treatment with biochar immobilized laccase material, and distilled water were injected into the culture dish as the control group. The filter paper was kept moist during seed germination. 30 seeds were sown in each dish, and each treatment was repeated 3 times, and cultured in a 28℃ incubator. The number of seed germination and root length values ​​were counted with a bud length of 1mm as the standard.

[0103] V. Analytical methods and instruments

[0104] The surface morphology of the prepared materials was characterized by field emission scanning electron microscopy (SEM Zeiss Gemini 300Germany); the surface element distribution of the prepared materials was analyzed by energy dispersive spectrometer (EDS Zeiss Gemini 300Germany). The specific surface area and pore size distribution of the prepared materials were determined by N2 adsorption and desorption measurements at liquid nitrogen temperature (77K) using a fully automatic specific surface and porosity analyzer (BET Micromeritics ASAP 2460USA). The functional groups on the surface of the materials were obtained using Fourier transform infrared spectroscopy (FTIR Thermo Fisher Niolet iN10). The intermediates and degradation pathways of the catalytic reaction were analyzed using an Agilent 1290-6550 series high performance liquid chromatography-mass spectrometer (HPLC-QTOF).

[0105] 6. Data Processing

[0106] The experimental data were processed using Excel 2021 and Origin 2021 software, and the data were expressed as the mean ± standard error of three measurements. DMASA was used for one-way analysis of variance with the least significant difference (LSD) post hoc comparison test and the Waller-Duncan test. The toxicity of PA and its intermediates was evaluated using ECOSAR software.

[0107] VII. Effect of modified biochar on the preparation of immobilized laccase

[0108] Specific surface area is an important factor affecting adsorption performance. In order to further explore the specific surface area and pore size of BC after modification using different methods, BET characterization of BC, CBC, KBC and NBC was completed. The base modification process may eliminate the substances blocking the pores and lead to the formation of additional pores, increasing the surface area of ​​BC, which has been confirmed by surface area measurement.

[0109] As shown in Table 1, the surface area of ​​untreated BC is 2.49 m 2 / g, the surface areas of CBC, KBC and NBC after different alkali etching were 644.95, 243.36 and 482.24 m 2 / g. The results showed that the surface areas of CBC, KBC and NBC increased significantly compared with BC. After Lac fixation, the BET surface area, average pore size and pore volume of LC-CBC were 179.472 m 2 / g, 2.41nm and 0.021cm 3 / g, the specific surface area decreased by 72.17% compared with CBC, but the average pore size increased by about 1.15 times. The reduction in BET surface area may be due to Lac occupying the surface area of ​​CBC, or Lac covalently cross-linked with GA and retained on the surface of CBC. The obvious reduction in BET surface area proves that Lac was successfully fixed to CBC.

[0110] Figure 1 The adsorption-desorption isotherms in the graph show that the adsorption-desorption isotherms of BC modified into CBC, KBC and NBC change from type I to type IV with H4 hysteresis loop, reflecting the structural characteristics of the four different porous materials. Figure 2 The pore size distribution in the pores is mainly concentrated in the micropores and mesopores between 0 and 10 nm. In order to select the best support for Lac immobilization, the same concentration of Lac was immobilized on biochars with different treatments, such as Figure 3 The results show that the immobilization of Lac on K2CO3, potassium citrate and KOH is significantly higher than that on the original biochar, and K2CO3 is higher than potassium citrate and KOH. The loading amounts of K2CO3, potassium citrate, KOH and the original biochar are 177.32U / g, 156.45U / g, 116.84U / g and 81.65U / g, respectively. Therefore, CBC is selected as the best support for Lac immobilization.

[0111] like Figure 4 As shown, the FTIR spectra showed important chemical bond characteristics of CBC, pure laccase and LC-CBC. For CBC, OH bond stretching of the hydroxyl functional group was observed at 3446 cm -1 The peak value is 2921cm -1 and 1619cm -1 Reflects the CH bonds and C=C bonds of the aromatic ring. For LC-CBC, at 3434cm -1 and 2918cm -1 The OH and CH bonds are located at 1633 cm -1 The peak at 1000-1400 cm is the C=O stretching vibration of amide I after Lac fixation. The CN bond and NH bond are at 1000-1400 cm -1 The stretching vibration peak at 1525 cm -1 Showing the presence of amide II structure, 500-650 cm -1 The appearance of the peak at 1000 nm confirmed the presence of aromatic skeleton structures amide I and amide II in LC-CBC. The appearance of the characteristic peak of Lac indicated that Lac was successfully immobilized on the carrier, while the peak of LC-CBC was similar to that of CBC, indicating that LC-CBC was mainly composed of carbon-based materials.

[0112] Table 1 Surface properties of biochar

[0113]

[0114] 8. Morphology and qualitative surface characteristics analysis

[0115] Figure 5 SEM images of BC, CBC and LC-CBC. Figure 5 (a) is the SEM image of BC. The pores are evenly distributed on the surface, which is formed during the biomass pyrolysis process. Figure 5 (b) is the SEM image of CBC after K2CO3 treatment. Compared with BC, the pores distributed on the surface of CBC are deeper and larger in size, which may be related to the effect of alkali treatment, which usually removes impurities present in the pores of biochar. Figure 5 (c) and (d) are SEM images of LC-CBC. In the photos of biochar containing immobilized enzymes, differentiation of the support surface can be observed, agglomeration appears on the surface, and a thick layer of enzyme coating exists, which may be due to the functionalization of glutaraldehyde on the surface and the immobilization of Lac. Figure 6 and Figure 7 EDS analysis showed that C, O, and N were the main elements of CBC and LC-CBC. The weight ratio of N in LC-CBC was 7.94 times that of CBC, indicating that laccase was successfully immobilized. Figure 8 and Fig. 9 The element mapping results in the graph intuitively show that the abundance of N element in LC-CBC is higher than that in CBC.

[0116] 9. pH stability

[0117] Solution pH can significantly affect enzyme activity and structure because it determines the ionization state of amino acids. The stability of Lac and LC-CBC was studied in the solution pH range of 3 to 8. Fig.10 As shown in the figure, Lac showed the highest activity at pH 5, while LC-CBC showed the best activity at pH 4. When the pH was lower or higher than the optimal pH, the relative activities of Lac and LC-CBC decreased. This phenomenon may be attributed to the fact that the combination of biochar and laccase affected the H + and OH - The distribution of laccase may be due to the reduced conformational flexibility and increased rigidity of laccase after immobilization. Compared with Lac, LC-CBC exhibits better environmental adaptability in a wider pH range.

[0118] 10. Temperature stability

[0119] In terms of temperature stability, the optimum temperatures for Lac and LC-CBC are 30°C and 40°C, respectively. Fig.11It can also be observed that with the increase of temperature, the activity of Lac and LC-CBC decreased, which is mainly due to structural changes and enzyme denaturation, resulting in loss of activity. LC-CBC is less sensitive to temperature changes. This enhancement in stability and resistance to denaturation can be attributed to the chelating interaction between Lac and CBC, which improves the structural stability of the immobilized enzyme molecules and protects the tertiary structure of the enzyme, allowing Lac to maintain its activity at high temperatures. The immobilized laccase retained 39% of its original activity at 70°C, while Lac only retained 9.17% of its original activity. The immobilized laccase had a higher activity at higher temperatures. The results show that CBC can be used as a carrier to reduce the degree of conformational changes of Lac and maintain greater activity under different environmental conditions.

[0120] 11. Storage stability

[0121] The rapid depletion of the catalytic activity of free laccase during storage and the difficulty in recovery after the reaction are major challenges as they may limit the potential of free laccase for large-scale application. Therefore, the storage stability of immobilized laccase is an important indicator for evaluating its effectiveness in practical applications. Lac and LC-CBC were stored at room temperature and refrigerated for 30 days, and their activities were tested regularly to evaluate their storage stability. The residual activities of Lac and LC-CBC during storage at 4°C and 25°C were shown in Figure 2. Fig.12 As shown. Immobilization of laccase improved the storage stability of the enzyme, and LC-CBC showed significantly higher stability than the free form. After 30 days, LC-CBC retained 78.5% and 70.6% of its activity at 4°C and 25°C, respectively, while Lac retained 50.6% and 32.3% of its activity. The results showed that immobilized laccase improved its storage stability. This may be because immobilization is beneficial to avoid conformational changes in the active site of the enzyme.

[0122] 12. Effect of pH on laccase degradation of PA

[0123] pH is one of the key parameters to be considered in enzyme-catalyzed reactions. The effect of pH on laccase activity is achieved by changing the dissociation state of the acid or base groups in the active part of laccase. When the pH is too high or too low, the interaction between pH and the electrostatic charge on the surface of the enzyme molecule may cause changes in the microstructure of the laccase and cause the laccase to become inactive. Therefore, according to the soil pH value, the pH range of 5.0-8.0 was selected to evaluate the effect of pH on the efficiency of LC-CBC in degrading PA. Fig.13It can be seen that when the pH of the reaction system is 5.0, free Lac and LC-CBC have the best degradation effect on PA, with degradation rates of 47.23% and 51.97%, respectively. This is consistent with the result that Lac has the best enzymatic activity in PBS buffer with a pH of 3.0 to 8.0. Deviating from this optimal pH, the degradation rate of PA decreases. When the pH is 8.0, the degradation rate of PA by LC-CBC is 31.67%, while the degradation rate of PA by free Lac is only 20.86%. This may be related to the strong electrostatic repulsion between oppositely charged groups in the active site of the enzyme, which leads to changes in the active site of the enzyme, loss of enzyme activity, and decreased degradation rate. Compared with free Lac, LC-CBC has a higher degradation effect in a wider pH range.

[0124] 13. Effect of temperature on laccase degradation of PA

[0125] The catalytic performance of most enzyme-catalyzed reactions is temperature-dependent. The effects of free Lac and LC-CBC on the degradation of PA at temperatures between 25 and 55 °C are shown in Table 1. Fig.14 As shown. The results show that at a temperature of 25°C, the degradation of PA by free Lac reaches a maximum value, with a degradation rate of 50%. As the temperature increases, the degradation rate shows a downward trend. In the range of 25-45°C, the degradation efficiency of PA by LC-CBC increases with increasing temperature, reaching a maximum of 57.63% at 45°C, and then shows a downward trend. At 55°C, the degradation rate of LC-CBC only decreased by 3.4%, while the degradation rate of free Lac decreased by 27%, indicating that the thermal stability of LC-CBC has been greatly improved compared to Lac, and PA can be removed in a wider temperature range. This is because after Lac is immobilized, the conformational changes of the laccase molecule at higher temperatures are prevented, thereby preventing the denaturation of the protein and retaining the activity of some enzymes.

[0126] 14. Effect of initial concentration on laccase degradation of PA

[0127] The effects of Lac and LC-CBC on the removal of PA at different initial concentrations were investigated. Fig.15 As shown in the figure, as the initial concentration of PA increases, the removal rate decreases. When the initial concentration of PA increases from 20 mg / L to 70 mg / L, the removal rate of PA by LC-CBC decreases from 54.54% to 24.57%. This may be due to the limited active sites available in the entire catalytic degradation system, which limits the catalytic degradation of high-concentration PA. On the other hand, as the initial PA concentration increases, the degradation products may increase, resulting in competition or inhibition of enzyme activity between the target pollutants and the degradation products, thereby leading to a decrease in degradation efficiency. As the initial concentration increases, the degradation rate shows a downward trend.

[0128] 15. Effect of dosage on laccase degradation of PA

[0129] The effect of the initial enzyme dosage on PA catalytic degradation was investigated by adjusting the initial enzyme dosage. Fig.16 It can be seen that the PA degradation rate increased from 44.34% to 100% when the dosage of LC-CBC increased from 1U / mL to 10U / mL. This may be because as the dosage of immobilized enzyme increases, the pollutants have more opportunities to contact the active sites of laccase, which is more conducive to the removal of PA. However, with the increase in the dosage of Lac, the degradation rate of PA did not increase significantly. This may be because when the dosage of Lac exceeds a certain amount, continuing to increase the dosage of Lac will increase the crowding of free Lac per unit volume, which may lead to mutual masking of Lac active centers, which is not conducive to the expression of enzyme activity, so that the extra Lac molecules cannot degrade PA.

[0130] 16. Effect of time on laccase degradation of PA

[0131] The present invention uses Lac and LC-CBC to study the degradation of PA. Fig.17 The degradation rates of Lac and LC-CBC in 24h are shown. The results show that the degradation rate of PA by Lac reached 100% at 12h. Compared with Lac, the degradation of PA by LC-CBC reached 100% at 10h. Before 10h, the degradation effect of LC-CBC on PA was higher than that of Lac. The use of LC-CBC achieved faster and higher degradation of PA. The low degradation rate of PA by LAC may be because PA occupies the active center of the enzyme and inactivates the enzyme in the biodegradation reaction. The high degradation rate of PA by LC-CBC can be attributed to the catalytic action of the enzyme leading to the degradation of PA and the adsorption of degradation products on biochar. At the same time, the adsorption process may promote the transfer of PA from the solution to the active sites of the enzyme on the surface of biochar, and a synergistic effect is generated between the carbon carrier and Lac, thereby enhancing the removal of PA.

[0132] 17. Effect of Laccase on Degradation of Phenolic Acid Substances

[0133] In order to further study the catalytic degradation ability of Lac, LC-CBC and LC-BC, they were also applied to the degradation of other phenolic acids, such as Fig.18 As shown in the figure, LC-CBC has better degradation performance on FA (100%), TC (100%), CA (87.23%) and HBA (81.4%) than Lac and LC-BC has good degradation performance on a wide range of phenolic acid substances, indicating that LC-CBC has universal applicability in the removal of various phenolic acid pollutants.

[0134] 18. LC-CBC Reusability

[0135] Reusability is one of the main advantages of immobilized laccase, as it can reduce the cost in practical applications compared with the free form. Fig.19 As shown in Figure 3, the activity of LC-CBC gradually weakened with the increase in the number of cycles, but its relative activity remained above 50% at the 7th cycle. The decrease in activity can be attributed to the destruction of the laccase structure leading to inactivation and the desorption of laccase during continuous washing.

[0136] The reusability performance of LC-CBC was evaluated by applying PA degradation for seven consecutive cycles. Fig.18 As shown in Figure 2, the PA degradation efficiency gradually decreased with the increase in the number of cycles. However, even in the 7th degradation cycle, LC-CBC still maintained a PA degradation efficiency of 49.64% after 6 h of reaction. The gradual decrease in PA degradation efficiency may be due to the partial inactivation of the immobilized laccase during repeated use or the partial desorption of the biocatalyst during the continuous washing and separation operations.

[0137] 19. Biological Experiments

[0138] In order to investigate the effect of PA degradation on pepper seed germination, pepper seeds were cultured using treated PA, untreated PA and control samples. Fig. 20 As shown, there was no significant difference in the root length of peppers grown in water treated with LC-CBC after PA application. It was found that PA had an inhibitory effect on the germination of pepper seeds, but the inhibitory effect on seed germination was reduced after PA was treated with LC-CBC. Taking distilled water as the control, the untreated PA reduced the germination rate of pepper seeds by 18% compared with the CK group, while the germination rate of the treated PA was not significantly different from that of the control treatment, indicating that PA treated with LC-CBC can reduce the inhibition of pepper seed germination and is beneficial to the germination of pepper seeds.

[0139] 20. Degradation pathway of PA

[0140] The reaction system of LC-CBC degradation of PA was taken after 4, 6 and 10 hours of reaction, and the degradation products were detected by LC-MS. Fig.21As shown in the figure, the degradation pathway of PA by LC-CBC is mainly divided into two steps. First, LC-CBC adsorbs PA in water onto the carrier and catalyzes the oxidation of PA. When laccase oxidizes the substrate, type I copper ions accept electrons from the substrate through the His-Cys-His pathway. The electrons are transferred to type II copper ions and type III copper ions in the laccase molecule to form a trinuclear copper cluster, and the molecular oxygen in the active center is reduced to water. The enzyme molecule becomes an oxidized laccase. At the same time, the substrate hydroxyl group that loses electrons is dehydrogenated to produce extremely unstable free radicals, and the subsequent ortho-quinone or para-quinone induces non-enzymatic polymerization, condensation and other reactions. The phenolic acid substances as substrates are first oxidized by laccase, and a carboxyl group is removed from PA to form substance B. At the same time, the free radical attacks the carboxyl group to form compound A. Substances A and B are further oxidized to open the carbon-oxygen double bond to form phenol. Phenol is oxidized and decomposed into catechol by molecular oxygen under the action of laccase. Catechol is ring-opened by the oxidized laccase to form substance I, and I is further oxidized to form small molecule substance J. Another degradation pathway of substance B is that the benzene ring of substance B is opened to form short-chain hydrocarbons, which are further oxidized into small molecule substance J.

[0141] 21. Toxicity analysis of PA and its intermediates

[0142] Fig. 22 and 23 The acute and chronic toxicity of PA and its intermediates to fish, water fleas and green algae are shown. It can be seen from the figure that during the degradation of PA by LC-CBC, some intermediates produce stronger acute or chronic toxicity than PA, indicating that the intermediates have more serious toxicity, but as the degradation continues, they are finally degraded into substances that are non-toxic or less toxic to the environment, indicating that the LC-CBC degradation process can reduce the harm of intermediate toxic products to the environment.

[0143] In this study, tobacco stem biochar etched with different alkalis was used as a carrier for immobilized laccase, and the optimal immobilized laccase carrier was determined by loading and enzyme activity. The specific surface area increased by 228.71 times under K2CO3 etching, and the laccase loading reached the optimal loading of 177.32U / g under the conditions of temperature 30℃ and pH 6. Immobilized laccase has the advantages of high pH, ​​high storage stability and high temperature. The immobilized laccase retained 51% of its activity after 7 cycles and had good stability. The degradation rate of PA within 10h under the conditions of pH 6 and temperature 30℃ was 100%, and it had good degradation effect on various phenolic acid substances. The degraded phthalic acid was more conducive to the germination of peppers. The phthalic acid pathway was confirmed by LC-MS and toxicity analysis, indicating that it was decomposed into small molecules with less toxicity. Therefore, biochar can be used as a carrier of immobilized laccase with high stability, providing a new solution for the removal of self-toxic phenolic acid substances in soil.

[0144] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop continuous cropping, characterized in that: The steps include: S1, placing tobacco straw powder in a sealed crucible in a muffle furnace for pyrolysis to obtain tobacco straw biochar BC; S2, soaking tobacco straw biochar and potassium carbonate in a mass ratio of 1:4 for 2-4 hours, drying, sealing the dried biochar crucible and pyrolyzing it in a muffle furnace, then air-cooling to room temperature, washing with deionized water, drying, sieving, and obtaining potassium carbonate-modified tobacco straw biochar CBC; S3, dispersing the potassium carbonate modified tobacco straw biochar CBC in HNO3 solution, mixing at 300rpm and 30℃ for 3-5h to obtain a functionalized biochar suspension, washing with deionized water to a pH of 7, drying overnight, and storing at room temperature to obtain functionalized CBC; S4, placing the functionalized CBC in step S3 in 50 mM phosphate buffer for 20-30 h, adding the same volume of 1.0% w / v glutaraldehyde, shaking at room temperature, washing the excess glutaraldehyde with deionized water, drying overnight, and storing to obtain the glutaraldehyde-containing CBC; S5. Mix the glutaraldehyde-containing CBC in step S4 with 5-10 U / mL laccase Lac solution at a solid-liquid ratio of 1 g:10 mL at 25-30° C. to immobilize the enzyme, thereby obtaining immobilized laccase LC-CBC.

2. The biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession according to claim 1, characterized in that: The pyrolysis temperature in the muffle furnace in step S1 is 300-600° C., and the pyrolysis time is 2-4 hours.

3. The biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession according to claim 1, characterized in that: The pyrolysis temperature of the muffle furnace in step S2 is 400-700° C., the pyrolysis time is 2-4 hours, and the pyrolysis rate is 5° C. / min.

4. The biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession according to claim 1, characterized in that: In step S4, the mixture was shaken at 300 rpm for 10-15 h at room temperature.

5. The biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession according to claim 1, characterized in that: The temperature for drying overnight is 25-45°C.

6. The biochar-immobilized laccase method for efficiently degrading phenolic acid self-toxic substances in crop succession according to claim 1, characterized in that: The concentration of the HNO3 solution is 75-95%.

7. A method for degrading phthalic acid by biochar-immobilized laccase, characterized in that: The steps are: Immobilized laccase is added to a 20 mg / L phthalic acid solution, and the solution is oscillated at an optimum temperature in a constant temperature oscillator. The sample is filtered through a 0.22 μ filter membrane, and the phthalic acid concentration of the obtained sample is determined by a high performance liquid chromatograph. The reaction pH is 4.0-6.0, the temperature is 25-35° C., the initial phthalic acid concentration is 20 mg / L, the immobilized laccase dosage is 1-4 U / mL, and the reaction time is 10-12 h. The immobilized laccase is the immobilized laccase LC-CBC prepared by the method of claim 1.

8. A method for removing phthalic acid and other similar phenolic acid substances in soil, characterized in that: The steps are: Immobilized laccase is added to soil, the pH of the soil is 3.0-7.0, the temperature is 15-45°C, the dosage of immobilized laccase is 10-50U / mL, the degradation time is 10-12h, and the immobilized laccase is the immobilized laccase LC-CBC prepared by the method of claim 1.

9. A method for increasing the germination rate of peppers in soil, characterized in that: The steps are: Immobilized laccase is added to the pepper soil solution containing phthalic acid, the pH of the solution is controlled to be 4-8, the temperature is 20-40°C, and the reaction is carried out for 10-12 hours. The immobilized laccase is the immobilized laccase LC-CBC prepared by the method of claim 1.

10. A soil conditioner for improving the germination rate of peppers in soil, characterized in that: The steps are: The immobilized laccase is mixed evenly with an inert filler and a binder in a mass ratio of 30-60:30-50:10-20, and the mixture is made into a granular or powdery soil conditioner product by extrusion and granulation, wherein the immobilized laccase is the immobilized laccase LC-CBC prepared by the method of claim 1; The inert filler is at least one of vermiculite, perlite, ceramsite or rock wool; and the binder is at least one of starch, bentonite or sodium carboxymethyl cellulose.

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