CN113930416A

CN113930416A - Preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals

Info

Publication number: CN113930416A
Application number: CN202111383171.3A
Authority: CN
Inventors: 刘维涛; 郑泽其; 李剑涛; 周启星
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-01-14
Anticipated expiration: 2041-11-22
Also published as: CN113930416B

Abstract

The invention discloses a preparation method of immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modified biochar. Then, fully mixing the alkali modified biochar with ferric chloride and ferrous chloride hydrate in an ethanol solution, and pyrolyzing the mixture in a tubular furnace at 500 ℃ to obtain magnetic porous biochar SBC @ Fe₃O₄Dispersing the SBC @ Fe in a glutaraldehyde solution to obtain SBC @ Fe₃O₄The magnetically modified glutaraldehyde of @ GA activates biochar. Finally, the carrier is subjected to a three-factor five-horizontal orthogonal experimental method according to different enzyme-carbon ratiosAdding laccase solutions with different concentration gradients, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe₃O₄The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading capacity of laccase. The invention has the advantages of low cost, suitability for large-scale preparation, less enzyme loss and the like.

Description

Preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals

Technical Field

The invention belongs to the field of laccase immobilization, relates to an immobilized laccase and a preparation method thereof, and particularly relates to a preparation method of a composite modified biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystals.

Background

Laccase (EC1.10.3.2) is a copper-rich oxidoreductase that degrades phenolic and non-phenolic complex compounds, often as catalysts for various aromatic compounds. The origin of laccases can be divided into four families, plant, fungus, bacteria and insect. Laccases were first found in the secretions of the plant sumac and co-exist in the cell walls of countless higher plants. However, the plant laccase is abundant, but the research on the fungal laccase is complete. Within a broad family of fungi there are numerous fungi that can produce laccases, such as ascomycetes, basidiomycetes and deuteromycetes. With the enhancement of awareness of human to environmental protection and the increase of the demand for resource utilization, laccase has wide application in aspects such as organic synthesis, dye decolorization, biosensor preparation, wastewater treatment, soil remediation and the like due to its high efficiency and specificity. However, free laccase still has obvious defects in the aspects of thermal stability, reusability, pollutant degradation efficiency and the like. Therefore, the immobilization by the combination of the immobilized laccase and a loading material, including adsorption, crosslinking, embedding, covalent bonding and the like, is one of the main strategies for improving the stability and reusability of the laccase nowadays. Meanwhile, the dual-functionality of adsorbing or degrading pollutants by laccase and materials can be realized by properly selecting the load materials, so that the removal efficiency of the pollutants is greatly improved.

Biochar is a carbon-containing solid substance prepared by biomass under the condition of limited oxygen, and becomes one of main immobilized support materials due to high porosity, large specific surface area and strong adsorption capacity to heavy metals and organic pollutants. However, the traditional biochar has low adsorption capacity to pollutants and is difficult to recover from the environment after application, so the pore structure of the traditional biochar can be further changed through modification, and the functional groups on the surface of the traditional biochar are increased, so that the traditional biochar has stronger adsorption and fixation capacity to pollutants. The alkali modification can reduce the ash content of the biochar and increase the number of hydroxyl and carboxyl. Magnetic modification can introduce magnetic substances into the biochar, so that the biochar is more convenient to separate and recycle from the environment, and the treatment cost is reduced. Therefore, the modified biochar is used as a load material of the immobilization technology, and has profound environmental significance for maintaining the activity of the enzyme, improving the stability of the immobilized enzyme and improving the reusability.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation method of a composite modified biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystals, which has low cost, is convenient to recycle from the environment and improves the storage stability and the operation stability of the enzyme. The invention aims to solve the defects that the existing immobilized enzymes are prepared by a plurality of methods, but a single immobilization technology is mainly used, such as embedding, adsorption, crosslinking and the like, and the methods are not favorable for maintaining the activity of the enzymes and have insufficient enzyme loading capacity. And the loss of enzyme activity is reduced, the loading capacity of the enzyme is increased, the recovery rate of the immobilized product is improved, and meanwhile, the immobilized product can be produced and applied in a large scale by selecting a low-cost loading material.

The purpose of the invention is realized by the following technical scheme: a preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals comprises the following steps:

(1) heating the dried wheat straw powder in a muffle furnace at 500 ℃ for 2.5 hours, and pyrolyzing to prepare biochar;

the average pore diameter of the wheat straw biochar is 3nm-16 nm;

the pore size of the wheat straw biochar is 0.001cm³/g-0.008cm³/g；

The specific surface area of the wheat straw biochar is 5.991m²/g。

(2) Preparing sodium hydroxide modified biochar:

and (2) sufficiently mixing 20g of the biochar (WBC) obtained in the step (1) with 4mol/L of NaOH solution according to a mass ratio of 1 to 3, stirring the mixture for two hours at room temperature by using a magnetic stirrer, and then drying the mixture in an oven at 105 ℃. And (3) placing the dried sample in a 250ml corundum boat, introducing 80-100 ml/min nitrogen, heating to 500 ℃ at 10 ℃/min in a tube furnace, heating to 800 ℃ at 5 ℃/min, keeping the temperature for 90min, and then cooling to room temperature. Soaking the prepared biochar in 1.5mol/L hydrochloric acid for two hours, then washing the biochar with deionized water for a plurality of times, drying the biochar in an oven, and homogenizing the biochar with a 100-mesh sieve to obtain sodium hydroxide modified biochar (SBC) which is stored in a glass drying dish for later use.

The average pore diameter of the sodium hydroxide modified biochar is 2nm-21 nm;

the pore size of the sodium hydroxide modified biochar is 0.01cm³/g-0.04cm³/g；

The specific surface area of the sodium hydroxide modified biochar is 41.285m²/g。

(3) Preparing magnetic modified ferroferric oxide crystal biochar:

mixing the alkali modified biochar obtained in the step (2) with 0.02mol FeCl₃·6H₂O and 0.01mol FeCl₂·4H₂O was mixed well in 200ml ethanol solution. And putting the mixture into a 250ml corundum boat, covering the corundum boat with tinfoil in a tube furnace, heating the corundum boat to 500 ℃ at the speed of 10 ℃/min, carrying out pyrolysis and heat preservation for 110min, introducing nitrogen at the speed of about 80-100 ml/min for protection, and cooling the corundum boat to room temperature. Then washing with deionized water for several times to prepare the magnetic porous biochar SBC @ Fe₃O₄。

The average pore diameter of the magnetic porous biochar is 2nm-6 nm;

the pore size of the magnetic porous biochar is 0.01cm³/g-0.06cm³/g；

The specific surface area of the magnetic porous biochar is 78.262m²/g。

(4) Preparing immobilized laccase: 1g of the magnetically modified biochar obtained in step (3) was dispersed in 25ml of glutaraldehyde solution for 2.5 hours. Subsequently, the activated support was washed several times with deionized water and citric acid-disodium hydrogen phosphate buffer pH 5, and dried in an oven at 105 deg.C to obtainTo SBC @ Fe₃O₄The magnetically modified glutaraldehyde of @ GA activates biochar. Adding laccase solutions with different concentration gradients into the carrier according to different enzyme-carbon ratios by a three-factor five-horizontal orthogonal experimental method, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe₃O₄The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading amount of the laccase. The product was washed several times with citric acid-disodium hydrogen phosphate buffer at pH 5, filtered with a magnet and stored in a refrigerator at-20 deg.C for further use.

The oven temperature in the selected step (1) is 105 ℃, and the heating time is 220 min.

The preparation conditions of the sodium hydroxide solution in the step (2) are as follows: 80g of NaOH powder was dissolved in 500ml of distilled water to prepare a 4mol/L NaOH solution.

The temperature of the oven in the step (2) is 105-120 ℃.

The ethanol solution in the step (3) is analytically pure ethanol.

The glutaraldehyde solution in the step (4) is a glutaraldehyde solution with the volume ratio of 2.5%.

The citric acid-disodium hydrogen phosphate buffer solution in the step (4) is prepared by mixing 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution.

And (4) adopting a 0.22 mu m organic nylon filter membrane as the suction filtration membrane in the step (4).

The three factors of the orthogonal experiment in the step (4) are enzyme-to-carbon ratio (mass ratio), concentration of laccase solution (U/mL) and immobilization time (h).

Wherein, the five level variables of the charcoal enzyme ratio are respectively 1:2, 1:3, 1:4, 1:5 and 1:6, the five level variables of the concentration of the laccase solution are respectively 2, 4, 6, 8 and 10, and the five level variables of the immobilization time are respectively 8, 12, 16, 20 and 24.

The technical progress of the invention is represented as follows: the method can realize the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal. Thereby overcoming the defect of short maintenance time of laccase activity.

1. The invention adopts the wheat straw as the biomass, and the wheat straw biochar as the raw material can not only relieve the pollution problem caused by straw burning, but also be used as an environmental modifier to be depended on the environment for a long time, and can synergistically adsorb and degrade pollutants with laccase.

2. The invention adopts the adsorption crosslinking composite immobilization technology to immobilize the laccase, has good immobilization effect, less laccase loss, reusability and improved thermal stability.

3. The invention adopts the magnetically modified biochar as the load material, has strong magnetism, can realize the recycling in the environment, has simple operation and low preparation cost, is suitable for large-scale production, and has obvious economic benefit and industrial value.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 is SEM images of magnetically modified ferriferrous oxide crystal biochar, magnetically modified glutaraldehyde activated biochar and immobilized laccase prepared by the invention. Wherein, (a) is magnetic modified ferroferric oxide crystal biochar, (b) is magnetic modified glutaraldehyde activated biochar, and (c) is immobilized laccase.

FIG. 2 is an X-ray diffraction diagram of magnetically modified ferroferric oxide crystal biochar, magnetically modified glutaraldehyde activated biochar and immobilized laccase prepared by the method.

FIG. 3 is N of the wheat straw biochar, the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar prepared by the invention₂Adsorption-desorption curves and pore size distribution plots.

FIG. 4 is a graph of the hysteresis regression of magnetically modified glutaraldehyde-activated biochar and immobilized laccase prepared according to the present invention.

FIG. 5 is a graph comparing the pH stability of free laccase and immobilized laccase.

FIG. 6 is a graph comparing the temperature stability of free laccase and immobilized laccase.

FIG. 7 is a graph of the operational stability of immobilized laccase.

Detailed Description

The invention is further described below with reference to the following figures and examples.

The laccase was selected in the examples of the present invention and purchased from a source leaf organism (80498-15-3) with a specification of 120U/g.

The equipment used in the method mainly comprises a muffle furnace, a tube furnace, a magnetic stirrer, a vacuum suction filter, an ultraviolet spectrophotometer, a field emission scanning electron microscope, a vibration sample magnetometer, an X-ray diffractometer, a full-automatic specific surface area and porosity analyzer and the like.

The invention will be further described with reference to the drawings and specific preferred embodiments without thereby departing from the scope of the invention.

Example 1:

according to the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal, laccase is immobilized on the modified porous charcoal through an adsorption crosslinking composite technology.

The preparation method of the biochar immobilized laccase based on the compound modification of the sodium hydroxide and the ferroferric oxide crystals in the embodiment comprises the following steps:

(1) preparation of wheat straw biochar

60g of natural wheat straw powder is taken, dried in an oven at 105 ℃, then pyrolyzed in a muffle furnace at 500 ℃ to obtain the wheat straw biochar, and the wheat straw biochar is placed in a glass drying dish for storage and standby.

(2) Preparation of sodium hydroxide modified biochar

And (2) fully mixing 20g of biochar (WBC) obtained in the step (1) with 4mol/L of NaOH solution according to the mass ratio of 1:3, stirring for two hours at room temperature by using a magnetic stirrer, and then drying in an oven at 105-120 ℃. The dried sample was placed in a 250ml corundum boat and charged with 80ml/min nitrogen, heated to 500 ℃ at 10 ℃/min in a tube furnace, heated to 800 ℃ at 5 ℃/min and held for 90min, and then cooled to room temperature. Soaking the prepared biochar in 1.5mol/L hydrochloric acid for two hours, then washing the biochar with deionized water for a plurality of times, drying the biochar in an oven, and homogenizing the biochar with a 100-mesh sieve to obtain sodium hydroxide modified biochar (SBC) which is stored in a glass drying dish for later use.

(3) Preparation of magnetic modified ferroferric oxide crystal biochar

Mixing SBC obtained in the step (2) with 0.02mol FeCl₃·6H₂O，0.01molFeCl₂·4H₂O was mixed well in 200ml of an analytically pure ethanol solution. Placing the mixture in a 250ml corundum boat, covering the corundum boat in a tube furnace by using tinfoil, heating the corundum boat to 500 ℃ at the speed of 10 ℃/min, carrying out pyrolysis and keeping the temperature for 110min, introducing nitrogen at the speed of about 80ml/min for protection, and cooling the corundum boat to room temperature. Then washing with deionized water for several times to prepare the magnetic porous biochar SBC @ Fe₃O₄And storing in a glass drying dish for later use.

(4) Preparation of immobilized laccase

1g of the magnetically modified biochar obtained in step (3) was dispersed in 25ml of glutaraldehyde solution for 2.5 hours. Subsequently, the activated support was washed several times with deionized water and citric acid-disodium hydrogen phosphate buffer at pH 5, and dried in an oven to obtain SBC @ Fe₃O₄The magnetically modified glutaraldehyde of @ GA activates biochar. Adding laccase solutions with different concentration gradients into the carrier according to different enzyme-carbon ratios by a three-factor five-horizontal orthogonal experimental method, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe₃O₄The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading amount of the laccase. The product was washed several times with citric acid-disodium hydrogen phosphate buffer at pH 5, filtered with a magnet and stored in a refrigerator at-20 deg.C for further use.

And (3) measuring the load of the immobilized enzyme:

weighing 0.05g of immobilized enzyme, adding 1mL of buffer solution, centrifuging for 10min (the temperature is 4 ℃, and the centrifugal force is 8000g), and taking supernatant as the liquid to be detected. Respectively adding 500 microliters of distilled water and 500 microliters of Coomassie brilliant blue indicator into a quartz cuvette as blanks, then respectively adding 500 microliters of solution to be detected and 500 microliters of Coomassie brilliant blue indicator into the quartz cuvette, measuring absorbance at 620nm, and obtaining the loading capacity (mg/g) of laccase by calculation, wherein the calculation formula is as follows:

protein concentration C of the initial laccase_i(mg/mL)＝0.07×(ΔA+0.0007)

Protein concentration of the immobilized laccaseC_f(mg/mL)＝0.07×(ΔA+0.0007)

Where Δ A represents the change in absorbance before and after the measurement.

Wherein, V₁Representing the amount of free laccase solution (mL) added during the initial laccase protein concentration determination, V₂Representing the amount of the test solution (mL) added during the determination of the concentration of the immobilized laccase protein.

And (3) enzyme activity determination:

taking about 15mg of free laccase or immobilized laccase in a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with the optimal pH value and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g and the temperature is 4 ℃) for 10min, taking 1ml of supernatant in a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, carrying out water bath at the optimal temperature for 3min, centrifuging again (the centrifugal force is 8000g and the temperature is 4 ℃) for 10min, taking 1ml of supernatant in the quartz slit cuvette, and measuring the absorbance again at 420 nm. The enzyme activity was calculated using the ABTS equation as follows:

wherein Δ A represents the increase in absorbance of ABTS, V_tRepresents the volume (L) of the reaction system, and ε represents the molar extinction coefficient (36000 L.M) of ABTS^-1·cm^-1) DeltaT represents the reaction time (min) and m represents the mass of the enzyme (g).

Example 2:

an orthogonal experimental assay study for the preparation of immobilized laccase in example 1:

according to the orthogonal experimental arrangement, 25 groups of experiments are carried out by changing different factors, and the measurement results are shown in the following table 1:

TABLE 1

The results of the range analysis are shown in the following table 2:

TABLE 2

Level of	Carbon to enzyme ratio (g/g)	Enzyme concentration (U/mL)	Immobilization time (h)
				1	0.3692	0.3864	0.2320
2	0.3137	0.3107	0.2301
				3	0.3323	0.3162	0.5150
4	0.3628	0.3338	0.2526
				5	0.3443	0.3753	0.4926
Delta	0.0554	0.0756	0.2849
				Rank of rank	3	2	1

According to the results of range analysis, the optimal immobilization conditions of the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal are as follows: the ratio of charcoal to enzyme is 1:2, the concentration of laccase is 2U/mL, and the immobilization time is 16 h. And the priority order of the three factors is immobilization time, laccase concentration and carbon-enzyme ratio.

Example 3:

the immobilized laccase was prepared according to the optimal immobilization conditions found in example 2 and subjected to performance characterization and testing:

the magnetic modified ferriferrous oxide crystal biochar, the magnetic modified glutaraldehyde activated biochar and the immobilized laccase in example 1 were subjected to field emission Scanning Electron Microscope (SEM) detection and analysis, and the results are shown in fig. 1. Wherein (a) is magnetic modified ferroferric oxide crystal biochar, (b) is magnetic modified glutaraldehyde activated biochar, and (c) is immobilized laccase. As can be seen from the graph (a), the pores of the biochar after the ferroferric oxide crystal is magnetically modified become more uniform, and as can be seen from the graph (b), the biochar is activated by the magnetically modified glutaraldehyde to enlarge part of the pores of the porous biochar, and as can be seen from the graph (c), the biochar activated by the magnetically modified glutaraldehyde is used as a carrier, the laccase is successfully immobilized on the carrier material, and the structure of the immobilized laccase obtained after the laccase is immobilized is unchanged.

The magnetic modified ferroferric oxide crystal biochar, the magnetic modified glutaraldehyde activated biochar and the immobilized laccase in example 1 were subjected to X-ray diffraction (XRD) analysis, wherein (a) is the magnetic modified ferroferric oxide crystal biochar, (b) is the magnetic modified glutaraldehyde activated biochar, and (c) is the immobilized laccase. As can be seen from FIG. 2, two diffraction peaks appeared at 30.1 ℃ and 35.6 ℃ in the pattern, which was designated as Fe according to PDF (89-0691)₃O₄Crystal, indicating that the magnetic modified charcoal is loaded with Fe₃O₄And (4) crystals.

The wheat straw biochar, the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar in the example 1 are subjected to full-automatic specific surface area and porosity analysis (BET). As can be seen from FIG. 3, the adsorption isotherms from wheat straw biochar to sodium hydroxide modified biochar to magnetic modified ferroferric oxide crystal biochar transition from type I to type II, the type II adsorption isotherm is mostly a non-porous or typical multi-molecular-layer physical adsorption process on a macroporous adsorbent, and the combination of the pore size distribution diagram shows that the mesoporous and macroporous ratios of the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar are increased.

The magnetically modified glutaraldehyde-activated biochar and immobilized laccase from example 1 were subjected to vibro-sample magnetic intensity analysis (VSM). As can be seen from FIG. 4, the saturation magnetic field intensity of the biochar activated by the magnetically modified glutaraldehyde serving as the load material is about 7.3emu/g, while the saturation magnetic field intensity of the immobilized laccase can be increased to about 17emu/g, which is increased by 1.33 times and belongs to strong magnetism. The loading of the laccase on the magnetic material is shown to not only not weaken the magnetism, but also enhance the magnetism, and is more beneficial to the adsorption effect and the recovery function of the immobilized laccase in the environment.

Example 4:

the immobilized laccase was prepared according to the optimal immobilization conditions obtained in example 2 and subjected to stability tests, including pH stability, temperature stability and handling stability, with the following steps and results:

(1) stability of pH

In the process of measuring the activities of free laccase and immobilized laccase, the pH value of a citric acid-disodium hydrogen phosphate buffer solution is changed to measure the enzyme activity under different pH environment gradients, so that the optimum pH value of the laccase activity is obtained, wherein the pH gradient is set to be 2.5, 3, 4, 5, 6 and 7. The specific method comprises the following steps: taking about 15mg of free laccase or immobilized laccase in a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with different pH gradients and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g,4 ℃) for 10min, taking 1ml of supernatant in a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, reacting at room temperature for 3min, centrifuging again (the centrifugal force is 8000g,4 ℃) for 10min, taking 1ml of supernatant in the quartz slit cuvette, and measuring the absorbance again at 420 nm. Calculating enzyme activity by using an ABTS method formula, and taking the ratio of the enzyme activity under different pH gradients to the highest enzyme activity as relative enzyme activity, and recording the relative enzyme activity in percentage.

As can be seen from FIG. 5, the optimum pH range of the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal is wider than 2.5-4, and the optimum pH values of 5 and 3 are respectively selected as the optimum pH values for determining the activities of the free laccase and the immobilized laccase.

(2) Temperature stability

In the process of measuring the activities of free laccase and immobilized laccase, the enzyme activities under different temperature environment gradients are measured by changing the reaction temperature of a water bath, so that the optimal temperature of the laccase activity is obtained, wherein the temperature gradients are set to be 20, 30, 40, 50, 60 and 70 ℃. The specific method comprises the following steps: and (2) adding about 15mg of free laccase or immobilized laccase into a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with the optimum pH value measured in the step (2) and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g and 4 ℃) for 10min, taking 1ml of supernatant into a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, reacting for 3min under different water bath temperature gradients, centrifuging again (the centrifugal force is 8000g and 4 ℃) for 10min, taking 1ml of supernatant into the quartz slit cuvette, and measuring the absorbance again at 420 nm. Calculating enzyme activity by using an ABTS method formula, and taking the ratio of the enzyme activity under different temperature gradients to the highest enzyme activity as relative enzyme activity, and recording the relative enzyme activity in percentage.

As can be seen from FIG. 6, the temperatures of 50 ℃ and 60 ℃ were selected as the optimum temperatures for determining the activities of the free laccase and the immobilized laccase, respectively.

(3) Stability of operation

Adsorbing the immobilized laccase after the enzyme activity is determined on the outer side of a centrifugal tube by using a magnet, pouring out the added buffer solution and an ABTS substrate solution, washing the immobilized laccase and the ABTS substrate solution by using deionized water for three times, continuously determining the activity of the immobilized laccase after the cyclic recovery according to the optimal temperature of the immobilized laccase obtained in the step (2) by using the method for determining the activity in the step (2), repeating the cycle for ten times, and taking the ratio of the enzyme activity determined each time to the first enzyme activity as the relative enzyme activity, and recording the relative enzyme activity in percentage.

As can be seen from FIG. 7, the enzyme activity was hardly decreased after the first three operations, and was maintained at about 90% until the fourth operation. The enzyme activity after the fifth time is reduced to about 60%, and the enzyme activity is stably maintained at about 30% from the sixth time to the tenth time. The immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal can be stably used for about four to five times.

Claims

1. a preparation method based on sodium hydroxide and ferric oxide crystal composite modified biochar immobilized laccase, is characterized in that, comprises the steps:

(1) Preparation of wheat straw biochar: take natural wheat straw powder, dry it in an oven, and then pyrolyze it in a muffle furnace to obtain wheat straw biochar WBC;

(2) Preparation of alkali-modified biochar: fully mix the biochar WBC obtained in step (1) with NaOH solution in a mass ratio of 1:3, stir with a magnetic stirrer at room temperature for two hours, and then place it in an oven to dry , put the dry sample in a corundum boat, and use nitrogen as the ambient atmosphere, pyrolyze in a tube furnace, and cool it to room temperature, soak the prepared biochar with 1.5mol/L hydrochloric acid for two hours, and then use Washed with ionized water several times, dried in an oven, and homogenized with a 100-mesh sieve to obtain alkali-modified biochar SBC, which was stored in a glass drying dish for use;

(3) Preparation of magnetically modified biochar: The alkali-modified biochar obtained in step (2) was thoroughly mixed with 0.02mol FeCl ₃ ·6H2O and 0.01mol FeCl ₂ _· 4H2O in an ethanol solution, and the mixture was placed in a corundum boat , covered with tin foil and pyrolyzed in a tube furnace, cooled to room temperature, and washed with deionized water for several times to obtain magnetic porous biochar SBC@Fe ₃ O ₄ ;

(4) Preparation of immobilized laccase: The magnetically modified biochar obtained in step (3) was dispersed in glutaraldehyde solution, and then the activated support was washed with deionized water and citric acid-disodium hydrogen phosphate buffer Several times, the magnetically modified glutaraldehyde-activated biochar of SBC@Fe ₃ O ₄ @GA was obtained by drying in an oven, and then the three-factor and five-level orthogonal experiment method was used to add different enzyme-carbon ratios to the above-mentioned carrier. The laccase solution with concentration gradient was stirred with a magnetic stirrer for several hours to obtain the SBC@Fe ₃ O ₄ @GA@Lac product, and the optimal immobilization method was selected according to the loading amount of laccase. The product was citric acid-phosphoric acid Wash several times with disodium hydrogen buffer, suction filtration with magnets, and store for later use.

2. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that,

In the step (1), the oven temperature is 105°C, and the holding time is 220min;

In the step (1), the heating temperature of the muffle furnace is 500°C, and the heating time is 2.5h;

In described step (2), sodium hydroxide solution concentration is 4mol/L;

In the step (2), the oven temperature is 105-120°C;

In the selected step (2), the nitrogen flow rate of the tubular furnace is 80-100ml/min, the heating rate is 10°C/min and the heating temperature is up to 500°C, and then the heating rate is 5°C/min and the heating temperature is up to 800°C, and the holding time is 90min;

In the step (3), the nitrogen flow rate of the tubular furnace is 80-100ml/min, the heating temperature is 500°C, the heating rate is 10°C/min, and the holding time is 110min;

In the selected step (4), the oven temperature is 105°C;

In the selected step (4), the pH of the citric acid-disodium hydrogen phosphate buffer is 5, which is formed by mixing 0.1 mol/L citric acid solution and 0.2 mol/L disodium hydrogen phosphate solution,

The volume ratio of the glutaraldehyde solution in the selected step (4) is 2.5%.

3. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, in described step (4), the orthogonal experiment method is set The three factors are: charcoal enzyme ratio mg/mg, laccase solution concentration U/mL, and immobilization time h.

4. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, in described step (4), enzyme carbon ratio mg/mg five The scale gradients are 1:2, 1:3, 1:4, 1:5, 1:6.

5. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, in described step (4), laccase solution concentration U/mL The five ratio gradients are 2, 4, 6, 8, 10 U/mL.

6. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, in described step (4), immobilization time h five ratios Gradients are 8, 12, 16, 20, 24.

7. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, the assay method of laccase load in described step (4) As follows: Weigh 0.05g of the immobilized product, add 1mL buffer, centrifuge for 10min at a temperature of 4°C and a centrifugal force of 8000g, take the supernatant as the liquid to be tested, and add 500 μl to the quartz cuvette respectively Distilled water and 500 microliters of Coomassie brilliant blue indicator were used as blanks, then 500 microliters of the liquid to be tested and 500 microliters of Coomassie brilliant blue indicator were added to the quartz cuvette respectively, and the absorbance was measured at 620 nm. Loading mg/g;

The formula is as follows:

Initial laccase protein concentration C _i (mg/mL)=0.07×(ΔA+0.0007)

The protein concentration of laccase after immobilization C _f (mg/mL)=0.07×(ΔA+0.0007)

Among them, ΔA represents the change of absorbance before and after the measurement,

Wherein, V ₁ represents the amount of free laccase solution added in mL during the initial laccase protein concentration determination, and V ₂ represents the amount of the solution to be measured added in mL during the immobilized laccase protein concentration determination process.

8. the preparation method of immobilized laccase based on sodium hydroxide and ferric oxide crystal composite modified biochar according to claim 1, is characterized in that, the assay method of enzymatic activity is as follows: get about 15mg free laccase or Immobilized laccase was placed in a 15ml centrifuge tube, followed by adding 2ml of citric acid-disodium hydrogen phosphate buffer with optimal pH and 2ml of 0.5mmol/LABTS solution, centrifuged immediately for 10min under the conditions of a centrifugal force of 8000g and 4°C, and took 1ml of The supernatant was placed in a quartz slit cuvette, the absorbance was measured at 420 nm, then fully shaken, water bathed at the optimum temperature for 3 min, centrifuged again at a centrifugal force of 8000 g and 4 °C for 10 min, and 1 ml of the supernatant was taken in In the quartz slit cuvette, measure the absorbance again at 420 nm, and calculate the enzyme activity with the ABTS method formula;

The formula is as follows:

Among them, ΔA represents the increase of the absorbance of ABTS, V _t represents the volume L of the reaction system, ε represents the molar extinction coefficient of ABTS, ΔT represents the reaction time min, and m represents the mass g of the enzyme.