Preparation method and application of immobilized laccase at room temperature

Abstract

The invention discloses a preparation method and application of immobilized laccase at room temperature, and the preparation method comprises the following steps: step 1, weighing laccase, dissolving the laccase in a sodium acetate-acetic acid buffer solution, carrying out magnetic stirring, and then adding a sodium hydroxide solution containing trimesic acid to form a mixed solution; step 2, adding an aqueous solution containing ferric ions into the mixed solution, magnetically stirring, standing and aging in a refrigerator, and centrifugally collecting a solid product; and step 3, re-suspending the washed precipitate in a polyvinylpyrrolidone aqueous solution, fully dispersing, and then drying to obtain the immobilized laccase, which is recorded as Lacc-Fe-BTC. The immobilized laccase prepared by the invention can be used for rapidly and efficiently degrading aflatoxin B1 and zearalenone, and recycling and reutilization of the enzyme after reaction can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of mycotoxin treatment in grain and oil foods, and in particular to a preparation method of immobilized laccase at room temperature and application thereof. Background Art

[0002] Mycotoxins are a type of secondary metabolites produced by various filamentous fungi during the planting, harvesting, transportation and storage of grains. They are widely present in foods and feeds such as corn, barley, and wheat, and are one of the most common and important contaminants in grain and oil foods. Aflatoxins are a type of carcinogenic fungal toxins produced by Aspergillus fungi such as Aspergillus flavus and Aspergillus parasiticus. They are commonly found in staple crops such as corn, rice, peanuts, dried fruits and spices, as well as dairy products. Zearalenone can also be detected in grains such as corn and wheat and their products. Mycotoxins pose a major threat to human and animal health, whether exposed through grains or indirectly through feed and animal-derived products. Bioenzymatic degradation can specifically degrade mycotoxins while maintaining the integrity of flavor substances and nutrients in food. Therefore, it is very effective in controlling and reducing crop mycotoxin contamination and has great application potential.

[0003] Laccase (EC.1.10.3.2) is a multi-copper monomer protein with the ability to oxidize phenolic compounds. Studies have found that laccases from different sources have good degradation effects on major fungal toxins such as aflatoxins and zearalenone. Among them, the laccase from Trametes versicolor can degrade aflatoxins and zearalenone at the same time. However, laccase has poor thermal stability, limited pH tolerance range, poor tolerance to organic solvents, slow reaction rate, low reuse rate and other problems during use, which greatly limits its practical application. The use of enzyme immobilization technology can enhance the catalytic activity, stability and recyclability of the enzyme, minimize enzyme contamination in the product, and occupy an important position in industrial enzyme catalysis applications. Metal-organic frameworks are periodic mesh nanomaterials formed by the composite of metal ions and organic ligands. They have adjustable pore size, large specific surface area, diverse functional sites, and good adsorption properties. Combining MOFs materials as immobilized carriers with fungal toxin-degrading enzymes such as laccase can not only effectively load the enzymes, improve the stability and reusability of the enzymes in specific environments, but also effectively improve the degradation performance of the enzymes on fungal toxins. Summary of the invention

[0004] In view of this, the present invention provides a method for preparing immobilized laccase at room temperature and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for preparing immobilized laccase at room temperature comprises the following steps:

[0007] Step 1: weighing laccase, dissolving it in a sodium acetate-acetic acid buffer solution, and performing magnetic stirring, and then adding a sodium hydroxide solution containing trimesic acid to form a mixed solution;

[0008] Step 2: adding an aqueous solution containing trivalent iron ions to the mixed solution, stirring the solution magnetically, placing the solution in a refrigerator for aging, and collecting the solid product by centrifugation;

[0009] Step 3: The washed precipitate was resuspended in a polyvinyl pyrrolidone aqueous solution, fully dispersed, and then dried to obtain immobilized laccase, which was recorded as Lac@Fe-BTC.

[0010] Preferably, in step 1, the pH of the sodium acetate-acetic acid buffer solution is 4.5-6.0, and the concentration of the buffer solution is 50-100 mM.

[0011] Preferably, in step 1, the concentration of trimesic acid in the sodium hydroxide solution containing trimesic acid is 0.42-0.56 mol / L.

[0012] Preferably, in step 2, the concentration of the aqueous solution of trivalent iron ions is 0.62-0.83 mol / L.

[0013] Preferably, in step 2, the stirring speed is 200-800 rpm, the stirring time at room temperature is 0.5-6 hours, the refrigerator temperature is 4° C., the static aging time is 3-12 hours, and the centrifugal speed is 5000-8000 rpm.

[0014] Preferably, the volume ratio of the sodium acetate-acetic acid buffer solution, the sodium hydroxide solution of trimesic acid and the aqueous solution of ferric ions is 15-20:1:1, at which time the molar ratio of ferric ions, trimesic acid and sodium hydroxide is 1.5:1:3, and the final concentration of laccase in the reaction system is 0.4-1.0 mg / mL.

[0015] Preferably, in step 3, the concentration of the polyvinyl pyrrolidone solution is 0.5%-2.0%.

[0016] Preferably, in step 3, the drying is freeze drying for 12-18 hours, or natural drying at room temperature for 24-30 hours.

[0017] A method for preparing immobilized laccase at room temperature and its application in degrading fungal toxins.

[0018] Preferably, the fungal toxin is one or more of AFB1 or ZEN, the reaction conditions are that the pH of the sodium acetate-acetic acid buffer solution is 4.5-6, or the pH of the Tris-Hcl buffer solution is 7-8, and 0.5-1mM of a laccase mediator substance is added, such as one of acetosyringone, tert-butylhydroquinone, ferulic acid, p-coumaric acid, and vanillin.

[0019] Compared with the prior art, the present invention has achieved the following technical effects:

[0020] (1) The present invention simultaneously introduces laccase during the formation of Fe-BTC MOF, and immobilization is achieved in one step, thus avoiding the problem of long MOF synthesis time; the synthesis process of the present invention is quick and simple, and the enzyme can be coated on Fe 3+ The interior of the MOF connected with trimesic acid provides shell protection for laccase;

[0021] (2) In the present invention, the laccase solution is dissolved in a buffer solution, and then mixed with an organic ligand solution and an iron ion solution in sequence. The buffer solution has a buffering capacity, which prevents the enzyme from being deformed and inactivated by an overly acidic or alkaline environment, so that the immobilization process is successfully prepared under relatively mild conditions, and the activity of the enzyme can be well maintained;

[0022] (3) During freeze drying, a certain concentration of polyvinyl pyrrolidone solution is added to prevent Lac@Fe-BTC from aggregating during the freeze drying process, and also plays a freeze drying protection role;

[0023] (4) The Lac@Fe-BTC prepared in the present invention has the ability to efficiently adsorb aflatoxins and zearalenone. The rapid enrichment of mycotoxins in Lac@Fe-BTC and the synergistic catalytic effect between mycotoxin-degrading enzymes can quickly and efficiently adsorb and degrade mycotoxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a SEM image of a method for preparing immobilized laccase at room temperature and its application;

[0025] Among them, (a) is the Fe-BTC MOF diagram; (b) is the Lac@Fe-BTC diagram;

[0026] Figure 2 It is a schematic diagram of a method for preparing immobilized laccase at room temperature of the present invention and the effect of the pH of the buffer solution used therein on the immobilization effect of the enzyme;

[0027] Figure 3 It is a schematic diagram of a method for preparing immobilized laccase at room temperature of the present invention and the pH stability of free laccase and its immobilized enzyme Lac@Fe-BTC used therein;

[0028] Figure 4 It is a schematic diagram of a method for preparing immobilized laccase at room temperature of the present invention and the temperature stability of free laccase and its immobilized enzyme Lac@Fe-BTC used therein;

[0029] Figure 5 A method for preparing immobilized laccase at room temperature and a diagram showing the effect of pH on the removal of AFB1 and ZEN by the immobilized enzyme Lac@Fe-BTC;

[0030] Among them, Figure (a) shows the effect of pH on the removal of AFB1 by the immobilized enzyme Lac@Fe-BTC;

[0031] Figure (b) shows the effect of pH on the removal of ZEN by the immobilized enzyme Lac@Fe-BTC;

[0032] Figure 6 A schematic diagram of a method for preparing immobilized laccase at room temperature and the reusability of the immobilized enzyme Lac@Fe-BTC used in the present invention for degrading AFB1 and ZEN;

[0033] Figure (a) is a schematic diagram of the reusability of the immobilized enzyme Lac@Fe-BTC for degradation of AFB1;

[0034] Figure (b) is a schematic diagram of the reusability of the immobilized enzyme Lac@Fe-BTC in degrading ZEN. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The present invention discloses a method for preparing immobilized laccase at room temperature, comprising the following steps:

[0037] Step 1: weighing laccase, dissolving it in a sodium acetate-acetic acid buffer solution with a pH of 4.5-6.0 and a concentration of 50-100 mM, and magnetically stirring, and then adding a sodium hydroxide solution of trimesic acid with a concentration of 0.42-0.56 mol / L to form a mixed solution;

[0038] Step 2: adding an aqueous solution containing trivalent iron ions at a concentration of 0.62-0.83 mol / L to the mixed solution, stirring magnetically at a speed of 200-800 rpm, placing in a 4°C refrigerator, aging for 3-12 hours, and centrifuging at a speed of 5000-8000 rpm to collect the solid product;

[0039] The volume ratio of sodium acetate-acetic acid buffer solution, sodium hydroxide solution of trimesic acid and aqueous solution of ferric ions is 15-20:1:1, the molar ratio of ferric ions, trimesic acid and sodium hydroxide is 1.5:1:3, and the final concentration of laccase in the reaction system is 0.4-1.0 mg / mL.

[0040] Step 3: The washed precipitate is resuspended in a polyvinyl pyrrolidone aqueous solution with a concentration of 0.5%-2.0%, fully dispersed, and then freeze-dried for 12-18 hours, or naturally dried at room temperature for 24-30 hours to obtain immobilized laccase, recorded as Lac@Fe-BTC.

[0041] The present invention also discloses a method for preparing immobilized laccase at room temperature and its application in the degradation of fungal toxins, wherein the fungal toxins are one or more of AFB1 or ZEN, the reaction conditions are a sodium acetate-acetic acid buffer solution with a pH of 4.5-6), or a Tris-HCl buffer solution with a pH of 7-8, and 0.5-1 mM of a laccase mediator substance is added, such as one of acetosyringone, tert-butylhydroquinone, ferulic acid, p-coumaric acid, and vanillin.

[0042] The properties of the immobilized enzyme obtained by the present invention were verified by the following methods:

[0043] (1) Determination of enzyme immobilization capacity: using BCA kit;

[0044] The percentage of immobilized enzyme and enzyme loading on the support was calculated by the difference between the initial laccase concentration and the laccase enzyme concentration in the centrifugation supernatant after immobilization.

[0045] (2) Determination of laccase activity:

[0046] Detection principle: ABTS can be oxidized by laccase to blue ABTS + , within a certain range, ABTS + The concentration is proportional to the color, so the absorbance is detected at 420nm using UV to further determine its concentration.

[0047] Preparation of substrate ABTS (1 mmol): 0.0548 g ABTS was dissolved in 100 mL HAc-NaAc solution at pH 4.5, followed by sonication in an ultrasonic cleaner to assist solubilization.

[0048] The activities of free laccase and immobilized laccase were calculated by measuring the UV absorbance of ABTS solution at 420 nm after oxidizing ABTS.

[0049] The specific steps are as follows: 0.45 mL of ABTS solution (1 mmol) was measured into two 1.5 mL EP tubes respectively, and then preheated at 40°C for 5 minutes. Then, 0.05 mL of the same amount of free laccase or immobilized laccase was added to one of the EP tubes and reacted in the solution at 40°C for 10 minutes. After the reaction, 0.5 mL of 0.8 mol / L NaF solution was added to terminate the reaction. Since the presence of solids in the solution after the reaction will affect the UV detection, in order not to affect the UV detection, the solids should be centrifuged and precipitated before detection.

[0050] The laccase activity unit was defined as the amount of enzyme required to oxidize 1 μmol of ABTS substrate per minute under the reaction conditions of 40°C and pH 4.5.

[0051]

[0052] Where A is the absorbance change after laccase oxidation of ABTS at time t, t is the reaction time (min), V is the volume of the reaction solution, and ε is the molar extinction coefficient of ABTS at 420 nm (36×10 3 M -1 cm -1 )

[0053] Example 1: Preparation of Lac@Fe-BTC and the effect of buffer solution pH on the immobilization effect of laccase

[0054] After accurately weighing 6 mg of laccase, add it to 9 mL of sodium acetate-acetic acid buffer solution and mix it thoroughly; then, under magnetic stirring conditions, add 0.5 mL of 0.56 mol / L trimesic acid solution to the solution and stir it evenly, then add 0.5 mL of 0.83 mol / L ferric chloride hexahydrate solution, and continue stirring until the reaction is uniform; let it stand in a 4°C refrigerator for aging for a certain period of time; after the reaction is completed, centrifuge and wash it repeatedly with an appropriate amount of buffer solution, the washed precipitate is resuspended in a polyvinyl pyrrolidone (PVP) aqueous solution, and after being fully dispersed, it is freeze-dried to finally obtain a solid product, which is stored in a 4°C refrigerator for use.

[0055] During the enzyme immobilization process, the enzyme may be denatured due to inappropriate pH values, so choosing a suitable buffer system is crucial to maintain enzyme activity, thereby improving its immobilization effect.

[0056] To this end, the present invention explores the synthesis process of Lac@Fe-BTC biocomposite materials under the condition of sodium acetate-acetic acid buffer solution with a pH range of 4.0-6.0;

[0057] like Figure 1 As shown, it is the SEM image of Fe-BTC MOF (a) and Lac@Fe-BTC (b) of the present invention; Figure 2 As shown, the composite material prepared in NaAc-HAc buffer solution with a pH of 5.5 has the highest relative activity, reaching 86.3%. After adding trimesic acid solution to NaAc-HAc buffer solution with a pH of 5.5, the pH of the system increased to 6.2; after adding ferric chloride hexahydrate solution, the pH of the system dropped to 4.53, ensuring the stable generation of laccase under relatively mild conditions;

[0058] According to literature reports, the isoelectric point of laccase is 4.2, and the final system pH is 4.53, slightly higher than the isoelectric point, and laccase is negatively charged. Therefore, laccase can interact with Fe 3+ The ions formed a strong electrostatic attraction and were effectively embedded in the Fe-BTC structure. Under this condition, the embedding rate of laccase reached 83.31%.

[0059] Example 2: Enzymatic properties of immobilized enzyme Lac@Fe-BTC

[0060] Enzymes are prone to denaturation under extreme environmental conditions (such as acidity, alkalinity or high temperature), resulting in the loss of their activity, which limits their effectiveness in industrial applications. In order to evaluate the stability of laccase and its immobilized form Lac@Fe-BTC under different acidic conditions, the present invention incubated the two under different pH conditions for 1 hour, and compared the relative retention of the enzyme activity of free laccase and Lac@Fe-BTC; the enzyme activities of laccase and Lac@Fe-BTC under the optimal pH conditions were set as 100%, and their residual activities were calculated to evaluate the pH stability.

[0061] like Figure 3 As shown in the figure, under acidic and alkaline pH conditions, the relative enzyme activity of free laccase decreased, and the free laccase activity was retained at the highest level under neutral conditions. At pH 8, the enzyme activity of Lac@Fe-BTC was relatively retained at the highest level, and at pH 4 and pH 6, the activity of Lac@Fe-BTC was also higher than that of free laccase.

[0062] Temperature is one of the important factors affecting the activity of laccase. Too high or too low temperature may lead to the inactivation of the enzyme. In order to study the thermal stability of laccase and its immobilized form Lac@Fe-BTC, the present invention incubated both in a water bath at 30℃-70℃ for 1 hour, and detected the relative retention of the enzyme activity of free laccase and Lac@Fe-BTC after incubation; the enzyme activities of laccase and Lac@Fe-BTC at the optimal temperature were set as 100%, and the thermal stability was evaluated by calculating the residual activity.

[0063] like Figure 4 As shown, the stability at a temperature of 60-70°C is significantly improved.

[0064] Example 3: Effect of Immobilized Enzyme Lac@Fe-BTC on Degradation of AFB1 and ZEN

[0065] The same amount of immobilized enzyme Lac@Fe-BTC or free laccase was added to a solution containing 2.5 μg / mL AFB1 or 5.0 μg / mL ZEN, and 0.5 mM acetosyringone was added as the reaction medium of laccase. After mixing evenly, the mixture was placed in an oscillating incubator for oscillation reaction. After the reaction, the immobilized enzyme Lac@Fe-BTC was removed by centrifugation, and the residual concentrations of AFB1 and ZEN in the sample were determined by high performance liquid chromatography (HPLC), and their removal rates were calculated.

[0066] The removal rate of toxins by immobilized enzyme Lac@Fe-BTC and free laccase can be calculated by the following formula:

[0067] Removal rate = (C0-C1) / C0

[0068] Among them, C0 and C1 are the concentrations of toxins in the solution before and after the reaction, respectively.

[0069] like Figure 5 As shown, the removal rates of AFB1 and ZEN by immobilized laccase and free laccase at 40°C for 1 hour in the pH range of 4 to 8 were demonstrated; the results showed that the removal rates of AFB1 and ZEN by immobilized laccase under different pH conditions could reach more than 80% and more than 90%, respectively, and its degradation performance was significantly higher than that of free laccase.

[0070] Specifically, the removal efficiency of AFB1 by the immobilized enzyme was the highest at pH 7, reaching 89.12%, while for ZEN, the overall removal efficiency was the best at pH 4, reaching 97.01%. In addition, at pH 6 to 8, the degradation efficiency of ZEN by the immobilized enzyme was significantly higher than that of free laccase, indicating that the application potential of laccase in neutral and alkaline environments has been further expanded.

[0071] Comprehensive analysis showed that immobilized laccase not only improved the degradation efficiency of AFB1 and ZEN, but also had good pH adaptability, showing broad potential in practical applications.

[0072] Example 5: Recycling of immobilized enzyme Lac@Fe-BTC

[0073] After the reaction, the immobilized enzyme was recycled; the reaction mixture was centrifuged at 4°C to separate the immobilized enzyme Lac@Fe-BTC precipitate; the precipitate was washed three times with NaAc-HAc buffer solution to remove the residual reactants, and then it could be used for the next round of degradation reaction.

[0074] like Figure 6 As shown, the immobilized laccase can still maintain a removal rate of more than 80% after being reused for 4 times, indicating that it has good reusability.

[0075] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing immobilized laccase at room temperature, characterized in that: The steps include: Step 1: weighing laccase, dissolving it in a sodium acetate-acetic acid buffer solution, and performing magnetic stirring, and then adding a sodium hydroxide solution containing trimesic acid to form a mixed solution; Step 2: adding an aqueous solution containing trivalent iron ions to the mixed solution, stirring the solution magnetically, placing the solution in a refrigerator for aging, and collecting the solid product by centrifugation; Step 3: The washed precipitate is resuspended in a polyvinyl pyrrolidone aqueous solution, fully dispersed, and then dried to obtain immobilized laccase, which is denoted as Lac@Fe-BTC.

2. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In the step 1, the pH of the sodium acetate-acetic acid buffer solution is 4.5-6.0, and the concentration of the buffer solution is 50-100 mM.

3. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In the step 1, the concentration of trimesic acid in the sodium hydroxide solution containing trimesic acid is 0.42-0.56 mol / L, and the molar ratio of trimesic acid to sodium hydroxide is 1:

3.

4. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In step 2, the concentration of the aqueous solution of trivalent iron ions is 0.62-0.83 mol / L.

5. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In the step 2, the stirring speed is 200-800 rpm, the stirring time at room temperature is 0.5-6 hours, the refrigerator temperature is 4° C., the static aging time is 3-12 hours, and the centrifugal speed is 5000-8000 rpm.

6. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: The volume ratio of the sodium acetate-acetic acid buffer solution, the sodium hydroxide solution of trimesic acid and the aqueous solution of ferric ions is 15-20:1:1, the molar ratio of ferric ions, trimesic acid and sodium hydroxide is 1.5:1:3, and the final concentration of laccase in the reaction system is 0.4-1.0 mg / mL.

7. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In the step 3, the concentration of the polyvinyl pyrrolidone solution is 0.5%-2.0%.

8. The method for preparing immobilized laccase at room temperature according to claim 1, characterized in that: In step 3, the drying is freeze drying for 12-18 hours, or natural drying at room temperature for 24-30 hours.

9. Use of the method for preparing immobilized laccase at room temperature according to any one of claims 1 to 8 in degrading fungal toxins.

10. The use of the method for preparing immobilized laccase at room temperature according to claim 9 in degrading fungal toxins, characterized in that: The fungal toxin is one or more of aflatoxin B1 or zearalenone, the reaction conditions are that the pH of the sodium acetate-acetic acid buffer solution is 4.5-6, or the pH of the Tris-Hcl buffer solution is 7-8, and 0.5-1mM of laccase mediator substance is added, such as one of acetosyringone, tert-butylhydroquinone, ferulic acid, p-coumaric acid, and vanillin.

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