Method for immobilizing laccase by utilizing mimic enzyme material, immobilized laccase obtained by method and application of immobilized laccase

Abstract

The invention discloses a method for immobilizing laccase by using a mimic enzyme material, the obtained immobilized laccase and application, and belongs to the technical field of biomimetic catalysis and biological catalysis. In order to solve the technical problems that the existing immobilized laccase is low in catalytic activity and cannot be recycled, the laccase is immobilized by utilizing a Cu < + > / Cu < 2 + >-HKUST-1 mimic enzyme material with a laccase-like active center, the laccase is recycled, and meanwhile, the immobilized material can further achieve a synergistic effect with the laccase active center to further improve the laccase activity; the activity of the immobilized laccase is 3.7 times that of free laccase with the same mass at most, and the synergistic catalytic activity can be improved by 36.8%. Therefore, the immobilized laccase and the preparation method thereof provided by the invention have important significance on industrial popularization and application of laccase.

Description

Technical Field

[0001] The invention belongs to the technical field of bionic catalysis and biocatalysis, and specifically relates to a method for immobilizing laccase using enzyme-mimicking materials, and the obtained immobilized laccase and its application. Background Art

[0002] Laccase (EC1.10.3.2) is a polyphenol oxidase containing copper, which is widely found in various plants, insects, bacteria and fungi. Laccase can oxidize substances with various structures such as polyphenols, polyamines and aromatic amines, and the final product of its reduction is only water. It is a green catalyst and has important application value and potential in many fields such as textiles, medicine, sewage treatment and biosensors.

[0003] Laccase has poor stability, cannot be recycled, has high cost, and has low catalytic efficiency, which limits its large-scale application. In order to improve the reusability of laccase and overcome the problem of poor stability of laccase, it is currently mainly immobilized by inorganic materials, bio-based materials, composite materials, etc. For example, various magnetic immobilization materials Fe3O4 / graphene, Fe3O4@MOF, Fe3O4@SiO2 (International Journal of Biological Macromolecules: 138, 1-12 (2019); Bioresource Technology: 317 (2020); Chin. J. Chem.: 30, 2849-2860 (2012)) have been reported to be used for immobilizing laccase, but laccase loses a lot of enzyme activity during the immobilization process, and the catalytic efficiency is reduced, which cannot be promoted on a large scale in industrial production. The bio-based materials used as laccase immobilization materials are mainly chitosan, alginate, etc. Metin et al. (Macromolecular Research, 2013, 21 (10): 1145-1152.) used active chitosan membrane grafted with polyethyleneimine to adsorb and immobilize laccase. The immobilized laccase maintained 72% of its initial activity at 55°C for 120 minutes, which is 1.84 times that of free laccase under the same conditions. However, bio-based materials have problems such as low mechanical strength, easy decomposition, short service life, and poor cycle performance. Metal organic framework materials (MOF materials) have become very promising laccase immobilization materials due to their adjustable structure, rich enzyme binding sites, and high specific surface area. Currently, the MOF materials reported in the literature for immobilizing laccase mainly include HKUST-1 (Critical Reviews in Environmental Science and Technology, 2020, 52(8): 1282-1324.), ZIF-8 (ater Air and Soil Pollution, 2015, 226(2): 10), MIL-53(Al) (Langmuir, 2022, 38(3): 1158-1169.), etc. Although this type of immobilized material can better control the immobilization amount, it does not solve the problem of low catalytic efficiency of laccase as an oxidation catalyst.

[0004] In recent years, MOF materials have also been reported as laccase mimics. Duke et al. (The Journal of Physical Chemistry C, 2015, 119 (49): 27457-27466.) synthesized mixed-valence Cu-MOF and found that the introduction of mixed-valence Cu (I) / Cu (II) sites changed the electronic properties of MOF, which can improve the conductivity through the electronic hopping mechanism and produce higher conductivity. It can better simulate the activity of laccase than the enzyme-mimicking materials containing only Cu (II). Patent application number CN202311447680.7 reports the macroporous and microporous MOF materials Cu + / Cu 2+ -HKUST-1 have good laccase mimetic activity. However, there is no report on the immobilization of laccase using MOF materials with laccase mimetic activity. Summary of the invention

[0005] In order to solve the technical problems of low catalytic activity and inability to recycle of existing immobilized laccase, the present invention utilizes Cu with laccase-like active centers. + / Cu 2+ -HKUST-1 enzyme-mimicking material immobilizes laccase. While realizing the recycling of laccase, the immobilized material can also synergize with the active center of laccase to further improve the activity of laccase, which is of great significance for the industrialization, promotion and application of laccase.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for immobilizing laccase on an enzyme-mimicking material, the method comprising the steps of:

[0008] Laccase was dissolved in acetic acid-sodium acetate buffer and then macroporous Cu + / Cu 2+ -HKUST-1 enzyme-mimicking material was placed in a shaker at 200 rpm for room temperature adsorption, and the precipitate obtained by centrifugation was the immobilized laccase.

[0009] In one embodiment of the present invention, the macroporous Cu + / Cu 2+ -The preparation method of HKUST-1 enzyme-mimicking material is as follows:

[0010] S1: Use phosphoric acid solution to treat microporous Cu 2+ -HKUST-1 was etched to obtain macroporous Cu 2+ -HKUST-1;

[0011] S2: The macroporous Cu was treated with hydroquinone aqueous solution. 2+-HKUST-1 was reduced to obtain macroporous Cu + / Cu 2+ -HKUST-1, a mixed-valence MOF material with laccase activity.

[0012] In one embodiment of the present invention, the phosphoric acid solution in S1 is composed of DMSO, methanol and phosphoric acid, has a pH value of 2-6, an etching temperature of 30-50° C., and a etching time of 4-48 hours.

[0013] In one embodiment of the present invention, the hydroquinone aqueous solution contains hydroquinone and macroporous Cu + / Cu 2+ -HKUST-1 has a mass ratio of (10-20):1.

[0014] In one embodiment of the present invention, the reduction temperature is 60-120° C. and the time is 14-18 hours.

[0015] In one embodiment of the present invention, the macroporous Cu + / Cu 2+ -The ratio of Cu(I):Cu(II) in HKUST-1 enzyme-mimicking material is 1:(3-10).

[0016] In one embodiment of the present invention, the pH of the acetic acid-sodium acetate buffer is 3-5.

[0017] In one embodiment of the present invention, the mass ratio of the enzyme-mimicking material to laccase is 1:(0.5-2).

[0018] In one embodiment of the present invention, the adsorption time is 0.5h-2h.

[0019] The second object of the present invention is to provide the immobilized laccase obtained by the above method.

[0020] The third object of the present invention is to provide a method for verifying the activity of the immobilized laccase obtained by the above method, wherein the method uses 2,4-dichlorophenol and 4-aminoantipyrine as substrates, and verifies the activity of the immobilized laccase by detecting the absorbance at a wavelength of 400-600nm every 1 hour in a Tris-HCl buffer solution using an ELISA reader.

[0021] In one embodiment of the present invention, the pH value of the Tris-HCl buffer solution is 4-6, and the mass ratio of 2,4-dichlorophenol, 4-aminoantipyrine, Tris-HCl buffer solution and immobilized laccase is 1 μg: 1 μg: 8 mL: (8-12) mg.

[0022] The fourth object of the present invention is to provide the use of the immobilized laccase obtained by the above method in oxidizing phenolic compounds.

[0023] In one embodiment of the present invention, the phenolic compounds include monophenols, diphenols and polyphenols.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention utilizes a macroporous MOF material Cu with enzyme-like activity. + / Cu 2+ -HKUST-1 was used to immobilize laccase, and the activity of the immobilized laccase was 3.7 times that of the free laccase of the same mass, which was further synergistically improved by 36.8% compared with the sum of the activities of free laccase and enzyme-mimicking materials.

[0026] (2) The present invention utilizes a macroporous MOF material Cu with enzyme-mimicking activity. + / Cu 2+ -HKUST-1 was used to immobilize laccase. After the immobilized laccase was recycled for 5 times, the enzyme activity still retained more than 80% of the initial activity, which has good recyclability and is of great significance for the further promotion and application of laccase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the infrared characterization result diagram of the immobilized laccase, enzyme-mimicking material and laccase in Example 1. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels by those skilled in the art.

[0030] The terms "comprising," "including," "having," "containing," or any other variations thereof, as used in the following examples, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0031] When equivalent, concentration or other value or parameter is represented by the range limited by range, preferred range or a series of upper preferred value and lower preferred value, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether the scope is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the scope is intended to include its end value and all integers and fractions within the scope. In the present application specification and claims, range limitation can be combined and / or interchanged, if these ranges are not otherwise stated, include all sub-ranges contained therein.

[0032] The endpoints and any values ​​of the ranges disclosed in the invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.

[0033] The laccase used in the following examples is commercial laccase derived from Coriolus versicolor, purchased from Sigma Chemical Reagent Co., Ltd., with product number 38429.

[0034] The macroporous Cu used in the following examples + / Cu 2+ -HKUST-1 enzyme-mimicking material was prepared with reference to the Chinese patent application with publication number CN117483005A, wherein the ratio of Cu(I):Cu(II) is 1:(3-10).

[0035] Embodiment 1:

[0036] Preparation of immobilized laccase: 25 mg of laccase and 20 mL of acetic acid-sodium acetate buffer (pH 4.5) were added to a 100 mL conical flask. After dissolving, a macroporous Cu(I):Cu(II) solution was added at a ratio of 1:10. + / Cu 2+ -HKUST-1 enzyme-mimicking material 40 mg, put the conical flask into a shaker (200 rpm) for adsorption at room temperature for 1.5 hours, and the precipitate obtained by centrifugation is the immobilized laccase after laccase immobilization. The precipitate was washed 3 times with distilled water, washed once with acetic acid-sodium acetate buffer, the filtrate was combined, and the laccase content in the filtrate was determined (100 μL total supernatant solution + 400 μL Coomassie Brilliant Blue G-250, absorbance measured at 595 nm), and the laccase immobilization amount was calculated to be 328 mg / g using the following formula.

[0037]

[0038] Note: v1 is the total volume of the supernatant solution, c1 is the total protein concentration of the supernatant solution, c0 is the protein concentration of the solution before immobilization, v0 is the volume of the immobilization solution, and m is the mass of the enzyme immobilization material.

[0039] Determination of immobilized laccase activity: Take a 50mL centrifuge tube, add 1mL of distilled water and 10mg of immobilized laccase, enzyme-mimicking material and free laccase of the same mass as the immobilized laccase, add 8mL of 200mM pH=5 Tris-HCl buffer solution, add 500μL of 2mg / L 4-AP and 2,4-DP. Take 200μL of the supernatant of the three reaction solutions every 1h, add it to a 96-well plate, put it into a microplate reader, and detect the absorbance at 510nm. The results show that the activity of 10mg immobilized laccase is 0.26 (AU), the activity of 10mg enzyme-mimicking material is 0.12 (AU), and the activity of 3.28mg free laccase (the same mass as the immobilized laccase) is 0.07 (AU). The activity of immobilized laccase is 3.7 times that of free laccase of the same mass, and the activity is 36.8% higher than the sum of the activities of enzyme-mimicking material and free laccase.

[0040] Embodiment 2:

[0041] Preparation of immobilized laccase: 25 mg of laccase and 20 mL of acetic acid-sodium acetate buffer (pH 4.4) were added to a 100 mL conical flask. After dissolving, a macroporous Cu(I):Cu(II) solution was added at a ratio of 1:6. + / Cu 2+ -HKUST-1 enzyme-mimicking material 40 mg, put the conical flask into a shaker (200 rpm) for adsorption at room temperature for 1.5 hours, and the precipitate obtained by centrifugation is the immobilized laccase after laccase immobilization. The precipitate was washed 3 times with distilled water, washed once with acetic acid-sodium acetate buffer, the filtrate was combined, and the laccase content in the filtrate was determined (100 μL total supernatant solution + 400 μL Coomassie Brilliant Blue G-250, absorbance measured at 595 nm), and the laccase immobilization amount was calculated to be 307 mg / g using the formula provided in Example 1.

[0042] Determination of immobilized laccase activity: Take a 50mL centrifuge tube, add 1mL of distilled water and 10mg of immobilized laccase, enzyme-mimicking material and free laccase of the same mass as the immobilized laccase, add 8mL of 200mM pH=5 Tris-HCl buffer solution, add 500μL of 2mg / L 4-AP and 2,4-DP. Take 200μL of the supernatant of the three reaction solutions every 1h, add it to a 96-well plate, put it into a microplate reader, and detect the absorbance at 510nm. The results show that the activity of 10mg immobilized laccase is 0.21 (AU), the activity of 10mg enzyme-mimicking material is 0.10 (AU), and the activity of 3.07mg free laccase (the same mass as the immobilized laccase) is 0.07 (AU). The activity of immobilized laccase is 3.0 times that of free laccase of the same mass, and the activity is 23.5% higher than the sum of the activities of enzyme-mimicking material and free laccase.

[0043] Embodiment 3:

[0044] Preparation of immobilized laccase: 30 mg of laccase and 20 mL of acetic acid-sodium acetate buffer (pH 4.5) were added to a 100 mL conical flask. After dissolving, a macroporous Cu(I):Cu(II) solution was added in a ratio of 1:3. + / Cu 2+ -HKUST-1 enzyme-mimicking material 40 mg, put the conical flask into a shaker (200 rpm) for adsorption at room temperature for 1 hour, and the precipitate obtained by centrifugation is the immobilized laccase after laccase immobilization. The precipitate was washed 3 times with distilled water, washed once with acetic acid-sodium acetate buffer, the filtrate was combined, and the laccase content in the filtrate was determined (100 μL total supernatant solution + 400 μL Coomassie Brilliant Blue G-250, absorbance measured at 595 nm), and the laccase immobilization amount was calculated using the formula provided in Example 1 to be 321 mg / g.

[0045] Determination of immobilized laccase activity: Take a 50mL centrifuge tube, add 1mL of distilled water and 10mg of immobilized laccase, enzyme-mimicking material and free laccase of the same mass as the immobilized laccase, add 8mL of 200mM Tris-HCl buffer solution of pH=5, add 500μL of 2mg / L 4-AP and 2,4-DP. Take 200μL of the supernatant of the three reaction solutions every 1h, add it to a 96-well plate, put it into a microplate reader, and detect the absorbance at 510nm. The results show that the activity of 10mg immobilized laccase is 0.20 (AU), the activity of 10mg enzyme-mimicking material is 0.09 (AU), and the activity of 3.21mg free laccase (the same mass as the immobilized laccase) is 0.07 (AU). The activity of immobilized laccase is 2.8 times that of free laccase of the same mass, and the activity is 25.0% higher than the sum of the activities of enzyme-mimicking material and free laccase.

[0046] Embodiment 4:

[0047] Preparation of immobilized laccase: 25 mg of laccase and 20 mL of acetic acid-sodium acetate buffer (pH 4.5) were added to a 100 mL conical flask. After dissolving, a macroporous Cu(I):Cu(II) solution was added at a ratio of 1:10. + / Cu 2+ -HKUST-1 enzyme-mimicking material 40 mg, put the conical flask into a shaker (200 rpm) for adsorption at room temperature for 0.5 hours, and the precipitate obtained by centrifugation is the immobilized laccase after laccase immobilization. The precipitate was washed 3 times with distilled water, washed once with acetic acid-sodium acetate buffer, the filtrate was combined, and the laccase content in the filtrate was determined (100 μL total supernatant solution + 400 μL Coomassie Brilliant Blue G-250, absorbance measured at 595 nm), and the laccase immobilization amount was calculated to be 295 mg / g using the formula provided in Example 1.

[0048] Determination of immobilized laccase activity: Take a 50mL centrifuge tube, add 1mL of distilled water and 10mg of immobilized laccase, enzyme-mimicking material and free laccase of the same mass as the immobilized laccase, add 8mL of 200mM pH=5 Tris-HCl buffer solution, add 500μL of 2mg / L 4-AP and 2,4-DP. Take 200μL of the supernatant of the three reaction solutions every 1h, add it to a 96-well plate, put it into a microplate reader, and detect the absorbance at 510nm. The results show that the activity of 10mg immobilized laccase is 0.20 (AU), the activity of 10mg enzyme-mimicking material is 0.12 (AU), and the activity of 2.95mg free laccase (the same mass as the immobilized laccase) is 0.07 (AU). The activity of immobilized laccase is 2.9 times that of free laccase of the same mass, and the activity is 5.2% higher than the sum of the activities of enzyme-mimicking material and free laccase.

[0049] Embodiment 5:

[0050] Preparation of immobilized laccase: 25 mg of laccase and 20 mL of acetic acid-sodium acetate buffer (pH 4.5) were added to a 100 mL conical flask. After dissolving, a macroporous Cu(I):Cu(II) solution was added at a ratio of 1:10. + / Cu 2+ -HKUST-1 enzyme-mimicking material 40 mg, put the conical flask into a shaker (200 rpm) for adsorption at room temperature for 1.5 hours, and the precipitate obtained by centrifugation is the immobilized laccase after laccase immobilization. The precipitate was washed 3 times with distilled water, washed once with acetic acid-sodium acetate buffer, the filtrate was combined, and the laccase content in the filtrate was determined (100 μL total supernatant solution + 400 μL Coomassie Brilliant Blue G-250, absorbance measured at 595 nm), and the laccase immobilization amount was calculated to be 328 mg / g using the formula provided in Example 1.

[0051] Determination of immobilized laccase activity: Take a 50mL centrifuge tube, add 1mL of distilled water and 8mg of immobilized laccase, enzyme-mimicking material and free laccase of the same mass as the immobilized laccase, add 8mL of 200mM Tris-HCl buffer solution with pH=5, and add 500μL of 2mg / L 4-AP and 2,4-DP. Take 200μL of the supernatant of the three reaction solutions every 1h, add it to a 96-well plate, put it into a microplate reader, and detect the absorbance at 510nm. The results show that the activity of 8mg immobilized laccase is 0.17 (AU), the activity of 8mg enzyme-mimicking material is 0.10 (AU), and the activity of 2.62mg free laccase (the same mass as the immobilized laccase) is 0.05 (AU). The activity of immobilized laccase is 3.3 times that of free laccase of the same mass, and the activity is 13.3% higher than the sum of the activities of enzyme-mimicking material and free laccase.

[0052] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for immobilizing laccase on enzyme-mimicking materials, characterized in that: Follow these steps: Laccase was dissolved in acetic acid-sodium acetate buffer and then macroporous Cu + / Cu 2+ -HKUST-1 enzyme-mimicking material was placed in a shaker at 200 rpm for room temperature adsorption, and the precipitate obtained by centrifugation was the immobilized laccase.

2. The method according to claim 1, characterized in that The macroporous Cu + / Cu 2+ -The ratio of Cu(I):Cu(II) in HKUST-1 enzyme-mimicking material is 1:(3-10).

3. The method according to claim 1, characterized in that The pH of the acetic acid-sodium acetate buffer is 3-5.

4. The method according to claim 1, characterized in that: The mass ratio of the enzyme-mimicking material to laccase is 1:(0.5-2).

5. The method according to claim 1, characterized in that The adsorption time is 0.5h-2h.

6. The immobilized laccase obtained by the method according to any one of claims 1 to 5.

7. A method for verifying the activity of immobilized laccase obtained by the method according to any one of claims 1 to 5, characterized in that: The activity of the immobilized laccase was verified by using 2,4-dichlorophenol and 4-aminoantipyrine as substrates in a Tris-HCl buffer solution and measuring the absorbance at a wavelength of 400-600 nm every 1 h using an ELISA reader.

8. The activity verification method according to claim 7, characterized in that: The pH value of the Tris-HCl buffer solution is 4-6, and the mass ratio of 2,4-dichlorophenol, 4-aminoantipyrine, Tris-HCl buffer solution and immobilized laccase is 1 μg: 1 μg: 8 mL: (8-12) mg.

9. Use of the immobilized laccase obtained by the method according to any one of claims 1 to 5 in oxidizing phenolic compounds.

10. The use according to claim 9, characterized in that: The phenolic compounds include monophenols, diphenols and polyphenols.

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