A method for preparing and verifying activity of a mixed valence MOF material having laccase activity

Abstract

The application relates to a preparation method and activity verification method of a mixed valence MOF material with laccase activity. The application belongs to the field of biomimetic catalysis. The application aims to solve the technical problem of low catalytic activity of existing laccase enzyme simulation materials. The application designs a kind of Cu + / Cu 2+ The application has the advantages of simple process, mild conditions, and the obtained Cu + / Cu 2+ The HKUST-1 material has higher laccase activity than the current Cu 2+ The enzyme simulation material has the potential of higher laccase activity, the catalytic activity of 2,3-dichlorophenol is up to 1.4 times that of the equal mass commercial laccase, has the advantages of high efficiency and low cost, and has good industrial application prospect. The application can be widely used for oxidizing phenolic compounds.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic catalysis, specifically relating to a method for preparing and verifying the activity of a mixed-valence MOF material with laccase activity. Background Technology

[0002] Laccase is a polyphenol oxidase (EC 1.10.3.2) containing four copper ions. It was first discovered in 1883 by Japanese scholar Yoshida in the lacquer sap of the Japanese lacquer tree and belongs to the copper blue oxidase family. After more than a century of research, it has been found that laccase is widely present in various fungi and plants and can catalyze the oxidation of a variety of substrates, including monophenols, diphenols, and polyphenols. It has found wide applications in various fields, such as bioremediation, nanobiotechnology, woodworking, pulp bleaching, textile dyeing, biotechnology applications in the food industry, biorefining, and wastewater treatment. However, the complex preparation and purification processes of commercial laccase lead to high costs, and its susceptibility to denaturation and degradation limits its further industrial application. Therefore, finding an enzyme-mimicking material that possesses the catalytic activity of natural laccase while also exhibiting high stability and low cost is of great significance.

[0003] The active center of laccase is relatively conserved, and its oxidation function relies heavily on electron transfer between the four copper ions within this center. In the redox cycle of laccase-catalyzed reactions, these four copper ions pass through monovalent copper (Cu)... + ) and divalent copper (Cu) 2+ The oxidative stress of copper laccase involves electron transfer through a change in valence state, simultaneously oxidizing the substrate into free radicals. Therefore, a series of laccase-like materials with copper as the catalytic center have been reported. For example, Liu et al. utilized guanosine monophosphate (GMP) and divalent copper (Cu)... 2+ Nanozymes with laccase activity were formed through coordination; Zhang et al. synthesized laccase analogs with high catalytic activity using divalent copper as the catalytic center and glutathione (GSH) as the ligand. These copper-centered enzymes have the advantage of high stability, but Cu... 2+ As the active center, it has low electron transfer efficiency and its catalytic activity is lower than that of laccase, making it unable to replace commercial laccase. Therefore, preparing a laccase mimic with good stability, high activity, and low cost would help promote the industrial application of laccase-related research. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and verifying the activity of mixed-valence MOF materials with laccase activity.

[0005] The technical solution of the present invention is as follows:

[0006] One objective of this invention is to provide a method for preparing a mixed-valence MOF material with laccase activity, the method comprising the following steps:

[0007] S1: Using phosphoric acid solution to treat microporous Cu 2+ -HKUST-1 was used for etching to obtain macroporous Cu. 2+ -HKUST-1;

[0008] S2: Apply hydroquinone aqueous solution to macroporous Cu 2+ -HKUST-1 was used for reduction to obtain macroporous Cu. + / Cu 2+ -HKUST-1 refers to a mixed-valence MOF material with laccase activity.

[0009] Further specifying, the phosphoric acid solution in S1 consists of dimethyl sulfoxide (DMSO), methanol, and phosphoric acid, with a pH value of 2-6.

[0010] Further specified, the etching temperature in S1 is 30-50℃, and the time is 4-48h.

[0011] Further specifying, in S2, hydroquinone in the aqueous solution reacts with macroporous Cu. 2+ The mass ratio of HKUST-1 is (10-20):1.

[0012] Further specified, the reduction temperature in S2 is 60-120℃, and the time is 14-18h.

[0013] The second objective of this invention is to provide a method for preparing a mixed-valence MOF material with laccase activity, the method comprising the following steps:

[0014] Hydroquinone aqueous solution was used to treat microporous Cu 2+ -HKUST-1 was used for reduction to obtain microporous Cu. + / Cu 2+ -HKUST-1 refers to a mixed-valence MOF material with laccase activity.

[0015] Further specifying, hydroquinone in hydroquinone aqueous solution reacts with microporous Cu 2+ The mass ratio of HKUST-1 is (10-20):1.

[0016] Further restrictions are placed on the reduction temperature, which is 60-120℃, and the time, which is 14-18h.

[0017] A third objective of this invention is to provide a mixed-valence MOF material with laccase activity obtained by the above method, wherein the material is macroporous Cu. + / Cu 2+ -HKUST-1 or microporous Cu+ / Cu 2+ -HKUST-1.

[0018] The fourth objective of this invention is to provide a method for verifying the laccase-mimicking activity of mixed-valence MOF materials with laccase activity obtained by the above method, wherein the method is carried out in the following steps:

[0019] Using 2,4-dichlorophenol and 4-aminoantipyrine as substrates, the absorbance of mixed-valence MOF materials was measured at a wavelength of 400-600 nm every 1 hour using a microplate reader in Tris-HCl buffer solution to verify the laccase-mimicking activity.

[0020] Further specifying, the pH value of the Tris-HCl buffer solution is 4-6.

[0021] Further specifying, the ratio of 2,4-dichlorophenol, 4-aminoantipyrine, Tris-HCl buffer solution and mixed-valence MOF material is 1 mg: 1 mg: 8 mL: (8-12) mg.

[0022] The fifth objective of this invention is to provide an application of a mixed-valence MOF material with laccase activity obtained by the above method in oxidized phenolic compounds.

[0023] To further define, phenolic compounds include monophenols, diphenols, and polyphenols.

[0024] The significant advantages of this invention compared to existing technologies are:

[0025] (1) This invention designs a Cu + / Cu 2+ Porous materials of mixed-valence copper exhibit better performance than current Cu materials. 2+ The potential of enzyme-mimicking materials to achieve higher laccase activity not only further promotes the industrial application of laccase but also has significant academic and applied value for expanding the research field of laccase.

[0026] (2) Cu prepared by this invention + / Cu 2+ -HKUST-1 material, simple process, mild conditions, Cu + / Cu 2+ -HKUST-1 material, as a laccase imitator, exhibits up to 1.4 times the catalytic activity of 2,3-dichlorophenol compared to the same mass of commercial laccase. It boasts advantages of high efficiency and low cost, and has promising prospects for industrial application. Attached Figure Description

[0027] Figure 1 This is a comparison chart showing the results of catalytic oxidation of 2,4-dichlorophenol by different enzyme-mimicking materials described in this invention and the activity of commercial laccase of equal mass. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

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

[0031] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range. In this specification and claims, range definitions may be combined and / or interchanged, unless otherwise stated, these ranges include all subranges contained therein.

[0032] The endpoints and any values ​​of the ranges disclosed in this 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] The microporous Cu used in the following examples 2+-HKUST-1 (HKUST-1CuBTC) was purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., with a particle size of 0.5μm-40μm and a micropore size of 0.9nm. The commercial laccase was sourced from Yunzhi and purchased from Sigma Chemical Reagent Co., Ltd., product number 38429.

[0034] Example 1

[0035] (1) Preparation of mixed valence microporous MOF materials: Take a 100mL thick-walled pressure-resistant bottle and add 400mg of dissolved divalent microporous Cu. 2+ HKUST-1 and 8g hydroquinone were dissolved in 40mL of distilled water, and the mixture was heated at 83°C for 16 hours. The product was filtered, and the residue was washed three times with 100mL of methanol. The residue was then vacuum dried at room temperature to obtain mixed-valence microporous Cu. + / Cu 2+ -HKUST-1, Cu(II):Cu(I)=12:1.

[0036] (2) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence state microporous Cu. + / Cu 2+ -HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=4) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 1.4 times that of an equivalent mass of commercial laccase.

[0037] Example 2

[0038] (1) Preparation of mixed valence microporous MOF materials: Take a 100mL thick-walled pressure-resistant bottle and add 400mg of dissolved divalent microporous Cu. 2+ HKUST-1 and 4g hydroquinone were dissolved in 40mL of distilled water, and the mixture was heated at 83°C for 16 hours. The product was filtered, and the residue was washed three times with 100mL of methanol. The residue was then vacuum dried at room temperature to obtain mixed-valence microporous Cu. + / Cu 2+ -HKUST-1, Cu(II):Cu(I)=16:1.

[0039] (2) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence state microporous Cu. + / Cu 2+-HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 1.2 times that of an equivalent mass of commercial laccase.

[0040] Comparative Example 1

[0041] Commercially available microporous Cu 2+ -HKUST-1 activity assay: Take a 50mL centrifuge tube and add 10mg of microporous Cu. 2+ -HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 0.42 times that of an equivalent mass of commercial laccase.

[0042] Example 3

[0043] (1) Preparation of divalent macroporous materials: Take a 50mL centrifuge tube and add 200mg of divalent microporous Cu. 2+ HKUST-1, 50 mL of DMSO and 50 mL of methanol were added, and the pH of the solution was adjusted to 2.6 with phosphoric acid. The solution was etched for 8 hours at 200 rpm in a shaker at 40 °C. The resulting product was washed with methanol and dried at 60 °C for 6 hours to obtain divalent macroporous Cu. 2+ -HKUST-1 exhibits uniformly distributed macropores with a diameter of 300-500nm.

[0044] (2) Preparation of mixed valence macroporous MOF materials: Take a 100mL thick-walled pressure-resistant bottle and add 400mg of dissolved divalent macroporous Cu. 2+ HKUST-1 and 4g hydroquinone were dissolved in 40mL of distilled water, and the mixture was heated at 85°C for 16 hours. The product was filtered, and the residue was washed three times with 100mL of methanol. The residue was then vacuum dried at room temperature to obtain mixed-valence microporous Cu. + / Cu 2+ -HKUST-1, Cu(II):Cu(I)=9:1.

[0045] (3) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence macroporous Cu. + / Cu 2+-HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 0.82 times that of an equivalent mass of commercial laccase.

[0046] Example 4

[0047] (1) Preparation of divalent macroporous materials: Take a 50mL centrifuge tube and add 200mg of divalent microporous Cu. 2+ HKUST-1, 50 mL of DMSO and 50 mL of methanol were added, and the pH of the solution was adjusted to 2.6 with phosphoric acid. The solution was etched for 8 hours at 200 rpm in a shaker at 40 °C. The resulting product was washed with methanol and dried at 60 °C for 6 hours to obtain divalent macroporous Cu. 2+ -HKUST-1 exhibits uniformly distributed macropores with a diameter of 300-500nm.

[0048] (2) Preparation of mixed valence macroporous MOF materials: Take a 100mL thick-walled pressure-resistant bottle and add 400mg of dissolved divalent macroporous Cu. 2+ HKUST-1 and 4g hydroquinone were dissolved in 40mL of distilled water, and the mixture was heated at 105°C for 16 hours. The product was filtered, and the residue was washed three times with 100mL of methanol. The residue was then vacuum dried at room temperature to obtain mixed-valence microporous Cu. + / Cu 2+ -HKUST-1, Cu(II):Cu(I)=5:1.

[0049] (3) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence macroporous Cu. + / Cu 2+ -HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 0.73 times that of an equivalent mass of commercial laccase.

[0050] Example 5

[0051] (1) Preparation of divalent macroporous materials: Take a 50mL centrifuge tube and add 200mg of divalent microporous Cu. 2+HKUST-1, 50 mL of DMSO and 50 mL of methanol were added, and the pH of the solution was adjusted to 2.6 with phosphoric acid. The solution was etched for 8 hours at 200 rpm in a shaker at 40 °C. The resulting product was washed with methanol and dried at 60 °C for 6 hours to obtain divalent macroporous Cu. 2+ -HKUST-1 exhibits uniformly distributed macropores with a diameter of 300-500nm.

[0052] (2) Preparation of mixed-valence macroporous MOF materials: Take a 100mL thick-walled pressure-resistant bottle and add 400mg of dissolved divalent macroporous Cu. 2+ HKUST-1 and 8g hydroquinone were dissolved in 40mL of distilled water, and the mixture was heated at 85°C for 16 hours. The product was filtered, and the residue was washed three times with 100mL of methanol. The residue was then vacuum dried at room temperature to obtain mixed-valence microporous Cu. + / Cu 2+ -HKUST-1, Cu(II):Cu(I)=3:1.

[0053] (3) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence macroporous Cu. + / Cu 2+ -HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 0.94 times that of an equivalent mass of commercial laccase.

[0054] Comparative Example 2

[0055] (1) Preparation of divalent macroporous materials: Take a 50mL centrifuge tube and add 200mg of divalent microporous Cu. 2+ HKUST-1, 50 mL of DMSO and 50 mL of methanol were added, and the pH of the solution was adjusted to 2.6 with phosphoric acid. The solution was etched for 8 hours at 200 rpm in a shaker at 40 °C. The resulting product was washed with methanol and dried at 60 °C for 6 hours to obtain divalent macroporous Cu. 2+ -HKUST-1 exhibits uniformly distributed macropores with a diameter of 300-500nm.

[0056] (2) Activity assay: Take a 50 mL centrifuge tube and add 10 mg of mixed valence macroporous Cu. + / Cu 2+-HKUST-1, 1 mg of 2,4-dichlorophenol and 1 mg of 4-aminoantipyrine, and 8 mL of Tris-HCl buffer solution (pH=5) were mixed in a centrifuge tube and placed in a shaker at 200 rpm at room temperature. Every 1 hour, 300 μL of the reaction solution was collected, centrifuged, and 200 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 510 nm using a microplate reader. The activity was 0.67 times that of an equivalent mass of commercial laccase.

[0057] The above description is merely a preferred embodiment of the present invention. These specific embodiments are different implementations based on the overall concept of the present invention, and the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a mixed-valence MOF material with laccase activity, characterized in that, Follow these steps: S1: Using phosphoric acid solution to treat microporous Cu 2+ -HKUST-1 was used for etching to obtain macroporous Cu. 2+ -HKUST-1; S2: Apply hydroquinone aqueous solution to macroporous Cu 2+ -HKUST-1 was used for reduction to obtain macroporous Cu. + / Cu 2+ -HKUST-1, namely, a mixed-valence MOF material with laccase activity; The phosphoric acid solution in S1 consists of DMSO, methanol, and phosphoric acid, with a pH of 2-6, an etching temperature of 30-50℃, and an etching time of 4-48 hours; hydroquinone aqueous solution contains hydroquinone and macroporous Cu. 2+ The mass ratio of -HKUST-1 is (10-20):1; the reduction temperature is 60-120℃, and the time is 14-18h.

2. A method for preparing a mixed-valence MOF material with laccase activity, characterized in that, Hydroquinone aqueous solution was used to treat microporous Cu 2+ -HKUST-1 was used for reduction to obtain microporous Cu. + / Cu 2+ -HKUST-1, namely, a mixed-valence MOF material with laccase activity; Hydroquinone in aqueous solution and microporous Cu 2+ The mass ratio of -HKUST-1 is (10-20):1, the reduction temperature is 60-120℃, and the time is 14-18h.

3. The mixed-valence MOF material with laccase activity obtained by the method of claim 1 or 2, characterized in that, The material is macroporous Cu. + / Cu 2+ -HKUST-1 or microporous Cu + / Cu 2+ -HKUST-1.

4. A method for verifying the laccase-mimicking activity of mixed-valence MOF materials with laccase activity obtained by the method of claim 1 or 2, characterized in that, Using 2,4-dichlorophenol and 4-aminoantipyrine as substrates, the absorbance of mixed-valence MOF materials was measured at a wavelength of 400-600 nm every 1 hour using a microplate reader in Tris-HCl buffer solution to verify the laccase-mimicking activity.

5. The method according to claim 4, characterized in that, The pH of the Tris-HCl buffer solution was 4-6, and the ratio of 2,4-dichlorophenol, 4-aminoantipyrine, Tris-HCl buffer solution and mixed valence MOF material was 1 mg: 1 mg: 8 mL: (8-12) mg.

6. The application of the mixed-valence MOF material with laccase activity obtained by the method of claim 1 or 2 in the oxidation of phenolic compounds.

7. The application according to claim 6, characterized in that, Phenolic compounds include monophenols, diphenols, and polyphenols.

Images