A laccase derived from fomes fomentarius and its use

Abstract

The application discloses a laccase from Inonotus winteri and application thereof. The laccase produced by liquid fermentation from Inonotus winteri with the preservation number CCTCC NO: M 2020809 can be applied to catalyze the polymerization of two types of film-forming monomers, i.e. organic compounds containing phenolic hydroxyl and organic compounds containing at least two amino groups, to generate organic copolymer films by self-assembly at different interfaces. The adhesion of the copolymer films to solid materials is water-resistant, and the copolymer films are resistant to acid, alkali, organic solvents and heat. The organic copolymer films can introduce a functional layer to the surface of the films through secondary reaction, so as to achieve the purpose of functionalization and modification of the surface of the substrate. The laccase is easy to realize industrial production, has low cost and suitable enzymatic properties, and is not sensitive to metal ions in the medium.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a laccase derived from winter polypores and its applications. Background Technology

[0002] Laccase (phenylene glycol:oxygen oxidoreductase, EC 1.10.3.2) is a copper-containing polyphenol oxidase belonging to the blue polycopper oxidase (MCOs) family, widely distributed in plants, fungi, some bacteria, and insects. Laccase can catalyze compounds such as monophenols, polyphenols, aniline, and polycyclic aromatic hydrocarbons, thus demonstrating significant application value in food, papermaking, textiles, green synthesis, bio-batteries, formaldehyde-free plywood bonding, and organic pollutant treatment. Laccase is one of the most promising oxidoreductases.

[0003] The surfaces of many products and equipment require coatings to achieve decoration, protection, or to impart new functions, which are crucial to the safety, performance, and lifespan of these products and equipment. Polymer coatings are essential functional materials for national economic development and defense security, as polymers are the film-forming agents in these coatings. Surface functionalization of materials has significant applications in many fields, and polymer coatings are one means of altering surface properties and achieving surface functionalization. The preparation of complex materials using simple methods and the design and synthesis of nano-coating materials are both hot topics in coating materials research and development. The creation, preparation methods, and applications of novel interface materials are also key areas of international scientific and technological competition. Summary of the Invention

[0004] The purpose of this invention is to provide an application of fungal laccase derived from Polyporus brumalis in catalyzing the polymerization of film-forming monomers at different interfaces to generate organic copolymer films.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a fungal laccase, prepared by liquid fermentation of a winter polyporus (Polyporus brumalis) with strain preservation number CCTCC NO: M 2020809, wherein the amino acid sequence of the fungal laccase is as follows (1) or (2):

[0007] (1) The amino acid sequence of the enzyme protein is shown in SEQ ID NO:1;

[0008] (2) An amino acid sequence that has undergone substitution, deletion or addition of one or more amino acids based on the amino acid sequence defined in (1) and has laccase activity.

[0009] The preparation method of the above-mentioned fungal laccase is as follows:

[0010] (1) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) strain CCTCC NO:M2020809 on a slant. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 150-200mL of liquid seed culture medium, and incubate at 25-35℃ and 150-200rpm for 3-5 days to prepare seed culture;

[0011] (2) Shake flask fermentation culture: The seed liquid is inoculated into the fermentation medium at an inoculation rate of 6% to 12% by volume. The fermentation medium is 125 to 175 mL in a 500 mL Erlenmeyer flask. The culture is shaken at 25 to 35 °C. The shaking speed is 100 to 150 rpm on the first to third day of fermentation, and 160 to 200 rpm after 3 days of fermentation. The culture is carried out for 11 to 14 days to obtain the fermentation broth.

[0012] Laccase accumulates in the extracellular fermentation broth, and can be collected, isolated, or purified from the fermentation broth without disrupting the cells;

[0013] (3) Preparation of crude enzyme solution: The fermentation broth was centrifuged at 10000×g at 4℃ for 10min, and the supernatant was the crude enzyme solution of laccase.

[0014] Preferably, the liquid seed culture medium is: corn flour 25-35 g / L, soybean meal 10-20 g / L, α-amylase 30-65 U / L, NaH2PO4 2-4 g / L, KCl 0.8-2 g / L, MgSO4·7H2O 1-2 g / L, with the remainder being water, pH 5.7-6.5, sterilized at 0.1 MPa for 25 min.

[0015] Preferably, the fermentation medium is specifically composed of the following substances: fructose 40-50 g / L, corn flour 10-15 g / L, soybean peptone 5.4-10.8 g / L, (NH4)2SO4 0-2 g / L, KCl 0.8-1.3 g / L, NaH2PO4 1.5-2 g / L, MgSO4·7H2O 0.25-1 g / L, CuSO4·5H2O 0.5-1.5 mmol / L, VB1 0.02-0.03 g / L, Tween-80 0.3-0.7 g / L, vanillin 0.4-1.3 mmol / L, and the remainder is water, pH 5.7-6.5, sterilized at 0.1 MPa for 25 min.

[0016] Secondly, the present invention provides the application of the above-mentioned fungal laccase in catalyzing the self-assembly of two types of film-forming monomers at different interfaces to generate organic copolymer films, characterized in that one type of film-forming monomer is an organic compound containing phenolic hydroxyl groups, and the other type of film-forming monomer is an organic compound containing at least two amino groups.

[0017] Preferably, the film-forming monomer is either an organic compound containing at least two phenolic hydroxyl groups or an organic compound containing at least two amino groups; or the film-forming monomer is either an organic compound containing at least one phenolic hydroxyl group and one carboxyl group or an aromatic amine organic compound containing at least two amino groups.

[0018] Preferably, the interface is a gas-liquid interface, a liquid-liquid interface, or a solid-liquid interface.

[0019] Beneficial effects of the present invention

[0020] This invention utilizes laccase produced by liquid fermentation of *Polyporus brumalis* (accession number CCTCC NO: M 2020809). This laccase can be applied to catalyze the self-assembly of two types of film-forming monomers—organic compounds containing phenolic hydroxyl groups and organic compounds containing at least two amino groups—at different interfaces to generate organic copolymer films. For example, it catalyzes the self-assembly of ferulic acid and p-phenylenediamine on solid material surfaces to generate organic copolymer films with extremely strong adhesion; similarly, it catalyzes the self-assembly of iris flavin and polyethyleneimine (PEI) on solid material surfaces to generate organic copolymer films with extremely strong adhesion. These copolymer films belong to novel functional polymer materials. These copolymer films exhibit water resistance to adhesion to solid materials and good resistance to acids, alkalis, organic solvents, and heat, providing feasible environmental tolerance for the development of novel functional copolymer adhesive layers for applications in multiple fields.

[0021] This organic copolymer membrane can be functionalized by introducing a functional layer onto its surface through a secondary reaction, thereby achieving surface functionalization and enabling the designability of the material. The laccase derived from winter-growing polypores is easily and industrially prepared at a low cost, does not form useless enzyme-substrate complexes, and water is the only byproduct; it is not highly sensitive to metal ions in the medium; and molecular oxygen can be used as the oxidant, thus eliminating the need to add hydrogen peroxide during the reaction.

[0022] Terminology Definitions and Explanations

[0023] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and integrated with each other.

[0024] The term "film-forming monomer" refers to organic compound molecules or molecular groups that become copolymer films through polymerization reactions.

[0025] The term "liquid-liquid interface" refers to the interface between an aqueous phase and a liquid phase that is immiscible with water.

[0026] The term "self-assembled membrane" refers to an ordered supramolecular system formed by the spontaneous adsorption of active molecules onto a heterogeneous interface through chemical bonds.

[0027] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Attached Figure Description

[0028] Figure 1 Infrared spectra of film-forming monomers and copolymer films.

[0029] The winter polyporus of this invention is classified and named Polyporus brumalis TIB.BPE.11072, which was deposited on November 30, 2020 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, 430072, abbreviated as CCTCC, with accession number CCTCC NO: M2020809. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0031] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under standard conditions or as recommended by the manufacturer.

[0032] method

[0033] 1. Methods for determining laccase activity

[0034] The sample cell temperature of the spectrophotometer was controlled at 55℃. 2.7 mL of 0.05 mol / L sodium citrate buffer (pH 3.0) and 0.2 mL of 1 mmol / L ABTS solution were placed in a cuvette with a path length of 1 cm. 0.1 mL of the enzyme solution diluted to an appropriate ratio was added to initiate the reaction. The absorbance of the reaction solution at 420 nm was measured during the first 3 minutes, and the rate of change of absorbance was calculated. An enzyme solution of the corresponding concentration, heated to boiling for 5 minutes, was used as a control.

[0035] Laccase activity is defined as the amount of enzyme required to oxidize 1 μmol of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) per minute, which is one unit of enzyme activity (U).

[0036] The formula for calculating enzyme activity is:

[0037]

[0038] In the formula, U is the enzyme activity of the test enzyme solution (U / mL); V is the total reaction volume (mL); ΔOD is the change in absorbance at 420 nm; n is the dilution factor of the test enzyme solution; ε420=36000M -1 cm -1 t is the molar extinction coefficient of ABTS; v is the volume (mL) of enzyme solution diluted to the appropriate factor; t is the reaction time (min); L is the optical path length of the cuvette (cm).

[0039] 2. Determination of protein content

[0040] Following Bradford's method, Coomassie Brilliant Blue G-250 was used to determine the laccase protein content using bovine serum albumin as a standard (Bradford MM. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1-2): 248-254.).

[0041] Example 1. Fermentation preparation of laccase

[0042] (1) Liquid seed culture medium: corn flour 30g / L, soybean meal 15g / L, α-amylase 54U / L, NaH2PO4 2.6g / L, KCl 2.25g / L, MgSO4·7H2O 1.5g / L, the remainder is water, pH 4.0, sterilized at 0.1MPa for 25min.

[0043] Fermentation medium: fructose 48 g / L, corn flour 12 g / L, soybean peptone 9 g / L, (NH4)2SO4 0.5 g / L, KCl 1 g / L, NaH2PO4 1.8 g / L, MgSO4·7H2O 0.25 g / L, CuSO4·5H2O 1 mmol / L, VB1 0.03 g / L, Tween-80 0.5 g / L, vanillin 1 mmol / L, the remainder being water, pH 6.5, sterilized at 0.1 MPa for 25 min.

[0044] (2) Liquid seed culture: Take about 3cm of slant culture of winter polyporus (Polyporus brumalis) CCTCC NO: M 2020809. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 150mL of liquid seed culture medium, and incubate at 30℃ and 150rpm for 4 days to obtain seed culture.

[0045] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 6%, with 150 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 30°C with shaking. The shaking speed was 150 rpm for days 1-3 of fermentation, and 200 rpm after day 3. The fermentation broth was obtained after 13 days of fermentation. The laccase content in the fermentation broth was 982 U / mL.

[0046] (3) Preparation of crude enzyme solution: The fermentation broth was centrifuged at 10000×g at 4℃ for 10min, and the supernatant was the crude enzyme solution of laccase.

[0047] (4) Isolation and purification of laccase:

[0048] Take 100 mL of crude laccase solution and slowly add ammonium sulfate in stages while stirring, gradually increasing the concentration of the ammonium sulfate solution to 60%. After sufficient precipitation, centrifuge at 12000×g for 10 min at 4℃, collect the precipitate, and dissolve it in an appropriate volume of pH 7.0, 0.02 mol / L citrate-disodium hydrogen phosphate buffer to obtain the salting-out solution. Dialyze the salting-out solution in the same buffer solution overnight, changing the dialysate every 10 h until the conductivity and pH of the liquid inside and outside the dialysis bag are the same, thus obtaining the laccase dialysate.

[0049] A DEAE-Sepharose Fast Flow ion exchange medium chromatography column was equilibrated with a pH 7.0, 0.02 mol / L citrate-disodium hydrogen phosphate buffer (Solution A). The dialyzed enzyme solution was filtered through a 0.22 μm filter and loaded with 10 mL at a flow rate of 2 mL / min. After loading, the column was equilibrated with Solution A until the baseline was zero. Then, a continuous gradient elution was performed with citrate-disodium hydrogen phosphate buffer (Solution B) containing 0–0.5 mol / L NaCl at a flow rate of 1 mL / min. The absorbance at 280 nm was recorded, and the active fraction was collected (2 mL per tube). Laccase activity and protein concentration were measured in the collected samples. The eluates containing laccase activity were combined to obtain the column-purified laccase (referred to as pure enzyme solution in this invention), which was stored at -20°C for later use. The results of each of the above separation and purification steps are shown in Table 1, where specific activity refers to the enzyme activity units contained per milligram of enzyme protein.

[0050] Table 1 Results of each separation and purification step

[0051] step Total enzyme activity (U) Total protein (mg) Enzyme activity (U / mg) Purification factor Recovery rate (%) Fermentation liquid 25168 / / / / crude enzyme solution 25168 27.61 911.45 1.00 100.00 Salting out liquid 16857 14.88 1132.91 1.24 66.98 Dialysate 16788 14.84 1131.55 1.24 66.71 Pure enzyme solution 15867 12.15 1305.96 1.43 63.04

[0052] Example 2. Enzymatic Properties Analysis of Laccase

[0053] (1) Optimal temperature

[0054] The activity of laccase in the pure enzyme solution prepared in Example 4 was measured at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ and 70℃, respectively, using ABTS, guaiacol, o-methylbenzidine, DMP, eugenol, catechol and p-phenylenediamine as substrates. The temperature at which the enzyme activity was highest was the optimal temperature for laccase to catalyze the substrate. The results are shown in Table 2.

[0055] (2) Optimal pH

[0056] At 55℃, laccase activity was measured at pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0, using ABTS, guaiacol, o-methylbenzidine, DMP, eugenol, catechol and p-phenylenediamine as substrates. The pH at which the enzyme activity was highest was the optimal pH for laccase to catalyze the substrate. The results are shown in Table 2.

[0057] Table 2. Optimal temperature and pH of laccase for different substrates

[0058] Substrate Optimal temperature (°C) Optimal pH ABTS 55 3 Guaiacol 55 5 o-Toluidine 55 5 DMP 55 4 Syringaldehyde azo 55 6 catechol 55 5 p-phenylenediamine 55 4

[0059] Example 3. Amino acid sequence of laccase

[0060] The amino acid sequence of the laccase prepared in Example 1 of this invention is shown in SEQ ID NO:1 (obtained by sequencing commissioned to Suzhou Genewiz Biotechnology Co., Ltd.). This laccase sequence shows 97% homology with related sequences in the NCBI database.

[0061] Example 4. Preparation of copolymer membranes (gas-liquid interface membranes) catalyzed by laccase

[0062] (1) Preparation of ferulic acid-p-phenylenediamine copolymer membrane by laccase catalysis:

[0063] 0.25 g ferulic acid and 0.25 g p-phenylenediamine were dissolved in 125 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 3.0), and 7.5 U of the laccase prepared in this invention were added. The mixture was shaken and mixed, and then placed in a 50 °C oven (Zhicheng, ZFD-5090, China) for 13 h. A "ferulic acid-p-phenylenediamine copolymer film" was formed on the upper surface of the solution (the interface between air and solution).

[0064] Infrared spectroscopy analysis: The film-forming monomers ferulic acid and p-phenylenediamine were analyzed using the KBr pellet method (approximately 2 mg of sample mixed with 300 mg of KBr) on an infrared spectrometer (Bio-Rad, FTS-6000, USA). The cleaned gas-liquid interface film was placed on a KBr wafer, air-dried at room temperature, and then analyzed on an infrared spectrometer (Bio-Rad, FTS-6000, USA).

[0065] Infrared spectra of the film-forming monomers ferulic acid, p-phenylenediamine, and the gas-liquid interface film (ferulic acid-p-phenylenediamine copolymer film) prepared in this example are shown in [reference needed]. Figure 1 .

[0066] exist Figure 1 In the middle, the stretching vibration peaks of -OH and -NH are in the range of 3200–3500 cm⁻¹. -1 Between the spectra of the copolymer film and the spectra of the monomers p-phenylenediamine and ferulic acid, the broadening at this point indicates a change in the hydroxyl groups in the ferulic acid structure and a partial transformation of the primary amine to secondary amine in the p-phenylenediamine structure, suggesting the presence of the Michael addition reaction and the formation of amide bonds. At 1653 cm⁻¹ -1 The presence of a peak representing the C=N stretching vibration indicates the formation of a Schiff base bond between the phenolic hydroxyl group of ferulic acid and the amino group of p-phenylenediamine. Furthermore, in the spectrum of monomeric ferulic acid, a peak at 1687 cm⁻¹... -1 A characteristic absorption peak of the carboxyl group appeared at [location missing], but this peak was significantly weakened in the spectrum of the gas-liquid interface film, while it was [value missing] at 1586 cm⁻¹. -1The presence of a characteristic absorption band for amides indicates that the carboxyl and amino groups react to form amide bonds. These changes in absorption peaks in the spectrum suggest that, under the catalytic oxidation of laccase, ferulic acid and p-phenylenediamine form Schiff base bonds and amide bonds, and then undergo covalent cross-linking via Michael addition to form a copolymer film.

[0067] (2) Preparation of Coine-arginine copolymer membranes by laccase catalysis:

[0068] 0.050 g of coine, 0.050 g of arginine, and 5.0 mg of vanillin were dissolved in a mixture of 35 mL of 0.05 mol / L sodium succinate-succinate buffer solution (pH 2.0) and 15 mL of acetone. 7.5 U of the laccase prepared in this invention was added, and the mixture was thoroughly mixed. The mixture was then incubated at 30 °C (Zhicheng, ZSD-1090, China) for 3 h. A "coine-arginine copolymer film" was formed on the upper surface of the solution.

[0069] The process of transferring the membrane onto the coverslip is as follows: use a syringe to draw out the solution under the membrane, re-inject distilled water, and repeat the liquid aspiration-injection-liquid aspiration operation several times; insert the coverslip into the liquid phase under the membrane, carefully move it to the bottom of the membrane, gently lift the membrane, and lay it flat on the coverslip to air dry at room temperature.

[0070] (3) Preparation of iris flavin-PEI copolymer membrane by laccase catalysis:

[0071] 0.250 g of iris flavin, 0.250 g of PEI, and 12.5 mg of ABTS were dissolved in a mixed solvent of 175 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) and 75 mL of ethanol. 50 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in a 60 °C oven (Zhicheng, ZFD-5090, China) for 8 h. An "iris flavin-PEI copolymer film" was formed on the upper surface of the solution.

[0072] Transfer method of "iris flavin-PEI copolymer film": Slowly drain the liquid at the bottom so that the film falls onto the substrate surface located at the bottom of the container.

[0073] (4) Preparation of rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer membrane by laccase catalysis:

[0074] 0.125 g of rhein, 0.125 g of 2,4-diamino-6-methyl-1,3,5-triazine, and 20 mg of syringaldehyde were dissolved in a mixed solvent of 175 mL of 0.05 mol / L sodium acetate-acetic acid buffer solution (pH 4.0) and 75 mL of methanol. 5 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in an oven at 50 °C (Zhicheng, ZFD-5090, China) for 10 h. A "rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer film" was formed on the upper surface of the solution.

[0075] (5) Preparation of p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film by laccase catalysis:

[0076] 0.125 g of p-hydroxybenzoic acid and 0.250 g of 2-fluoro-p-phenylenediamine were dissolved in 125 mL of distilled water, the pH was adjusted to 6.0 with phosphoric acid, and 0.125 U of the laccase prepared in this invention was added. The mixture was stirred and allowed to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 48 h. A "p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film" was formed on the upper surface of the solution.

[0077] (6) Preparation of 1,6-dihydroxynaphthalene-diethylenetriamine copolymer membrane by laccase catalysis:

[0078] 0.125 g of 1,6-dihydroxynaphthalene, 0.250 g of diethylenetriamine, and 25 mg of eugenol were dissolved in a mixed solvent of 200 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 50 mL of acetone. 10 U of the laccase prepared in this invention was added, mixed well, and incubated at 37 °C in an incubator (Zhicheng, ZSD-1090, China) for 10 h. A "1,6-dihydroxynaphthalene-diethylenetriamine copolymer film" was formed on the upper surface of the solution.

[0079] (7) Preparation of shikonin-lysine copolymer membrane by laccase catalysis:

[0080] 0.125g of shikonin, 0.125g of lysine, and 12.5mg of ABTS were dissolved in a mixed solvent of 175mL of 0.05mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 3.0) and 75mL of ethanol. 15U of the laccase prepared in this invention was added, mixed well, and allowed to stand in an oven at 45℃ (Zhicheng, ZFD-5090, China) for 5h. A "shikonin-lysine copolymer film" was formed on the upper surface of the solution.

[0081] (8) Preparation of gallic blue-2,4,6-triaminopyrimidine copolymer membrane by laccase catalysis:

[0082] 0.250 g of gallic blue and 0.250 g of 2,4,6-triaminopyrimidine were dissolved in 250 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0), and 15 U of the laccase prepared in this invention was added. The mixture was stirred and allowed to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 10 h. A "gallic blue-2,4,6-triaminopyrimidine copolymer film" was formed on the upper surface of the solution.

[0083] (9) Preparation of 4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer membrane by laccase catalysis:

[0084] 0.125 g of 4,4'-dihydroxybiphenyl, 0.125 g of 2,3-diaminonaphthalene, and 15 mg of ABTS were dissolved in a mixed solvent of 215 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) and 35 mL of acetone. 5 U of the laccase prepared in this invention was added, and the mixture was left to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 24 h. A "4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer film" was formed on the upper surface of the solution.

[0085] (10) Preparation of bisphenol A-proflavin copolymer membrane by laccase catalysis:

[0086] 0.250 g of bisphenol A, 0.250 g of proflavin, and 15 mg of ABTS were dissolved in a mixed solvent of 175 mL of 0.1 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 75 mL of ethanol. 3 U of the laccase prepared in this invention was added, mixed well, and incubated at 45 °C in an incubator (Zhicheng, ZSD-1090, China) for 10 h. A "bisphenol A-proflavin copolymer film" was formed on the upper surface of the solution.

[0087] (11) Preparation of 1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer membrane by laccase catalysis:

[0088] 0.040 g of 1,8,9-trihydroxyanthracene, 0.060 g of 4,4'-diaminodiphenyl sulfone, and 20 mg of ABTS were dissolved in a mixed solvent of 140 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) and 60 mL of ethanol. 10 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in a 45 °C oven (Zhicheng, ZFD-5090, China) for 10 h. A "1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer film" was formed on the upper surface of the solution.

[0089] (12) Preparation of geraniol-1,6-hexanediamine copolymer membrane by laccase catalysis:

[0090] 0.025 g of geraniol and 0.050 g of 1,6-hexanediamine were dissolved in a mixed solvent of 225 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 4.0) and 25 mL of dimethyl sulfoxide. 15 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in a 30 °C incubator (Zhicheng, ZSD-1090, China) for 5 h. A "geraniol-1,6-hexanediamine copolymer film" was formed on the upper surface of the solution.

[0091] (13) Preparation of Pine flavonoid-spermine copolymer membrane by laccase catalysis:

[0092] 0.050g of Jinsong Biflavonoids and 0.100g of spermine were dissolved in a mixed solvent of 70mL of 0.05mol / L trisodium phosphate-phosphate buffer solution (pH 3.0) and 30mL of ethanol. 2U of the laccase prepared in this invention was added, mixed well, and allowed to stand in an oven at 40℃ (Zhicheng, ZFD-5090, China) for 6h. A "Jinsong Biflavonoid-Spermine copolymer film" was formed on the upper surface of the solution.

[0093] (14) Preparation of narcissin-2,4-diaminoanisole copolymer membrane by laccase catalysis:

[0094] 0.075 g of narcissin and 0.125 g of 2,4-diaminoanisole were dissolved in a mixed solvent of 200 mL of 0.05 mol / L sodium malonate-malonic acid buffer solution (pH 4.0) and 50 mL of ethanol. 5 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in an incubator at 10 °C (Zhicheng, ZSD-1090, China) for 10 h. A "narcissin-2,4-diaminoanisole copolymer film" was formed on the upper surface of the solution.

[0095] (15) Preparation of hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer membrane by laccase catalysis:

[0096] 0.020 g of hesperidin and 0.020 g of 6-hydroxy-2,4,5-triaminopyrimidine were dissolved in a mixed solvent of 140 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 5.0) and 60 mL of ethanol. 10 U of the laccase prepared in this invention was added, mixed well, and allowed to stand in an oven at 55 °C (Zhicheng, ZFD-5090, China) for 7 h. A "hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer film" was formed on the upper surface of the solution.

[0097] Example 5. Preparation of copolymer membranes (liquid-liquid interface membranes) catalyzed by laccase

[0098] (1) Preparation of Coine-arginine copolymer membranes by laccase catalysis:

[0099] 0.050 g of coine, 0.050 g of arginine, and 5.0 mg of vanillin were dissolved in a mixed solvent of 35 mL of 0.05 mol / L sodium succinate-succinate buffer solution (pH 2.0) and 15 mL of acetone. 7.5 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and incubated at 30 °C (Zhicheng, ZSD-1090, China) for 3 h. A "coine-arginine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase. The transfer process of the "Coyin-arginine copolymer membrane" is as follows: Use a syringe to aspirate most of the liquid paraffin on the upper side and the aqueous phase on the lower side of the "Coyin-arginine copolymer membrane", and re-inject distilled water into the lower side of the "Coyin-arginine copolymer membrane". Repeat the liquid aspiration-injection-liquid aspiration operation several times. Insert a coverslip into the aqueous phase under the membrane, carefully move it to the bottom of the "Coyin-arginine copolymer membrane", gently lift it up, use filter paper to absorb the residual liquid paraffin on the membrane surface, and let it air dry at room temperature.

[0100] (2) Preparation of ferulic acid-p-phenylenediamine copolymer membrane by laccase catalysis:

[0101] 0.25 g ferulic acid and 0.25 g p-phenylenediamine were dissolved in 125 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 3.0), and 7.5 U of the laccase prepared in this invention was added. The mixture was thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and allowed to stand in a 50 °C oven (Zhicheng, ZFD-5090, China) for 13 h. A "ferulic acid-p-phenylenediamine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0102] (3) Preparation of iris flavin-PEI copolymer membrane by laccase catalysis:

[0103] 0.250 g of iris flavin, 0.250 g of PEI, and 12.5 mg of ABTS were dissolved in a mixed solvent of 175 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) and 75 mL of ethanol. 50 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was then placed on the aqueous phase and allowed to react for 8 hours in a 60 °C oven (Zhicheng, ZFD-5090, China). An "iris flavin-PEI copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0104] (4) Preparation of rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer membrane by laccase catalysis:

[0105] 0.125 g of rhein, 0.125 g of 2,4-diamino-6-methyl-1,3,5-triazine, and 20 mg of eugenol were dissolved in a mixed solvent of 175 mL of 0.05 mol / L sodium acetate-acetic acid buffer solution (pH 4.0) and 75 mL of methanol. 5 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was allowed to stand in a 50 °C oven (Zhicheng, ZFD-5090, China) for 10 h. A "rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0106] (5) Preparation of p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film by laccase catalysis:

[0107] 0.125 g of p-hydroxybenzoic acid and 0.250 g of 2-fluoro-p-phenylenediamine were dissolved in 125 mL of distilled water, and the pH was adjusted to 6.0 with phosphoric acid. 0.125 U of the laccase prepared in this invention was added and mixed thoroughly to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was allowed to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 48 h. A "p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0108] (6) Preparation of 1,6-dihydroxynaphthalene-diethylenetriamine copolymer membrane by laccase catalysis:

[0109] 0.125 g of 1,6-dihydroxynaphthalene, 0.250 g of diethylenetriamine, and 25 mg of eugenol were dissolved in a mixed solvent of 200 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 50 mL of acetone. 10 U of the laccase prepared in this invention was added and thoroughly mixed to obtain the laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and incubated at 37 °C (Zhicheng, ZSD-1090, China) for 10 h. A "1,6-dihydroxynaphthalene-diethylenetriamine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0110] (7) Preparation of shikonin-lysine copolymer membrane by laccase catalysis:

[0111] 0.125 g of shikonin, 0.125 g of lysine, and 12.5 mg of ABTS were dissolved in a mixed solvent of 175 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 3.0) and 75 mL of ethanol. 15 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was then placed on the aqueous phase and allowed to react for 5 h in a 45 °C oven (Zhicheng, ZFD-5090, China). A "shikonin-lysine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0112] (8) Preparation of gallic blue-2,4,6-triaminopyrimidine copolymer membrane by laccase catalysis:

[0113] 0.250 g of gallic blue and 0.250 g of 2,4,6-triaminopyrimidine were dissolved in 250 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0), and 15 U of the laccase prepared in this invention was added. The mixture was thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was allowed to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 10 h. A "gallic blue-2,4,6-triaminopyrimidine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0114] (9) Preparation of 4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer membrane by laccase catalysis:

[0115] 0.125 g of 4,4'-dihydroxybiphenyl, 0.125 g of 2,3-diaminonaphthalene, and 15 mg of ABTS were dissolved in a mixed solvent of 215 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) and 35 mL of acetone. 5 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and allowed to stand for 24 h in a 40 °C oven (Zhicheng, ZFD-5090, China). A "4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0116] (10) Preparation of bisphenol A-proflavin copolymer membrane by laccase catalysis:

[0117] 0.250 g of bisphenol A, 0.250 g of proflavin, and 15 mg of ABTS were dissolved in a mixed solvent of 175 mL of 0.1 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 75 mL of ethanol. 3 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and incubated at 45 °C (Zhicheng, ZSD-1090, China) for 10 h. A "bisphenol A-proflavin copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0118] (11) Preparation of 1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer membrane by laccase catalysis:

[0119] 0.040 g of 1,8,9-trihydroxyanthracene, 0.060 g of 4,4'-diaminodiphenyl sulfone, and 20 mg of ABTS were dissolved in a mixed solvent of 140 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) and 60 mL of ethanol. 10 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and allowed to stand for 10 h in a 45 °C oven (Zhicheng, ZFD-5090, China). A "1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer membrane" was formed at the interface between the liquid paraffin and the aqueous phase.

[0120] (12) Preparation of geraniol-1,6-hexanediamine copolymer membrane by laccase catalysis:

[0121] 0.025 g of geraniol and 0.050 g of 1,6-hexanediamine were dissolved in a mixed solvent of 225 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 4.0) and 25 mL of dimethyl sulfoxide. 15 U of the laccase prepared in this invention was added and thoroughly mixed to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was incubated at 30 °C (Zhicheng, ZSD-1090, China) for 5 h. A "geraniol-1,6-hexanediamine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0122] (13) Preparation of Pine flavonoid-spermine copolymer membrane by laccase catalysis:

[0123] 0.050 g of Jinsong Biflavonoids and 0.100 g of spermine were dissolved in a mixed solvent of 70 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 3.0) and 30 mL of ethanol. 2 U of the laccase prepared in this invention was added and mixed thoroughly to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase and the mixture was allowed to stand in a 40 °C oven (Zhicheng, ZFD-5090, China) for 6 h. A "Jinsong Biflavonoid-Spermine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0124] (14) Preparation of narcissin-2,4-diaminoanisole copolymer membrane by laccase catalysis:

[0125] 0.075 g of narcissin and 0.125 g of 2,4-diaminoanisole were dissolved in a mixed solvent of 200 mL of 0.05 mol / L sodium malonate-malonic acid buffer solution (pH 4.0) and 50 mL of ethanol. 5 U of the laccase prepared in this invention was added and mixed thoroughly to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was incubated at 10 °C (Zhicheng, ZSD-1090, China) for 10 h. A "narcissin-2,4-diaminoanisole copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0126] (15) Preparation of hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer membrane by laccase catalysis:

[0127] 0.020 g of hesperidin and 0.020 g of 6-hydroxy-2,4,5-triaminopyrimidine were dissolved in a mixed solvent of 140 mL of 0.05 mol / L trisodium phosphate-phosphate buffer (pH 5.0) and 60 mL of ethanol. 10 U of the laccase prepared in this invention was added and mixed thoroughly to obtain a laccase catalytic system (aqueous phase). A 3 mm thick layer of liquid paraffin was placed on the aqueous phase, and the mixture was allowed to stand for 7 h in a 55 °C oven (Zhicheng, ZFD-5090, China). A "hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer film" was formed at the interface between the liquid paraffin and the aqueous phase.

[0128] Example 6. Preparation of copolymer membranes (liquid-solid interface membranes) catalyzed by laccase

[0129] (1) Preparation of ferulic acid-p-phenylenediamine copolymer membrane by laccase catalysis

[0130] 0.25 g ferulic acid and 0.25 g p-phenylenediamine were placed in a 250 mL Erlenmeyer flask, and 125 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 3.0) was added. A polyethylene (PE) plate, after removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 7.5 U of the laccase prepared in this invention was added, and the mixture was stirred at 100 rpm for 13 h at 50 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a ferulic acid-p-phenylenediamine copolymer film was formed on the surface of the PE plate. The film was washed three times with ethanol and distilled water, and then air-dried at room temperature.

[0131] Adhesion test of the film to the substrate: The critical adhesion of the ferulic acid-p-phenylenediamine copolymer film to the surface of a PE board was determined using a nanoindenter (Bruker, Hysitron TI 980, USA). The horizontal movement speed of the sample stage was 5 μm / s. The scratching process consisted of two steps: First, tilt correction, pre-scanning with a constant small force from a tangential displacement of 0 to 150 μm. Second, scratching, with a linearly increasing load of up to 10 mN, scratching back from a tangential displacement of 150 μm to 0 μm. The critical normal force and critical tangential force of the ferulic acid-p-phenylenediamine copolymer film on the PE board surface obtained by the above determination were 2054 μN and 1264 μN, respectively. The ferulic acid-p-phenylenediamine copolymer film prepared by laccase catalysis produced by Polyporus brumalis (CCTCC NO: M 2020809) of this invention has a very strong adhesion to the substrate.

[0132] (2) Preparation of iris flavin-PEI copolymer membrane by laccase catalysis

[0133] 0.100g iris flavonoids, 0.100g PEI, and 5mg ABTS were placed in a 250mL Erlenmeyer flask. A mixed solvent of 70mL 0.05mol / L sodium succinate-succinate buffer solution (pH 2.0) and 30mL ethanol was added. A PE plate (pre-treated to remove dust, oil, sweat, and molding lubricant) was placed in the solution in the Erlenmeyer flask. 40U of laccase prepared according to this invention was added and mixed well. The mixture was shaken at 100rpm for 3 hours at 60℃ in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). Iris flavonoids-PEI copolymer films were formed on the inner wall surface of the Erlenmeyer flask and the surface of the PE plate, respectively. The films were washed three times with ethanol and distilled water, respectively, and then air-dried at room temperature.

[0134] Stability tests of the membrane against acids, alkalis, and organic solvents: Immersing the iris-PEI copolymer membrane in Erlenmeyer flasks coated with the membrane were performed using the following solutions: 0.02% NaOH solution, 0.04% NaOH solution, 30% H₂SO₄ solution, 40% H₂SO₄ solution, ethanol, 50% ethanol aqueous solution, methanol, 50% methanol aqueous solution, acetonitrile, 50% acetonitrile aqueous solution, dimethyl sulfoxide (DMSO), and 90% dimethyl sulfoxide aqueous solution. The membranes were placed at 25°C for 24 hours, manually shaken 10 times during this period, and the membrane detachment was observed. The mass loss rate of the membrane was calculated by weighing. The mass loss rate of the membrane was calculated using the following formula:

[0135]

[0136] In the formula: w0 is the mass (g) of the empty Erlenmeyer flask before the experiment after being kept at constant weight at 105℃; w1 is the mass (g) of the Erlenmeyer flask with inner wall coating after being kept at constant weight at 105℃; w2 is the mass of the Erlenmeyer flask with inner wall coating after being soaked in different solvents, then washed with distilled water, dried and kept at constant weight at 105℃.

[0137] The experimental results are shown in Table 3. The membrane showed no significant changes after standing at 25°C for 24 hours in 0.02% NaOH solution, 30% H2SO4 solution, 100% ethanol, 100% methanol, 100% acetonitrile and 90% dimethyl sulfoxide solution, and exhibited strong resistance to acids, alkalis and organic solvents.

[0138] Table 3. Mass loss rate of iris flavin-PEI copolymer film after immersion in acid, alkali and organic solvents.

[0139]

[0140] The copolymer film incorporates sorbitol, a molecule containing hydroxyl groups in its structure: The PE plate coated with the iris flavin-PEI copolymer film prepared in Example 6(2) was placed in 250 mL of trisodium phosphate-phosphate buffer solution (0.05 mol / L, pH 5.0) containing 2 g / L sorbitol. Laccase 3 U and tetramethylpiperidine (TEMPO) prepared in this invention were added. The mixture was shaken at 40 °C and 100 rpm for 24 h. The plate was then removed, washed several times with anhydrous ethanol, rinsed with distilled water, and air-dried at room temperature.

[0141] The elemental composition of the iris flavin-PEI copolymer membrane surface, including whether it covalently bonds with sorbitol, was determined using X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Escalab 250xi, USA). After reaction with sorbitol, the N / O ratio on the membrane surface decreased from 0.699 to 0.522. The iris flavin-PEI copolymer membrane surface contains four elements: C, H, N, and O. Since sorbitol does not contain N, the decrease in the N / O ratio on the membrane surface confirms that the membrane binds sorbitol molecules.

[0142] Citric acid, a molecule containing a carboxyl group, was introduced into the copolymer membrane structure: The PE plate coated with the iris flavin-PEI copolymer membrane prepared in Example 6(2) was placed in a trisodium phosphate-phosphate buffer solution (0.05mol / L, pH 5.5) containing 2g / L citric acid. 0.25g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.15g of N-hydroxysuccinimide (NHS) were added to activate the carboxyl group. The reaction was carried out at 25°C and 100rpm for 24h. The plate was then removed, washed several times with anhydrous ethanol, rinsed with distilled water, and air-dried at room temperature.

[0143] The elemental composition of the iris flavin-PEI copolymer membrane surface, including whether it covalently bonds to citric acid, was determined using X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Escalab 250xi, USA). After reaction with citric acid, the N / O ratio on the membrane surface decreased from 0.699 to 0.640, indicating that the iris flavin-PEI copolymer membrane surface contains four elements: C, H, N, and O. Citric acid, on the other hand, contains only three elements: C, H, and O, and no N. The decrease in the N / O ratio on the membrane surface after the reaction demonstrates that the membrane binds to citric acid molecules.

[0144] (3) Preparation of Coine-arginine copolymer membranes by laccase catalysis:

[0145] 0.050 g of coine, 0.050 g of arginine, and 5.0 mg of vanillin were placed in a 250 mL Erlenmeyer flask. A mixed solvent of 35 mL of 0.05 mol / L sodium succinate-succinate buffer solution (pH 2.0) and 15 mL of acetone was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 7.5 U of the laccase prepared in this invention was added, and the mixture was thoroughly mixed. The mixture was then shaken at 100 rpm for 3 h at a constant temperature of 30 °C in a Crystal (IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a coine-arginine copolymer film was formed on the surface of the PE plate.

[0146] (4) Preparation of rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer membrane by laccase catalysis:

[0147] 0.125g of rhein, 0.125g of 2,4-diamino-6-methyl-1,3,5-triazine, and 20mg of eugenol were placed in a 500mL Erlenmeyer flask. A mixed solvent of 175mL of 0.05mol / L sodium acetate-acetic acid buffer solution (pH 4.0) and 75mL of methanol was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 5U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100rpm for 10h at 50℃ in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a rhein-2,4-diamino-6-methyl-1,3,5-triazine copolymer film was formed on the surface of the PE plate.

[0148] (5) Preparation of p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film by laccase catalysis:

[0149] 0.125 g of p-hydroxybenzoic acid and 0.250 g of 2-fluoro-p-phenylenediamine were placed in a 500 mL Erlenmeyer flask. 125 mL of distilled water was added, and the pH was adjusted to 6.0 with phosphoric acid. A PE plate, after being cleaned of surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 0.125 U of the laccase prepared in this invention was added, and the mixture was stirred at 100 rpm for 48 h in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a p-hydroxybenzoic acid-2-fluoro-p-phenylenediamine copolymer film was formed on the surface of the PE plate.

[0150] (6) Preparation of 1,6-dihydroxynaphthalene-diethylenetriamine copolymer membrane by laccase catalysis:

[0151] 0.125 g of 1,6-dihydroxynaphthalene, 0.250 g of diethylenetriamine, and 25 mg of eugenol were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 200 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 50 mL of acetone was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 10 U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100 rpm for 10 h at 37 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a 1,6-dihydroxynaphthalene-diethylenetriamine copolymer film was formed on the surface of the PE plate.

[0152] (7) Preparation of shikonin-lysine copolymer membrane by laccase catalysis:

[0153] 0.125g of shikonin, 0.125g of lysine, and 12.5mg of ABTS were placed in a 500mL Erlenmeyer flask. A mixed solvent of 175mL of 0.05mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 3.0) and 75mL of ethanol was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 15U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100rpm for 5 hours at 45℃ in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a shikonin-lysine copolymer film was formed on the surface of the PE plate.

[0154] (8) Preparation of gallic blue-2,4,6-triaminopyrimidine copolymer membrane by laccase catalysis:

[0155] 0.250 g of gallic blue and 0.250 g of 2,4,6-triaminopyrimidine were placed in a 500 mL Erlenmeyer flask. 250 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) was added. A PE plate, after removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 15 U of the laccase prepared in this invention was added, and the mixture was stirred at 100 rpm for 10 h at a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a gallic blue-2,4,6-triaminopyrimidine copolymer film was formed on the surface of the PE plate.

[0156] (9) Preparation of 4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer membrane by laccase catalysis:

[0157] 0.125 g of 4,4'-dihydroxybiphenyl, 0.125 g of 2,3-diaminonaphthalene, and 15 mg of ABTS were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 215 mL of 0.05 mol / L potassium hydrogen phthalate-sodium hydroxide buffer solution (pH 5.0) and 35 mL of acetone was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 5 U of the laccase prepared in this invention was added. The mixture was shaken at 100 rpm for 24 h at 40 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a 4,4'-dihydroxybiphenyl-2,3-diaminonaphthalene copolymer film was formed on the surface of the PE plate.

[0158] (10) Preparation of bisphenol A-proflavin copolymer membrane by laccase catalysis:

[0159] 0.250 g of bisphenol A, 0.250 g of proflavin, and 15 mg of ABTS were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 175 mL of 0.1 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 4.0) and 75 mL of ethanol was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 3 U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100 rpm for 10 h at 45 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a bisphenol A-proflavin copolymer film was formed on the surface of the PE plate.

[0160] (11) Preparation of 1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer membrane by laccase catalysis:

[0161] 0.040 g of 1,8,9-trihydroxyanthracene, 0.060 g of 4,4'-diaminodiphenyl sulfone, and 20 mg of ABTS were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 140 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) and 60 mL of ethanol was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 10 U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100 rpm for 10 h at 45 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a 1,8,9-trihydroxyanthracene-4,4'-diaminodiphenyl sulfone copolymer film was formed on the surface of the PE plate.

[0162] (12) Preparation of geraniol-1,6-hexanediamine copolymer membrane by laccase catalysis:

[0163] 0.025 g of geraniol and 0.050 g of 1,6-hexanediamine were placed in a 500 mL Erlenmeyer flask. 225 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 4.0) and 25 mL of dimethyl sulfoxide were added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 15 U of the laccase prepared in this invention was added, and the mixture was stirred at 100 rpm for 5 h at a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a geraniol-1,6-hexanediamine copolymer film was formed on the surface of the PE plate.

[0164] (13) Preparation of Pine flavonoid-spermine copolymer membrane by laccase catalysis:

[0165] 0.050g of Jinsong Biflavonoids and 0.100g of spermine were placed in a 500mL Erlenmeyer flask. A mixed solvent of 70mL of 0.05mol / L trisodium phosphate-phosphate buffer solution (pH 3.0) and 30mL of ethanol was added. A PE plate, after removing dust, oil, sweat and molding lubricant from its surface, was placed in the solution in the Erlenmeyer flask. 2U of the laccase prepared in this invention was added and mixed well. The mixture was shaken at 100rpm for 6 hours at 40℃ in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a Jinsong Biflavonoid-Spermine copolymer film was formed on the surface of the PE plate.

[0166] (14) Preparation of narcissin-2,4-diaminoanisole copolymer membrane by laccase catalysis:

[0167] 0.075 g of narcissin and 0.125 g of 2,4-diaminoanisole were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 200 mL of 0.05 mol / L sodium malonate-malonic acid buffer solution (pH 4.0) and 50 mL of ethanol was added. A PE plate, after removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 5 U of the laccase prepared in this invention was added and mixed well. The mixture was shaken at 100 rpm for 10 h at a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a narcissin-2,4-diaminoanisole copolymer film was formed on the surface of the PE plate.

[0168] (15) Preparation of hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer membrane by laccase catalysis:

[0169] 0.020 g of hesperidin and 0.020 g of 6-hydroxy-2,4,5-triaminopyrimidine were placed in a 500 mL Erlenmeyer flask. A mixed solvent of 140 mL of 0.05 mol / L trisodium phosphate-phosphate buffer solution (pH 5.0) and 60 mL of ethanol was added. A PE plate, after pre-removing surface dust, oil, sweat, and molding lubricant, was placed in the solution in the Erlenmeyer flask. 10 U of the laccase prepared in this invention was added, and the mixture was mixed. The mixture was then shaken at 100 rpm for 7 h at 55 °C in a constant temperature shaker (Crystal, IS-RDV3, Crystal Microsystems, Inc., USA). After the reaction was completed, a hesperidin-6-hydroxy-2,4,5-triaminopyrimidine copolymer film was formed on the surface of the PE plate.

[0170] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A laccase derived from winter polypores and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 520 <212> PRT <213> Winter-growing polyporus (Polyporus brumalis) <400> 1 Met Ala Arg Phe Gln Ser Leu Leu Ser Tyr Val Thr Leu Leu Phe Val 1 5 10 15 Ala Ser Ala Tyr Ala Ala Ile Gly Pro Val Thr Asp Leu Thr Val Thr 20 25 30 Asp Ala Asn Ile Ser Pro Asp Gly Phe Glu Arg Ala Gly Ile Val Val 35 40 45 Asn Lys Val Phe Pro Ala Pro Leu Ile Thr Gly Gln Lys Gly Asp Arg 50 55 60 Phe Gln Leu Asn Leu Val Asn Gln Met Thr Asn His Thr Met Leu Lys 65 70 75 80 Thr Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp 85 90 95 Ala Asp Gly Pro Ala Phe Val Asn Gln Cys Pro Ile Ala Ser Gly Asn 100 105 110 Ser Phe Leu Tyr Asp Phe Gln Val Pro Asp Gln Ala Gly Thr Phe Trp 115 120 125 Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro 130 135 140 Phe Val Val Tyr Asp Pro Thr Asp Pro His Leu Ala Leu Tyr Asp Val 145 150 155 160 Asp Asp Asp Ser Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Ala 165 170 175 Ala Arg Gly Pro Arg Phe Pro Leu Gly Ala Asp Ser Thr Leu Ile Asn 180 185 190 Gly Leu Gly Arg Ser Thr Ala Thr Pro Thr Ala Asp Leu Ala Val Ile 195 200 205 Ser Val Thr Lys Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser 210 215 220 Cys Asp Pro Asn His Thr Phe Ser Ile Asp Gly His Lys Leu Thr Val 225 230 235 240 Ile Glu Ala Asp Gly Ile Ser Thr Gln Pro Val Thr Gly Ile Asp Ser 245 250 255 Ile Gln Ile Phe Ala Ala Gln Arg Tyr Ser Phe Val Leu Thr Ala Asp 260 265 270 Gln Asp Val Asp Asn Tyr Trp Val Arg Ala Asn Pro Asn Phe Gly Thr 275 280 285 Thr Gly Phe Ala Gly Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Gly 290 295 300 Ala Pro Ala Val Glu Pro Thr Thr Ser Gln Thr Gly Thr Asn Leu Leu 305 310 315 320 Val Glu Thr Asp Leu His Pro Leu Ser Val Met Pro Val Pro Gly Leu 325 330 335 Pro Thr Gln Gly Gly Ala Asp Phe Asn Leu Asn Leu Ala Phe Asn Phe 340 345 350 Asn Gly Ser Asp Phe Phe Ile Asn Gly Ala Thr Phe Thr Pro Pro Thr 355 360 365 Val Pro Val Leu Leu Gln Ile Ile Ser Gly Ala Asn Ser Ala Gln Asp 370 375 380 Leu Leu Pro Ala Gly Ser Val Tyr Ala Leu Pro Ser Asn Ser Ser Ile 385 390 395 400 Glu Leu Thr Phe Pro Ala Thr Ala Ala Ala Pro Gly Ala Pro His Pro 405 410 415 Phe His Leu His Gly His Ala Phe Ala Val Val Arg Ser Ala Gly Ser 420 425 430 Thr Val Tyr Asn Tyr Val Asp Pro Val Tyr Arg Asp Val Val Ser Thr 435 440 445 Gly Thr Pro Ala Ala Gly Asp Asn Val Thr Ile Arg Phe Gln Thr Asp 450 455 460 Asn Pro Gly Pro Trp Phe Leu His Cys His Ile Asp Phe His Leu Asp 465 470 475 480 Ala Gly Phe Ala Val Val Phe Ala Glu Asp Leu Pro Asp Val Val Ser 485 490 495 Ala Asn Pro Val Pro Gln Ala Trp Ser Asp Leu Cys Pro Ile Tyr Asn 500 505 510 Ala Leu Asp Pro Ser Asp Gln Xaa 515 520

Claims

1. A fungal laccase, characterized in that, The fungal laccase was prepared by liquid fermentation of a winter polypore strain with accession number CCTCC NO: M 2020809, and the amino acid sequence of the fungal laccase is shown in SEQ ID NO:

1.

2. The application of the fungal laccase according to claim 1 in catalyzing the self-assembly of two types of film-forming monomers at different interfaces to generate organic copolymer films, characterized in that, The interface is a gas-liquid interface, a liquid-liquid interface, or a solid-liquid interface; the film-forming monomer... One type is organic compounds containing at least one phenolic hydroxyl group and one carboxyl group; the other type of film-forming monomers are aromatic amine organic compounds containing at least two amino groups, specifically any of the following (1)-(15) as film-forming monomers: (1) Ferulic acid and p-phenylenediamine; (2) Coenzyme and arginine; (3) Irisflavin and PEI; (4) Rhein and 2,4-diamino-6-methyl-1,3,5-triazine copolymer film; (5) Hydroxybenzoic acid and 2-fluoro-p-phenylenediamine; (6) 1,6-Dihydroxynaphthalene and diethylenetriamine; (7) Shikonin and lysine; (8) Gallic blue and 2,4,6-triaminopyrimidine; (9) 4,4'-dihydroxybiphenyl and 2,3-diaminonaphthalene; (10) Bisphenol A and proflavin; (11) 1,8,9-trihydroxyanthracene and 4,4'-diaminodiphenyl sulfone; (12) Geraniol and 1,6-hexanediamine; (13) Jinsong double flavonoids and spermine; (14) Narcissin and 2,4-diaminoanisole; (15) Hesperidin and 6-hydroxy-2,4,5-triaminopyrimidine.

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