A laccase-producing polyporus piceinus and a method for producing laccase

Abstract

The application discloses a laccase-producing polyporus brumalis and a method for preparing laccase. The application provides polyporus brumalis CCTCC NO: M 2020809 for fermenting and preparing laccase, and the amino acid sequence of the laccase is shown as SEQ ID NO: 2. The fermentation level of the laccase is high, the fermentation level in a flask reaches 980 U / mL, and the fermentation level in a 70L fermenter reaches 880 U / mL. In order to ensure the food safety of the laccase, the strain and the preferred medium components of the laccase are edible. In addition, the laccase obtained by the application can catalyze the polymerization of film-forming monomers to generate a copolymer film, and is a method for synthesizing a copolymer film by using a biological enzyme catalyst.

Description

Technical Field

[0001] This invention relates to the field of microbial enzymology, and in particular to a winter polypore strain that produces laccase and a method for preparing laccase. Background Technology

[0002] Laccase (phenylene glycol:oxygen oxidoreductase, EC 1.10.3.2) catalyzes compounds such as monophenols, polyphenols, aniline, and polycyclic aromatic hydrocarbons, thus demonstrating significant application value in food, papermaking, textiles, bio-batteries, formaldehyde-free plywood bonding, and organic pollutant remediation. Laccase is one of the most promising oxidoreductases, therefore, the demand for high-yielding laccase strains and low-cost laccase preparation technologies is increasingly urgent. However, due to the low redox potential of bacterial laccase and the low heterologous expression level of fungal laccase, research on laccase molecular modification and large-scale efficient preparation is still in the research and development stage both domestically and internationally, and has not yet achieved satisfactory results.

[0003] *Polyporus brumalis*, also known as hairy fairy microporous fungus or winter pseudopolyporus, belongs to the phylum Basidiomycota, subphylum Agaricomycotina, class Agaricomycetes, order Polyporales, family Polyporaceae, and genus *Polyporus*. It is a woody fungus. The young fruiting bodies of *Polyporus brumalis* are edible [Li Ruguang. *Jilin Province Fungi* (Volume 1, Basidiomycota) [M]. Changchun: Northeast Normal University Press, 1991: 202.]. To date, there have been no reports of winter polyporous fungi that produce high levels of laccase. Summary of the Invention

[0004] The purpose of this invention is to provide a laccase-producing winter polypore fungus and a method for preparing laccase using this strain. The method for preparing laccase provided by this invention yields high-quality laccase, and the winter polypore laccase provided by this invention catalyzes the self-assembly of film-forming monomers to generate organic copolymer membranes.

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

[0006] In a first aspect, the winter polyporus (Polyporus brumalis) TIB.BPE.11072 provided by the present invention was deposited at the China Center for Type Culture Collection on November 30, 2020, with the biological accession number CCTCC NO: M 2020809.

[0007] Secondly, the present invention provides the application of the winter-growing polyporus (Polyporus brumalis) TIB.BPE.11072 in the preparation of laccase, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0008] Thirdly, the present invention provides a method for preparing laccase from the winter-growing polyporus (Polyporus brumalis) TIB.BPE.11072, wherein the amino acid sequence of the laccase is shown in SEQ ID NO: 2, and the method comprises the following steps:

[0009] (1) Liquid seed culture: Take about 1-3 cm of slant mycelium of Polyporus brumalis strain CCTCC NO: M2020809. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 100-250mL of liquid seed culture medium, and incubate at 25-35℃ on a shaker at 150-200rpm for 3-5 days to prepare seed culture;

[0010] (2) Shake flask fermentation culture: The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% to 14% by volume. The fermentation medium is filled into a 500mL Erlenmeyer flask with a liquid volume of 100 to 250mL. The culture is shaken at 25 to 35℃. The shaking speed is 100 to 150rpm on the first to third day of fermentation, and 160 to 250rpm after 3 days of fermentation to obtain the fermentation broth.

[0011] Laccase accumulates in the extracellular fermentation broth, and can be collected, isolated, or purified from the fermentation broth without disrupting the cells.

[0012] (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.

[0013] The liquid seed culture medium consisted of: corn flour 10–50 g / L, soybean meal 5–35 g / L, α-amylase 20–80 U / L, NaH2PO4 1–5 g / L, KCl 0.5–2.5 g / L, MgSO4·7H2O 0.2–5 g / L, with the remainder being water. The pH was 4.0–6.5, and the medium was sterilized at 0.1 MPa for 25 min.

[0014] The fermentation medium contains a carbon source, a nitrogen source, inorganic components, and a laccase fermentation promoter.

[0015] 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.

[0016] Preferably, the carbon source of the fermentation medium includes, but is not limited to, one or more of the following: corn flour, fructose, lactose, galactose, xylose, maltose, glucose, and maltodextrin. Fructose is preferred as the carbon source.

[0017] More preferably, the carbon source of the fermentation medium is selected as a composite carbon source of fructose and corn flour, and the mass ratio of fructose to corn flour is (10-5):(0-5).

[0018] Preferably, the organic nitrogen source of the fermentation medium includes, but is not limited to, casein peptone or soybean peptone; soybean peptone is preferred.

[0019] The "casein peptone" described in this invention is a substance obtained by enzymatic hydrolysis of casein with food-grade bromelain, papain, trypsin, pepsin, or a combination thereof.

[0020] The "soy peptone" described in this invention is a substance obtained by enzymatic hydrolysis of soybean protein with food-grade bromelain, papain, trypsin, pepsin, or a combination thereof.

[0021] Preferably, the inorganic nitrogen source of the fermentation medium includes, but is not limited to, NH4H2PO4, (NH4)2SO4 or NH4NO3; preferably (NH4)2SO4.

[0022] More preferably, the nitrogen source of the fermentation medium is a composite nitrogen source of soybean peptone and (NH4)2SO4, and the mass ratio of soybean peptone to (NH4)2SO4 is (10-5):(0-5).

[0023] Preferably, the inorganic components of the fermentation medium include, but are not limited to, copper ions, and the concentration of added copper ions is 0.5–2.5 mmol / L.

[0024] Preferably, the fermentation medium further comprises a laccase fermentation promoter, which includes, but is not limited to, one or more of the following: vitamin B1, Tween-80, Triton X-100, benzoic acid, coumaric acid, vanillin, gallic acid, and ethanol.

[0025] More preferably, the laccase fermentation promoter is one or more of vitamin B1, Tween-80, or vanillin.

[0026] The "laccase fermentation promoter" described in this invention refers to a substance that can promote the production of laccase by microorganisms during the laccase fermentation process, thereby improving the laccase fermentation level.

[0027] Preferably, the fermentation medium is specifically composed of the following substances: fructose 30-60 g / L, corn flour 0-30 g / L, soybean peptone 4.5-18 g / L, (NH4)2SO4 0-5 g / L, KCl 0.5-2 g / L, NaH2PO4 1-2.5 g / L, MgSO4·7H2O 0.25-2 g / L, CuSO4·5H2O 0.5-2.5 mmol / L, VB1 0.02-0.04 g / L, Tween-80 0.01-0.7 g / L, vanillin 0.1-1.3 mmol / L, and the remainder is water, pH 4.0-6.5, sterilized at 0.1 MPa for 25 min.

[0028] 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.

[0029] The preferred step (1) above is to inoculate approximately 3 cm of the slant culture of *Polyporus brumalis* strain CCTCC NO: M2020809. 2 Inoculate into liquid seed culture medium and culture in a shaker at 25–35°C and 150–200 rpm for 3–4.5 days to prepare seed solution.

[0030] The preferred step (2) above is as follows: the seed liquid is inoculated into the fermentation medium at an inoculation rate of 6% to 12% by volume, the fermentation medium is filled in 125 to 175 mL in a 500 mL Erlenmeyer flask, and the mixture 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 mixture is cultured for 11 to 14 days to obtain the fermentation broth.

[0031] The beneficial effects of this invention are:

[0032] This invention provides a non-genetically modified winter polypore strain with accession number CCTCC NO:M 2020809, and a method for preparing laccase using this strain. To ensure the food safety of the prepared laccase, the strain and preferred culture medium components are both edible. The fermentation level is high, reaching 980 U / mL in shake flask fermentation and 880 U / mL in a 70L fermenter (ABTS method, where one enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute).

[0033] Furthermore, the laccase obtained in this invention (the amino acid sequence of laccase is shown in SEQ ID NO: 2) can catalyze the polymerization and self-assembly of film-forming monomers, such as ferulic acid and p-phenylenediamine, coine and arginine, on the surface of solid materials to generate organic copolymer films with extremely strong adhesion. It can also catalyze the polymerization and self-assembly of film-forming monomers at the interface between gas and reaction solution and at the interface between reaction solution and immiscible liquid to generate organic copolymer films. This belongs to a new type of functional polymer material and is a method of synthesizing copolymers using biological enzyme catalysis.

[0034] Biological Preservation Instructions

[0035] A winter-growing polyporus strain: Classification and nomenclature: Polyporus brumalis TIB.BPE.11072, deposited on November 30, 2020 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number CCTCC NO:M 2020809. Attached Figure Description

[0036] Figure 1 A phylogenetic tree constructed based on the sequence analysis of the internal transcriptional spacer region;

[0037] Figure 2 Infrared spectra of film-forming monomers and copolymer films. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Methods for determining laccase activity

[0042] The temperature of the spectrophotometer sample cell 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 1 cm path length. 0.1 mL of the enzyme solution diluted to an appropriate concentration 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 in absorbance was calculated. An enzyme solution of the corresponding concentration, heated to boiling for 5 minutes, was used as a control.

[0043] One enzyme activity unit (U) 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.

[0044] The formula for calculating enzyme activity is:

[0045]

[0046] 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; ε 420 =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).

[0047] Example 1. Laccase-producing winter polyporus (Polyporus brumalis) CCTCC NO: M 2020809

[0048] The laccase-producing winter polyporus (Polyporus brumalis) strain CCTCC NO:M 2020809 was deposited on November 30, 2020, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China.

[0049] The strain was identified by gene sequencing. The sequence of the internal transcribed spacers (ITS) located between the 3' end of the 18S rDNA and the 5' end of the 28S rDNA was determined (PCR primers used: ITS4: 5'-TCCTCCGCTTATTGATATGC-3'; ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3'). The region sequence of the internal transcribed spacers is shown in SEQ ID NO:1. This sequence was compared for homology with the GenBank nucleic acid sequence database, and a phylogenetic tree was constructed. Figure 1 It was identified as a winter polyporus (Polyporus brumalis (Pers.) Fr.).

[0050] Example 2. Preparation of Laccase

[0051] (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.

[0052] 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.

[0053] (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.

[0054] 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.

[0055] (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.

[0056] Example 3. Preparation of laccase

[0057] (1) Liquid seed culture medium: 50 g / L corn flour, 35 g / L soybean meal, 80 U / L α-amylase, 5 g / L NaH2PO4, 2.5 g / L KCl, 5 g / L MgSO4·7H2O, the remainder being water, pH 6.5, sterilized at 0.1 MPa for 25 min.

[0058] Fermentation medium: fructose 60 g / L, corn flour 30 g / L, soybean peptone 18 g / L, (NH4)2SO4 5 g / L, KCl 2 g / L, NaH2PO4 2.5 g / L, MgSO4·7H2O 2 g / L, CuSO4·5H2O 2.5 mmol / L, VB1 0.04 g / L, Tween-80 0.7 g / L, vanillin 1.3 mmol / L, the remainder being water, pH 5.7, sterilized at 0.1 MPa for 25 min.

[0059] (2) Liquid seed culture: Take about 1 cm of slant culture of winter polyporus (Polyporus brumalis) CCTCC NO: M 2020809. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 100mL of liquid seed culture medium, and incubate at 25℃ and 150rpm for 3 days to obtain seed culture.

[0060] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 12%, with the fermentation medium filling 125 mL of a 500 mL Erlenmeyer flask. The culture was incubated at 25°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 11 days of fermentation. The laccase content in the fermentation broth was 574 U / mL.

[0061] (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.

[0062] Example 4. Preparation of Laccase

[0063] (1) Liquid seed culture medium: corn flour 10g / L, soybean meal 5g / L, α-amylase 20U / L, NaH2PO4 1g / L, KCl 0.5g / L, MgSO4·7H2O 0.2g / L, the remainder is water, pH 5.7, sterilized at 0.1MPa for 25min.

[0064] Fermentation medium: fructose 30 g / L, soybean peptone 4.5 g / L, KCl 0.5 g / L, NaH2PO4 1 g / L, MgSO4·7H2O 0.25 g / L, CuSO4·5H2O 0.5 mmol / L, VB1 0.02 g / L, Tween-80 0.01 g / L, vanillin 0.1 mmol / L, the remainder being water, pH 6.5, sterilized at 0.1 MPa for 25 min.

[0065] (2) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (CCTCC NO: M 2020809). 2 Inoculate into 500mL Erlenmeyer flasks, each containing 250mL of liquid seed culture medium, and incubate at 35℃ and 200rpm for 5 days to obtain seed culture.

[0066] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 12 days of fermentation. The laccase content in the fermentation broth was 482 U / mL.

[0067] (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.

[0068] Example 5. Preparation of laccase

[0069] (1) Liquid seed culture medium: corn flour 30g / L, soybean meal 15g / L, α-amylase 40U / L, NaH2PO4 2g / L, KCl 1.5g / L, MgSO4·7H2O 1g / L, the remainder is water, pH 5.3, sterilized at 0.1MPa for 25min.

[0070] Fermentation medium: fructose 30 g / L, corn flour 30 g / L, soybean peptone 4.5 g / L, (NH4)2SO4 4.5 g / L, KCl 0.8 g / L, NaH2PO4 1.5 g / L, MgSO4·7H2O 1 g / L, CuSO4·5H2O 2.5 mmol / L, Triton X-100 0.5 g / L, benzoic acid 1 mmol / L, the remainder being water, pH 5.3, sterilized at 0.1 MPa for 25 min.

[0071] (2) Liquid seed culture: Take about 1 cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (CCTCC NO: M 2020809). 2Inoculate into 500mL Erlenmeyer flasks, each containing 250mL of liquid seed culture medium, and incubate at 25℃ and 200rpm for 3 days on a shaker to obtain seed culture.

[0072] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 12 days of fermentation. The laccase content in the fermentation broth was 528 U / mL.

[0073] (3) Preparation of crude enzyme solution: The fermentation broth was centrifuged at 10000×g at 6℃ for 10min, and the supernatant was the crude enzyme solution of laccase.

[0074] Example 6. Preparation of Laccase

[0075] (1) Liquid seed culture medium: corn flour 10g / L, soybean meal 5g / L, α-amylase 20U / L, NaH2PO4 1g / L, KCl 0.5g / L, MgSO4·7H2O 0.2g / L, the remainder is water, pH 6.0, sterilized at 0.1MPa for 25min.

[0076] Fermentation medium: lactose 30 g / L, soybean peptone 5 g / L, (NH4)2SO4 5 g / L, KCl 1.3 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 1 g / L, CuSO4·5H2O 0.5 mmol / L, Triton X-100 0.5 g / L, benzoic acid 0.1 mmol / L, the remainder being water, pH 6.2, sterilized at 0.1 MPa for 25 min.

[0077] (2) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (CCTCC NO: M 2020809). 2 Inoculate into 500mL Erlenmeyer flasks, each containing 100mL of liquid seed culture medium, and incubate at 35℃ and 150rpm for 5 days to obtain seed culture.

[0078] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 11 days of fermentation. The laccase content in the fermentation broth was 478 U / mL.

[0079] (3) Preparation of crude enzyme solution: The fermentation broth was centrifuged at 8℃ and 10000×g for 10 min, and the supernatant was the crude enzyme solution of laccase.

[0080] Example 7. Preparation of Laccase

[0081] (1) Liquid seed culture medium: corn flour 35g / L, soybean meal 20g / L, α-amylase 65U / L, NaH2PO4 4g / L, KCl 2g / L, MgSO4·7H2O 2g / L, the remainder is water, pH 4.4, sterilized at 0.1MPa for 25min.

[0082] Fermentation medium: galactose 40 g / L, casein peptone 5.4 g / L, KCl 0.8 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 0.25 g / L, CuSO4·5H2O 1.5 mmol / L, VB1 0.02 g / L, coumaric acid 0.1 mmol / L, the remainder being water, pH 4.4, sterilized at 0.1 MPa for 25 min.

[0083] (2) Liquid seed culture: Take about 2cm of slant culture of winter polyporus (Polyporus brumalis) CCTCC NO: M 2020809. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 200mL of liquid seed culture medium, and incubate at 30℃ and 170rpm for 4 days to obtain seed culture.

[0084] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The flask was incubated at 35°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 676 U / mL.

[0085] (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.

[0086] Example 8. Preparation of laccase

[0087] (1) Liquid seed culture medium: corn flour 25g / L, soybean meal 10g / L, α-amylase 30U / L, NaH2PO4 2g / L, KCl 0.8g / L, MgSO4·7H2O 1g / L, the remainder is water, pH 6.5, sterilized at 0.1MPa for 25min.

[0088] Fermentation medium: xylose 50 g / L, NH4H2PO4 10 g / L, KCl 0.8 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 1 g / L, VB1 0.03 g / L, Tween-80 0.7 g / L, gallic acid 0.5 mmol / L, the remainder being water, pH 6.5, sterilized at 0.1 MPa for 25 min.

[0089] (2) Liquid seed culture: Take about 1 cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (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 3 days to obtain seed culture.

[0090] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 12 days of fermentation. The laccase content in the fermentation broth was 619 U / mL.

[0091] (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.

[0092] Example 9. Preparation of laccase

[0093] (1) Liquid seed culture medium: corn flour 30g / L, soybean meal 15g / L, α-amylase 45U / L, NaH2PO4 3g / L, KCl 1.5g / L, MgSO4·7H2O 1.5g / L, the remainder is water, pH 5.7, sterilized at 0.1MPa for 25min.

[0094] Fermentation medium: maltose 45 g / L, (NH4)2SO4 10 g / L, KCl 0.8 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 0.25 g / L, CuSO4·5H2O 1 mmol / L, VB1 0.03 g / L, Tween-80 0.3 g / L, ethanol 5 mmol / L, the remainder being water, pH 5.7, sterilized at 0.1 MPa for 25 min.

[0095] (2) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (CCTCC NO: M 2020809). 2Inoculate into 500mL Erlenmeyer flasks, each containing 250mL of liquid seed culture medium, and incubate at 35℃ and 200rpm for 5 days to obtain seed culture.

[0096] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 639 U / mL.

[0097] (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.

[0098] Example 10. Preparation of Laccase

[0099] (1) Liquid seed culture medium: 25 g / L corn flour, 10 g / L soybean meal, 30 U / L α-amylase, 2 g / L NaH2PO4, 0.8 g / L KCl, 1 g / L MgSO4·7H2O, the remainder being water, pH 5.0, sterilized at 0.1 MPa for 25 min.

[0100] Fermentation medium: glucose 40 g / L, soybean peptone 5.4 g / L, KCl 0.8 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 0.25 g / L, CuSO4·5H2O 0.5 mmol / L, VB1 0.02 g / L, Tween-80 0.3 g / L, vanillin 0.4 mmol / L, the remainder being water, pH 4.5, sterilized at 0.1 MPa for 25 min.

[0101] (2) Liquid seed culture: Take about 2cm of slant culture of winter polyporus (Polyporus brumalis) CCTCC NO: M 2020809. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 250mL of liquid seed culture medium, and incubate at 35℃ and 200rpm for 5 days to obtain seed culture.

[0102] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 728 U / mL.

[0103] (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.

[0104] Example 11. Preparation of Laccase

[0105] (1) Liquid seed culture medium: corn flour 35g / L, soybean meal 20g / L, α-amylase 65U / L, NaH2PO4 4g / L, K2SO4 2.5g / L, MgSO4·7H2O 2g / L, the remainder is water, pH 6.2, sterilized at 0.1MPa for 25min.

[0106] Fermentation medium: maltodextrin 50 g / L, NH4NO3 10 g / L, KCl 0.8 g / L, NaH2PO4 2 g / L, MgSO4·7H2O 1 g / L, CuSO4·5H2O 1.5 mmol / L, VB1 0.03 g / L, Triton X-100 0.7 g / L, coumaric acid 0.1 mmol / L, the remainder being water, pH 6.1, sterilized at 0.1 MPa for 25 min.

[0107] (2) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (CCTCC NO: M 2020809). 2 Inoculate into 500mL Erlenmeyer flasks, each containing 250mL of liquid seed culture medium, and incubate at 35℃ and 200rpm for 5 days to obtain seed culture.

[0108] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 3%, with 200 mL of the fermentation medium in a 500 mL Erlenmeyer flask. The culture was incubated at 35°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 11 days of fermentation. The laccase content in the fermentation broth was 487 U / mL.

[0109] (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.

[0110] Example 12. Preparation of laccase (70L fermenter)

[0111] (1) Liquid seed culture medium: corn flour 30g / L, soybean meal 15g / L, α-amylase 54U / L, NaH2PO4 2.6g / L, KCl 1.5g / L, MgSO4·7H2O 1.5g / L, the remainder is water, pH 6.5, sterilized at 0.1MPa for 25min.

[0112] 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 5.7, sterilized at 0.1 MPa for 25 min.

[0113] (2) Liquid seed culture: Take about 3cm of the mycelial growth of winter polyporus (Polyporus brumalis) slant (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 3 days to obtain seed culture.

[0114] Fermentation in a 70L fermenter: A 70L fermenter (Applikon, pilot system 70L, Netherlands) was filled with 47L of fermentation medium, sterilized, and cooled to 32°C. 3L of seed culture was added, and the fermentation temperature was set to 30°C, the pressure to 0.5 bar, and the stirring speed to 120 rpm. The initial aeration rate was 20L / min, which was increased to 60L / min after three days. Fermentation broth was obtained after 14 days. The laccase content in the fermentation broth was 882 U / mL.

[0115] (3) Preparation of crude enzyme solution: The fermentation broth is filtered to obtain crude enzyme solution of laccase.

[0116] Example 13. Purification of laccase

[0117] 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.

[0118] 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 patent), which was stored at -20°C for later use.

[0119] Example 14. Application of laccase (gas-liquid interface membrane)

[0120] Laccase catalyzes the interfacial polymerization of ferulic acid and p-phenylenediamine between aqueous solution and air to form a ferulic acid-p-phenylenediamine copolymer film.

[0121] (1) Preparation method of gas-liquid interface film

[0122] 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 laccase prepared in this patent was 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).

[0123] (2) Infrared spectral analysis of film-forming monomers and copolymer films

[0124] Infrared spectral analysis of film-forming monomers ferulic acid and p-phenylenediamine: The KBr pellet method (approximately 2 mg of sample mixed with 300 mg of KBr) was used for detection and analysis on an infrared spectrometer (Bio-Rad, FTS-6000, USA).

[0125] Infrared spectral analysis of gas-liquid interface membrane: The cleaned gas-liquid interface membrane was placed on a KBr crystal, dried at room temperature, and then detected and analyzed on an infrared spectrometer (Bio-Rad, FTS-6000, USA).

[0126] 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 2 .

[0127] exist Figure 2In 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. -1 The 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.

[0128] Example 15. Application of laccase (liquid-liquid interface membrane)

[0129] Preparation method of immiscible liquid interface copolymer film: 0.050 g coine, 0.050 g arginine, and 5.0 mg vanillin were dissolved in a mixed solvent of 35 mL 0.05 mol / L sodium succinate-succinate buffer solution (pH 2.0) and 15 mL acetone. 7.5 U of 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 in an incubator (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.

[0130] Example 16. Application of laccase (liquid-solid interface membrane)

[0131] In aqueous solution, laccase catalyzes the polymerization of ferulic acid and p-phenylenediamine on the surface of a solid material (substrate) to form a ferulic acid-p-phenylenediamine copolymer film (liquid-solid interface film), which has strong adhesion to the substrate.

[0132] (1) Preparation method of liquid-solid interface film

[0133] 0.25 g ferulic acid and 0.25 g p-phenylenediamine were placed in a 250 mL Erlenmeyer flask. 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 laccase prepared according to this patent was added and mixed well. The mixture was shaken 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.

[0134] (2) Adhesion test of membrane to substrate material

[0135] The critical adhesion of the ferulic acid-p-phenylenediamine copolymer film to the PE board surface 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, a pre-scan with a constant small force from a tangential displacement of 0–150 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) exhibits strong adhesion to the substrate.

[0136] 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 winter polypore fungus that produces laccase and a method for preparing laccase. <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 622 <212> DNA <213> Winter-growing polyporus (Polyporus brumalis) <400> 1 gagacattat cgagttctga acgggttgta gctggccttc cgaggcatgt gcacgccctg 60 ctcatccact ctacacctgt gcacttactg tgggtttcaa gagcttcgaa gcgggggctt 120 aatcgctctc gccgagttgt tactgggcct acgtttatca caaacacttt aaagtaacag 180 aatgtaatcg cgtctaacgc atctatatac aactttcagc aacggatctc ttggctctcg 240 catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc 300 atcgaatctt tgaacgcacc ttgcgctcct tggtattccg aggagcatgc ctgtttgagt 360 gtcatgaaat tctcaaccta acgggttctt aaccggactt gcttaggctt ggacttggag 420 gcttgtcggc tctagcagtc ggctcctctc aaatgcatta gcttggttcc ttgcggatcg 480 gctcacggtg tgataattgt ctacgccgcg accgttgaag cgttttaatg gccagcttct 540 aatcgtctct tgcgagacag cattcatcga actctgacct caaatcaggt aggactaccc 600 gctgaactta agcatatcat aa 622 <210> 2 <211> 520 <212> PRT <213> Polyporus brumalis <400> 2 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 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tcctccgctt attgatatgc 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ggaagtaaaa gtcgtaacaa gg 22

Claims

1. A winter-growing polypore fungus ( Polyporus brumalis TIB.BPE.11072, characterized in that, The winter polypore has the accession number CCTCC NO: M 2020809 at the China Center for Type Culture Collection.

2. A winter polypore fungus based on claim 1 ( Polyporus brumalis The method for preparing laccase according to TIB.BPE.11072 is characterized by, The amino acid sequence of the laccase is shown in SEQ ID NO: 2, and the steps include the following: (1) Liquid seed culture: Take about 1-3 cm of slant mycelium from the winter polypore strain TIB.BPE.11072. 2 Inoculate into 500mL Erlenmeyer flasks, each containing 100-250mL of liquid seed culture medium, and incubate at 25-35℃ and 150-200rpm for 3-5 days to prepare seed culture; (2) Shake flask fermentation culture: The seed liquid is inoculated into the fermentation medium at a volume ratio of 5% to 14%. The volume of the fermentation medium in the 500 mL Erlenmeyer flask is 100 to 250 mL. 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 250 rpm after 3 days of fermentation to obtain the fermentation broth. (3) Preparation of crude enzyme solution: The fermentation broth was centrifuged at 10000×g for 10 min at 4℃, and the supernatant was the crude enzyme solution of laccase; The liquid seed culture medium consisted of: corn flour 10-50 g / L, soybean meal 5-35 g / L, α-amylase 20-80 U / L, NaH2PO4 1-5 g / L, KCl 0.5-2.5 g / L, MgSO4·7H2O 0.2-5 g / L, with the remainder being water. The pH was 4.0-6.5, and the medium was sterilized at 0.1 MPa for 25 min. The fermentation medium consists of the following components: fructose 30-60 g / L, corn flour 0-30 g / L, soybean peptone 4.5-18 g / L, (NH4)2SO4 0-5 g / L, KCl 0.5-2 g / L, NaH2PO4 1-2.5 g / L, MgSO4·7H2O 0.25-2 g / L, CuSO4·5H2O 0.5-2.5 mmol / L, VB1 0.02-0.04 g / L, Tween-80 0.01-0.7 g / L, vanillin 0.1-1.3 mmol / L, with the remainder being water. The pH is 4.0-6.5, and the medium is sterilized at 0.1 MPa for 25 min.

3. The method according to claim 2, characterized in that, The liquid seed culture medium consists of: 25-35 g / L corn flour, 10-20 g / L soybean meal, 30-65 U / L α-amylase, 2-4 g / L NaH2PO4, 0.8-2 g / L KCl, 1-2 g / L MgSO4·7H2O, with the remainder being water, pH 5.7-6.5, sterilized at 0.1 MPa for 25 min.

4. The method according to claim 2, characterized in that, 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.

5. The method according to any one of claims 2-4, characterized in that, Step (1) involves inoculating the fungus with winter polypores ( Polyporus brumalis The mycelial growth on the slant of strain CCTCC NO: M 2020809 was approximately 3 cm. 2 Inoculate into liquid seed culture medium and culture in a shaker at 25-35℃ and 150-200 rpm for 3-4.5 days to prepare seed solution.

6. The method according to any one of claims 2-4, characterized in that, Step (2) involves inoculating the seed culture into the fermentation medium at a volume ratio of 6% to 12%. The fermentation medium is filled with 125 to 175 mL in a 500 mL Erlenmeyer flask. The culture is carried out at 25 to 35 °C with shaking. The shaking speed is 100 to 150 rpm on the first to third day of fermentation, and 160 to 200 rpm after the third day of fermentation. The culture is carried out for 11 to 14 days to obtain the fermentation broth.

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