Heat-resistant, organic solvent-resistant, highly active laccase and use thereof

Abstract

The invention provides heat-resistant, organic solvent-resistant and high-activity laccase and application thereof. The laccase is any one of the following items: (1) a sequence shown as SEQ ID NO.1; (2) a sequence as shown in SEQ ID NO. 2; (3) a sequence as shown in SEQ ID NO.3; (4) a sequence as shown in SEQ ID NO.4; (5) a sequence shown in SEQ ID NO. 5; and (5) a sequence as shown in SEQ ID NO. 6. The research finds that the laccase has the characteristics of heat resistance and organic solvent resistance, and has the capability of removing environmental pollutants in a mediator-free system, including the capability of decolorizing dye reactive brilliant blue KN-R and the capability of degrading chloramphenicol and aflatoxin B1. Reactive brilliant blue KN-R, chloramphenicol and aflatoxin B1 are widely existing environmental pollutants, have stable properties and threaten ecological safety and human health.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a heat-resistant, organic solvent-resistant, highly active laccase and application thereof. Background Art

[0002] Laccase is a multi-copper oxidase that can oxidize polyphenols, aromatic amines and their derivatives. During the oxidation process, the copper ions of laccase take electrons from the substrate and eventually transfer the electrons to oxygen, reducing the oxygen to water. In the electron transfer process, there is no need to add coenzymes to transfer electrons like other oxidoreductases, and the only byproduct is water. Therefore, laccase has the advantages of being green and environmentally friendly. Laccase has been used in drug synthesis, degradation of harmful pollutants in the environment, and strengthening of flour in the food industry. Laccase is widely present in higher plants, fungi, and bacteria. The laccase currently used in industry is mainly produced by fungi, but the fermentation cycle of fungal laccase is long, it is difficult to express heterologously in Escherichia coli, and the enzymatic properties are difficult to meet the harsh industrial conditions. Therefore, the exploration of prokaryotic sources, stable properties, and high-activity laccases has received widespread attention. Summary of the invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a heat-resistant, organic solvent-resistant, highly active laccase and use thereof.

[0004] To this end, the present invention provides the following technical solutions:

[0005] A laccase, wherein the laccase is any one of the following:

[0006] (1) comprising the sequence shown in SEQ ID NO.1;

[0007] (2) comprising the sequence shown in SEQ ID NO.2;

[0008] (3) comprising the sequence shown in SEQ ID NO.3;

[0009] (4) comprising the sequence shown in SEQ ID NO.4;

[0010] (5) comprising the sequence shown in SEQ ID NO.5;

[0011] (5) comprising the sequence shown in SEQ ID NO.6.

[0012] Optionally, the laccase is any one of the following:

[0013] (1) the sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto;

[0014] (2) the sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto;

[0015] (3) the sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto;

[0016] (4) the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto;

[0017] (5) The sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto.

[0018] (6) The sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto.

[0019] A polynucleotide encodes the nucleotide sequence of the laccase.

[0020] Optionally, it comprises a nucleotide sequence shown in any one of SEQ ID NOs. 7 to 12.

[0021] A vector comprises the polynucleotide.

[0022] A host cell, comprising:

[0023] expressing the laccase;

[0024] and / or, comprising said polynucleotide;

[0025] and / or, comprising the vector.

[0026] A polypeptide molecule contains the laccase.

[0027] The laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule are used in decolorization and bleaching of the dye Reactive Brilliant Blue KN-R (CAS No.: 2580-78-1).

[0028] Use of the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule in the degradation of chloramphenicol (CAS number: 56-75-7).

[0029] The use of the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule in the degradation of aflatoxin B1 (CAS number: 1162-65-8).

[0030] Optionally, the method comprises use in preparing a heat-resistant, organic solvent-resistant, highly active or mediator-free decolorizing agent, bleaching agent, chloramphenicol degrading agent or aflatoxin B1 degrading agent;

[0031] And / or, use in bleaching of the dye Reactive Brilliant Blue KN-R.

[0032] A decolorizing agent, a bleaching agent, a chloramphenicol degrading agent or an aflatoxin B1 degrading agent, comprising the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule, with or without the addition of auxiliary materials permitted by the process.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. A laccase provided by the present invention, wherein the laccase is any one of the following: (1) comprising the sequence shown in SEQ ID NO.1; (2) comprising the sequence shown in SEQ ID NO.2; (3) comprising the sequence shown in SEQ ID NO.3; (4) comprising the sequence shown in SEQ ID NO.4; (5) comprising the sequence shown in SEQ ID NO.5; (6) comprising the sequence shown in SEQ ID NO.6. The present invention has found that the above laccases are derived from Bacillus freudenreichii, Thermosediminibacter oceani, Sulfurimonas sp., Streptomyces clavuligerus, Mesocricetibacter intestinalis, and Bhargavaea cecembensis. These laccases exhibit heat resistance and organic solvent resistance, as well as the ability to degrade reactive brilliant blue KN-R, chloramphenicol, and aflatoxin B1 in a mediator-free system. Reactive brilliant blue KN-R is a dye commonly used in printing and dyeing processes. The dye will produce a large amount of toxic wastewater during production and use, causing great pollution to the environment. Chloramphenicol is a broad spectrum antibiotic widely detected in aquaculture wastewater, which seriously threatens ecological and environmental safety and human health. Aflatoxin B1 is a common highly toxic and carcinogenic fungal toxin in grains. Therefore, the laccase of the present invention shows important application value in the fields of environmental protection and food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 is the plasmid map in Example 1 of the present invention;

[0037] Figure 2 is the temperature tolerance result of laccase in Experimental Example 1 of the present invention;

[0038] Figure 3 is the result of the tolerance of laccase to organic solvents in Experimental Example 2 of the present invention;

[0039] Figure 4 It is the degradation result of reactive brilliant blue KN-R by laccase in Application Example 1 of the present invention;

[0040] Figure 5 This is the degradation result of chloramphenicol by laccase in Application Example 2 of the present invention;

[0041] Figure 6 This is the degradation result of aflatoxin B1 by laccase in Application Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0043] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0044] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences.

[0045] As used herein, "vector" refers to a construct capable of delivering one or more target genes or sequences to a host cell, preferably expressed in a host cell. Vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0046] As used herein, the term "decolorizing agent or bleaching agent without a mediator system" means that it does not require an intermediate mediator, can act directly, and avoids additional consumption or the introduction of new pollutants. Generally, laccase is used for dye decolorization and requires the use of ABTS or some polyphenolic small molecule substrates as intermediate mediators to decolorize the target substrate (such as Reactive Brilliant Blue KN-R). The laccase in the present invention can decolorize the target substrate without an intermediate mediator.

[0047] In a first aspect, the present invention provides a laccase, wherein the laccase is any one of the following:

[0048] (1) comprising the sequence shown in SEQ ID NO.1;

[0049] (2) comprising the sequence shown in SEQ ID NO.2;

[0050] (3) comprising the sequence shown in SEQ ID NO.3;

[0051] (4) comprising the sequence shown in SEQ ID NO.4;

[0052] (5) comprising the sequence shown in SEQ ID NO.5;

[0053] (6) comprising the sequence shown in SEQ ID NO.6.

[0054] In some preferred but non-limiting specific embodiments, the laccase is any one of the following: (1) a sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith; (2) a sequence as shown in SEQ ID NO.2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith; (3) a sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith; (4) a sequence as shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith; (5) a sequence as shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith; (6) a sequence as shown in SEQ ID The sequence shown in NO.6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto.

[0055] The laccase of the present invention can replace, delete and / or add at least one amino acid on the basis of the specific amino acid sequence listed above, and the obtained laccase has the activity of degrading dyes, chloramphenicol or aflatoxin B1. The number of the above-mentioned substituted, deleted or added amino acids can be any value, such as 1, 5, 10, 15 and above, so that the sequence identity of the changed amino acid sequence and its respective corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0056] In a second aspect, the present invention provides a polynucleotide encoding the nucleotide sequence of the laccase.

[0057] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably and include DNA molecules or RNA molecules. DNA molecules can be single-stranded or double-stranded.

[0058] Due to the degeneracy of the genetic code, a large number of polynucleotides that can be used to encode the laccase of the present invention can be obtained. Therefore, when a specific amino acid sequence has been identified, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. A more preferred polynucleotide can be selected through codon optimization according to the preference of the host cell used in the actual preparation process.

[0059] The polynucleotide can be obtained by conventional methods, such as PCR amplification or artificial synthesis, etc. Currently, the polynucleotide sequence can be obtained completely by chemical synthesis.

[0060] In some preferred but non-limiting embodiments, the nucleotide sequence comprises any one of SEQ ID NOs. 7 to 11.

[0061] A vector comprising the polynucleotide. The vector may be a recombinant vector or an expression vector, and the vector may be used to transform a host cell to express the polynucleotide or the laccase.

[0062] A host cell, comprising: expressing the laccase; and / or comprising the polynucleotide; and / or comprising the vector.

[0063] In some preferred but non-limiting embodiments, since Sulfurimonas sp., Bacillus freudenreichii, Streptomyces clavuligerus, Mesocricetibacterintestinalis, Thermosediminibacter oceani, Bhargavaea cecembensis express the laccase of the present invention respectively, the host cell expressing the laccase can be selected from the above cells. In addition, the host cell can also be selected from other prokaryotic cells, such as Escherichia coli, Bacillus subtilis, etc.

[0064] In a fifth aspect, the present invention provides a polypeptide molecule comprising the laccase. The laccase may be fused with other functional polypeptides.

[0065] In a sixth aspect, the present invention provides uses of the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule in dye decolorization and bleaching.

[0066] Optionally, the invention includes use in preparing a decolorizing agent or bleaching agent for the dye Reactive Brilliant Blue KN-R which is heat-resistant, organic solvent-resistant, highly active or mediator-free;

[0067] In a seventh aspect, the present invention provides the use of the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule in the degradation of chloramphenicol or the treatment of pollutants containing chloramphenicol.

[0068] Optionally, the method includes use in preparing a chloramphenicol degrading agent that is heat-resistant, organic solvent-resistant, highly active or mediator-free.

[0069] In an eighth aspect, the present invention provides the use of the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule in the degradation of aflatoxin B1 or the treatment of pollutants containing aflatoxin B1.

[0070] Optionally, the method includes use in preparing a heat-resistant, organic solvent-resistant, highly active or mediator-free aflatoxin B1 degrading agent.

[0071] In a ninth aspect, the present invention comprises the laccase, the polynucleotide, the vector, the host cell or the polypeptide molecule, with or without the addition of process-permitted auxiliary materials.

[0072] LB medium: peptone 10.0 g, yeast extract 5.0 g, NaCl 10 g, distilled water 1000 ml, pH 7.3, solid medium plus agar 20.0 g.

[0073] Example 1 Preparation of laccase

[0074] (1) Construction of recombinant plasmid: Synthesize the gene sequences shown in SEQ ID NO.7-12 (the gene sequences correspond to the amino acid sequences shown in SEQ ID NO.1-6), insert the products into the BamH I and Hind III restriction sites of the vector pET28a, and use the enzyme digestion system as follows: 10 μg of plasmid, 2 μl of enzyme (BamH I and Hind III enzyme), 50 μl of total volume, and digest at 37°C for 30 min. Ligation system: 50 ng of gene fragment and 200 ng of plasmid vector, 10 μl of total volume, and T4 ligase reaction at 37°C for 1 h to complete plasmid ligation, and obtain expression plasmids pET28a-Bfre, pET28a-Tocean, pET28a-Sulfur; pET28a-Scla, pET28a-Mint, pET28a-Bcece. The plasmid maps are as follows: Figure 1 shown.

[0075] (2) Construction of genetic engineering bacteria for heterologous expression of laccase: The expression plasmid was transformed into Escherichia coli BL21 (DE3). The transformation conditions were as follows: 5 μl of the recombinant plasmid and 100 μl of competent Escherichia coli BL21 (DE3) were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, 1 ml of LB liquid medium was added, incubated at 37°C for 1 h, and coated on LB kanamycin-resistant plates. The genetic engineering bacteria containing the corresponding laccase gene recombinant plasmid were obtained.

[0076] (3) Select and inoculate a single clone of the genetically engineered E. coli obtained in step (2) into LB medium containing 50 μg / ml kanamycin, and culture at 37°C, 220 rpm overnight. Inoculate 1 v / v% of the bacterial solution into LB medium containing 50 μg / ml kanamycin, and add CuCl2 to a final concentration of 1 mM. Culture in a shaker at 37°C, 220 rpm until OD 600 When the pH reaches 0.6, IPTG with a final concentration of 0.1 mM is added to induce protein expression, and the mixture is cultured at 16°C and 160 rpm for 16 hours. After the fermentation is terminated, the cells are collected by centrifugation, resuspended with Tris-HCl buffer, ultrasonically disrupted, and centrifuged at 12000 rpm for 30 minutes to remove the precipitate to obtain Escherichia coli disrupted liquid. The supernatant is purified by Ni-NTA affinity chromatography column, and imidazole is removed by ultrafiltration to obtain the laccase Bfre, Tocean, Sulfur, Scla, Mint, and Bcece of the present invention (the amino acid sequences of the above 6 laccases are respectively consistent with SEQ ID NO.1 to 6 after detection), and the concentration is about 1 mg / ml.

[0077] Experimental Example 1 Temperature Tolerance

[0078] This experimental example investigated the temperature tolerance of laccases Bfre, Tocean, Sulfur, Scla, Mint and Bcece prepared in Example 1.

[0079] Methods: The substrate ABTS was dissolved in pH 4.0 citrate buffer with a final concentration of 0.5 mM ABTS, heated to 30, 40, 50, 60, 70, 80, 90 °C, and then mixed with 10 μl of laccase (each adjusted to 1 mg / ml) Bfre, Tocean, Sulfur, Scla, Mint or Bcece. The activity of the above laccases was then determined by the following method: Laccase activity was determined at the above different temperatures using ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) as a substrate. The assay mixture contained 0.5 mM ABTS and 50 mM citrate / phosphate buffer. The enzyme activity was quantified by the absorbance increase at 420 nm due to the oxidation of ABTS, and the absorbance change at 420 nm per minute was used.

[0080] The results are as follows Figure 2 As shown, it can be seen that the optimal reaction temperature of the laccases Bfre, Sulfur, and Scla is 80°C, the optimal reaction temperature of Tocean is 90°C, and the optimal reaction temperature of Bcece is 70°C, indicating that these laccases can work at higher temperatures. Although the activity of laccase Mint decreases with increasing temperature and its optimal reaction temperature is 30°C, it maintains a high activity in the range of 30°C-60°C.

[0081] Experimental Example 2 Tolerance to organic solvents

[0082] This experimental example investigated the organic solvent tolerance of laccases Bfre, Tocean, Sulfur, Scla, Mint and Bcece prepared in Example 1.

[0083] Method: The laccase Bfre, Tocean, Sulfur, Scla, Mint and Bcece (each with a volume of 0.5 ml and each with a concentration adjusted to 1 mg / ml) were added to methanol, ethanol, acetonitrile and acetone organic solvents, respectively, and the final concentrations of methanol, ethanol, acetonitrile and acetone in the obtained mixed solution were all 50 v / v%, and then incubated for 48 hours. The activity of the above laccases was then detected by the following method: Laccase activity was determined at 37°C using ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) as a substrate. The assay mixture contained 0.5 mM ABTS, 50 mM citrate / phosphate buffer. The absorbance increase caused by oxidation of ABTS at 420 nm was measured.

[0084] The results are as follows Figure 3 As shown, the residual activity of the laccase Bfre, Tocean, Sulfur, and Scla reached more than 80%, which fully demonstrated that the laccase had tolerance to the four organic solvents, and the residual activity of Bcece to ethanol reached more than 80%, and the residual activity to the other three organic solvents was between 60-80%, indicating that Bcece had a certain tolerance to the four organic solvents. The residual activity of Mint enzyme to methanol reached more than 60%, indicating that Mint had a certain tolerance to methanol.

[0085] Application Example 1

[0086] In this application example, 1 μM of the laccase Bfre, Tocean, Sulfur, Scla, Mint, and Bcece prepared in Example 1 were mixed with the dye Reactive Brilliant Blue KN-R at a final concentration of 1:100 molar ratio, and the resulting mixture was heated at 85°C for 10 minutes. The mixed solution was centrifuged at 6000 rpm for 30 minutes, the dye absorbance was detected at an ultraviolet wavelength of 594 nm, and the decolorization rate was calculated. The dye not treated with enzymes was set as the control group.

[0087] Decolorization rate (%) = absorbance of experimental group / absorbance of control group × 100%.

[0088] The results are as follows Figure 4 As shown, laccases Bfre, Tocean, Sulfur, Scla, Mint, and Bcece can degrade the dye Reactive Brilliant Blue KN-R, among which Bfre, Tocean, and Bcece have the best degradation effects, and Sulfur and Mint have the best degradation effects reaching more than 40%.

[0089] Application Example 2

[0090] In this application example, 1 μM of the laccase Bfre, Tocean, Sulfur, Scla, Mint, and Bcece prepared in Example 1 were mixed with chloramphenicol at a final concentration of 1:100 molar ratio, and reacted at room temperature of 25°C for 48 hours. After 48 hours, the mixed solution was heated at 95°C for 10 minutes to terminate the reaction. Chloramphenicol degradation was detected by high performance liquid chromatography, where the chromatographic column was C 18 Column (ZORBAX Eclipse Plus C18, 3.0x 150mm, 5μm), the mobile phase was a mixed solution of water: methanol: glacial acetic acid (55:45:0.1, v / v / v), isocratic elution, column temperature was 30℃, detection wavelength was 278nm, chloramphenicol was measured by characteristic peak area and degradation rate was calculated. Chloramphenicol without enzyme treatment was set as the control group.

[0091] Degradation rate (%) = peak area of ​​experimental group / peak area of ​​control group × 100%;

[0092] The results are as follows Figure 5 As shown, laccases Bfre, Tocean, Sulfur, Scla, Mint and Bcece all showed certain ability to degrade chloramphenicol, among which Bfre had a poor degradation effect, while Sulfur and Bcece had better degradation effects, with an average value of more than 20%.

[0093] Application Example 3

[0094] In this application example, 1 μM of laccase Bfre, Tocean, Sulfur, Scla, Mint, and Bcece prepared in Example 1 were mixed with aflatoxin B1 at a final concentration of 1:10 molar ratio, and reacted at 25°C for 48 hours. The degradation of aflatoxin B1 was detected by high performance liquid chromatography, where the chromatographic column was C 18 Column (ZORBAX Eclipse Plus C18, 3.0x 150mm,5μm)with C 18 Guard column (ZORBAX Eclipse Plus C18, 4.6x 12.5mm, 5um), the mobile phase was a mixed solution of acetonitrile: methanol: water (1:1:2, v / v / v), isocratic elution, column temperature was 30°C, detection wavelength was 365nm, aflatoxin B1 was measured by characteristic peak area and degradation rate was calculated. Aflatoxin B1 without enzyme treatment was set as the control group.

[0095] Degradation rate (%) = peak area of ​​experimental group / peak area of ​​control group × 100%;

[0096] The results are as follows Figure 6 As shown, laccases Bfre, Tocean, Sulfur, Scla, Mint and Bcece all showed a degradation capacity of more than 20% for aflatoxin B1, among which Bfre, Tocean, Mint and Bcece had better degradation effects, with an average value of more than 30%.

[0097] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A laccase, characterized in that The laccase is any one of the following: (1) comprising the sequence shown in SEQ ID NO.1; (2) comprising the sequence shown in SEQ ID NO.2; (3) comprising the sequence shown in SEQ ID NO.3; (4) comprising the sequence shown in SEQ ID NO.4; (5) comprising the sequence shown in SEQ ID NO.5; (6) comprising the sequence shown in SEQ ID NO.

6.

2. The laccase according to claim 1, characterized in that The laccase is any one of the following: (1) the sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto; (2) the sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto; (3) the sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto; (4) the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto; (5) the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto; (6) The sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity thereto.

3. A polynucleotide, characterized in that A nucleotide sequence encoding the laccase according to any one of claims 1 or 2.

4. The polynucleotide according to claim 3, characterized in that It comprises the nucleotide sequence shown in any one of SEQ ID NOs. 7 to 12.

5. A carrier, characterized in that Comprising the polynucleotide according to claim 3 or 4.

6. A host cell, characterized in that The host cell comprises: Expressing the laccase according to any one of claims 1 to 2; and / or, comprising the polynucleotide of claim 3 or 4; And / or, comprising the vector according to claim 5.

7. A polypeptide molecule, characterized in that Contains the laccase according to any one of claims 1-2.

8. Use of the laccase according to claim 1 or 2, the polynucleotide according to claim 3 or 4, the vector according to claim 5, the host cell according to claim 6 or the polypeptide molecule according to claim 7 in dye decolorization, bleaching, chloramphenicol degradation or aflatoxin B1 degradation.

9. The use according to claim 8, characterized in that Including use in preparing heat-resistant, organic solvent-resistant, highly active or mediator-free decolorizing agents, bleaching agents, chloramphenicol degrading agents or aflatoxin B1 degrading agents; And / or, use in decolorization of the dye Reactive Brilliant Blue KN-R or bleaching of the dye Reactive Brilliant Blue KN-R.

10. A decolorizing agent, a bleaching agent, a chloramphenicol degrading agent or an aflatoxin B1 degrading agent, characterized in that: The method comprises the laccase according to claim 1 or 2, the polynucleotide according to claim 3 or 4, the vector according to claim 5, the host cell according to claim 6 or the polypeptide molecule according to claim 7, with or without the addition of auxiliary materials permitted by the process.

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