High-temperature-resistant laccase, gene and application of high-temperature-resistant laccase in mycotoxin degradation

Abstract

The invention belongs to the technical field of gene engineering, and relates to high-temperature-resistant laccase, a gene and application of the high-temperature-resistant laccase in mycotoxin degradation. The amino acid sequence of the laccase TtLac-4 is as shown in SEQ ID NO: 1. On the basis of wild laccase, site-directed mutagenesis modification is carried out through artificial rational design, and an amino acid sequence of the modified high-temperature-resistant laccase and a nucleotide sequence of the artificially optimized high-temperature-resistant laccase gene are obtained. The high-temperature-resistant laccase obtained through gene coding of the high-temperature-resistant laccase has the remarkably-improved high-temperature-resistant characteristic, and the optimum temperature reaches 90 DEG C. The degradation rate of the laccase TtLac-4 obtained by the invention on mycotoxin aflatoxin B1 and zearalenone reaches 100%. The invention lays a foundation for a green degradation process and application of mycotoxin degradation.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a high temperature resistant laccase and a gene and application thereof in mycotoxin degradation. Background Art

[0002] Laccase (EC 1.10.3.2) is a type of copper-containing polyphenol oxidase widely found in nature. It belongs to the multi-copper oxidase family and is widely distributed in plants, fungi, bacteria and other organisms. It has important biological functions. It not only participates in the degradation of wood and plant cell walls, but is also closely related to a variety of metabolic pathways and ecological functions. Laccase can catalyze the oxidation reaction of phenolic and non-phenolic compounds and reduce molecular oxygen to water. Due to its oxidase activity and environmental friendliness, laccase has been widely used in pollutant degradation, bioremediation and food processing in recent years. In the field of pollutant degradation and bioremediation, laccase can be used to degrade organic pollutants in water and soil, such as pesticides, active pharmaceutical ingredients and nitrogen-containing azo dyes in wastewater, thereby achieving environmental purification. In food and feed processing, laccase is mainly used to improve the color, flavor, taste, antioxidant and shelf life of food. Laccase can also be used to remove or transform certain undesirable substances, such as polyphenols, bitter components and pigments, to improve the sensory properties of food. Currently, laccase shows good prospects for the detoxification of mycotoxins in food and feed.

[0003] Mycotoxins include aflatoxins, zearalenone, vomitoxin, fumonisin and patulin, which are secondary metabolites produced by molds. They mainly contaminate grains, nuts and feed, and damage the liver, kidneys and immune system. For example, aflatoxin B1 (AFB1) is a Class I carcinogen defined by the International Agency for Research on Cancer (IARC), and zearalenone (ZEN) has an estrogenic effect on animals. The toxicity of mycotoxins is stable and difficult to completely remove through conventional food processing, so more effective detoxification technology needs to be developed.

[0004] Detoxification methods can be roughly divided into physical methods, chemical methods and biological methods. Physical methods mainly include sorting and separation, high temperature treatment and adsorption. Sorting and separation can effectively reduce the level of mycotoxins in contaminated grains, but the efficiency is low. High temperature treatment has limited effect on some mycotoxins (such as vomitoxin), but has no significant effect on aflatoxin. Adsorption can adsorb some mycotoxins, but may also reduce the nutritional content in food. Chemical methods include ozonation and organic acid methods. Ozonation can effectively oxidize aflatoxin, but the reaction conditions need to be strictly controlled to avoid damage to food quality. Organic acids can partially degrade zearalenone, but the efficiency is limited. Biological methods use microorganisms and their metabolites to degrade mycotoxins. The detoxification conditions are mild and effective, environmentally friendly, and the degradation products have low toxicity. It has become a research hotspot. However, biological methods may only be effective for specific types of mycotoxins, and different microorganisms may compete with each other in the same environment, thereby affecting the degradation effect of mycotoxins. In addition, when using microorganisms to degrade toxins, it is necessary to ensure that there is no contamination by pathogens or other harmful microorganisms to avoid secondary pollution, and biological detoxification is limited.

[0005] Laccase plays a particularly prominent role in mycotoxin detoxification. Studies have shown that laccase, as a biological enzyme that performs well in mycotoxin detoxification, can significantly degrade aflatoxin B1 (AFB1) and reduce its toxicity and carcinogenicity while retaining the nutritional value of food. Laccase has great potential in the field of mycotoxin detoxification.

[0006] Although laccase has broad application prospects in the field of food safety, its industrial application still faces problems such as insufficient enzyme activity stability, rapid activity decline in high temperature or high pH environment, and high cost. To address the above problems, genetic engineering is used to modify laccase to improve enzyme activity and environmental adaptability, and to further screen efficient laccase-producing strains to meet the needs of industrial applications. Summary of the invention

[0007] The main purpose of the present invention is to transform laccase, and provide a high temperature resistant laccase TtLac-4 and its encoding gene ttlac-4 and application. The original laccase TtLac and its amino acid sequence, the sequence for encoding the laccase gene ttlac, etc. are improved to improve its heat resistance and stability. The present invention can realize the application of the laccase TtLac-4 in industry, and can obtain a laccase production strain with good application prospects, which is very beneficial to the promotion and application of laccase TtLac-4. The laccase TtLac-4 obtained by the present invention is a high temperature resistant laccase that can be applied to the field of mycotoxin degradation, and can be particularly applied to the degradation of aflatoxin B1 and zearalenone.

[0008] In order to achieve the above object, the first aspect of the present invention provides a thermostable laccase TtLac-4, wherein the amino acid sequence of the laccase TtLac-4 is as shown in SEQ ID NO: ： As shown in 1.

[0009] The second aspect of the present invention provides a method for obtaining the above-mentioned laccase TtLac-4, which comprises: mutating the arginine (R) at position 54 of the wild-type laccase TtLac to glycine (G), mutating the proline (P) at position 121 to glutamate (E), mutating the leucine (L) at position 199 to tryptophan (W), and mutating the valine (V) at position 376 to threonine (T); the amino acid sequence of the wild-type laccase TtLac is shown in SEQ ID NO: 3.

[0010] The third aspect of the present invention provides a thermostable laccase gene ttlac-4, the base sequence of which is more than 95% identical to the base sequence shown in SEQ ID NO: 2. Preferably, the base sequence of the laccase gene ttlac-4 is as shown in SEQ ID NO: 2.

[0011] Compared with the prior art, the above technical solution conceived by the present invention can effectively improve the heat resistance and catalytic efficiency of the existing laccase due to the improvement of the amino acid sequence of the original laccase TtLac and the base sequence encoding the laccase gene ttlac.

[0012] The fourth aspect of the present invention provides a recombinant expression vector comprising the above-mentioned laccase gene ttlac-4.

[0013] The fifth aspect of the present invention provides a method for constructing the above-mentioned recombinant expression vector, comprising the following steps: introducing restriction endonuclease cutting sites XbaⅠ and EcoRⅠ at both ends of the above-mentioned laccase gene ttlac-4, respectively; inserting the laccase gene double-digested by XbaⅠ and EcoRⅠ into the yeast expression vector pPICZαA double-digested by XbaⅠ and EcoRⅠ, to obtain the laccase recombinant expression vector pPICZαA-ttlac-4.

[0014] The sixth aspect of the present invention provides a recombinant expression strain, comprising the above-mentioned recombinant expression vector, wherein the host cell of the recombinant expression vector is yeast.

[0015] The seventh aspect of the present invention provides a method for preparing the above-mentioned recombinant expression strain, comprising the following steps: introducing the above-mentioned laccase recombinant expression vector into a host cell to obtain a recombinant expression strain.

[0016] According to a specific embodiment of the present invention, the method for preparing the recombinant expression strain comprises the following steps: linearizing the recombinant expression vector pPICZαA-ttlac-4 with the restriction endonuclease BglⅡ; introducing the linearized recombinant expression vector pPICZαA-ttlac-4 into yeast by electroporation, and screening positive clones on a YPD plate containing Zeocin resistance to obtain the recombinant expression strain.

[0017] The eighth aspect of the present invention provides a method for producing the above-mentioned thermostable laccase, which comprises culturing the above-mentioned recombinant expression strain and obtaining the laccase from the culture.

[0018] Specifically include the following steps:

[0019] (1) transforming a host cell with the above-mentioned recombinant vector to obtain a recombinant strain;

[0020] (2) fermenting the recombinant strain to obtain the expressed laccase TtLac-4;

[0021] (3) Collecting the expressed laccase TtLac-4.

[0022] According to a specific embodiment of the present invention, the method for preparing laccase by the above-mentioned recombinant expression strain comprises the following steps:

[0023] Pick a single colony of the recombinant expression strain and inoculate it into 5mL YPD liquid medium, culture it overnight at 28℃, 200rpm as seed liquid, and then inoculate 1mL of seed liquid into a shake flask containing 25mL BMGY. The culture conditions are 28℃, 200rpm, methanol is added every 24h to make the final concentration of 1%, and 1mM copper ions are added at the same time to induce laccase expression for 96h, collect the supernatant by centrifugation, and store it in a refrigerator at 4℃. The fermentation supernatant is taken to determine the activity of laccase, and the protein content is determined by BCA method.

[0024] The ninth aspect of the present invention provides the use of the above laccase in the degradation of mycotoxins.

[0025] Specifically, the application method is to contact the laccase with mycotoxins; the mycotoxins include but are not limited to aflatoxin B1 and / or zearalenone.

[0026] The present invention significantly improves the high temperature resistance and activity of laccase by modifying the amino acid sequence of laccase TtLac and the key sites of the gene ttlac sequence encoding the laccase TtLac. In addition, the preparation process of laccase is simple, and the production cost is reduced. The laccase of the present invention has outstanding high temperature resistance, the optimum temperature reaches 90°C, can be used for the degradation of mycotoxins, and the degradation rate of mycotoxins aflatoxin B1 and zearalenone reaches 100%. The present invention lays a foundation for the green degradation process and application of mycotoxin degradation.

[0027] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0029] Figure 1 This is a diagram showing the positions of the four amino acids used for mutation of the original laccase in the example of the present invention: glycine (G) at position 54, glutamic acid (E) at position 121, tryptophan (W) at position 199, and threonine (T) at position 376 in the three-dimensional structure.

[0030] Figure 2 The gel images are of the laccase production and protein analysis of the wild-type laccase TtLac recombinant strain and the modified laccase TtLac-4 recombinant strain in shake flasks in the examples of the present invention.

[0031] Figure 3 This is a graph showing the optimum temperature for the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention.

[0032] Figure 4 This is a graph showing the optimum pH for the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention.

[0033] Figure 5 This is a graph showing the temperature stability of the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention.

[0034] Figure 6 This is a graph showing the pH stability of the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention.

[0035] Figure 7 This is a bar graph comparing the degradation rates of aflatoxin B1 and zearalenone by the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention at 70°C.

[0036] Figure 8 These are the HPLC results of the degradation of aflatoxin B1 (AFB1) catalyzed by the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention, respectively.

[0037] Fig. 9 These are the HPLC results of the degradation of zearalenone (ZEN) catalyzed by the thermostable laccase TtLac-4 and the wild-type laccase TtLac in the examples of the present invention, respectively. DETAILED DESCRIPTION

[0038] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0039] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0040] Example 1

[0041] This example is used to illustrate the transformation of wild-type laccase TtLac into thermostable laccase TtLac-4 by site-directed mutagenesis. The specific transformation process includes:

[0042] By analyzing the three-dimensional structure of laccase, the arginine (R) at position 54 of the wild-type Thermus thermophilus laccase (WP_201350935) was mutated to glycine (G), the proline (P) at position 121 was mutated to glutamate (E), the leucine (L) at position 199 was mutated to tryptophan (W), and the valine (V) at position 376 was mutated to threonine (T). Figure 1 shown.

[0043] The site-directed mutagenesis technique is a conventional method in the art, and its specific operation steps are well known to those skilled in the art. The amino acid sequence of the modified laccase TtLac-4 is shown in SEQ ID NO: 1. The amino acid sequence of the wild-type laccase TtLac is shown in SEQ ID NO: 3.

[0044] Example 2

[0045] This example is used to illustrate the sequence characteristics of the modified thermostable laccase gene.

[0046] In this embodiment, the nucleotide sequence of the laccase gene ttlac-4 is redesigned based on the amino acid sequence of the mutated laccase TtLac-4. Specifically, it includes replacing low-frequency codons with high-frequency codons; reducing the complexity and minimum free energy of the secondary structure of the gene-encoded mRNA. The GC content and distribution in the gene are balanced, and the repetitive sequences and cis-acting elements in the gene are removed. The laccase gene sequence is designed, and the laccase gene fragment is obtained by artificial synthesis. The technology for artificially synthesizing genes is a conventional method in the art, and its specific operating steps are well known to those skilled in the art. The base sequence of the laccase gene ttlac-4 is shown in SEQ ID NO: 2, and the base sequence of the wild-type laccase gene ttlac is shown in SEQ ID NO: 4.

[0047] Example 3

[0048] This example is used to illustrate the construction of a wild-type laccase TtLac recombinant expression vector and a modified thermostable laccase TtLac-4 recombinant expression vector.

[0049] (1) Add restriction sites EcoRⅠ and XbaⅠ at both ends of the designed thermostable laccase gene ttlac-4, digest the gene with EcoRⅠ and XbaⅠ and collect the digestion products, and digest the vector pPICZαA with EcoRⅠ and XbaⅠ at the same time and collect the digestion products. The digestion system is: 50μL DNA, 2μL EcoRⅠ, 2μL XbaⅠ, 30μL buffer, add water to make up to 300μL volume, and digest at 37℃ for 4 hours.

[0050] (2) The digestion product of the laccase gene ttlac-4 and the digestion product of the vector pPICZαA were connected with T4 DNA ligase. The connection system was: 4.5 μL of gene fragment, 1.5 μL of pPICZαA fragment, 1 μL of T4 ligase buffer, 1 μL of T4 DNA ligase, and water was added to make up to 10 μL. After the connection was completed, the recombinant expression vector pPICZαA-ttlac-4 was obtained.

[0051] The above method was used to construct the wild-type laccase TtLac recombinant vector pPICZαA-ttLac.

[0052] Example 4

[0053] This example is used to illustrate the acquisition and expression of the recombinant strains of the wild-type laccase TtLac and the modified thermostable laccase TtLac-4.

[0054] (1) The recombinant expression vector pPICZαA-ttlac-4 obtained in Example 3 was transformed into yeast. The electroporation method was as follows: 10 μL of the linearized product of the above recombinant expression plasmid was mixed with 90 μL of fresh yeast competent cells and placed on an ice box for 5 min; the electroporator was turned on and the electroporator parameters were adjusted to 1550 V; the linearized product and competent cell mixture in the ice bath was transferred to an electroporation cup precooled on ice for electroporation; 800 μL of YPD preheated at 28°C was quickly added to the electroporation cup, transferred to a 1.5 mL sterile centrifuge tube, and placed on a 28°C shaker for 2 h; 200 uL of bacterial solution was spread on a YPD plate containing Zeocin resistance and inverted in a 28°C incubator for 3 days. After a single colony was grown, a yeast recombinant strain containing the ttlac-4 gene was obtained.

[0055] (2) Construct a wild-type laccase TtLac recombinant strain according to the method of step (1).

[0056] (3) The above recombinant strain was inoculated into 4 mL of YPD medium and cultured overnight in a constant temperature shaker at 28°C as seed liquid. Then 1 mL of the seed liquid was inoculated into a shake flask containing 25 mL of BMGY. The culture conditions were 28°C, 200 rpm, methanol was added every 24 hours to make its final concentration 1%, and 1 mM copper ions were added at the same time to induce laccase expression for 96 hours. Next, the cultured bacterial liquid was centrifuged in a low-temperature refrigerated centrifuge for 5 minutes at 8000 r / min, the supernatant was collected, and the fermentation supernatant was taken for SDS-PAGE detection. The results are as follows Figure 2 . Figure 2 Shown are gel images of laccase production and protein analysis of the wild-type laccase TtLac recombinant strain and the modified laccase TtLac-4 recombinant strain in shake flasks.

[0057] (4) Determination of laccase activity: Add 200 μL of pH 4 BR Buffer to a 2 mL EP tube, add 10 μL of 0.1 M A BTS, vortex to mix, preheat both the experimental group and the control group on a 70 °C metal bath for 2 min, then add 3 μL of enzyme solution to the experimental group in sequence, with the same interval of 15 s, and then react on the metal bath for 5 min. Take out the tubes in sequence at the same interval and immediately put them in an ice bath to terminate the reaction. Take 200 μL of the reaction solution after termination and add it to a 96-well plate. Read the OD420 value on a microplate reader. Calculate the enzyme activity based on the OD420 values ​​of the experimental group and the control group.

[0058] The enzyme activity calculation formula is:

[0059] ΔOD: Absorbance change before and after reaction

[0060] ξ:36000M -1cm -1

[0061] l: measured by ELISA, 0.58cm

[0062] T: time unit: min

[0063] E: Protein content μg / mL

[0064] V 酶 :Enzyme dosage volume

[0065] V 反应体系 : The volume of the entire reaction system

[0066] According to the operation procedures described in this example, wild-type laccase TtLac and redesigned laccase TtLac-4 recombinant strains were obtained, and enzyme production fermentation was carried out in shake flasks.

[0067] Example 5

[0068] This example is used to measure and analyze the enzymatic properties of the wild-type laccase TtLac and the redesigned laccase TtLac-4.

[0069] (1) Determination of the optimum temperature

[0070] Under the condition of pH 7, the enzyme activities of wild-type laccase TtLac and optimized laccase TtLac-4 were measured at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, respectively, and the highest enzyme activity was set as 100% to determine the optimal temperature. According to the above method, the optimal temperatures of wild-type laccase TtLac and optimized laccase TtLac-4 were determined. The results are shown in Figure 3 As shown, the optimum temperature of the wild-type laccase TtLac is 70°C, and the optimum temperature of the modified laccase TtLac-4 is 90°C, which is 20°C higher than the optimum temperature of the wild-type laccase TtLac.

[0071] (2) Determination of the optimal pH

[0072] Under the conditions of the optimum temperature, the enzyme activities of the wild-type laccase TtLac and the optimized laccase TtLac-4 were measured at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and the highest enzyme activity was set as 100% to determine the optimum pH. According to the above method, the optimum pH of the wild-type laccase TtLac and the optimized laccase TtLac-4 was determined. The results are shown in Figure 4 As shown, the optimal pH of the modified laccase TtLac-4 is 4, while the optimal pH of the wild-type laccase TtLac is 5.

[0073] (3) Temperature stability

[0074] The wild-type laccase TtLac and the optimized laccase TtLac-4 were stored at 50-90°C for 30 min, and the enzyme activity of the treated samples was determined. The enzyme activity of the untreated enzyme solution was set as 100%, and the enzyme activity of the samples treated at different temperatures was calculated. According to the above method, the heat resistance of the wild-type laccase TtLac and the optimized laccase TtLac-4 was determined. The results are shown in Figure 5 As shown, the thermostability of the modified laccase TtLac-4 is significantly better than that of the wild-type laccase TtLac.

[0075] (4) pH stability

[0076] The wild-type laccase TtLac and the optimized laccase TtLac-4 were stored at pH 2.0-12.0 for 30 min, and the enzyme activity of the treated samples was determined. The enzyme activity of the untreated enzyme solution was set as 100%, and the enzyme activity of the samples treated with different pH values ​​was calculated. According to the above method, the pH stability of the wild-type laccase TtLac and the optimized laccase TtLac-4 was determined. The results are shown in Figure 6 As shown, the pH stability of the modified laccase TtLac-4 is significantly better than that of the wild-type laccase TtLac.

[0077] Example 6

[0078] This example is used to analyze the degradation ability of wild-type laccase TtLac and redesigned laccase TtLac-4 on aflatoxin B1.

[0079] (1) The wild-type laccase TtLac and the optimized laccase TtLac-4 were placed in contact with 50 μg / mL aflatoxin B1 at 70°C to degrade it.

[0080] (2) According to the above method, the degradation efficiency of wild-type laccase TtLac and optimized laccase TtLac-4 on aflatoxin B1 was determined. Figure 7 As shown in the figure, in the same time, the ability of the modified laccase TtLac-4 to degrade aflatoxin B1 is significantly better than that of the wild-type laccase TtLac. The degradation rate of aflatoxin B1 by the wild-type laccase TtLac is about 75% at 15h, while the modified laccase TtLac-4 completely degrades aflatoxin B1 in the system within 15h, with a degradation rate of 100%. Figure 8 These are the HPLC results of AFB1 degradation catalyzed by wild-type laccase TtLac and modified laccase TtLac-4, respectively.

[0081] Example 7

[0082] This example is used to analyze the degradation ability of wild-type laccase TtLac and redesigned laccase TtLac-4 on zearalenone.

[0083] (1) The wild-type laccase TtLac and the optimized laccase TtLac-4 were placed in contact with 50 μg / mL of zearalenone at 70°C to degrade them.

[0084] (2) According to the above method, the degradation efficiency of wild-type laccase TtLac and optimized laccase TtLac-4 on zearalenone was determined. Figure 7 As shown in the figure, in the same time, the ability of the modified laccase TtLac-4 to degrade zearalenone is significantly better than that of the wild-type laccase TtLac. The wild-type laccase TtLac only degraded about 23% of zearalenone at 15h, while the modified laccase TtLac-4 completely degraded zearalenone in the system at 15h, with a degradation rate of 100%. Fig. 9 These are the HPLC results of ZEN degradation catalyzed by wild-type laccase TtLac and modified laccase TtLac-4, respectively.

[0085] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A thermostable laccase TtLac-4, characterized in that: The amino acid sequence of the laccase TtLac-4 is shown in SEQ ID NO:

1.

2. A method for obtaining the laccase TtLac-4 according to claim 1, characterized in that: The method comprises: mutating the arginine (R) at position 54 of the wild-type laccase TtLac into glycine (G), mutating the proline (P) at position 121 into glutamic acid (E), mutating the leucine (L) at position 199 into tryptophan (W), and mutating the valine (V) at position 376 into threonine (T); the amino acid sequence of the wild-type laccase TtLac is shown in SEQ ID NO:

3.

3. A thermostable laccase gene ttlac-4, characterized in that: The base sequence of the laccase gene ttlac-4 has more than 95% identity with the base sequence shown in SEQ ID NO:

2. Preferably, the base sequence of the laccase gene ttlac-4 is as shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The method comprises the laccase gene ttlac-4 as claimed in claim 3.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that: The method comprises the following steps: introducing restriction endonuclease sites XbaⅠ and EcoRI at both ends of the laccase gene ttlac-4 described in claim 3; inserting the laccase gene double-digested by XbaⅠ and EcoRI into the yeast expression vector pPICZαA double-digested by XbaⅠ and EcoRI to obtain the laccase recombinant expression vector pPICZαA-ttlac-4.

6. A recombinant expression strain, characterized in that: The invention comprises the recombinant expression vector according to claim 4, wherein the host cell of the recombinant expression vector is yeast.

7. The method for preparing the recombinant expression strain according to claim 6, characterized in that: The method comprises the following steps: introducing the laccase recombinant expression vector according to claim 4 into a host cell to obtain a recombinant expression strain.

8. The method for producing the thermostable laccase according to claim 1, characterized in that: Cultivate the recombinant expression strain according to claim 6 and obtain laccase from the culture.

9. Use of the laccase according to claim 1 in the degradation of mycotoxins.

10. The use according to claim 9, characterized in that: The laccase described in claim 1 is contacted with a mycotoxin; the mycotoxin is aflatoxin B1 and / or zearalenone.

Images