A method for constructing a high-efficiency zearalenone-degrading enzyme

Abstract

The application discloses a construction method of high-efficiency zearalenone degrading enzyme and belongs to the field of enzyme engineering. The zearalenone degrading enzyme mutant with improved thermal stability and acid condition enzyme activity is obtained by performing the combined mutation of D92K / G114L / D117W / V140R / V153M / D157V / S220W on the wild-type zearalenone degrading enzyme with the amino acid sequence shown in SEQ ID NO. 1 and fusing the AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP polypeptide at the N end of the zearalenone degrading enzyme. The thermal stability and the enzyme activity under the animal stomach condition (37 DEG C, pH 4.2) of the zearalenone degrading enzyme mutant constructed by the application are significantly improved compared with those of the wild-type zearalenone degrading enzyme, and the zearalenone degrading enzyme mutant can be used for preparing food additives or feed additives to reduce the content of zearalenone in the animal body and improve the quality of meat quality in the breeding industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering, and particularly relates to a construction method of a zearalenone degrading enzyme with higher degradation efficiency in an animal body, and a zearalenone degrading enzyme mutant with improved thermal stability and acid condition enzyme activity and a preparation method and application thereof. BACKGROUND

[0002] In today's relatively developed productivity, the basic living materials needed by human beings are relatively sufficient. Under the domination of relatively high productivity, people have the basis to pursue a higher quality of life. The pursuit of a higher quality of life is health first. Health is the most important basis for personal development. The external influencing factors of personal health are environment, food, etc. Among them, the safety of food is closely related to human health.

[0003] China has a large population and high grain yield. Because the air humidity and temperature in most parts of China are suitable for the growth and reproduction of molds, corn, wheat, soybean and the like are main raw materials for livestock breeding. The phenomenon of mold contamination is relatively common. Corn, wheat, soybean and the like generally contain mycotoxins, and zearalenone is one of the mycotoxins, which is harmful to chickens, pigs, cattle, sheep and the like, reduces the quality of meat in the breeding industry, and potentially harms the human body.

[0004] Enzymatic degradation of zearalenone is a green and environmentally friendly method. After pigs eat, the pH in the stomach is generally about 4.2, and the existing zearalenone degrading enzyme has low enzyme activity under the condition of the stomach, and cannot efficiently degrade zearalenone. It is a scientific and feasible method to achieve efficient degradation of zearalenone in the stomach of pigs.

[0005] Therefore, how to provide a zearalenone degrading enzyme with high efficiency in the animal body is a problem to be solved in the art. SUMMARY

[0006] The present application aims to provide a construction method of a zearalenone degrading enzyme with higher degradation efficiency in an animal body, and provide a zearalenone degrading enzyme mutant with improved thermal stability and acid condition enzyme activity and a preparation method and application thereof.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The application discloses a construction method of a zearalenone degrading enzyme with higher degradation efficiency in an animal body, and the method comprises the following steps: performing the following combined mutation D92K / G114L / D117W / V140R / V153M / D157V / S220W or the following combined mutation D92K / K94D / G114L / D117W / V140R / V153M / D157V / S220W on a wild type zearalenone degrading enzyme, wherein the amino acid sequence of the wild type zearalenone degrading enzyme is shown in SEQ ID NO. 1.

[0009] Further, the method further comprises fusing the following polypeptide AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP to the N terminal of the wild type or the zearalenone degrading enzyme subjected to the mutation.

[0010] The application discloses a zearalenone degrading enzyme mutant, which is a mutant M1 with an amino acid sequence shown in SEQ ID NO. 3, a mutant M2 with an amino acid sequence shown in SEQ ID NO. 5 or a mutant M3 with an amino acid sequence shown in SEQ ID NO. 7.

[0011] The application discloses a zearalenone degrading enzyme mutant, which is a mutant M1 with an amino acid sequence shown in SEQ ID NO. 3, a mutant M2 with an amino acid sequence shown in SEQ ID NO. 5 or a mutant M3 with an amino acid sequence shown in SEQ ID NO. 7.

[0012] The application discloses a zearalenone degrading enzyme mutant, which is a mutant M1 with an amino acid sequence shown in SEQ ID NO. 3, a mutant M2 with an amino acid sequence shown in SEQ ID NO. 5 or a mutant M3 with an amino acid sequence shown in SEQ ID NO. 7.

[0013] The application discloses a zearalenone degrading enzyme mutant, which is a mutant M1 with an amino acid sequence shown in SEQ ID NO. 3, a mutant M2 with an amino acid sequence shown in SEQ ID NO. 5 or a mutant M3 with an amino acid sequence shown in SEQ ID NO. 7.

[0014] The preparation method of the zearalenone degrading enzyme mutant comprises the following steps: culturing the recombinant strain expressing the zearalenone degrading enzyme mutant, and inducing expression to obtain a fermentation product containing the zearalenone degrading enzyme mutant. Further, the preparation method further comprises the steps of crushing and purifying.

[0015] The zearalenone degrading enzyme mutant is applied to the degradation of zearalenone or the preparation of a product for degrading zearalenone.

[0016] The zearalenone degrading enzyme mutant is applied to the preparation of a food additive.

[0017] The zearalenone degrading enzyme mutant is applied to the preparation of a feed additive.

[0018] An additive for degrading zearalenone comprises the zearalenone degrading enzyme mutant.

[0019] Advantages and beneficial effects of the present application: the zearalenone degrading enzyme is subjected to combined mutation (D92K / K94D / G114L / D117W / V140R / V153M / D157V / S220W), and a specific polypeptide is fused at the N terminal, so that the thermal stability and acid degradation performance of the target enzyme are improved. The thermal stability and specific enzyme activity of the zearalenone degrading enzyme mutant obtained by the present application under the animal stomach condition (37℃, pH 4.2) are significantly improved compared with the initial wild-type zearalenone degrading enzyme, and the zearalenone degrading enzyme mutant can be used for preparing a food additive or a feed additive to reduce the content of zearalenone in the animal body and improve the quality of meat quality in the breeding industry. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the plasmid map of the wild-type zearalenone degrading enzyme expression vector pET28a(+)-ZHD.

[0021] Figure 2 It is the nucleic acid electrophoresis map of pET28a(+)-ZHD double enzyme digestion.

[0022] Figure 3 It is the schematic diagram of multi-base mutation process.

[0023] Figure 4 It is the SDS-PAGE diagram of wild-type zearalenone degrading enzyme expression and purification; Figure A: Lane M: Maker; Lane 1: supernatant after ultrasonic crushing of the bacterial body; Lane 2: precipitate after ultrasonic crushing of the bacterial body; Figure B: Lane M: Maker; Lane 1: purified wild-type zearalenone degrading enzyme.

[0024] Figure 5SDS-PAGE figure of purified zearalenone degrading enzyme mutants M1 and M2. Lane M: Marker; Lane 1: Purified mutant M1; Lane 2: Purified mutant M2. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0026] The experimental methods not specified in the following examples are basically operated according to the conditions described in the common molecular cloning manual. The experimental methods not specified in the following examples are selected according to the conventional methods and conditions, or according to the product instructions.

[0027] Example 1 Construction of zearalenone degrading enzyme and its mutant expression strain

[0028] 1. Construction of wild-type zearalenone degrading enzyme expression strain

[0029] The amino acid sequence of zearalenone degrading enzyme (SEQ ID NO. 1) is obtained from NCBI. According to the codon preference, the gene sequence of zearalenone degrading enzyme is optimized and entrusted to a biological company for synthesis, and the optimized sequence is shown as SEQ ID NO. 2. The synthesized sequence is used as a template, and the primers ZHD-F / ZHD-R are used for PCR amplification. After identification by agarose gel electrophoresis, the target band is recovered, and double enzyme digestion is performed using R I and d III. The recovered enzyme digestion fragments and linearized plasmids are connected using ligase. The ligation product is transformed into E. coli DH5α, and pET28a(+)-ZHD (after verification by colony PCR) is obtained. Then, it is transformed into BL21 (DE3) to express zearalenone degrading enzyme. E. coli Eco R I and d III are used for double enzyme digestion, and the recovered enzyme digestion fragments are recovered. The recovered enzyme digestion fragments and linearized plasmids are connected using ligase. The ligation product is transformed into E. coli DH5α, and pET28a(+)-ZHD (after verification by colony PCR) is obtained. Then, it is transformed into BL21 (DE3) to express zearalenone degrading enzyme. Hin Eco R I and d III are used for double enzyme digestion, and the recovered enzyme digestion fragments are recovered. The recovered enzyme digestion fragments and linearized plasmids are connected using ligase. The ligation product is transformed into E. coli DH5α, and pET28a(+)-ZHD (after verification by colony PCR) is obtained. Then, it is transformed into BL21 (DE3) to express zearalenone degrading enzyme. Hin Figure 1 R I and d III are used for double enzyme digestion, and the recovered enzyme digestion fragments are recovered. The recovered enzyme digestion fragments and linearized plasmids are connected using ligase. The ligation product is transformed into E. coli DH5α, and pET28a(+)-ZHD (after verification by colony PCR) is obtained. Then, it is transformed into BL21 (DE3) to express zearalenone degrading enzyme. E. coli

[0030] Table 1 Zearalenone degrading enzyme amplification primers

[0031]

[0032] The steps of transforming the ligation product into E. coli competent cells are as follows: ​​​​

[0033] 1) Turn on the constant temperature water bath in advance, and set the temperature to 42°C.

[0034] 2) Take the competent cells DH5a / BL21(DE3) from -80°C refrigerator and place them in an ice box containing ice to thaw naturally. After thawing, they can be transformed.

[0035] 3) Add 2-5 μL of ligation product or plasmid to an EP tube containing 100 μL of competent cells DH5a / BL21(DE3), and insert the EP tube into an ice bath for 20 min. The purpose of this step is to allow the plasmid to approach and adsorb to the surface of the competent cells under low temperature conditions.

[0036] 4) Insert the EP tube into a foam float and place it in a 42°C water bath for 90 s. The purpose of this step is to change the temperature from low to 42°C, which changes the permeability of the cell membrane of the competent cells, making the plasmid enter the interior of the competent cells.

[0037] 5) Immediately after the heat shock, insert the EP tube into ice and ice bath for 3-5 min.

[0038] 6) Add 800 μL of antibiotic-free LB to the EP tube and place it in a 37°C constant temperature shaker at 250 rpm for 45 min.

[0039] 7) Centrifuge at 12000 rpm for 1 min, then discard 700 μL of supernatant, resuspend the competent cells with the remaining 200 μL of LB, and gently blow and mix evenly. Use a pipette to transfer the resuspended bacterial solution to a solid LB plate containing 50 μg / mL, and use a burned coating rod to evenly spread the bacterial solution. Place it upright for 30 min until the bacterial solution is completely absorbed. Then place it upside down in a 37°C constant temperature incubator for overnight culture.

[0040] Perform double enzyme digestion on the grown single colonies: pick single colonies grown on the above LB solid medium, inoculate 5 mL of liquid LB medium containing kanamycin, and incubate at 37°C, 200 r / min overnight. Store the bacterial solution in a glycerol tube, and extract the recombinant plasmid according to the instructions of the plasmid extraction kit. Perform double enzyme digestion on the extracted recombinant plasmid with Eco RI and Hin d III, and analyze the target gene fragment and vector fragment after double enzyme digestion using 1% agarose gel electrophoresis. Figure 2

[0041] 2. Construction of zearalenone degrading enzyme first stage mutant M1 expression strain

[0042] ​Based on previous research results, the following combination of mutations was performed on wild-type zearalenone degrading enzyme to improve its thermal stability: D92K / G114L / D117W / V140R / V153M / D157V / S220W.

[0043] Since this experiment required combined mutations at all mutation sites, the method adopted was to first mutate aspartic acid at position 92 to lysine, and then verify this through sequencing. Because the amino acids at positions 114 and 117 are close together, sequential multi-base site-directed mutagenesis was used. Using the plasmid with the mutated amino acid at position 92 as a template, glycine and aspartic acid at positions 114 and 117 were mutated to leucine and tryptophan, respectively. This process was repeated sequentially, mutating valine at position 140, valine at position 153, aspartic acid at position 157, and serine at position 220 to arginine, methionine, valine, and tryptophan, respectively. Finally, the amino acid sequence of the zearalenone-degrading enzyme mutant M1, as shown in SEQ ID NO.3, was obtained, and its corresponding coding sequence is shown in SEQ ID NO.4.

[0044] Table 2. Design of Mutant Primers

[0045]

[0046] Single-base and consecutive multi-base mutations were performed using the Mut Express® II Rapid Mutation Kit V2 from Novizan Biotechnology Co., Ltd. Single-base mutations were performed by direct reverse amplification of the original plasmid using reverse complementary primers (Table 2), while multi-base mutations (… Figure 3 This requires designing partially reverse complementary primers for the two mutation sites, A and B. The two primer pairs are then used interchangeably to obtain segments AB and BA. Dpn I. Digest the amplification product. Mix the AB and BA segments in a specific ratio and react at 37°C for 30 min. Complete the recombination reaction under Exnase II catalysis to achieve in vitro DNA circularization. The recombinant product is directly transformed, and clones formed on plates are positively screened. The specific reaction systems involved in the mutation are shown in Table 3-5 below. The construction of the zearalenone-degrading enzyme mutant M1 expression strain is the same as above.

[0047] Table 3 Plasmid amplification reaction system

[0048]

[0049] Table 4 Dpn I. Plasmid template digestion reaction system

[0050]

[0051] Table 5 Recombinant Reaction In Vitro Cycling

[0052]

[0053] 3. Construction of the expression strain of the second-stage mutant M2 of zearalenone-degrading enzyme

[0054] Based on the zearalenone-degrading enzyme mutant M1, the surface solvent accessibility of the mutant M1 was predicted by the NetSurfP3.0 server. The 94th charged surface residue lysine with the highest exposure rate and the most solvent contact was mutated to aspartic acid with a negative polarity, obtaining the zearalenone-degrading enzyme mutant M2 (the amino acid sequence is shown as SEQ ID NO. 5, and the coding sequence is shown as SEQ ID NO. 6). The primer design for the K94D mutation is shown in Table 6, and the mutation method, construction of the expression strain, and other operations are the same as above.

[0055] Table 6 Design of site-directed mutation primers

[0056]

[0057] 4. Construction of the expression strain of the third-stage mutant M3 of zearalenone-degrading enzyme

[0058] Based on the zearalenone-degrading enzyme mutant M2, a short peptide was fused to the N segment to improve its stability under acidic conditions. The sequence of the fused short peptide is: AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP. The amino acid sequence of the zearalenone-degrading enzyme mutant M3 after fusion is shown as SEQ ID NO. 7, and the coding sequence is shown as SEQ ID NO. 8.

[0059] The coding sequence of the zearalenone-degrading enzyme mutant M3 was synthesized, and after PCR amplification using the primers SZHD-F and SZHD-R (Table 7) as the template, the expression strain of the zearalenone-degrading enzyme mutant M3 was constructed by enzyme digestion, ligation, and transformation, and the related operations were the same as above.

[0060] Table 7 Amplification primers for the fused zearalenone-degrading enzyme M3

[0061]

[0062] Example 2 Expression and purification of zearalenone-degrading enzyme and its mutants

[0063] 1. Expression of zearalenone-degrading enzyme or its mutants

[0064] (1) Preparation of seed liquid: In the clean bench, use a loop to take the strain preserved at -20°C E. coliBL21(DE3) recombinant expression strain, three-zone streak on LB plate containing kanamycin, inverted culture in 37℃ constant temperature incubator for 12h. Single colony on the plate was inoculated into 50mL LB containing kanamycin.

[0065] (2) Induced culture: 1mL seed liquid was added to LB shake flask culture medium containing kanamycin, and placed in a 37℃, 220rpm shaker for culture. When OD 600 was 0.6, 0.5mM IPTG was added to induce protein expression in recombinant bacteria, and the temperature was reduced to 18℃ and the speed was reduced to 120rpm. Protein expression was induced at low temperature for 16h, and the induced bacteria were collected by centrifugation at 6500rpm for 5min. The bacteria were washed twice with 0.9% physiological saline. At the same time, the recombinant bacteria without IPTG were used as control and the same operation was performed.

[0066] (3) Ultrasonic disruption: wet bacteria and bacteria disruption buffer (50mM Tris-HCl, pH 8.0) were mixed at a ratio of 1:10, and the bacteria were fully suspended by stirring; the bacteria were disrupted by ultrasonic under ice bath conditions, the ultrasonic conditions were: power 70W, working 20min, running 2s, stopping 3s; ultrasonic twice, centrifugation at 12000rpm, 4℃ for 30min, and the supernatant and precipitate were collected respectively. A small amount of supernatant and precipitate was taken for SDS-PAGE analysis of zearalenone degrading enzyme expression. The control group was also operated.

[0067] 2. Purification of zearalenone degrading enzyme or its mutant

[0068] After SDS-PAGE verification, the expressed zearalenone degrading enzyme and its mutant were soluble proteins.

[0069] The recombinant protein has 6×histidine at the C-terminal, and the imidazole ring of histidine can bind to metal ions. The recombinant protein can be purified by Ni 2+ affinity chromatography column. The target protein selectively binds to nickel filler and separates from impurities. High concentration of imidazole competitively binds to Ni 2+ , thereby achieving the effect of eluting the target protein. The specific steps of Ni-column affinity chromatography are as follows:

[0070] (1) Remove the protective ethanol in the column with 3-5 times the column volume of distilled water;

[0071] (2) Equilibrate the column with at least 5 times the column volume of binding buffer (0.5M NaCl, 20mM Tris-HCl, pH 8.0);

[0072] (3) Add all the supernatant to the column, and collect the effluent again to add to the column, so that the target protein is fully combined with the filler;

[0073] (4) Wash the column with at least 5 column volumes of binding buffer until no yellow liquid flows out;

[0074] (5) Elute with 10-20 column volumes of different gradient elution buffer (0.5M NaCl, 20mM Tris-HCl, 5-500mM imidazole, pH 8.0), and collect the eluate respectively;

[0075] (6) After elution, wash the equilibrated nickel column with 3-5 column volumes of binding buffer, and it is ready for the next purification;

[0076] (7) After purification, wash the column with 3-5 column volumes of ddH2O, fill the column with 20% ethanol, and store it at 4°C for later use.

[0077] The collected eluate is concentrated by using an ultrafiltration concentration tube with a molecular weight cut-off of 10.0 kDa. Centrifuge at 4°C, 3000-3500 x g / min for 20 min, collect about 1.5 mL of target enzyme solution, discard the liquid in the collection tube, replace it with Buffer Desalting (50mM Tris-HCl, pH 8.0), repeat the above centrifugation steps 3-5 times, and then collect the protein in the ultrafiltration tube into an EP tube. Place it in liquid nitrogen for 20 seconds, then take it out immediately, and store it at -80°C.

[0078] The SDS-PAGE results of the expression and purification of wild-type zearalenone-degrading enzyme and mutants M1 and M2 are shown in Figure 4 and 5 .

[0079] Example 3 Enzyme activity and stability determination of zearalenone-degrading enzyme and its mutants

[0080] Enzyme activity is defined as the amount of enzyme required to consume 1 μg of substrate per unit time, which is 1 U. Specific enzyme activity is defined as the enzyme activity per milligram of enzyme protein, with the unit being U / mg.

[0081] The enzyme reaction system (500 μL) for determination contains 10 μL of enzyme solution (concentration 0.5 mg / mL), 10 μL of substrate ZEN (concentration 1 mg / mL), and 480 μL of buffer (50 mM Tris-HCl buffer, pH 9.0).

[0082] Determination of the optimum temperature of the enzyme: The above enzyme reaction system is placed at different temperatures (30-60°C) for 10 min, then immediately inactivated with 500 μL of methanol, ice-bathed for 10 min, filtered through a 0.22 μm organic filter membrane, and the residual amount of zearalenone is determined by HPLC to calculate the activity of zearalenone-degrading enzyme and determine the optimum temperature of zearalenone-degrading enzyme and its mutants.

[0083] Determination of the optimum pH of the enzyme: The enzyme reaction system at different pH (4.2-11.0) was placed at the optimum temperature for 10 min, then immediately inactivated with 500 μL of methanol, ice-bathed for 10 min, filtered with a 0.22 μm organic filter membrane, and the enzyme activity was determined by HPLC to determine the optimum pH of the zearalenone degrading enzyme and its mutants.

[0084] The HPLC detection conditions were as follows: the detector was a fluorescence detector, the excitation wavelength was 274 nm, the emission wavelength was 440 nm, the chromatographic column was a WondaSil-C18 chromatographic column (150 x 4.6 mmol / L, 5 μm), the mobile phase was acetonitrile: water = 5:5, the flow rate was 1 mL / min, and the column temperature was 30 °C.

[0085] Determination of the thermal stability of the enzyme: The zearalenone degrading enzyme or its mutants were incubated at 50 °C for 0 min, 2 min, 5 min, 7 min, and 10 min, respectively, and then the enzyme activity was measured after ice-bathing for 60 s. The calculation method of thermal stability: the enzyme activity under the optimum reaction conditions before incubation was taken as 100%, the residual activity was measured under the optimum conditions after incubation at 50 °C for different times, and the results were used to draw a standard curve, and the half-life of the enzyme at 50 °C was calculated by the standard curve equation.

[0086] Determination of the enzyme activity under the in-gastric conditions (37 °C, pH 4.2): The zearalenone degrading enzyme or its mutants were incubated in a reaction system with pH 4.2 at 37 °C for 10 min, then immediately inactivated with 500 μL of methanol, ice-bathed for 10 min, and the enzyme activity was measured.

[0087] The determination results of the zearalenone degrading enzyme and its mutants are as follows:

[0088] The optimum temperature of the wild-type zearalenone degrading enzyme (the amino acid sequence is shown in SEQ ID NO. 1) was 37 °C, the optimum pH was 9.0, the specific enzyme activity under the optimum conditions was 132.80 U / mg, the half-life at 50 °C was 2 min, and the specific enzyme activity under the conditions of 37 °C and pH 4.2 was 7.69 U / mg.

[0089] The optimum temperature of the zearalenone degrading enzyme mutant M1 (the amino acid sequence is shown in SEQ ID NO. 3) was 37 °C, the optimum pH was 9.0, the specific enzyme activity under the optimum conditions was 143.66 U / mg, the half-life at 50 °C was 6.90 min, and the specific enzyme activity under the conditions of 37 °C and pH 4.2 was 10.25 U / mg.

[0090] The optimal temperature of the zearalenone degrading enzyme mutant M2 (amino acid sequence as shown in SEQ ID NO. 5) is 37℃, the optimal pH is 9.0, the specific enzyme activity under the optimal condition is 152.99 U / mg, the half-life at 50℃ is 7.87 min, and the specific enzyme activity at 37℃ and pH 4.2 is 38.67 U / mg.

[0091] The optimal temperature of the zearalenone degrading enzyme mutant M3 (amino acid sequence as shown in SEQ ID NO. 7) is 37℃, the optimal pH is 9.0, the specific enzyme activity under the optimal condition is 158.85 U / mg, the half-life at 50℃ is 8.22 min, and the specific enzyme activity at 37℃ and pH 4.2 is 43.55 U / mg.

[0092] The above examples are only used to help illustrate the present application, and the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for constructing a zearalenone-degrading enzyme with higher degradation efficiency, characterized in that, include: The wild-type zearalenone degrading enzyme was subjected to the following combined mutations: D92K / K94D / G114L / D117W / V140R / V153M / D157V / S220W. The amino acid sequence of the wild-type zearalenone degrading enzyme is shown in SEQ ID NO.1, and the amino acid sequence of the mutated zearalenone degrading enzyme is shown in SEQ ID NO.

5.

2. A mutant of zearalenone-degrading enzyme, characterized in that: It is mutant M2 with the amino acid sequence shown in SEQ ID NO.

5.

3. The encoding gene of the zearalenone-degrading enzyme mutant according to claim 2, characterized in that: The nucleotide sequence of the gene encoding mutant M2 is shown in SEQ ID NO.

6.

4. A recombinant vector expressing the zearalenone-degrading enzyme mutant of claim 2, characterized in that: The gene encoding the zearalenone-degrading enzyme mutant contained therein.

5. A recombinant strain expressing the zearalenone-degrading enzyme mutant of claim 2, characterized in that: The gene encoding the zearalenone-degrading enzyme mutant, or the recombinant vector according to claim 4.

6. A method for preparing the zearalenone-degrading enzyme mutant according to claim 2, characterized in that, The process includes the following steps: culturing and inducing expression of the recombinant strain described in claim 5 to obtain a fermentation product containing a zearalenone-degrading enzyme mutant.

7. The application of the zearalenone-degrading enzyme mutant according to claim 2 in the degradation of zearalenone.

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