Construction method of efficient zearalenone degrading enzyme

Abstract

The invention discloses a construction method of an efficient zearalenone degrading enzyme, and belongs to the field of enzyme engineering. According to the present invention, D92K / G114L / D117W / V140R / V153M / D157V / S220W combined mutation is performed on a wild type zearalenone degrading enzyme represented by an amino acid sequence such as SEQ ID NO.1, and an AEAEAAHAEAEAAHAGGGSGGGGSEP polypeptide is fused at the N end of the wild type zearalenone degrading enzyme, such that the thermal stability and the enzymatic activity under the acidic condition are improved; compared with a wild type zearalenone degrading enzyme, the thermal stability of the constructed zearalenone degrading enzyme mutant and the enzyme activity under animal stomach conditions (37 DEG C, pH 4.2) are obviously improved; the zearalenone can be used for preparing food additives or feed additives so as to reduce the content of zearalenone in animal bodies and improve the quality of meat in the breeding industry.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and specifically relates to a method for constructing a zearalenone-degrading enzyme with higher degradation efficiency in an animal body, a zearalenone-degrading enzyme mutant with improved thermal stability and enzyme activity under acidic conditions, and a preparation method and application thereof. Background Art

[0002] Today, with the development of productivity, the basic living materials needed by human beings are relatively sufficient. Under the control of relatively high productivity, people have the foundation to pursue a higher quality of life. In the pursuit of a higher quality of life, health is the first priority. Health has become the most important foundation for personal development. The external factors that affect personal health are: environment, food, etc. Among them, food safety is closely related to human health.

[0003] China has a large population and high grain production. Since the air humidity and temperature in most parts of China are suitable for the growth and reproduction of mold, corn, wheat, soybeans, etc. are the main raw materials for animal husbandry. Mold contamination is quite common. Corn, wheat, soybeans, etc. generally contain mycotoxins. Zearalenone is a type of mycotoxin, which is very harmful to chickens, pigs, cattle, sheep, etc., reducing the quality of meat in the breeding industry and potentially causing harm to the human body.

[0004] Enzymatic degradation of zearalenone is a green and environmentally friendly method. After pigs eat, the pH in their stomach is generally around 4.2. Existing zearalenone-degrading enzymes have low enzymatic activity under stomach conditions and cannot efficiently degrade zearalenone. Achieving efficient degradation of zearalenone in the pig's stomach is a scientifically feasible method.

[0005] Therefore, how to provide an enzyme that can efficiently degrade zearalenone in animals is an urgent problem to be solved in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a zearalenone-degrading enzyme with higher degradation efficiency in animals, as well as to provide a zearalenone-degrading enzyme mutant with improved thermal stability and enzyme activity under acidic conditions, and a preparation method and application thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for constructing a zearalenone-degrading enzyme with higher degradation efficiency in an animal, comprising: subjecting a wild-type zearalenone-degrading enzyme to the following combined mutations D92K / G114L / D117W / V140R / V153M / D157V / S220W or the following combined mutations D92K / K94D / G114L / D117W / V140R / V153M / D157V / S220W, wherein the amino acid sequence of the wild-type zearalenone-degrading enzyme is shown in SEQ ID NO.1.

[0009] Furthermore, the method also includes fusing the following polypeptide to the N-terminus of the wild-type or mutated zearalenone degrading enzyme: AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP.

[0010] A mutant of corn zearalenone degrading enzyme, which is a mutant M1 with an amino acid sequence as shown in SEQ ID NO.3, a mutant M2 with an amino acid sequence as shown in SEQ ID NO.5, or a mutant M3 with an amino acid sequence as shown in SEQ ID NO.7. Compared with the wild-type corn zearalenone degrading enzyme, the mutant M1 has a combined mutation of D92K / G114L / D117W / V140R / V153M / D157V / S220W; the mutant M2 has a further K94D mutation on the basis of the mutant M1; and the mutant M3 has a fusion polypeptide AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP at the N-terminus of the mutant M2.

[0011] A gene encoding the above-mentioned zearalenone degrading enzyme mutant, wherein the nucleotide sequence of the gene encoding mutant M1 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding mutant M2 is shown in SEQ ID NO.6, and the nucleotide sequence of the gene encoding mutant M3 is shown in SEQ ID NO.8.

[0012] A recombinant vector expressing the above-mentioned zearalenone degrading enzyme mutant contains the coding gene of the above-mentioned zearalenone degrading enzyme mutant. Furthermore, the recombinant expression vector is based on pET-28a.

[0013] A recombinant strain expressing the above-mentioned zearalenone degrading enzyme mutant contains the above-mentioned coding gene or the above-mentioned recombinant vector. Furthermore, the recombinant strain uses Escherichia coli as a host.

[0014] The preparation method of the above-mentioned zearalenone degrading enzyme mutant comprises the following steps: culturing and inducing the expression of the recombinant strain expressing the above-mentioned zearalenone degrading enzyme mutant to obtain a fermentation product containing the zearalenone degrading enzyme mutant. Furthermore, the preparation method also comprises the steps of crushing and purification.

[0015] Application of the above-mentioned zearalenone-degrading enzyme mutant in degrading zearalenone or preparing products of degrading zearalenone.

[0016] Application of the above-mentioned zearalenone-degrading enzyme mutant in the preparation of food additives.

[0017] Application of the above-mentioned zearalenone-degrading enzyme mutant in the preparation of feed additives.

[0018] An additive for degrading zearalenone, comprising the zearalenone-degrading enzyme mutant.

[0019] Advantages and beneficial effects of the present invention: The present invention performs combined mutations (D92K / K94D / G114L / D117W / V140R / V153M / D157V / S220W) on the zearalenone degrading enzyme, and fuses a specific polypeptide at the N-terminus, thereby improving the thermal stability and acidic degradation performance of the target enzyme. The thermal stability of the zearalenone degrading enzyme mutant obtained by the present invention and the specific enzyme activity under the conditions in the stomach of animals (37°C, pH 4.2) are significantly improved compared with the initial wild-type zearalenone degrading enzyme, and can be used to prepare food additives or feed additives to reduce the content of zearalenone in animals and improve the quality of meat in the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0022] Figure 3 Schematic diagram of the multi-base mutation process.

[0023] Figure 4 This is an SDS-PAGE diagram of the expression and purification of wild-type corn zearalenone-degrading enzyme; Figure A: Lane M: Maker; Lane 1: supernatant after ultrasonic disruption of bacteria; Lane 2: precipitate after ultrasonic disruption of bacteria; Figure B: Lane M: Maker; Lane 1: purified wild-type corn zearalenone-degrading enzyme.

[0024] Figure 5The SDS-PAGE image of the purified mutants M1 and M2 of zearalenone-degrading enzyme. Lane M: Maker; Lane 1: purified mutant M1; Lane 2: purified mutant M2. DETAILED DESCRIPTION

[0025] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods in the following examples without specific conditions are basically operated according to the conditions described in common molecular cloning manuals. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0027] Example 1 Construction of strains expressing zearalenone-degrading enzyme and its mutants

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

[0029] The amino acid sequence of corn zearalenone degrading enzyme (SEQ ID NO.1) was obtained from NCBI. According to the preference of E. coli codons, the gene sequence of corn zearalenone degrading enzyme was optimized and commissioned to a biological company for synthesis. The optimized sequence is shown in SEQ ID NO.2. Using the synthesized sequence as a template, PCR amplification was performed using primers ZHD-F / ZHD-R. After identification by agarose gel electrophoresis, the target band was recovered by gel excision, and double enzyme digestion and recovery were performed using EcoR I and HindⅢ. The plasmid pET-28a(+) was double-digested with EcoR I and HindⅢ, and the linearized plasmid after enzyme digestion was excised and recovered. The recovered enzyme-digested fragment and the linearized plasmid were connected with ligase. The ligation product was transferred into Escherichia coli DH5α, and pET28a(+)-ZHD( Figure 1 ) and then transferred into E. coli BL21 (DE3) to express zearalenone-degrading enzyme.

[0030] Table 1 Primers for amplification of target gene zearalenone-degrading enzyme

[0031]

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

[0033] 1) Open the constant temperature water bath in advance and adjust the temperature to 42°C.

[0034] 2) Take the competent cells DH5α / BL21(DE3) out of the -80℃ freezer in advance and place them in an ice box filled with ice to thaw naturally. After thawing, transformation can be performed.

[0035] 3) Add 2-5 μL of ligation product or plasmid to an EP tube containing 100 μL of competent cells DH5α / BL21 (DE3), and place the EP tube in an ice bath for 20 minutes. The purpose of this step is to allow the plasmid to approach the competent cells under low temperature conditions and adsorb on the surface of the competent cells.

[0036] 4) Insert the EP tube into the foam float and heat shock it in a 42℃ water bath for 90s. The purpose of this step is to change the cell membrane permeability of the competent cells from low temperature to 42℃, so that the plasmid can enter the competent cells.

[0037] 5) After the heat shock is over, immediately insert the EP tube into ice and keep it in an ice bath for 3-5 minutes.

[0038] 6) Add 800 μL of antibody-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 12000rpm for 1min, discard 700μL of supernatant, resuspend the competent cell pellet with the remaining 200μL LB and gently blow evenly. Use a pipette to transfer the resuspended bacterial solution to a solid LB plate containing 50μg / mL, spread the bacterial solution evenly with a burned spreading stick, and place it upright for 30min until the bacterial solution is completely absorbed. Then place it upside down in a 37℃ constant temperature incubator for overnight culture.

[0040] The grown single colonies were double-digested for identification: single colonies grown on the above LB solid medium were picked and inoculated into 5 mL of liquid LB medium containing kanamycin, and cultured overnight at 37°C and 200 r / min. Part of the bacterial solution was taken and stored in a glycerol tube. An appropriate amount of bacterial solution was taken from each of them to extract the recombinant plasmid according to the instructions of the plasmid extraction kit. The extracted recombinant plasmid was double-digested with EcoR I and HindⅢ, and the products after double-digestion were analyzed by 1% agarose gel electrophoresis to determine the target gene fragment and the vector fragment ( Figure 2 ).

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

[0042] According to the results of previous studies, the following combined mutations were made to the wild-type corn zearalenone-degrading enzyme to improve thermal stability: D92K / G114L / D117W / V140R / V153M / D157V / S220W.

[0043] Since all mutation sites need to be combined and mutated in this experiment, the method adopted is to first mutate the aspartic acid at position 92 to lysine and sequence verification. Because the amino acids at positions 114 and 117 are close to each other, continuous multi-base site-directed mutagenesis can be used, and the plasmid with the mutated amino acid at position 92 is used as a template to mutate the glycine and aspartic acid at positions 114 and 117 to leucine and tryptophan. Similarly, the valine at position 140, the valine at position 153, the aspartic acid at position 157, and the serine at position 220 are gradually mutated to arginine, methionine, valine, and tryptophan, respectively. Finally, the amino acid sequence of the corn zearalenone degrading enzyme mutant M1 shown in SEQ ID NO.3 is obtained, and its corresponding coding sequence is shown in SEQ ID NO.4.

[0044] Table 2 Mutation primer design

[0045]

[0046]

[0047] Single-base and continuous multi-base mutations were performed using the Mut II Rapid Mutation Kit V2. Single-base mutations were directly reverse-amplified from the original plasmid using reverse complementary primers (Table 2), while multi-base mutations ( Figure 3 ) It is necessary to first design partially reverse complementary primers at the two mutation sites A and B. The two pairs of primers are used crosswise to obtain AB segment and BA segment, and the amplified product is digested with Dpn I. The AB segment and the BA segment are mixed in proportion, reacted at 37°C for 30 minutes, and the recombination reaction is completed under the catalysis of Exnase II to achieve in vitro cyclization of DNA. The recombinant product is directly transformed, and the clones formed on the plate are positively screened. The reaction system involved in the mutation is specifically shown in Table 3-5 below. The construction of the expression strain of the corn zearalenone degrading enzyme mutant M1 is the same as above.

[0048] Table 3 Plasmid amplification reaction system

[0049]

[0050] Table 4 Dpn I digestion plasmid template reaction system

[0051]

[0052] Table 5 Recombination reaction in vitro cyclization

[0053]

[0054] 3. Construction of the expression strain of the second-stage mutant M2 of zearalenone-degrading enzyme

[0055] Based on the corn zearalenone degrading enzyme mutant M1, the surface solvent accessibility of mutant M1 was predicted by NetSurfP3.0 server. Lysine, the 94th charged surface residue with the highest exposure rate and the easiest to be contacted by solvent, was selected to mutate into polar negatively charged aspartic acid to obtain corn zearalenone degrading enzyme mutant M2 (amino acid sequence as shown in SEQ ID NO.5, coding sequence as shown in SEQ ID NO.6). The primer design for the above K94D mutation is shown in Table 6, and the mutation method, construction of expression strain and other operations are the same as above.

[0056] Table 6 Site-directed mutagenesis primer design

[0057]

[0058] 4. Construction of the expression strain of the third-stage mutant M3 of zearalenone-degrading enzyme

[0059] Based on the corn zearalenone degrading enzyme mutant M2, a short peptide was fused to the N segment to improve its stability under acidic conditions. The fused short peptide sequence is: AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP. The amino acid sequence of the fused corn zearalenone degrading enzyme mutant M3 is shown in SEQ ID NO.7, and the coding sequence is shown in SEQ ID NO.8.

[0060] The coding sequence of the corn zearalenone degrading enzyme mutant M3 was synthesized, and PCR amplification was performed using the sequence as a template using primers SZHD-F and SZHD-R (Table 7). Then, the expression strain of the corn zearalenone degrading enzyme mutant M3 was constructed by enzyme digestion, ligation, and transformation, and the relevant operations were the same as above.

[0061] Table 7 Amplification primers for fusion of zearalenone degrading enzyme M3

[0062]

[0063]

[0064] Example 2 Expression and purification of zearalenone-degrading enzyme and its mutants

[0065] 1. Expression of zearalenone-degrading enzyme or its mutant

[0066] (1) Preparation of seed solution: Use an inoculation loop to pick up the E. coli BL21 (DE3) recombinant expression strain stored at -20°C in a clean bench, streak three zones on an LB plate containing kanamycin, and incubate upside down in a constant temperature incubator at 37°C for 12 hours. Pick a single colony on the plate and inoculate it into 50 mL of LB containing kanamycin.

[0067] (2) Induction culture: Add 1 mL of seed solution to LB shake flask medium containing kanamycin and culture in a shaker at 37°C and 220 rpm until OD 600 When the pH was 0.6, IPTG with a final concentration of 0.5 mM was added to induce the recombinant bacteria to express proteins, the temperature was lowered to 18 ° C, and the speed was reduced to 120 rpm. Protein expression was induced at low temperature for 16 hours, and the induced bacteria were collected by centrifugation at 6500 rpm for 5 minutes. The bacteria were washed twice with 0.9% saline. At the same time, the recombinant bacteria without IPTG were used as the control, and the operation was the same.

[0068] (3) Ultrasonic disruption: Mix wet bacteria and lysis buffer (50mM Tris-HCL, pH8.0) at a ratio of 1:10, stir thoroughly to fully suspend the bacteria; ultrasonically disrupt the bacteria in an ice bath under the following conditions: power 70W, working for 20min, running for 2s, and stopping for 3s; ultrasonicate twice, centrifuge at 12000rpm and 4℃ for 30min, and collect the supernatant and precipitate respectively. Take a small amount of supernatant and precipitate for SDS-PAGE analysis of the expression of zearalenone degrading enzyme in corn. The control group is operated in the same way.

[0069] 2. Purification of zearalenone-degrading enzyme or its mutant

[0070] SDS-PAGE confirmed that the expressed zearalenone-degrading enzyme and its mutants were soluble proteins.

[0071] The recombinant protein has 6× histidine at the C-terminus. The imidazole ring of histidine can bind to metal ions. 2+ Affinity chromatography columns can be used to purify the target protein. The target protein selectively binds to the nickel filler and is separated from the impurities, while high concentrations of imidazole compete with Ni. 2+ Binding, thereby achieving the effect of eluting the target protein. The specific steps of Ni-column affinity chromatography are as follows:

[0072] (1) Remove the protective ethanol in the column with 3-5 column volumes of distilled water;

[0073] (2) Equilibrate the column with at least 5 column volumes of binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 8.0);

[0074] (3) Add all the supernatant to the column, and add the collected effluent to the column again to allow the target protein to fully bind to the filler;

[0075] (4) Wash the column with at least 5 column volumes of binding buffer until no yellow liquid flows out;

[0076] (5) eluting with 10-20 column volumes of different gradient elution buffer (0.5 M NaCl, 20 mM Tris-HCl, 5-500 mM imidazole, pH 8.0), and collecting the eluates respectively;

[0077] (6) After elution is completed, wash the equilibrated nickel column with 3-5 column volumes of binding buffer and then use it for the next purification;

[0078] (7) After purification, rinse the column with 3-5 column volumes of ddH2O, fill the column with 20% ethanol, and store at 4°C for later use.

[0079] The collected eluate was concentrated and collected using an ultrafiltration concentration tube with a molecular weight cutoff of 10.0 kDa. The target protein was centrifuged at 4°C and 3000-3500×g / min for 20 min to collect about 1.5 mL of the target enzyme solution. The liquid in the collection tube was discarded, and the desalting buffer (50 mM Tris-HCl, pH 8.0) was replaced. The above centrifugation steps were repeated 3-5 times. After the end, the protein in the ultrafiltration tube was aspirated into an EP tube, which was quickly frozen in liquid nitrogen for 20 s and then taken out immediately and stored at -80°C.

[0080] 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 .

[0081] Example 3 Determination of the enzyme activity and stability of zearalenone-degrading enzyme and its mutants

[0082] Enzyme activity is defined as: the amount of enzyme required to consume 1 μg of substrate per unit time is 1 U. Specific enzyme activity is defined as: the enzyme activity per milligram of enzyme protein, the unit is U / mg.

[0083] The enzyme reaction system (500 μL) for the measurement contained: 10 μL enzyme solution (concentration 0.5 mg / mL), 10 μL substrate ZEN (concentration 1 mg / mL), and 480 μL buffer (50 mM, pH 9.0 Tris-HCl buffer).

[0084] Determination of the optimal temperature of the enzyme: The above enzyme reaction system was placed at different temperatures (30-60°C) for 10 minutes and then immediately inactivated with 500 μL of methanol, ice-bathed for 10 minutes, filtered with a 0.22 μm organic filter membrane, and the residual amount of zearalenone was determined by HPLC. The activity of the zearalenone-degrading enzyme was calculated, and the optimal temperature of the zearalenone-degrading enzyme and its mutants was determined.

[0085] Determination of the optimal pH of the enzyme: The enzyme reaction systems with different pH (4.2-11.0) were placed at the optimal temperature for 10 minutes, then immediately inactivated with 500 μL of methanol, ice-bathed for 10 minutes, filtered with a 0.22 μm organic filter membrane, and the enzyme activity was detected by HPLC to determine the optimal pH of the zearalenone-degrading enzyme and its mutants.

[0086] 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 column (150×4.6 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.

[0087] Determination of enzyme thermal stability: Zearalenone-degrading enzyme or its mutants were incubated at 50°C for 0min, 2min, 5min, 7min and 10min, and then the enzyme activity was measured after 60s of ice bath. Calculation method of thermal stability: The enzyme activity under the optimal reaction conditions before incubation was 100%, and the residual activity was measured under the optimal conditions after incubation at 50°C for different time periods. The standard curve was drawn based on the results of the determination, and the half-life of the enzyme at 50°C could be calculated using the standard curve equation.

[0088] Determination of enzyme activity under gastric conditions (37°C, pH 4.2): Zearalenone-degrading enzyme or its mutant was reacted in a reaction system with a pH of pH 4.2 at 37°C for 10 min, then immediately inactivated with 500 μL of methanol and the enzyme activity was measured in an ice bath for 10 min.

[0089] The above test results of zearalenone degrading enzyme and its mutants are as follows:

[0090] The optimum temperature of the wild-type corn zearalenone degrading enzyme (amino acid sequence as shown in SEQ ID NO.1) is 37°C and the optimum pH is 9.0; the specific enzyme activity under the optimum conditions is 132.80U / mg; the half-life at 50°C is 2min; the specific enzyme activity at 37°C and pH 4.2 is 7.69U / mg.

[0091] The optimum temperature of the zearalenone degrading enzyme mutant M1 (amino acid sequence as shown in SEQ ID NO.3) is 37°C and the optimum pH is 9.0; the specific enzyme activity under the optimum conditions is 143.66U / mg; the half-life at 50°C is 6.90min; and the specific enzyme activity at 37°C and pH 4.2 is 10.25U / mg.

[0092] The optimum temperature of the zearalenone degrading enzyme mutant M2 (amino acid sequence as shown in SEQ ID NO.5) is 37°C and the optimum pH is 9.0; the specific enzyme activity under the optimum conditions is 152.99 U / mg; the half-life at 50°C is 7.87 min; and the specific enzyme activity at 37°C and pH 4.2 is 38.67 U / mg.

[0093] The optimum temperature of the zearalenone degrading enzyme mutant M3 (amino acid sequence as shown in SEQ ID NO.7) is 37°C and the optimum pH is 9.0; the specific enzyme activity under the optimum conditions is 158.85 U / mg; the half-life at 50°C is 8.22 min; and the specific enzyme activity at 37°C and pH 4.2 is 43.55 U / mg.

[0094] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

1.

2. The construction method according to claim 1, characterized in that: It also includes fusing the following polypeptide to the N-terminus of the wild-type or mutated zearalenone degrading enzyme: AEAEAHAHAEAEAHAHGGGGSGGGSGGGSEP.

3. A zearalenone-degrading enzyme mutant, characterized in that: It is a mutant M1 with an amino acid sequence as shown in SEQ ID NO.3, a mutant M2 with an amino acid sequence as shown in SEQ ID NO.5, or a mutant M3 with an amino acid sequence as shown in SEQ ID NO.

7.

4. A gene encoding a zearalenone-degrading enzyme according to claim 3, characterized in that: The nucleotide sequence of the gene encoding mutant M1 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding mutant M2 is shown in SEQ ID NO.6, and the nucleotide sequence of the gene encoding mutant M3 is shown in SEQ ID NO.

8.

5. A recombinant vector expressing the zearalenone degrading enzyme mutant according to claim 3, characterized in that: Contains the coding gene of the zearalenone-degrading enzyme mutant.

6. A recombinant strain expressing the zearalenone degrading enzyme mutant according to claim 3, characterized in that: A gene encoding the zearalenone degrading enzyme mutant, or a recombinant vector according to claim 5.

7. A method for preparing the zearalenone-degrading enzyme mutant according to claim 3, characterized in that: The method comprises the following steps: culturing the recombinant strain according to claim 6, inducing expression, and obtaining a fermentation product containing a zearalenone-degrading enzyme mutant.

8. Use of the zearalenone-degrading enzyme mutant according to claim 3 in degrading zearalenone or preparing a product of degrading zearalenone.

9. The use according to claim 8, characterized in that: The products include food additives and feed additives.

10. An additive for degrading zearalenone, characterized in that: Comprising the zearalenone-degrading enzyme mutant according to claim 3.

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