Method for improving thermal stability and acidic degradation performance of ochratoxin A degrading enzyme

Abstract

The invention discloses a method for improving thermal stability and acidic degradation performance of ochratoxin A degrading enzyme, and belongs to the field of enzyme engineering. Wild type ochratoxin A degrading enzyme with the amino acid sequence shown as SEQ ID NO.1 is subjected to Q113P / S210 M / A235Y / G372R combined mutation, the ochratoxin A degrading enzyme mutant with improved thermal stability and acidic degradation performance is obtained, and compared with the wild type ochratoxin A degrading enzyme, the enzyme activity of the mutant is greatly improved under the conditions of 37 DEG C and pH 4.2 in the stomach; the ochratoxin A can be used for preparing food additives or feed additives so as to reduce the biological accumulation efficiency of the ochratoxin A.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and relates to the modification of enzyme activity, and specifically relates to a method for improving the thermal stability and acid degradation performance of an ochratoxin A degrading enzyme, and an ochratoxin A degrading enzyme mutant with improved thermal stability and acid degradation performance, and a preparation method and application thereof. Background Art

[0002] With the continuous progress of society, people's quality of life is also improving, and they have higher requirements for food hygiene and safety. Mycotoxins are secondary metabolites synthesized by filamentous fungi such as Aspergillus, Fusarium and Penicillium, which can cause serious diseases in humans and animals. These fungi that produce mycotoxins can contaminate food and feed, causing socioeconomic and health impacts. Ochratoxins are an important class of symbiotic toxins. More than 20 types of ochratoxins have been discovered, among which ochratoxin A (OTA) is the most toxic. The incidence of ochratoxin A is highest in grains, followed by plant foods (beer, coffee, etc.). Because ochratoxin A has strong stability and exists in tissues, animals and dairy products, ochratoxin A has a high bioaccumulation efficiency and spreads in the food chain. It is carcinogenic, nephrotoxic, hepatotoxic, genotoxic and neurotoxic to humans and animals.

[0003] Enzymatic degradation of ochratoxin A is a green and environmentally friendly method. The enzymes that degrade ochratoxin A generally have higher enzymatic activity under neutral or weakly alkaline conditions. After pigs eat, the pH in the stomach is generally around 4.2. At this time, the enzyme activity of existing ochratoxin A degrading enzymes is low and cannot efficiently degrade ochratoxin A. Achieving efficient degradation of ochratoxin A in the pig's stomach is a scientific and feasible method. Compared with physical or chemical methods to degrade ochratoxin A, enzymatic degradation has the advantages of low cost, no pollution, easy handling, and convenient operation, and has more prospects for achieving efficient degradation.

[0004] Therefore, how to provide an enzyme that can efficiently degrade ochratoxin A in animals is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art, provide a method for improving the thermal stability and acid degradation performance of ochratoxin A degrading enzyme, and provide an ochratoxin A degrading enzyme mutant with improved thermal stability and acid degradation performance, and a preparation method and application thereof.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for improving the thermal stability and acid degradation performance of an ochratoxin A degrading enzyme, comprising mutating the 113th glutamine of a wild-type ochratoxin A degrading enzyme to proline, the 210th serine to methionine, the 235th alanine to tyrosine, and the 372nd glycine to arginine, wherein the amino acid sequence of the wild-type ochratoxin A degrading enzyme is shown in SEQ ID NO.1.

[0008] A method for improving the thermal stability of an ochratoxin A degrading enzyme comprises mutating the 113th glutamine of a wild-type ochratoxin A degrading enzyme to proline, the 210th serine to methionine, and the 235th alanine to tyrosine, wherein the amino acid sequence of the wild-type ochratoxin A degrading enzyme is shown in SEQ ID NO.1.

[0009] A mutant of ochratoxin A degrading enzyme, which is mutant E1 or mutant E2-2. The amino acid sequence of the mutant E1 is shown in SEQ ID NO.3, and the mutant has a combined mutation of Q113P / S210M / A235Y compared with the wild-type ochratoxin A degrading enzyme. The amino acid sequence of the mutant E2-2 is shown in SEQ ID NO.7, and the mutant has a combined mutation of Q113P / S210M / A235Y / G372R compared with the wild-type ochratoxin A degrading enzyme.

[0010] A gene encoding the above-mentioned mutant of ochratoxin A degrading enzyme. The nucleotide sequence of the gene encoding the mutant E1 is preferably shown as SEQ ID NO. 4. The nucleotide sequence of the gene encoding the mutant E2-2 is preferably shown as SEQ ID NO. 8.

[0011] A recombinant vector for expressing the above-mentioned ochratoxin A degrading enzyme mutant contains the coding gene of the ochratoxin A degrading enzyme mutant. Furthermore, the recombinant expression vector is based on pET-28a.

[0012] A recombinant strain expressing an ochratoxin A degrading enzyme mutant contains a coding gene for the ochratoxin A degrading enzyme mutant or contains the above-mentioned recombinant vector. Furthermore, the recombinant expression strain uses Escherichia coli as a host.

[0013] The method for preparing the ochratoxin A degrading enzyme mutant comprises the following steps: culturing and inducing the expression of a recombinant strain expressing the ochratoxin A degrading enzyme mutant to obtain a fermentation product containing the ochratoxin A degrading enzyme mutant. Furthermore, the preparation method further comprises the steps of crushing and purification.

[0014] Application of the ochratoxin A degrading enzyme mutant in degrading ochratoxin A or preparing a product of degrading ochratoxin A.

[0015] Application of the above ochratoxin A degrading enzyme mutant in the preparation of food additives.

[0016] Application of the above ochratoxin A degrading enzyme mutant in the preparation of feed additives.

[0017] An additive for degrading ochratoxin A, comprising the above-mentioned ochratoxin A degrading enzyme mutant.

[0018] Advantages and beneficial effects of the present invention: The present invention performs Q113P / S210M / A235Y / G372R combined mutations on ochratoxin A degrading enzyme, thereby improving the thermal stability and acid degradation performance of the mutant enzyme. The thermal stability of the ochratoxin A degrading enzyme mutant E2-2 provided by the present invention at 60°C is extended by nearly 3.4 minutes compared with the half-life of the initial wild-type ochratoxin A degrading enzyme, and the specific enzyme activity under the conditions of stomach (37°C, pH 4.2) is increased from 2.62U / mg to 18.81U / mg. The mutant can be used to prepare food additives or feed additives to reduce the bioaccumulation efficiency of ochratoxin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the plasmid map of the ochratoxin A degrading enzyme expression vector pET28a(+)-AFOTase.

[0020] Figure 2 This is an electrophoresis diagram of colony PCR verification of E. coli DH5α transformed with pET28a(+)-AFOTase using T7 primers. In the figure, lane M: Maker; lanes 1-8: PCR products of eight single colonies picked.

[0021] Figure 3 The SDS-PAGE results of the supernatant, precipitate (A) and supernatant purification (B) of the bacterial cell expressing ochratoxin A degrading enzyme E2-2. In Figure A, lane M: Maker; lane 1: precipitate after ultrasonic disruption; lane 2: supernatant after ultrasonic disruption. In Figure B, lane M: Maker; lane 1: purified protein.

[0022] Figure 4 The SDS-PAGE results of the supernatant and precipitate of the bacterial cell expressing ochratoxin A degrading enzyme E2-1 are shown in FIG. In the figure, lane M: Maker; lane 1: supernatant after ultrasonic disruption; lane 2: precipitate after ultrasonic disruption. DETAILED DESCRIPTION

[0023] The present invention is further illustrated by the following examples. The experimental methods in the following examples without specifying specific conditions are basically performed according to the conditions described in common molecular cloning manuals.

[0024] Example 1 Construction of strains expressing ochratoxin A degrading enzyme and its mutants

[0025] 1. Construction of initial ochratoxin A degrading enzyme expression strain

[0026] (1) Synthesis of ochratoxin A degrading enzyme coding sequence

[0027] According to the codon preference of E. coli, the coding sequence of ochratoxin A degrading enzyme (AFOTAse) with an amino acid sequence as shown in SEQ ID NO.1 was optimized (the optimized coding sequence is shown in SEQ ID NO.2), and Qingke Biotechnology Co., Ltd. was commissioned to synthesize the coding sequence with restriction enzyme sites Bam HI and HindⅢ as shown in SEQ ID NO.13.

[0028] (2) Connection and conversion

[0029] Plasmid pET-28a(+) and the synthetic ochratoxin A degrading enzyme coding sequence were double-digested with Bam HI and Hind III, and the digested pET-28a(+) and the ochratoxin A degrading enzyme coding sequence were recovered and then connected with a ligase.

[0030] The ligation product was transformed into E. coli DH5α by chemical transformation to construct the ochratoxin A degrading enzyme expression plasmid pET28a(+)-AFOTase. After the plasmid was extracted, it was chemically transformed into E. coli BL21 to obtain the expression strain of ochratoxin A degrading enzyme.

[0031] The chemical transformation method transforms the ligation product into E.coli DH5α competent cells. The transformation steps are as follows:

[0032] 1) First adjust the temperature of the thermostatic water bath to 42°C.

[0033] 2) Take out a tube (100 μL) of competent bacteria from the -80°C ultra-low temperature freezer, insert it into ice, and place it in an ice bath for 10 minutes.

[0034] 3) Add 10 μL of the ligation product, shake gently and place on ice for 20 min.

[0035] 4) After gently shaking, place it in a 42°C water bath for 90 seconds of heat shock, then quickly return it to ice and let it stand for 5 minutes.

[0036] 5) Add 700 μL of LB medium without antibiotics to each of the above tubes and mix gently, then shake on a shaker at 37°C for 50 min.

[0037] 6) Add 300 μL of the above transformation mixture taken out from the clean bench to each solid LB plate culture dish containing the appropriate antibiotic, and evenly apply it using a glass coating rod that has been burned with an alcohol lamp and cooled.

[0038] The marked culture dish (with kanamycin resistance) is placed in a 37°C constant temperature incubator for 30-60 minutes. After the surface liquid has penetrated into the culture medium, it is placed in a 37°C constant temperature incubator overnight. The grown single colonies are used for recombinant plasmid identification: single colonies grown on the above LB solid culture medium are selected, and T7 primers are used for colony PCR. PCR primers are shown in Table 1. After the PCR is completed, 1% agarose gel electrophoresis is used for analysis and verification (such as Figure 2 As shown). The verified single colony was inoculated into 5 mL of liquid LB medium containing kanamycin, and cultured overnight at 37°C and 200 r / min. A portion of the bacterial solution was taken to store the strain in a glycerol tube. A portion of the bacterial solution was taken for plasmid extraction and then transformed into E. coli BL21. The transformation method was the same as above.

[0039] Table 1 Colony PCR primers

[0040]

[0041] 2. Construction of strain expressing mutant E1 of ochratoxin A degrading enzyme

[0042] The method of single-point mutation by roller amplification is used to sequentially perform Q113P / S210M / A235Y mutations on the initial ochratoxin A degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 to improve its thermal stability. That is, using the pET28a(+)-AFOTase plasmid as a template, the 113th glutamine is first mutated to proline by PCR, and sequencing is verified. After sequencing is correct, the PCR product is transformed into E.coli DH5α competent cells by chemical transformation, and the transformation and colony PCR verification operations are the same as above. The successfully transformed strain is selected for overnight culture, and then the plasmid is extracted for standby use. Then, using the plasmid with the 113th amino acid mutation as a template, the 210th serine is mutated to methionine by PCR, and sequencing is verified. After sequencing is correct, the operation is the same as above. Using the plasmid with the 113th amino acid mutation as a template, the 235th alanine is mutated to tyrosine by PCR, and sequencing is verified. After sequencing is correct, the operation is the same as above. Finally, the plasmid pET28a(+)-AFOTase-Q113P / S210M / A235Y with mutations at all three sites was transformed into E. coli BL21.

[0043] The primer sequences for the above-mentioned Q113P, S210M, and A235Y mutations are shown in Table 2 below.

[0044] Table 2 Mutation primers

[0045]

[0046]

[0047] The pET28a(+)-AFOTase-Q113P / S210M / A235Y plasmid was transformed into E. coli DH5α, the plasmid was extracted, and then transformed into E. coli BL21 to obtain an ochratoxin A degrading enzyme mutant E1 expression strain, and the specific operation was the same as above. The amino acid sequence of the ochratoxin A degrading enzyme mutant E1 is shown in SEQ ID NO.3, and the coding sequence is shown in SEQ ID NO.4.

[0048] 3. Construction of strain expressing ochratoxin A degrading enzyme mutant E2

[0049] (1) Mutation site selection and mutation primer design

[0050] Based on the ochratoxin A degrading enzyme mutant E1, the mutation sites were further screened by molecular docking to find the residues near the active center ( The neutral amino acid residues I216, G372, L392 or N405 in the mitochondria (containing) were mutated into arginine to produce the following ochratoxin A degrading enzyme mutants E2-1, E2-2, E2-3, and E2-4.

[0051] Ochratoxin A degrading enzyme mutant E2-1 (Q113P / S210M / A235Y / I216R), the amino acid sequence is shown in SEQ ID NO.5, and the coding sequence is shown in SEQ ID NO.6;

[0052] Ochratoxin A degrading enzyme mutant E2-2 (Q113P / S210M / A235Y / G372R), the amino acid sequence of which is shown in SEQ ID NO.7, and the coding sequence of which is shown in SEQ ID NO.8;

[0053] Ochratoxin A degrading enzyme mutant E2-3 (Q113P / S210M / A235Y / L392R), the amino acid sequence is shown in SEQ ID NO.9, and the coding sequence is shown in SEQ ID NO.10;

[0054] The ochratoxin A degrading enzyme mutant E2-4 (Q113P / S210M / A235Y / N405R), the amino acid sequence is shown in SEQ ID NO.11, and the coding sequence is shown in SEQ ID NO.12.

[0055] Using the above-mentioned pET28a(+)-AFOTase-Q113P / S210M / A235Y plasmid as a template, single-site mutation was performed by roller amplification. The primers for each point mutation of I216R, G372R, L392R, and N405R are shown in Tables 3-6 below.

[0056] Table 3 Mutation primers

[0057]

[0058] Table 4 Mutation primers

[0059]

[0060]

[0061] Table 5 Mutation primers

[0062]

[0063] Table 6 Mutation primers

[0064]

[0065] Each mutant plasmid was transformed to obtain expression strains of ochratoxin A degrading enzyme mutants E2-1, E2-2, E2-3, and E2-4, respectively.

[0066] Example 2 Expression and purification of ochratoxin A degrading enzyme and its mutants

[0067] 1. Expression of ochratoxin A degrading enzyme and its mutants

[0068] (1) Preparation of seed solution: Use an inoculation loop to pick up the recombinant expression strain stored at -20°C in a clean bench, draw three lines on an LB plate containing kanamycin, and incubate inverted 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.

[0069] (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 The pH was 0.6, and a final concentration of 0.5 mM IPTG was added to induce the recombinant bacteria to express proteins. The temperature was lowered to 18°C ​​and the speed was reduced to 150 rpm. Protein expression was induced at low temperature for 18 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 strain without IPTG was used as a control.

[0070] (3) Ultrasonic disruption: The wet cells were mixed with a lysis buffer (20 mM Tris-HCl, pH 7.4) at a ratio of 1:10 and stirred thoroughly to fully suspend the cells; the cells were ultrasonically disrupted in an ice bath under the following conditions: power 60 W, working for 20 min, running for 3 s, and stopping for 5 s; ultrasonication was repeated twice, and the cells were centrifuged at 12,000 rpm and 4°C for 30 min, and the supernatant and precipitate were collected respectively. A small amount of the supernatant and precipitate were used for SDS-PAGE analysis of the expression of ochratoxin A hydrolase.

[0071] 2. Purification of ochratoxin A degrading enzyme and its mutants

[0072] 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:

[0073] (1) Remove the protective ethanol in the column with 3-5 column volumes of distilled water;

[0074] (2) Equilibrate the column with at least 5 column volumes of binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 8.0);

[0075] (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;

[0076] (4) Wash the column with at least 5 column volumes of binding buffer until no yellow liquid flows out;

[0077] (5) Elution was performed using 10-20 column volumes of different gradient elution buffer (0.5 M NaCl, 20 mM Tris-HCl, 5-500 mM imidazole, pH 7.4), and the eluates were collected separately.

[0078] The collected eluate was concentrated and collected using an ultrafiltration concentrator with a molecular weight cutoff of 10 kDa. The target protein was centrifuged at 4°C, 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 Buffer (20 mM Tris-HCl, pH 7.4) was replaced for desalting. The above centrifugation steps were repeated 3-5 times. After the end, the protein in the ultrafiltration tube was drawn into an EP tube, placed in liquid nitrogen for quick freezing for 20 seconds, and then immediately taken out and stored at -80°C. A small amount of the purified product was taken for SDS-PAGE analysis.

[0079] SDS-PAGE analysis revealed that the vast majority of ochratoxin A degrading enzyme E2-1 was present in the precipitate, while the content in the supernatant was very low and could not be purified. The ochratoxin A degrading enzyme and other mutants were expressed in large amounts in the supernatant and could all be purified. The SDS-PAGE results of the supernatant, precipitate, and supernatant purification of the bacterial cell expressing ochratoxin A degrading enzyme E2-2 are shown in Figure 2. Figure 3 ; The SDS-PAGE results of the supernatant and precipitate of the bacterial cell expressing ochratoxin A degrading enzyme E2-1 are as follows Figure 4 .

[0080] Example 3 Performance determination of ochratoxin A degrading enzyme and its mutants

[0081] 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.

[0082] The enzyme reaction system (500 μL) for the assay contained: 10 μL of 0.15 mg / mL enzyme solution, 10 μL of 1 mg / mL substrate ochratoxin A (OTA, purchased from Qingdao Purebond Biotechnology Co., Ltd.), and 480 μL of 20 mM Tris-HCl buffer at pH 8.0.

[0083] Determination of the optimal temperature of the enzyme: The above enzyme reaction system was placed at different temperatures (30-50°C) for 60 minutes and then immediately inactivated with 500 μL acetonitrile, ice-bathed for 10 minutes, filtered with a 0.22 μm organic filter membrane, and the ochratoxin A residue was detected by HPLC. The activity of the ochratoxin A degrading enzyme or its mutant was calculated to determine the optimal temperature of the ochratoxin A degrading enzyme and its mutant.

[0084] Determination of the optimal pH of the enzyme: The enzyme reaction systems with different pH values ​​(4.2-9.0) were placed at the optimal temperature for reaction for 60 min, then immediately inactivated with 500 μL acetonitrile, placed in an ice bath for 10 min, filtered with a 0.22 μm organic filter membrane, and the ochratoxin A residue was detected by HPLC. The activity of the ochratoxin A degrading enzyme or its mutant was calculated to determine the optimal temperature of the ochratoxin A degrading enzyme and its mutant.

[0085] The HPLC detection conditions are as follows: the detector is a fluorescence detector, the excitation wavelength is 330 nm, the emission wavelength is 460 nm, the chromatographic column is a WondaSil-C18 chromatographic column (150×4.6 mmol / L, 5 μm), the mobile phase is acetonitrile:1% glacial acetic acid=5:5, the flow rate is 1 mL / min, and the column temperature is 30°C.

[0086] Determination of thermal stability: Ochratoxin A degrading enzyme or its mutants were incubated at 60°C for 0min, 5min, 10min, 15min, 20min, 25min, and 30min, respectively, and then the enzyme activity was measured after 60s of ice bath. Calculation method of thermal stability: The specific enzyme activity under the optimal reaction conditions before incubation was 100%, and the residual activity was measured under the optimal conditions after incubation at 60°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 60°C can be calculated by the standard curve equation.

[0087] Determination of enzyme activity under gastric conditions (37°C, pH 4.2): Ochratoxin A degrading enzyme or its mutant was reacted in a reaction system with a pH of pH 4.2 at 37°C for 60 minutes, then immediately inactivated with 500 μL acetonitrile and the enzyme activity was measured after an ice bath for 10 minutes.

[0088] After the above determinations, the relevant properties of ochratoxin A degrading enzyme and its different mutants are as follows.

[0089] The optimal temperature of the initial ochratoxin A degrading enzyme (amino acid sequence as shown in SEQ ID NO.1) is 40°C, and the optimal pH is 8; the specific enzyme activity under the optimal conditions is 45.99U / mg; the half-life at 60°C is 6.86min; and the specific enzyme activity at 37°C and pH 4.2 is 2.62U / mg.

[0090] The optimal temperature of the mutant ochratoxin A degrading enzyme E1 (amino acid sequence as shown in SEQ ID NO.3) is 40°C, the optimal pH is 8; the specific enzyme activity under the optimal conditions is 54.12U / mg; the half-life at 60°C is 12.11min; the specific enzyme activity at 37°C and pH 4.2 is 2.93U / mg. Compared with the original ochratoxin A degrading enzyme, the half-life of ochratoxin A degrading enzyme E1 at 60°C increased from 6.86min to 12.11min, and its specific enzyme activity at 37°C and pH 4.2 did not increase significantly.

[0091] The optimum temperature of the mutant ochratoxin A degrading enzyme E2-2 (amino acid sequence as shown in SEQ ID NO.7) is 40°C, the optimum pH is 7.5; the specific enzyme activity under the optimum conditions is 50.84U / mg; the half-life at 60°C is 10.25min; the specific enzyme activity at 37°C and pH 4.2 is 18.81U / mg. Compared with the original ochratoxin A degrading enzyme, the half-life of ochratoxin A degrading enzyme E2-2 at 60°C increased from 6.86min to 10.25min, and the specific enzyme activity at 37°C and pH 4.2 increased from 2.62U / mg to 18.81U / mg.

[0092] No activity was detected for the mutant ochratoxin A degrading enzyme E2-3 (amino acid sequence shown in SEQ ID NO.9) and the mutant ochratoxin A degrading enzyme E2-4 (amino acid sequence shown in SEQ ID.11).

[0093] The above embodiments are only used to help illustrate the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for improving the thermal stability and acid degradation performance of ochratoxin A degrading enzyme, characterized in that: include: The glutamine at position 113 of the wild-type ochratoxin A degrading enzyme is mutated to proline, the serine at position 210 is mutated to methionine, the alanine at position 235 is mutated to tyrosine, and the glycine at position 372 is mutated to arginine. The amino acid sequence of the wild-type ochratoxin A degrading enzyme is shown in SEQ ID NO.

1.

2. A method for improving the thermal stability of ochratoxin A degrading enzyme, characterized in that: include: The glutamine at position 113 of the wild-type ochratoxin A degrading enzyme is mutated to proline, the serine at position 210 is mutated to methionine, and the alanine at position 235 is mutated to tyrosine. The amino acid sequence of the wild-type ochratoxin A degrading enzyme is shown in SEQ ID NO.

1.

3. An ochratoxin A degrading enzyme mutant, characterized in that: The mutant is a mutant E1 whose amino acid sequence is shown in SEQ ID NO.3, or the mutant is a mutant E2-2 whose amino acid sequence is shown in SEQ ID NO.

7.

4. A gene encoding an ochratoxin A degrading enzyme mutant according to claim 3, characterized in that: The nucleotide sequence of the mutant E1 encoding gene is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the mutant E2-2 is shown in SEQ ID NO.

8.

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

6. A recombinant strain expressing the ochratoxin A degrading enzyme mutant according to claim 3, characterized in that: Contains the coding gene of the ochratoxin A degrading enzyme mutant, or contains the recombinant vector according to claim 5.

7. A method for preparing the ochratoxin A 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 an ochratoxin A degrading enzyme mutant.

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

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

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

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