A method for improving the thermal stability and acid degradation performance of ochratoxin A degrading enzyme

Abstract

The present invention discloses a method for improving the thermal stability and acid degradation performance of an ochratoxin A degrading enzyme, belonging to the field of enzyme engineering. The present invention obtains an ochratoxin A degrading enzyme mutant with improved thermal stability and acid degradation performance by subjecting the wild-type ochratoxin A degrading enzyme with the amino acid sequence shown in SEQ ID NO. 1 to a combined mutation of Q113P / S210M / A235Y / G372R. The mutant exhibits significantly improved enzymatic activity under gastric conditions (37°C, pH 4.2) relative to the wild-type ochratoxin A degrading enzyme. The mutant can be used to prepare a food additive or feed additive to reduce the bioaccumulation efficiency of ochratoxin A.

Description

Technical Field

[0001] The present 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, as well as an ochratoxin A degrading enzyme mutant with improved thermal stability and acid degradation performance, and a preparation method and application thereof. Background Art

[0002] As society progresses, people's quality of life improves, leading to higher demands for food hygiene and safety. Mycotoxins are secondary metabolites synthesized by filamentous fungi such as Aspergillus, Fusarium, and Penicillium, and can cause serious diseases in humans and animals. These mycotoxins can contaminate food and feed, resulting in socioeconomic and health impacts. Ochratoxins are an important class of symbiotic toxins. More than 20 ochratoxins have been identified, of which ochratoxin A (OTA) is the most toxic. Its highest incidence is in cereals, followed by plant-based foods (such as beer and coffee). Due to its robust stability, ochratoxin A persists in tissues, animals, and dairy products. Consequently, ochratoxin A has a high bioaccumulation rate and is transmitted through the food chain. It is carcinogenic, nephrotoxic, hepatotoxic, genotoxic, and neurotoxic to both humans and animals.

[0003] Enzymatic degradation of ochratoxin A is a green and environmentally friendly method. Current enzymes that degrade ochratoxin A generally have high activity under neutral or weakly alkaline conditions. However, after pigs eat, the pH in their stomachs is generally around 4.2. At this level, existing ochratoxin A-degrading enzymes have low activity and are unable to effectively degrade ochratoxin A. Achieving efficient degradation of ochratoxin A in the pig stomach is a scientifically feasible approach. Compared with physical or chemical methods for degrading ochratoxin A, enzymatic degradation offers advantages such as low cost, no pollution, easy handling, and convenient operation, with the potential for achieving more efficient degradation.

[0004] Therefore, how to provide an enzyme that can efficiently degrade ochratoxin A in animals is an urgent problem that needs 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, as well as 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 acidic degradation performance of an ochratoxin A degrading enzyme, comprising mutating glutamine at position 113 of a wild-type ochratoxin A degrading enzyme to proline, mutating serine at position 210 to methionine, mutating alanine at position 235 to tyrosine, and mutating glycine at position 372 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 glutamine at position 113 to proline, serine at position 210 to methionine, and alanine at position 235 to tyrosine in a wild-type ochratoxin A degrading enzyme, wherein the amino acid sequence of the wild-type ochratoxin A degrading enzyme is shown in SEQ ID NO.1.

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

[0010] A gene encoding the aforementioned ochratoxin A degrading enzyme mutant. 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 aforementioned ochratoxin A degrading enzyme mutant contains a gene encoding 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, comprising a gene encoding the ochratoxin A degrading enzyme mutant or 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 a recombinant strain expressing the ochratoxin A-degrading enzyme mutant and inducing expression to obtain a fermentation product containing the ochratoxin A-degrading enzyme mutant. Furthermore, the method further comprises the steps of fragmentation and purification.

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

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

[0016] Application of the above-mentioned 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 incorporates the Q113P / S210M / A235Y / G372R combined mutations into an ochratoxin A-degrading enzyme, improving the mutant's thermal stability and acidic degradation performance. The ochratoxin A-degrading enzyme mutant E2-2 provided by the present invention exhibits a thermal stability at 60°C that is approximately 3.4 minutes longer than the half-life of the original wild-type ochratoxin A-degrading enzyme. Its specific enzyme activity in the stomach (37°C, pH 4.2) increases from 2.62 U / mg to 18.81 U / mg. This mutant can be used to prepare food 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 Figure 1 shows SDS-PAGE results of the supernatant, precipitate (A), and supernatant purification (B) of cells expressing ochratoxin A-degrading enzyme E2-2. In Figure A, lane M: Maker; lane 1: ultrasonic precipitate; lane 2: ultrasonic supernatant. In Figure B, lane M: Maker; lane 1: purified protein.

[0022] Figure 4 This is an SDS-PAGE image of the supernatant and precipitate of cells expressing ochratoxin A-degrading enzyme E2-1. 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 were 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 the 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 Hind III as shown in SEQ ID NO.13.

[0028] (2) Connection and transformation

[0029] Plasmid pET-28a(+) and the synthetic ochratoxin A degrading enzyme coding sequence were double-digested with Bam HI and Hind III. The digested pET-28a(+) and the ochratoxin A degrading enzyme coding sequence were recovered and ligated with 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 an ochratoxin A degrading enzyme expression strain.

[0031] The ligation product was transformed into E. coli DH5α competent cells by chemical transformation. The transformation steps are as follows:

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

[0033] 2) Take one tube (100 μL) of competent bacteria out of a -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 ligation product, shake gently, and place on ice for 20 minutes.

[0035] 4) Shake gently and place in a 42°C water bath for 90 seconds for heat shock. Then quickly return to ice and let 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 minutes.

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

[0038] Mark the coated culture dish (with kanamycin resistance) and place it in a 37°C constant temperature incubator for 30-60 minutes. After the surface liquid has penetrated into the culture medium, turn it upside down and place it in a 37°C constant temperature incubator overnight. Identify the recombinant plasmid by picking a single colony grown on the LB solid medium and performing colony PCR using T7 primers. PCR primers are shown in Table 1. After the PCR is completed, analyze and verify by 1% agarose gel electrophoresis (see Table 1). Figure 2 Inoculate a single, verified colony into 5 mL of liquid LB medium containing kanamycin and incubate overnight at 37°C and 200 rpm. Store a portion of the culture in a glycerol tube. Extract the plasmid from a portion of the culture and transform it into E. coli BL21 using the same method as above.

[0039] Table 1 Colony PCR primers

[0040]

[0041] 2. Construction of an expression strain for the ochratoxin A degrading enzyme mutant E1

[0042] Using roller wheel amplification for single-point mutagenesis, the original ochratoxin A degrading enzyme (SEQ ID NO. 1) was subjected to the Q113P / S210M / A235Y mutations to improve its thermal stability. Using the pET28a(+)-AFOTase plasmid as a template, glutamine at position 113 was first mutated to proline by PCR, and sequencing was performed to verify the mutation. After sequencing, the PCR product was chemically transformed into E. coli DH5α competent cells. The transformation and colony PCR verification procedures were the same as above. Successfully transformed strains were cultured overnight, and the plasmids were extracted and set aside. Next, using the plasmid with the mutated amino acid at position 113 as a template, serine at position 210 was mutated to methionine by PCR, and sequencing was performed to verify the mutation. Once sequencing was correct, the same procedures were repeated. Finally, using the plasmid with the mutated amino acid at positions 113 and 210 as templates, alanine at position 235 was mutated to tyrosine by PCR, and sequencing was performed to verify the mutation. Once sequencing was correct, the same procedures were repeated. Finally, the plasmid pET28a(+)-AFOTase-Q113P / S210M / A235Y with all three sites mutated 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] E. coli DH5α was transformed with the pET28a(+)-AFOTase-Q113P / S210M / A235Y plasmid. The plasmid was extracted and then transformed into E. coli BL21 to generate an expression strain for the ochratoxin A degrading enzyme mutant E1, following the same procedures 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 an expression strain for the ochratoxin A degrading enzyme mutant E2

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

[0050] Based on the mutant E1 of ochratoxin A degrading enzyme, we further screened the mutation sites by molecular docking to find the residues near the active center ( The neutral amino acid residues I216, G372, L392 or N405 in the mitochondrial compartment were mutated to 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 of which is shown in SEQ ID NO. 5, and the coding sequence of which 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 of which is shown in SEQ ID NO. 9 and the coding sequence of which is shown in SEQ ID NO. 10;

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

[0055] Using the pET28a(+)-AFOTase-Q113P / S210M / A235Y plasmid as a template, single-site mutagenesis was performed using roller wheel amplification. Primers for the I216R, G372R, L392R, and N405R point mutations are listed 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) Prepare seed solution: Use an inoculating loop to pick up the recombinant expression strain stored at -20°C in a clean bench, streak three lines on an LB plate containing kanamycin, and incubate inverted in a 37°C incubator for 12 h. Pick a single colony from the plate and inoculate it into 50 mL of LB plate containing kanamycin.

[0069] (2) Induction culture: Add 1 mL of seed solution to LB shake flask culture medium containing kanamycin, place it in a shaker at 37°C and 220 rpm, and wait until the OD 600 The recombinant bacteria were induced to express protein after adding IPTG to a final concentration of 0.5 mM. The temperature was lowered to 18°C ​​and the rotation speed was reduced to 150 rpm. Protein expression was induced at low temperature for 18 hours. The induced bacteria were then collected by centrifugation at 6500 rpm for 5 minutes. The cells were washed twice with 0.9% saline. A strain without IPTG was also used as a control.

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

[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 using an ultrafiltration concentrator with a molecular weight cutoff of 10 kDa to collect the target protein. Centrifuge at 4°C, 3000-3500 g / min for 20 min to collect approximately 1.5 mL of the target enzyme solution. The liquid in the collection tube was discarded and replaced with 20 mM Tris-HCl buffer (pH 7.4) for desalting. Repeat the centrifugation step 3-5 times. After completion, the protein in the ultrafiltration tube was transferred to an EP tube, quickly frozen in liquid nitrogen for 20 seconds, and immediately removed and stored frozen at -80°C. A small amount of the purified product was used 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. Ochratoxin A degrading enzyme and other mutants were expressed in high 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 bacteria 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, which is 1 U. Specific enzyme activity is defined as the enzyme activity per milligram of enzyme protein, with the unit being 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 Puruibang 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, then immediately inactivated with 500 μL of acetonitrile, placed in an ice bath for 10 minutes, and filtered through a 0.22 μm organic filter membrane. The residual ochratoxin A was detected by HPLC, and 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 60 minutes, then immediately inactivated with 500 μL of acetonitrile, placed in an ice bath for 10 minutes, and filtered through a 0.22 μm organic filter membrane. The residual ochratoxin A was detected by HPLC, and 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 were as follows: the detector was a fluorescence detector, the excitation wavelength was 330 nm, the emission wavelength was 460 nm, the chromatographic column was a WondaSil-C18 column (150×4.6 mmol / L, 5 μm), the mobile phase was acetonitrile:1% glacial acetic acid = 5:5, the flow rate was 1 mL / min, and the column temperature was 30°C.

[0086] Thermal stability determination: Ochratoxin A-degrading enzyme or its mutants were incubated at 60°C for 0, 5, 10, 15, 20, 25, and 30 minutes, followed by measurement of enzyme activity after incubation on ice for 60 seconds. Thermal stability was calculated by taking the specific enzyme activity under optimal reaction conditions before incubation as 100%. After incubation at 60°C for different times, the residual activity was measured under optimal conditions. A standard curve was constructed based on the results, and the enzyme half-life at 60°C was calculated using 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 optimal conditions is 45.99 U / mg; the half-life at 60°C is 6.86 min; and the specific enzyme activity at 37°C and pH 4.2 is 2.62 U / mg.

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

[0091] The mutant ochratoxin A-degrading enzyme E2-2 (amino acid sequence shown in SEQ ID NO. 7) has an optimal temperature of 40°C and an optimal pH of 7.5. Under these optimal conditions, its specific enzyme activity is 50.84 U / mg, its half-life at 60°C is 10.25 minutes, and its specific enzyme activity at 37°C and pH 4.2 is 18.81 U / mg. Compared with the original ochratoxin A-degrading enzyme, the half-life of ochratoxin A-degrading enzyme E2-2 at 60°C was increased from 6.86 minutes to 10.25 minutes, and its specific enzyme activity at 37°C and pH 4.2 increased from 2.62 U / mg to 18.81 U / 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 an ochratoxin A-degrading enzyme, characterized in that: Only the 113th glutamine of the wild-type ochratoxin A degrading enzyme is mutated to proline, the 210th serine is mutated to methionine, the 235th alanine is mutated to tyrosine, and the 372nd glycine 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 an ochratoxin A-degrading enzyme, characterized in that: Only the 113th glutamine of the wild-type ochratoxin A degrading enzyme is mutated to proline, the 210th serine is mutated to methionine, and the 235th alanine 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 the mutant E1 whose amino acid sequence is shown in SEQ ID NO.3, or the mutant is the mutant E2-2 whose amino acid sequence is shown in SEQ ID NO.

7.

4. A gene encoding the 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 a gene encoding 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 and inducing expression to obtain 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 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: It comprises the ochratoxin A degrading enzyme mutant according to claim 3.

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