Ochratoxin-degrading enzyme bnotase4, gene thereof and application thereof

Abstract

The present application belongs to the field of agricultural biotechnology, and relates to ochratoxin A (OTA) and ochratoxin B (OTB) degradation enzyme BnOTase4, a coding gene thereof and application. The present application provides degradation enzyme BnOTase4, the amino acid sequence of which is shown as SEQ ID NO. 1; provides a gene coding the above degradation enzyme, the base sequence of which is shown as SEQ ID NO. 2; and also provides application of the above degradation enzyme in degrading OTA and OTB. The BnOTase4 degradation enzyme has OTA and OTB degradation activity, and the degradation rate reaches 100%. In a degradation system at 37℃ and pH 7.4, the Michaelis constant K m is 1.09 and 0.64 μmol / L, respectively, when OTA and OTB are used as substrates. The degradation enzyme BnOTase4 of the present application can be applied in the fields of food, feed and agriculture, and reduces the health hazards of ochratoxin to humans and animals.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a degrading enzyme BnOTase4 that degrades ochratoxin A and ochratoxin B, its encoding gene, and its applications. Background Technology

[0002] Ochratoxins are fungal toxins produced by molds such as *Aspergillus* spp. and *Penicillium* spp., and possess nephrotoxicity, hepatotoxicity, immunotoxicity, and carcinogenicity. Agricultural products such as grains, legumes, grapes, coffee, spices, nuts, oilseeds, and dairy products are susceptible to ochratoxin contamination. Currently, more than 20 types of ochratoxins have been identified, among which ochratoxin A (OTA) and ochratoxin B (OTB) have a wide range of contamination, high toxicity, and high contamination rates. Co-contamination with OTA and OTB has been found in various products (such as red wine, wheat, and dried fruit). The key to ochratoxin contamination control lies in simultaneously removing multiple ochratoxins from food and feed.

[0003] Currently, mycotoxin detoxification includes three methods: physical, chemical, and biological detoxification. Physical detoxification uses adsorbents or irradiation degradation, but its specificity is low, easily affecting the sensory qualities and quality of food. Chemical detoxification uses acidic compounds, alkaline compounds, salts, and oxidants to destroy the toxic groups of the toxin or change its solubility, then removes the detoxifying compounds through various processes. This method has problems such as reagent residues and damage to nutrients and flavor. Biological detoxification uses microbial preparations and enzymes to adsorb and decompose mycotoxins, offering advantages such as high specificity, high removal efficiency, environmental friendliness, and no damage to nutrients. Currently, abundant microbial resources have been found to possess OTA-degrading activity, but only a few OTA-degrading enzymes have been reported, including carboxypeptidase from Bacillus amyloliquefaciens (Chang X et al., Food Addit Contam Part A-Chem, 2015, 32:564-71), amidase from Oligotrophozoites (Zhou Yu et al., Patent Application No. 201910004511.3), Alcaligenes faecalis (Zhang H et al., Toxins, 2019, 11:518), and Aspergillus niger (Dobritzsch D et al., Biochem J, 2014, 462:441-52). Enzymes that simultaneously degrade OTA and OTB are even rarer. Extensive exploration of microbial ochratoxin-degrading enzymes and their genes can enrich detoxification research in the field of ochratoxin pollution control and is the foundation for the protein engineering modification and efficient expression application of detoxification enzymes. Summary of the Invention

[0004] To address the technical problems existing in the prior art, the present invention aims to provide a degradative enzyme with the function of degrading ochratoxin A (OTA) and ochratoxin B (OTB), its encoding gene, and its applications.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The present invention provides BnOTase4, an ochratoxin A and ochratoxin B degrading enzyme from the strain Brevundimonas naejangsanensis ML17, having an amino acid sequence as shown in SEQ ID NO.1; or having at least 90% sequence identity, preferably 95%, with the amino acid sequence shown in SEQ ID NO.1, and having the same or similar function as the amino acid sequence shown in SEQ ID NO.1.

[0007] The present invention provides a gene encoding BnOTase4, an enzyme that degrades ochratoxin A and ochratoxin B, wherein the gene has a nucleotide sequence as shown in SEQ ID NO.2; or a nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO.2, but is different from the sequence of SEQ ID NO.2 due to the degeneracy of the genetic code; or a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO.2, preferably 95% sequence identity.

[0008] This invention provides a recombinant expression vector, characterized in that it carries the encoding gene for the degradation enzyme BnOTase4. The vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus; the preferred recombinant expression vector is pET-28a(+).

[0009] The present invention provides a host for expressing the gene encoding the degradation enzyme BnOTase4, characterized in that the host is a bacterial, fungal, plant, insect or animal cell, and the preferred host is Escherichia coli BL21(DE3) strain.

[0010] This invention provides an enzyme preparation product containing the degrading enzyme BnOTase4.

[0011] This invention provides the application of the degrading enzyme BnOTase4 in the degradation of ochratoxin A and ochratoxin B, the application including the degradation of ochratoxin A and ochratoxin B in food, feed or agricultural products.

[0012] The beneficial effects of this invention are as follows: the degrading enzyme BnOTase4 can degrade ochratoxin A and ochratoxin B with a degradation rate of 100%. In a degradation system at 37°C and pH 7.4, the Michaelis constant K of the degrading enzyme BnOTase4 was determined using OTA and OTB as substrates, respectively. m The concentrations were 1.09 and 0.64 μmol / L. The degrading enzyme BnOTase4 of this invention is a novel ochratoxin degrading enzyme. The amino acid sequence similarity of this enzyme with currently discovered OTA degrading enzymes is less than 25%, for example, 17.3% similarity with the amino acid sequence of the Aspergillus niger OTA degrading enzyme reported by Dobritzsch et al. (2014); 24.4% similarity with the amino acid sequence of the Oligotrophozoites OTA degrading enzyme reported by Zhou Yu et al. (patent application number 201910004511.3); and 11.4% similarity with the amino acid sequence of the Alcaligenes faecalis OTA degrading enzyme reported by Zhang et al. (2019). The ochratoxin A and ochratoxin B degrading enzyme BnOTase4 can be applied in food, feed, and agriculture to reduce the health hazards of ochratoxins to humans and animals. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0014] Figure 1 Example 1 shows the double digestion verification of the recombinant enzyme BnOTase4 expression vector;

[0015] Figure 2 Example 2 shows the SDS-PAGE electrophoresis and Western blot analysis of the BnOTase4-induced expression product.

[0016] Figure 3 SDS-PAGE electrophoresis image of the nickel column purified fraction of recombinant enzyme BnOTase4 in Example 3;

[0017] Figure 4 Example 4 shows the HPLC chromatograms of the degradation of OTA and OTB by the recombinant enzyme BnOTase4;

[0018] Figure 5 Example 5 shows the Lineweaver-Burk plot equation for the determination of recombinant enzyme BnOTase4 using OTA and OTB as substrates. Detailed Implementation

[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] Example 1: Construction of recombinant vector and recombinant strain for the degrading enzyme BnOTase4

[0022] Extracellular enzyme solutions from the shortwave monoclonal bacterium *Brevundimonas naejangsanensis* ML17 strain were purified using a protein separation and purification system. The OTA-degrading active components were identified by mass spectrometry and compared with the whole genome sequence of ML17 strain, yielding the OTA-degrading enzyme BnOTase4, which belongs to the amide hydrolase family. The amino acid sequence of this enzyme is shown in SEQ ID NO.1, and the encoding gene sequence is shown in SEQ ID NO.2. The physicochemical parameters of BnOTase4 were predicted using ProtParam software (http: / / web.expasy.org / protparam / ). The enzyme has 447 amino acid residues, a theoretical molecular weight of 45685.08 Da, and a theoretical isoelectric point pI = 7.04. The signal peptide of BnOTase4 degradative enzyme was predicted using the SignalP 5.0 Server online software (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0). The signal peptide of this enzyme is amino acids 1-22, and the mature peptide is amino acids 23-447.

[0023] Using pET-28a(+) plasmid as a vector and *Escherichia coli* BL21(DE3) strain as the host, the mature peptide gene of the BnOTase4 degrading enzyme was expressed. The nucleotide sequence of the target gene was optimized based on the codon bias of *E. coli*, and a 6×His tag was added to the N-terminus for recombinase purification. This tag was inserted between the BamHI and NotI restriction sites of the expression plasmid. After plasmid construction, it was transformed into *E. coli* DH5α strain for amplification and preservation. The recombinant plasmid was extracted from *E. coli* DH5α strain and transformed into competent *E. coli* BL21(DE3) strain. Positive clones were screened using LB agar plates containing kanamycin. Positive clones were cultured in LB liquid medium (containing kanamycin) to the logarithmic growth phase, and the cells were collected by centrifugation. The plasmid was extracted and digested with BamHI and NotI. The digestion products were verified by 1% agarose gel electrophoresis (see Appendix). Figure 1 ).

[0024] Example 2: Induced expression of recombinase BnOTase4

[0025] Single colonies of recombinant BL21(DE3) bacteria were picked from LB agar plates containing 10 μg / mL kanamycin and inoculated into 50 mL of LB liquid medium (containing 10 μg / mL kanamycin) and cultured at 200 rpm (37°C) for 8 h (logarithmic growth phase). The BL21(DE3) recombinant strain cultured to the logarithmic growth phase was then inoculated at 1% volume into 200 mL of LB liquid medium (containing 10 μg / mL kanamycin) and cultured at 200 rpm (37°C) until the OD reached 0.4-0.5 (early logarithmic growth phase, approximately 3-4 h). 0.2 mmol / L IPTG inducer was added, and the culture was incubated at 160 rpm (20°C) for 20 h. The induced recombinant BL21(DE3) bacterial culture was centrifuged at 8500g for 5 min (4°C) to collect the bacterial cells. The bacterial cells were reconstituted in 20 mL of PBS (pH 7.4) buffer. Lysozyme (final concentration 0.1 mg / mL) was added and the mixture was treated for 30 min (37℃) to disrupt the cell wall and increase bacterial permeability. A 50 mL centrifuge tube containing 20 mL of bacterial suspension was placed on ice and sonicated to disrupt the bacterial cells. The sonication conditions were: 200 W power, 2 s intervals (to prevent overheating), and a total working time of 30 min. The sonication probe was always kept below the liquid surface to prevent foaming. Sonication continued until the solution became clear. After sonication, the suspension was centrifuged at 12,000 rpm for 5 min (4℃). The supernatant contained the intracellular soluble expression component, and the insoluble matter contained the inclusion body expression component. SDS-PAGE electrophoresis and Western blot analysis were used to analyze the expression level of the target gene. Target bands were observed in both intracellular soluble and inclusion body components near 45 kDa (see Appendix). Figure 2 ).

[0026] Example 3: Nickel column purification of recombinant enzyme BnOTase4

[0027] The recombinase BnOTase4 was purified using a nickel affinity chromatography column. The recombinase contains six His tags at its N-terminus and is loaded with Ni. 2+ The affinity chromatography column selectively adsorbs histidine residues on the surface of proteins; recombinant proteins containing the His tag are adsorbed onto Ni-containing... 2+ The recombinase was eluted onto an affinity chromatography column using a high concentration of imidazole elution buffer.

[0028] (1) Sample preparation: After centrifuging the protein sample at 12000 rpm for 5 min (4℃) to remove the precipitate, add imidazole to make the final concentration 20 mmol / L. Mix the protein sample with the nickel packing material and stir, then incubate for about half an hour before loading the sample (this process is to allow the nickel packing material to fully bind with the target protein). The ratio of nickel packing material to protein sample should not exceed 2 / 3 of the maximum nickel loading.

[0029] (2) Column packing: After thoroughly stirring the mixture of nickel packing material and protein solution, pour it into the chromatography column.

[0030] (3) Washing and equilibration: Pour the equilibration buffer into the column containing the nickel packing and protein mixture, and wash thoroughly until the baseline signal value of the detector (A280nm) is close to zero.

[0031] (4) Washing: Use washing buffer to wash off the impurities at a flow rate of 3 mL / min. Collect 6 mL of the eluent in each tube and continue to collect the eluent until the detector signal value is close to zero.

[0032] (5) Elution: The target protein was eluted with elution buffer at a flow rate of 3 mL / min. 6 mL of elution buffer was collected from each tube and collected continuously until the detector signal value at 280 nm approached zero.

[0033] (6) SDS-PAGE analysis was performed on the collected liquids in each tube.

[0034] Note: Equilibration buffer: 20 mmol / L NaH2PO4·2H2O, 0.3 mol / L NaCl, 20 mmol / L imidazole, pH 7.4; Wash buffer: 20 mmol / L NaH2PO4·2H2O, 0.3 mol / L NaCl, 100 mmol / L imidazole, pH 7.4; Elution buffer: 20 mmol / L NaH2PO4·2H2O, 0.3 mol / L NaCl, 300 mmol / L imidazole, pH 7.4.

[0035] After washing with 100 mmol / L imidazole, the recombinant enzyme BnOTase4 was further eluted with 300 mmol / L imidazole to obtain a relatively pure target protein with a molecular weight close to 45 kDa (see Appendix). Figure 3 ).

[0036] Example 4: OTA and OTB degradation activity of recombinant enzyme BnOTase4

[0037] 300 μL of the induced expression product of recombinant enzyme BnOTase4 was reacted with OTA or OTB (final concentration 1 μg / mL) for 12 h. After the reaction, 10 μL of 6M hydrochloric acid was added to acidify (pH approximately 2-3), and then 300 μL of CHCl3 was added to extract OTA, OTB, and their degradation products from the reaction system. The CHCl3 extraction was performed twice, and the CHCl3 extracts were combined, dried, dissolved in 300 μL of methanol, and filtered through a 0.22 μm organic microporous membrane for later use. The control group (CK) consisted of PBS buffer with the same amount of OTA or OTB as the experimental group. OTα produced by the degradation of OTA by commercial carboxypeptidase A (CPA) and OTβ produced by the degradation of OTB were used as controls for the degradation products of OTA and OTB, respectively.

[0038] HPLC detection parameters: Column: C18 Spherisorb S5 ODS2 column (Waters Corporation, USA), 150 × 4.6 nm, particle size 3.5 μm. Mobile phase: water (containing 0.1% formic acid, phase A) and acetonitrile (containing 0.1% formic acid, phase B). Elution mode: isogradient; total flow rate: 1 mL / min (0.3 mL / min for phase A, 0.7 mL / min for phase B); sample loading volume: 10 μL. Fluorescence detector: excitation wavelength 333 nm, emission wavelength 460 nm. Shimadzu LabSolutions software was used to analyze OTA and OTB detection results. OTA / OTB degradation rate calculation formula: Degradation rate (%) = (1 - sample peak area / control peak area) × 100%.

[0039] The retention times (RT) of OTA and OTα were 4.27 and 3.427 min, respectively. BnOTase4 completely degraded OTA, generating a product with an RT of 3.402, presumably OTα. The retention times (RT) of OTB and OTβ were 3.443 and 2.899 min, respectively. BnOTase4 completely degraded OTB, generating a product with an RT of 2.9012, presumably OTβ (see Appendix). Figure 4 ).

[0040] Example 5: Michaelis constant K of recombinant enzyme BnOTase4 m value

[0041] The reaction rate of recombinant enzyme BnOTase4 was determined using OTA and OTB as substrates. BnOTase4 was dissolved in PBS buffer at pH 7.4 to prepare 300 μL of OTA or OTB enzymatic reaction systems at concentrations of 62.5, 125, 250, 500, and 1000 ng / mL. The reactions were carried out at 37°C for 12 h. After the reaction, the amount of residual OTA or OTB in the system was determined by HPLC. K was calculated using the double reciprocal plot method (i.e., the Lineweaver-Burk plot equation). m and V max The calculation formula is as follows:

[0042]

[0043] The Lineweaver-Burk graph equation for recombinant enzyme BnOTase4 with OTA as a substrate is y =

[0044] 374.09x + 0.8499, Michaelis constant K m The value is 1.09 μmol / L, and the maximum reaction rate V max The concentration was 1.18 ng / min; the equation with OTB as the substrate was y = 222.77x + 0.9414, and the Michaelis constant K was... m The value is 0.64 μmol / L, and the maximum rate V max It is 1.06 ng / min (see appendix) Figure 5 ).

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> China Agricultural University <120> Ochratoxin-degrading enzyme BnOTase4, its gene, and its applications <130> MP21036509Z <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 447 <212> PRT <213> Brevundimonas naejangsanensis ML17 <400> 1 Met Arg Ile Lys Thr Leu Leu Ala Gly Ala Val Ala Ala Phe Ala Leu 1 5 10 15 Thr Gly Pro Ala Leu Ala Gln Asp Val Ala Ile Thr Gly Gly Gln Val 20 25 30 Leu Thr Gly Thr Ser Val Ile Glu Asn Gly Thr Val Val Ile Arg Asn 35 40 45 Gly Lys Val Val Ser Val Gly Thr Gly Ala Ala Pro Ala Gly Leu Arg 50 55 60 Val Ile Asp Ala Arg Gly Lys Ile Val Thr Pro Gly Phe Val Ala Val 65 70 75 80 Asp Ser Gly Leu Gly Gly Thr Glu Val Gly Ser Val Arg Gly Thr Asn 85 90 95 Asp Leu Ala Asn Arg Ala Asn Thr Leu Thr Ala Ala Phe Asp Leu Ser 100 105 110 Tyr Gly Leu Asp Pro Trp Ser Phe Thr Leu Pro Val Ala Arg Leu Gly 115 120 125 Gly Val Thr Arg Ala Val Val Thr Pro Gln His Gly Gly Ser Gly Gly 130 135 140 Gly His Ser His Asp Asp Ser Asp Phe Ala Gly Ala Gly Gln Gly Gly 145 150 155 160 Phe Gln Thr Pro Gly Leu Phe Ala Gly Gln Ala Ala Val Ile Lys Leu 165 170 175 Gly Gly Ala Asp Ile Leu Val Lys Pro Arg Val Ala Met Val Ala Pro 180 185 190 Phe Gly Glu Ala Gly Ala Ala Val Ala Gly Gly Ala Arg Gly Ala Glu 195 200 205 Phe Val Leu Phe Lys Glu Thr Leu Ala Glu Val Arg Ala Tyr Ala Arg 210 215 220 Asn Lys Ala Ala Tyr Glu Arg Ala Asp Met Arg Ala Leu Ser Leu Ser 225 230 235 240 Arg Ala Asp Met Glu Ala Leu Ile Pro Val Ala Asn Gly Ala Met Pro 245 250 255 Leu Ile Val Thr Val Asn Arg Ala Ser Asp Ile Gln Gln Val Leu Arg 260 265 270 Leu Ala Arg Glu Glu Gly Val Lys Val Ile Leu Asp Gly Ala Ala Glu 275 280 285 Gly Trp Leu Val Ala Asp Glu Ile Ala Ala Ala Lys Val Pro Val Ile 290 295 300 Leu His Pro Thr Thr Asn Leu Pro Ser Asn Phe Glu Met Arg Ala Ala 305 310 315 320 Arg Met Gln Asn Ala Ala Ala Leu Asn Ala Ala Gly Val Val Ile Ala 325 330 335 Ile Lys Gly Asn Glu Gly Ser Ala His Arg Ala Arg Asp Ile Arg Tyr 340 345 350 Asn Ala Gly Asn Ala Val Ser His Gly Leu Pro Phe Ala Ala Ala Ile 355 360 365 Gln Ala Ile Thr Val Asn Pro Ala Arg Ile Phe Gly Phe Asp Gly Gln 370 375 380 Phe Gly Glu Leu Lys Ala Gly Ala Ala Gly Asp Val Val Val Trp Ser 385 390 395 400 Gly Asp Pro Leu Glu Pro Leu Ser Gln Pro Ser Ala Val Leu Ile Asp 405 410 415 Gly Val Glu Gln Pro Leu Gln Ala Arg Asn Leu Leu Leu Arg Asp Arg 420 425 430 Tyr Arg Thr Gly Gly Glu Gly Ala Met Pro Pro Ala Tyr Gly Asn 435 440 445 <210> 2 <211> 1344 <212> DNA <213> Brevundimonas naejangsanensis ML17 <400> 2 atgcgtatca aaactctgct tgcgggcgca gttgcagcct tcgcactgac cggccccgcc 60 ctggcccagg acgtcgccat caccggcggc caggtgctga ccggaacctc ggtgatcgag 120 aacggcaccg tcgtcatccg taacggcaag gtcgtttcgg tcggaaccgg cgcggccccg 180 gccggcctgc gcgtcatcga cgcgcgcggc aagatcgtca cgccgggctt cgtcgccgta 240 gattccggcc ttggcggcac cgaggtcggc tcggtgcgcg ggaccaacga cctggccaac 300 cgcgccaaca ccctgacggc ggccttcgac ctgtcctatg gcctggaccc ctggtccttc 360 accctgccgg tggcccgtct gggcggcgtc acccgcgccg tcgtcacgcc ccagcacggc 420 ggctcgggcg gcggccacag ccacgacgac agcgacttcg ccggcgccgg tcagggcggc 480 ttccagacgc cgggcctgtt cgcgggccag gcggcggtca tcaagctggg cggcgccgac 540 atcctggtga agccgcgcgt ggccatggtc gcgcccttcg gcgaggccgg ggcggcggtg 600 gcgggcggcg cccgcggcgc cgagttcgtc ctgttcaagg agaccctggc cgaggtacgg 660 gcctacgccc ggaacaaggc ggcctatgag cgggccgaca tgcgcgctct gtcgctgtct 720 cgcgccgata tggaggccct gatcccggtg gcgaacggcg ccatgccgct gatcgtcacc 780 gtcaaccgcg cctcggacat ccagcaggtg ctgcgtctgg cgcgcgagga aggcgtcaag 840 gtcattctgg acggcgccgc cgaaggctgg ctggtggccg atgagatcgc ggcggccaag 900 gttccggtca tcctgcaccc gaccaccaac ctgccctcga acttcgagat gcgggcggcg 960 cggatgcaga atgcggcggc cttgaacgcg gcgggcgtgg tcatcgccat caagggcaac 1020 gagggttcgg cccaccgcgc tcgcgacatc cgctacaacg cgggcaacgc cgtgtcgcac 1080 ggcctgccgt tcgcggcggc catccaggcg atcacggtca acccggcgcg catcttcggc 1140 ttcgacggcc agttcggcga gctgaaggcc ggagcggcgg gcgacgtcgt ggtctggtcc 1200 ggcgatccgc tggagccgct gagccagccc tcggccgtgc tgatcgacgg tgtggagcag 1260 ccgctgcagg cgcgcaacct gctgctgcgc gaccgttacc gcaccggcgg cgaaggggcg 1320 atgccgcccg cctacggcaa ctga 1344

Claims

1. A BnOTase4 enzyme, which degrades ochratoxin A and ochratoxin B, characterized in that, This degrading enzyme is a protein composed of the amino acid sequence shown in SEQ ID NO.1, and is capable of degrading ochratoxin A and ochratoxin B.

2. A gene encoding BnOTase4, an ochratoxin A and ochratoxin B degrading enzyme, characterized in that, The encoding gene encodes the BnOTase4 degrading enzyme of claim 1, which has the activity of degrading ochratoxin A and ochratoxin B.

3. The gene encoding BnOTase4, the ochratoxin A and ochratoxin B degrading enzyme, according to claim 2, is characterized in that... The gene consists of a nucleotide sequence as shown in SEQ ID NO.2 and is capable of encoding a protein with activity of degrading ochratoxin A and ochratoxin B; or a nucleotide sequence with the same amino acid sequence as SEQ ID NO.2, but different from the sequence of SEQ ID NO.2 due to the degeneracy of the genetic code, and is capable of encoding a protein with activity of degrading ochratoxin A and ochratoxin B.

4. A recombinant expression vector, characterized in that, Carrying the nucleotide sequence of the gene described in claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or mammalian cell viruses.

6. A recombinant bacterium or recombinant cell, characterized in that, The recombinant bacteria or recombinant cells express the ochratoxin A and ochratoxin B degrading enzyme BnOTase4 as described in claim 1.

7. The recombinant bacteria or recombinant cells according to claim 6, characterized in that, The hosts for recombinant bacteria are selected from bacteria and fungi; the hosts for recombinant cells are selected from plant and animal cells.

8. An enzyme preparation, characterized in that, The enzyme preparation comprises the ochratoxin A and ochratoxin B degrading enzyme BnOTase4 as described in claim 1.

9. The use of the degrading enzyme BnOTase4 according to claim 1 in the degradation of ochratoxin A and ochratoxin B.

10. The application according to claim 9, characterized in that, The applications include the degradation of ochratoxin A and ochratoxin B in food, feed, or agricultural products.

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