Cloning, expression and application of a vomitoxin detoxifying enzyme gene

Abstract

The present application relates to the cloning, expression and application of a vomitoxin detoxifying enzyme gene, and belongs to the technical field of biology. The present application discloses vomitoxin detoxifying enzyme genes dadh and akr13b3 from Devosia strain, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 3 respectively, and the genes encode vomitoxin (DON) detoxifying enzymes DADH and AKR13B3. In the genes, DADH oxidizes DON to form low-toxic 3-keto-DON, and AKR13B3 reduces 3-keto-DON to form non-toxic product 3-epi-DON, which proves that DADH and AKR13B3 have complete detoxifying ability for DON, can effectively solve the problem of vomitoxin pollution in grain raw materials and feed, and have potential industrial application prospect.

Description

1. Technical Field

[0001] The present invention relates to the cloning, expression and application of a vomitoxin detoxification enzyme gene, and belongs to the field of biotechnology. 2. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin (VT), is a toxic secondary metabolite produced by fungi such as Fusarium graminearum and Fusarium graminearum infecting grains. It is widely present in grain raw materials such as wheat and corn and their products, causing huge economic losses to the grain and animal husbandry industries, and causing serious harm to the health of humans and animals. Therefore, how to effectively control and solve DON contamination in grain and feed has become a research hotspot in the field of food processing and food safety at home and abroad.

[0003] In recent years, the method of mycotoxin biodegradation has attracted much attention. Mycotoxin biodegradation refers to the process of destroying the toxic groups in the molecular structure of the toxin by microorganisms and their metabolically produced enzymes, making it a non-toxic or low-toxic metabolite. Compared with traditional physical and chemical detoxification methods, enzyme degradation has the advantages of high detoxification efficiency, strong specificity, no pollution to food, feed and environment, etc., and thus has shown great application value in the field of food processing. Current research on DON biodegradation mainly includes microbial degradation and enzymatic detoxification. Existing studies have shown that the epoxy group at C12,13 and the hydroxyl group at C3 in the DON molecule are the main toxic groups of DON and the main research sites for biodegradation. Microorganisms can reduce the epoxy group to a C9,12 diene structure to form a highly toxic de-epoxy product, DOM-1 (Zhai et al., Front Microbial., 2019, 10, 1-12). However, the degradation mechanism of the microbial de-epoxidation pathway of DON has not been fully elucidated, and there has been no report on any DON detoxification enzyme. In addition to the epoxy group, the C3 hydroxyl group biotransformation includes oxidation, acetylation, glycosylation, and isomerization (Michlmayr et al., Toxins., 2015, 7, 2685-2700; Tian et al., Toxins., 2016, 8, 1-15; Zhai et al., Front Microbial., 2019, 10, 1-12). Among them, the pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase DepA / QDDH from Devosia sp. can oxidize the C3 hydroxyl group of DON to generate low-toxic 3-keto-deoxynivalenol (3-keto-DON), which is then reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent aldehyde-ketone reductase DepB / AKR13B2 / AKR6D1 to generate non-toxic 3-epi-deoxynivalenol (3-epi-DON), i.e., isomerization at C3 (Carere et al., Microbbiotechnol., 2018, 11, 1106-1111; Carere et al., Front Microbial., 2018, 9, 1-9; He et al., Food Chem., 2020, 321, 126703), which is the most detailed DON degradation mechanism described so far, with high specificity and single-direction irreversibility.

[0004] Current research on DON biodegradation is just getting started, and there is little research on DON detoxification enzymes and recombinant enzymes. Therefore, it is of great practical significance to explore new and high-performance DON detoxification enzymes. 3. Summary of the Invention

[0005] Technical issues

[0006] The purpose of the present invention is to use genetic engineering technology to explore a new, high-performance DON detoxification enzyme gene and DON detoxification enzyme, which can be used in degrading vomitoxin.

[0007] Technical Solution

[0008] A detoxification enzyme gene for vomitoxin (DON), characterized by comprising the detoxification enzyme genes dadh and akr13b3, whose nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.3, respectively.

[0009] The vomitoxin detoxification enzyme encoded by the vomitoxin detoxification enzyme gene is characterized in that the vomitoxin detoxification enzymes DADH and AKR13B3 encoded by the vomitoxin detoxification enzyme genes dadh and akr13b3 have amino acid sequences as shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0010] The method for preparing the vomitoxin detoxification enzyme is characterized by comprising the following steps:

[0011] (1) PCR amplification of the DON detoxification enzyme genes dadh and akr13b3;

[0012] (2) Construction of prokaryotic expression vectors for the detoxification enzyme genes dadh and akr13b3;

[0013] (3) The detoxifying enzymes DADH and AKR13B3 of vomitoxin were expressed in prokaryotic host cells.

[0014] Among them, the prokaryotic expression vector used in step (2) refers to one or more of an Escherichia coli expression vector, a Bacillus subtilis expression vector, a Bacillus licheniformis expression vector, a Bacillus megaterium expression vector, a Bacillus brevis expression vector, a lactic acid bacteria expression vector, a yeast expression vector, a Streptomyces expression vector and a filamentous fungus expression vector.

[0015] The prokaryotic host cell used in step (3) refers to an Escherichia coli host cell, a Bacillus subtilis host cell, a Bacillus licheniformis host cell, a Bacillus megaterium host cell, a Bacillus brevis host cell, a lactic acid bacteria host cell, a yeast host cell, a Streptomyces host cell or a filamentous fungus host cell.

[0016] The detoxifying enzyme for vomitoxin can be used in the biodegradation of vomitoxin.

[0017] Beneficial effects

[0018] The present invention isolated a Devosia strain A6-243 from soil in Taiyuan, Shanxi Province, and classified it as Devosia strain. It has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC NO: 21800.

[0019] 1. The present invention discovered the novel DON detoxification enzyme genes dadh and akr13b3 from Devosia strain A6-243, achieved their heterologous expression in prokaryotic cells, and constructed a genetically engineered strain that efficiently expresses DADH and AKR13B3.

[0020] 2. This study identified pure novel DON-degrading detoxification enzymes, DADH and AKR13B3, from the complete genome of Devosia sp. A6-243 strain isolated from soil, which exhibits DON-degrading activity. 10-20 μg of DADH and AKR13B3 can completely degrade 45-150 μg / mL of DON into the non-toxic product 3-epi-DON within 2-18 hours. Sequence comparison revealed that DADH shared 55.94% sequence similarity with previously reported dehydrogenases in the NCBI database, while AKR13B3 shared only 36.47% sequence identity with previously reported aldehyde-keto reductases, demonstrating its novel DON detoxification enzyme.

[0021] 3. DADH and AKR13B3 of the present invention exhibit excellent thermal stability. After treatment at 50°C for 3 hours, both DADH and AKR13B3 retained over 55% of their activity. After treatment at pH 6-10 for 12 hours, DADH retained over 60% of its activity, while AKR13B3 retained over 80% of its activity after treatment at pH 6-7.5 for 12 hours. Therefore, DADH and AKR13B3 have potential for industrial application.

[0022] 4. The present invention's DADH can oxidize DON to the low-toxic 3-keto-DON, while AKR13B3 can reduce 3-keto-DON to the non-toxic product 3-epi-DON. Furthermore, the combined reaction of DADH and AKR13B3 can degrade 45-150 μg / mL of DON into the non-toxic product 3-epi-DON at a 100% degradation rate. This effectively addresses the issue of vomitoxin contamination in grain raw materials and feed, reduces grain losses, and ensures food and feed safety. It also has potential for industrial application. IV. Description of the Figures

[0023] Figure 1This is the agarose gel electrophoresis pattern of the DON detoxification enzyme gene fragment from Devosia A6-243.

[0024] (A) M: DNA Marker DL2000; 1: DON detoxification enzyme gene fragment dadh from Devosulia A6-243. (B) M: DNA Marker DL2000; 1: DON detoxification enzyme gene fragment akr13b3 from Devosulia A6-243.

[0025] Figure 2 Schematic diagram of electrophoresis for purified DADH and AKR13B3 proteins.

[0026] (A) M: protein marker 26610; 1: E. coli fragment containing the empty plasmid pET28a; 2: crude DADH enzyme sample; 3: purified DADH sample. (B) M: protein marker 26610; 1: E. coli fragment containing the empty plasmid pET28a; 2: crude AKR13B3 enzyme sample; 3: purified AKR13B3 sample.

[0027] Figure 3 To study the enzymatic properties of DADH.

[0028] (A) Optimal reaction temperature of DADH; (B) Temperature stability of DADH; (C) Optimal reaction pH of DADH; (D) pH stability of DADH

[0029] Figure 4 To study the enzymatic properties of AKR13B3.

[0030] (A) Optimal reaction temperature of AKR13B3; (B) Temperature stability of AKR13B3; (C) Optimal reaction pH of AKR13B3; (D) pH stability of AKR13B3

[0031] Figure 5 The detoxification ability of DADH and AKR13B3 on DON.

[0032] (A) Detoxification ability of DADH for DON; (B) Detoxification ability of AKR13B3 for 3-keto-DON

[0033] Figure 6 The detoxification effect of the combination of DADH and AKR13B3 on DON. V. Specific Implementation Methods

[0034] The present application isolates a strain of Devosia A6-243 from the soil in Taiyuan, Shanxi, which is classified and named as Devosia strain, has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yuan, Beijing, Institute of Microbiology of the Chinese Academy of Sciences, the strain preservation number is CGMCC NO: 21800. The preservation date is February 1, 2021.

[0035] Example 1: Cloning of Devosia strain A6-243 DON detoxification enzyme gene

[0036] (1) Extraction of the whole genome of Devosia strain A6-243: centrifugal collection of Devosia strain A6-243 bacterial cells, using the Bacterial DNA kit of OMGA company to extract the whole genome of bacteria.

[0037] (2) Primer design: according to the whole genome sequencing results of Devosia strain A6-243, through comparative genomics and transcriptomics, the DON detoxification enzyme gene is determined, and the PCR upstream and downstream primers of DON detoxification enzyme gene DADH-F, DADH-R, AKR13B3-F, AKR13B3-R, and the linearization primers V-F and V-R of the vector are designed:

[0038] V-F: GATCCGAATTCGAGCTCCG;

[0039] V-R: CATGGTATATCTCCTTCTTAAAGTTAAACA;

[0040] DADH-F: TAAGAAGGAGATATACCATGCAGGTCGATATCAGTGCGTTGC;

[0041] DADH-R: ACGGAGCTCGAATTCGGATCCTTGGCAGCGGCCTCAGG;

[0042] AKR13B3-F: GCCTGGTGCCGCGCGGCAGCATGACCAAGCTCGACGCATC;

[0043] AKR13B3-R: ACGGAGCTCGAATTCGGATCGGCGCGACCAATGGCATC.

[0044] (3) Cloning of the target enzyme gene: The whole genome of Devosia A6-243 was used as a template, and the primers designed above were used for PCR amplification of the target fragment. The PCR amplification system was as follows: 2× Taq Master Mix 25 μL, DNA template 1 μL, ddH2O 20 μL, and 2 μL of upstream and downstream primers respectively; PCR amplification conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, for 30 cycles; and extension at 72°C for 10 min.

[0045] The vector linearization PCR amplification system in step (1) was as follows: 2× Taq Master Mix 25 μL, DNA template 1 μL, ddH2O 20 μL, and 2 μL of upstream and downstream primers; PCR amplification conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 4 min, for 30 cycles; and extension at 72°C for 10 min.

[0046] The gel recovery kit was used for gel recovery and purification. The purified product was connected with the linearized pET28a vector and transformed into competent cells E. coli BL21 by heat shock method. The product was spread on kanamycin resistance plates and positive transformants were picked for bacterial growth verification (such as Figure 1 The DNA was sent to GENEWIZ for sequencing verification.

[0047] According to the sequencing results, the DON detoxification enzyme dadh is 1764bp in length (as shown in SEQ ID NO.1), and the akr13b3 is 987bp in length (as shown in SEQ ID NO.3). That is, the DON detoxification enzyme DADH of Devospasella A6-243 encodes a protein composed of 587 amino acids (as shown in SEQ ID NO.2), and the DON detoxification enzyme AKR13B3 of Devospasella A6-243 encodes a protein composed of 328 amino acids (as shown in SEQ ID NO.4).

[0048] Example 2: Expression and purification of DON detoxification enzyme in Escherichia coli

[0049] (1) Expression vector construction: The target fragments of dadh and akr13b3 amplified by PCR were ligated with the linearized vector pET28a by homologous methods, and then transformed into competent cells E. coli BL21 and plated on kanamycin-resistant plates. Positive transformants were picked for bacterial growth and sequenced. The correctly sequenced plasmids were named pET28a-DADH and pET28a-AKR13B3, respectively.

[0050] (2) Expression of recombinant enzymes: The recombinant bacteria pET28a-DADH and pET28a-AKR13B3 were inoculated into LB liquid medium containing 50 μg / mL Kana and cultured overnight at 37°C, 180 rpm to prepare seed liquid. A 1% inoculum was then transferred to 100 mL of the corresponding LB liquid medium containing antibiotics and incubated at 37°C until the OD value reached between 0.6 and 0.8. 100 μl of IPTG (100 mg / mL) was added and expression was induced at 16°C overnight.

[0051] (3) Purification of recombinant enzyme: collect the cells by low-temperature centrifugation, suspend the cells in buffer and ultrasonically break them, centrifuge again and take the supernatant to obtain the crude enzyme solution. Since the target protein is fused with a histidine tag during plasmid construction, Ni 2+ -NTA affinity column purification of DON detoxification enzymes DADH and AKR13B3. The method is as follows:

[0052] Sample loading: The final concentration of the sample was adjusted to 5 mM imidazole to enhance specific adsorption and passed through the membrane; Ni 2+ The -NTA affinity column was equilibrated with 10 column volumes of 5 mM imidazole buffer; the sample was loaded and adsorbed three times in a cycle; after loading, unadsorbed proteins were eluted first with 5 mM imidazole buffer and then with 50 mM imidazole buffer;

[0053] Elution: After the impurities are eluted, the target protein is eluted with 10 times the volume of the medium 200mM imidazole buffer, and the effluent is collected to obtain the purified sample. The imidazole is removed by dialysis to obtain the purified protein. The purified protein is verified by SDS-PAGE electrophoresis. Figure 2 As shown in A, there is a specific band near 66 kDa, which is consistent with the theoretical molecular weight of DADH of 63.4 kDa; Figure 2 As shown in B, a specific band was found near 36 kDa, which was consistent with the theoretical molecular weight of AKR13B3 of 35.9 kDa.

[0054] Example 3: Study on the Enzymatic Properties of DADH and AKR13B3

[0055] (1) Optimal reaction temperature and temperature stability of DADH and AKR13B3

[0056] The enzyme activities of DADH and AKR13B3 were measured at different temperatures (20, 25, 30, 35, 40, 45, 50, 55 and 60°C) to determine the optimal reaction temperatures of DADH and AKR13B3. The results showed that the optimal reaction temperatures of DADH and AKR13B3 were 40 and 45°C, respectively ( Figure 3 A, as shown in 4A).

[0057] DADH and AKR13B3 were treated at 4°C, 20°C, 30°C, 40°C, 50°C and 60°C for 0h, 1h, 2h, 3h, 4h, 5h and 6h, respectively, and then the enzyme activity was measured, with the untreated sample being 100%. The results showed that DADH and AKR13B3 had very good thermal stability. After treatment at 50°C for 3h, both DADH and AKR13B3 could retain more than 55% of their activity (such as Figure 3 B, as shown in 4B).

[0058] (2) Optimal reaction pH and pH stability of DADH and AKR13B3

[0059] The enzyme activities of DADH and AKR13B3 were measured at different pH values ​​(4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10) to determine the optimal reaction pH for DADH and AKR13B3. The results showed that the optimal pH for both DADH and AKR13B3 was 6.5 (e.g. Figure 3 C, as shown in 4C).

[0060] DADH and AKR13B3 were treated at pH 4, 5, 6, 7, 8, 9, and 10 for 12 hours, and then the enzyme activity was measured, with the untreated condition being 100%. The results showed that DADH and AKR13B3 had very good pH stability. DADH could retain more than 60% of its activity after treatment at pH 6-10 for 12 hours, and AKR13B3 could retain more than 80% of its activity after treatment at pH 6-7.5 for 12 hours (e.g. Figure 3 D, as shown in 4D).

[0061] Example 4: Detoxification ability of DADH and AKR13B3 for DON

[0062] (1) Using DON as substrate (final concentration 150 μg / mL), 10 μg of DADH, 1 μL of 0.1 M CaCl2, 1 μL of 10 mM PQQ, and pH 7.5 Tris-HCl were added to 100 μL. The mixture was reacted at 37°C for 12 h, and then an equal volume of acidified methanol was added to terminate the reaction. The changes in DON concentration before and after the reaction were detected by high performance liquid chromatography (HPLC). The results showed that DADH could effectively convert DON into low-toxic 3-keto-DON (such as Figure 3 shown).

[0063] (2) with 3-keto-DON as substrate (final concentration 300 μM), AKR13B3 10 μg, 10 mM NADPH 1 μL, Tris-HCl pH = 7.5 to 100 μL, reaction at 37 °C for 12 h, and the change of 3-keto-DON concentration before and after reaction was detected by high performance liquid chromatography (HPLC), the results showed that AKR13B3 could completely convert 3-keto-DON into non-toxic 3-epi-DON (as shown in Figure 3

[0064] Example 5: Combined reaction of DADH and AKR13B3

[0065] with DON as substrate (final concentration 150 μg / mL), 10 μg DADH, 0.1 M CaCl2 1 μL, 10 mM PQQ 1 μL, 10 μg AKR13B3, 10 mM NADPH 5 μL, Tris-HCl pH = 7.5 to 100 μL, reaction at 37 °C for 18 h, then add the same volume of acidified methanol to terminate the reaction, and the change of DON concentration before and after reaction was detected by high performance liquid chromatography (HPLC). The results showed that DADH and AKR13B3 could completely convert DON into non-toxic 3-epi-DON (as shown in Figure 4

[0066] The Dvorinella A6-243 with DON degrading activity screened from soil has identified a new detoxification enzyme DADH and AKR13B3 with DON degrading activity from the whole genome, which can completely degrade 45-150 μg / mL of DON into non-toxic product 3-epi-DON within 18 h. By sequence alignment, it was found that the sequence similarity of DADH with the previously reported dehydrogenase in NCBI database was 55.94%, and the sequence identity of AKR13B3 with the reported aldehyde-ketone reductase was only 36.47%, which was a new type of DON detoxification enzyme.

[0067] ​​In addition, enzymatic property studies showed that the optimal reaction temperature and pH of DADH were 40°C and 6.5, respectively, which were consistent with the reported QDDH; the optimal reaction temperature and pH of AKR13B3 were 45°C and 6.5, respectively, which were consistent with the reported AKR13B2; and DADH and AKR13B3 had very good thermal stability and pH stability. After treatment at 50°C for 3 hours, both DADH and AKR13B3 could retain more than 55% of their activity; DADH could retain more than 60% of its activity after treatment at pH 6-10 for 12 hours, and AKR13B3 could retain more than 80% of its activity after treatment at pH 6-7.5 for 12 hours, indicating that DADH and AKR13B3 have potential industrial application prospects.

[0068] SEQ ID NO.1:

[0069]

[0070] SEQ ID NO.2:

[0071] MKSKISVLLASAAMLSVSSVAYAQVDISALPMVTDEILANPDAGDWPSYGRDVMNYRYSPLDQINKDNVGNLTMVWGRALEPGNLQSAPLEFGGVMFIAAPGDVVQAIDAATGQLVWEYRRTLPDRETLNSLGENKRGIALYEDKIYMVSWDNFIVALDAKTGQVAWESDRGGGADMISNTTGPIVADGVVVAGSTCQFSEFGCYVTGHDAATGEELWRNTFIPKAGEEGDDTWGDSTEDQRWMTGAWGQMTYDPVTGLVFYGSTGAGPAAEFQRNTVGGTLYGSNTRFAVKPKTGEIVWRHQVLPRDNWDQECTYEMIPVDINSNPSADMEGLLALGTATPGEKRVLTGVPCKTGVMWQFDAQTGEFIYARDTVQENLIEKVDETGLVTVNEAAIPTEVDTPTFMCPTYLGGRDWPPTAFNPETKVMFVPLTNMCANATVLDQEPTGLDVYNTELEYILPEGVTHAGRIDAINVETGKTVWSWTDQTPLYAPIVSTAGGLIFVGGTDRKFKAIDQETGEVVWSTTLPSRATGHPISYEVDGRQYIAIPAGGPGYASLFLEASGTTADTVSGSNAVYVFALPEAAAK

[0072] SEQ ID NO.3:

[0073] atgaccaagctcgacgcatccctgtcggggaggttttccattggcggtgatctcaaggtcaaccgcctcggcttcggcgccatgcgcctcaccggtgatggcatctggggtccgcccaaggatcgtgacgaagccattcgcgtgctcaagcgcctgcccgagatcggggtcgacttcatcgacaccgccgagagctatggcccctatgtcagcgaagagctgatcggggaggcgctggcgccctatgacaagggcaccatcattgccaccaagagcgggctgacccgcagcggtcccaatcaatggccgccgctggggcgtccggaattcctgcgccagggcgtcatgaccagcctgcgccggctcaagctcgagcgcctcgatctctggcaattgcaccgcatcgacgccaagacgccgcgcgccgagcagttcgaggtgattgccgcgatgcagaaagaaggcctgattcgccatgccggcctttccgaggtcagcgtcgccgacatcgaggaggccagcaaatatttcaaggtcacaacggtgcagaacctttacaacttcgccaatcgcaagagcgaagcggtgctcgactattgcgaaaagcacggcatcggtttcatcccctggttcccgctggccggcggtgatctggtggagggtcatgaaaaggcccgcgccgtcatggacaagcatggcgccagcggcagccagatcgccctggcgtggctgctcaagcgctcccccgtcatgctgcccattcccggcaccagcaaggtcaagcatctcgaggacaatgtggccgctgccgccatcgatctcagcgacgaggatttcgccgcgctcgatgccattggtcgcgcctga

[0074] SEQ ID NO.4:

[0075] MTKLDASLSGRFSIGGDLKVNRLGFGAMRLTGDGIWGPPKDRDEAIRVLKRLPEIGVDFIDTAESYGPYVSEELIGEALPYDKGTIIATKSGLTRSGPNQWPPLGRPEFLRQGVMTSLRRLKLERLDLWQLHRIDAKTPRAEQFEVIAAMQKEGLIRHAGLSEVSVADIEEASKYFKVTTVQNLYNFANRKSEAVLDYCEKHGIGFIPWFPLAGGDLVEGHEKARAVMDKHGASGSQIALAWLLKRSPVMLPIPGTSKVKHLEDNVAAAAIDLSDEDFAALDAIGRA.

Claims

1. A vomitoxin detoxification enzyme gene composition, characterized in that: including the detoxification enzyme gene for vomitoxin dadh and akr13b3, The nucleotide sequences thereof are shown in SEQ ID NO. 1 and SEQ ID NO. 3, respectively.

2. The vomitoxin detoxification enzyme composition encoded by the vomitoxin detoxification enzyme gene composition according to claim 1, characterized in that: Deoxynivalenol detoxification enzyme gene dadh and akr13b3 The amino acid sequences of the encoded vomitoxin detoxification enzymes DADH and AKR13B3 are shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

3. The method for preparing the vomitoxin detoxification enzyme composition according to claim 2, characterized in that: The following steps are involved: (1) The detoxification enzyme gene of claim 1 dadh and akr13b3 Perform PCR amplification; (2) Construction of the DON detoxification enzyme gene dadh and akr13b3 expression vector; (3) The detoxifying enzymes DADH and AKR13B3 of vomitoxin are expressed in host cells.

4. The method for preparing the vomitoxin detoxification enzyme composition according to claim 3, wherein: The expression vector used in step (2) refers to one or more of an Escherichia coli expression vector, a Bacillus subtilis expression vector, a Bacillus licheniformis expression vector, a Bacillus megaterium expression vector, a Bacillus brevis expression vector, a lactic acid bacteria expression vector, a yeast expression vector, a Streptomyces expression vector and a filamentous fungus expression vector.

5. The method for preparing the vomitoxin detoxification enzyme composition according to claim 3 or 4, characterized in that: The host cell used in step (3) refers to an Escherichia coli host cell, a Bacillus subtilis host cell, a Bacillus licheniformis host cell, a Bacillus megaterium host cell, a Bacillus brevis host cell, a lactic acid bacteria host cell, a yeast host cell, a Streptomyces host cell or a filamentous fungus host cell.

6. Use of the vomitoxin detoxification enzyme composition according to claim 2 in the biodegradation of vomitoxin.

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