A type of Bacillus amyloliquefaciens GB3-2 and its application

Abstract

This invention discloses a Bacillus amyloliquefaciens GB3-2 and its applications, belonging to the field of microbial technology. The Bacillus amyloliquefaciens GB3-2 was deposited on October 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28643. This invention discloses the application of Bacillus amyloliquefaciens GB3-2 and its produced deoxynivalenol degrading enzyme 3-2AKR in the preparation of deoxynivalenol biodegrading agents. This invention is the first to report that an enzyme produced by Bacillus amyloliquefaciens can be used to degrade deoxynivalenol. This enzyme has high catalytic efficiency and has broad application prospects in the field of biological detoxification of deoxynivalenol toxins in feed and grain raw materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a Bacillus amyloliquefaciens GB3-2 and its applications. Background Technology

[0002] Deoxynivalenol (DON) is one of the most widely distributed mycotoxins globally, primarily produced during Fusarium head blight, a disease caused by Fusarium wilt, affecting wheat and other grains including barley, corn, oats, and rye. Fusarium head blight is a serious fungal disease, causing significant yield and economic losses annually in wheat and barley during field and storage. DON exhibits various toxic effects, including acute, chronic, cytotoxic, and immunotoxic effects. It damages plant tissues, destroys grains, and acts as a virulence factor stimulating fungal colonization in plants. DON-contaminated grains enter the food chain, including humans and animals, posing a serious health threat. Utilizing plant resistance genetics is insufficient to address the harm caused by DON contamination; therefore, new technologies for efficiently eliminating DON toxicity are urgently needed.

[0003] Currently, the control of Fusarium head blight mainly relies on the use of fungicides in the field, which leads to the emergence of fungicide-resistant strains and an increase in DON biosynthesis. Traditional DON detoxification methods include physical and chemical methods, but these two methods have problems such as unstable effectiveness, incomplete detoxification, and difficulty in large-scale production, and may reduce the nutritional quality of raw food. Therefore, research on other detoxification methods is crucial.

[0004] Microbial degradation has been proven to be a highly efficient, safe, and pollution-free method for DON detoxification, representing a significant breakthrough in the field of DON degradation research. However, currently available DON-degrading microorganisms are not suitable for practical production applications. Therefore, this invention aims to identify and screen strains capable of efficiently degrading DON, particularly beneficial microorganisms that can be directly added to animal feed, and to study their probiotic properties, stress resistance, and in vivo detoxification effects, laying the foundation for the development of highly efficient, safe, and environmentally friendly DON detoxification microecological preparations. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a Bacillus amyloliquefaciens GB3-2 and its applications. Bacillus amyloliquefaciens GB3-2 can produce enzyme 3-2AKR, which has the function of degrading deoxynivalenol and can be used for detoxification of agricultural products.

[0006] To achieve the above objectives, the present invention provides a Bacillus amyloliquefaciens GB3-2, which was deposited on October 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28643.

[0007] The present invention also provides the application of the aforementioned Bacillus amyloliquefaciens GB3-2 in the preparation of deoxynivalenol biodegradable agents.

[0008] The present invention also provides a deoxynivalenol degrading enzyme 3-2AKR, which is produced by Bacillus amyloliquefaciens GB3-2, and the amino acid sequence of the deoxynivalenol degrading enzyme 3-2AKR is shown in SEQ ID NO.4.

[0009] Preferably, the nucleotide sequence of the gene encoding the deoxynivalenol degrading enzyme 3-2AKR is shown in SEQ ID NO. 3.

[0010] The present invention also provides a recombinant plasmid comprising the gene encoding the deoxynivalenol degrading enzyme 3-2AKR of Fusarium nivale and a plasmid vector.

[0011] The present invention also provides a recombinant strain, which is prepared by transforming the recombinant plasmid into a host strain.

[0012] The present invention also provides the application of the deoxynivalenol degrading enzyme 3-2AKR, the recombinant plasmid, or the recombinant strain in the preparation of deoxynivalenol biodegrading agents.

[0013] The present invention also provides the application of the deoxynivalenol degrading enzyme 3-2AKR, the recombinant plasmid, or the recombinant strain in the degradation of deoxynivalenol in agricultural products.

[0014] The present invention also provides a deoxynivalenol biodegrading agent, comprising one of the following: Bacillus amyloliquefaciens GB3-2, the deoxynivalenol degrading enzyme 3-2AKR, the recombinant plasmid, or the recombinant strain.

[0015] Preferably, the amount of the deoxynivalenol biodegradable agent used is calculated based on the quality of the agricultural product, and the amount used is 0.1-5% of the quality of the agricultural product.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] This invention relates to a Bacillus amyloliquefaciens GB3-2 and its produced deoxynivalenol degrading enzyme 3-2AKR. The deoxynivalenol degrading enzyme 3-2AKR has the function of degrading deoxynivalenol. This invention is the first to report that an enzyme produced by Bacillus amyloliquefaciens can be used to degrade deoxynivalenol. This enzyme has high catalytic efficiency and has broad application prospects in the field of biological detoxification of deoxynivalenol toxins in feed and grain raw materials. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the purified SDS-PAGE of the expression product of recombinant plasmid pET-28a(+)-AKR. Lane 1 represents the purified recombinant deoxynivalenol degrading enzyme 3-2AKR, and lane M represents the protein molecular weight standards (66.4, 44.3, 29, 20.1, 14.3 kD).

[0020] Figure 2 The degradation of DON by recombinant deoxynivalenol degrading enzyme 3-2AKR under different temperature conditions;

[0021] Figure 3 To investigate the degradation of DON by recombinant deoxynivalenol degrading enzyme 3-2AKR under different pH conditions;

[0022] Figure 4 The degradation of DON in corn steep liquor by recombinant deoxynivalenol degrading enzyme 3-2AKR is shown in Figure 1. In Figure 2, A represents the DON content before enzyme treatment, and B represents the DON content after enzyme treatment. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The main experimental materials and reagents used in the embodiments of this invention are as follows: the *E. coli* expression vector pET-28a(+) and the cloning strain *E. coli* DH5α were preserved by the Laboratory of Basic and Applied Microbiology, Shenyang Agricultural University. The expression strain *E. coli* BL21(DE3) was purchased from Shanghai Sangon Biotech Co., Ltd. Restriction endonucleases and DN polymerases were purchased from Takara, deoxynivalenol was purchased from Acmec, and the DON ELISA kit was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other reagents were domestically produced analytical grade.

[0029] The Bacillus amyloliquefaciens GB3-2 provided by this invention was obtained by isolating rice tissue. This strain was obtained through primary screening using phenyl ethylene oxide as the sole carbon source and secondary screening using a unique method.

[0030] The initial screening medium formula (g / L) was as follows: Na2SO4 1.6g, KH2PO4 4g, MgSO4 0.5g, NaNO3 0.5g, (NH4)2SO4 0.5g, CaCl2 0.025g, pH 7.0. After sterilization, filtered sterilized phenyl ethylene oxide was added to make the final concentration 10~20 mmol / L. The medium was then used for secondary screening with a liquid medium containing deoxynivalenol to finally obtain strains that can degrade deoxynivalenol.

[0031] The secondary screening medium formula (g / L) is as follows: Na2SO4 1.6g, KH2PO4 4g, MgSO4 0.5g, NaNO3 0.5g, (NH4)2SO4 0.5g, CaCl2 0.025g, pH 7.0; after the medium is cooled to 40℃, deoxynivalenol is added to bring the final concentration to 1ug / mL.

[0032] GB3-2 bacterial suspension was inoculated onto primary screening medium using the streak plate method and incubated at 37°C for 24 h before growth was observed. The primary screening medium used in this experiment was a selective medium with phenyl ethylene oxide as the sole carbon source. Phenyl ethylene oxide has the same epoxy structure as DON, and strains grown on this selective medium have the potential to degrade the DON epoxy structure.

[0033] Single colonies that grew in the primary screening medium were picked out and inoculated into the secondary screening medium. After incubation at 37°C and 200 rpm for 24 h, 100 μL of 1000 μg / mL DON was added to 900 pL of the fermentation broth (1 x 10⁻⁶). 9 The DON content in the fermentation broth was measured at CFU / mL and incubated at 37°C for 48 h (n=200 rpm). A sterile screening medium containing an equal amount of DON was used as a control, and the broth was incubated under the same conditions for 48 h. The DON content in the fermentation broth was detected using a DON ELISA kit, and strain GB3-2, which exhibited high DON degradation capabilities, was screened.

[0034] The strain GB3-2 was identified by morphological and molecular biological analysis as belonging to Bacillus amyloliquefaciens. It was deposited on October 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.28643.

[0035] Example 1

[0036] 1. Obtaining and expressing deoxynivalenol-degrading enzyme 3-2AKR protein from Fusarium deoxynivalenols.

[0037] Using Bacillus amyloliquefaciens GB3-2 genomic DNA as a template, the coding gene for the 3-2AKR protein was amplified. A recombinant expression vector containing the 3-2AKR protein coding gene sequence and its engineered strain were constructed, and the 3-2AKR protein was expressed. The specific steps are as follows:

[0038] Take 50 μL of *Bacillus amyloliquefaciens* GB3-2 culture from a glycerol tube and streak it onto an LB agar plate. Incubate at 37°C for 12 h. Pick a single colony from the LB agar plate and inoculate it into 5 mL of liquid LB medium. Incubate at 180 rpm for 12 h at 37°C. Aliquot the culture into sterile 1.5 mL microcentrifuge tubes and centrifuge at 12000 rpm for 1 min to collect the cells. Discard the supernatant and freeze the cells in liquid nitrogen. Grind the cells thoroughly with a pre-frozen grinding rod in liquid nitrogen, keeping the sample frozen continuously in liquid nitrogen. Add 600 μL of preheated CTAB solution, shake vigorously to mix, and lyse at 60°C for 1 h, mixing up and down every 10 min. Add an equal volume of a pre-prepared chloroform-isoamyl alcohol mixture to the centrifuge tubes. Mix the solutions (volume ratio 24:1) thoroughly and centrifuge at 12000 rpm for 15 min at 4 °C. Transfer 400 μL of the supernatant to a new centrifuge tube, add an equal volume of chloroform-isoamyl alcohol mixture, mix gently, and centrifuge at 12000 rpm for 15 min at 4 °C. Transfer the supernatant to a new centrifuge tube, add twice the volume of -20 °C pre-cooled anhydrous ethanol, mix gently, and precipitate at -20 °C for 20 min. Centrifuge at 12000 rpm for 15 min at 4 °C. Discard the supernatant, wash the precipitate with 1000 μL of 70% ethanol aqueous solution, and centrifuge at 7500 rpm for 5 min at 4 °C. Remove excess liquid from the bottom of the centrifuge tube and air dry for 10 min. Dissolve the precipitate in 100 μL of sterile H2O to obtain genomic DNA, which will be used for subsequent experiments and stored at -20 °C.

[0039] 2. Amplification of the gene encoding deoxynivalenol degrading enzyme 3-2AKR from Fusarium nivale.

[0040] The gene encoding the deoxynivalenol degrading enzyme 3-2AKR from *Fusarium nivale* was amplified using the following steps: Based on the multiple cloning site of the vector pET-28a(+), EcoRI and XhoI were selected as restriction enzyme sites. Upstream primer P1 and downstream primer P2 were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. The sequences of upstream primer P1 and downstream primer P2 are as follows:

[0041] Upstream primer P1: GGAATTCATGGATGAGACACGTGCG (SEQ ID NO.1);

[0042] Downstream primer P2: CCGCTCGAGCTTGAAGATGTCACTGATATATTG (SEQ ID NO.2).

[0043] PCR amplification was performed using Bacillus amyloliquefaciens GB3-2 genomic DNA as a template, and the reaction system is shown in Table 1.

[0044] Table 1 PCR amplification reaction system

[0045] Prime STAR MAX 25μL forward primer 1.0μL reverse primer 1.0μL DNA template 1.0μL <![CDATA[3-2AKR2O]]> Up to 50μL

[0046] Reaction procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 2 min, 35 cycles; 72℃ final extension for 10 min. PCR amplification products were subjected to 1% agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit.

[0047] The gene sequence encoding deoxynivalenol degrading enzyme 3-2AKR (SEQ ID NO.3): ATGGATGAGACACGTGCGCTGGGCAGGACGGATTTACGGGTAAAGCGTATCGGCTTTGGCGCAAATGCGGTCGGAGGACATAACCTGTTTCCGAATCTTAATGATGAAACGGGAAAAGAACTCATCCGCACAGCTTTGGATGGCGGCGTCAATTTTATTGATACGGCGTTTATATACGGCTTGGGACGTTCCGAAGAGTTAATCGGTGAAGTCGTCGCAGAGCGCGGCGTTCGGGACAGCCTCGTGATTGCGTCAAAAGGTGCGCATAAAGAGGTCAACGGACAAATCGAGCTGGAT AACAGCCGTGAATTTCTTCGGAGTGAGGCGGAAAAAAGCCTGAAGCGGCTGAAAACAGATGTGATTGATTTGTATTACGTTCACTTCCCTGACGGCAAAACACCGCTGGCCGAAGTGGCAGGCACACTAAAAGAACTGAAGGACGAAGGGA AAATCAGAGCAATCGGCGCCTCCAATCTCAGCTTCCGGCAATTGCAGGAGTTCAACGCTGACGGCTATTTAGAGGTTTTTTCAATCGGAATATTCACTGATTCAGCGCGAGGCGGAAAAAGATCTGCTGCCTTACTGTGAAAAGAACGGCATT

[0048] TCCTTTATCCCTTATTTCCCGCTTGCTTCCGGCTTGCTGACAGGAAAGTTCACAAAAGACACAGTCTTTGAGGACAGCCGAAAGAATAGGCCGCCGTTTCAGGGAGAAGCTTTTCTCCAAAACTTGGAGAGAGTGGATAAGCTGAAGTCCGTTGCGGAAGAAAAA GGCGCGCATGCAGCGCATATCGCACTCGCATGGCTTCTCGCAGCCGGCTGTGGATGCGATCATTCCCGGAGCCAAACGCCCTGACCAGCTGAAACACAATCTGGGCGCGCTTGATGTGCAATTAACCGAAGACGAGGTTCAATATATCAGTGACATCTTCAAG

[0049] The amino acid sequence of deoxynivalenol degrading enzyme 3-2AKR (SEQ ID NO.4): MDETRALGRTDLRVKRIGFGANAVGGHNLFPNLNDETGKELIRTALDGGVNFIDTAFIYGLGRSEELIGEVVAERGVRDSLVIASKGAHKEVNGQIELDNSREFLRSEAEKSLKRLKTDVIDLYYVHFPDGKTPLAEVAGTLKELKDEGKIRAIGASNLSFRQLQEFNADGYLEVFQSEYSLIQREAEKDLLPYCEKNGISFIPYFPLASGLLTGKFTKDTVFEDSRKNRPPFQGEAFLQNLERVDKLKSVAEEKGAHAAHIALAWLLSQPAVDAIIPGAKRPDQLKHNLGALDVQLTEDEVQYISDIFK.

[0050] Preparation of linearized vectors for recombinant expression vectors containing the gene sequence encoding deoxynivalenol degrading enzyme 3-2AKR: pET-28a(+) plasmid was digested with EcoRI and XhoI, and the digestion system is shown in Table 2.

[0051] Table 2 Enzyme digestion system

[0052] Pet28a (+) 30μL EcoRI 2μL XhoI 2μL RNase A 2μL 10×Buffer 10μL <![CDATA[3-2AKR2O]]> Up to 100μL

[0053] Enzyme digestion conditions: 37℃ water bath for 30 min. The digestion products were subjected to 1% agarose gel electrophoresis, and the plasmid digested fragments were recovered using an agarose gel DNA recovery kit.

[0054] Homologous recombination cloning: The reaction system is shown in Table 3.

[0055] Table 3 Homologous recombination cloning reaction system

[0056] Solution I 5μL Target fragment 4.5μL carrier Up to 10μL

[0057] Reaction conditions: Mix gently and react at 16℃ for 12 h; after the reaction, place the PCR tube on ice and directly transform the ligation product into E. coli competent cells DH5α. After Kana resistance screening, positive transformants were picked, recombinant plasmids were extracted, and single and double enzyme digestion verification and sequencing were performed to confirm that the correct recombinant strain DH5α / pET-28a(+)-AKR was constructed. Then, the correct recombinant plasmid pET-28a(+)-AKR was transformed into E. coli BL21(DE3).

[0058] 3. Induction, expression, and purification of deoxynivalenol degrading enzyme 3-2AKR in Escherichia coli

[0059] Recombinant Escherichia coli BL21(DE3) transformed with pET-28a(+)-AKR plasmid was inoculated into 5 mL of liquid LB medium and activated overnight. The culture was then transferred at a volume ratio of 1:100 to a 500 mL Erlenmeyer flask containing 250 mL of the medium and cultured at 180 rpm at 37 °C until OD500. 600 The concentration was 0.7, and IPTG was added to a final concentration of 0.4 mM to induce the expression of the target protein.

[0060] Purification of deoxynivalenol degrading enzyme 3-2AKR from Fusarium nivale: Induced bacterial cultures, whose expression was confirmed by SDS-PAGE electrophoresis, were aliquoted into 50 mL centrifuge tubes and collected by centrifugation at 5000 rpm for 15 min at 40 °C. The supernatant was discarded, and the precipitate was washed with 15 mL of PBS buffer in each tube. The precipitate was centrifuged at 5000 rpm for 10 min at 40 °C, and this process was repeated twice. 10 mL of lysis buffer was added to the precipitate, and the precipitate was resuspended and sonicated. The disrupted bacterial culture was centrifuged at 12000 rpm for 30 min at 40 °C, and the supernatant was retained. The protective buffer in a Ni-NTA purification resin pre-packed column was allowed to flow out naturally, followed by rinsing the pre-packed column with 10 column volumes of Ni-Native-O buffer at a flow rate of approximately 1 mL / min. The disrupted supernatant was added to the pre-packed column and slowly passed through at a flow rate of 1 mL / min. After all the supernatant had passed through the column, the column was washed with 10 mL of 150 mM imidazole Ni-Native-buffer, and the flow-through was collected at a flow rate of 1 mL / min. The proteins collected at different concentrations were analyzed by SDS-PAGE, and the target protein was successfully purified.

[0061] After purification, the expression product of recombinant plasmid pET-28a(+)-AKR was subjected to SDS-PAGE electrophoresis. The electrophoresis image is shown below. Figure 1 As shown.

[0062] Example 2

[0063] Effect of temperature on the activity of deoxynivalenol degrading enzyme 3-2AKR in degrading DON

[0064] Solid deoxynivalenol was dissolved in acetonitrile to prepare a stock solution of 500 μg / mL. The cofactor NAD... + Nicotinamide adenine dinucleotide (NAD) was dissolved in ultrapure water to prepare a 1 mM stock solution. The experiment was conducted using the following 400 μL reaction system: 330 μL Tris-HCl buffer (0.05 M, pH 8.0), 40 μL 3-2AKR protein (23.5 μg), and 10 μL NAD+. + The solution contained 20 μL of DON. A system without 3-2AKR protein was used as a control. The reaction was carried out at different temperatures (20, 25, 30, 35, 40, 45 °C) for 12 h, and then 400 μL of methanol was added to terminate the reaction. The system was centrifuged at 12000 rpm for 1 min, and the supernatant was filtered through a Millex-GV membrane (0.22 μm). The residual DON content in the system was detected by high performance liquid chromatography.

[0065] The chromatographic conditions for the detection of DON by high performance liquid chromatography were as follows: column: Agilent C18 column, 4.6 mm × 150 mm × 5 μm; mobile phase: acetonitrile:water (volume ratio 1:9); flow rate: 1 mL / min; pump pressure: 100 bar; injection volume: 10 μL; UV detector wavelength: 218 nm; acquisition time: 30 min.

[0066] DON degradation rate = [(1 - amount of DON remaining in the treatment group) / amount of DON in the control group] × 100%

[0067] The results are expressed as follows: the DON degradation rate at 40℃ is 100%, and the degradation rate at other temperatures is expressed as a relative rate. Figure 2 As shown, the optimal temperature for the degradation of DON by deoxynivalenol degrading enzyme 3-2AKR is 40℃.

[0068] Example 3

[0069] Effect of pH on the activity of deoxynivalenol degrading enzyme 3-2AKR in degrading DON

[0070] The effect of deoxynivalenol degrading enzyme 3-2AKR on the degradation activity of DON under different pH conditions was tested. The reaction system was as follows: 330 μL Tris-HCl buffer (0.05 M, pH 8.0), 40 μL 3-2AKR protein (23.5 μg), and 10 μL NAD+. + Solution: 20 μL DON solution. A system without 3-2AKR protein was used as a control. The reaction was carried out at 40 °C for 12 h at different pH values ​​(5, 6, 7, 8, 9, 10, 11, 12), and then 400 μL methanol was added to terminate the reaction. The system was centrifuged at 12000 rpm for 1 min, and the supernatant was filtered through a Millex-GV membrane (0.22 μm). The residual DON content in the system was detected using the method described in Example 2.

[0071] The results are expressed as follows: the DON degradation rate at pH 10 is 100%, and the degradation rate at other pH conditions is expressed as a relative rate. Figure 3 As shown, the optimal pH for the degradation of DON by deoxynivalenol degrading enzyme 3-2AKR is 10.

[0072] Example 4

[0073] Effect of deoxynivalenol degrading enzyme 3-2AKR on the activity of corn steep liquor in degrading DON

[0074] The reaction system used was as follows: 310 μL Tris-HCl buffer (0.05 M, pH 8.0), 40 μL 3-2AKR protein (23.5 μg), and 10 μL NAD+. + Solution: 40 μL corn steep liquor. A system without 3-2AKR protein was used as a control. After the reaction was carried out at 40 °C for 12 h, 400 μL methanol was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 1 min, and the supernatant was filtered through a Millex-GV membrane (0.22 μm). The residual DON content in the system was detected using the method described in Example 2.

[0075] After the reaction was completed, the degradation rate of DON was measured, and the results are as follows: Figure 4 As shown: the degradation rate of DON in corn steep liquor was 37.02% ( Figure 4 ).

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of deoxynivalenol-degrading enzyme 3-2 AKR, a recombinant plasmid containing a gene encoding the deoxynivalenol-degrading enzyme 3-2 AKR, and a recombinant strain containing the recombinant plasmid in the preparation of a deoxynivalenol biodegradation agent, characterized in that, the amino acid sequence of the deoxynivalenol-degrading enzyme 3-2 AKR is shown as SEQ ID NO. 4; and the nucleotide sequence of the gene encoding the deoxynivalenol-degrading enzyme 3-2 AKR is shown as SEQ ID NO.

3.

2. Use of deoxynivalenol-degrading enzyme 3-2 AKR, a recombinant plasmid containing a gene encoding the deoxynivalenol-degrading enzyme 3-2 AKR, and a recombinant strain containing the recombinant plasmid in the degradation of deoxynivalenol in agricultural products, characterized in that, the amino acid sequence of the deoxynivalenol-degrading enzyme 3-2 AKR is shown as SEQ ID NO. 4; and the nucleotide sequence of the gene encoding the deoxynivalenol-degrading enzyme 3-2 AKR is shown as SEQ ID NO.

3. ​ ​ ​ ​

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