A deoxynivalenol-degrading enzyme and its encoding gene, preparation method and application

Abstract

A degrading enzyme for vomitoxin and its encoding gene, preparation method and application, belonging to the field of microorganism and genetic engineering technology. In order to solve the technical problem that the technology of biodegradation of vomitoxin mostly depends on a mild operating environment, the present invention provides a degrading enzyme for vomitoxin with an amino acid sequence as shown in SEQ ID NO.1, as well as a gene encoding the above-mentioned degrading enzyme for vomitoxin, a recombinant vector and a recombinant bacterium carrying the gene, and a specific method for preparing the degrading enzyme for vomitoxin. In addition, the present invention uses the enzyme for the degradation of vomitoxin, and it is found that it can play a degradation role under the conditions of 30-70°C and pH 2-9. The enzyme does not depend on a mild operating environment and has broad application prospects in the degradation of vomitoxin.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and genetic engineering, and specifically relates to a vomitoxin-degrading enzyme and a coding gene thereof, a preparation method and an application thereof. Background Art

[0002] Vomitoxin (VT), also known as deoxynivalenol (DON), has the chemical name 3α,7α,15-trihydroxy-12,13-epoxytrichothecenes-9-ene-8-one. Vomitoxin is mainly produced by Fusarium graminearum ( Fusarium gramineae ) and Fusarium oxysporum ( Fusarium wilt ) when fungi infect grains (such as wheat, barley, oats, corn, etc.). These fungi will multiply and produce toxins in suitable environmental conditions, such as humid and warm climates, thereby contaminating grains. Deoxynivalenol has a significant impact on animal growth performance, resulting in reduced animal feed intake, reduced growth performance, diarrhea and increased mortality. It also affects the liver health of animals and may lead to reduced reproductive performance. In addition, deoxynivalenol reduces the effectiveness of pig vaccine immunity and increases the incidence of infectious and severe diseases. After humans eat foods containing deoxynivalenol, they may experience symptoms such as stomach discomfort, abdominal distension, dizziness, headache, nausea, vomiting, numbness of hands and feet, general fatigue, and facial flushing. In severe cases, deoxynivalenol may also damage the hematopoietic system and even cause death. Deoxynivalenol is a global public health issue and poses a major challenge to food and feed safety. Its high detection rate and excess rate also make it a serious threat to animal and human health, which further highlights the urgency of reducing or eliminating the toxicity of deoxynivalenol. Current strategies for detoxification of deoxynivalenol include physical, chemical and biological methods. Although physical and chemical methods are effective, their low efficiency, nutrient loss and high cost limit their widespread application. In contrast, biological methods are considered the most promising detoxification method because of their high efficiency, safety, environmental protection, and ability to maintain the quality and nutrition of raw materials under mild conditions. However, existing biodegradation technologies mostly rely on mild operating environments and perform poorly when faced with high temperature environments or extreme acid-base conditions, which greatly limits the breadth of their applications. Therefore, if we can explore a biodegradation method that is efficient, safe, and can adapt to high temperatures and harsh acid-base conditions, it will broaden the application conditions of vomitoxin bioremediation, which has extremely important practical significance and broad application prospects. Summary of the invention

[0003] In order to solve the technical problem that the existing biodegradation technologies of vomitoxin mostly rely on mild operating environments, perform poorly when facing high temperature environments or extreme acid-base conditions, and greatly limit the breadth of its application, the present invention provides a vomitoxin degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1, as well as a gene encoding the above-mentioned vomitoxin degrading enzyme, a recombinant vector carrying the gene, and a recombinant bacterium. In addition, the present invention also covers a specific method for preparing the vomitoxin degrading enzyme, and explores the degradation effect of the vomitoxin degrading enzyme in degrading vomitoxin. The results showed that the enzyme was used for the degradation of vomitoxin, and it could play a degradation role under the reaction conditions of 30-70°C and pH 2-9. The enzyme does not rely on a mild operating environment and can be added to feed as a feed additive. It has broad application prospects in the degradation of vomitoxin and can ensure the safety of food and feed.

[0004] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0005] The first object of the present invention is to provide a deoxynivalenol-degrading enzyme, the amino acid sequence of the deoxynivalenol-degrading enzyme is shown in SEQ ID NO.1.

[0006] The second object of the present invention is to provide a gene encoding the above-mentioned vomitoxin degrading enzyme, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] The third object of the present invention is to provide a recombinant vector containing the above gene.

[0008] In one embodiment of the present invention, the plasmid selected for constructing the recombinant vector is any one of pET24a, pHY300PLK, pBE-S, pub110, YEp352 and pPIC9.

[0009] The fourth object of the present invention is to provide a recombinant bacterium containing the above gene or the above recombinant vector.

[0010] In one embodiment of the present invention, the host bacteria selected for the construction of the recombinant bacteria is Escherichia coli ( Escherichia coli )、Bacillus subtilis( Bacillus subtilis )、Bacillus licheniformis( Bacillus lincheniform )、Pichia pastoris( Shepherd's pie ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae )

[0011] A fifth object of the present invention is to provide an additive containing the above-mentioned DON-degrading enzyme.

[0012] In one embodiment of the present invention, the core components of the additive include the above-mentioned vomitoxin degrading enzyme, and / or contain beneficial substances produced during the fermentation process of the above-mentioned specific recombinant bacteria. The effects of these components can effectively promote the degradation of vomitoxin and improve the safety of feed.

[0013] A sixth object of the present invention is to provide a feed containing the above-mentioned vomitoxin-degrading enzyme or the above-mentioned additive.

[0014] The seventh object of the present invention is to provide a method for preparing the above-mentioned vomitoxin-degrading enzyme, which comprises constructing a recombinant vector containing the above-mentioned gene, introducing the recombinant vector into a host bacterium to obtain a recombinant bacterium; culturing the recombinant bacterium in a liquid culture medium to obtain a bacterial liquid, and obtaining bacterial bodies by centrifugation; collecting the supernatant as a crude enzyme liquid by ultrasonic disruption and centrifugation; and obtaining a purified enzyme liquid by affinity chromatography on a Ni column.

[0015] In one embodiment of the present invention, the host bacteria is a prokaryotic bacterium or a eukaryotic bacterium.

[0016] In one embodiment of the present invention, the host bacteria is Escherichia coli ( Escherichia coli )、Bacillus subtilis( Bacillus subtilis )、Bacillus licheniformis( Bacillus lincheniformis )、Pichia pastoris( Shepherd's pie ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae )

[0017] The eighth object of the present invention is to provide the use of the above-mentioned vomiting detoxification enzyme, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant bacteria or the above-mentioned additive in degrading vomiting toxin in feed.

[0018] The ninth object of the present invention is to provide a method for degrading vomitoxin, which comprises adding the vomitoxin degrading enzyme or the additive to a sample to be treated containing vomitoxin, and performing enzymolysis for 1-60 h under reaction conditions of 30-70° C. and pH 2-9.

[0019] It is further defined that the reaction conditions are 30-60° C., pH 4-9 and enzymatic hydrolysis for 10-60 h.

[0020] Preferably, the reaction conditions are 30-50° C., pH 6-8, and enzymatic hydrolysis for 30-60 h.

[0021] Beneficial effects of the present invention:

[0022] The vomitoxin degrading enzyme provided by the present invention exhibits excellent performance in degrading vomitoxin. It can not only decompose vomitoxin efficiently and rapidly, but also has excellent acid, alkali and high temperature resistance. It can exert its degradation effect under the reaction conditions of 30-70°C and pH 2-9, and is not dependent on a mild operating environment. It provides the possibility for more diversified application scenarios, has broad application prospects, and can ensure the safety of food and feed.

[0023] In addition, traditional techniques generally use Escherichia coli ( Escherichia coli ) as the host, the present invention uses Escherichia coli, Bacillus subtilis, Bacillus licheniformis and Saccharomyces cerevisiae as host bacteria to produce the vomitoxin degrading enzyme provided by the present invention, and it is found that there is no significant difference in the degradation effect of the vomitoxin degrading enzyme obtained by using Bacillus subtilis, Bacillus licheniformis or Saccharomyces cerevisiae as the host bacteria compared with that by using Escherichia coli as the host. The use of food-grade safe strains (such as Bacillus subtilis, Bacillus licheniformis or Saccharomyces cerevisiae, etc.) as host bacteria to produce vomitoxin degrading enzyme can make the application of vomitoxin degrading enzyme in feed more advantageous. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be clear that the specific embodiments shown here are only for the purpose of illustrating and explaining the core content and principles of the present invention, and are not intended to limit or restrict the present invention in any form. The numerical values ​​disclosed in the following examples are all deemed to include a range of variation close to these numerical values, and the individual point values ​​can be freely combined to form new ranges, and these new ranges are all deemed to be the contents clearly disclosed by the present invention.

[0025] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0026] The vomitoxin standard substances used in the present invention were all purchased from Sigma, ensuring the consistency of the experimental materials.

[0027] The phosphate buffer solution (PBS buffer solution) used in the present invention has the following composition: 0.24 g / L potassium dihydrogen phosphate, 1.44 g / L disodium hydrogen phosphate, 8 g / L sodium chloride, 0.2 g / L potassium chloride, and the balance is water, with a pH of 7.2.

[0028] The culture medium involved in the present invention is as follows:

[0029] LB liquid medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the balance is water.

[0030] LB solid medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar, and the balance is water.

[0031] TB liquid culture medium: 24 g / L yeast powder, 12 g / L peptone, 16.4 g / L potassium hydrogen phosphate trihydrate, 2.3 g / L potassium dihydrogen phosphate, 5 g / L glycerol, and the balance is water.

[0032] When testing the vomitoxin content, we followed the national standard "GB / T 30956-2014 Determination of deoxynivalenol in feed - Immunoaffinity column cleanup-High performance liquid chromatography" to ensure the accuracy and reliability of the test results.

[0033] As for the evaluation of the degradation efficiency of DON, the following calculation formula is used for quantification: Degradation rate (%) = (total amount of DON in the sample before reaction - amount of DON remaining in the sample after reaction) / total amount of DON in the sample before reaction × 100%. This indicator intuitively reflects the ability of the enzyme preparation to degrade DON.

[0034] Comparative Example:

[0035] This comparative example is used to illustrate the wild-type vomitoxin-degrading enzyme and its preparation method and application.

[0036] 1. Wild-type DON-degrading enzyme

[0037] The amino acid sequence of the wild-type vomitoxin-degrading enzyme provided in this comparative example is shown in SEQ ID NO.3.

[0038] SEQ ID NO.3:

[0039] MGALDRRGFNRRVLLGGAAVATSLSLAPEARSDAGPAQAAPGGEVRRIKLYAERLADGQMGYGLEKGRATIPGPLIELNEGDTLHIEFENTMDVRASLHVHGLDYEVSSDGTTLNKSDVEPGGTRTYTWRTHAPGRRSDGTWRAGSAGYWHYHDHVVGTEHG TGGIRKGLYGPVIVRRKGDVLPDATHTIVFNDMLINNRPAHSGPNFEATVGDRVEFVMITHGEYYHTFHMHGHRWADNRTGMLTGPDDPSQVVDNKIVGPADSFGFQVIAGEGVGAGAWMYHCHVQSHSDMGMVGLFLVKKTDGTIPGYEPHEHSGQRAEHHH

[0040] (II) Preparation method of wild-type vomitoxin-degrading enzyme

[0041] (1) Obtaining the gene fragment encoding the deoxynivalenol-degrading enzyme

[0042] A BamH I restriction site (GGATCC) was added to the 5' end of the nucleotide sequence encoding the above-mentioned wild-type vomitoxin-degrading enzyme as shown in SEQ ID NO.4, and a Sal I restriction site (GTCGAC) was added to the 3' end. GeneWeichi Co., Ltd. was commissioned to synthesize the above-mentioned gene fragment through artificial chemical methods.

[0043] SEQ ID NO.4:

[0044] ATGGGCGCTCTTGATCGTCGTGGCTTCAACCGTCGTGTTCTTCTTGGCGGCGCTGCTGTTGCTACATCTCTTTCTCTTGCTCCTGAAGCTCGTTCTGATGCTGGCCCTGCTCAAGCTGCTCCTGGCGGCGAAGTTCGTCGTATCAAACTTTACGCTGAACGTCTTGCTGATGGCCAAATGGGCTACGGCCTTGAAAAAGGCCGTGCTACAATCCCTGGCCCTCTTATCGAACTTAACGAAGGCGATACACTTCATATCGAATTCGAAAACACAATGGATGTTCGTGCTTCTCTTCATGTTCATGGCCTTGATTACGAAGTTTCTTCTGATGGCACAACACTTAACAAATCTGATGTTGAACCTGGCGGCACACGTACATACACATGGCGTACACATGCTCCTGGCCGTCGTTCTGATGGCACATGGCGTGCTGGCTCTGCTGGCTACTGGCATTACCATGATCATGTTGTTGGCACAGAACATGGCACAGGCGGCATCCGTAAAGGCCTTTACGGCCCTGTTATCGTTCGTCGTAAAGGCGATGTTCTTCCTGATGCTACACATACAATCGTTTTCAACGATATGCTTATCAACAACCGTCCTGCTCATTCTGGCCCTAACTTCGAAGCTACAGTTGGCGATCGTGTTGAATTCGTTATGATCACACATGGCGAATACTACCATACATTCCATATGCATGGCCATCGTTGGGCTGATAACCGTACAGGCATGCTTACAGGCCCTGATGATCCTTCTCAAGTTGTTGATAACAAAATCGTTGGCCCTGCTGATTCTTTCGGCTTCCAAGTTATCGCTGGCGAAGGCGTTGGCGCTGGCGCTTGGATGTACCATTGCCATGTTCAATCTCATTCTGATATGGGCATGGTTGGCCTTTTCCTTGTTAAAAAAACAGATGGCACAATCCCTGGCTACGAACCTCATGAACATTCTGGCCAACGTGCTGAACATCATCATTAA

[0045] (2) Construction of recombinant vector

[0046] The gene fragment synthesized in step (1) and plasmid pET24a were double-digested using restriction endonucleases BamH I and Sal I (see Table 1 for the double-digestion reaction system), i.e., reacted at 37°C for 2 h, and then nucleic acid gel recovery was performed.

[0047] Table 1 Double enzyme digestion reaction system

[0048]

[0049] The gene fragment after double restriction digestion was connected with the plasmid pET24a after the same double restriction digestion using T4 ligase (see Table 2 for the ligation system). The ligation reaction conditions were 16°C for 4 h.

[0050] Table 2 Ligation reaction system

[0051]

[0052] The ligated product was transformed into DH5α competent cells by heat shock method, and then coated on a solid LB plate containing karamycin and cultured overnight at 37°C; the obtained colonies were verified by PCR, specifically, the monoclonal strain containing the nucleotide sequence shown in SEQ ID NO.4 was verified by PCR using primers F1 (nucleotide sequence as shown in SEQ ID NO.5: 5'-ATGGGCGCTCTTGATCGTCG-3') and R1 (nucleotide sequence as shown in SEQ ID NO.6: 5'-TTAATGATGATGTTCAGCACG-3'). The PCR reaction program was as follows: 98°C, 10 min; (98°C, 30s; 55°C, 30s; 72°C, 1-2min) × 30 cycles; 72°C, 10 min. The size of the recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.4 was verified by nucleic acid gel, and the verification results showed that the target band size of the recombinant plasmid was 1953 bp, which was consistent with the expected size. Then the corresponding monoclonal strain is selected, cultured, and the plasmid is extracted for sequencing to confirm that the sequence is correct.

[0053] (3) Construction of recombinant strains

[0054] The recombinant vector verified by sequencing in step (2) and the empty vector pET24a were electroporated (voltage 1500 V, capacitance 25 μF, resistance 200 Ω) into BL21(DE3) to obtain a recombinant strain expressing the vomitoxin-degrading enzyme encoded by the nucleotide sequence shown in SEQ ID NO.4 (denoted as BL21(DE3) / pET24a-0) and a control strain BL21(DE3) / pET24a.

[0055] (4) Enzyme preparation

[0056] The recombinant strain BL21(DE3) / pET24a-0 obtained in step (3) was inoculated into a 100 mL Erlenmeyer flask containing 10 mL LB liquid medium and cultured at 37°C for 14 h. 2 mL of the activated bacterial solution was inoculated into 50 mL TB liquid medium and cultured at 37°C for 28 h. The cells were collected by centrifugation. The cells were then suspended in phosphate buffer and ultrasonically disrupted. The cells were centrifuged at 10,000 rpm for 20 min at 4°C and the supernatant was collected to obtain a crude enzyme solution.

[0057] The obtained crude enzyme solution was subjected to affinity chromatography using a Ni column to obtain a highly pure enzyme solution.

[0058] In the process of affinity chromatography purification using Ni column, since the target recombinant enzyme is fused with His tag, the Ni column is first equilibrated with a specific buffer (50 mM NaCl and 10 mM imidazole, pH 7.4) at a flow rate of 1 mL / min; then, the sample containing the crude enzyme solution is cyclically loaded on the Ni column at a flow rate of 1 mL / min. This step is intended to allow the recombinant protein carrying the His tag to fully bind. The above-mentioned specific buffer is used for washing at a flow rate of 1 mL / min to remove impurity proteins that are non-specifically bound or not firmly adsorbed. This step helps to improve the selectivity and efficiency of subsequent purification steps. Finally, in order to elute and collect the target protein, imidazole buffer (50 mM NaCl, 500 mM imidazole, pH 7.4) is used for elution to obtain a high-purity enzyme solution.

[0059] 3. Application of wild-type DON-degrading enzyme

[0060] Enzyme activity assay:

[0061] Take 1000 µL of the high-purity enzyme solution obtained in step (4) and transfer it to a 1.5 mL centrifuge tube. Then, add the standard solution of vomitoxin to the centrifuge tube and mix thoroughly to ensure that the two are evenly mixed to form a mixed solution. The final concentration of vomitoxin in this mixed solution is 60 ppm.

[0062] 1) Effect of reaction time on enzyme activity

[0063] The reaction conditions were 37°C and pH 7. The mixed solution was incubated, and 20 µL samples were collected at the 1st hour, 10th hour, 20th hour, 30th hour, 40th hour, 50th hour, and 60th hour after the start of the reaction to detect the residual level of vomitoxin (see Table 3). According to the data listed in Table 3, it can be clearly seen that when the reaction lasted for 40 hours, the degradation efficiency of vomitoxin exceeded 47.8%, and the degradation efficiency increased slowly as the reaction time increased. Based on this, subsequent experiments will select 40 hours as the reaction time for further research.

[0064] 2) Effect of temperature on enzyme activity

[0065] Under the condition of pH 7, the above 7 identical mixed solutions were placed in 7 different temperature environments of 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 40 hours of reaction. Afterwards, 20 μL samples were taken from each reaction system to detect the residual amount of vomitoxin, and the results are shown in Table 4. According to the data listed in Table 4, it can be clearly seen that when the reaction temperature is 40℃, the degradation efficiency of vomitoxin is the highest; when the reaction temperature is 30℃, 50℃, 60℃ or 70℃, a good degradation effect can also be obtained; when the reaction temperature exceeds 70℃, although the degradation effect is reduced, it still has a degradation effect. Based on this, the subsequent experiments will select 40℃ as the reaction temperature for further research.

[0066] 3) Effect of pH on enzyme activity

[0067] At a constant temperature of 40°C, 8 identical mixed solutions were adjusted to different pH environments of 2, 3, 4, 5, 6, 7, 8 and 9 for 40 hours of reaction. After the reaction, 20 µL of the mixed solution under each experimental condition was taken as a sample to determine the residual concentration of DON, and the results are shown in Table 5. According to the data listed in Table 5, it can be clearly seen that when the pH of the reaction environment is 7, the degradation efficiency of DON is the highest; when the pH of the reaction environment is 6, 8 or 9, a good degradation effect can also be obtained; when the pH of the reaction environment is lower than 5, although the degradation effect is reduced, it still has a degradation effect.

[0068] Embodiment 1:

[0069] This example is used to illustrate a vomitoxin degrading enzyme having an amino acid sequence as shown in SEQ ID NO.1 provided by the present invention, and a preparation method and application thereof. The amino acid sequence of the vomitoxin degrading enzyme is mutated from SEQ ID NO.3 in the comparative example, and the mutation site is underlined.

[0070] SEQ ID NO.1:

[0071] MGALDRRGFNRRVLLGGAAVATSLSLAPEARSDAGPAQAAPGGEVRRIKLYAERLADGQMGYGLEKGRATIPGPLIELNEGDTLHIEFENTMDVRASLHVHGLDYEVSSDGTTLNKSDVEPGGTRTYTWRTHAPGRRSDGTWRAGSAGYWHYHDHVVGTEHGTGGIRKGLYGPVIVRRKGDVLPDATHTIVFNDMLIN C RPAHSGPNFEATVGDRVEFVMITHGEYYHTFHMHGHRWADNRTGMLTGPDDPSQVV H NKIVGPADSFGFQVIAGEGVGAGAWMYHCHVGSHSDMMGMVGLFLVKKTDGTIPGYEPHEHSGQRAEHHH

[0072] The preparation method of the deoxynivalenol degrading enzyme provided in this embodiment is different from that in the comparative example only in that the nucleotide sequence encoding the deoxynivalenol degrading enzyme in step (1) is shown in SEQ ID NO.2. In this embodiment, the same method is used to add restriction sites at both ends of SEQ ID NO.2 and artificially synthesize gene fragments, and then the recombinant strain BL21 (DE3) / pET24a-1 is obtained through the construction of the recombinant vector and the construction of the recombinant strain. Then, the recombinant strain is used to prepare a high-purity enzyme solution in the same manner as the comparative example, and then the degradation efficiency of deoxynivalenol is detected under different reaction times, temperatures and pH conditions to evaluate the enzyme activity. The detection results of the degradation rate of deoxynivalenol at different reaction times are shown in Table 3, the detection results of the degradation rate of deoxynivalenol at different reaction temperatures are shown in Table 4, and the detection results of the degradation rate of deoxynivalenol at different pH values ​​are shown in Table 5.

[0073] SEQ ID NO.2:

[0074] ATGGGCGCTCTTGATCGTCGTGGCTTCAACCGTCGTGTTCTTCTTGGCGGCGCTGCTGTTGCTACATCTCTTTCTCTTGCTCCTGAAGCTCGTTCTGATGCTGGCCCTGCTCAAGCTGCTCCTGGCGGCGAAGTTCGTCGTATCAAACTTTACGCTGAACGTCTTGCTGATGGCCAAATGGGCTACGGCCTTGAAAAAGGCCGTGCTACAATCCCTGGCCCTCTTATCGAACTTAACGAAGGCGATACACTTCATATCGAATTCGAAAACACAATGGATGTTCGTGCTTCTCTTCATGTTCATGGCCTTGATTACGAAGTTTCTTCTGATGGCACAACACTTAACAAATCTGATGTTGAACCTGGCGGCACACGTACATACACATGGCGTACACATGCTCCTGGCCGTCGTTCTGATGGCACATGGCGTGCTGGCTCTGCTGGCTACTGGCATTACCATGATCATGTTGTTGGCACAGAACATGGCACAGGCGGCATCCGTAAAGGCCTTTACGGCCCTGTTATCGTTCGTCGTAAAGGCGATGTTCTTCCTGATGCTACACATACAATCGTTTTCAACGATATGCTTATCAAC TGC CGTCCTGCTCATTCTGGCCCTAACTTCGAAGCTACAGTTGGCGATCGTGTTGAATTCGTTATGATCACACATGGCGAATACTACCATACATTCCATATGCATGGCCATCGTTGGGCTGATAACCGTACAGGCATGCTTACAGGCCCTGATGATCCTTCTCAAGTTGTT CAT AACAAAATCGTTGGCCCTGCTGATTCTTTCGGCTTCCAAGTTATCGCTGGCGAAGGCGTTGGCGCTGGCGCTTGGATGTACCATTGCCATGTTGGATCTCATTCTGATATGGGCATGGTTGGCCTTTTCCTTGTTAAAAAAACAGATGGCACAATCCCTGGCTACGAACCTCATGAACATTCTGGCCAACGTGCTGAACATCATCATTAA

[0075] Embodiment 2:

[0076] This embodiment provides a method for preparing and using a vomitoxin-degrading enzyme having an amino acid sequence as shown in SEQ ID NO.1.

[0077] The preparation method of the vomitoxin degrading enzyme provided in this embodiment is different from that in Example 1, the only difference is that the plasmid selected in the process of constructing the recombinant vector in step (2) is the Bacillus subtilis expression vector pBE-S, and then in step (3) the obtained recombinant vector is transformed into Bacillus subtilis ( Bacillus subtilis ) WB600 to obtain a recombinant strain (recorded as WB600 / pBE-S-1). Then, the recombinant strain was used to prepare a high-purity enzyme solution in the same manner as the comparative example, and then the degradation efficiency of vomitoxin was detected under different reaction times, temperatures and pH conditions to evaluate the enzyme activity. The detection results of the degradation rate of vomitoxin at different reaction times are shown in Table 3, the detection results of the degradation rate of vomitoxin at different reaction temperatures are shown in Table 4, and the detection results of the degradation rate of vomitoxin at different pH values ​​are shown in Table 5.

[0078] Embodiment 3:

[0079] This embodiment provides a method for preparing and using a vomitoxin-degrading enzyme having an amino acid sequence as shown in SEQ ID NO.1.

[0080] The preparation method of the vomitoxin degrading enzyme provided in this embodiment is different from that in Example 1, except that the plasmid selected in step (2) for constructing the recombinant vector is Bacillus licheniformis ( Bacillus licheniformis ) expression vector pHY300PLK, and then in step (3), the obtained recombinant vector is transformed into Bacillus licheniformis to obtain a recombinant strain (denoted as BL / pHY300PLK-1). Then, the recombinant strain was used to prepare a high-purity enzyme solution in the same manner as in the comparative example, and then the degradation efficiency of vomitoxin was detected under different reaction times, temperatures and pH conditions to evaluate the enzyme activity. The detection results of the degradation rate of vomitoxin at different reaction times are shown in Table 3, the detection results of the degradation rate of vomitoxin at different reaction temperatures are shown in Table 4, and the detection results of the degradation rate of vomitoxin at different pH values ​​are shown in Table 5.

[0081] Embodiment 4:

[0082] This embodiment provides a method for preparing and using a vomitoxin-degrading enzyme having an amino acid sequence as shown in SEQ ID NO.1.

[0083] The preparation method of the deoxynivalenol degrading enzyme provided in this embodiment is different from that in Example 1, the only difference is that the plasmid selected in the process of constructing the recombinant vector in step (2) is the yeast expression vector YEp352, and then in step (3) the obtained recombinant vector is transformed into Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) to obtain the recombinant strain (denoted as SC / YEp352-1). Then, the recombinant strain was used to prepare a high-purity enzyme solution in the same manner as in the comparative example, and then the degradation efficiency of vomitoxin was detected under different reaction times, temperatures and pH conditions to evaluate the enzyme activity. The detection results of the degradation rate of vomitoxin at different reaction times are shown in Table 3, the detection results of the degradation rate of vomitoxin at different reaction temperatures are shown in Table 4, and the detection results of the degradation rate of vomitoxin at different pH values ​​are shown in Table 5.

[0084] Table 3 Detection results of vomitoxin degradation rate at different reaction times

[0085]

[0086] Table 4 Detection results of DON degradation rate at different reaction temperatures

[0087]

[0088] Table 5 Detection results of DON degradation rate at different pH values

[0089]

[0090] The high-purity enzyme solutions from Examples 1-4 were mixed with corn slurry containing 20 ppm of vomitoxin, and the concentration of the enzyme in the pure enzyme was precisely controlled based on the mass of the corn slurry to ensure that the final concentration of the enzyme in the mixture was 5 ppm. Subsequently, the reaction treatment was carried out at 40°C and pH 7 for 40 hours. The test results are shown in Table 6. The degradation efficiency of the pure enzymes obtained in Examples 1-4 for the vomitoxin in corn slurry reached 82.9%, 81.2%, 82.5% and 82.8%, respectively. Compared with the use of Escherichia coli as the host bacteria in Example 1, the high-purity enzyme solutions obtained by using Bacillus subtilis, Bacillus licheniformis and Saccharomyces cerevisiae as host bacteria to produce the vomitoxin degrading enzyme with the amino acid sequence described in SEQ ID NO.1 in Examples 2-4 did not have significant differences in the degradation efficiency of the vomitoxin in corn slurry. It can be seen that the use of food-grade safe strains (Bacillus subtilis, Bacillus licheniformis or Saccharomyces cerevisiae, etc.) as host bacteria to produce vomitoxin degrading enzymes can make the application of vomitoxin degrading enzymes in feed more advantageous.

[0091] In addition, the degradation efficiency of the wild-type vomitoxin degrading enzyme high-purity enzyme solution in the comparative example and the vomitoxin degrading enzyme high-purity enzyme solution obtained in Example 1 for vomitoxin in corn steep liquor reached 35.4% and 82.9% respectively (see Table 6). As shown in Table 6, the degradation efficiency of the vomitoxin degrading enzyme obtained in Example 1 was increased by 134.18% compared with the degradation efficiency of the wild-type vomitoxin degrading enzyme obtained in the comparative example, indicating that the vomitoxin degrading enzyme provided in Example 1 greatly improved the degradation effect of vomitoxin in corn steep liquor compared with the wild-type vomitoxin degrading enzyme.

[0092] Table 6 Detection results of the degradation rate of DON in corn steep liquor by enzyme solution at 40℃ and pH 7 for 40 h

[0093]

[0094] The high-purity enzyme solutions from the comparative example and Example 1 were mixed with corn slurry containing 20 ppm of vomitoxin, respectively. Based on the mass of the corn slurry, the concentration of the enzyme in the pure enzyme was precisely controlled to ensure that the final concentration of the enzyme in the mixture was 5 ppm. Subsequently, the reaction treatment was carried out for 40 hours at 60°C and a pH of 4.0, and then the degradation rate of vomitoxin was detected. The test results showed that the degradation efficiency of the vomitoxin-degrading enzyme solution obtained in Example 1 was 362.86% higher than that of the wild-type vomitoxin-degrading enzyme solution obtained in the comparative example (see Table 7), indicating that the vomitoxin-degrading enzyme provided in Example 1 can still show a good ability to degrade vomitoxin in corn slurry under high temperature and low pH conditions, indicating that the enzyme has excellent application potential in the industrial field.

[0095] Table 7 Detection results of the degradation rate of DON in corn steep liquor by enzyme solution at 60℃ and pH 4.0 for 40 h

[0096]

[0097] In addition, various embodiments of the present invention may be combined in any way, and as long as they do not violate the concept of the present invention, they shall also be regarded as the contents disclosed by the present invention. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with the technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the definition of the claims.

Claims

1. A deoxynivalenol-degrading enzyme, characterized in that: The amino acid sequence of the vomitoxin degrading enzyme is shown in SEQ ID NO.

1.

2. A gene encoding the vomitoxin-degrading enzyme according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacteria contains the gene according to claim 2 or the recombinant vector according to claim 3.

5. An additive, characterized in that: The additive contains the DON-degrading enzyme according to claim 1.

6. A feed, characterized in that: The feed contains the vomitoxin-degrading enzyme according to claim 1 or the additive according to claim 5.

7. A method for preparing the vomitoxin-degrading enzyme according to claim 1, characterized in that: Constructing a recombinant vector containing the gene of claim 2, and introducing the recombinant vector into a host bacterium to obtain a recombinant bacterium; culturing the recombinant bacterium in a liquid culture medium to obtain a bacterial liquid, and centrifuging to obtain bacterial cells; ultrasonically disrupting and centrifuging to collect the supernatant as a crude enzyme solution; The purified enzyme solution was obtained by affinity chromatography on a Ni column.

8. Use of the vomiting detoxification enzyme according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant bacteria according to claim 4 or the additive according to claim 5, characterized in that: The application is to degrade vomitoxin in feed.

9. A method for degrading vomitoxin, characterized in that: The deoxynivalenol degrading enzyme according to claim 1 or the additive according to claim 5 is added to a sample to be treated containing deoxynivalenol, and enzymolysis is carried out for 1-60 hours under reaction conditions of 30-70° C. and pH 2-9.

10. The method according to claim 9, characterized in that The reaction conditions are 30-60° C., pH 4-9 and enzymolysis for 10-60 h.

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