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

Abstract

The present invention provides a zearalenone degrading enzyme and its encoding gene, preparation method and application. The amino acid sequence of the degrading enzyme includes: 1) a protein as shown in SEQ ID NO.1; 2) a mutant of the protein as shown in SEQ ID NO.1. The degrading enzyme and its mutant protein provided by the present application have the activity of zearalenone degrading enzyme in an acidic environment. The present invention also constructs a plasmid and an engineering strain capable of expressing the degrading enzyme, and realizes heterologous expression of the enzyme in Escherichia coli BL21 (DE3). The enzyme can be used in industrial fields such as agriculture, feed and food to reduce the harm to human and animal health caused by zearalenone pollution.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to a protein with zearalenone-degrading activity, a mutant modified based on a zearalenone-degrading enzyme, a gene encoding the protein, a recombinant vector inserted with the gene, a transformant containing the gene, a method for preparing the zearalenone-degrading enzyme, and application of the zearalenone-degrading enzyme in detoxification of agricultural products. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is an estrogen-like fungal toxin produced by Fusarium. ZEN and its reduction products have the effect of promoting animal growth. Earlier, its structural analogue zearalenol was used as a growth promoter added to feed, but its toxicity was later known. In 1988, the European Union announced a ban on the use of zearalenol as a growth promoter. ZEN has strong reproductive toxicity and can bind to estrogen receptors to show estrogen activity, causing reproductive hormone disorders in animals, thereby destroying the reproductive development system of animals. Among animals, pigs are the most sensitive to ZEN. Zearalenone can be detoxified by physical and chemical methods, but these methods will introduce many uncertain factors while detoxifying, and will also adsorb or change the nutrients in food. The biodegradation method for degrading zearalenone is mild, green and safe, and has strong specificity, and has a very broad application prospect.

[0003] In recent years, the ZEN content of cereals and their processed products detected in various regions across the country has exceeded the standard, which not only causes a waste of precious food resources, but also poses a great threat to human and animal health. At present, the research on ZEN degrading enzymes mainly focuses on the lactone hydrolase ZHD101 from Clonostachys rosea and its derivatives. This type of degrading enzyme can effectively convert ZEN into non-toxic products, and the optimal reaction pH of this type of enzyme is 8.0-10.0 (Table 1).

[0004] Table 1 Comparison of properties of ZEN lactone hydrolases from different microbial sources

[0005]

[0006]

[0007] However, in the feed industry, it is usually necessary to add 1%-3% citric acid or lactic acid to acidify the feed to compensate for the indigestion caused by lack of gastric acid in livestock, so the pH value of the feed is generally around 5.0. The existing ZEN degrading enzymes have the problem of low enzyme activity in practical applications (acidic environment). Therefore, providing a new enzyme resource that can achieve efficient degradation of zearalenone in an acidic environment has become a technical problem that needs to be solved in this field. Summary of the invention

[0008] In response to the above problems, the present invention provides a protein with the activity of degrading zearalenone, a mutant modified based on zearalenone degrading enzyme, a gene encoding the protein, a recombinant vector inserted with the gene, and a transformant containing the gene. The protein and its mutants have high enzyme activity in an acidic environment and can be used to degrade zearalenone in agricultural products and feed.

[0009] Specifically, the present application is implemented through the following technical solutions:

[0010] First, the present application provides a zearalenone-degrading enzyme, which comprises any of the following proteins:

[0011] 1) A protein having an amino acid sequence as shown in SEQ ID NO.1;

[0012] 2) a mutant of a protein whose amino acid sequence is shown in SEQ ID NO. 1, wherein the mutant protein has the activity of degrading zearalenone;

[0013] The mutant refers to a protein obtained by mutation of at least one of the amino acids at position 51 (S51E), position 105 (Q105L), position 131 (L131M), position 157 (T157S), position 164 (H164P), position 168 (N168L), position 182 (Y182W), position 184 (A184G), position 215 (A215P), position 223 (D223E), position 234 (T234R), position 241 (A241T), position 257 (A257G), position 277 (E277S), and position 286 (D286S) of the amino acid sequence shown in SEQ ID NO.1; the mutation refers to the substitution, deletion, or addition of one or two amino acids at the corresponding position.

[0014] Preferably, the above-mentioned mutant is preferably a protein obtained by mutation of at least one of the amino acids at positions 182, 184 and 257 of the amino acid sequence as shown in SEQ ID NO.1; in particular, any one of the combined mutations of Y182W and A184G, the combined mutations of Y182W and A257G, the combined mutations of A184G and A257G, and the combined mutations of Y182W, A184G and A257G, which improve the catalytic activity of the degradation enzyme at pH 5.0.

[0015] Preferably, the amino acid sequence of the mutant protein includes any one of the proteins shown in the amino acid sequences of SEQ ID NO.3 to SEQ ID NO.9.

[0016] Secondly, the present application also provides a gene encoding the above-mentioned zearalenone degrading enzyme, the nucleotide sequence of which includes any of the following:

[0017] 1) the nucleotide sequence shown in SEQ ID NO.2;

[0018] 2) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases from bases 50 to 300 of the nucleotide sequence shown in SEQ ID NO.2.

[0019] Third, the present application provides a method for preparing the above-mentioned zearalenone-degrading enzyme, and the specific steps are as follows:

[0020] (1) constructing a recombinant plasmid containing the zearalenone degrading enzyme gene, and transforming it into a host cell to obtain a transformant;

[0021] The recombinant plasmid comprises one of the following nucleotide sequences: a) the nucleotide sequence shown in SEQ ID NO.2; B) the nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases from the 50th to the 300th base of the nucleotide sequence shown in SEQ ID NO.2;

[0022] (2) culturing the transformants to induce the expression of zearalenone-degrading enzymes;

[0023] (3) Isolate and purify the zearalenone-degrading enzyme.

[0024] Preferably, in the above-mentioned method for preparing zearalenone-degrading enzyme, the host cell comprises at least one of Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae.

[0025] In the above preparation method, the construction of recombinant plasmids, preparation of transformants, expression, separation and purification techniques all adopt conventional methods in the field, such as patent CN110283804A, literature "Cloning and expression of polychlorinated biphenyl degradation genes of Rhodococcus pyridinophilus, Sun Yan et al., "China Environmental Science", 2004", "Gene cloning and recombinant expression of organophosphorus pesticide degrading enzymes, Du Xinjun et al., "China Health Inspection Journal", 2009", "Cloning and expression of 3-phenoxybenzoic acid degradation genes of Acinetobacter, Liang Junshi et al., "Progress in Biotechnology", 2015", "Expression of zearalenone degradation enzyme gene zly-6 in Bacillus subtilis, Fu Haodong et al., "Journal of Nuclear Agricultural Sciences", 2022".

[0026] Fourthly, the present application also provides the use of the above-mentioned zearalenone-degrading enzyme in degrading zearalenone in agricultural products and feed.

[0027] Preferably, the application method of the zearalenone degrading enzyme is to add the above-mentioned zearalenone degrading enzyme at an addition amount of 1% (m / m) to agricultural products or feed contaminated with (zearalenone), preferably adding 10% (m / m) or more pure water or pH 5.0-8.0 buffer solution at the same time, stirring evenly at room temperature and standing for 30 minutes to achieve the degradation of zearalenone in agricultural products or feed.

[0028] In this application, the term "agricultural products" includes materials that provide the nutritional needs of livestock and do not cause harmful phenomena under reasonable feeding, such as rice, sorghum, peanuts, corn, wheat, and local specialties of various regions.

[0029] Fifth, the present application also provides a recombinant plasmid, the recombinant plasmid comprising any one of the following nucleotide sequences: a) a nucleotide sequence as shown in SEQ ID NO.2;

[0030] b) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases from bases 50 to 300 of the nucleotide sequence shown in SEQ ID NO.2;

[0031] Sixth, the present application also provides a transformant, which comprises the above-mentioned recombinant plasmid.

[0032] In view of the problem that the existing ZEN degrading enzyme has low enzyme activity in an acidic environment, the present application screens and obtains a zearalenone degrading enzyme ZENH with high enzyme activity in an acidic environment, obtains its coding gene sequence and amino acid sequence, and designs mutations at multiple different sites of amino acids 50 to 300 for the amino acid sequence of ZENH (SEQ ID NO.1), and investigates the properties of these mutants, and finds that mutations at these sites will improve the enzyme activity, stability and optimal pH of ZENH. Compared with the existing zearalenone degrading enzyme, the zearalenone degrading enzyme ZENH and its mutants of the embodiment of the present invention can maintain high enzyme activity, or improve enzyme stability, or improve substrate adaptability under weakly acidic conditions (pH 5.0), and are more suitable for the practical application of ZEN degradation in agricultural products and feed, and provide new biological agents for the degradation of zearalenone in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention, but do not constitute a limitation of the present invention.

[0034] Figure 1 This is the SDS-PAGE purification diagram of the zearalenone-degrading enzyme ZENH;

[0035] Wherein, M: Marker; S: zearalenone-degrading enzyme ZENH.

[0036] Figure 2 Schematic diagram of the optimal reaction temperature of zearalenone-degrading enzyme ZENH.

[0037] Figure 3 Schematic diagram of the optimal reaction pH of zearalenone-degrading enzyme ZENH.

[0038] Figure 4 This is the liquid chromatogram of protein ZENH degrading zearalenone.

[0039] Figure 5 This is a schematic diagram of the sequence comparison analysis results of the degradation enzymes ZENH and ZHD101. DETAILED DESCRIPTION

[0040] The specific implementation of the present invention is described in detail below in conjunction with the experimental materials and reagents attached. The specific implementation described below is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0041] The present invention uses conventional techniques and methods used in the field of molecular biology. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field based on the technical scheme described in the present invention, without being limited to the specific embodiments of the present invention.

[0042] Strains and vectors:

[0043] The Escherichia coli expression vector pET29a(+) was stored in our laboratory, and the strain BL21(DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0044] LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0045] The preparation method of recombinant Escherichia coli is as follows:

[0046] First, the genomic DNA of the BL21 (DE3) strain was used as a template, and primers ZENH-F (nucleotide sequence as shown in SEQ ID NO.10) and ZENH-R (nucleotide sequence as shown in SEQ ID NO.11) were used to amplify the ZENH gene (nucleotide sequence as shown in SEQ ID NO.1). The PCR amplification procedure was shown in Table 2, and the PCR product was purified by a PCR product purification kit for later use.

[0047] Table 2

[0048]

[0049] The purified zearalenone degrading enzyme gene zenH and expression vector pET29a were double-digested with BamH I and HindIII to obtain sticky ends, and the degrading enzyme gene zenH and pET29a vector with the same sticky ends were connected by T4 ligase to construct the Escherichia coli expression vector pET29a-zenH. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli strain BL21-pET29a-zenH.

[0050] Example 1 Preparation of Zearalenone Degrading Enzyme ZENH

[0051] Take the recombinant E. coli BL21-pET29a-zenH, inoculate it into 100 mL LB medium, and culture it at 37°C and 200 rpm for 2-3 hours. 600 When the pH was 0.6, add IPTG at a final concentration of 0.5 mM, induce culture at 18°C ​​for 15 h, then collect the bacteria by centrifugation at 4°C for later use.

[0052] Purification of zearalenone-degrading enzyme ZENH by affinity chromatography: Resuspend the bacteria with 10mL Binding Buffer (pH 7.4), place in a beaker containing an ice-water mixture, and ultrasonically disrupt the bacteria for 10min to 15min. The instrument program is set as follows: ultrasonic for 1.5s and stop for 2s, power 350W. Centrifuge at 10000g for 15min to collect the supernatant, and the supernatant at this time is the crude enzyme solution. Take a nickel column, wash the nickel column with deionized water for 10 column volumes, and then balance the column with Binding Buffer for 5 column volumes at a flow rate of 1mL / min; then allow the supernatant to flow through the nickel column at a flow rate of 1mL / min. Finally, use the prepared Washing Buffer containing 0.3M imidazole to elute the zearalenone-degrading enzyme on the nickel column. Collect the purified zearalenone-degrading enzyme, use dialysis to remove the excess imidazole and NaCl in the solution, and the resulting protein solution is the finished ZENH enzyme solution.

[0053] The ZENH gene in this experiment can be expressed in a soluble form in E. coli BL21 (DE3). Figure 1 As shown, the results of polyacrylamide gel electrophoresis (SDS-PAGE) showed a single protein band at 40 kDa (containing the S-Tag on the vector), indicating that the degradation enzyme ZENH was successfully expressed in Escherichia coli BL21 (DE3). Figure 1 The middle lane S is the purified degradation enzyme ZENH.

[0054] The amino acid sequence of the zearalenone-degrading enzyme ZENH obtained in this example is shown in SEQ ID NO.1, and the encoding nucleotide sequence is shown in SEQ ID NO.2.

[0055] Example 2 Determination of properties of zearalenone-degrading enzyme ZENH

[0056] The enzyme activity of zearalenone-degrading enzyme ZENH was detected by high performance liquid chromatography. The specific method is as follows:

[0057] (1) Preparation of zearalenone stock solution: Weigh 1 mg of zearalenone standard and dissolve it in 100 μL of chromatographic grade methanol to prepare a stock solution with a concentration of 10 mg / mL and store at -20°C.

[0058] (2) Sample pretreatment: 2 μL of ZEN stock solution was added to 1 mL of phosphate buffer (pH 7.0) to make the final concentration of zearalenone 20 μg / mL, followed by the addition of 5 μL of purified zearalenone-degrading enzyme. The mixture was incubated at 30°C for 10 min, and an equal volume of ethyl acetate was added. The mixture was vortexed for 30 s to terminate the enzyme reaction.

[0059] (3) Sample inspection: Extract the zearalenone in the reaction solution with an equal volume of ethyl acetate, dry it with nitrogen, and then reconstitute it with 1 mL of chromatographic methanol. Pass it through a 0.22 μm filter membrane for testing. Waters high performance liquid chromatography was used to quantitatively detect the ZEN concentration, and the detection conditions were as follows: Agilent C18 chromatographic column (4.6 mm × 250 mm × 5 μm); mobile phase: methanol: water = 4:1 (v / v); flow rate: 0.8 mL / min; injection volume: 10 μL; detection wavelength: λ = 218 nm. The enzyme activity of the zearalenone-degrading enzyme ZENH was determined by comparing the spectrum with that of the zearalenone control group.

[0060] 1. Determination of the optimal reaction temperature of zearalenone-degrading enzyme ZENH

[0061] Add 2 μL of ZEN stock solution to 1 mL of 50 mM phosphate buffer (pH 7.0) to make the final concentration of zearalenone 20 μg / mL, and preheat at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C for 10 min, then add 5 μL of purified zearalenone degrading enzyme to react for 10 min, add an equal volume of ethyl acetate, and vortex for 30 seconds to terminate the enzyme reaction. After pretreating the sample according to the above method, the zearalenone content was detected by high performance liquid chromatography. The test results are as follows: Figure 2 As shown, the optimal reaction temperature of zearalenone-degrading enzyme ZENH is 55°C.

[0062] 2. Determination of the optimal reaction temperature and pH of zearalenone-degrading enzyme ZENH

[0063] 5 μL of purified ZENH was added to different pH buffers containing 20 μg / mL zearalenone for enzymatic reaction to determine its optimal pH. The selected buffers were: 50 mM sodium citrate buffer (pH 4.0-7.0, SSC), 50 mM phosphate buffer (pH 7.0-8.0, PBS), 50 mM glycine-sodium hydroxide buffer (pH 8.0-10.0, GNB). The zearalenone-degrading enzyme ZENH reacted with ZEN at 55°C for 10 min in the above buffers of different pH, and an equal volume of ethyl acetate was added and vortexed for 30 s to terminate the enzyme reaction. After the samples were pretreated according to the above method, the zearalenone content was detected by high performance liquid chromatography.

[0064] Test results such as Figure 3 As shown, the optimum reaction pH of zearalenone-degrading enzyme ZENH is 7.0, and the enzyme activity can be maintained above 80% in the pH range of 6.0 to 8.0.

[0065] Example 3 Preliminary application of zearalenone-degrading enzyme in ZEN contamination of corn distiller's grains

[0066] Naturally contaminated corn kernels were selected, and corn distiller's grains were prepared according to the method described by Ye Junfang in "Evaluation of Fermentation Quality of Fuel Ethanol Produced from Corn", in which the final concentration of zearalenone was 5 mg / kg. 10 g of corn distiller's grains were weighed and placed in a 250 mL conical flask, and 50 mL of 20 mM PBS (pH 7.0) solution was added, followed by preheating at 55 °C for 10 min. 1 mL of crude enzyme solution containing zearalenone-degrading enzyme ZENH was added to the corn distiller's grains solution, and samples were taken every 15 min to detect the ZEN content in the samples. The liquid chromatogram of zearalenone degradation by protein ZENH is shown in the figure below. Figure 4 shown.

[0067] Table 3 Degradation rate of ZEN by ZENH at different time

[0068] Time (min) Degradation rate (%) 15 47.88％±0.22 30 80.25％±0.34 45 98.5％±0.11

[0069] The degradation rates of ZEN at different incubation times are shown in Table 3 . ZENH can degrade more than 98% of ZEN after incubation for 45 min.

[0070] It can be seen that the optimum temperature of the zearalenone-degrading enzyme ZENH obtained in Example 1 is 55°C, and it still has enzyme activity at room temperature (25°C-37°C). In the detoxification treatment of corn distiller's grains, the same detoxification effect as at 55°C can be achieved by prolonging the reaction time.

[0071] The zearalenone degrading enzyme gene sequence and amino acid sequence were compared by BLAST in GenBank. Figure 5 1 is a schematic diagram of sequence comparison analysis results of the degradation enzyme ZENH and ZHD101. It can be seen that the amino acid sequence similarity between the degradation enzyme ZENH sequence and the zearalenone degradation enzyme ZHD101 from Clonostachys rosea is 15.6%, indicating that the zearalenone degradation enzyme ZENH obtained in Example 1 is a new type of ZEN degradation enzyme.

[0072] Example 4 Zearalenone-degrading enzyme with single point mutation (S51E)

[0073] 1. Construction of the mutant gene of zearalenone-degrading enzyme in corn

[0074] In order to improve the stability of the zearalenone-degrading enzyme ZENH in Example 1 and the enzyme activity under acidic conditions (pH 5.0), this example performed homology modeling on ZENH on ColabFold (AlphaFold2.ipynb-Colaboratory), created a PDB file of ZENH, analyzed the three-dimensional structure of ZENH using Pymol software, and designed amino acid mutation sites near the substrate binding site, active site, and enzyme molecule surface of ZENH, thereby realizing the modification of the enzyme.

[0075] The S51E site was subjected to point mutation, and the specific steps are as follows:

[0076] Using the recombinant plasmid pET29a-zenH inserted with the zearalenone degrading enzyme gene as a template, mutations were introduced through primers, and forward and reverse primers containing the S51E site were designed, and their nucleotide sequences were shown in SEQ ID NO.12 and SEQ ID NO.13, respectively. The template, primers and Mix were mixed to form a PCR system, and the system is shown in Table 4 below:

[0077] Table 4

[0078]

[0079] The amplification conditions of the PCR reaction are shown in Table 5 below:

[0080] Table 5

[0081]

[0082] Preparation of recombinant plasmid: Recover PCR product (6291 bp) from gel and determine product concentration;

[0083] Bluting Kination reaction: Prepare the following reaction solution (Table 6) in a microcentrifuge tube, and use the reaction to blunt the ends of the DNA fragments and phosphorylate the 5' end.

[0084] Table 6

[0085]

[0086]

[0087] Preparation of recombinant plasmid: Take 5 μL of DNA fragment after Bluting Kination reaction, add 5 μL Solution I, mix well, and react at 16℃ for 1 hour;

[0088] Transformation and screening: The recombinant plasmid was transformed into E. coli BL21 (DE3) by heat shock method, cultured in a 37°C incubator for 14 h, single clones were selected, and positive clones were selected by bacterial liquid PCR;

[0089] Induce expression: activate positive clones and culture at 37℃ and 200rpm for 2-3h. 600 When the pH value was 0.6, add IPTG at a final concentration of 0.5 mM, induce culture at 18°C ​​for 15 h, and collect the cells by centrifugation at 4°C for later use;

[0090] Preparation of crude enzyme solution: resuspend the bacteria in 50 mM PBS (pH 7.4), disrupt the bacteria in an ice-water bath using an ultrasonic disruptor, and obtain the crude enzyme solution after centrifugation;

[0091] Activity verification: 2 μL of ZEN stock solution was added to 1 mL of 50 mM phosphate buffer (pH 5.0) to make the final concentration of zearalenone 20 μg / mL, followed by adding 5 μL of crude enzyme solution and reacting at 55 °C for 10 min. An equal volume of ethyl acetate was added and vortexed for 30 s to terminate the enzyme reaction.

[0092] Example 5 Zearalenone-degrading enzyme with a single point mutation (Q105L);

[0093] To introduce mutations, forward and reverse primers containing the Q105L site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 14 and SEQ ID NO. 15, respectively. Other methods and steps were the same as those in Example 4.

[0094] Example 6 Zearalenone-degrading enzyme with a single point mutation (L131M);

[0095] To introduce mutations, forward and reverse primers containing the L131M site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 16 and SEQ ID NO. 17, respectively. Other methods and steps were the same as those in Example 4.

[0096] Example 7 Zearalenone-degrading enzyme with single point mutation (T157S);

[0097] To introduce mutations, forward and reverse primers containing the T157S site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively. Other methods and steps were the same as those in Example 4.

[0098] Example 8: Zearalenone-degrading enzyme with single point mutation (H164P);

[0099] To introduce mutations, forward and reverse primers containing the H164P site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 20 and SEQ ID NO. 21, respectively. Other methods and steps were the same as those in Example 4.

[0100] Example 9: Zearalenone-degrading enzyme with single point mutation (N168L);

[0101] To introduce mutations, forward and reverse primers containing the N168L site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 22 and SEQ ID NO. 23, respectively. Other methods and steps were the same as in Example 4.

[0102] Example 10 Zearalenone-degrading enzyme with a single point mutation (Y182W);

[0103] To introduce mutations, forward and reverse primers containing the Y182W site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 24 and SEQ ID NO. 25, respectively. Other methods and steps were the same as in Example 4.

[0104] The amino acid sequence of the mutant obtained in this example is shown in SEQ ID NO.3.

[0105] Example 11 Zearalenone-degrading enzyme with a single point mutation (A184G);

[0106] To introduce mutations, forward and reverse primers containing the A184G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 26 and SEQ ID NO. 27, respectively. Other methods and steps were the same as in Example 4.

[0107] The amino acid sequence of the mutant obtained in this example is shown in SEQ ID NO.4.

[0108] Example 12 Zearalenone-degrading enzyme with a single point mutation (A215P);

[0109] To introduce mutations, forward and reverse primers containing the A215P site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 28 and SEQ ID NO. 29, respectively. Other methods and steps were the same as in Example 4.

[0110] Example 13: Zearalenone-degrading enzyme with single point mutation (D223E);

[0111] To introduce mutations, forward and reverse primers containing the D223E site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 30 and SEQ ID NO. 31, respectively. Other methods and steps were the same as in Example 4.

[0112] Example 14 Zearalenone-degrading enzyme with a single point mutation (T234R);

[0113] To introduce mutations, forward and reverse primers containing the T234R site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 32 and SEQ ID NO. 33, respectively. Other methods and steps were the same as those in Example 4.

[0114] Example 15 Zearalenone-degrading enzyme with a single point mutation (A241T);

[0115] To introduce mutations, forward and reverse primers containing the A241T site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 34 and SEQ ID NO. 35, respectively. Other methods and steps were the same as those in Example 4.

[0116] Example 16 Zearalenone-degrading enzyme with a single point mutation (A257G);

[0117] To introduce mutations, forward and reverse primers containing the A257G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 36 and SEQ ID NO. 37, respectively. Other methods and steps were the same as in Example 4.

[0118] The amino acid sequence of the mutant obtained in this example is shown in SEQ ID NO.5.

[0119] Example 17 Zearalenone-degrading enzyme with a single point mutation (E277S);

[0120] To introduce mutations, forward and reverse primers containing the E277S site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 38 and SEQ ID NO. 39, respectively. Other methods and steps were the same as those in Example 4.

[0121] Example 18: Zearalenone-degrading enzyme with a single point mutation (D286S);

[0122] To introduce a mutation, forward and reverse primers containing the D286S site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 40 and SEQ ID NO. 41, respectively. Other methods and steps were the same as in Example 4.

[0123] The results of enzyme activity detection are shown in Table 7 (the enzyme activity detection method is the same as in Example 2):

[0124] Table 7 Enzyme activity of zearalenone-degrading enzyme mutants

[0125]

[0126] As can be seen from Table 7, with the unmutated degradation enzyme (the degradation enzyme ZENH obtained in Example 1) as the control, the enzyme activity of the transformant with a single point mutation was improved compared with the template, but the expected effect was not achieved. Although the enzyme activity of the mutants of Example 4 (S51E), Example 7 (T157S), and Example 13 (D223E) decreased, the substrate range was expanded after mutation, and α-zearalenol could be degraded; although the catalytic activity of the mutant of Example 14 (T234R) decreased, the enzyme stability after mutation was significantly improved; generally speaking, it is difficult for a mutant with a single point mutation to have a very significant improvement in enzyme performance compared with the parent, and a better mutant can be obtained by combining mutation points.

[0127] Example 19 Zearalenone-degrading enzyme with two point mutations (Y182W+A184G);

[0128] Using the single point mutation plasmid of Example 10 (Y182W) as a template, the mutation was introduced by primers, and forward and reverse primers containing the A184G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 42 and SEQ ID NO. 43, respectively. Other methods and steps were the same as in Example 4.

[0129] The amino acid sequence of the mutant protein obtained in this example is shown in SEQ ID NO.6.

[0130] Example 20 Zearalenone-degrading enzyme with two point mutations (Y182W+A257G);

[0131] Using the single point mutation plasmid of Example 10 (Y182W) as a template, the mutation was introduced by primers, and forward and reverse primers containing the A257G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 44 and SEQ ID NO. 45, respectively. Other methods and steps were the same as in Example 4.

[0132] The amino acid sequence of the mutant protein obtained in this example is shown in SEQ ID NO.7.

[0133] Example 21 Zearalenone-degrading enzyme with two point mutations (A184G+A257G);

[0134] Using the single point mutation plasmid of Example 8 (A184G) as a template, the mutation was introduced by primers, and forward and reverse primers containing the A257G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 46 and SEQ ID NO. 47, respectively. Other methods and steps were the same as in Example 4.

[0135] The amino acid sequence of the mutant protein obtained in this example is shown in SEQ ID NO.8.

[0136] Example 22 Zearalenone-degrading enzyme with three point mutations (Y182W+A184G+A257G);

[0137] Using the double-point mutation plasmid of Example 17 (Y182W+A184G) as a template, mutations were introduced by primers, and forward and reverse primers containing the A257G site were designed, and the nucleotide sequences of the primers were shown in SEQ ID NO. 48 and SEQ ID NO. 49, respectively. Other methods and steps were the same as in Example 4.

[0138] The amino acid sequence of the mutant protein obtained in this example is shown in SEQ ID NO.9.

[0139] Enzyme activity detection is shown in Table 8:

[0140] Table 8 Enzyme activity of zearalenone-degrading enzyme mutants

[0141]

[0142] As shown in Table 8, among the multiple point mutations, only the combination of double point mutations Y182W+A184G can significantly improve the enzyme activity of zearalenone-degrading enzyme ZENH at pH 5.0, while the introduction of A257G mutation site on the basis of single point mutation cannot significantly improve the enzyme activity of ZENH under acidic conditions.

[0143] Example 23 Toxicity evaluation of zearalenone-degrading enzyme and its mutants in mice

[0144] The test was conducted with reference to the limit method in the "National Food Safety Standard Acute Oral Toxicity Test". 72 SPF male KM mice weighing 18-22g were randomly divided into a control group and 8 treatment groups, with 8 mice in each group. The treatment group was gavaged with the saline solution of the zeaxenone-degrading enzyme ZENH or its mutant obtained in the above example (with a protein concentration of 50mg / mL), and the control group was gavaged with an equal volume of saline at 9:00 and 15:00 every day, and the test period was 10d. The temperature in the experimental mouse breeding room was maintained at (22±3)℃, the relative humidity was 40%-70%, and the light and dark were 12h each; they were all fed with standard mouse food, and the sterilized bedding was changed every 2d. After the gavage, the weight, poisoning symptoms, and number of deaths of the mice were observed and recorded.

[0145] Table 9 Mouse acute test measurement analysis table

[0146]

[0147]

[0148] As shown in Table 9, the lethal dose (LD 50) is higher than 800 mg / kg.BW. According to the acute toxicity dose classification standard, zearalenone-degrading enzyme is actually non-toxic under the test conditions.

[0149] Example 24 Evaluation of Zearalenone Degrading Enzyme on Growth Performance of Mice

[0150] The test was conducted with reference to the limit method in the "National Food Safety Standard Acute Oral Toxicity Test". 60 SPF male KM mice weighing 18-22g were randomly divided into a control group, a low-dose group, a medium-dose group, and a high-dose group, with 5 replicates in each group and 3 mice in each replicate. The control group (daily diet) was fed with DDGS (purchased from Shijiazhuang Jiahui Feed Factory), while the low-dose group, the medium-dose group, and the high-dose group were mixed with 25%, 50%, and 100% of DDGS treated with zearalenone degrading enzyme ZENH in the control group diet, respectively. The pre-test period was 5d and the test period was 28d. Feeding management was the same as in Example 23. The mice were weighed on an empty stomach before the test and on the 14th and 28th days of the test. The daily feed consumption, diarrhea number, and death number were recorded, and the average daily weight gain, diarrhea rate, and mortality rate from 0 to 14d and 15 to 28d were calculated.

[0151] The preparation method of the above-mentioned "DDGS treated with zearalenone-degrading enzyme" is as follows: add zearalenone-degrading enzyme ZENH to DDGS at an addition amount of 1% (m / m), and add 10% (m / m) pure water at the same time, stir evenly at room temperature and let stand for 30 minutes to obtain DDGS treated with zearalenone-degrading enzyme. In the experiment, the feed was then dried and added to the diet according to the proportion.

[0152] Table 10 Effect of zearalenone-degrading enzyme on growth performance of KM mice

[0153]

[0154] As shown in Table 10, there was no significant difference in the average daily weight gain of each group of KM mice fed with corn distiller's grains treated with zearalenone-degrading enzyme, and the diarrhea rate and mortality rate were both 0. Therefore, the zearalenone-degrading enzyme had no side effects such as toxicity to mice.

[0155] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0156] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0157] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by this patent. Sequence Listing <110> Jiangsu Academy of Agricultural Sciences <120> A corn zearalenone-degrading enzyme and its encoding gene, preparation method and application <130> ywx0-01-20220510-01-NO0020 <141> 2022-05-10 <160> 49 <170> SIPOSequenceListing 1.0 <210> 1 <211> 316 <212> PRT <213> Artificial Sequence <400> 1 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Tyr Glu Ala Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Ala Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 2 <211> 951 <212> DNA <213> Artificial Sequence <400> 2 atgaacgaca ggtcccccac ccgacacgga gcaccggacg acgtgttcgt cgcgcacggc 60 ttcgaggaga agctcgtgaa cctcggcgag atcgacatga actacgcgga ggccggatca 120 cccacgaagc ccgccctgct tctcctgccg tcccagagcg agtcgtggtg gggctacgaa 180 gaggtcatgc atctgctcac ggacgacttc catgtcttcg cggtggacat gcgcggacag 240 gggcggagca cgtggactcc gggccgatac agcctcgaca acttcggcaa cgacctcgtc 300 cggttcatcg accaggtgat cggtcgcccc gtcatcgtgg cgggggaactc gtcgggcgga 360 ctgatcgccg cgtggctggc ggcgtactcc ctcccgggac agatccgtgc cgcgttcgcc 420 gcggatgctc ccttcttcgc ctccgagctc actccgaagg tcggacacac gatccgacag 480 gctgccggcc acatcttcgt caactggcgt gacttctcg gagaccagtg gtgcgtgggg 540 gactatgagg cgtacctgaa ggccatgcgc aactccgaga tccccatgct ccggcaggtg 600 ccgctccccg acgaagcccc gcagaacctg aaggatacg acgccgagtg ggctcgagcc 660 ttctacgacg gcacggtcgc ccagacctgc cctcaccaca cgatgctggc gcaggtcaag 720 gctcccgtcc tcgtgacgca ccacttcgc ctcatcgacc ccaccaccgc tggactgatg 780 ggcgccatgt cggccctcca ggccgagaag gcgatggagc tcatgcgtga agccggcgtg 840 aaggtcgact acgtcgatgc gcccgacgcg ccgcacatca tgcacgcgct cgagcccgag 900 cgctacgtcg gcatcctgcg ggactgggtc tcgacgctgc cgcaggcctg a 951 <210> 3 <211> 316 <212> PRT <213> Artificial Sequence <400> 3 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Trp Glu Ala Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Ala Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 4 <211> 316 <212> PRT <213> Artificial Sequence <400> 4 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Tyr Glu Gly Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Ala Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 5 <211> 316 <212> PRT <213> Artificial Sequence <400> 5 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Tyr Glu Ala Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Gly Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 6 <211> 316 <212> PRT <213> Artificial Sequence <400> 6 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Trp Glu Gly Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Ala Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 7 <211> 316 <212> PRT <213> Artificial Sequence <400> 7 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Trp Glu Ala Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Gly Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 8 <211> 316 <212> PRT <213> Artificial Sequence <400> 8 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Tyr Glu Gly Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Gly Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 9 <211> 316 <212> PRT <213> Artificial Sequence <400> 9 Met Asn Asp Arg Ser Pro Thr Arg His Gly Ala Pro Asp Asp Val Phe 1 5 10 15 Val Ala His Gly Phe Glu Glu Lys Leu Val Asn Leu Gly Glu Ile Asp 20 25 30 Met Asn Tyr Ala Glu Ala Gly Ser Pro Thr Lys Pro Ala Leu Leu Leu 35 40 45 Leu Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Val Met His 50 55 60 Leu Leu Thr Asp Asp Phe His Val Phe Ala Val Asp Met Arg Gly Gln 65 70 75 80 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 85 90 95 Asn Asp Leu Val Arg Phe Ile Asp Gln Val Ile Gly Arg Pro Val Ile 100 105 110 Val Ala Gly Asn Ser Ser Gly Gly Leu Ile Ala Ala Trp Leu Ala Ala 115 120 125 Tyr Ser Leu Pro Gly Gln Ile Arg Ala Ala Phe Ala Ala Asp Ala Pro 130 135 140 Phe Phe Ala Ser Glu Leu Thr Pro Lys Val Gly His Thr Ile Arg Gln 145 150 155 160 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Phe Leu Gly Asp Gln 165 170 175 Trp Cys Val Gly Asp Trp Glu Gly Tyr Leu Lys Ala Met Arg Asn Ser 180 185 190 Glu Ile Pro Met Leu Arg Gln Val Pro Leu Pro Asp Glu Ala Pro Gln 195 200 205 Asn Leu Lys Glu Tyr Asp Ala Glu Trp Ala Arg Ala Phe Tyr Asp Gly 210 215 220 Thr Val Ala Gln Thr Cys Pro His His Thr Met Leu Ala Gln Val Lys 225 230 235 240 Ala Pro Val Leu Val Thr His His Phe Arg Leu Ile Asp Pro Thr Thr 245 250 255 Gly Gly Leu Met Gly Ala Met Ser Ala Leu Gln Ala Glu Lys Ala Met 260 265 270 Glu Leu Met Arg Glu Ala Gly Val Lys Val Asp Tyr Val Asp Ala Pro 275 280 285 Asp Ala Pro His Ile Met His Ala Leu Glu Pro Glu Arg Tyr Val Gly 290 295 300 Ile Leu Arg Asp Trp Val Ser Thr Leu Pro Gln Ala 305 310 315 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <400> 10 cgggatccat gaacgacagg tcccccac 28 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <400> 11 cccaagcttg gcctgcggca gcgtc 25 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 gaacagagcg agtcgtggtg 20 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <400> 13 cggcaggaga agcagg 16 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 ctcgtgatcg gtcgccccgt 20 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <400> 15 gtcgatgaac cggacgag 18 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 atgccgggac agatccgtgc 20 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <400> 17 ggagtacgcc gccagc 16 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 tccatccgac aggctgccg 19 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <400> 19 gtgtccgacc ttcggagtg 19 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <400> 20 cccatcttcg tcaactggcg tg 22 <210> twenty one <211> 15 <212> DNA <213> Artificial Sequence <400> twenty one gccggcagcc tgtcg 15 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two ctctggcgtg acttcctcgg a 21 <210> twenty three <211> 17 <212> DNA <213> Artificial Sequence <400> twenty three gacgaagatg tggccgg 17 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty four tgggaggcgt acctgaaggc c 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 gtcccccacg caccactggt 20 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <400> 26 gggtacctga aggccatgcg c 21 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <400> 27 ctcatagtcc cccacgc 17 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <400> 28 ttcgagtggg ctcgagcct 19 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 gtcgtactcc ttcaggttct 20 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <400> 30 gagggcacgg tcgcccaga 19 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <400> 31 gtagaaggct cgagcccac 19 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <400> 32 cgcatgctgg cgcaggtcaa g 21 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <400> 33 gtggtgaggg caggtctg 18 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <400> 34 acgcccgtcc tcgtgacgc 19 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <400> 35 cttgacctgc gccagcat 18 <210> 36 <211> twenty one <212> DNA <213> Artificial Sequence <400> 36 ggcggactga tgggcgccat g 21 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <400> 37 ggtggtgggg tcgatgagg 19 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <400> 38 tcggccggcg tgaaggtcg 19 <210> 39 <211> 17 <212> DNA <213> Artificial Sequence <400> 39 acgcatgagc tccatcg 17 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <400> 40 tcggcgcccg acgcgccg 18 <210> 41 <211> twenty three <212> DNA <213> Artificial Sequence <400> 41 gacgtagtcg accttcacgc cgg 23 <210> 42 <211> twenty one <212> DNA <213> Artificial Sequence <400> 42 gggtacctga aggccatgcg c 21 <210> 43 <211> 17 <212> DNA <213> Artificial Sequence <400> 43 ctcccagtcc cccacgc 17 <210> 44 <211> twenty one <212> DNA <213> Artificial Sequence <400> 44 ggcggactga tgggcgccat g 21 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <400> 45 ggtggtgggg tcgatgagg 19 <210> 46 <211> twenty one <212> DNA <213> Artificial Sequence <400> 46 ggcggactga tgggcgccat g 21 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <400> 47 ggtggtgggg tcgatgagg 19 <210> 48 <211> twenty one <212> DNA <213> Artificial Sequence <400> 48 ggcggactga tgggcgccat g 21 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <400> 49 ggtggtgggg tcgatgagg 19

Claims

1. A zearalenone-degrading enzyme, It is characterized in that The degrading enzyme is any of the following: 1) A protein having an amino acid sequence as shown in SEQ ID NO.1; 2) a mutant of the protein whose amino acid sequence is shown in SEQ ID NO.1; The mutant is at least one of the proteins having amino acid sequences as shown in SEQ ID NO.3 to SEQ ID NO.

9.

2. A gene encoding a zearalenone-degrading enzyme in corn, It is characterized in that The nucleotide sequence of the coding gene is any one of the following: 1) The nucleotide sequence shown in SEQ ID NO.2; 2) The gene encoding the protein whose amino acid sequence is shown as SEQ ID NO.3 to SEQ ID NO.

9.

3. A method for preparing a zearalenone-degrading enzyme, It is characterized in that The specific steps are as follows: (1) constructing a recombinant plasmid containing a zearalenone-degrading enzyme gene, and transforming it into a host cell to obtain a transformant; the recombinant plasmid contains the coding gene as described in claim 2; (2) Cultivating transformants and inducing the expression of zearalenone-degrading enzymes; (3) Isolate and purify the zearalenone-degrading enzyme.

4. The method for preparing the zearalenone-degrading enzyme according to claim 3, It is characterized in that The host cell is at least one of Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae.

5. Use of the zearalenone degrading enzyme according to claim 1 in degrading zearalenone in agricultural products or feed.

6. The use according to claim 5, It is characterized in that The application refers to adding the zearalenone degrading enzyme to agricultural products or feed at a mass ratio of 0.1%-5%, thereby completing the degradation of zearalenone.

7. A recombinant plasmid comprising the coding gene as claimed in claim 2.

8. A transformant comprising the recombinant plasmid according to claim 7.

Images