Trichothecene toxin degrading enzyme ZHDDH4 as well as mutant and application thereof

Abstract

The invention discloses a degrading enzyme ZHDDH4 with activity of degrading trichothecene toxin as well as a mutant and application of the degrading enzyme ZHDDH4. The invention firstly provides the trichothecene toxin degrading enzyme ZHDDH4, and the amino acid sequence of the trichothecene toxin degrading enzyme ZHDDH4 is shown as SEQ ID No. 1 or SEQ ID No. 2. According to the invention, the trichothecenes toxin degrading enzyme ZHDDH4 is further subjected to single-site mutation of any one of amino acids of A43M, N95M, H140F, G278L or D515R to obtain a single-site mutant. The trichothecenes toxin degrading enzyme ZHDDH4 or each mutant provided by the invention has relatively high activity of degrading the trichothecenes toxin, and can be used for biodegradation of the trichothecenes toxin in various biological matrixes; the method has a huge application prospect in the fields of enzyme preparation preparation, feed, food, Chinese herbal medicine, biomass energy production and sewage treatment.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to a coding gene and application of a trichothecene toxin-degrading enzyme ZHDDH4 and its mutants, especially its application in the biodegradation of trichothecene toxins such as vomitoxin, 15-acetyldeoxynivalenol, and nivalenol in the processes of feed, food, Chinese herbal medicine, biomass energy production, and sewage treatment. Background Art

[0002] Trichothecenes are the most important and numerous class of toxins produced by Fusarium spp., primarily a group of structurally related sesquiterpenoid mycotoxins. All trichothecenes share a double bond at carbon positions 9 and 10 and an epoxide cluster at carbon positions 12 and 13, but the epoxidation varies. They can be divided into four main subclasses, of which Classes A and B are the most important. Class A trichothecenes, primarily produced by Fusarium sporeans and Fusarium pyrifolium, include T-2 toxin, HT-2 toxin, and fusaric acid. Class B trichothecenes, primarily produced by Fusarium graminearum and Fusarium graminearum, include deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3ADON) or 15-acetyl-deoxynivalenol (15ADON), nivalenol (NIV), and fusarinone-X (FUS). Trichothecenes have a wide range of toxic effects on humans and animals and can severely damage the immune system. Low doses of trichothecenes can rapidly activate the early expression of immune-related inflammatory cytokine genes, causing immune stimulation. High doses of trichothecenes can severely damage immune organs such as the lymph nodes and spleen, induce leukocyte apoptosis, and lead to a decrease in leukocyte count, causing immunosuppression. Trichothecenes are widespread contaminants and pose significant risks to animals. Therefore, the feed and food industries require efficient and safe mycotoxin detoxification methods. Bioenzymes have attracted considerable attention due to their high catalytic efficiency, safety, environmental friendliness, and minimal impact on feed and food matrices. Currently, few reports exist on enzymes that degrade trichothecenes. Studies have shown that DON is converted to its epimer, 3-epi-DON, via isomerization of the C3 hydroxyl group. This isomerization process consists of two steps: the first is the dehydrogenation of DON to 3-keto-DON, primarily involving enzymes such as the PQQ-dependent dehydrogenase DepA and the aldehyde-keto reductase AKR18A1. The second step is the hydrogenation reduction of 3-keto-DON to 3-epi-DON, primarily involving enzymes such as the dehydratase DepB and the aldehyde-keto reductase AKR13B2.

[0003] Most existing trichothecene-degrading enzymes suffer from low catalytic efficiency, low expression levels, and poor temperature tolerance, limiting their industrial application. Therefore, it is necessary to identify new trichothecene-degrading enzymes with high catalytic activity and high temperature tolerance to promote their widespread application in food, feed, traditional Chinese medicine, biomass energy production, and wastewater treatment. Summary of the Invention

[0004] The object of the present invention is to provide a trichothecene toxin-degrading enzyme ZHDDH4.

[0005] Another object of the present invention is to provide a mutant of the trichothecene toxin-degrading enzyme ZHDDH4.

[0006] Another object of the present invention is to provide a gene encoding the above-mentioned trichothecene toxin degrading enzyme ZHDDH4 or a mutant thereof.

[0007] Another object of the present invention is to provide a recombinant expression vector comprising the gene encoding the trichothecene toxin-degrading enzyme ZHDDH4 or its mutant, and a recombinant host cell containing the recombinant expression vector.

[0008] Another object of the present invention is to provide a method for preparing the trichothecene toxin-degrading enzyme ZHDDH4 or a mutant thereof.

[0009] Another object of the present invention is to provide the use of the trichothecene toxin degrading enzyme ZHDDH4 or its mutant in the biodegradation of trichothecene toxins.

[0010] Another object of the present invention is to provide the use of the above-mentioned trichothecene toxin degrading enzyme ZHDDH4 or its mutants in the fields of enzyme preparation, food, feed, Chinese herbal medicine, biomass energy production and sewage treatment.

[0011] Another object of the present invention is to provide the use of the above-mentioned trichothecene toxin degrading enzyme ZHDDH4 or its mutants in the biodegradation of vomitoxin, 15-acetyldeoxynivalenol and nivalenol in food, feed, Chinese herbal medicine, biofuel production and sewage treatment processes.

[0012] To achieve the above objectives, the present invention provides the following technical solutions:

[0013] One aspect of the present invention is to discover a trichothecene toxin degrading enzyme ZHDDH4 capable of degrading trichothecene toxins from the genome of a nitrate-reducing bacterium using genome sequencing technology, and its amino acid sequence is shown in SEQ ID No. 1 or SEQ ID NO. 2.

[0014] The trichothecene toxin degrading enzyme ZHDDH4 encoding gene described in the present invention also falls within the protection scope of the present invention.

[0015] Another aspect of the present invention is to provide a single-site mutant of the trichothecene toxin degrading enzyme ZHDDH4, wherein the single-site mutant of the trichothecene toxin degrading enzyme ZHDDH4 is a single-site mutant obtained by subjecting the amino acid sequence of the trichothecene toxin degrading enzyme ZHDDH4 to any one of the amino acid single-site mutations A43M, N95M, H140F, G278L or D515R; preferably, the mutant is a single-site mutant obtained by subjecting the amino acid sequence of the trichothecene toxin degrading enzyme ZHDDH4 to any one of the amino acid single-site mutations N95M, H140F or D515R; more preferably, the mutant is a single-site mutant obtained by subjecting the amino acid sequence of the trichothecene toxin degrading enzyme ZHDDH4 to the N95M single-site mutation.

[0016] The amino acid single-site mutation "N95M" of the present invention indicates that the 95th amino acid of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 is mutated from asparagine (N) to methionine (M); the expressions of the remaining single-site mutations are similar.

[0017] The coding genes of the single-site mutants described in the present invention also fall within the scope of protection of the present invention.

[0018] Another aspect of the present invention is to provide a recombinant expression vector or a recombinant host cell containing the trichothecene toxin degrading enzyme ZHDDH4 encoding gene or each mutant encoding gene of the trichothecene toxin degrading enzyme ZHDDH4; wherein the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0019] Another aspect of the present invention is to provide a method for preparing a trichothecene toxin degrading enzyme ZHDDH4 or a mutant of a trichothecene toxin degrading enzyme ZHDDH4.

[0020] include:

[0021] (1) operably connecting a gene encoding a trichothecene toxin degrading enzyme ZHDDH4 or a gene encoding a mutant of a trichothecene toxin degrading enzyme ZHDDH4 with an expression regulatory element to construct a recombinant expression vector;

[0022] (2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying the recombinant protein.

[0023] The present invention also provides the use of the above-mentioned trichothecene toxin degrading enzyme ZHDDH4 and its respective single-site mutants, especially in the biodegradation of trichothecene toxins. The trichothecene toxins include but are not limited to vomitoxin, 15-acetyldeoxynivalenol and nivalenol.

[0024] Advantages and beneficial effects of the present invention:

[0025] The trichothecene toxin degrading enzyme ZHDDH4 or each unit point mutant of the trichothecene toxin degrading enzyme ZHDDH4 provided by the present invention has high activity and strong biodegradation ability for trichothecene toxins such as vomitoxin, 15-acetyldeoxynivalenol, and nivalenol, and has broad application prospects in the production of enzyme preparations, food, feed, Chinese herbal medicine, biomass energy production or mycotoxin biodegradation in sewage treatment processes.

[0026] Definitions of terms used in this invention

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally understood by those skilled in the art.

[0029] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome.

[0030] The term "transformation" refers to the process by which a eukaryotic cell acquires new genetic markers as a result of the incorporation of foreign DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art:

[0032] Figure 1 Shown is the SDS-PAGE electrophoresis analysis of the purified expression product of the recombinant plasmid pET-31b-ZHDDH4;

[0033] Figure 2The figure shows the degradation of vomitoxin by trichothecene toxin-degrading enzyme ZHDDH4 under different temperature and pH conditions;

[0034] Figure 3 Shown is the stability effect diagram of trichothecene toxin degrading enzyme ZHDDH4 at different temperatures and pH;

[0035] Figure 4 Shown are LC-MS spectra of the degradation of 15-acetyldeoxynivalenol by the trichothecene toxin-degrading enzyme ZHDDH4 and its single-site mutant N95M;

[0036] Figure 5 Shown are the LC-MS spectra of nivalenol degradation by the trichothecene toxin-degrading enzyme ZHDDH4 and its single-site mutant N95M. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific embodiments. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0038] Example 1 Acquisition and expression of trichothecene toxin-degrading enzyme ZHDDH4

[0039] The optimized nucleotide sequence was artificially synthesized according to conventional techniques in the art, and the gene was inserted into the vector pET-31b.

[0040] The plasmid pET-31b-ZHDDH4 containing the ZHDDH4 gene was transformed into Escherichia coli competent cells DH5α, and ampicillin resistance was screened. Positive transformants were picked, the recombinant plasmid was extracted, and single and double enzyme digestion verification and sequencing were performed to confirm that the correct recombinant strain was constructed. The correct recombinant plasmid was then transformed into Escherichia coli TSBL21 (DE3) pLysS.

[0041] The recombinant Escherichia coli TSBL21 (DE3) pLysS transformed with the pET-31b-ZHDDH4 plasmid was inoculated into 5 mL of liquid LB medium for activation overnight, and then transferred to a 500 mL Erlenmeyer flask with a liquid volume of 1 L at a ratio of 1:100. The culture was carried out at 180 r / min and 37 ° C until the OD600 reached 0.7. A final concentration of 0.4 mM IPTG was added to induce protein expression.

[0042] The fermentation broth was collected, centrifuged at 4°C, 12000r / min for 30min, and the supernatant was discarded; the bacteria were resuspended with PBS solution of pH 7.4, centrifuged at 4°C, 12000r / min for 30min, the supernatant was discarded, and the washing of the bacteria was repeated three times. The bacterial cells were then resuspended in binding buffer, ultrasonically disrupted, centrifuged at 4°C, 12000r / min for 10min, the supernatant was collected and filtered. Since the expressed ZHDDH4 protein has 6 His tags at the C-terminus, a nickel ion affinity chromatography column was used to purify the protein. For the steps of equilibration, loading, elution, etc., please refer to the Qiagen user manual. The purified protein was ultrafiltered with a cutoff tube (30kDa) to remove the imidazole contained therein, and the target band was detected by SDS-PAGE. The results are shown as follows. Figure 1 As shown, lane 4 is the expression product, and the arrow indicates the target band, which indicates that the molecular weight of the protein expressed by the recombinant strain is about 63 kDa, which is consistent with the theoretical molecular weight.

[0043] Example 2 Determination of the Optimal Reaction Temperature and pH for Degradation of DON by Trichothecene Degrading Enzyme ZHDDH4

[0044] Solid DON was dissolved in acetonitrile to create a 5 mg / mL stock solution, and the prosthetic group pyrroloquinoline quinone (PQQ) was dissolved in ultrapure water to create a 10 mM stock solution. The effects of different temperatures on the degrading activity of the trichothecene toxin-degrading enzyme ZHDDH4 (ZHDDH4) were determined using the following 500 μL reaction system: 200 μL sodium phosphate buffer (100 mM, pH 7), 270 μL purified ZHDDH4 protein (100 μg), 25 μL PQQ solution, and 5 μL DON solution. A control without ZHDDH4 protein was used. Reactions were carried out at different temperatures (20, 25, 30, 35, 40, and 45°C) for 24 hours. The reaction was terminated by adding 500 μL of methanol, centrifuged at 12,000 rpm for 1 minute, and the supernatant filtered through a Millex-GV filter (0.22 μm). The residual DON content in the reaction was determined by high-performance liquid chromatography.

[0045] The chromatographic conditions for the high-performance liquid chromatography detection of vomitoxin were as follows: chromatographic column: Agilent C18 column, 4.6 mm × 150 mm × 5 μm; methanol-water (15:85); flow rate: 1 mL / min; pump pressure: 78 bar; injection volume: 20 μL; UV detector detection wavelength: λ = 218 nm; acquisition time: 10 min.

[0046] Degradation rate of DON (%) = (1-the amount of DON remaining in the treatment group / the amount of DON in the control group) × 100%

[0047] The degradation rate of DON at the optimal temperature was 100%, and the relative activity of trichothecene-degrading enzyme ZHDDH4 in degrading DON under other conditions was calculated.

[0048] The results are as follows Figure 2 As shown in A, the optimal temperature for the degradation of vomitoxin by the trichothecene toxin-degrading enzyme ZHDDH4 is 30℃.

[0049] To test the effect of different pH conditions on the degrading activity of the trichothecene toxin-degrading enzyme ZHDDH4 on vomitoxin, a 500 μL reaction system was used: 200 μL of different pH buffers (100 mM sodium citrate buffer, pH 4-6; 100 mM sodium phosphate buffer, pH 7-8; 100 mM glycine-NaOH buffer, pH 9-10), 270 μL of purified ZHDDH4 protein (100 μg), 25 μL of prosthetic group PQQ solution, and 5 μL of vomitoxin solution. The reaction was incubated at 37°C for 24 hours, then terminated with 500 μL of methanol. The reaction was centrifuged at 12,000 rpm for 1 minute, and the supernatant was filtered through a Millex-GV filter (0.22 μm). The residual vomitoxin content in the system was determined by high-performance liquid chromatography.

[0050] The degradation rate of DON at the optimal pH was 100%, and the relative activity of trichothecene-degrading enzyme ZHDDH4 in degrading DON under other conditions was calculated.

[0051] The results are as follows Figure 2 As shown in B, the optimal pH for the degradation of vomitoxin by the trichothecene toxin-degrading enzyme ZHDDH4 is 7.0.

[0052] Example 3 Temperature and pH Stability of Trichothecene Degrading Enzyme ZHDDH4

[0053] To test the temperature stability of the trichothecene-degrading enzyme ZHDDH4, the residual activity of the recombinant ZHDDH4 protein in degrading vomitoxin was determined after treatment at different temperatures and for different times. Six temperature levels (25, 30, 35, 40, 45, and 50°C) and two treatment times (1 hour and 2 hours) were used. After treatment at different temperatures and for different times, the sample was placed on ice for 1 minute, and the residual activity was determined under the optimal conditions. The reaction system (500 μL) consisted of 200 μL of pH 7 buffer (100 mM sodium phosphate buffer), 270 μL of 100 μg of ZHDDH4, 25 μL of PQQ, and 5 μL of vomitoxin solution (final concentration 50 ppm). The reaction was incubated at 37°C for 24 hours and terminated by the addition of 500 μL of HPLC-grade methanol. The vomitoxin content in the samples was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate at 0 minutes was defined as 100%, and the relative activity under different conditions was calculated.

[0054] The results are as follows Figure 3 As shown in A, the trichothecene toxin-degrading enzyme ZHDDH4 is relatively unstable above 40°C. After treatment at 40°C for 1 h and 2 h, 42.1% and 39.4% relative activities remain, respectively. After treatment at 45°C for 1 h and 2 h, 33.1% and 0% relative activities remain, respectively. After treatment at 50°C for 1 h, the enzyme is completely inactivated.

[0055] To test the pH stability of alcohol dehydrogenase (ZHDDH), the residual activity of ZHDDH4 recombinant protein in degrading vomitoxin was determined after treatment in different pH buffers for varying durations. Seven pH levels (3, 4, 5, 6, 7, 8, and 9) were set, with two treatment durations (12 h and 24 h). After treatment at different pH conditions for varying durations, the residual activity was determined under optimal conditions. The reaction system (500 μL) consisted of 200 μL of pH 7 buffer (100 mM sodium phosphate buffer), 270 μL of trichothecene toxin-degrading enzyme ZHDDH4 (100 μg), 25 μL of the prosthetic group PQQ, and 5 μL of vomitoxin solution (final concentration 50 ppm). The reaction was incubated at 37°C for 24 h, then terminated by the addition of 500 μL of HPLC-grade methanol. The vomitoxin content in the samples was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate at 0 min was defined as 100%, and the relative activity under different conditions was calculated.

[0056] The results are as follows Figure 3 As shown in Figure B, the trichothecene toxin-degrading enzyme ZHDDH4 has good pH stability in a neutral environment. The enzyme still retains 58.7% relative activity after being treated in a pH = 7 buffer for 24 hours.

[0057] Example 4 Design, Construction and Expression of Trichothecene Degrading Enzyme ZHDDH4 Mutants

[0058] The ΔΔG values of the trichothecene-degrading enzyme ZHDDH4 at the mutation sites were calculated and analyzed using the DUET, DeepDDG, MAESTROweb, and DynaMut2 online servers, respectively. A ΔΔG value greater than 0 predicted by the DUET, DeepDDG, and DynaMut2 online servers indicated a positive mutation, indicating improved stability compared to the wild-type trichothecene-degrading enzyme ZHDDH4. A ΔΔG value less than 0 predicted by the MAESTROweb online server indicated a positive mutation. Single-site mutants with improved stability were screened sequentially, ultimately resulting in the identification of 10 mutants with potential for improving the stability of the trichothecene-degrading enzyme ZHDDH4.

[0059] Five single-site mutants of the trichothecene-degrading enzyme ZHDDH4 were constructed using a two-step PCR method and inserted into the Escherichia coli expression vector pET31b. After correct sequencing, the mutants were transformed into the E. coli TSBL21(DE3)pLysS expression strain for induced expression. After ultrasonication and centrifugation, the supernatant was purified using a nickel affinity chromatography column to obtain single-site mutants of the dehydrogenase ZHDDH4. The single-site mutants were treated at 40, 50, 60, and 70°C for 1 hour, and their degradation rates of DON were subsequently measured at 37°C.

[0060] The results are shown in Table 1. According to the test results, all single-site mutants can degrade DON. Among them, there are 6 mutants whose enzyme activity is not significantly reduced (Table 1). These 6 single-site mutants are A43M, Q68I, N95M, H140F, G278L, and D515R. Among them, A43M, N95M, H140F, G278L, and D515R have improved stability compared with the wild-type trichothecene toxin-degrading enzyme ZHDDH4. Mutants N95M and H140F can still degrade 59.87% and 32.33% of DON, respectively, after treatment at 60°C for 1 hour.

[0061] Table 1 Degradation rates of DON by wild-type trichothecene-degrading enzyme ZHDDH4 and its mutants before treatment and after treatment at different temperatures for 1 h (%)

[0062]

[0063] Example 5 Degradation Activity of Trichothecene Degrading Enzyme ZHDDH4 and Its Single-Point Mutant N95M Against 15-Acetyldeoxynivalenol

[0064] 15-Acetyldeoxynivalenol was dissolved in acetonitrile to a 1 mg / mL stock solution. The following 500 μL reaction system was used: 200 μL sodium phosphate buffer (100 mM, pH 7), 270 μL 100 μg of the trichothecene-degrading enzyme ZHDDH4 or its single-site mutant, 25 μL of the prosthetic group PQQ solution, and 5 μL 15-acetyldeoxynivalenol (final concentration: 10 μg / mL). A control was used without the addition of the trichothecene-degrading enzyme ZHDDH4 or its single-site mutant. The reaction was incubated at 37°C for 48 hours, then terminated with an equal volume of methanol. The 15-acetyldeoxynivalenol content was determined by liquid chromatography-tandem mass spectrometry. The results showed that the efficiency of trichothecene toxin-degrading enzyme ZHDDH4 in catalyzing the degradation of 15-acetyldeoxynivalenol was 78.6%, and the efficiency of single-site mutant N95M in catalyzing the degradation of 15-acetyldeoxynivalenol was 99.5%. Figure 4 ).

[0065] Example 6 Degradation Activity of Trichothecene Degrading Enzyme ZHDDH4 and Its Single-Point Mutant N95M Against Nivalenol

[0066] Nivalenol was dissolved in acetonitrile to prepare a 1 mg / mL stock solution, and the experiment was carried out in a 500 μL reaction system as follows: 200 μL sodium phosphate buffer (100 mM, pH 7), 270 μL trichothecene degrading enzyme ZHDDH4 or its single-site mutant (100 μg), 25 μL prosthetic group PQQ solution, and 5 μL 15-acetyldeoxynivalenol (final concentration of 10 μg / mL). A system without the addition of trichothecene degrading enzyme ZHDDH4 or its single-site mutant was used as a control. After 48 hours of reaction at 37°C, an equal volume of methanol was added to terminate the reaction, and the content of nivalenol in the sample was detected by liquid chromatography-tandem mass spectrometry. The results showed that the efficiency of trichothecene degrading enzyme ZHDDH4 in catalyzing the degradation of nivalenol was 34.8%, and the efficiency of the single-site mutant N95M in catalyzing the degradation of nivalenol was 51.6% ( Figure 5 ).

[0067] Example 7 Detoxification Effect of Trichothecene Toxin Degrading Enzyme ZHDDH4 and Its Single-Point Mutant N95M on Trichothecene Toxins in Feed

[0068] The trichothecene toxin degrading enzyme ZHDDH4 or its single-point mutant N95M described in Example 1 or Example 4 was used to treat feed containing trichothecenes (5 ppm of vomitoxin, 1 ppm of 15-acetyldeoxynivalenol, and 1 ppm of nivalenol) at a ratio of 0.1%, and the mixture was digested in simulated animal gastrointestinal fluid in vitro for 24 hours to degrade the trichothecenes in the feed.

[0069] Simulated gastric fluid: Accurately weigh 2 g of feed containing vomitoxin, 15-acetyldeoxynivalenol, and nivalenol, add 2 mg of the trichothecene-degrading enzyme ZHDDH4 or its single-site mutant N95M, place in a 100 mL Erlenmeyer flask, add 25 mL of 0.1 M PBS (pH 6.0), adjust the pH to 6.8, and mix thoroughly. Add 1 mL of prepared amylase solution and digest at 39°C, 150 rpm, for 2 h. Add 10 mL of 0.2 M HCl, adjust the pH to 2.0 with 1 M HCl or 1 M NaOH solution, add 1 mL of freshly prepared acid protease (50,000 U / g), mix thoroughly, seal with parafilm, and incubate in a shaker at 39°C, 150 rpm, for 6 h.

[0070] Simulated small intestinal fluid: After incubation with simulated gastric fluid for 6 h, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH solution, add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 h (150 rpm).

[0071] After the reaction, the degradation rates of trichothecene toxins by the trichothecene-degrading enzyme ZHDDH4 and its single-point mutant N95M were measured. The results showed that the trichothecene-degrading enzyme ZHDDH4 had a degradation rate of 80.37% for vomitoxin, 91.95% for 15-acetyldeoxynivalenol, and 33.10% for nivalenol. The single-point mutant N95M had a degradation rate of 85.40% for vomitoxin, 94.90% for 15-acetyldeoxynivalenol, and 37.44% for nivalenol.

[0072] Example 8 Detoxification Effect of Trichothecene Degrading Enzyme ZHDDH4 and Its Single-Point Mutant H140F on Trichothecene Toxins in Corn

[0073] Corn containing trichothecenes (4.5 ppm of vomitoxin, 0.8 ppm of 15-acetyldeoxynivalenol, and 0.9 ppm of nivalenol) was treated with the trichothecene-degrading enzyme ZHDDH4 or its single-point mutant H140F described in Example 1 or Example 4, respectively, and mixed at a ratio of 0.1%. The degradation efficiency of the trichothecenes in corn was determined by the in vitro simulated animal gastrointestinal fluid method described in Example 7.

[0074] The results showed that the degradation rate of trichothecene toxin-degrading enzyme ZHDDH4 for vomitoxin was 82.40%, the degradation rate for 15-acetyldeoxynivalenol was 90.64%, and the degradation rate for nivalenol was 31.34%; the degradation rate of single-point mutant H140F for vomitoxin was 84.34%, the degradation rate for 15-acetyldeoxynivalenol was 91.65%, and the degradation rate for nivalenol was 32.39%.

[0075] Example 9: Detoxification Effect of Trichothecene Degrading Enzyme ZHDDH4 and Its Single-Point Mutant D515R on Trichothecene Toxins in Wheat

[0076] Wheat containing trichothecenes (5.2 ppm of vomitoxin, 1.1 ppm of 15-acetyldeoxynivalenol, and 0.9 ppm of nivalenol) was treated with the trichothecene-degrading enzyme ZHDDH4 or its single-point mutant D515R described in Example 1 or Example 4, respectively, and mixed at a ratio of 0.1%. The degradation efficiency of the trichothecenes in wheat was determined according to the in vitro simulated animal gastrointestinal fluid method described in Example 7.

[0077] The results showed that the degradation rate of trichothecene toxin-degrading enzyme ZHDDH4 for vomitoxin was 91.28%, the degradation rate for 15-acetyldeoxynivalenol was 97.22%, and the degradation rate for nivalenol was 39.32%; the degradation rate of single-point mutant D515R for vomitoxin was 72.46%, the degradation rate for 15-acetyldeoxynivalenol was 84.10%, and the degradation rate for nivalenol was 32.37%.

[0078] Example 10 Detoxification Effect of Trichothecene Degrading Enzyme ZHDDH4 and Its Single-Point Mutant N95M on Trichothecene Toxins in Ethanol Fermentation Byproduct DDGS

[0079] The trichothecene-degrading enzyme ZHDDH4 or its single-point mutant N95M described in Example 1 or Example 4 was used to treat DDGS, a byproduct of ethanol fermentation containing trichothecenes (6 ppm of vomitoxin, 1.5 ppm of 15-acetyldeoxynivalenol, and 1 ppm of nivalenol), respectively, and mixed at a ratio of 0.1%. The degradation efficiency of the trichothecenes in wheat was determined by the in vitro simulated animal gastrointestinal fluid method described in Example 7.

[0080] The results showed that the degradation rate of trichothecene toxin-degrading enzyme ZHDDH4 for vomitoxin was 86.98%, the degradation rate for 15-acetyldeoxynivalenol was 90.09%, and the degradation rate for nivalenol was 28.14%; the degradation rate of single-point mutant N95M for vomitoxin was 85.13%, the degradation rate for 15-acetyldeoxynivalenol was 73.52%, and the degradation rate for nivalenol was 32.90%.

[0081] Example 11 Detoxification Effect of Trichothecene Toxin Degrading Enzyme ZHDDH4 and Its Single-Point Mutant N95M on Trichothecene Toxins in Wastewater

[0082] The trichothecene toxin degrading enzyme ZHDDH4 or its single-point mutant N95M described in Example 1 or Example 4 was used to treat sewage containing trichothecenes (4.3 ppm of vomitoxin, 0.5 ppm of 15-acetyldeoxynivalenol, and 0.6 ppm of nivalenol), respectively, and the mixture was mixed at a ratio of 0.1% to determine the degradation efficiency of the trichothecenes in the sewage.

[0083] The results showed that the degradation rate of trichothecene toxin-degrading enzyme ZHDDH4 for vomitoxin was 90.23%, the degradation rate for 15-acetyldeoxynivalenol was 95.31%, and the degradation rate for nivalenol was 38.46%; the degradation rate of single-point mutant N95M for vomitoxin was 95.33%, the degradation rate for 15-acetyldeoxynivalenol was 97.63%, and the degradation rate for nivalenol was 40.86%.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A trichothecene toxin-degrading enzyme ZHDDH4 with biological detoxification function, characterized in that: Its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The gene encoding the trichothecene toxin degrading enzyme ZHDDH4 according to claim 1, characterized in that: Its nucleotide sequence is as shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The single-site mutant of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1, characterized in that: The single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence of the trichothecene toxin degrading enzyme ZHDDH4 according to claim 1 to any one of the amino acid single-site mutations A43M, N95M, H140F, G278L or D515R. The gene encoding the single-site mutant according to claim 3 .

5. A recombinant expression vector containing the coding gene according to claim 2 or claim 4 and a recombinant host cell containing the recombinant expression vector.

6. A method for preparing the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1, comprising: (1) The gene encoding the trichothecene toxin-degrading enzyme ZHDDH4 is operably linked to an expression regulatory element to construct a recombinant expression vector; (2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying the recombinant protein.

7. A method for preparing a single-site mutant of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 3, comprising: (1) operably connecting the coding gene of the single-site mutant of the trichothecene toxin-degrading enzyme ZHDDH4 to an expression regulatory element to construct a recombinant expression vector; (2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying the recombinant protein.

8. Use of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1 or the single-site mutant according to claim 3 in the biodegradation of trichothecene toxins.

9. Use of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1 or the single-site mutant according to claim 3 in enzyme preparation, feed, food, Chinese herbal medicine, biomass energy production and sewage treatment.

10. Use of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1 or the single-site mutant according to claim 3 in the biodegradation of vomitoxin in feed, food, Chinese herbal medicine, biomass energy by-products and sewage.

11. Use of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1 or the single-site mutant according to claim 3 in the biodegradation of 15-acetyldeoxynivalenol in feed, food, Chinese herbal medicine, biomass energy by-products and wastewater.

12. Use of the trichothecene toxin-degrading enzyme ZHDDH4 according to claim 1 or the single-site mutant according to claim 3 in the biodegradation of nivalenol in feed, food, Chinese herbal medicine, biomass energy by-products and sewage.

Images