T-2 toxin degrading enzyme and use thereof

Abstract

The present invention relates to the technical field of genetic engineering, and in particular, to a T-2 toxin degrading enzyme and use thereof. The T-2 toxin degrading enzyme comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5. The use comprises: degrading trichothecenes. By means of ancestral sequence reconstruction, multiple degrading enzymes with significantly improved thermostability and activity are obtained, which have broad-spectrum substrate adaptability, can efficiently utilize GSH as a substrate, and catalyze the destruction of key toxic groups in T-2 toxin and deoxynivalenol, thereby reducing the toxicity of the toxins. The T-2 toxin degrading enzyme provided can serve as a genetic element or a detoxification agent, playing an important detoxification role in the fields of feed, biomedicine, and crop breeding, thus possessing broad application prospects.

Description

A T-2 toxin-degrading enzyme and its application

[0001] Cross-application

[0002] This application claims priority to Chinese Patent Application No. 2025103721306, filed on March 27, 2025, entitled “A T-2 toxin degrading enzyme and its application thereto,” the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention relates to the field of genetic engineering technology, and in particular to a T-2 toxin degrading enzyme and its application. Background Technology

[0004] Trichothecenes are secondary metabolites produced by fungi such as *Fusarium* spp., which can contaminate crops such as grains, corn, and wheat. These toxins not only cause severe losses to agricultural production but also pose a significant threat to human and animal health. Trichothecenes are highly toxic, causing acute or chronic poisoning, manifesting as symptoms such as loss of appetite, vomiting, immunosuppression, and visceral damage. Among them, T-2 toxin is the most acutely toxic of all trichothecenes, and its high toxicity poses a significant risk to food safety and animal health.

[0005] Currently, methods for treating T-2 toxin mainly include chemical degradation, physical adsorption, and ultraviolet irradiation. However, these methods suffer from high costs, low efficiency, and environmental pollution, making them unsuitable for practical applications. In contrast, enzyme-catalyzed degradation technology has attracted significant attention due to its high efficiency, high specificity, and environmental friendliness.

[0006] Existing research indicates that Fhb7, a member of the glutathione transferase (GST) family, possesses some degradation activity against trichothecene toxins such as DON, but its degradation efficiency against T-2 toxin is low. Although studies have attempted to modify Fhb7 through genetic engineering to improve its catalytic activity and thermostability, an enzyme capable of efficiently degrading T-2 toxin has not yet been developed. Furthermore, while some microorganisms possess the ability to degrade T-2 toxin, their low degradation efficiency and unclear biosafety limit their industrial application. Therefore, developing a T-2 toxin-degrading enzyme with high thermostability and efficient catalytic activity is of significant practical importance. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention provides a T-2 toxin degrading enzyme and its application.

[0008] This invention developed five Fhb7-derived enzymes, ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266, using ancestral sequence reconstruction (ASR) technology. These enzymes exhibit significant degradation activity and thermostability against T-2 toxin, providing a new solution for the biodegradation of trichothecene toxins.

[0009] In a first aspect, the present invention provides a T-2 toxin degrading enzyme, wherein the T-2 toxin degrading enzyme comprises any one of the following amino acid sequences:

[0010] (1) The amino acid sequence as described in any one of SEQ ID NO. 1-5;

[0011] (2) An amino acid sequence of a protein having the same function, obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in any of SEQ ID NO.1-5.

[0012] The present invention has found that when the amino acid sequence is as described above or has high homology and the same activity, the thermal stability and catalytic activity of the degrading enzyme are more excellent.

[0013] The amino acid sequence shown in SEQ ID NO.1 (ASR256):

[0014] The amino acid sequence shown in SEQ ID NO.2 (ASR263):

[0015] The amino acid sequence shown in SEQ ID NO.3 (ASR264):

[0016] The amino acid sequence shown in SEQ ID NO.4 (ASR265):

[0017] The amino acid sequence shown in SEQ ID NO.5 (ASR266):

[0018] In a second aspect, the present invention provides a nucleic acid for encoding the aforementioned T-2 toxin degrading enzyme.

[0019] Furthermore, the nucleic acid includes any one of the following nucleic acid sequences:

[0020] (1) A nucleotide sequence as shown in any one of SEQ ID NO. 6-10;

[0021] (2) A nucleotide sequence that encodes a protein having the same function, obtained by substituting, deleting or inserting one or more nucleotides of the nucleotide sequence shown in any of SEQ ID NO. 6-10.

[0022] (3) A nucleotide sequence that can hybridize with any of the nucleotide sequences shown in SEQ ID NO. 6-10 under strict conditions.

[0023] The nucleotide sequence shown in SEQ ID NO.6 (ASR256) is as follows:

[0024] The nucleotide sequence shown in SEQ ID NO.7 (ASR263) is as follows:

[0025] The nucleotide sequence shown in SEQ ID NO.8 (ASR264):

[0026] The nucleotide sequence shown in SEQ ID NO.9 (ASR265):

[0027] The nucleotide sequence shown in SEQ ID NO.10 (ASR266):

[0028] Thirdly, the present invention provides a biological material comprising the aforementioned T-2 toxin degrading enzyme or the aforementioned nucleic acid; the biological material being an expression cassette, a vector, or a transgenic cell.

[0029] The transgenic cells described in this invention do not include transgenic cells capable of independently developing into a complete individual, that is, they do not include plant varieties.

[0030] Further, the vector is a pQlinkHx vector or a pET28a vector; and / or, the transgenic cell is *Escherichia coli* or yeast. Preferably, it is a recombinant K1 series *Escherichia coli* or a recombinant Pichia pastoris strain.

[0031] When using microorganisms as hosts, any commercially available plasmid can be used as a vector, as long as it can stably maintain the expression of the target gene. When using E. coli as a host, the pET28a or pQlinkHx plasmids are preferred.

[0032] In one specific embodiment of this invention, the obtained gene (DNA fragment) is ligated downstream of a specific promoter sequence to induce expression and produce a large quantity of the target protein in *E. coli*. For example, the pET28a plasmid carries an inducible T7 promoter, and by adding isopropyl-β-D-thiogalactopyranoside (IPTG) during culture, *E. coli* can be efficiently induced to express the target degrading enzyme.

[0033] Transformants were cultured using standard methods. For example, when using Bacillus or Escherichia coli, LB medium (1% tryptone, 0.5% yeast extract, 0.5% saline) or 2xYT medium (1.6% tryptone, 1% yeast extract, 0.5% saline) could be used. After inoculating the strain into the medium, the culture was stirred and incubated at 36–38°C for 4–8 hours. Then, IPTG was added to a final concentration of 0.1–1 mM, and the culture was continued at 16–20°C for 12–20 hours. After incubation, the culture was separated by centrifugation, and the microbial cells were recovered.

[0034] Furthermore, the aforementioned recovered microbial cells can be disrupted using any of the following known methods: mechanical disruption (e.g., rotary mixer, Freund's crusher, homogenizer, mortar and pestle), freeze-thaw cycle, autolysis, drying (e.g., freeze-drying, air-drying), enzyme treatment (e.g., using lysozyme), ultrasonic treatment, chemical treatment (e.g., acid or alkali treatment). The supernatant after centrifugation, etc., is used as crude enzyme solution for the degradation of T-2 toxin. Additionally, depending on the circumstances, components with degrading activity for trichothecene toxins, including T-2 toxin and vomitoxin, can be further purified from the recovered microbial cells using conventional enzyme purification methods (e.g., salting out, isoelectric point precipitation, organic solvent precipitation, dialysis, various chromatography methods, etc.) to prepare partially purified enzymes or purified enzymes for use. The enzyme added to the reaction system can be in any form, provided it possesses the desired activity, including but not limited to microbial cells (including transformants), the processed products of these cells, or enzymes obtained from these processed products.

[0035] Fourthly, the present invention provides a kit comprising the aforementioned T-2 toxin degrading enzyme, or the aforementioned nucleic acid, or the aforementioned biological material.

[0036] Fifthly, the present invention provides the application of the aforementioned T-2 toxin degrading enzyme, or the aforementioned nucleic acid, or the aforementioned biological material, or the aforementioned kit in the degradation of trichothecene toxins.

[0037] Furthermore, the trichothecene toxin is a trichothecene toxin containing an epoxy group.

[0038] Preferably, the trichothecene toxin is at least one of T-2 toxin, vomitoxin, NIV toxin, 15-acylated vomitoxin, or DAS. More preferably, it is T-2 toxin or vomitoxin.

[0039] Furthermore, in the degradation of T-2 toxin, preferably, the initial concentration of T-2 toxin is 0.01–1 mM, more preferably 0.01–0.1 mM. Preferably, the initial concentration of glutathione (GSH) is 1–10 mM, more preferably 0.2–2 mM. Preferably, the reaction conditions are a pH of 4–10, more preferably pH 7. The temperature is 10°C–60°C, more preferably 37°C. The reaction time is preferably 0.1–2 hours, and stirring is maintained during the reaction to ensure thorough mixing.

[0040] In a sixth aspect, the present invention provides the use of the aforementioned T-2 toxin degrading enzyme, or the aforementioned nucleic acid, or the aforementioned biological material, or the aforementioned kit in any of the following:

[0041] (1) Prepare antidotes or drugs for inhibiting trichothecene toxins;

[0042] (2) Develop feed additives;

[0043] (3) Prevent and control microbial contamination that produces trichothecene toxins;

[0044] (4) Construct microorganisms capable of degrading trichothecene toxins;

[0045] (5) Breeding of wheat scab resistant crops.

[0046] Furthermore, the drugs used in (1) include antidotes.

[0047] Furthermore, the microorganisms used in (4) are engineered bacteria.

[0048] The engineered bacteria include, but are not limited to, Bacillus, yeast, Lactobacillus, Streptococcus, and other beneficial bacteria permitted for use in the feed industry or pharmaceutical industry.

[0049] Based on the principles of synthetic biology, the aforementioned T-2 toxin degrading enzyme, nucleic acid, or biological material of this invention can be used as functional elements for breeding wheat scab-resistant crops or for constructing beneficial engineered bacteria that can degrade trichothecene toxins, including T-2 toxin and vomitoxin.

[0050] The present invention has the following beneficial effects:

[0051] This invention uses the Fusarium head blight resistance protein Fhb7 from *Thinopyrum elongatum* as a template. Through ancestral sequence reconstruction technology, five ancestral protein sequences were reconstructed that can simultaneously and efficiently degrade T-2 toxin and vomitoxin. Furthermore, these proteins exhibit higher thermal stability and catalytic activity than Fhb7 and its high-thermal-stability mutant M10, enabling their widespread industrial application as detoxification enzymes. The degrading enzymes provided by this invention can serve as gene elements or detoxifying agents, playing a crucial detoxification role in the fields of feed, biomedicine, and crop breeding, and possess broad application prospects. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0053] Figure 1 is a diagram illustrating the mechanism of T-2 toxin degradation by the T-2 toxin-degrading enzyme provided in Example 1 of the present invention.

[0054] Figure 2 is a protein electrophoresis diagram of T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 provided in Example 2 of the present invention.

[0055] Figure 3 shows the activity test results of T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 provided in Example 2 of the present invention.

[0056] Figure 4 shows the melting temperature (Tm value) test results of the T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 provided in Example 3 of the present invention.

[0057] Figure 5 shows the optimal reaction temperature test results of T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 provided in Example 4 of the present invention.

[0058] Figure 6 shows the temperature tolerance test results of the T-2 toxin degrading enzymes ASR_256, ASR_263 and ASR_264 provided in Example 5 of the present invention.

[0059] Figure 7 shows the half-life test results of T-2 toxin degrading enzymes ASR_256, ASR_263, and ASR_264 provided in Example 6 of this invention. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0062] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available, for example:

[0063] The DNA modulation, restriction enzyme cleavage, T4 DNA ligase ligation, and E. coli transformation methods in the examples were all performed according to the second edition of *Molecular Cloning* (Maniatis Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)). Furthermore, the quantification of T-2 toxin, vomitoxin, and glutathione adduct in the reaction solution was performed using UPLC-MS. Separation was performed using a Waters BEH-C18 column, with elution using a gradient of acetonitrile and water (containing 0.1% formic acid).

[0064] Example 1

[0065] 1. This invention developed five Fhb7-derived enzymes (also known as T-2 toxin degrading enzymes): ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266, using ancestral sequence reconstruction technology. These enzymes exhibit significant degradation activity against T-2 toxin (the degradation mechanism is shown in Figure 1, using glutathione (GSH) as an antidote, enzymatically opening the key toxic C12 / C13 epoxy groups of trichothecene toxins to achieve degradation). The amino acid sequence of ASR_256 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.6. The amino acid sequence of ASR_263 is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.7. The amino acid sequence of ASR_264 is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.8. The amino acid sequence of ASR_265 is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.9. The amino acid sequence of ASR_266 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.10.

[0066] The genes for each of the above T-2 toxin degrading enzymes were obtained through gene synthesis. Using the synthesized genes as templates, primers were designed to amplify the target gene fragments. The target gene and the linearized pQlinkHx vector were then ligated by homologous recombination and transformed into Escherichia coli DH5α. Plasmids pQlinkHx-ASR256, pQlinkHx-ASR263, pQlinkHx-ASR264, pQlinkHx-ASR265, and pQlinkHx-ASR266 were isolated from the obtained ampicillin-resistant transformants.

[0067] 2. Taking pQlinkHx-ASR256 as an example, the construction method is as follows:

[0068] (1) Amplification of gene fragments

[0069] Primer pairs used:

[0070] Fhb7_ASR256_F:5'-GTATTTTCAGGGATCCACCAGCATGAGCTCTTC-3',

[0071] Fhb7_ASR256_R:5'-GGCGGCCGCTTATTTCACCTCCGCGTATTTTTC-3'.

[0072] The PCR reaction system is as follows:

[0073] Table 1 PCR reaction system

[0074] Table 2 PCR reaction procedure

[0075] (2) Obtaining the linear vector pQlinkHx: Using a plasmid containing the pQlinkHx vector as a template, the linearized vector of pQlinkHx was obtained by PCR using the following primers.

[0076] Primer pairs:

[0077] Fhb7_ASR256_pQ_F:5'-AATACGCGGAGGTGAAATAAGCGGCCGCCTAG-3',

[0078] Fhb7_ASR256_pQ_R:5'-CATGCTGGTGGATCCCTGAAAATACAGGTTTTC-3'.

[0079] Table 3 PCR reaction system

[0080] Table 4 PCR reaction procedure

[0081] (3) After amplification of each gene fragment, the PCR products were subjected to 1% agarose gel electrophoresis to purify each DNA fragment. Homologous recombination was used to ligate the target gene fragment and the linearized vector. The ligation method is as follows:

[0082] The pQlinkHx-ASR256 plasmid was constructed using Vazyme homologous recombinase. The 10 μL ligation system consisted of: 1 μL of enzyme, 2 μL of 5×CE buffer, 2 μL of linearized vector pQlinkHx, and 5 μL of gel-recovered target gene fragment.

[0083] The ligation reaction solution was completely transformed into Escherichia coli DH5α, and plasmid pQlinkHx-ASR256 was isolated from the obtained ampicillin-resistant transformant.

[0084] 3. The other plasmids were constructed using the same method, with the following differences:

[0085] (1) The primer pairs used for constructing pQlinkHx-ASR263 are as follows:

[0086] Fhb7_ASR263_F:5'-GTATTTTCAGGGATCCATGGCCACTTCATCCTC-3',

[0087] Fhb7_ASR256_R:5'-GGCGGCCGCTTATTTCACCTCCGCGTATTTTTC-3'.

[0088] Fhb7_ASR256_pQ_F:5'-AATACGCGGAGGTGAAATAAGCGGCCGCCTAG-3',

[0089] Fhb7_ASR263_pQ_R: 5'-GTGGCCATGGATCCCTGAAAATACAGGTTTTC-3'.

[0090] (2) The primer pairs used for constructing pQlinkHx-ASR264 are as follows:

[0091] Fhb7_ASR264_F:5'-GTATTTTCAGGGATCCATGGCCACCTCATCATC-3',

[0092] Fhb7_ASR264_R: 5'-CGGCCGCTTATTTAACCTCCGCATATTTCTCCAG-3'.

[0093] Fhb7_ASR264_pQ_F: 5'-GAAATATGCGGAGGTTAAATAAGCGGCCGCCTAGG-3',

[0094] Fhb7_ASR264_pQ_R: 5'-GAGGTGGCCATGGATCCCTGAAAATACAGGTTTTC-3'.

[0095] (3) The primer pairs used for constructing pQlinkHx-ASR265 are as follows:

[0096] Fhb7_ASR265_F: 5'-GTATTTTCAGGGATCCATGGCAACCTCAAGTCCC-3',

[0097] Fhb7_ASR265_R: 5'-GGCCGCTTATTTTACCTCAGCATATTTTTCCAGTGC-3'.

[0098] Fhb7_ASR265_pQ_F: 5'-GAAAAATATGCTGAGGTAAAATAAGCGGCCGCCTAGG-3',

[0099] Fhb7_ASR265_pQ_R: GGTTGCCATGGATCCCTGAAAATACAGGTTTTC-3'.

[0100] (4) The primer pairs used for constructing pQlinkHx-ASR266 are as follows:

[0101] Fhb7_ASR266_F:GTATTTTCAGGGATCCATGGCCACCTCCAGCAG-3',

[0102] Fhb7_ASR266_R: GGCGGCCGCTTACTTCACCTCCGCGTATTTC-3'.

[0103] Fhb7_ASR266_pQ_F:CGGAGGTGAAGTAAGCGGCCGCCTAGGAC-3',

[0104] Fhb7_ASR264_pQ_R:GAGGTGGCCATGGATCCCTGAAAATACAGGTTTTC-3'.

[0105] Example 2

[0106] The present invention further expresses, purifies, and tests the activity of the T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266 obtained in Example 1, as follows:

[0107] 1. Inoculate *E. coli* DH5α strains containing plasmids pQlink-ASR256, pQlinkHx-ASR263, pQlinkHx-ASR264, pQlinkHx-ASR265, and pQlinkHx-ASR266 into liquid LB medium for culture. Then, transfer 10 mL of the culture to 1 L of LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking at 220 rpm until the bacterial culture reaches OD. 600The concentration of the enzyme was 0.8–1.0. IPTG was added to a final concentration of 0.5 mM, and induction was performed at 20°C for 16–20 h. After incubation, the bacterial cells were recovered by centrifugation (4500 rpm, 20 min) and resuspended in 15 mL of lysis buffer (20 mM NaH₂PO₄-Na₂HPO₄, pH 7.3). The cells were then sonicated to disrupt their structure (500 W, 37% power, sonication for 5 s, pause for 5 s, for a total disruption of 25 min). The disrupted cells were aliquoted into 2 mL EP tubes and centrifuged again (15000 rpm, 20 min) to remove the cells. The supernatant was collected and filtered through a 0.22 μm filter to obtain the crude enzyme solution. After equilibrating a gravity nickel column to 5 column volumes with lysis buffer, the crude enzyme solution was slowly added to the column, allowing it to flow gradually through the column. Wash the contaminating protein with washing buffer (20mM NaH2PO4-Na2HPO4, 0.2mM NaCl, 40mM imidazole, pH 8.5), and then elute the target protein with elution buffer (20mM NaH2PO4-Na2HPO4, 0.2mM NaCl, 300mM imidazole, pH 8.5).

[0108] The expression levels of these five T-2 toxin-degrading enzymes in *E. coli* were as high as 30-60 mg / L. The eluted target protein was concentrated to a volume of 1-2 mL using a 10 kDa ultrafiltration tube. Replacement buffer (20 mM NaH₂PO₄-Na₂HPO₄, 0.2 mM NaCl, pH 8.5) was added, followed by centrifugation to remove imidazole. The protein concentration was reduced to approximately 10 mg / mL. 3 μL of the purified protein solution was added to 3 μL of 5× protein loading buffer, followed by 9 μL of deionized water. The mixture was heated at 100°C for 5 min and then analyzed by 10% polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue staining. The protein band size was approximately 31 kDa (as shown in Figure 2).

[0109] 2. Determination of the activity of Fhb7 ancestral sequence expressed proteins: The enzymatic reaction system consisted of 100 μL, including 5 μg of ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 proteins, 1 mM glutathione, and 50 μM T-2 toxin. The reaction was carried out at 37℃ for 5 minutes.

[0110] Detection method: Immediately after the reaction, quench the reaction by adding 100 μL of acetonitrile in an ice bath. After filtration through a 0.22 μm filter membrane, perform UPLC-MS detection using the following liquid chromatography method:

[0111] 0-0.5 min: 0% acetonitrile, 100% water (containing 0.1% formic acid);

[0112] 0.5-1 min: Acetonitrile concentration gradient increases to 2%, aqueous phase concentration gradient decreases to 98%;

[0113] 1-2.5 min: Acetonitrile concentration gradient increases to 5%, aqueous phase concentration gradient decreases to 95%;

[0114] 2.5-4.5 min: Acetonitrile concentration gradient increases to 40%, aqueous phase concentration gradient decreases to 60%;

[0115] 4.5-8 min: Acetonitrile concentration gradient increases to 90%, aqueous phase concentration gradient decreases to 10%.

[0116] 3. The results showed that the ancestral sequences ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 all had T-2 degradation activity, among which ASR_263 had the best activity (as shown in Figure 3).

[0117] Example 3

[0118] The present invention further determines the melting temperature Tm values ​​of the purified T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266 obtained in Example 2, using the following specific method:

[0119] 1. Preparation of the assay system (25 μL): Take 10 μg of the protein purified in Example 2 and add buffer (20 mM NaH2PO4-Na2HPO4, 0.2 mM NaCl, pH 8.5) to a final volume of 25 μL. After mixing with a pipette, heat the 25 μL assay system at different temperatures (0℃, 30℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃) for 3 min. Perform three replicates for each sample. Centrifuge the heated samples at 20000g for 20 min, collect 12.5 μL of supernatant (approximately 5 μg of protein), add 3 μL of 5× protein loading buffer, heat at 100℃ for 5 min, centrifuge at 5000 rpm for 3 s, and collect 15 μL of supernatant for gel electrophoresis. Perform 10% SDS-PAGE detection and Coomassie Brilliant Blue staining.

[0120] 2. The relative abundance of proteins after heating was calculated using ImageJ, and the Tm values ​​of ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 were fitted using Graphpad. The results showed that the Tm values ​​of T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265 and ASR_266 were between 52-63℃, with ASR_264 having the highest Tm value of 62.73℃ (as shown in Figure 4).

[0121] Example 4

[0122] The present invention further determines the optimal temperature for the purified T-2 toxin degrading enzymes ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266 obtained in Example 2, including the following procedure:

[0123] The 100 μL reaction system included: 5 μg of ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266 proteins obtained in Example 2, 1 mM glutathione, 50 μM T-2 toxin, and buffer (20 mM NaH2PO4-Na2HPO4, 0.2 mM NaCl, pH 8.5) to a final volume of 100 μL. The reaction temperatures were changed to 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃, respectively. Three replicates were set up for each sample. Enzyme activity was evaluated based on the amount of T-2 toxin and glutathione adduct generated; the temperature with the highest conversion rate was considered the optimal temperature for the enzyme. ASR_256, ASR_263, and ASR_265 exhibited the best catalytic activity at 35-40℃, while ASR_264 and ASR_266 showed the best catalytic activity at 45℃ (as shown in Figure 5).

[0124] Example 5

[0125] The present invention further evaluates the temperature tolerance of the purified ASR_256, ASR_263 and ASR_264 from Example 2, including the following procedure:

[0126] 1. Protein heat treatment: Dilute the protein to 1 mg / mL with buffer (20 mM NaH2PO4-Na2HPO4, 0.2 mM NaCl, pH 8.5). Take 5 μL of protein and incubate at different temperatures of 0℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ for 5 min. Immediately after incubation, place the system on ice.

[0127] 2. Preparation of the assay system (100 μL): After incubation, add 5 μL of protein, 1 mM glutathione, and 50 μM T-2 toxin to buffer (20 mM NaH2PO4-Na2HPO4, 0.2 mM NaCl, pH 8.5) to a final volume of 100 μL.

[0128] 3. Residual Enzyme Activity Test: The above assay system was reacted at 37℃ for 5 min. After the reaction, 100 μL of acetonitrile in an ice bath was immediately added to quench the reaction. After filtration through a 0.22 μm filter membrane, the amount of T-2 toxin and glutathione adduct produced in the sample was analyzed by UPLC-MS to evaluate enzyme activity. Using protein incubated at 0℃ as a control, the amount of product produced in the heat-treated sample relative to the amount of product in the untreated sample was calculated as the residual activity of the protein after heat treatment.

[0129] 4. The results showed that ASR_256 protein showed almost no loss of activity after heat treatment at 35℃-55℃ for 5 minutes, but its activity dropped sharply to 1% at 55℃, and it completely lost its catalytic activity at 60℃. ASR_263 protein showed almost no loss of activity after heat treatment at 35℃-40℃ for 5 minutes, but its activity decreased significantly after heat treatment at 45℃-55℃, dropping to 9% at 55℃, and it lost all activity at 60℃. ASR_264 protein exhibited better heat tolerance and showed significant thermal activation within the 35℃-45℃ temperature range. Its activity increased significantly after incubation within this temperature range, reaching a maximum of 255% of its pre-heat treatment activity. It retained more than 51% of its residual activity after heat treatment at 50℃, and still maintained 4% of its activity at 60℃ (as shown in Figure 6).

[0130] Example 6

[0131] The present invention further investigates the half-life t of the ASR_256, ASR_263, and ASR_264 proteins involved in Example 2. 1 / 2 The determination includes the following procedures:

[0132] 1. Protein heat treatment: Dilute the protein to 1 mg / mL with buffer (20 mM NaH2PO4-Na2HPO4, 0.2 mM NaCl, pH 8.5). Take 100 μL of the diluted protein and heat treat it in a metal bath at 37 °C for different times (0 h, 6 h, 18 h, 30 h, 48 h, 68 h, 90 h). Take 5 μL of the protein after each heat treatment for the determination of enzyme residual activity.

[0133] 2. Determination of residual enzyme activity after heat treatment: The assay system and method for detecting residual enzyme activity were prepared according to Example 5. The half-life t of ASR_256, ASR_263, and ASR_264 proteins were calculated based on the residual enzyme activity at different treatment times. 1 / 2 (That is, the time required to reduce enzyme activity to 50% of its original value by heating at 37°C for different times).

[0134] 3. The results show that the half-life of ASR_256 at 37℃ is between 65 and 70 hours; the half-life of ASR_263 and ASR_264 at 37℃ is greater than 90 hours (as shown in Figure 7). Industrial applicability

[0135] This invention developed five Fhb7-derived enzymes, ASR_256, ASR_263, ASR_264, ASR_265, and ASR_266, using ancestral sequence reconstruction technology. These five enzymes exhibit high degradation efficiency for trichothecene toxins, especially T-2 toxin and vomitoxin. Furthermore, these proteins possess higher thermostability and catalytic activity than Fhb7 and its thermostable mutant M10, enabling their widespread industrial application as detoxification enzymes. The degrading enzymes provided by this invention can serve as gene elements or detoxifying agents, playing a crucial detoxification role in the fields of feed, biomedicine, and crop breeding, and have broad application prospects.

Claims

1. A T-2 toxin-degrading enzyme, characterized in that, The T-2 toxin degrading enzyme comprises any one of the following amino acid sequences: (1) The amino acid sequence as described in any one of SEQ ID NO. 1-5; (2) An amino acid sequence of a protein having the same function, obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in any of SEQ ID NO.1-5.

2. A nucleic acid, characterized in that, The nucleic acid is used to encode the T-2 toxin degrading enzyme of claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleic acid includes any of the following nucleic acid sequences: (1) A nucleotide sequence as shown in any one of SEQ ID NO. 6-10; (2) A nucleotide sequence that encodes a protein having the same function, obtained by substituting, deleting or inserting one or more nucleotides of the nucleotide sequence shown in any of SEQ ID NO. 6-10. (3) A nucleotide sequence that can hybridize with any of the nucleotide sequences shown in SEQ ID NO. 6-10 under strict conditions.

4. A biomaterial, characterized in that, include: The T-2 toxin degrading enzyme of claim 1, or the nucleic acid of claim 2 or 3; The biological material is an expression cassette, vector, or transgenic cell.

5. The biomaterial according to claim 4, characterized in that, The vector is a pQlinkHx vector; and / or, the transgenic cells are Escherichia coli or yeast.

6. A reagent kit, characterized in that, The kit comprises: the T-2 toxin degrading enzyme of claim 1, or the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5.

7. The use of the T-2 toxin degrading enzyme of claim 1, or the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5, or the kit of claim 6 in the degradation of trichothecene toxins.

8. The application according to claim 7, characterized in that, The trichothecene toxin is a trichothecene toxin containing an epoxy group, preferably T-2 toxin or vomitoxin.

9. The use of the T-2 toxin degrading enzyme of claim 1, or the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5, or the kit of claim 6, in any of the following: (1) Prepare antidotes or drugs for inhibiting trichothecene toxins; (2) Develop feed additives; (3) Prevent and control microbial contamination that produces trichothecene toxins; (4) Construct microorganisms capable of degrading trichothecene toxins; (5) Breeding of wheat scab resistant crops.

10. The application according to claim 9, characterized in that, The drug in application (1) is an antidote; and / or, the microbial contamination in application (3) is microbial contamination of grains and feeds that have the ability to produce trichothecene toxins during storage and transportation; and / or, the microorganisms in application (4) include one or more of Bacillus, yeast, Lactobacillus or Streptococcus.

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