High-thermal-stability vomitoxin detoxification fusion enzyme mutant and application thereof

Abstract

The application belongs to the technical field of protein engineering, and particularly relates to a high-thermal-stability vomitoxin detoxification fusion enzyme mutant and application thereof. With an artificially constructed vomitoxin detoxification fusion enzyme ADDE as a parent, a protein language model SaProt is combined with three-dimensional structure analysis to identify that a proline at a 103th position of the vomitoxin detoxification enzyme is a key regulation site of thermal stability, and the proline is mutated into histidine to obtain a mutant P103H. The half-life of the mutant at 50 DEG C reaches more than 5 times of that of a wild type, while a high catalytic activity is maintained, and the mutant has a good application prospect in high-temperature environments such as feed processing and food industry.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, specifically relating to a highly thermally stable vomitoxin detoxification fusion enzyme mutant and its applications. Background Technology

[0002] Deoxynivalenol (DON) is a type B trichothecene fungal toxin produced by Fusarium spp., and is one of the most frequently detected and prevalent fungal toxins in grains and by-products worldwide. DON primarily contaminates wheat, barley, corn, oats, and their processed products. After entering the human and animal body through the food chain, it can cause various toxic effects, including vomiting, diarrhea, growth retardation, immunosuppression, liver and kidney damage, and intestinal barrier disruption. It is listed as a significant food safety hazard by the World Health Organization and the Food and Agriculture Organization of the United Nations. With the advancement of antibiotic-free feed policies and increasingly stringent food quality and safety standards, the effective detoxification and control of DON has become a common and critical technical challenge that urgently needs to be addressed in the grain processing and livestock industries.

[0003] Currently, detoxification methods for DON mainly fall into three categories: physical adsorption, chemical treatment, and enzymatic degradation. Among these, enzymatic degradation is widely recognized as the most promising green detoxification strategy due to its mild operating conditions, strong catalytic specificity, irreversible destruction of the active groups of toxin molecules, and absence of harmful byproducts. Of the known DON biodegradation pathways, the oxidation / isomerization reaction acting on the C3 hydroxyl group is the most thoroughly studied and the most complete irreversible detoxification pathway. Various aldehyde-ketone reductases, quinone-dependent dehydrogenases, and artificial fusion enzymes derived from microorganisms have been reported to possess DON degradation activity.

[0004] However, existing reported vomitoxin detoxifying enzymes generally suffer from a fatal flaw: insufficient thermal stability. In typical industrial processing steps such as feed pelleting, extrusion, cooking, and steam conditioning, materials often need to withstand high-temperature treatments of 55°C to 90°C. Most wild-type vomitoxin detoxifying enzymes have extremely short half-lives within this temperature range, typically undergoing irreversible inactivation within tens of minutes of treatment at 50°C. This shortcoming in thermal stability severely restricts the application of these enzyme preparations in practical production scenarios, becoming a core bottleneck for the industrialization of DON's biological detoxification technology.

[0005] To address the aforementioned issues, improving the thermostability of existing vomitoxin detoxification enzymes through protein engineering has become a key breakthrough for promoting the industrialization of this technology. Traditional enzyme modification strategies, such as directed evolution, rational design, and common sequence analysis, while achieving some progress, generally suffer from limitations in screening throughput, insufficient predictive accuracy, or mild modification effects. Therefore, developing a vomitoxin detoxification enzyme mutant with significantly improved thermostability is of urgent and significant importance for promoting its industrial application. Summary of the Invention

[0006] This invention primarily provides a vomitoxin detoxification fusion enzyme mutant that significantly improves thermal stability while maintaining its original catalytic activity, as well as an efficient method for constructing this mutant, thereby addressing the problem of insufficient stability of wild-type enzymes in existing technologies. The technical solution is as follows:

[0007] A highly thermostable vomitoxin detoxification fusion enzyme mutant, wherein the mutant is obtained by mutation based on the amino acid sequence of the vomitoxin detoxification fusion enzyme shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid proline at position 103 of the amino acid sequence shown in SEQ ID NO.1 was mutated to histidine; the amino acid sequence of the mutant is shown in SEQ ID NO.2.

[0009] SEQ ID NO.1 is:

[0010] MTKLDASLSGRFSIGGDLKVNRLGFGAMRLTGDGIWGPPKDRDEAIRVLKRLPEIGVDFIDTAESYGPYVSEELIGEALAPYDKGTIIATKSGLTRSGPNQWPPLGRPEFLRQGVMTSLRRLKLERLDLWQLHRIDAKTPRAEQFEVIAAMQKEGLIRHAGLSEVSVADIEEASKYFKVTTVQNLYNFANRKSEAVLDYCEKHGIGFIPWFPLAGGDLVEGHEKARAVMDKHGASGSQIALAWLLKRSPVMLPIPGTSKVKHLEDNVAAAAIDLSDEDFAALDAIGRADPNSSSVDKLAAALEGSGSGSGSGSGSGSGMQVDISALPMVTDEILANPDAGDWPSYGRDVMNYRYSPLDQINKDNVGNLTMVWGRALEPGNLQSAPLEFGGVMFIAAPGDVVQAIDAATGQLVWEYRRTLPDRETLNSLGENKRGIALYEDKIYMVSWDNFIVALDAKTGQVAWESDRGGGADMISNTTGPIVADGVVVAGSTCQFSEFGCYVTGHDAATGEELWRNTFIPKAGEEGDDTWGDSTEDQRWMTGAWGQMTYDPVTGLVFYGSTGAGPAAEFQRNTVGGTLYGSNTRFAVKPKTGEIVWRHQVLPRDNWDQECTYEMIPVDINSNPSADMEGLLALGTATPGEKRVLTGVPCKTGVMWQFDAQTGEFIYARDTVQENLIEKVDETGLVTVNEAAIPTEVDTPTFMCPTYLGGRDWPPTAFNPETKVMFVPLTNMCANATVLDQEPTGLDVYNTELEYILPEGVTHAGRIDAINVETGKTVWSWTDQTPLYAPIVSTAGGLIFVGGTDRKFKAIDQETGEVVWSTTLPSRATGHPISYEVDGRQYIAIPAGGPGYASLFLEASGTTADTVSGSNAVYVFALPEAAAK。

[0011] SEQ ID NO. 2 is:

[0012] 。

[0013] Further, the half-dead period of the mutant body at 50℃ is not lower than 5 times that of the wild type.

[0014] Biomaterials related to the aforementioned highly heat-stable vomitoxin detoxification fusion enzyme mutant include any one of the following:

[0015] A) The nucleic acid molecule encoding the aforementioned highly thermostable vomitoxin detoxification fusion enzyme mutant;

[0016] B) An expression cassette containing the nucleic acid molecule described in A);

[0017] C) A recombinant vector containing the nucleic acid molecule described in A), or a recombinant vector containing the expression cassette described in B;

[0018] D) Recombinant microorganisms containing the nucleic acid molecules described in A), or recombinant microorganisms containing the expression cassette described in B), or recombinant microorganisms containing the recombinant vector described in C).

[0019] A method for preparing the above-mentioned highly thermostable vomitoxin detoxification fusion enzyme mutant includes the following steps: performing point mutation on wild-type vomitoxin detoxification fusion enzyme; obtaining a nucleic acid molecule encoding the highly thermostable vomitoxin detoxification fusion enzyme mutant; ligating the nucleic acid molecule encoding the highly thermostable vomitoxin detoxification fusion enzyme mutant into a vector to obtain a recombinant expression vector; transforming host cells and expressing the target protein.

[0020] Further, the method includes the following steps: the vector includes one or more of the following: Escherichia coli expression vector, Bacillus subtilis expression vector, Bacillus licheniformis expression vector, Bacillus megaterium expression vector, Bacillus brevis expression vector, lactic acid bacteria expression vector, yeast expression vector, Streptomyces expression vector, or filamentous fungal expression vector.

[0021] Furthermore, the host cell is one or more of the following: Escherichia coli host cell, Bacillus subtilis host cell, Bacillus licheniformis host cell, Bacillus megaterium host cell, Bacillus brevis host cell, lactic acid bacteria host cell, yeast host cell, Streptomyces host cell, or filamentous fungal host cell.

[0022] Furthermore, point mutations were performed on the wild-type vomitoxin detoxification fusion enzyme using primer pairs with sequences shown in SEQ ID NO.49 and SEQ ID NO.50.

[0023] The application of the aforementioned highly thermally stable vomitoxin detoxification fusion enzyme mutant in degrading vomitoxin or reducing the activity of vomitoxin.

[0024] The above-mentioned highly thermally stable vomitoxin detoxification fusion enzyme mutants are used in the preparation of food processing agents, food flavor regulators, texture improvers, preservatives, feed, pharmaceuticals, or pollutant treatment.

[0025] By adopting the above scheme, the method of the present invention has the following advantages:

[0026] 1. The thermostability of the vomitoxin detoxification fusion enzyme mutant obtained in this invention is significantly improved: compared with the wild type, the half-life of mutant P103H at 50℃ is extended from 35.56 minutes to 185.78 minutes, an increase of 5.22 times. The half-lives at 55℃ and 60℃ are extended to 45.94 minutes and 18.47 minutes, respectively, effectively alleviating the problem of poor thermostability of existing vomitoxin detoxification enzymes.

[0027] 2. The vomitoxin detoxification fusion enzyme mutant obtained by this invention can meet the requirements of high-temperature industrial processing environments and has good application prospects in high-temperature environments such as feed processing and food industry. Attached Figure Description

[0028] Figure 1 A comparison of the optimal reaction temperatures for the wild-type vomitoxin detoxification fusion enzyme and the mutant P103H of this invention.

[0029] Figure 2 A comparison of the optimal reaction pH for the wild-type vomitoxin detoxification fusion enzyme and the mutant P103H of this invention.

[0030] Figure 3 A comparison of the temperature stability of the wild-type vomitoxin detoxification fusion enzyme and the mutant P103H of this invention at 50°C.

[0031] Figure 4 A comparison of the temperature stability of the wild-type vomitoxin detoxification fusion enzyme and the mutant P103H of this invention at 55°C.

[0032] Figure 5 A comparison of the temperature stability of the wild-type vomitoxin detoxification fusion enzyme and the mutant P103H of this invention at 60°C. Detailed Implementation

[0033] Example: 1. Identification of key mutation sites for the thermostability of SaProt-based vomitoxin detoxification fusion enzyme. Thirty mutations that have a beneficial effect on protein stability or function were selected, involving 14 mutation sites (including D83, P103, V147, F188, H232, E297, M319, E387, W447, N516, L578, E609, Q656, and E673). To further focus on strategies to improve thermostability by enhancing the rigidity of flexible regions, mutation sites located inside the α-helix and β-sheet (V147, H232, E297, E387, N516, and Q656) were excluded. Finally, eight sites located in the flexible loop region—D83, P103, F188, M319, W447, L578, E609, and E673—were selected as candidates.

[0034] 2. Construction, expression, and preliminary screening of candidate vomitoxin detoxification fusion enzyme mutants

[0035] For the eight identified candidate sites, site-directed mutagenesis primers were designed (primer sequences are shown in Table 1). Using the pET-28a(+)-ADDE plasmid carrying the wild-type vomitoxin detoxification fusion enzyme gene as a template, corresponding point mutations (including D83P, P103A, P103E, P103G, P103Q, P103S, P103T, P103V, F188L, M319P, W447A, W447G, W447I, W447L, W447P, W447S, W447T, W447V, L578N, E609D, E673Q, etc.) were introduced by whole-plasmid PCR.

[0036] The PCR amplification system was as follows: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 12.5 μL of 2×Phanta MaxMaster Mix, 0.5 μL of template DNA (10 ng / μL), and ddH2O to a final volume of 25 μL.

[0037] The PCR amplification program was as follows: pre-denaturation at 95℃ for 3 min; then 30 cycles, each cycle consisting of denaturation at 95℃ for 15 s, annealing at 65℃ for 15 s, extension at 72℃ for 4 min; and finally, complete extension at 72℃ for 5 min.

[0038] The PCR product was digested with Dpn I to remove the template. The digestion system was: 8.5 μL of PCR product, 0.5 μL of QuickCut Dpn I, and 1 μL of 10×QuickCut Buffer. The digestion was carried out at 37℃ for 15 min.

[0039] The PCR product after template digestion was subjected to recombination reaction. The recombination system consisted of: 1 μL Exnase II, 2 μL 5×CE II Buffer, 2 μL ddH2O, and 5 μL PCR product. The reaction was carried out at 37℃ for 30 min.

[0040] Transform the recombinant product into E. coli BL21(DE3) competent cells: Thaw competent cells on ice, add 10 μL of recombinant product to 100 μL of competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Then heat shock in a 42℃ water bath for 45 s, and immediately transfer to ice to cool for 2-3 min. Add 600 μL of LB medium and shake at 37℃ and 200 rpm for 1 h. Spread 100 μL of the bacterial culture onto LB agar plates containing kanamycin (50 μg / mL) and incubate upside down in a 37℃ incubator for 12 h.

[0041] After overnight culture, single clones should grow on the recombinant reaction transformation plate. Select single clones for sequencing verification.

[0042] Table 1: Primer sequences

[0043] Primer Name ID No. Sequence (5′→3′) D83P-F SEQ ID NO.3 TATCCGAAGGGCACCATCATTGCCACCAAGAG D83P-R SEQ ID NO.4 ATGGTGCCCTTCGGATAGGGCGCCAGCGCCTC E609D-F SEQ ID NO.5 GGACCAGGATTGTACCTATGAAATGATCCCGGT E609D-R SEQ ID NO.6 AGGTACAATCCTGGTCCCAGTTGTCGCGCGGC E673Q-F SEQ ID NO.7 GTCCAGCAGAACCTGATCGAGAAGGTCGACGA E673Q-R SEQ ID NO.8 ATCAGGTTCTGCTGGACGGTATCACGAGCGTA F188L-F SEQ ID NO.9 TTACAACCTGGCCAATCGCAAGAGCGAAGCGG F188L-R SEQ ID NO.10 GATTGGCCAGGTTGTAAAGGTTCTGCACCGTTG L578N-F SEQ ID NO.11 TATGGTTCGAACACTCGCTTCGCAGTGAA L578N-R SEQ ID NO.12 GAGTGTTCGAACCATAATTCGTGCCGCCAACGGTATTGCG M319P-F SEQ ID NO.13 TCCGCAGGTCGATATCAGTGCGTTGCCGATGG M319P-R SEQ ID NO.14 TGATATCGACCTGCGGACCACTACCAGAACCGCTTCC P103A-F SEQ ID NO.15 AATCAATGGGCACCGCTGGGGCGTCCGGAATT P103A-R SEQ ID NO.16 AGCGGTGCCCATTGATTGGGACCGCTGCGGGT P103E-F SEQ ID NO.17 AATCAATGGGAACCGCTGGGGCGTCCGGAATT P103E-R SEQ ID NO.18 AGCGGTTCCCATTGATTGGGACCGCTGCGGGTC P103G-F SEQ ID NO.19 AATCAATGGGGTCCGCTGGGGCGTCCGGAATT P103G-R SEQ ID NO.20 AGCGGACCCCATTGATTGGGACCGCTGCGGGT P103Q-F SEQ ID NO.61 AATCAATGGCAGCCGCTGGGGCGTCCGGAATT P103Q-R SEQ ID NO.62 AGCGGCTGCCATTGATTGGGACCGCTGCGGGT P103S-F SEQ ID NO.21 AATCAATGGAGCCCGCTGGGGCGTCCGGAATT P103S-R SEQ ID NO.22 AGCGGGCTCCATTGATTGGGACCGCTGCGGGT P103T-F SEQ ID NO.23 AATCAATGGACCCCGCTGGGGCGTCCGGAATT P103T-R SEQ ID NO.24 AGCGGGGTCCATTGATTGGGACCGCTGCGGGT P103V-F SEQ ID NO.25 ATCAATGGGTTCCGCTGGGGCGTCCGGAATTC P103V-R SEQ ID NO.26 CAGCGGAACCCATTGATTGGGACCGCTGCGGG W447A-F SEQ ID NO.27 GTCTCCGCAGACAACTTCATCGTTGCCCTCGA W447A-R SEQ ID NO.28 AAGTTGTCTGCGGAGACCATGTAGATCTTGTCTTCAT W447G-F SEQ ID NO.29 GTCTCCGGTGACAACTTCATCGTTGCCCTCGA W447G-R SEQ ID NO.30 AAGTTGTCACCGGAGACCATGTAGATCTTGTCTTCAT W447I-F SEQ ID NO.31 GGTCTCCATTGACAACTTCATCGTTGCCCTCG W447I-R SEQ ID NO.32 AGTTGTCAATGGAGACCATGTAGATCTTGTCTTCAT W447L-F SEQ ID NO.33 GTCTCCCTGGACAACTTCATCGTTGCCCTCGA W447L-R SEQ ID NO.34 AAGTTGTCCAGGGAGACCATGTAGATCTTGTCTTCAT W447P-F SEQ ID NO.35 TCCCCGGACAACTTCATCGTTGCCCTCGACGC W447P-R SEQ ID NO.36 ATGAAGTTGTCCGGGGAGACCATGTAGATCTTGTCTTCAT W447S-F SEQ ID NO.37 GTCTCCAGCGACAACTTCATCGTTGCCCTCGA W447S-R SEQ ID NO.38 AAGTTGTCGCTGGAGACCATGTAGATCTTGTCTTCAT W447T-F SEQ ID NO.39 GTCTCCACCGACAACTTCATCGTTGCCCTCGA W447T-R SEQ ID NO.40 AAGTTGTCGGTGGAGACCATGTAGATCTTGTCTTCAT W447V-F SEQ ID NO.41 GGTCTCCGTTGACAACTTCATCGTTGCCCTCG W447V-R SEQ ID NO.42 AGTTGTCAACGGAGACCATGTAGATCTTGTCTTCAT

[0044] Positive clones with correct sequencing were inoculated into LB liquid medium and cultured at 37°C with shaking at 200 rpm until OD. 600 The concentration of the enzyme was 0.6–0.8. IPTG was added to a final concentration of 0.2 mM, and expression was induced at 16°C for 16 hours. The bacterial cells were collected by centrifugation, resuspended in 20 mM Tris-HCl buffer (containing 200 mM NaCl, pH 8.0), and sonicated. The enzymes were then purified by nickel affinity chromatography to obtain the wild-type and each mutant purified enzyme. SDS-PAGE electrophoresis analysis showed that all mutants expressed well, and their molecular weights were consistent with the wild-type.

[0045] The initial catalytic activity of each mutant against vomitoxin was determined at 37℃ and pH 7.5. The enzyme reaction system was: 50 µg protein, 1 mM Ca 2+The reaction mixture consisted of 50 µM PQQ, 50 µM DON, and 50 mM Tris-HCl (pH 7.5). After reacting at 37°C for 2 hours, the reaction was terminated by heating at 95°C for 5 minutes. The reaction solution was filtered through a 0.22 µm filter membrane, and the residual vomitoxin content was determined by high-performance liquid chromatography (HPLC). The chromatographic column was a ZORBAX Eclipse Plus C18, the mobile phase was 5% acetonitrile and 95% water, isocratic elution was performed for 20 minutes, and the detection wavelength was 218 nm. The results showed that most mutants retained more than 80% of the wild-type enzyme activity, while the activity of all W447 site mutants was significantly reduced (only 5-30% of the wild-type), indicating that W447 is crucial for catalytic activity. Thermostability was assessed in mutants (D83P, P103A, P103E, P103G, P103S, P103T, P103V, F188L, M319P, E609D, E673Q) that maintained high activity (>80%). After incubating the purified enzymes at 50°C for 120 minutes, residual activity was measured. The results showed that the wild-type residual activity was only 12.27%, while the residual activities of all P103 site mutants were above 17%. Among them, P103A, P103E, P103Q, and P103S had residual activities as high as 31-40%, significantly better than the wild-type and other site mutants. This confirms that proline at position 103 is a key site regulating the thermostability of the vomitoxin detoxification enzyme.

[0046] 3. Construction of P103 site saturated mutant library and screening of mutants to improve thermal stability:

[0047] To further explore the effect of amino acid residue 103 on thermal stability, primers were designed to perform site-directed saturation mutagenesis at this site. A mutant library covering all 19 amino acids was constructed using the same method as in step 2, and successfully expressed and purified.

[0048] Table 2 Primer sequences for the P103 saturated mutant

[0049] Primer Name ID No. Sequence (5′→3′) P103C-F SEQ ID NO.43 ATCAATGGTGTCCGCTGGGGCGTCCGGAATTC P103C-R SEQ ID NO.44 CAGCGGACACCATTGATTGGGACCGCTGCGGG P103D-F SEQ ID NO.45 ATCAATGGGATCCGCTGGGGCGTCCGGAATTC P103D-R SEQ ID NO.46 CAGCGGATCCCATTGATTGGGACCGCTGCGGG P103F-F SEQ ID NO.47 TCAATGGTTTCCGCTGGGGCGTCCGGAATTCC P103F-R SEQ ID NO.48 CCAGCGGAAACCATTGATTGGGACCGCTGCGG P103H-F SEQ ID NO.49 ATCAATGGCATCCGCTGGGGCGTCCGGAATTC P103H-R SEQ ID NO.50 CAGCGGATGCCATTGATTGGGACCGCTGCGGG P103I-F SEQ ID NO.51 TCAATGGATTCCGCTGGGGCGTCCGGAATTCC P103I-R SEQ ID NO.52 CCAGCGGAATCCATTGATTGGGACCGCTGCGG P103K-F SEQ ID NO.53 TCAATGGAAACCGCTGGGGCGTCCGGAATTCC P103K-R SEQ ID NO.54 CCAGCGGTTTCCATTGATTGGGACCGCTGCGG P103L-F SEQ ID NO.55 AATCAATGGCTGCCGCTGGGGCGTCCGGAATT P103L-R SEQ ID NO.56 AGCGGCAGCCATTGATTGGGACCGCTGCGGGT P103M-F SEQ ID NO.57 ATCAATGGATGCCGCTGGGGCGTCCGGAATTC P103M-R SEQ ID NO. 58 CAGCGGCATCCATTGATTGGGACCGCTGCGGG P103N-F SEQ ID NO. 59 TCAATGGAATCCGCTGGGGCGTCCGGAATTCC P103N-R SEQ ID NO. 60 CCAGCGGATTCCATTGATTGGGACCGCTGCGG P103R-F SEQ ID NO. 63 AATCAATGGCGTCCGCTGGGGCGTCCGGAATT P103R-R SEQ ID NO. 64 AGCGGACGCCATTGATTGGGACCGCTGCGGGT P103W-F SEQ ID NO. 65 AATCAATGGTGGCCGCTGGGGCGTCCGGAATT P103W-R SEQ ID NO. 66 AGCGGCCACCATTGATTGGGACCGCTGCGGGT P103Y-F SEQ ID NO. 67 TCAATGGTATCCGCTGGGGCGTCCGGAATTCC P103Y-R SEQ ID NO. 68 CCAGCGGATACCATTGATTGGGACCGCTGCGG

[0050] Thermodynamics of wild-type vomitoxin detoxification fusion enzyme and all P103 saturated mutants were determined at 50°C. Half-life (t) 1 / 2 The assay method was as follows: 0.5 µg / mL of purified enzyme was incubated at 50 °C for 0, 30, 60, 90, and 120 min, respectively, followed by placement on ice for 30 min. The residual activities of the wild-type and mutant enzymes were then determined according to the method described in Example 2, and the half-life was calculated.

[0051] The results showed that the wild-type vomitoxin detoxification fusion enzyme t 1 / 2The half-life was 35.56 minutes. All P103 site mutants showed varying degrees of increased half-life, ranging from 46 to 185 minutes. Among them, mutant P103H exhibited the most significant improvement, with a half-life of 185.78 minutes, which is 5.22 times that of the wild type. Therefore, P103H is the preferred mutant with improved thermal stability.

[0052] 4. Enzymatic characterization of mutant P103H;

[0053] The enzymatic properties of the wild type and the optimal mutant P103H were systematically characterized.

[0054] (1) Optimal temperature: Refer to the enzyme reaction system and enzyme reaction conditions in step 2, adjust the relative activity of wild-type and mutant enzymes at different reaction temperatures (15-50℃). Figure 1 The results showed that the optimal temperature for both was 35℃. Above 40℃, P103H exhibited significantly higher relative activity than the wild type. For example, at 50℃, the residual activity of P103H was 55.79%, while that of the wild type was only 21.50%.

[0055] (2) Optimal pH: Refer to the enzyme reaction system and enzyme reaction conditions in step 2, adjust the relative activities of wild-type and mutant enzymes in different pH buffers (pH=5.0~8.5). Figure 2 The results showed that both had an optimal pH of 7.5 and were stable in the pH range of 7.0 to 8.0.

[0056] (3) Thermal stability: Referring to the thermal deactivation mechanics determination method in step 3, the enzyme solution was incubated at 50℃, 55℃ and 60℃ for different times and the residual activity was measured. Figures 3 to 5 The results showed that P103H exhibited significantly better stability than the wild type at various temperatures. For example, Figure 3 In the study, after treatment at 50℃ for 120 minutes, the residual activity of the wild type was only 12.27%, while P103H still retained 64.08% residual activity. Figure 4 In the study, after treatment at 55℃ for 20 minutes, the residual activity of the wild-type was only 27.98%, while that of P103H was still 72.55%. Figure 5 In the study, after treatment at 60℃ for 10 minutes, the residual activity of the wild type was 15.22%, and that of P103H was 60.15%.

[0057] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A highly heat-stable vomitoxin detoxification fusion enzyme mutant, characterized in that, The mutant was obtained by mutating the amino acid sequence of the vomiting toxin detoxification fusion enzyme shown in SEQ ID NO.

1.

2. The high-thermal-stability vomitoxin detoxification fusion enzyme mutant according to claim 1, characterized in that, The amino acid proline at position 103 of the amino acid sequence shown in SEQ ID NO.1 was mutated to histidine; the amino acid sequence of the mutant is shown in SEQ ID NO.

2.

3. The high-thermal-stability vomitoxin detoxification fusion enzyme mutant according to claim 1, characterized in that, The mutant has a half-life of no less than 5 times that of the wild type at 50°C.

4. A biomaterial related to the high thermal stability vomitoxin detoxification fusion enzyme mutant of claim 1, characterized in that, The biomaterials include any one of the following: A) A nucleic acid molecule encoding the high-thermal-stability vomitoxin detoxification fusion enzyme mutant according to any one of claims 1 to 3; B) An expression cassette containing the nucleic acid molecule described in A); C) A recombinant vector containing the nucleic acid molecule described in A), or a recombinant vector containing the expression cassette described in B; D) Recombinant microorganisms containing the nucleic acid molecules described in A), or recombinant microorganisms containing the expression cassette described in B), or recombinant microorganisms containing the recombinant vector described in C).

5. A method for preparing a highly thermostable vomitoxin detoxification fusion enzyme mutant according to any one of claims 1 to 3, characterized in that, Includes the following steps: A point mutation was performed on the wild-type vomitoxin detoxification fusion enzyme to obtain a nucleic acid molecule encoding the mutant of the highly thermostable vomitoxin detoxification fusion enzyme; the nucleic acid molecule encoding the mutant of the highly thermostable vomitoxin detoxification fusion enzyme was ligated into a vector to obtain a recombinant expression vector; the vector was then transformed into host cells and the target protein was expressed.

6. The method for preparing the high-thermal-stable vomitoxin detoxification fusion enzyme mutant according to claim 5, characterized in that, Includes the following steps: The vector includes one or more of the following: Escherichia coli expression vector, Bacillus subtilis expression vector, Bacillus licheniformis expression vector, Bacillus megaterium expression vector, Bacillus brevis expression vector, lactic acid bacteria expression vector, yeast expression vector, Streptomyces expression vector, or filamentous fungal expression vector.

7. The method for preparing the highly heat-stable vomitoxin detoxification fusion enzyme mutant according to claim 5, characterized in that, The host cell is one or more of the following: Escherichia coli host cell, Bacillus subtilis host cell, Bacillus licheniformis host cell, Bacillus megaterium host cell, Bacillus brevis host cell, lactic acid bacteria host cell, yeast host cell, Streptomyces host cell, or filamentous fungal host cell.

8. The method for preparing the highly heat-stable vomitoxin detoxification fusion enzyme mutant according to claim 5, characterized in that, Point mutations were performed on the wild-type vomitoxin detoxification fusion enzyme using primer pairs with sequences shown in SEQ ID NO.49 and SEQ ID NO.

50.

9. The use of the high thermal stability vomitoxin detoxification fusion enzyme mutant according to any one of claims 1 to 3 in the degradation of vomitoxin or reduction of vomitoxin activity.

10. The use of the high thermal stability vomitoxin detoxification fusion enzyme mutant according to any one of claims 1 to 3 in the preparation of food processing agents, food flavor regulators, texture improvers, preservatives, feed, pharmaceuticals or contaminant treatment.

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