Vomitoxin hydrolyzing enzyme and method of degrading vomitoxin

Abstract

The application provides a vomitoxin hydrolytic enzyme and a method for degrading vomitoxin. The vomitoxin hydrolytic enzyme has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence with more than 70% homology with SEQ ID NO: 1. The problem that the effect of the hydrolytic enzyme preparation for degrading vomitoxin in the prior art is poor in degrading DON can be solved, and the application is suitable for the field of degrading vomitoxin.

Description

Technical Field

[0001] This invention relates to the field of degrading vomitoxin, and more specifically, to a vomitoxin hydrolase and a method for degrading vomitoxin. Background Technology

[0002] Deoxynivalenol (DON), a type B trichothecene compound produced by Fusarium, poses a significant challenge to food safety due to its unique pressure and heat resistance. DON remains stable below 125°C, moderately stable within the 125-200°C range, but its structure begins to partially decompose at 200-250°C. In pH environments, DON is stable under weakly acidic conditions, but undergoes partial degradation to form the derivative DOM under strongly acidic conditions, while exhibiting instability under alkaline conditions. These physicochemical properties mean that traditional food processing or cooking methods cannot effectively eliminate DON, and may even generate new toxic derivatives during heat treatments such as baking.

[0003] Given its widespread distribution and potential threat to public health, DON has been classified as a Group 3 carcinogen by the International Agency for Research on Cancer. Its ability to induce acute gastroenteritis, loss of appetite, growth inhibition, and anemia in animals and humans, coupled with its ability to accelerate apoptosis, decrease immune function, and even cause serious consequences such as bleeding, esophageal perforation, and shock at high doses, makes DON a key target for food safety monitoring. The toxic mechanism of DON mainly involves its specific binding to the 60S subunit peptidyl transferase center of eukaryotic ribosomes. This binding occurs through the formation of three hydrogen bonds: the first between the hydrogen of the uracil U2873 sugar chain and the epoxy group at C-12 of DON; the second between the hydrogen of guanine G2403 and the hydroxyl group at C15 of DON; and the third between the oxygen of uracil U2869 and the hydrogen of the C3 group of DON. This binding hinders ribosome function, thereby affecting protein synthesis. Weakening or disrupting these hydrogen bonds can significantly reduce the toxicity of DON to eukaryotic cells.

[0004] Enzyme preparations, as a novel technology for decomposing mycotoxins (i.e., vomitoxin), are gradually becoming a leading method for controlling DON contamination. For example, existing enzyme preparations can reduce the toxicity of DON through oxidation or modification of specific chemical groups, making them suitable for detoxification treatment in the feed and food industries. However, current technologies face challenges including insignificant degradation effects (partial degradation less than 90%), unclear degradation sites (e.g., modification of only the 3-OH group fails to destroy the main toxic C12 and C13 epoxy groups), and high residual toxicity of post-degradation metabolites. Summary of the Invention

[0005] The main objective of this invention is to provide a vomitoxin hydrolase and a method for degrading vomitoxin, in order to solve the problem that the hydrolase preparations for degrading vomitoxin in the prior art have poor efficacy in degrading DON.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a vomitoxin hydrolase is provided, the sequence of which is shown in SEQ ID NO: 1, or the sequence of which has more than 70% homology with SEQ ID NO: 1.

[0007] Furthermore, the vomitoxin hydrolase also includes a vomitoxin hydrolase mutant based on the amino acid sequence shown in SEQ ID NO: 1, with the mutation site being G128.

[0008] Furthermore, the mutation type of the mutant is G128E, and the sequence of the mutant is shown in SEQ ID NO: 3.

[0009] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes the aforementioned vomitoxin hydrolase.

[0010] Furthermore, the DNA molecule has a nucleotide sequence as shown in SEQ ID NO:2 or SEQ ID NO:4, or a nucleotide sequence that is more than 70% homologous to SEQ ID NO:2 or SEQ ID NO:4.

[0011] To achieve the above objectives, according to a third aspect of the present invention, a recombinant vector is provided, which contains the aforementioned DNA molecule.

[0012] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, wherein the engineered bacteria contains the aforementioned DNA molecule or the aforementioned recombinant vector.

[0013] Furthermore, host cells include prokaryotic cells.

[0014] Furthermore, prokaryotic cells include Escherichia coli.

[0015] Furthermore, Escherichia coli includes BL21(DE3), DH5α, or TOP10.

[0016] To achieve the above objectives, according to a fifth aspect of the present invention, a method for degrading vomitoxin is provided, the method comprising: mixing the above-mentioned vomitoxin hydrolase or the above-mentioned host cell with the target substance to be degraded to obtain a mixed system, and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substance to be degraded.

[0017] Furthermore, the method also includes: mixing the above-mentioned vomitoxin hydrolase, or the above-mentioned host cells, carriers and target substances to be degraded to obtain a mixed system, and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substances to be degraded.

[0018] Furthermore, the carrier includes one or more of yeast polysaccharides, montmorillonite, or attapulgite.

[0019] Furthermore, the system parameters for the degradation reaction are as follows: the temperature of the degradation reaction is 15~45℃, the humidity of the degradation reaction is 70~80%RH, and the degradation reaction time is 2~6h.

[0020] Furthermore, the mixture also includes reaction aids.

[0021] Furthermore, the reaction aids include amino acids and / or metal compounds.

[0022] Furthermore, the metallic element in the metallic compound includes one or more of iron, copper, or zinc.

[0023] Furthermore, the amino acid includes cysteine.

[0024] By applying the technical solution of the present invention, the hydrolase for degrading vomitoxin in this application having an amino acid sequence as shown in SEQ ID NO: 1, or a hydrolase for degrading vomitoxin having an amino acid sequence with more than 70% homology to SEQ ID NO: 1, has strong activity and strong degradation ability in the degradation reaction with the target substance to be degraded. The degradation effect is better than that of the hydrolase for degrading vomitoxin in the prior art, and it is more suitable for promotion and application. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 A flowchart illustrating the method for degrading vomitoxin as described in this application specification is shown.

[0027] Figure 2 A three-dimensional schematic diagram of the secondary structure of the DON degrading enzyme of Example 1 in this application specification is shown.

[0028] Figure 3 The DON standard curve of test example 1 in this application specification is shown. Detailed Implementation

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0030] As mentioned in the background art, existing enzyme preparations have problems such as poor DON degradation effect, unclear degradation sites, and high residual toxicity of metabolites generated during the degradation reaction. Based on this, the inventors have discovered a new hydrolytic enzyme for degrading vomitoxin in this application, and thus proposed a series of protection schemes in this application.

[0031] In a first typical embodiment of this application, a vomitoxin hydrolase is provided, the sequence of which is shown in SEQ ID NO: 1, or an amino acid sequence having more than 70% homology with the sequence of SEQ ID NO: 1 of the vomitoxin hydrolase.

[0032] SEQ ID NO: 1: MALAHSNIPSGTTVIPSPFQVHVSDEQIEELQLLVKLSKLAPPTYEGLQQDRKYGITNEWLANAKEAWKSLDWRSAESRINSFPQFTYDIEGLTIHFVALFSERKDAIPIVLLHGWPGSFLEFLPALT SIRDKYSPETLPYHIVIPSLPGFTFSSSGPPLDVNFTGVDTARVINKVMLNLGFEDGYVAQGGDIGSRIGRILAVDHESCKAVHLNACYMGKPSNVPDTAITELVRRHYAVLATVSSSYPPPSGRSPYITHPS.

[0033] Currently, several technical solutions exist for degrading vomitoxin (hereinafter referred to as DON) using enzymes that degrade vomitoxin. These solutions involve enzyme screening, genetic engineering, expression system optimization, and industrial applications. For example, existing technologies include methods for degrading vomitoxin by genetically engineering sorbitol dehydrogenase, which exhibits superior degradation efficiency and thermal stability. However, this reaction system requires the addition of the expensive cofactor PQQ (Pyrroloquinoline Quinone, as shown in Formula I). This increases the cost of the reaction; or it can reduce the toxicity of DON by oxidizing or modifying the 3-OH and C9=C10 sites of DON through the vomitoxin-degrading enzyme DDH. However, DDH only modifies the side chain groups of DON (such as 3-OH), oxidizing them to generate 3-keto-DON (residual toxicity >20%), and cannot destroy the C12 and C13 epoxy groups (the main toxic structure of DON), resulting in incomplete detoxification.

[0034] Existing technologies also include strains isolated from marine sediments that detoxify DON by producing specific degrading enzymes that destroy the C12-13 epoxy groups. However, after 72 hours of degradation, their DON degradation rate only reaches about 85%, far lower than other DON-degrading bacteria (other DON-degrading enzymes can reach over 45% within almost 4 hours). Alternatively, highly efficient degradation of vomitoxin can be achieved through the design of specific amino acid sequences, but these enzymes mainly reduce toxicity by modifying the 3-OH hydroxyl groups or C9=C10 double bonds in the vomitoxin molecule (such as oxidation, acetylation, or glycosylation), and there is also the possibility of incomplete detoxification.

[0035] In summary, although existing technologies include enzyme preparations and microbial techniques for degrading DON, their efficiency, cost-effectiveness, and the harmless treatment of degradation products still need improvement in practical applications. This application, through extensive screening, discovered a novel hydrolytic enzyme derived from *Aspergillus tabineus* that degrades vomitoxin. Aspergillus tubingensis This enzyme exhibits good DON degradation activity and can precisely target the C12 and C13 epoxy groups on DON, reducing the toxicity of DON degradation products in the system to 1% of the original DON toxicity. It provides a highly efficient, low-cost DON-degrading hydrolase with safe degradation products, filling a gap in existing technologies and providing protection for food safety and public health. In this specification, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0036] The protein provided by the amino acid sequence of SEQ ID NO: 1 has 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) homology and has the same function. Its active site, active pocket, activity mechanism, protein structure, etc. are likely the same as those of the protein provided by SEQ ID NO: 1, and it is a homologous protein obtained through amino acid mutation.

[0037] Obtaining sequences with the aforementioned homology can be achieved through amino acid substitution and replacement. Generally, substitutions between amino acids with similar properties produce similar effects. For ease of description, the abbreviations of the amino acid residues are listed below: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0038] Amino acid substitutions or replacements, for example, can occur in the aforementioned homologous proteins, where conserved amino acid substitutions may take place. "Conserved amino acid substitutions" include, but are not limited to:

[0039] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0040] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0041] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0042] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0043] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0044] In a preferred embodiment, the vomitoxin hydrolase further includes a vomitoxin hydrolase mutant based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation site of the mutant is G128.

[0045] In a preferred embodiment, the mutation type of the mutant is G128E. Preferably, the sequence of the mutant is shown in SEQ ID NO: 3.

[0046] SEQ ID NO: 3: MALAHSNIPSGTTVIPSPFQVHVSDEQIEELQLLVKLSKLAPPTYEGLQQDRKYGITNEWLANAKEAWKSLDWRSAESRINSFPQFTYDIGGLTIHFVALFSERKDAIPIVLLHGWPGSFLEFLPALT SIRDKYSPETLPYHIVIPSLPGFTFSSSGPPLDVNFTGVDTARVINKVMLNLGFEDGYVAQGGDIGSRIGRILAVDHESCKAVHLNACYMGKPSNVPDTAITELVRRHYAVLATVSSSYPPPSGRSPYITHPS.

[0047] The aforementioned amino acid mutations were experimentally investigated in the embodiments of this application. They exhibited the activity of the hydrolase described above, and when applied to the degradation of DON, their catalytic ability was enhanced compared to the wild-type hydrolase, reaching 60% after 4 hours and 90% after 72 hours. The mutation sites were located around the active amino acid sites of the wild-type hydrolase. Such mutations resulted in better degradation ability, while mutations located far from the active sites had a smaller impact on enhancing the activity.

[0048] In a second typical embodiment of this application, a DNA molecule is provided that encodes the aforementioned hydrolytic enzyme that degrades vomiting toxins.

[0049] In a preferred embodiment, the DNA molecule has a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a nucleotide sequence having more than 70% homology with SEQ ID NO: 4.

[0050] SEQ IN NO:2:atggcactcgctcacagcaacattccctcgggtacgaccgtcatcccatcccctttccaggtccatgtttcggacgaacaaatcgaggagctacagctgttggtcaagctgtcgaagctcgcacctccaacatacgaaggtcttcagcaggatcgtaaatatggcataaccaacgaatggcttgccaatgcaaaggaagcttggaagagcttagactggcgatcggcagaaagccgtatcaacagcttccctcagttcacgtatgatatcgagggcctgaccattcactttgtggctttattctccgagagaaaggatgcaatccctattgttcttctccacggctggccaggcagctttctcgagtttcttcccgctctgacttcaattcgggacaaatatagcccagaaaccttgccataccatatagtgattccgtcgcttccggggttcacgttctcatctggtcctccgctggatgtcaacttcactggcgttgatacggcccgcgtcatcaacaaggtgatgctcaatctcggtttcgaggatggctatgtggcacaaggtggagacattggatcaaggatcggtcgcatacttgcagtagatcatgaatcttgcaaagccgtgcatttgaatgcgtgctatatgggaaagccctccaacgtaccagacaccgctattactgagttggtccgtagacattatgcggtattagctaccgtttccagtagttatcccccgccatcaggcagatccccatacattactcacccgtcc。

[0051] SEQ ID NO: 4:

[0052] The aforementioned DNA can encode the aforementioned hydrolytic enzyme protein that degrades vomitoxin and can be linked to a recombinant plasmid to form a circular DNA. Both the aforementioned DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, and tRNA to obtain the aforementioned hydrolytic enzyme protein that degrades vomitoxin.

[0053] In a third typical embodiment of this application, a recombinant vector is provided, which contains the aforementioned DNA molecule.

[0054] In a fourth typical embodiment of this application, a host cell is provided, which contains the aforementioned DNA molecule or the aforementioned recombinant vector.

[0055] The host cells of this application include engineered bacteria capable of producing hydrolytic enzymes that degrade vomitoxin. The hydrolytic enzymes have high degradation efficiency and can degrade the C12 and C13 epoxy structures in DON to obtain deepoxyvomitoxin (DOM-1). Furthermore, the vomitoxin hydrolytic enzymes of this application can reduce the toxicity level of the DON degradation products in the system to 1% of the original DON toxicity, which is more conducive to sustainable development.

[0056] In a preferred embodiment, the host cell of the engineered bacteria includes a prokaryotic cell.

[0057] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large quantity of hydrolytic enzyme proteins that degrade vomitoxin. Hydrolytic enzyme proteins that degrade vomitoxin can also be obtained using existing technologies, such as disrupting host cells, purifying the disrupted proteins, or other methods.

[0058] In a preferred embodiment, the prokaryotic cells include Escherichia coli.

[0059] In a preferred embodiment, the Escherichia coli includes BL21(DE3), DH5α, or TOP10, preferably BL21(DE3).

[0060] In a fifth typical embodiment of this application, a method for degrading vomitoxin is provided. The method includes: mixing the aforementioned vomitoxin hydrolase or the aforementioned host cells with a target substance to be degraded to obtain a mixed system; and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substance. The target substance to be degraded includes DON-contaminated grains or feed. Preferably, the mass ratio of the host cells to the target substance to be degraded is (1-2):(5-10), more preferably 1:2.5-10, including but not limited to 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:9, 1:8, or 1:10.

[0061] The flowchart of the method for degrading vomitoxin in this application is shown below. Figure 1 As shown, in the method for degrading vomitoxin according to this application, vomitoxin hydrolase or the aforementioned host cells are mixed with the target substance to be degraded. The concentration of the bacterial solution and the mixing ratio need to be optimized based on the concentration of DON in the target substance and the activity of the engineered bacterial enzyme to ensure the efficient conduct of the degradation reaction.

[0062] In a preferred embodiment, the method for degrading vomitoxin further includes: mixing the above-mentioned vomitoxin hydrolase, or the above-mentioned host cell, carrier and target substance to be degraded to obtain a mixed system, and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substance to be degraded.

[0063] In a preferred embodiment, the carrier includes, but is not limited to, one or more of yeast polysaccharides, montmorillonite, or attapulgite.

[0064] The method for degrading vomitoxin in this application further includes mixing the crude enzyme solution of the aforementioned vomitoxin hydrolase with a carrier to obtain an enzyme-carrier complex. The carrier in this application (including but not limited to the yeast polysaccharide, montmorillonite, or attapulgite mentioned above, and can also be replaced with any porous carrier well known to those skilled in the art, with the dosage adjusted according to actual needs) utilizes its natural porous structure to immobilize the target enzyme molecules in the crude enzyme solution through covalent bonding or physical adsorption, forming a uniform and stable enzyme-carrier complex. This complex retains the enzyme's catalytic activity and, in addition to having specific adsorption capacity for DON, also exhibits synergistic adsorption of other structurally similar toxins, thereby achieving enzyme immobilization and removal of multiple toxins in one step. Compared with the conventional process of purifying first and then loading the enzyme, the addition of the carrier simplifies the individual purification step, further reducing purification costs; and the carrier-enzyme complex can directly enter the subsequent drying process. Because the carrier framework provides mechanical support and increases the evaporation surface area, the drying time can be shortened, the unit energy consumption can be reduced accordingly, and the overall process cost can be lowered.

[0065] In a preferred embodiment, the mixture further includes a reaction aid. The reaction aid enhances the activity and stability of the hydrolytic enzyme, thereby accelerating the degradation process of DON and improving degradation efficiency. Those skilled in the art can flexibly select this type of reaction aid according to actual production needs.

[0066] In a preferred embodiment, the degradation reaction temperature is 15~45℃ (including but not limited to 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 37.5℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃).

[0067] In a preferred embodiment, the humidity of the degradation reaction is 70-80%RH (including but not limited to 70, 75 or 80%RH).

[0068] In a preferred embodiment, the degradation reaction time is 2 to 6 hours (including but not limited to 2, 3, 4, 5 or 6 hours).

[0069] Controlling the parameters of the degradation reaction in this application within the above-mentioned range is beneficial to the occurrence of the degradation reaction, promotes the forward progress of the degradation reaction, improves the efficiency of the reaction, enables the above-mentioned hydrolytic enzyme to maintain a high activity in the reaction system, exerts catalytic activity, and makes the degradation effect more stable.

[0070] In a preferred embodiment, the reaction aid includes, but is not limited to, amino acids and / or metal compounds. Preferably, the amino acid includes cysteine. Preferably, in the above-mentioned reaction aid, the concentration of metal ions in the metal compounds is 0.1-1.0 mM (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mM), and the concentration of amino acids is 0.5-5.0 mM (including but not limited to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 mM).

[0071] The reaction aids used in this application are inexpensive and readily available, and can promote the degradation reaction, improving reaction efficiency. Compared with existing systems, they offer higher efficiency and lower cost, making them suitable for large-scale promotion and application in fields requiring DON degradation. The aforementioned cysteine ​​enhances the stability of the DON-degrading enzyme structure, reducing the risk of enzyme denaturation. Furthermore, the addition of cysteine ​​regulates the pH and charge of the reaction microenvironment, resulting in a more stable degradation reaction system.

[0072] In a preferred embodiment, the metal element in the metal compound includes one or more of iron, copper, zinc, manganese, or aluminum. Preferably, the metal compound includes a metal sulfate compound. Preferably, the amino acid includes cysteine.

[0073] This application provides a rapid, efficient, and cost-effective method for degrading DON. Using the engineered bacteria and their expressed hydrolytic enzymes described in this application, DON is effectively degraded under optimized reaction conditions. This method for degrading vomitoxin is not only suitable for pretreatment in the feed and food industries but also provides a solution to DON contamination problems in grains and other agricultural products. It helps improve food safety standards, reduce economic losses caused by mycotoxins, and is environmentally friendly, demonstrating promising application prospects.

[0074] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0075] The reagents and detection methods involved in the embodiments of this application are as follows. Unless otherwise specified, the reagents in this application are all conventional commercially available products:

[0076] Types of reagents:

[0077] DON stock solution: Accurately weigh 10 mg DON (Beijing Meizheng Testing Technology Co., Ltd., MSS0333) and dilute to 10 mL with acetonitrile.

[0078] DON working solution: Accurately transfer an appropriate amount of DON standard stock solution and dilute it with phosphate buffer solutions of different pH values ​​to the concentration required for actual operation.

[0079] instrument:

[0080] Electronic balance: Model DF-200A, Changshu Weighing Instrument Factory, Jiangsu Province;

[0081] Electronic balance: Model AUW2200, Shimadzu, Japan;

[0082] Analytical balance: Model CPA225D, Sartorius;

[0083] Ultra-low temperature freezer: Model U570, NBS, USA;

[0084] Electric heating forced-air drying oven: Model DGX-9243B-1, Shanghai Fuma;

[0085] Thermostatic water bath: Model HH, Hubei Qintai Medical Instrument Factory;

[0086] Spray dryer: Model PW2000B, Shanghai Xiniu;

[0087] High performance liquid chromatograph: Model Alliance E2695, Beckman Coulter;

[0088] Fourier transform infrared spectrometer: Model Nicolet IS50, Thermo Scientific;

[0089] Specific surface area and porosity analyzer: Model ASAP 2460, Agilent Technologies;

[0090] Nanoparticle size potential analyzer: Model Zetasizer Nano ZS, Malvern;

[0091] Analytical balance: CPA225D, Sartorius Scientific Instruments Ltd. (Max = 220 g, min = 1 mg, d = 0.01 mg);

[0092] Constant temperature water bath: HH type, Hubei Qintai Medical Instrument Factory;

[0093] Refrigerator: Model BCD-236H, Haier Group Co., Ltd.;

[0094] Ultrasonic cleaning machine: Model KQ3200, Kunshan Ultrasonic Instrument Co., Ltd.;

[0095] Full-temperature shaking incubator: Model HZQ-F160, Suzhou Peiying;

[0096] Ultrapure water system: Milli-Q, Academic model, Millipure, USA;

[0097] Miniature vortex mixer: Model XW-80A, Shanghai Huxi Analytical Instrument Factory Co., Ltd.;

[0098] Ultra-low temperature freezer: Model U570, NBS, USA;

[0099] Automatic double pure water distiller: Model SZ-93, Shanghai Yarong Biochemical Instrument Factory;

[0100] Benchtop low-speed centrifuge: Model TDL-5C, Shanghai Fichar Analytical Instruments Co., Ltd.;

[0101] Refrigerated benchtop high-speed centrifuge: Model 3K15, Sigma;

[0102] Full-temperature shaking incubator: Model HZQ-F160, Suzhou Peiying Experimental Equipment Co., Ltd.;

[0103] pH meter: PHS-3C type, Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.;

[0104] High performance liquid chromatograph: Model Alliance E2695, Beckman Coulter;

[0105] Biological microscope: Model Nexcope, Ningbo Yongxin Optics Co., Ltd.

[0106] Example 1

[0107] 1. Enzyme screening

[0108] The candidate gene was cloned into an expression vector (e.g., pET28a(+)) and induced to be expressed in E. coli.

[0109] The recombinant enzyme was purified, and the DON degradation rate was quantitatively determined in an in vitro reaction system (pH 7.0, 30℃) to confirm its catalytic efficiency.

[0110] In vitro reaction system: Take 50 μL of crude enzyme solution and place it in a 1.5 mL centrifuge tube. Add 0.1 mL (100 ppm) of DON and 900 μL of ultrapure water. Incubate at 37 ℃ with shaking for 4 h. After the reaction is complete, place the sample in a 98 ℃ water bath for 2 min to inactivate the enzyme. Cool the sample to room temperature and store at 4 ℃ for later testing.

[0111] Degradation rate detection method: The degradation products are detected by HPLC;

[0112] HPLC detection method (the rest of the examples are the same as here): a) Liquid chromatography column: Chrom Core C18 (column length 150 mm, column inner diameter 4.6 mm, packing particle size 5 μm, Nano Chrom); b) Mobile phase: methanol + water (volume ratio 30:70); c) Flow rate: 0.8 mL / min; d) Column temperature: 35°C; e) Injection volume: 10 μL; f) Detection wavelength: 218 nm.

[0113] Screening results: The enzyme with the amino acid sequence shown in SEQ ID NO: 1 was found to have the best degradation effect, and it was further analyzed.

[0114] 2. Enzyme structure and mutation

[0115] The enzyme with the amino acid sequence SEQ ID NO: 1 was predicted using AI (AlphaFold) to obtain its three-dimensional structure, locate the substrate binding pocket and catalytic center, and its secondary structure diagram is shown below. Figure 2 As shown.

[0116] The structures of DON and its ligand degrading enzyme (SEQ ID NO: 1) were imported into SYBYL software for molecular docking. A strong interaction was observed between the EH enzyme and the DON molecule. Hydrogen bonds were formed between DON and the degrading enzyme's GLN84, ARG122, SER167, and TYR479 bonds, with bond lengths of 2.710 Å, 1.860 Å, 1.865 Å, and 1.986 Å, respectively. The S-value for DON docking with the EH enzyme was -5.8229, which is less than -5, indicating a strong binding affinity between DON and the degrading enzyme. The RMSD value for DON docking with the EH enzyme was 0.110, which is less than 0.5, indicating accurate docking results.

[0117] Replacement of key amino acids: Molecular dynamics simulations (MD) were used to assess the binding free energy of mutants with DON and to predict the optimal mutant combination.

[0118] 3. Enzyme expression

[0119] The selected Escherichia coli BL21(DE3) was used as the host, carrying pET28a(+)-EH.

[0120] LB medium, amplify bacterial cells to OD at 37°C. 600 =0.6, add 0.5 mM IPTG to induce expression (16℃, 18 h).

[0121] The bacterial cells were collected by centrifugation and then ultrasonically disrupted to obtain crude enzyme solution.

[0122] Preparation method of purified enzyme: Ni 2+ -NTA column captures His-tagged enzyme, eluted with imidazole gradient (20-500 mM). DEAE column separates impurities, eluted with NaCl gradient (0-1 M).

[0123] Aggregates were removed using a Superdex 200 column to obtain a homogeneous enzyme preparation. Purity ≥95% (SDS-PAGE assay), specific activity ≥200 U / mg, store at -80℃ for later use.

[0124] Example 2

[0125] Verification of enhanced activity in wild-type and mutant enzymes:

[0126] Degradative enzyme modification: Mutant enzymes were obtained by site mutation targeting the key amino acid in the binding pocket (glycine at position 128 → glu of glutamate) using the Mut ExpressFast Mutagenesis Kit V2.

[0127] Both wild-type and mutant enzymes underwent enzyme activity verification.

[0128] Enzyme activity verification method: 20 μL of crude enzyme solution was added to a 300 μL reaction system (pH 7.0) containing 50 mmol / L Tris-HCl, 150 μmol / L NADPH, and 100 μmol / L 3-keto-DON. The reaction system was then incubated at 30 °C for 5 min. The decrease in absorbance at 340 nm was detected using a UV-Vis spectrophotometer. One unit of enzyme activity (U) is defined as the amount of enzyme required to consume 1 μmol of NADPH per minute.

[0129] Methods for calculating enzyme activity:

[0130] Enzyme activity (U / mL) = [ΔA×V] 总 × N] / [ε×L×V 酶 ×t];

[0131] Parameter definitions:

[0132] ΔA: The decrease in absorbance at 340 nm within 5 min of the reaction (measured value, e.g., ΔA = 0.311 for the original enzyme and ΔA = 0.778 for the mutant).

[0133] V 总 Total volume of the reaction system (300 μL = 0.3 mL);

[0134] N: Enzyme solution dilution factor (N=1 if undiluted);

[0135] ε: Molar absorptivity of NADPH at 340 nm (6220 L) mo ¹ c ¹);

[0136] L: Optical path of the cuvette (1 cm);

[0137] V 酶 : The volume of crude enzyme solution added to the reaction system (20 μ = 0.02 mL).

[0138] t: reaction time (5 min).

[0139] In this reaction, the conversion of 3-keto-DON and the consumption of NADPH are in a 1:1 molar ratio. This enzyme activity unit directly reflects the enzyme's catalytic efficiency for 3-keto-DON.

[0140] Based on the enzyme activity calculated above, the relative enzyme activity of wild-type enzyme and mutant enzyme is calculated based on the reference enzyme activity. The formula is: relative enzyme activity (%) = (measured enzyme activity of the sample to be tested ÷ reference enzyme activity) × 100%.

[0141] The above-mentioned reference enzyme activity (negative control) was obtained by replacing the uninactivated crude enzyme solution of the experimental group with 20 μL of inactivated crude enzyme solution (e.g., boiling at 100 °C for 15 min to completely destroy enzyme activity). The other reaction system components (50 mmol / L Tris-HCl, 150 μmol / L NADPH, 100 μmol / L 3-keto-DON, pH 7.0) and incubation and detection conditions were completely consistent with those of the experimental group.

[0142] Based on the above calculations and conversions, the relative enzyme activity of the wild-type enzyme in this embodiment is 32.4%.

[0143] The relative enzyme activity of the mutant enzyme is 48.6%.

[0144] The mutant enzyme has 1.5 times higher activity than the original enzyme.

[0145] Example 3

[0146] Degradation reaction system: Take 50 μL of crude enzyme solution and place it in a 1.5 mL centrifuge tube. Add 0.1 mL (100 ppm) of DON, 900 μL of ultrapure water, and reaction aids. Incubate at 37 ℃ with shaking for 4 h at 70% RH. After the reaction is complete, place the sample in a 98 ℃ water bath for 2 min to inactivate the enzyme. Cool the sample to room temperature and store at 4 ℃ for analysis.

[0147] The mutant enzyme obtained in Example 2 was subjected to degradation reaction using the above-described degradation reaction system.

[0148] In this embodiment, the added reaction aids were 0.5 mM ferric sulfate (ferric ions) and 2.5 mM cysteine, and the reaction was carried out for 4 hours.

[0149] Example 4

[0150] The only difference from Example 3 is that the reaction aids in this example are copper sulfate (divalent copper ions) and cysteine. The other reaction conditions and steps are completely the same as in Example 3.

[0151] Example 5

[0152] The only difference from Example 3 is that the reaction aids in this example are zinc sulfate (divalent zinc ions) and cysteine. The remaining reaction conditions and steps are completely the same as in Example 3.

[0153] Comparative Example 1

[0154] The DON degradation experiment was conducted according to the optimal experimental conditions recommended in the instructions provided by the manufacturer of the commercially available product (Henan Yiwan Zhongyuan Biotechnology Co., Ltd., Meilijie) (the same applies to the comparative examples below). 10.01 mL of the commercially available product was accurately measured into a 50 mL centrifuge tube. 1.5 mL of PBS buffer solution, 1 mL of PQQ solution, 1 mL of calcium chloride solution, and 6.39 mL of pure water were added. 0.1 mL of DON standard working solution (100 ppm) was accurately added, and the mixture was shaken well. The mixture was placed in a shaker at 37 ℃ and 200 r / min and reacted for 4 h. After the reaction was complete, the sample was placed in a 98 ℃ water bath for 2 min to inactivate the enzyme. The sample was then cooled to room temperature.

[0155] Comparative Example 2

[0156] Accurately weigh 100 mg (accurate to 0.1 mg) of commercially available product 2 (Jiangxi Jidi Muju Biotechnology Co., Ltd., vomitoxin degrading enzyme). Perform two replicates for each sample, placing each sample into a 30 mL heat-resistant glass tube with a screw cap. Add 1.0 mL of 50 ppm DON working solution quantitatively, followed by 9.0 mL of pure water. Carefully cap the tube and place it in a 37°C constant-temperature shaker at 150 rpm for 4 hours. Then centrifuge at 12000 rpm for 5 minutes and collect the supernatant for later use.

[0157] At the same time, a blank control without the addition of toxin adsorbent was prepared.

[0158] Comparative Example 3

[0159] Accurately weigh 100 mg (accurate to 0.1 mg) of commercially available product 3 (Sichuan Jilongda Biotechnology Group Co., Ltd., Vodqing), with two replicates for each sample. Place each sample in a 30 mL heat-resistant glass tube with a screw cap. Add 1.0 mL of 50 ppm DON working solution quantitatively, followed by 9.0 mL of pure water. Carefully cap the tube and place it in a 37°C constant temperature shaker at 150 rpm for 4 hours. Then centrifuge at 12000 rpm for 5 minutes and collect the supernatant for later use.

[0160] At the same time, a blank control without the addition of toxin adsorbent was prepared.

[0161] Comparative Example 4

[0162] Accurately weigh 100 mg (accurate to 0.1 mg) of commercially available product 4 (Jiangsu Hanle Biotechnology Co., Ltd., Mycotoxin Enhanced Degrading Enzyme), with two replicates for each sample. Place each sample into a 30 mL heat-resistant glass tube with a screw cap. Quantitatively add 1.0 mL of 50 ppm DON working solution, followed by 9.0 mL of pure water. Carefully seal the tube and place it in a 37°C constant-temperature shaker at 150 rpm for 4 hours. Then centrifuge at 12000 rpm for 5 minutes and collect the supernatant for later use.

[0163] At the same time, a blank control without the addition of toxin adsorbent was prepared.

[0164] Detection Example 1

[0165] The degradation results of Examples 3-5 and Comparative Examples 1-4 were tested and compared, and the results are shown in Table 1.

[0166] Methods for detecting degradation results:

[0167] Construction of DON curves: Take an appropriate amount of DON (100 μg / mL) working solution and prepare standard solutions of 0.00 μg / mL, 0.50 μg / mL, 1.00 μg / mL, 2.50 μg / mL, 5.00 μg / mL, 10.00 μg / mL, and 20.00 μg / mL. Detect DON using HPLC and construct a standard curve of DON based on the peak area.

[0168] After the degradation reaction was completed, the sample was pretreated and analyzed by the HPLC described above. The remaining DON content in the sample was calculated based on the DON standard curve, as shown in the schematic diagram of the standard curve. Figure 3 As shown.

[0169] The method for calculating the degradation effect is as follows:

[0170] Standard curve equation: y = ax + b;

[0171] C 测 =(y 样 -b) / a;

[0172] C 测 The concentration of DON in the sample solution (μg / mL);

[0173] y 样 This represents the peak area of ​​DON in the sample solution.

[0174] Methods for detecting degradation sites:

[0175] The molecular weights of DON metabolites were identified using HPLC-ITTof-MS with the following parameters: HPLC parameters: Mobile phase: ultrapure water and acetonitrile (chromatographic grade); 0–3 min, 5%–30% acetonitrile; 4–7 min, 100% acetonitrile; 7.01–10 min, 5% acetonitrile; Flow rate: 0.3 mL / min; Injection volume: 5 μL. Mass spectrometry parameters: Ion source: ESI. + Scanning mode: cation mode; collision voltage: 15~40 V; cone voltage: 40 V; molecular weight range: 50~500 Da.

[0176] Table 1

[0177]

[0178] Test Example 2

[0179] Thermal stability testing: The thermostat was tested in a solution containing 50 μg DON, 50 mmol / L Tris-HCl (pH 7.0), and 1 mmol / L CaC. Add 10 μg of purified DON hydrolase (dissolved in 10 μL of the same buffer) to a 490 μL buffer system and mix well to make a total volume of 500 μL.

[0180] The reaction system was placed in water baths at 15 ℃, 37 ℃, and 45 ℃ for 4 h, respectively, to terminate the reaction. The residual concentration of DON in the reaction solution was determined using high-performance liquid chromatography (HPLC). Using the 0-hour reaction sample as a control, the DON conversion rate after 4 hours at each temperature was calculated to characterize the enzyme's sustained catalytic efficiency at different temperatures. The results showed that the DON conversion rate was 88.36% at 37 ℃. DON could be degraded at both 15 ℃ (low temperature) and 45 ℃ (high temperature), with efficiencies of 27.36% and 30.23%, respectively. This demonstrates that the DON hydrolase of this application possesses certain thermostability.

[0181] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: Using the wild-type vomitoxin-degrading hydrolase of this application to degrade vomitoxin achieves a good degradation effect without the need for additional expensive adjuvants. Mutagenesis of this wild-type vomitoxin-degrading hydrolase yields the G128E mutant, which enhances enzyme activity and degradation efficiency. Furthermore, it enables precise degradation of the C12 and C13 epoxide structures of DON in the target analyte, reducing residual toxicity during the degradation reaction. The degradation reaction system involving the vomitoxin-degrading hydrolase and its mutant of this application has clearly defined degradation sites, significant degradation effects, reduced overall reaction costs, and low residual toxicity of the metabolites. It is compatible with various degradation systems, making it more suitable for widespread application in feed, food processing, and other systems.

[0182] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A vomitoxin hydrolase, characterized in that, The sequence of the vomitoxin hydrolase is shown in SEQ ID NO:

1.

2. A vomitoxin hydrolase mutant, characterized in that, The sequence of the mutant is shown in SEQ ID NO:

3.

3. A DNA molecule, characterized in that, The DNA molecule encodes the vomitoxin hydrolase of claim 1 or the vomitoxin hydrolase mutant of claim 2.

4. The DNA molecule according to claim 3, characterized in that, The DNA molecule has a nucleotide sequence as shown in SEQ ID NO:2 or SEQ ID NO:

4.

5. A recombinant vector, characterized in that, The recombinant vector contains the DNA molecule as described in any one of claims 3 or 4.

6. A host cell, characterized in that, The host cell contains the DNA molecule of any one of claims 3 or 4 or the recombinant vector of claim 5.

7. The host cell according to claim 6, characterized in that, The host cells include prokaryotic cells.

8. The host cell according to claim 7, characterized in that, The prokaryotic cells include Escherichia coli.

9. The host cell according to claim 8, characterized in that, The Escherichia coli include BL21(DE3), DH5α, or TOP10.

10. A method for degrading vomitoxin, characterized in that, The method includes: mixing the vomitoxin hydrolase of claim 1, or the vomitoxin hydrolase mutant of claim 2, or the host cell of any one of claims 6 to 9 with the target substance to be degraded to obtain a mixed system, and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substance to be degraded.

11. The method according to claim 10, characterized in that, The method further includes: mixing the vomitoxin hydrolase of claim 1, or the vomitoxin hydrolase mutant of claim 2, or the host cell, vector, and target substance to be degraded according to any one of claims 6 to 9 to obtain a mixed system, and carrying out a degradation reaction in the mixed system to degrade the vomitoxin in the target substance to be degraded.

12. The method according to claim 11, characterized in that, The carrier includes one or more of yeast polysaccharides, montmorillonite, or attapulgite.

13. The method according to claim 12, characterized in that, The parameters of the degradation reaction are as follows: the temperature of the degradation reaction is 15~45℃, the humidity of the degradation reaction is 70~80%RH, and the time of the degradation reaction is 2~6h.

14. The method according to claim 12, characterized in that, The mixture also includes reaction aids.

15. The method according to claim 13, characterized in that, The reaction aids include amino acids and / or metal compounds.

16. The method according to claim 15, characterized in that, The metallic element in the metal compound includes one or more of iron, copper, or zinc.

17. The method according to claim 15, characterized in that, The amino acid includes cysteine.

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