A vomitoxin-detoxifying enzyme and its application
By digging out the new DON detoxification enzyme gene aadh from the phylum of Acidobacteria, a highly expressed genetically engineered strain was constructed, and efficient degradation of DON was achieved, and the problems of incomplete detoxification and secondary contamination in the existing technology were solved. It has good thermal stability and pH stability, and is suitable for applications in the food and feed industry.
Patent Information
- Application Number
- CN202411469671.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-06-10
- Estimated Expiration
- 2044-10-21
AI Technical Summary
When the prior art deals with the mycotoxin deoxyfusarcinol (DON), which is widely present in grains and feeds, there are problems such as incomplete detoxification, large loss of nutrients, and possible secondary pollution, which limits its widespread application in the food and feed industries.
By digging out the new DON detoxification enzyme gene aadh from Acidobacteriota, a genetically engineered strain that efficiently expresses aadh was constructed, and the heterologous expression of the DON detoxification enzyme AADH in prokaryotic cells was achieved, with high catalytic activity, good thermal stability and pH stability.
The efficient degradation of DON is achieved. 10-20μg AADH can degrade 45-150μg/mL of DON into the low-toxic product 3-keto-DON within 2-16 hours, with a degradation rate of more than 80%, and has potential industrial application prospects.
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Figure CN119144623B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a vomitoxin detoxifying enzyme and its application. Background Art
[0002] Mycotoxins are toxic secondary metabolites produced by fungi (such as Fusarium, Alternaria, Aspergillus, and Penicillium) infecting plant products during field or storage processes, including aflatoxins, zearalenone, ochratoxin, deoxynivalenol, and patulin, etc. Among them, deoxynivalenol (DON) widely exists in cereal raw materials and their products such as wheat, barley, and corn, and is the most widely distributed mycotoxin globally. By detecting DON in 608 samples of wheat and its products from 26 provinces and cities in seven major regions across the country, the results showed that the annual detection rates of DON in wheat and its by-products were 89.0% and 94.8% respectively, which not only caused huge economic losses, but also seriously endangered the health of humans and livestock. Therefore, how to efficiently and comprehensively control and solve the problem of DON pollution in grains and feeds is the key to promoting the sustainable development of agriculture and ensuring food safety.
[0003] In recent years, a variety of physical and chemical methods have been used for the detoxification of DON, such as adsorption, irradiation, heat treatment, light treatment, ozone treatment, and alkali treatment, etc. However, traditional physical and chemical detoxification methods have problems such as incomplete detoxification, large loss of nutrients, and possible secondary pollution, which limit their wide application in the food and feed industries. The biological detoxification methods based on microorganisms or enzymes have the advantages of high detoxification efficiency, strong specificity, no pollution to the environment, and little damage to the nutrients of feeds and foods, so they have received more and more attention and application. Research shows that the epoxy groups at C12 and C13 positions and the hydroxyl group at C3 position in the DON molecule are the main groups determining the toxicity of DON and are also the key sites for biological detoxification. At present, a variety of microorganisms capable of destroying the epoxy groups at C12 and C13 positions of DON have been screened out, which can convert DON into a de-epoxidized product DOM-1 with extremely low toxicity. However, the degradation mechanism of the biological de-epoxidation pathway of DON has not been fully elucidated, and no report on any DON detoxifying enzyme has been seen. In addition, the research team of Lei Xiaoguang used glutathione transferase (Fhb7) to achieve the ring-opening detoxification of DON under the action of glutathione (GSH), but the toxicity of its product has not been identified. In addition to the epoxy group, the oxidation, glycosylation, acetylation, and isomerization of the hydroxyl group at C3 position of DON are also the main pathways for DON detoxification. Among them, Devosia DevosiaThe pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase DepA / QDDH / DADH / DDH from (sp.) can oxidize the C3 hydroxyl group of DON to generate the less toxic 3-keto-deoxynivalenol (3-keto-DON), and then the NADPH-dependent aldo-keto reductase DepB / AKR6D1 / AKR13B2 / AKR13B3 reduces 3-keto-DON to generate the non-toxic 3-iso-deoxynivalenol (3- epi -DON), that is, the isomerization at the C3 position. This is the most thoroughly studied detoxification method at present, which is safe, efficient, and has stable effects.
[0004] Studies have shown that the toxicity of the degradation products of DON is lower than that of DON. Among them, the toxicity of 3-keto-DON is 1 / 10 of that of DON, while the toxicity of 3- epi -DON is 1 / 1181 of that of DON. In this enzymatic cascade reaction, the first step is crucial because it determines the initial rate and direction of the reaction and directly determines the overall performance of the entire catalytic process. However, the currently identified first-step DON detoxifying enzymes have problems such as low catalytic efficiency and poor thermal stability, which limit their further application in the food and feed industries. Since enzymes from different microorganisms have different enzymatic properties, it is of great practical significance to explore novel DON detoxifying enzymes with high catalytic activity, high thermal stability, and high substrate specificity. SUMMARY OF THE INVENTION
[0005] The object of the present invention is to overcome the deficiencies in the prior art and provide a vomitoxin detoxifying enzyme and its application.
[0006] To achieve the object of the present invention, the following technical solutions will be adopted to implement the present invention.
[0007] A DON detoxifying enzyme gene aadh has a nucleotide sequence as shown in SEQ ID NO.1.
[0008] A DON detoxifying enzyme is a protein encoded by the DON detoxifying enzyme gene aadh and has an amino acid sequence as shown in SEQ ID NO.2.
[0009] A preparation method of a DON detoxifying enzyme includes the following steps:
[0010] S1. Perform codon optimization and gene synthesis on the DON detoxifying enzyme gene in claim 1 aadh ;
[0011] S2. Construct a prokaryotic expression vector of the DON detoxifying enzyme gene aadh ;
[0012] S3. Express the prokaryotic expression vector in a prokaryotic host cell.
[0013] As a preferred embodiment of the present invention, the prokaryotic expression vector is one or more of an Escherichia coli expression vector, a Bacillus subtilis expression vector, a Bacillus licheniformis expression vector, a Bacillus megaterium expression vector, a Brevibacillus brevis expression vector, a lactic acid bacteria expression vector, a yeast expression vector, a Streptomyces expression vector, and a filamentous fungus expression vector.
[0014] As a preferred embodiment of the present invention, the prokaryotic host cell is an Escherichia coli host cell, a Bacillus subtilis host cell, a Bacillus licheniformis host cell, a Bacillus megaterium host cell, a Brevibacillus brevis host cell, a lactic acid bacteria host cell, a yeast host cell, a Streptomyces host cell, or a filamentous fungus host cell.
[0015] Application of a DON detoxifying enzyme in degrading vomitoxin.
[0016] As a preferred embodiment of the present invention, the DON detoxifying enzyme is applied to removing mold toxins from cereal raw materials and feeds. Beneficial effects
[0017] 1. The present invention has discovered a novel DON detoxifying enzyme gene derived from the phylum Acidobacteria ( Acidobacteriota ), constructed a genetically engineered strain for efficient expression aadh , and achieved heterologous expression of the DON detoxifying enzyme AADH in prokaryotic cells; aadh
[0018] 2. The PQQ-dependent alcohol dehydrogenase AADH identified in the present invention, which is derived from Acidobacteriota , has DON degradation activity. 10 - 20 μg of AADH can degrade 45 - 150 μg / mL of DON into the low-toxic product 3-keto-DON within 2 - 16 h. Through multiple sequence alignment, it is found that the sequence similarity of AADH with the currently reported DON detoxifying enzymes DepA, QDDH, DDH, and DADH is 56.55%, 56.55%, 50.43, and 48.56% respectively. Therefore, AADH is a novel DON detoxifying enzyme;
[0019] 3. The AADH of the present invention has good thermal stability and pH stability. When treated at 40 °C for 3 h, AADH can retain more than 50% of its residual activity; in addition, AADH can retain more than 60% of its residual activity when treated at pH 5.5 - 10 for 12 h. Therefore, AADH has potential industrial application potential;
[0020] 4. The AADH of the present invention can oxidize DON to produce low-toxic 3-keto-DON. Meanwhile, treating moldy wheat with AADH can degrade DON in the moldy wheat into 3-keto-DON, and the degradation rate reaches more than 80%, providing a new option for effectively solving the problem of vomitoxin pollution in grain raw materials and feeds, reducing grain losses, and ensuring food and feed safety. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is a schematic diagram of SDS-PAGE electrophoresis and Western blot electrophoresis of purified AADH protein, where: M: protein Marker; 1: cell lysate of Escherichia coli BL21 containing empty plasmid pET28a; 2: crude AADH enzyme sample; 3: purified AADH protein sample;
[0022] Figure 2 It is a curve graph of the detoxification ability of AADH to DON, where: (A) graph is the HPLC analysis of DON; (B) graph is the HPLC analysis of the degradation product 3-keto-DON of DON;
[0023] Figure 3 It is a curve graph of the detoxification effect of AADH on DON;
[0024] Figure 4 It is a curve graph of the enzymatic properties of AADH, where: (A) graph is the optimal reaction temperature of AADH; (B) graph is the temperature stability of AADH; (C) graph is the optimal reaction pH of AADH; (D) graph is the pH stability of AADH;
[0025] Figure 5 It is a bar graph comparing the detoxification effects of AADH in moldy wheat. DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be further described in conjunction with the accompanying drawings and embodiments.
[0027] As an embodiment of the present invention, cloning of the DON detoxifying enzyme gene of Acidobacteria
[0028] Taking PQQ-dependent alcohol dehydrogenase DepA as a template, uploading it to NCBI for multiple sequence alignment, an amino acid sequence AADH derived from Acidobacteria ( Acidobacteriota ) with a sequence identity of 56.55% to the DepA sequence and a functional annotation of PQQ-dependent alcohol dehydrogenase was obtained. This sequence was sent to GenScript Biotech Corporation for codon optimization and gene synthesis, and the synthesized gene sequence was integrated into the multiple cloning site of the Escherichia coli expression vector pET28a to obtain the expression vector pET28a-AADH.
[0029] According to the gene synthesis results, the full length of the DON detoxifying enzyme aadh is 1608 bp, and its nucleotide sequence is shown in SEQ ID NO.1, that is, the DON detoxifying enzyme AADH of Acidobacteria Acidobacteriota ), encoding a protein composed of 536 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.
[0030] As an example of the present invention, the expression and purification of DON detoxifying enzyme in Escherichia coli
[0031] (1) Construction of expression vector: The constructed expression vector pET28a-AADH was transformed into Escherichia coli BL21 competent cells for the expression of recombinant protein.
[0032] (2) Expression of recombinant enzyme: The recombinant bacterium pET28a-AADH was inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin (Kana), and cultured at 37 °C and 180 rpm for 16 h to obtain a seed solution. Then, the seed solution was transferred to 100 mL of fresh LB liquid medium (containing 50 μg / mL Kana) at an inoculation amount of 1%, and cultured at 37 °C and 180 rpm until the OD600 reached between 0.6 and 0.8. Then, 100 μL of IPTG (100 mg / mL) was added, and protein expression was induced at 16 °C and 180 rpm.
[0033] (3) Purification of recombinant enzyme: After fermentation culture, the cells were collected by centrifugation at 4 °C and 8000 rpm, and the cells were washed 3 times with 50 mM Tris-HCl (200 mM NaCl, pH = 7.5), and then resuspended in this buffer. The resuspended cells were sonicated and centrifuged (4 °C, 8000 rpm, 20 min) to remove the precipitate, and the supernatant was the crude enzyme solution. Since the C-terminus of the target protein was fused with a histidine tag during vector construction, Ni 2+ -NTA affinity column can be used to purify DON detoxifying enzyme AADH, and the method is as follows:
[0034] Loading: 10 mM imidazole was added to the supernatant to enhance specific adsorption, and then the supernatant was filtered through a 0.22 μM aqueous filter membrane to remove impurities. Before loading, the column was equilibrated with buffer (50 mM Tris-HCl, 200 mM NaCl, pH = 7.5), and then the sample was loaded in a cyclic manner 3 times.
[0035] Elution: After loading, the unadsorbed impurity proteins were first eluted with 5 column volumes of buffer containing 25 mM imidazole, and finally the target protein was eluted with 5 column volumes of buffer containing 250 mM imidazole. The eluate was collected to obtain the purified sample, and the imidazole was removed by dialysis to obtain the purified protein. The purified protein was verified by SDS-PAGE electrophoresis and Western blot. AsFigure 2 As shown in Figure A in Figure 1 , there is a specific band near 60 kDa, which is consistent with the theoretical molecular weight of AADH, 60.3 kDa, as
[0036] As an example of the present invention, the function of AADH was verified
[0037] Using DON as the substrate (final concentration 100 μg / mL), add 20 μg of AADH, 1 μL of 0.1 M CaCl 2 2, 2.5 mL of 10 mM phenazine methosulfate (PMS), 1 μL of 10 mM PQQ, and finally make up to 100 μL with Tris-HCl (50 mM, pH = 7.5), and react at 37 °C for 12 h. After the reaction, add 100 μL of methanol to terminate the reaction, and use high performance liquid chromatography (HPLC) to detect the change in the concentration of DON before and after the reaction. The elution conditions for DON are: elute with water / methanol (70:30, v / v) at 0.6 mL / min for 15 min, detection wavelength 218 nm; the elution conditions for 3-keto-DON are: elute with water / acetonitrile (90:10, v / v) at 1 mL / min for 15 min, detection wavelength 218 nm. The results show that AADH can effectively convert DON into less toxic 3-keto-DON, as Figure 2 shown in Figure B in
[0038] To further study the change law of DON degradation over time, samples were taken at different reaction times for DON concentration determination. It was found that as the reaction time extended, the concentration of the substrate DON gradually decreased, while the product 3-keto-DON gradually accumulated and reached equilibrium at the 14th h. After the reaction ended, the degradation rate of DON could reach more than 90%, as Figure 3 shown in
[0039] As an example of the present invention, the enzymatic properties of AADH were studied
[0040] (1) Optimal reaction temperature and temperature stability of AADH
[0041] To determine the optimal reaction temperature of AADH, the purified AADH protein samples were assayed for enzyme activity at different temperatures (20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C and 55 °C). The results show that the optimal reaction temperature of AADH is 35 °C, as Figure 4 shown in Figure A in
[0042] To determine the temperature stability of AADH, the pure AADH enzyme was treated at different temperatures (4 °C, 20 °C, 30 °C, 40 °C, and 50 °C) for 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively, and then the enzyme activity was measured, with the untreated sample taken as 100%. The results showed that AADH had good thermal stability. After being treated at 40 °C for 3 h, more than 50% of its catalytic activity could be retained, as shown in Figure 4 Figure B in
[0043] (2) Optimal reaction pH and pH stability of AADH
[0044] To determine the optimal reaction pH of AADH, the purified AADH protein samples were measured for enzyme activity at different pH values (pH 4.0 - 10.0). The results showed that the optimal reaction pH of AADH was 7.5, as shown in Figure 4 Figure C in
[0045] To determine the pH stability of AADH, the pure AADH enzyme was treated at different pH values (pH 5.0 - 10.0) for 12 h, and then the enzyme activity was measured, with the untreated sample taken as 100%. The results showed that AADH had good pH stability. After being treated within the range of pH 5.0 - 10.0 for 12 h, more than 50% of its catalytic activity could be retained, as shown in Figure 4 Figure D in
[0046] As an example of the present invention, the application of AADH in detoxifying mildewed wheat
[0047] The mildewed wheat grains were ground to 40 mesh. After being treated by high-pressure sterilization (121 °C, 18 min), 1 g was weighed and placed in a 10 mL centrifuge tube. Then, 200 μg of pure enzyme AADH, 40 μL of 0.1 M CaCl 2 40 μL, 40 μL of 10 mM PQQ, and 100 μL of 10 mM PMS were added. Then, 50 mM Tris-HCl (pH = 7.5) was added to make up to 4 mL, and the reaction was carried out at 37 °C for 24 h. After the reaction ended, the freeze-dried sample was extracted three times with 10 mL of acetonitrile / water solution (84:16, v / v), and the upper layer solution was taken. The supernatant was evaporated to dryness by a rotary evaporator, and finally the sample was resuspended with 200 μL of methanol. The change in the DON concentration in the wheat sample before and after the reaction was detected by high-performance liquid chromatography. It was found that AADH had the ability to degrade DON in mildewed wheat, could convert DON in mildewed wheat into low-toxic 3-keto-DON, and its degradation rate was over 80%, as shown in Figure 5 shown in
[0048] The present invention screened and identified a strain from the NCBI database, which is derived from the phylum Acidobacteria ( AcidobacteriotaA novel detoxifying enzyme AADH with DON degradation activity, which can degrade 25 - 200 μg / mL of DON into the low-toxic product 3-keto-DON within 16 h. Through sequence alignment, the sequence similarities of AADH with the currently reported DON detoxifying enzymes DepA, QDDH, DDH, and DADH are 56.55%, 56.55%, 50.43, and 48.56% respectively. Therefore, AADH is a novel DON detoxifying enzyme.
[0049] Enzymatic property studies showed that the optimal reaction temperature and optimal reaction pH of AADH were 35 °C and 7.5 respectively. It could retain more than 70% of its relative activity within the range of 30 - 45 °C, indicating that AADH had good functional adaptability within a relatively wide temperature range. AADH had good thermal stability and pH stability. When treated at 40 °C for 3 h, AADH could retain more than 50% of its residual activity; AADH could retain more than 60% of its residual activity when treated at pH 5.5 - 10 for 12 h.
[0050] In addition, when AADH was used to treat mildewed wheat, it had the ability to convert DON in mildewed wheat into low-toxic 3-keto-DON, and its conversion rate was over 80%, indicating its potential industrial application prospects and having important guiding significance for the development and application of DON detoxifying enzyme industrial enzyme preparations.
[0051] SEQ ID NO.1:
[0052]
[0053] SEQ ID NO.2:
[0054] MRAPKPDDWLIHRGNYQGWGYSPLDQINKSNVKTLQLVWSRAMEPGANEATPLVYNGVMYVGNPGDVIQAIDAASGDLMWEYRRPLPPVASMRNGLGQRKRSIALYGDQIYFVTWDNFVVSLGARSGELVWQTDRGGDLYVSNSSGPIVANGVVIAGSTCQYSSKGCYVTGHDARTGRELWRNEMIPRPGQPGDETWAGSSFESRWMTGVWGQLTYDPELDLVYYGSSGVGPASEAQRNMPGATMAGTNTRFAIRPKTGEVVWKHQVLPRDNWDQECTFEMMIINTPVNPGAPDMLSRKTLTGVPCKTGIAWSFDAANGEFLWAKPTTEQNIVARIDPKGLVTVNENAVLKEVGKTYHVCPTYNGGRDWPQGAYNPKSNVMYIPLSNLCIDSTARTDRKAAPEFVYNTTNVGKFATGKDKVGRIDAISVETGRTLWSWETRVSNYSPVLATGGGLLFNGSMDRYLRALDADRGQVLWQTRLPSQAVGGAVTYSINGRQFIAIAAGGGPIAALGTGLTPEADTFSGSNAMYVFALPQ。
[0055] The preferred embodiments of the embodiments of the present application have been described above with reference to the accompanying drawings, which does not limit the scope of the rights of the embodiments of the present application. Any modifications, equivalent replacements, and improvements made by those skilled in the art without departing from the scope and essence of the embodiments of the present application shall be within the scope of the rights of the embodiments of the present application.
Claims
1. Use of a DON detoxifying enzyme in degrading vomitoxin, characterized in that: The DON detoxification enzyme is a DON detoxification enzyme gene aadh Protein encoded by; the DON detoxification enzyme gene aadh It is derived from the phylum Acidobacteria, and its nucleotide sequence is shown in SEQ ID NO.1; the DON detoxifying enzyme has thermal stability, pH stability and the ability to degrade DON in moldy wheat.
2. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 1, characterized in that: The DON detoxifying enzyme can remove vomitoxin from grain raw materials and feed.
3. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 2, characterized in that: The amino acid sequence of the DON detoxifying enzyme is shown in SEQ ID NO.
2.
4. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 3, characterized in that: The preparation method of the DON detoxifying enzyme comprises the following steps: S1. The DON detoxification enzyme gene in claim 1 aadh performed codon optimization and gene synthesis; S2. Construction of DON detoxification enzyme gene aadh Prokaryotic expression vector; S3. Expressing the prokaryotic expression vector in prokaryotic host cells.
5. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 4, characterized in that: The prokaryotic expression vector is one or more of an Escherichia coli expression vector, a Bacillus subtilis expression vector, a Bacillus licheniformis expression vector, a Bacillus megaterium expression vector, a Bacillus brevis expression vector, a lactic acid bacteria expression vector, a yeast expression vector, a Streptomyces expression vector and a filamentous fungus expression vector.
6. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 4, characterized in that: The prokaryotic host cell is an Escherichia coli host cell, a Bacillus subtilis host cell, a Bacillus licheniformis host cell, a Bacillus megaterium host cell, a Bacillus brevis host cell, a lactic acid bacteria host cell, a yeast host cell, a Streptomyces host cell or a filamentous fungus host cell.