Vomitoxin detoxifying enzyme and application thereof

Abstract

The invention belongs to the technical field of biology, particularly relates to a vomitoxin detoxification enzyme and application thereof, and discloses a DON detoxification enzyme gene aadh sequence derived from acidobacteria, a DON detoxification enzyme coded by the DON detoxification enzyme gene aadh, a preparation method of the DON detoxification enzyme and application of the DON detoxification enzyme in degradation of vomitoxin. The preparation method comprises the following steps: performing codon optimization and gene synthesis on a DON detoxifying enzyme gene aadh; constructing a prokaryotic expression vector of the DON detoxifying enzyme gene aadh; expressing the prokaryotic expression vector in a prokaryotic host cell; the DON detoxifying enzyme provided by the invention can degrade vomitoxin, and can remove toxins in mildewed cereal raw materials and feeds.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a vomitoxin detoxification enzyme and an application thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by fungi (such as Fusarium, Alternaria, Aspergillus and Penicillium) infecting plant products in the field or during storage, including aflatoxins, zearalenone, ochratoxin, deoxynivalenol and patulin. Among them, deoxynivalenol (DON) is widely present in cereal raw materials such as wheat, barley and corn and their products, and is the most widely distributed mycotoxin in the world. Through the detection of DON in 608 samples of wheat and its products in 26 provinces and cities in seven major regions of China, 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 pollution problem of DON in food and feed is the key to promoting sustainable agricultural development 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. However, the traditional physical and chemical detoxification methods have problems such as incomplete detoxification, large loss of nutrients, and possible secondary pollution, which limits their wide application in the food and feed industry. The biological detoxification method based on microorganisms or enzymes has the advantages of high detoxification efficiency, strong specificity, no pollution to the environment, and little damage to the nutrients of feed and food. Therefore, it has received more and more attention and application. Studies have shown that the epoxy groups at the C12 and 13 positions and the hydroxyl group at the C3 position in the DON molecule are the main groups that determine the toxicity of DON and are also the key sites for biological detoxification. At present, a variety of microorganisms that can destroy the epoxy groups at the C12 and 13 positions of DON have been screened, which can convert DON into a de-epoxy 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 DON detoxification enzyme has been reported. In addition, Lei Xiaoguang's research team 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 yet been identified. In addition to the epoxy group, the oxidation, glycosylation, acetylation, and isomerization of the hydroxyl group at the C3 position of DON are also the main pathways for DON detoxification. Among them, the pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase DepA / QDDH / DADH / DDH from Devosia sp. can oxidize the C3 hydroxyl group of DON to generate low-toxic 3-keto-deoxynivalenol (3-keto-DON), and then the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent aldehyde-keto reductase DepB / AKR6D1 / AKR13B2 / AKR13B3 reduces 3-keto-DON to generate non-toxic 3-isomerized deoxynivalenol (3-epi-DON), i.e., isomerization at the C3 position. This is the most thoroughly studied detoxification method, which is safe, efficient, and has stable effects.

[0004] Studies have shown that the toxicity of DON degradation products is lower than that of DON, among which the toxicity of 3-keto-DON is 1 / 10 of that of DON, and 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 first step DON detoxification enzymes identified so far have problems such as low catalytic efficiency and poor thermal stability, which limits their further application in the food and feed industry. Since enzymes from different microorganisms have different enzymatic properties, it is of great practical significance to explore new DON detoxification enzymes with high catalytic activity, high thermal stability and high substrate specificity. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a vomitoxin detoxification enzyme and its application.

[0006] In order to achieve the purpose of the present invention, the present invention will be implemented by adopting the technical solution described below.

[0007] The nucleotide sequence of a DON detoxification enzyme gene aadh is shown in SEQ ID NO.1.

[0008] A DON detoxifying enzyme is a protein encoded by the DON detoxifying enzyme gene aadh, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] A method for preparing a DON detoxifying enzyme comprises the following steps:

[0010] S1. Codon optimization and gene synthesis of a DON detoxification enzyme gene aadh according to claim 1;

[0011] S2. Construction of a prokaryotic expression vector of DON detoxification enzyme gene aadh;

[0012] S3. Expressing the prokaryotic expression vector in prokaryotic host cells.

[0013] As a preferred embodiment of the present invention, the prokaryotic expression vector includes 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.

[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 Bacillus 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 used in removing mycotoxins from grain raw materials and feed.

[0017] Beneficial Effects

[0018] 1. The present invention discovered a novel DON detoxification enzyme gene aadh from Acidobacteriota, constructed a genetically engineered strain that efficiently expressed aadh, and achieved heterologous expression of DON detoxification enzyme AADH in prokaryotic cells;

[0019] 2. The PQQ-dependent alcohol dehydrogenase AAD H from Acidobacteriota identified in the present invention has DON degradation activity. 10-20 μg AADH can degrade 45-150 μg / mL DON into a low-toxic product 3-keto-DON within 2-16 hours. Through multiple sequence alignment, it was found that the sequence similarities of AADH with the currently reported DON detoxification enzymes DepA, QDDH, DDH and DADH were 56.55%, 56.55%, 50.43% and 48.56%, respectively. Therefore, AADH is a new type of DON detoxification enzyme;

[0020] 3. The AADH of the present invention has good thermal stability and pH stability. After being treated at 40°C for 3 hours, the AADH can retain more than 50% of its residual activity. In addition, after being treated at pH 5.5-10 for 12 hours, the AADH can retain more than 60% of its residual activity. Therefore, the AADH has potential for industrial application.

[0021] 4. The AADH of the present invention can oxidize DON to generate low-toxic 3-keto-DON. At the same time, the use of AA DH to treat moldy wheat can degrade DON in the moldy wheat into 3-keto-DON, with a degradation rate of more than 80%, which effectively solves the problem of vomitoxin contamination in grain raw materials and feed, reduces food losses, and ensures the safety of food and feed. Provides a new option. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The schematic diagram of SDS-PAGE electrophoresis and Western blot electrophoresis of purified AADH protein, where: M: protein marker; 1: E. coli BL21 cell fragment containing empty plasmid pET28a; 2: AADH crude enzyme sample; 3: AADH purified protein sample;

[0023] Figure 2 The graph is a curve of the detoxification ability of AADH to DON, wherein: (A) is an HPLC analysis of DON; (B) is an HPLC analysis of 3-keto-DON, a degradation product of DON;

[0024] Figure 3 It is a curve diagram of the detoxification effect of AADH on DON;

[0025] Figure 4 The enzymatic property curve of AADH, wherein: (A) shows the optimum reaction temperature of AADH; (B) shows the temperature stability of AADH; (C) shows the optimum reaction pH of AADH; (D) shows the pH stability of AADH;

[0026] Figure 5 This is a bar chart comparing the detoxification effect of AADH in moldy wheat. DETAILED DESCRIPTION

[0027] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0028] As an embodiment of the present invention, cloning of the DON detoxification enzyme gene of the Acidobacteria

[0029] Using PQQ-dependent alcohol dehydrogenase DepA as a template, it was uploaded to NCB I for multiple sequence alignment, and an amino acid sequence AADH from Acidobacteriota was obtained, which was 56.55% identical to DepA and functionally annotated as PQQ-dependent alcohol dehydrogenase. The sequence was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. 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.

[0030] According to the gene synthesis results, the DON detoxification enzyme aadh is 1608 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1, namely, Acidobacteriota DON detoxification enzyme AADH, encoding a protein composed of 536 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0031] As an embodiment of the present invention, expression and purification of DON detoxification enzyme in Escherichia coli

[0032] (1) Expression vector construction: The constructed expression vector pET28a-AADH was transformed into Escherichia coli BL21 competent cells for the expression of recombinant protein.

[0033] (2) Expression of recombinant enzyme: The recombinant bacteria pET28a-AADH were inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin (Kana), and cultured at 37°C, 180 rpm for 16 h to obtain seed solution. The seed solution was then transferred to 100 mL of fresh LB liquid medium (containing 50 μg / mL Kana) at an inoculum of 1%, and cultured at 37°C, 180 rpm until OD600 was between 0.6-0.8, and 100 μL of IPTG (100 mg / mL) was added to induce protein expression at 16°C, 180 rpm.

[0034] (3) Purification of recombinant enzyme: After fermentation, the cells were collected by centrifugation at 4°C, 8000 rpm, washed 3 times with 50 mM Tris-HCl (200 mM NaCl, pH = 7.5), and then resuspended in the buffer. The resuspended cells were ultrasonically disrupted and centrifuged (4°C, 8000 rpm, 20 min) to remove the precipitate. The supernatant was the crude enzyme solution. Since the C-terminus of the target protein was fused with a histidine tag during vector construction, the DON detoxification enzyme AADH can be purified using a Ni2+-NTA affinity column as follows:

[0035] 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 to remove impurities. Before loading, the column was equilibrated with a buffer solution (50 mM Tris-HCl, 200 mM NaCl, pH=7.5), and the sample was cycled and loaded three times.

[0036] Elution: After loading, use 5 column volumes of buffer containing 25 mM imidazole to elute the unabsorbed impurities, and finally use 5 column volumes of buffer containing 250 mM imidazole to elute the target protein. Collect the effluent to obtain the purified sample, and dialyze to remove the imidazole to obtain the purified protein. The purified protein was verified by SDS-PAGE electrophoresis and Western blotting. Figure 2 As shown in Figure A, there is a specific band near 60 kDa, which is consistent with the theoretical molecular weight of AADH, 60.3 kDa. Figure 1 shown.

[0037] As an embodiment of the present invention, the function of AADH is verified

[0038] With DON as substrate (final concentration 100μg / mL), add 20μg of AADH, 21μL of 0.1M CaCl, 2.5mL of 10mM phenazine methyl sulfate (PMS), 1μL of 10mM PQQ, and finally add Tris-HCl (50mM, pH=7.5) to 100μL, and react at 37℃ for 12h. After the reaction, add 100μL of methanol to terminate the reaction, and use high performance liquid chromatography (HPLC) to detect the concentration change of DON before and after the reaction. The elution conditions of DON are: 0.6mL / min water / methanol (70:30, v / v) elution for 15min, detection wavelength 218nm; the elution conditions of 3-keto-DON are: 1mL / min water / acetonitrile (90:10, v / v) elution for 15min, detection wavelength 218nm. The results showed that AADH can effectively convert DON into low-toxic 3-keto-DON. Figure 2 As shown in Figure B.

[0039] In order to further study the change of DON degradation over time, samples were taken at different reaction times to measure DON concentration. It was found that with the extension of reaction time, the concentration of substrate DON gradually decreased, while the product 3-keto-DON gradually accumulated and reached equilibrium at the 14th hour. After the reaction, the degradation rate of DON could reach more than 90%. Figure 3 shown.

[0040] As an embodiment of the present invention, the enzymatic properties of AADH are studied

[0041] (1) Optimal reaction temperature and temperature stability of AADH

[0042] To determine the optimal reaction temperature of AADH, the purified AADH protein samples were subjected to enzyme activity assays at different temperatures (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and 55°C). The results showed that the optimal reaction temperature of AADH was 35°C. Figure 4 As shown in Figure A.

[0043] To determine the temperature stability of AADH, pure AADH enzyme was treated at different temperatures (4°C, 20°C, 30°C, 40°C and 50°C) for 0h, 1h, 2h, 3h, 4h, 5h, and 6h, and then the enzyme activity was measured, with the untreated enzyme as 100%. The results showed that AADH has good thermal stability and can retain more than 50% of its catalytic activity when treated at 40°C for 3h. Figure 4 As shown in Figure B.

[0044] (2) Optimal reaction pH and pH stability of AADH

[0045] To determine the optimal reaction pH of AADH, the purified AADH protein samples were subjected to enzyme activity assays at different pH conditions (pH 4.0-10.0). The results showed that the optimal reaction pH of AADH was 7.5. Figure 4 As shown in Figure C.

[0046] To determine the pH stability of AADH, pure AADH enzyme was treated at different pH conditions (pH 5.0-10.0) for 12 hours, and then the enzyme activity was measured, with the untreated enzyme as 100%. The results showed that AADH has good pH stability and can retain more than 50% of its catalytic activity when treated for 12 hours in the pH range of 5.0-10.0. Figure 4 As shown in Figure D.

[0047] As an embodiment of the present invention, the application of AADH in detoxification of moldy wheat

[0048] The moldy wheat grains were crushed to 40 meshes, sterilized by high pressure (121°C, 18 min), and 1 g was weighed into a 10 mL centrifuge tube. 200 μg of pure enzyme AADH, 240 μL of 0.1 M CaCl, 40 μL of 10 mM PQQ and 100 μL of 10 mM PMS were added, and then 50 mM Tris-HCl (pH = 7.5) was added to make up to 4 mL, and the mixture was reacted at 37°C for 24 h. After the reaction, 10 mL of acetonitrile / water solution (84:16, v / v) was added to the freeze-dried sample and extracted three times, and the upper 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 changes in DON concentration in the wheat samples before and after the reaction were detected by high performance liquid chromatography. It was found that AADH has the ability to degrade DON in moldy wheat and can convert DON in moldy wheat into low-toxic 3-keto-DON, and its degradation rate is over 80%. Figure 5 shown.

[0049] The present invention screened and identified a novel detoxification enzyme AADH with DON degradation activity from Acidobacteriota from the NCB I database, which can degrade 25-200 μg / mL of DON into a low-toxic product 3-keto-DON within 16 hours. Through sequence comparison, it was found that the sequence similarities of AADH with the currently reported DON detoxification enzymes DepA, QDDH, DDH and DADH were 56.55%, 56.55%, 50.43% and 48.56%, respectively, so AADH is a novel DON detoxification enzyme.

[0050] Studies on the enzymatic properties show that the optimal reaction temperature and optimal reaction pH of AADH are 35°C and 7.5, respectively. It can retain more than 70% of its relative activity in the range of 30-45°C, indicating that AADH has good functional adaptability in a wide temperature range. AADH has good thermal stability and pH stability. When treated at 40°C for 3 hours, AADH can retain more than 50% of its residual activity; when treated at pH 5.5-10 for 12 hours, AADH can retain more than 60% of its residual activity.

[0051] In addition, AADH treated moldy wheat has the ability to convert DON in moldy wheat into low-toxic 3-keto-DON, with a conversion rate of more than 80%, indicating that it has potential industrial application prospects and has important guiding significance for the development and application of DON detoxification enzyme industrial enzyme preparations.

[0052] SEQ ID NO.1:

[0053]

[0054] SEQ ID NO.2:

[0055] MRAPKPDDWL I HRGNYQGWGYSPLDQI NKSNVKTLQLVWSRAMEPGANEEATPLVYNGVMYVGNPGDVIQAIDAASGDLMWEYRRPLPPVASMRNGLGQRKRS IALYGDQIYFVTWDNFVVSLGARSGELVWQTDRGGDLYVSNSSGPIVANGVVIAGSTCQYSSKGCYVTGHDARTGRELWRNEM IPRPGQPGDETWAGSSFESRWMTGVWGQLTYDPELDLVYYGSSGVGPASEAQRNMPGATMAGTNTRFAI RPKTGEVVWKHQVLPRDNWDQECTFEMMIINTPVNPGAPDMLSRKTLTGVPCKTG IAWSFDAANGEFLWAKPTTEQN IVARI DPKGLVTVNENAVLKEVGKTYHVCPTYNGGRDWPQGAYNPKSNVMY IPLSNLCIDSTARTDRKAAPEFVYNTTNVGKFATGKDKVGR IDAI SVETGRTLWSWETRVSNYSPVLATGGGLFNGSMDRYLRALDADRGQVLWQTRLPSQAVGGAVTYS I NGRQF IAIAAGGGPIAALGTGLTPEADTFSGSNAMYVFALPQ.

[0056] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but the scope of the rights of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by a person skilled in the art without departing from the scope and essence of the present invention should be within the scope of the rights of the present invention.

Claims

1. A DON detoxification enzyme gene aadh, characterized in that The DON detoxification enzyme gene aadh is derived from Acidobacteriota, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A DON detoxification enzyme, characterized in that The DON detoxification enzyme is a protein encoded by the DON detoxification enzyme gene aadh described in claim 1, and its amino acid sequence is shown in SEQ ID NO.

2.

3. A method for preparing a DON detoxifying enzyme, characterized in that: The steps include: S1. Codon optimization and gene synthesis of a DON detoxification enzyme gene aadh according to claim 1; S2. Construction of a prokaryotic expression vector of DON detoxification enzyme gene aadh; S3. Expressing the prokaryotic expression vector in prokaryotic host cells.

4. The method for preparing a DON detoxifying enzyme according to claim 3, characterized in that: The prokaryotic expression vector includes 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.

5. The method for preparing a DON detoxifying enzyme according to claim 3, 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.

6. Use of a DON detoxifying enzyme in degrading vomitoxin, characterized in that: The DON detoxification enzyme gene is the DON detoxification enzyme gene aadh according to claim 1.

7. The use of a DON detoxifying enzyme in degrading vomitoxin according to claim 6, characterized in that: The DON detoxifying enzyme is used in removing vomitoxin from grain raw materials and feed.

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