Aflatoxin oxidase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological catalysis, in particular to aflatoxin oxidase as well as a preparation method and application thereof. The new aflatoxin oxidase DnD-AFO is designed from the beginning by utilizing an autonomously designed artificial intelligence enzyme design workflow, and compared with the traditional wild type AFO, the DnD-AFO has the advantages that the activity is improved, the thermal stability is better (after high-temperature granulation, the activity is far superior to that of the wild type), the pepsin resistance and the acid resistance are improved, and the like. And the aflatoxin detoxification efficiency is further improved, and the aflatoxin detoxification agent can be well used as a food additive or a feed additive to be applied to related industries such as agriculture and food engineering, and has huge commercial value and added value.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and in particular to an aflatoxin oxidase and a preparation method and application thereof. Background Art

[0002] Due to the limited storage conditions of grain and feed, it is easy to mold. Aflatoxin is a strong carcinogenic toxin produced by Aspergillus flavus and Aspergillus parasiticus. It is the main toxin that contaminates food and feed. In order to detoxify aflatoxins in feed, many methods have been proposed, among which the use of aflatoxin oxidase (AFO) for detoxification is considered to be a green, environmentally friendly, efficient and irreversible method.

[0003] In 1999, Professor Wang Guanglin and his research team reported an activated substance purified from the edible fungus Armillariella tabescens that can remove the toxicity of aflatoxin, which was named aflatoxion-Oxidase (AFO). After studying the mechanism of action of the enzyme, it was found that as an oxidoreductase, it can undergo oxidation reactions with structural analogs such as aflatoxin (AFB1), destroying the chemical bonds of the bifuran ring in the aflatoxin structure and detoxifying aflatoxin (AFB1).

[0004] At present, AFO has been widely used and promoted as an antidote for removing aflatoxins remaining in food and feed. Compared with other means, it has become an excellent method for removing the toxicity of aflatoxins due to its advantages of being green and environmentally friendly, relatively efficient, low economic cost, and thorough detoxification. However, due to the long action time of AFO, it needs to take effect in the body of the livestock and needs to meet the high-temperature granulation standards. The existing wild-type AFO enzyme is used in the production of feed to improve the safety and quality of the feed, but it still has the disadvantages of low activity, poor thermal stability (almost loss of activity when heated at 85°C for three minutes (high-temperature granulation standard)), anti-pepsin and general acid resistance.

[0005] In addition, patent CN115851633A discloses an aflatoxin oxidase with improved pepsin resistance and its application, which uses protein engineering technology to modify the key amino acid residues in the wild-type aflatoxin oxidase molecule and screens out a mutant aflatoxin oxidase with improved pepsin resistance. The mutant aflatoxin oxidase (AFO) described in the patent acts on aflatoxin (AFB1) G355I / G475V) has a half-life of pepsin resistance that is 2.72 times that of wild-type AFO. G355I / G475V It retains more than 40% activity at 10-40°C, and the optimal reaction pH is 7.5.

[0006] So far, no AFO enzymes with high activity, good thermal stability (retaining activity after heating at 85°C for three minutes (high temperature pelleting standard)), anti-pepsin and high acid resistance have been reported. Enzymes with the above advantages can be better used in feed production to complete the task of detoxification of aflatoxin in an efficient, green and stable manner. Therefore, the existing technology needs to be improved and developed. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an aflatoxin oxidase and a preparation method and application thereof, aiming to solve the problems of low activity, poor thermal stability, and average pepsin resistance and acid resistance of the existing aflatoxin oxidase.

[0008] The technical solution of the present invention is as follows:

[0009] In a first aspect of the present invention, aflatoxin oxidase (DnD-AFO) is provided, wherein the amino acid sequence of the aflatoxin oxidase is SEQ ID NO.1.

[0010] The second aspect of the present invention provides a gene encoding the aflatoxin oxidase described in the present invention, wherein the nucleotide sequence of the gene encoding is SEQ ID NO.2.

[0011] The third aspect of the present invention provides a recombinant vector comprising the encoding gene of the present invention.

[0012] The fourth aspect of the present invention provides a recombinant bacterium comprising the encoding gene of the present invention.

[0013] The fifth aspect of the present invention provides a method for preparing the aflatoxin oxidase of the present invention, which comprises the steps of:

[0014] S1, constructing a recombinant vector expressing aflatoxin oxidase, and transfecting cells with the recombinant vector to obtain recombinant bacteria;

[0015] S2, culturing the recombinant bacteria to induce the expression of aflatoxin oxidase;

[0016] S3. Separate and purify aflatoxin oxidase.

[0017] Optionally, the step S1 specifically includes:

[0018] The coding gene of aflatoxin oxidase was introduced into pET28a plasmid, with restriction sites NdeI and EcoRI to obtain a recombinant plasmid, and then the recombinant plasmid was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium.

[0019] Optionally, the step S2 specifically includes:

[0020] The recombinant bacteria were inoculated into LB medium containing kanamycin and cultured at 25-40°C and 150-500rpm. When the absorbance density of the bacterial solution reached 0.6-0.8, isopropyl-β-d-thiogalactoside was added to induce the expression of aflatoxin oxidase.

[0021] Optionally, the concentration of kanamycin in the LB medium is 25-75 μg / mL, and the concentration of isopropyl-β-d-thiogalactoside is 0.05-0.15 mmol / L.

[0022] Optionally, the induction temperature is 10-25°C, and the induction time is 15-30h.

[0023] The sixth aspect of the present invention provides a use of the aflatoxin oxidase of the present invention in food additives or feed additives.

[0024] Beneficial effects: Compared with the traditional wild-type aflatoxin oxidase AFO, the aflatoxin oxidase DnD-AFO provided by the present invention has the advantages of improved activity, better thermal stability (after high-temperature granulation, the activity is much better than the wild type), improved anti-pepsin and acid resistance, etc., and further improves the detoxification efficiency of aflatoxin. It can be well used as a food additive or feed additive in agriculture, food engineering and other related industries, and has huge commercial value and added value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SDS-PAGE protein electrophoresis diagram in Example 1.

[0026] Figure 2 This is the SDS-PAGE electrophoresis diagram of the wild-type AFO in Example 2.

[0027] Figure 3 This is the SDS-PAGE electrophoresis diagram of aflatoxin oxidase DnD-AFO in Example 2.

[0028] Figure 4 This is a comparison chart of the enzyme activities of the DnD-AFO experimental group, the wild-type AFO experimental group and the blank control group in Example 4. DETAILED DESCRIPTION

[0029] The present invention provides an aflatoxin oxidase and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] The present invention obtains the range of conserved sites of AFO enzyme and the position information of key amino acids involved in the reaction based on comprehensive exploration and calculation of the AFO enzymatic reaction mechanism and protein multiple sequence alignment. Based on this information, a protein de novo design model ProteinMPNN is used to generate a large number of new protein sequences while ensuring that important structural positions do not change, and introduces functional attribute predictions of multiple artificial intelligence models (DLKcat, UNiKP, DeepDetect, etc.) to the generated sequences. In view of the functional attribute needs, the candidate mutants are sorted and selected using the independently designed scoring function with experimental data and multi-model fusion as influencing factors, and excellent aflatoxin oxidases with multiple functions are screened out from the generated proteins.

[0031] Specifically, the present invention uses a self-designed artificial intelligence enzyme design workflow to design new enzyme individuals from scratch based on a comprehensive exploration and calculation of the AFO enzymatic reaction mechanism and protein multiple sequence alignment. The self-designed artificial intelligence de novo design workflow is as follows:

[0032] (1) Using a variety of sequence processing and chemical and biological computational methods such as molecular dynamics, molecular docking, and multiple sequence alignment, we explored the key domains and conserved sequences of the aflatoxin oxidase to be designed from scratch, thereby obtaining enough prior knowledge to constrain the AI ​​protein generation model, thereby ensuring that the excellent functional properties of the generated new sequence are retained.

[0033] (2) Based on the advantages that the desired design sequence should have high activity, good thermal stability (retain activity when heated at 85°C for 3 min (high temperature granulation standard)), resistance to pepsin and high acid resistance, the protein de novo design model ProteinMPNN was selected for de novo design, and its design results were analyzed to ensure that the method for generating protein sequences is efficient and accurate.

[0034] (3) A scoring function was designed based on the required functions and properties of the target aflatoxin oxidase (AFO) (taking into account the AI ​​models involved in the prediction and screening of functional properties, such as DLKcat, UNiKP, and DeepDetect), and the simulation calculation results in the computer were standardized. The independently designed scoring function was used to select the best candidates from the candidate sequence.

[0035] The aflatoxin oxidase designed from scratch by the above workflow of the present invention is named DnD-AFO. Conventional activity assay, activity assay after high temperature granulation standard treatment, and resistance to hydrolysis in gastric fluid simulation fluid were performed on the DnD-AFO. Compared with the wild-type AFO, its multiple properties are superior to the wild-type AFO.

[0036] Based on this, an embodiment of the present invention provides an aflatoxin oxidase (DnD-AFO), wherein the amino acid sequence of the aflatoxin oxidase is SEQ ID NO.1.

[0037] The amino acid sequence of SEQ ID NO.1 is as follows:

[0038] MATTTVDNSRFLADKDVPLCGLNVRESFEKLTEQEKLYAHYMSKAAWAGARIIQRQWTEEAVKLYRLLLLTFSKNGKLVDLEKLKKASGLSEDEWNDVLSYAVEVLSNLSNFRRFGFRKIVPRAPAEKFRKVVEASENPEEAMALFDE LKDYIYSLEPYESLYIGKREEGHISNYYLGEPMDDEEVKAIQKVLEKLKISPRNTFRKVGEGEYELLVASAEKKPDKKYDFEINGKPATLTIKYGDYAEELKKVVAHLKEAVKYAQNENQKKAIEKYIESFTTGSVELHIEGSREWVK DKGPVVESYIGFEETYVDPDGFRAEWEGFTAIVNKELSKKYQELVDAAPELRKTLPWIEDYEIKEFRKPDFTALELIDFATGGIPAGINYPNYYEIREEVGFKNVSLANILAAKVPNEEVTFIHPDDLELYLEWDSDAFELQVAIHEL LGHGSGKLFREDENGKLNFDPEKVKNPLTVKKIDSWYKPGQTPETVLGEVSSSMEECRAETVALYLISNETILKIFGYTEPEKRENIIYVSFLLMARAGLRALEYYDPETGKHGQAHMQARLGITNHLIEAGIASLEKVYDSNGNLVNI YVRVDREKVLTEGREVVGKLLVELQVRKATADAEGARGFYTALTTPPEGWTGEIRDIVLAKKLPRKIFVQPNLKIENGKVVLTEYPLTYEGVIQSFIDRNLL

[0039] The aflatoxin oxidase DnD-AFO provided in the embodiment of the present invention has the advantages of improved activity, better thermal stability (after high-temperature granulation, the activity is much better than the wild type), and improved anti-pepsin and acid resistance compared to the traditional wild-type AFO. First, the macroscopic anti-pepsin effect of the DnD-AFO of the present invention is far better than that of the wild-type AFO. Through quantitative determination, the amount of residual enzyme of the DnD-AFO of the present invention is 3.36 times that of the wild type after adding gastric juice simulation liquid for 1 hour. Second, without heating, the amount of aflatoxin remaining within the prescribed time is used as the basis for determination, and the detoxification efficiency of the DnD-AFO of the present invention is 1.12 times that of the wild type. Third, after the wild-type AFO and the DnD-AFO of the present invention are heated in an 85°C metal bath for 3 minutes, the detoxification efficiency of the DnD-AFO of the present invention is 4.106 times that of the wild type.

[0040] In the present invention, based on the DnD-AFO amino acid sequence, aflatoxin oxidase sequence variants with similar functions can be designed, for example, by conservative mutation or retaining the key regions of the sequence and optimizing other regions to enhance the functional stability or activity of the sequence. Such aflatoxin oxidase sequence variants with similar functions also fall within the scope of protection of the present application.

[0041] The aflatoxin oxidase provided in the embodiments of the present invention can also be modified or labeled, and chemical modifications or fluorescent markers, affinity tags, etc. are introduced into the sequence to facilitate the detection or separation of the sequence in specific applications. For example, by adding a histidine tag at the end of the sequence to achieve binding to a nickel column, it can be used for purification or protein interaction research.

[0042] An embodiment of the present invention provides a gene encoding the aflatoxin oxidase DnD-AFO described in the aforementioned embodiment, wherein the nucleotide sequence of the encoding gene is SEQ ID NO.2.

[0043] The nucleotide sequence of SEQ ID NO.2 is as follows:

[0044]

[0045] The embodiment of the present invention provides a recombinant vector comprising the encoding gene described in the above embodiment.

[0046] The embodiment of the present invention provides a recombinant bacterium comprising the encoding gene described in the above embodiment.

[0047] The present invention provides a method for preparing the aflatoxin oxidase described in the above embodiment, which comprises the steps of:

[0048] S1, constructing a recombinant vector expressing aflatoxin oxidase, and transfecting cells with the recombinant vector to obtain recombinant bacteria;

[0049] S2, culturing the recombinant bacteria to induce the expression of aflatoxin oxidase;

[0050] S3. Separate and purify aflatoxin oxidase.

[0051] In some implementations, the step S1 specifically includes:

[0052] The coding gene of aflatoxin oxidase was introduced into pET28a plasmid with restriction sites of NdeI and EcoRI to obtain a recombinant plasmid, and then the recombinant plasmid was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria E. coli BL21 (DE3) / pET28a-DnD-AFO.

[0053] In some implementations, step S2 specifically includes:

[0054] The recombinant bacteria E. coli BL21 (DE3) / pET28a-DnD-AFO are inoculated into LB medium containing kanamycin, and cultured at 25-40°C (preferably 37°C) and 150-500rpm (preferably 200rpm). When the absorbance density of the bacterial liquid reaches 0.6-0.8, isopropyl-β-d-thiogalactoside is added to induce the expression of aflatoxin oxidase.

[0055] In some embodiments, the concentration of kanamycin in the LB medium is 25-75 μg / mL (preferably 50 μg / mL), and the concentration of isopropyl-β-d-thiogalactoside is 0.05-0.15 mmol / L (preferably 0.1 mmol / L).

[0056] In some embodiments, the induction temperature is 10-25°C (preferably 16°C), and the induction time is 15-30h (preferably 20h).

[0057] In the present invention, the separation and purification in step S3 is a conventional technical means in the art and will not be described in detail here.

[0058] An embodiment of the present invention provides a use of the aflatoxin oxidase described in the above embodiment in a food additive or a feed additive.

[0059] The present invention will be further described below by means of specific examples.

[0060] Example 1

[0061] This example provides a method for preparing aflatoxin oxidase DnD-AFO, which is as follows:

[0062] 1. Construction, culture and expression of genetically engineered bacteria E. coli BL21(DE3) / pET28a-DnD-AFO

[0063] The coding gene sequence of aflatoxin oxidase DnD-AFO as shown in SEQ ID NO.2 was introduced into the pET28a plasmid, with restriction sites NdeI and EcoRI to obtain the recombinant plasmid pET28a-DnD-AFO, and then the recombinant plasmid pET28a-DnD-AFO was introduced into Escherichia coli E.coli BL21 (DE3), the genetically engineered bacteria E.coli BL21 (DE3) / pET28a-DnD-AFO.

[0064] The genetically engineered bacteria E. coli BL21 (DE3) / pET28a-DnD-AFO constructed above were inoculated into LB medium containing kanamycin (concentration of 50 μg / mL), and cultured at 37 ° C, 200 rpm. When the absorbance density (OD600) of the bacterial liquid reached 0.6-0.8, 0.1 mmol / L of isopropyl-β-d-thiogalactoside (IPTG) was added for induction. The induction temperature was 16 ° C and the induction time was 20 h. After the culture was completed, the wet cells of the genetically engineered bacteria that efficiently expressed the aflatoxin oxidase DnD-AFO were obtained after centrifugation.

[0065] 2. Isolation and purification of aflatoxin oxidase DnD-AFO

[0066] The wet cells of the genetically engineered bacteria that efficiently express aflatoxin oxidase DnD-AFO obtained above were resuspended in 50mmol / L pH 8.0 sodium phosphate buffer (containing 500mmol / L NaCl), high-pressure homogenized and broken, centrifuged (12000rpm, 20min), and the supernatant was collected to obtain a crude enzyme solution of aflatoxin oxidase DnD-AFO;

[0067] Purification of aflatoxin oxidase DnD-AFO:

[0068] (1) Equilibrate the Ni column with 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl);

[0069] (2) The crude enzyme solution of aflatoxin oxidase DnD-AFO obtained above was passed through a Ni column at a flow rate of 1 mL / min to allow the target protein to be mounted on the Ni column;

[0070] (3) Use 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl) containing 10 mmol / L and 25 mmol / L imidazole to elute the impurities that have no binding ability to the Ni column;

[0071] (4) Elute the target protein with 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl) containing 250 mmol / L imidazole;

[0072] (5) The protein solution was concentrated by centrifugation using an ultrafiltration tube (4000 rpm, 20 min) to one-tenth of the original volume, and the volume was supplemented with 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl) to the original volume. This process was repeated three times to reduce the imidazole concentration in the protein solution;

[0073] (6) Use a vertical electrophoresis apparatus to perform SDS-PAGE on the collected protein samples. Take 20 μL of sample and add 5 μL of 5×SDS loading buffer for treatment. The loading volume is 10 μL. The standard molecular weight protein (Protein Molecular Weight Marker) is purchased from Thermo Company in the United States. The SDS-PAGE gel concentration is 12%. The voltage of 110V is used for concentration electrophoresis, and then the voltage is changed to 200V for separation electrophoresis. Purification obtains high-purity aflatoxin oxidase DnD-AFO. The SDS-PAGE protein electrophoresis diagram is shown in the figure. Figure 1 As shown, the black box is aflatoxin oxidase DnD-AFO, with a molecular weight of about 76 kDa, lane 1 is the crushed precipitate, lane 2 is the Ni column effluent, lane 3 is the elution using 10 mM imidazole, lane 4 is the elution using 25 mM imidazole, and lane 5 is the elution using 250 mM imidazole.

[0074] Example 2

[0075] This example explores the comparison of the anti-pepsin and anti-acid effects of the aflatoxin oxidase DnD-AFO prepared in Example 1 above and the wild-type AFO, as follows:

[0076] The aflatoxin oxidase DnD-AFO (1 mg / mL) and wild-type AFO (1 mg / mL) prepared in Example 1 were added to gastric juice simulation solution (20ug / mL pepsin, 10mM NaCl buffer adjusted to pH 2.5 with concentrated hydrochloric acid) for 1h, and the above system was added to a 2mL EP tube (total system is 1mL) and incubated at 37°C on a shaker for 1h. Samples were taken at 0min, 10min, 20min, 30min, 40min, 50min, and 60min to run SDS-PAGE protein gel (the sampling volume was 20uL, and 5uL 5× loading buffer was added, mixed evenly, and heated at 100°C for 5min, and the sample volume for running gel was 10uL).

[0077] According to the SDS-PAGE electrophoresis diagram, Image J software was used for quantitative analysis of WB bands. The residual enzyme amounts of aflatoxin oxidase DnD-AFO and wild-type AFO were obtained by quantitative calculation, and the anti-pepsin and anti-acid effects of aflatoxin oxidase DnD-AFO and wild-type AFO were compared.

[0078] Figure 2 This is the SDS-PAGE electrophoresis of wild-type AFO, where the black box is the remaining wild-type AFO, lane 1 is sampled at 0 min, lane 2 is sampled at 10 min, lane 3 is sampled at 20 min, lane 4 is sampled at 30 min, lane 5 is sampled at 40 min, lane 6 is sampled at 50 min, and lane 7 is sampled at 60 min. Figure 2 Grayscale analysis of protein bands was performed at different positions according to the time frame. The grayscale analysis result of the initial protein amount was 200363, and the grayscale analysis result of the remaining protein amount after 60 minutes was 24302. Comparing the two, it was found that the remaining enzyme amount of wild-type AFO after passing through gastric juice simulation fluid was 12.11% of the initial enzyme amount.

[0079] Figure 3 This is the SDS-PAGE electrophoresis diagram of aflatoxin oxidase DnD-AFO, where the black box is the remaining aflatoxin oxidase DnD-AFO, lane 1 is sampled at 0 min, lane 2 is sampled at 10 min, lane 3 is sampled at 20 min, lane 4 is sampled at 30 min, lane 5 is sampled at 40 min, lane 6 is sampled at 50 min, and lane 7 is sampled at 60 min. Figure 3 Grayscale analysis of protein bands was performed at different positions according to the time frame. The grayscale analysis result of the initial protein amount was 205061, and the grayscale analysis result of the remaining protein amount after 60 minutes was 83455. By comparing the two, it was found that the remaining enzyme amount of DnD-AFO after passing through the gastric juice simulation fluid was 40.69% of the initial enzyme amount.

[0080] The experimental results show that the amount of residual enzyme of the wild-type AFO was 12.11% of the input amount 1 hour after adding the gastric juice simulated liquid, while the amount of residual enzyme of the aflatoxin oxidase (DnD-AFO) of the present invention was 40.69% of the input amount 1 hour after adding the gastric juice simulated liquid, and the residual enzyme amount was 3.36 times that of the wild-type, indicating that the DnD-AFO of the present invention has significantly improved anti-pepsin and anti-acid properties compared with the wild-type AFO.

[0081] Example 3

[0082] This example explores the effect comparison of the normal enzyme activity of the aflatoxin oxidase DnD-AFO prepared in the above Example 1 and the wild-type AFO, as follows:

[0083] DnD-AFO experimental group: The reaction system components were: AFB1 (0.5 μg / mL), aflatoxin oxidase DnD-AFO (1 mg / mL) prepared in Example 1, CuSO4 (0.6 mmol / L), low salt buffer (pH 7.4, components: NaCl 0.5844 g, 0.2 mol / L Na2HPO4 81 mL, 0.2 mol / L NaH2PO4 19 mL, ddH2O was added to make the volume 1 L), the total volume of the reaction system was 3 mL, the reaction was closed in a 5 mL centrifuge tube, aflatoxin AFB1 was oxidized, and the amount of aflatoxin AFB1 remaining in the reaction system after 4 hours was detected (the detection conditions were FLD detector excitation 365 nm, emission 440 nm).

[0084] Wild-type AFO experimental group: The method was the same as that of the DnD-AFO experimental group, except that the aflatoxin oxidase DnD-AFO in the reaction system was replaced by wild-type AFO.

[0085] Blank control group: The method was the same as that of the DnD-AFO experimental group, except that aflatoxin oxidase DnD-AFO was not added.

[0086] The experimental results show that the remaining amount of aflatoxin in the reaction system with the addition of wild-type AFO is 64.44% of the input amount, while the remaining amount of aflatoxin in the reaction system with the addition of the aflatoxin oxidase DnD-AFO of the present invention is 59.92% of the input amount. It can be seen that the detoxification efficiency of the aflatoxin oxidase DnD-AFO of the present invention is 1.12 times that of the wild-type AFO, indicating that the DnD-AFO of the present invention has a significantly improved detoxification efficiency compared with the wild-type AFO.

[0087] Example 4

[0088] This example explores the thermal stability comparison between the aflatoxin oxidase DnD-AFO prepared in Example 1 and the wild-type AFO, as follows:

[0089] The aflatoxin oxidase DnD-AFO and wild-type AFO prepared in Example 1 were heated in a metal bath at 85° C. for 3 min, and then the enzyme activity was measured.

[0090] Experimental group with DnD-AFO heat treatment: The reaction system components were: AFB1 (0.5 μg / mL), aflatoxin oxidase DnD-AFO (1 mg / mL) prepared in Example 1 after being heated in a metal bath at 85°C for 3 min, CuSO4 (0.6 mmol / L), and low salt buffer (pH 7.4, components: NaCl 0.5844 g, 0.2 mol / L Na2HPO4 81 mL, 0.2 mol / L NaH2PO4 19 mL, fixed to 1 L with ddH2O), the total volume of the reaction system was 3 mL, the reaction was closed in a 5 mL centrifuge tube, aflatoxin AFB1 was oxidized, and the amount of aflatoxin AFB1 remaining in the reaction system after 4 hours was detected (the detection conditions were FLD detector excitation 365 nm, emission 440 nm).

[0091] Experimental group of heat treatment of wild-type AFO: The method was the same as that of the DnD-AFO experimental group, except that the aflatoxin oxidase DnD-AFO in the reaction system was replaced by wild-type AFO that had been heated in a metal bath at 85°C for 3 minutes.

[0092] Blank heat-treated control group: The method was the same as that of the DnD-AFO experimental group, except that aflatoxin oxidase DnD-AFO was not added.

[0093] Figure 4 It is a comparison chart of enzyme activities of the experimental group of DnD-AFO heat treatment, the experimental group of wild-type AFO heat treatment and the control group of blank heat treatment. The experimental results show that the residual amount of aflatoxin in the reaction system with the addition of heat-treated wild-type AFO is 87.34% of the input amount, while the residual amount of aflatoxin in the reaction system with the addition of the heat-treated aflatoxin oxidase DnD-AFO of the present invention is 48.04% of the input amount. It can be seen that the detoxification and degradation rate of the aflatoxin oxidase DnD-AFO of the present invention is 4.106 times that of the wild-type AFO, indicating that the DnD-AFO of the present invention has better thermal stability than the wild-type AFO, and has a significantly improved detoxification efficiency after being heated in a metal bath at 85°C for 3 minutes.

[0094] In summary, the aflatoxin oxidase DnD-AFO provided by the present invention has the advantages of improved activity, better thermal stability (after high-temperature granulation, the activity is much better than the wild type), improved anti-pepsin and acid resistance, etc. compared with the traditional wild-type aflatoxin oxidase AFO. It can be well used as a food additive or feed additive in agriculture, food engineering and other related industries, and has huge commercial value and added value.

[0095] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An aflatoxin oxidase, characterized in that: The amino acid sequence of the aflatoxin oxidase is SEQ ID NO.

1.

2. A gene encoding aflatoxin oxidase according to claim 1, characterized in that: The nucleotide sequence of the coding gene is SEQ ID NO.

2.

3. A recombinant vector comprising the coding gene as claimed in claim 2.

4. A recombinant bacterium comprising the coding gene as claimed in claim 2.

5. A method for preparing aflatoxin oxidase according to claim 1, characterized in that: Includes steps: S1, constructing a recombinant vector expressing aflatoxin oxidase, and transfecting cells with the recombinant vector to obtain recombinant bacteria; S2, culturing the recombinant bacteria to induce the expression of aflatoxin oxidase; S3. Separate and purify aflatoxin oxidase.

6. The method for preparing aflatoxin oxidase according to claim 5, characterized in that: The step S1 specifically includes: The coding gene of aflatoxin oxidase was introduced into pET28a plasmid, with restriction sites NdeI and EcoRI to obtain a recombinant plasmid, and then the recombinant plasmid was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium.

7. The method for preparing aflatoxin oxidase according to claim 5, characterized in that: The step S2 specifically includes: The recombinant bacteria were inoculated into LB medium containing kanamycin and cultured at 25-40°C and 150-500rpm. When the absorbance density of the bacterial solution reached 0.6-0.8, isopropyl-β-d-thiogalactoside was added to induce the expression of aflatoxin oxidase.

8. The method for preparing aflatoxin oxidase according to claim 7, characterized in that: The concentration of kanamycin in the LB medium is 25-75 μg / mL, and the concentration of isopropyl-β-d-thiogalactoside is 0.05-0.15 mmol / L.

9. The method for preparing aflatoxin oxidase according to claim 7, characterized in that: The induction temperature is 10-25°C, and the induction time is 15-30h.

10. Use of the aflatoxin oxidase according to claim 1 in food additives or feed additives.

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