Aflatoxin oxidase and its preparation method and application

Abstract

This invention relates to the field of biocatalysis technology, and more particularly to an aflatoxin oxidase, its preparation method, and its applications. This invention utilizes a self-designed artificial intelligence enzyme design workflow to de novo design a novel aflatoxin oxidase, DnD-AFO. Compared to the traditional wild-type AFO, DnD-AFO exhibits enhanced activity, better thermal stability (its activity is significantly superior to the wild-type after high-temperature granulation), and improved resistance to pepsin and acid. Furthermore, it further enhances the detoxification efficiency against aflatoxin, making it well-suited as a food or feed additive for applications in agriculture and food engineering, among other related industries, demonstrating significant commercial and added value.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to an aflatoxin oxidase, its preparation method, and its application. Background Technology

[0002] Due to limited storage conditions, grains and feeds are prone to mold growth. Aflatoxin, a potent carcinogen produced by Aspergillus flavus and Aspergillus parasiticus, is a major contaminant of food and feed. Various methods have been proposed to detoxify aflatoxin in feed, among which the use of aflatoxin oxidase (AFO) is considered a green, environmentally friendly, efficient, and irreversible method.

[0003] In 1999, Professor Wang Guanglin and his research team reported internationally an activator purified from the edible fungus *Armillariella tabescens* that could remove aflatoxin toxicity, named aflatoxin oxidase (AFO). Further research into the enzyme's mechanism of action revealed that, as an oxidoreductase, it can oxidize aflatoxin (AFB1) and other structural analogs, breaking the chemical bonds of the difuran ring in the aflatoxin structure and thus detoxifying it.

[0004] Currently, AFO (aflatoxin-free enzyme) has been widely used and promoted as an antidote for removing aflatoxin residues in food and feed. Compared to other methods, it has become an excellent method for eliminating aflatoxin toxicity due to its advantages such as being environmentally friendly, relatively efficient, economical, and thorough. However, because AFO has a long action time, it needs to take effect in the livestock being raised and must meet high-temperature pelleting standards. Existing wild-type AFO enzymes have improved feed safety and quality in feed production, but they still have disadvantages such as low activity, poor thermal stability (almost losing activity when heated to 85°C for three minutes (high-temperature pelleting standard), resistance to pepsin, and moderate acid resistance.

[0005] In addition, patent CN115851633A discloses an aflatoxin oxidase with enhanced pepsin resistance and its application. This patent utilizes protein engineering technology to modify key amino acid residues within the wild-type aflatoxin oxidase molecule, resulting in a mutant aflatoxin oxidase with enhanced pepsin resistance. The mutant aflatoxin oxidase (AFO) described in this patent acts on aflatoxin (AFB1). G355I / G475VThe resistance half-life to pepsin in mutant AFO is 2.72 times that of wild-type AFO. Compared to wild-type, mutant AFO... G355I / G475V It retains more than 40% of its activity at 10–40°C, and the optimal reaction pH is 7.5.

[0006] To date, no AFO enzyme has been reported that simultaneously possesses high activity, good thermal stability (activity retained after heating at 85℃ for three minutes (high-temperature pelleting standard), resistance to pepsin, and high acid resistance. Enzymes with multiple advantages are better suited for feed production to achieve efficient, green, and stable aflatoxin detoxification. Therefore, existing technologies require further improvement and development. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide an aflatoxin oxidase, its preparation method and application, in order to solve the problems of low activity, poor thermal stability, and general resistance to pepsin and acid in existing aflatoxin oxidases.

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

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

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

[0011] A third aspect of the present invention provides a recombinant vector comprising the coding gene described herein.

[0012] In a fourth aspect, the present invention provides a recombinant bacterium comprising the coding gene described herein.

[0013] A fifth aspect of the present invention provides a method for preparing the aflatoxin oxidase described herein, comprising the steps of:

[0014] S1. Construct a recombinant vector expressing aflatoxin oxidase, and transfect the recombinant vector into cells to obtain recombinant bacteria;

[0015] S2. Culture recombinant bacteria to induce the expression of aflatoxin oxidase;

[0016] S3. Aflatoxin oxidase was obtained by separation and purification.

[0017] Optionally, step S1 specifically includes:

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

[0019] Optionally, step S2 specifically includes:

[0020] The recombinant bacteria were inoculated into LB medium containing kanamycin and cultured at 25-40℃ and 150-500 rpm. When the absorbance 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℃, and the induction time is 15-30h.

[0023] In a sixth aspect, the present invention provides the application of the aflatoxin oxidase described herein in a food additive or feed additive.

[0024] Beneficial effects: Compared with the traditional wild-type aflatoxin oxidase AFO, the aflatoxin oxidase DnD-AFO provided by this invention has the advantages of improved activity, better thermal stability (after high-temperature granulation, the activity is far superior to the wild type), and improved anti-pepsin and acid-resistant properties. It also further improves the detoxification efficiency of aflatoxin. It can be well used as a food additive or feed additive in agriculture and food engineering and other related industries, and has great commercial value and added value. Attached Figure Description

[0025] Figure 1 This is an SDS-PAGE protein electrophoresis image from Example 1.

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

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

[0028] Figure 4 This is a comparison diagram of enzyme activity between the DnD-AFO experimental group, the wild-type AFO experimental group, and the blank control group in Example 4. Detailed Implementation

[0029] This invention provides an aflatoxin oxidase, its preparation method, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] This invention, based on a comprehensive exploration and computation of the AFO enzymatic reaction mechanism and protein multiple sequence alignment, obtains information on the conserved site range of AFO enzymes and the positions of key amino acids involved in the reaction. Building upon this information, the de novo protein design model ProteinMPNN is used to generate a large number of novel protein sequences while ensuring that important structural positions remain unchanged. The generated sequences are then used to predict their functional attributes using various artificial intelligence models (DLKcat, UNiKP, DeepDetect, etc.). For the required functional attributes, a self-designed scoring function, which integrates experimental data with multiple models as influencing factors, is used to rank and select candidate mutants, ultimately identifying high-quality aflatoxin oxidases with multiple functions from the generated proteins.

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

[0032] (1) By employing various sequence processing and chemical biological computation methods such as molecular dynamics, molecular docking and multiple sequence alignment, the key domains and conserved sequences of the aflatoxin oxidase to be designed from scratch are identified, thereby obtaining enough prior knowledge to constrain the AI ​​protein generation model, so as to ensure that the excellent functional properties of the generated new sequence are preserved.

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

[0034] (3) Design a scoring function for the required functions and properties of the target aflatoxin oxidase (AFO) (considering AI models participating in the functional property prediction screening, such as DLKcat, UNiKP, DeepDetect, etc.), standardize the simulation calculation results in the computer, and use the self-designed scoring function to select the best candidates from the candidate sequence.

[0035] The aflatoxin oxidase designed de novo using the above workflow is named DnD-AFO. Conventional activity assays, activity assays after high-temperature granulation standard treatment, and hydrolysis assays using simulated gastric juice were performed on DnD-AFO. Compared with wild-type AFO, it exhibits superior properties in several aspects.

[0036] Based on this, embodiments of the present invention provide 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 this invention has advantages over traditional wild-type AFO, including enhanced activity, better thermal stability (its activity is far superior to wild-type after high-temperature granulation), and improved resistance to pepsin and acid. First, the macroscopic anti-pepsin effect of DnD-AFO of this invention is far superior to wild-type AFO. Quantitative determination shows that after 1 hour of addition to a simulated gastric juice, the residual enzyme amount of DnD-AFO of this invention is 3.36 times that of wild-type. Second, without heating, based on the residual aflatoxin amount within a specified time, the detoxification efficiency of DnD-AFO of this invention is 1.12 times that of wild-type. Third, after heating both wild-type AFO and DnD-AFO of this invention in an 85°C metal bath for 3 minutes, based on the residual aflatoxin amount within a specified time, the detoxification efficiency of DnD-AFO of this invention is 4.106 times that of wild-type.

[0040] In this invention, based on the DnD-AFO amino acid sequence, aflatoxin oxidase sequence variants with similar functions can be designed. For example, by conserving mutations or by preserving key regions of the sequence and optimizing other regions, the functional stability or activity of the sequence can be enhanced. Such aflatoxin oxidase sequence variants with similar functions are also within the scope of protection of this application.

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

[0042] This invention provides a coding gene for the aflatoxin oxidase DnD-AFO described in the foregoing embodiments, wherein the nucleotide sequence of the coding gene is SEQ ID NO.2.

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

[0044]

[0045] This invention provides a recombinant vector containing the coding gene described in the foregoing embodiments.

[0046] This invention provides a recombinant bacterium containing the coding gene described in the foregoing embodiments.

[0047] This invention provides a method for preparing aflatoxin oxidase as described in the foregoing embodiments, comprising the following steps:

[0048] S1. Construct a recombinant vector expressing aflatoxin oxidase, and transfect the recombinant vector into cells to obtain recombinant bacteria;

[0049] S2. Culture recombinant bacteria to induce the expression of aflatoxin oxidase;

[0050] S3. Aflatoxin oxidase was obtained by separation and purification.

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

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

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

[0054] The recombinant strain E. coli BL21(DE3) / pET28a-DnD-AFO was inoculated into LB medium containing kanamycin and cultured at 25-40℃ (preferably 37℃) and 150-500 rpm (preferably 200 rpm). When the absorbance of the bacterial solution reached 0.6-0.8, isopropyl-β-d-thiogalactoside was 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 15-30 h (preferably 20 h).

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

[0058] This invention provides an application of the aflatoxin oxidase described in the foregoing embodiments in food additives or feed additives.

[0059] The present invention will be further described below through specific embodiments.

[0060] Example 1

[0061] This embodiment provides a method for preparing aflatoxin oxidase DnD-AFO, as detailed below:

[0062] 1. Construction, culture and expression of genetically engineered 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 NdeI and EcoRI restriction sites to obtain the recombinant plasmid pET28a-DnD-AFO. Then, the recombinant plasmid pET28a-DnD-AFO was introduced into E. coli BL21(DE3) to form the genetically engineered strain E. coli BL21(DE3) / pET28a-DnD-AFO.

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

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

[0066] The above-obtained high-efficiency aflatoxin oxidase DnD-AFO-expressing genetically engineered bacterial wet cells were resuspended in 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl), homogenized under high pressure, centrifuged (12000 rpm, 20 min), and the supernatant was collected to obtain the 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) Pass the crude enzyme solution of aflatoxin oxidase DnD-AFO obtained above through a Ni column at a flow rate of 1 mL / min to load the target protein onto 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 do not bind 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) Centrifuge the protein solution using an ultrafiltration tube (4000 rpm, 20 min) to one-tenth of the original volume, and replenish it to the original volume with 50 mmol / L pH 8.0 sodium phosphate buffer (containing 500 mmol / L NaCl). Repeat three times to reduce the imidazole concentration in the protein solution.

[0073] (6) The collected protein samples were analyzed by SDS-PAGE using a vertical electrophoresis apparatus. 20 μL of sample was added to 5 μL of 5×SDS loading buffer for processing. The loading volume was 10 μL for all samples. Protein Molecular Weight Markers were purchased from Thermo Fisher Scientific, USA. The SDS-PAGE gel concentration was 12%. Concentration electrophoresis was performed at 110V, followed by separation electrophoresis at 200V to purify the aflatoxin oxidase DnD-AFO to high purity. The SDS-PAGE protein electrophoresis image is shown below. Figure 1 As shown, the black box contains aflatoxin oxidase DnD-AFO, with a molecular weight of approximately 76 kDa. Lane 1 is for precipitate fragmentation, lane 2 is for Ni column elution, lane 3 is for elution with 10 mM imidazole, lane 4 is for elution with 25 mM imidazole, and lane 5 is for elution with 250 mM imidazole.

[0074] Example 2

[0075] This embodiment investigates the comparative effects of aflatoxin oxidase DnD-AFO prepared in Example 1 with wild-type AFO on its anti-pepsin and anti-acid activity, as detailed below:

[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 (20 μg / mL pepsin, 10 mM NaCl adjusted to pH 2.5 with concentrated hydrochloric acid) and treated for 1 h. The above system was added to 2 mL EP tubes (total system 1 mL) and incubated on a shaker at 37 °C for 1 h. Samples were taken at 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min and run on SDS-PAGE protein gel (sample volume 20 μL, with 5 μL of 5× loading buffer added, mixed well, heated at 100 °C for 5 min, and the sample loading volume was 10 μL).

[0077] Based on the SDS-PAGE electrophoresis image, Western blot analysis was performed using Image J software. The residual enzyme amounts of aflatoxin oxidase DnD-AFO and wild-type AFO were calculated quantitatively to compare their effects on anti-pepsin and anti-acidity.

[0078] Figure 2 This is an SDS-PAGE electrophoresis image of wild-type AFOs. The black boxes represent the remaining wild-type AFOs. Lane 1 was sampled at 0 min, lane 2 at 10 min, lane 3 at 20 min, lane 4 at 30 min, lane 5 at 40 min, lane 6 at 50 min, and lane 7 at 60 min. Figure 2 The grayscale analysis of protein bands was performed at different positions defined by time frame. The initial protein amount grayscale analysis result was 200363, and the remaining protein amount grayscale analysis result after 60 min was 24302. The comparison between the two results showed that the remaining enzyme amount of wild-type AFO after passing through gastric juice simulation solution was 12.11% of the initial enzyme amount.

[0079] Figure 3 This is an SDS-PAGE electrophoresis image of aflatoxin oxidase DnD-AFO. The black boxes represent the remaining aflatoxin oxidase DnD-AFO. Lane 1 was sampled at 0 min, lane 2 at 10 min, lane 3 at 20 min, lane 4 at 30 min, lane 5 at 40 min, lane 6 at 50 min, and lane 7 at 60 min. Figure 3 The protein band grayscale analysis was performed at different positions according to the time frame. The initial protein amount grayscale analysis result was 205061, and the remaining protein amount grayscale analysis result after 60 min was 83455. The comparison between the two results showed that the remaining enzyme amount of DnD-AFO after passing through the gastric juice simulation solution was 40.69% of the initial enzyme amount.

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

[0081] Example 3

[0082] This embodiment investigates the effect of the aflatoxin oxidase DnD-AFO prepared in Example 1 on the normal enzyme activity of wild-type AFO, as detailed below:

[0083] DnD-AFO experimental group: The reaction system consisted of: AFB1 (0.5 μg / mL), aflatoxin oxidase DnD-AFO (1 mg / mL) prepared in Example 1, 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, and ddH2O to a final volume of 1 L). The total volume of the reaction system was 3 mL. The reaction was blocked in a 5 mL centrifuge tube to oxidize aflatoxin AFB1. The amount of aflatoxin AFB1 remaining in the reaction system after 4 h was detected (detection conditions: FLD detector excitation 365 nm, emission 440 nm).

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

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

[0086] Experimental results show that the residual amount of aflatoxin in the reaction system with wild-type AFO was 64.44% of the input amount, while the residual amount of aflatoxin in the reaction system with the aflatoxin oxidase DnD-AFO of the present invention was 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 wild-type AFO, indicating that DnD-AFO of the present invention has a significantly improved detoxification efficiency compared with wild-type AFO.

[0087] Example 4

[0088] This embodiment investigates the comparison of the thermal stability of aflatoxin oxidase DnD-AFO prepared in Example 1 above with that of wild-type AFO, as detailed below:

[0089] The aflatoxin oxidase DnD-AFO prepared in Example 1 was subjected to heating in a metal bath at 85°C for 3 minutes, and then the enzyme activity was measured.

[0090] The experimental group subjected to heat treatment with DnD-AFO: The reaction system consisted of: 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, and ddH2O added to bring the volume to 1 L). The total volume of the reaction system was 3 mL. The reaction was blocked in a 5 mL centrifuge tube to oxidize aflatoxin AFB1. The amount of aflatoxin AFB1 remaining in the reaction system after 4 h was detected (detection conditions: FLD detector excitation at 365 nm and emission at 440 nm).

[0091] Wild-type AFO heat treatment experimental group: The method is the same as that of the DnD-AFO experimental group, except that the aflatoxin oxidase DnD-AFO in the reaction system is replaced with wild-type AFO that has been treated with heating in a metal bath at 85°C for 3 min.

[0092] The blank heat-treated control group was treated in the same way as the DnD-AFO experimental group, except that the aflatoxin oxidase DnD-AFO was not added.

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

[0094] In summary, the aflatoxin oxidase DnD-AFO provided by this invention has advantages over traditional wild-type aflatoxin oxidase AFO, such as improved activity, better thermal stability (its activity is far superior to the wild type after high-temperature granulation), and improved resistance to pepsin and acid. It can be well used as a food additive or feed additive in agriculture and food engineering and other related industries, and has great commercial value and added value.

[0095] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

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 the aflatoxin oxidase as described in claim 1, characterized in that, The nucleotide sequence of the encoded gene is SEQ ID NO.

2.

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

4. A recombinant bacterium comprising the encoding gene as described in claim 2.

5. A method for preparing aflatoxin oxidase as described in claim 1, characterized in that, Including the following steps: S1. Construct a recombinant vector expressing aflatoxin oxidase, and transfect the recombinant vector into cells to obtain recombinant bacteria, specifically including: The gene encoding aflatoxin oxidase was introduced into the pET28a plasmid with NdeI and EcoRI restriction sites to obtain a recombinant plasmid. The recombinant plasmid was then introduced into Escherichia coli BL21(DE3) to obtain a recombinant bacterium. The nucleotide sequence of the gene encoding aflatoxin oxidase is SEQ ID NO.

2. S2. Cultivating recombinant bacteria to induce the expression of aflatoxin oxidase, specifically including: The recombinant bacteria were inoculated into LB medium containing kanamycin and cultured at 25-40℃ and 150-500 rpm. When the absorbance of the bacterial solution reached 0.6-0.8, isopropyl-β-d-thiogalactoside was added to induce the expression of aflatoxin oxidase. S3. Aflatoxin oxidase was obtained by separation and purification.

6. The method for preparing aflatoxin oxidase according to claim 5, characterized in that, The concentration of kanamycin in the LB medium was 25-75 μg / mL, and the concentration of isopropyl-β-d-thiogalactoside was 0.05-0.15 mmol / L.

7. The method for preparing aflatoxin oxidase according to claim 5, characterized in that, The induction temperature is 10-25℃, and the induction time is 15-30h.

8. The use of the aflatoxin oxidase as described in claim 1 in a food additive or feed additive.

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