Gene for degrading aflatoxin B1 and application thereof

Abstract

The invention discloses a gene for degrading aflatoxin B1 and application of the gene. The nucleotide sequence of the gene is shown as SEQ ID No. 1. Aspergillus niger A327 is used as a research object, a candidate gene of aflatoxin B1 degrading enzyme in aspergillus niger is explored, a genetically engineered bacterium with high degradation capacity on aflatoxin B1 is obtained through homologous expression, the strain can efficiently degrade aflatoxin B1, the degradation rate on aflatoxin can reach 100%, and meanwhile, the strain has the advantages of being environmentally friendly and efficient.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and particularly relates to a gene for degrading aflatoxin B1 and its application. Background Art

[0002] Aflatoxin B1 (AFB1) is a secondary toxic metabolite mainly produced by Aspergillus flavus and Aspergillus parasiticus. AFB1 consists of a bifuran ring and a coumarin ring, is fluorescent, and emits blue light under ultraviolet light. It is the most toxic aflatoxin and also the most potent known natural chemical carcinogen, and the furan ring is considered the key structure responsible for its toxicity and carcinogenicity. Currently, more than 5 billion people worldwide are at risk of long-term exposure to AFB1 in food, which poses a serious threat to human health.

[0003] AFB1 is widely present in contaminated agricultural products such as grains, nuts, and spices, and its contamination problem is widespread globally. Especially in warm and humid regions, due to suitable growth conditions, Aspergillus flavus and Aspergillus parasiticus are prone to reproduce, resulting in more serious AFB1 contamination. The high toxicity and carcinogenicity of AFB1 pose a major threat to human health, and how to effectively remove AFB1 from food has become a global public health issue of concern.

[0004] Currently, the methods for removing AFB1 from food mainly include physical methods (such as screening, washing, irradiation, etc.), chemical methods (such as using oxidants, alkali treatment, etc.), and biological methods (such as using microorganisms or enzymes for degradation). However, these methods all have certain limitations. Physical methods may not be able to completely remove AFB1 and may affect the quality of food; chemical methods may introduce new harmful substances and pose potential risks to the environment and health; although biological methods have the advantages of environmental friendliness and high efficiency, no microorganisms or enzymes capable of efficiently degrading AFB1 have been found yet.

[0005] Aspergillus niger is a widely used industrial microorganism with multiple enzyme systems and strong metabolic capabilities. Existing studies have shown that Aspergillus niger has a certain ability to degrade certain toxic substances. Based on these characteristics of Aspergillus niger, researchers have begun to explore whether it has the potential to degrade AFB1. By discovering the genes in Aspergillus niger that can degrade AFB1 and using genetic engineering techniques for modification and optimization, it is expected to obtain genetically engineered bacteria capable of efficiently degrading AFB1. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a gene for degrading aflatoxin B1 and its application.

[0007] To achieve the above object, the present invention provides a gene for degrading aflatoxin B1, and its nucleotide sequence is shown as SEQ ID No.1.

[0008] The present invention also provides an aflatoxin degrading enzyme encoded by the above gene.

[0009] The present invention also provides a recombinant vector containing the above gene for degrading aflatoxin B1.

[0010] The present invention also provides a genetically engineered bacterium containing the above gene for degrading aflatoxin B1.

[0011] Furthermore, the starting strain of the genetically engineered bacterium is Aspergillus niger A327.

[0012] The present invention also provides the application of the above gene for degrading aflatoxin B1, the above aflatoxin degrading enzyme or the above recombinant vector in degrading aflatoxin B1.

[0013] The present invention also provides the application of the above genetically engineered bacterium in degrading aflatoxin B1.

[0014] The present invention also provides a method for degrading aflatoxin B1, comprising:

[0015] Inoculating the above genetically engineered bacterium into a contaminated substrate containing aflatoxin B1 and culturing.

[0016] Furthermore, the volume ratio of the inoculation is 5%; the culture conditions are: temperature 32 °C, pH 6.5, rotation speed 180 rmp / min, and culture time 84 - 90 h.

[0017] Furthermore, the mass concentration of aflatoxin B1 in the contaminated substrate containing aflatoxin B1 is 120 - 960 ng / mL.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] The present invention takes Aspergillus niger A327 as the research object, discovers the candidate gene of aflatoxin B1 degrading enzyme in Aspergillus niger, and obtains a genetically engineered bacterium with high degradation ability for aflatoxin B1 through homologous expression. This strain can efficiently degrade aflatoxin B1, and the degradation rate of aflatoxin B1 can reach 100%. At the same time, it has the advantages of environmental friendliness and high efficiency. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the kinetic curve of AFB1 degradation by Aspergillus niger A327;

[0022] Figure 2 It is the phylogenetic tree analysis of aflatoxin-degrading enzyme and AN-AFB1D in fungi;

[0023] Figure 3 It is the result of Aspergillus niger genomic DNA extraction;

[0024] Figure 4 It is the result of candidate gene amplification;

[0025] Figure 5 It is the result of cDNA sequence amplification;

[0026] Figure 6 It is the result of vector linearization;

[0027] Figure 7 It is the result of colony PCR;

[0028] Figure 8 It is the result of plasmid double digestion;

[0029] Figure 9 It is the effect of different mass concentrations of AFB1 on AFB1 degradation by Aspergillus niger;

[0030] Figure 10 It is the kinetic curve of AFB1 degradation by Aspergillus niger A327-15 under optimized fermentation conditions. Detailed Embodiments

[0031] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1 Experimental materials

[0038] 1.1 Strains

[0039] Aspergillus niger A327, preserved by the Ion Beam Bioengineering Laboratory of Inner Mongolia University.

[0040] 1.2 Preparation methods of culture media and solutions

[0041] The culture media and solutions involved in this experiment were prepared as follows:

[0042] (1) PDA slant medium: 200 g of potato (peeled), 20 g / L of glucose, 15 g / L of agar powder, natural pH.

[0043] (2) PDB medium: 200 g of potato (peeled), 20 g / L of glucose, natural pH.

[0044] (3) Czapek slant medium: 3% sucrose, 0.1% K2HPO4, 0.05% KCL, 0.3% NaNO3, 0.05% MgSO4, 0.001% FeSO4·7H2O, 1.2% agar, natural pH.

[0045] (4) LB liquid medium: 10 g / L of peptone, 10 g / L of sodium chloride, 5 g / L of yeast extract powder, pH adjusted to 7.4.

[0046] (5) LB solid medium: peptone 10 g / L, sodium chloride 10 g / L, yeast extract powder 5 g / L, agar powder 15 g / L, pH adjusted to 7.4.

[0047] (6) S3Y2 medium: yeast extract powder 2%, soluble starch 3%, potassium dihydrogen phosphate 0.5%, add 0.5% corn grits before sterilization.

[0048] (7) 100 mg / mL ampicillin solution: weigh 1 g penicillin powder, add 8 mL of sterile water, stir well until completely dissolved, continue to add sterile water to make up the volume to 10 mL, filter the solution using a 0.22 μL sterile filter and syringe, and store in a -20 °C refrigerator after aliquoting.

[0049] (8) LB selective medium: when the temperature of the LB solid medium drops to about 50 °C - 60 °C, add the antibiotic stock solution under sterile conditions to a final concentration of 50 μg / mL, and shake well. Pour the LB solid selective medium into a sterile petri dish and wait for it to solidify to form a plate; for the liquid medium, add the antibiotic stock solution after it has completely cooled. Store the prepared selective medium at 4 °C and check its sterility before use.

[0050] (9) 0.8 M MgSO4 stock solution: weigh 96.3 g of magnesium sulfate and add it to a container, add an appropriate amount of distilled water, stir with a glass rod to accelerate dissolution, after the magnesium sulfate is completely dissolved, transfer the solution to a volumetric flask, and continue to add distilled water until the liquid level reaches the 1 L mark of the volumetric flask.

[0051] (10) 0.8 M MgSO4 solution: measure 95 mL of the 0.8 M MgSO4 stock solution and 5 mL of the phosphate buffer solution, mix well with a glass rod.

[0052] (11) Phosphate buffer solution: First, prepare solution A by weighing 3.12 g of NaH2PO4 and making up the volume to 1 L with distilled water. Second, prepare solution B by weighing 7.16 g of Na2HPO4 and making up the volume to 1 L with distilled water. When using, take 810 mL of solution A and 190 mL of solution B, set the pH to 7.4, and if the pH increases or decreases, fine-tuning can be carried out.

[0053] (12) amdS transformation screening upper layer medium: glucose 2%, acetamide 15 mM, sucrose 0.8 M, dipotassium hydrogen phosphate 0.1%, CsCl 20 mM, trace elements 5 mL, agar powder 0.35%.

[0054] (13) amdS transformation screening lower layer medium: based on the amdS transformation screening upper layer medium, change to adding 1.2% agar powder.

[0055] (14) 10× Inorganic Salt Solution: Potassium Chloride 0.05%, Magnesium Sulfate 0.05%, Ferrous Sulfate 0.001%.

[0056] (15) 200 ml Sorbital Solution: Sorbital 1.2 M, CaCl₂ 0.05 M, Tris·Cl (pH adjusted to 7.5) 10 mM.

[0057] (16) 50 ml PEG Buffer: PEG4000 50%, CaCl₂ 0.05 M, Tris·Cl (pH adjusted to 7.5) 0.01 M.

[0058] (17) Preparation of 1% Agarose Gel: Measure 15 mL of 1× TAE buffer solution, then weigh 0.15 g of agarose with a balance and add it to a conical flask. Gently shake it. After weighing with the balance, place it in a microwave oven to heat. Weigh it again, add sterile water to make up for the evaporated liquid. Wait until the conical flask is not hot to the touch, add 1 μL of nucleic acid dye, and gently shake until the liquid is evenly mixed. Pour it into the gel-making tank and cool it at room temperature. Use it after solidification.

[0059] (18) AFB1 Stock Solution: Dissolve AFB1 solid powder with acetonitrile to a concentration of 100 μg / mL.

[0060] 1.3 Primer Design

[0061] The primers required for this experiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The detailed information of the primer names and sequences used is shown in Table 1.

[0062] Table 1 Primers Used for Expression Vector Construction

[0063]

[0064]

[0065] 2 Experimental Methods

[0066] 2.1 Extraction of Aspergillus niger Genomic DNA

[0067] Aspergillus niger was activated on a PDA slant. It was eluted from the slant with sterile water to make a spore suspension, and then inoculated into the precursor medium with an inoculation amount of 1 mL. It was cultured at 30 °C and 200 rpm / min with constant shaking for 14 - 16 h, and operated according to the genomic DNA extraction kit instructions.

[0068] 2.2 Acquisition of Target Gene

[0069] First, obtain the amino acid sequences of AFB1-degrading enzymes in other fungi from relevant literature, query the corresponding sequences in NCBI according to the amino acid sequence numbers (https: / / www.ncbi.nlm.nih.gov / protein), and determine the conserved domains and corresponding sequences through NCBI (CD-search), and search for conserved sequences by combining DNAman multiple sequence alignment; use the currently published sequence data of three groups of Aspergillus niger (Aspergillus niger ATCC 1015, Aspergillus niger CBS513.88, and Aspergillus niger RAF106) to establish local BLAST databases of nucleotides and amino acids respectively, align the amino acid conserved sequences with the Aspergillus niger sequences in NCBI local BLAST, construct a phylogenetic tree by combining the neighbor-joining (NJ) method of biological evolutionary distance to determine candidate genes, then search and download the sequences of the found sequences in the NCBI database, and finally synthesize the cDNA sequence of the target gene derived from Aspergillus niger, and name this candidate gene AN-AFB1D, and its nucleotide sequence is shown in SEQ ID No.1.

[0070] SEQ ID No.1:

[0071] >ACJE01000016.1:79519-81783Aspergillus niger ATCC 1015,whole genomeshotgun sequence

[0072]

[0073] 2.3 PCR Amplification Reaction

[0074] 2.3.1 Amplification of Gene AN-AFB1D

[0075] Using the cDNA sequence of the AN-AFB1D gene as a template, primers were synthesized through the Sangon Biotech platform. The reaction conditions were: 94°C for 3 min; 94°C for 30 s, 47°C for 30 s, 72°C for 2.07 min; extension at 72°C for 5 min; number of cycles: 30; amplification was carried out according to the PCR reaction system in Table 2. The amplified products were detected by agarose gel electrophoresis.

[0076] Table 2 PCR Reaction System

[0077]

[0078] 2.4 Gel Extraction of PCR Products

[0079] Under ultraviolet light, the target DNA band in the agarose gel was cut with a sterile blade (try to cut off the excess as much as possible), placed in a centrifuge tube and weighed (try to shorten the cutting process, and the ultraviolet irradiation time should not exceed 30 seconds). Equal volume of solubilization solution PN was added to the gel block, 100 μL of solubilization solution PN was added per 0.1 g of gel, and it was dissolved in a 50°C water bath. In order to ensure complete dissolution of the gel block, the centrifuge tube needed to be gently and continuously inverted during the process. The obtained solubilization solution was transferred to an adsorption column, left at room temperature for 2 min, and then the subsequent steps of rinsing the residual solution and eluting the DNA were carried out. Then the concentration of the recovered DNA fragment was detected with a ultra-micro spectrophotometer. The specific steps refer to the instruction manual of the ordinary agarose gel DNA recovery kit. The obtained DNA fragment can be directly used for subsequent experiments or stored at -20°C. All steps should be operated in a clean environment to prevent DNA contamination and degradation.

[0080] 2.5 Vector Linearization

[0081] Vector linearization was achieved by PCR amplification. The reaction system is shown in Table 3.

[0082] Table 3 PCR Reaction System

[0083]

[0084] Agarose gel electrophoresis experiment was carried out to detect the PCR products, and then gel extraction experiment was carried out.

[0085] 2.6 Seamless Cloning Ligation Reaction

[0086] The target gene of AN-AFB1D was ligated into the expression vector pUC19-acs by seamless cloning technology. The addition of the seamless cloning system and the experimental steps were operated according to the instructions. The reaction system is shown in Table 4.

[0087] Table 4 PCR reaction system

[0088]

[0089] 2.7 Transformation of Escherichia coli competent cells

[0090] Take out the Escherichia coli competent cells DH5α from the ultra-low temperature refrigerator (-80 °C), melt them in an ice bath, and the action should be as gentle as possible; add 4 μL of the ligation product to a centrifuge tube containing 50 μL of competent cells, mix gently, and incubate on ice for 30 minutes; then transfer the centrifuge tube to a 42 °C water bath for heat shock for 90 seconds, quickly transfer the centrifuge tube to an ice bath, and cool for 5 minutes. Do not shake the centrifuge tube during this process; add 500 μL of SOC liquid medium, incubate at 37 °C for 45 - 60 min, centrifuge at 4000 rpm for 3 min to collect the bacteria; transfer to a laminar flow hood, spread a certain amount of bacteria evenly on an LB plate, invert the plate, and incubate statically at 37 °C for 12 hours to obtain single colonies. The specific steps refer to the instruction manual of the Sangon seamless cloning kit and the usage method of Escherichia coli competent cells DH5α.

[0091] 2.8 Identification of positive clone strains

[0092] After culturing for 12 hours, transfer to a laminar flow hood, observe the colony morphology, select a single colony with good growth and appropriate size, transfer the colony to a centrifuge tube containing 10 μL of sterile water with a sterilized toothpick, and mix well to make a bacterial suspension. Take 1 μL of the bacterial suspension as a template, add it to the PCR system, load the obtained product on a 1% agarose gel, perform electrophoresis and observe the electrophoresis results.

[0093] 2.9 Extraction of plasmids from positive clone strains

[0094] Amplify the single colony containing the target gene. Transfer the single colony into an LB medium (containing antibiotics) for selective culture, and incubate at 37 °C with constant shaking overnight; take 5 ml of the bacterial solution, centrifuge at 8000 rpm for 2 min at room temperature to collect the bacteria; perform lysis, centrifugation, taking the supernatant, washing, and elution steps in sequence; the detailed experimental steps refer to the instruction manual of the SanPrep column plasmid miniprep kit, and the plasmid DNA obtained by extraction is verified by double digestion.

[0095] 2.10 PEG-protoplast transformation of Aspergillus niger

[0096] 2.10.1 Preparation of protoplasts

[0097] First, activate Aspergillus niger A327. Subsequently, inoculate the eluted bacterial suspension into S3Y2 liquid medium (30 ml), and culture it at 30 °C and 200 rpm / min with constant temperature oscillation for 12 hours; filter it with four layers of sterilized gauze, collect the mycelia, first rinse them with sterile water three times, and then rinse them with 0.8 M / L MgSO4 solution once to help improve the osmotic pressure of the mycelia; dissolve it with Sorbital solution to prepare the lytic enzyme solution, and filter and sterilize it with a 0.22 μm membrane to prevent microbial contamination; at 30 °C and 79 rpm / min, oscillate for about 1 hour 30 minutes to 1 hour 50 minutes, and continuously take samples during this period to observe the enzymatic hydrolysis of the mycelia under the microscope. When most of the mycelia have been successfully enzymatically hydrolyzed, stop the reaction; next, add an equal volume of 1.2 M Sorbital buffer to it and filter it; centrifuge the filtrate at 2800 rpm for 6 minutes at room temperature; after centrifugation, discard the supernatant, rinse it twice with Sorbital buffer, and centrifuge at 2800 rpm for 6 minutes at room temperature; finally, suspend the protoplasts in 200 μL of Sorbital buffer for transformation experiments.

[0098] 2.10.2 Protoplast transformation and screening

[0099] Take 200 μL of the protoplast solution in a sterile centrifuge tube, slowly rotate the centrifuge tube to mix it well; add 10 μL of plasmid DNA to the centrifuge tube, and add 50 μL of PEG buffer, and incubate it on ice for 30 minutes; add 1 mL of PEG buffer to the centrifuge tube, mix it slowly, place the test tube at room temperature, and let it stand for 20 minutes; then add 1 mL of Sorbital solution to the test tube, mix it well, take an appropriate volume of liquid from the transformation mixture, mix it with the upper soft agar medium that has been melted and cooled to 55 °C, and pour it onto the bottom solid medium that has already solidified. The operation should be slow, gentle and accurate to ensure even spreading and no air bubbles are generated; let it stand at room temperature for 30 minutes, wait for the upper medium to solidify naturally, transfer the culture dish to a constant temperature incubator at 30 °C, and let it stand and culture for 3 - 7 days. During this process, the selection medium with acetamide as the sole nitrogen source has a screening effect on the transformants. When single colonies are formed and grow well, select 100 transformants with good growth and regular morphology for subsequent experiments.

[0100] Transfer the selected transformants one by one to the pre-prepared slant medium. After the mycelia have fully grown on the slant and covered the slant, elute the slant and collect the spores on the mycelia; inoculate the spore suspension into PDB medium, add AFB1 to a final mass concentration of 120 ng / μL, and culture it at 30 °C and 200 rpm / min with constant temperature oscillation for 96 hours; after 96 hours, take samples and dilute the samples, and then detect the concentration of aflatoxin B1.

[0101] 2.11 Detection method for aflatoxin B1 concentration

[0102] In this experiment, the enzyme-linked immunosorbent assay was used to determine the concentration of aflatoxin B1:

[0103] (1) Add 50 μL / well of the standard or the sample to be tested to the wells of the enzyme-linked immunosorbent assay (ELISA) plate.

[0104] (2) Add 50 μL / well of the enzyme-labeled substance and 50 μL / well of the anti-reagent, mix gently, cover with a plate membrane, place in a light-proof drawer, and react at room temperature for 30 min.

[0105] (3) Remove the plate membrane, shake off the liquid in the wells, add 300 μL / well of the washing working solution, wash thoroughly for 30 s; discard the liquid in the wells, repeat the washing four times, and finally pat dry with absorbent paper.

[0106] (4) Add 100 μL / well of the chromogenic solution, cover with a plate membrane, place in a light-proof drawer at room temperature, and react for 15 min.

[0107] (5) Remove the plate membrane, add 50 μL / well of the stop solution, and the color changes from blue to yellow.

[0108] (6) Mix gently and read the absorbance value at 450 nm (reference wavelength 630 nm) using an ELISA reader (Note: The color can remain stable for 10 min).

[0109] 3 Experimental results

[0110] 3.1 Kinetic curve of AFB1 degradation by the starting strain

[0111] The degradation ability of Aspergillus niger strain A327 preserved in the laboratory to aflatoxin B1 in PDB medium was determined. The strain was inoculated into 30 mL of the medium, and then AFB1 was added to a final mass concentration of 120 ng / mL. Each group had three parallels and was cultured at 30 °C and 200 rmp / min. Samples were taken at corresponding times of 24 h, 48 h, 60 h, 72 h, 84 h, and 96 h, and samples to be tested were prepared. The residual amount of aflatoxin B1 at each moment was measured using an ELISA kit, and the degradation rate at each moment was obtained according to the following formula (1):

[0112] I = (I0 - I1) / I0 × 100% (1)

[0113] Where: I is the AFB1 degradation rate; I0 is the AFB1 concentration in the control group; I1 is the AFB1 concentration at each moment, with the unit ng / mL.

[0114] As Figure 1As shown in the figure, it is the situation of strain A327 degrading AFB1 in PDB medium. It is found that Aspergillus niger strain A327 has a strong ability to degrade aflatoxin B1 in PDB medium; after 96 hours of fermentation, the degradation rate reaches 37.19%; therefore, Aspergillus niger A327 is used as the starting strain for experiments, and PDB medium is used as the basic fermentation medium.

[0115] 3.2 Acquisition of target gene

[0116] First, search the literature for the AFB1 degradation enzyme of fungi, download the corresponding amino acid sequence and nucleotide sequence, and use NCBI (CD-search) to determine the conserved domain and the corresponding conserved sequence; use NCBI to search for three sets of Aspergillus niger whole genome sequences, and download all their amino acid and nucleotide sequences; respectively establish local BLAST databases of the amino acid sequences and nucleotide sequences of the three sets of Aspergillus niger, and use the NCBI local BLAST program to compare the conserved sequence with the above-established libraries, and use the similarity between the two sequences as a reference basis to preliminarily determine the candidate genes of AFB1 degradation enzyme in Aspergillus niger.

[0117] Then construct a phylogenetic tree by the neighbor-joining (NJ) method of biological evolutionary distance, including the candidate amino acid sequences of aflatoxin degradation enzymes in Aspergillus niger found above and the amino acid sequences of aflatoxin degradation enzymes in other fungi; use the homology between the candidate amino acid of aflatoxin B1 degradation enzyme in Aspergillus niger and the aflatoxin degradation enzymes in other fungi as a reference basis to further determine the candidate genes. As Figure 2 shown, use MEGA to construct a phylogenetic tree for the determined candidate sequences of aflatoxin degradation enzymes in Aspergillus niger and the aflatoxin degradation enzyme sequences in other fungi. It can be seen from the phylogenetic tree that the candidate amino acid sequence of aflatoxin B1 degradation enzyme derived from Aspergillus niger has the highest homology with the aflatoxin degradation enzyme of Mycena polygramma; in addition, it also has relatively high homology with other aflatoxin degradation enzymes such as Wolfiporia cocos, Laetiporus sulphureus, Trametes versicolor, Trametes punicea, and Lentinula detonsa, and the confidence of each branch is high.

[0118] Finally, the amino acid sequence (EHA20345.1, SEQ ID No. 3) in Aspergillus niger was used as the final candidate sequence for subsequent research. The similarity of this amino acid sequence to the aflatoxin B1 degrading enzyme amino acid sequences from other fungi was about 42%. The similarity to the amino acid sequence of the aflatoxin B1 degrading enzyme (RDX55369.1) from Polyporus brumalis was the highest, at 42.98%. The coding sequence (CDS) of this gene was synthesized, and the nucleotide sequence is shown in SEQ ID No. 2, with a length of 2121 bp.

[0119] SEQ ID No. 2:

[0120] >ACJE01000016.1:

[0121]

[0122] SEQ ID No.3:

[0123] >EHA20345.1 hypothetical proteinASPNIDRAFT_203804,partial

[0124] MDAQVKQHYLADSPPTVVRLEVKSHFDNLTDPKLRKYAHYLSRAAFEGTRITLRQVSPESEPIYDLILELHRACDGNWSELAQKTNVSDEHLRYFLEYATQFLGNCGNYKGFGDSKFIPRLPVEAFQALASATPKTKAAFDLANSTGGGIYETDEQSRMHLGYPEGGHMTTYYPDSPSITKDEITAIGDLMEAKGLPLENTRLKKTESGDFQLLIASGVSSPPVRDRDLGDAEIFELDGKLKGKTLRLVFGDYREEMAKVAHSVKQAGLNAANENQKRMLDAYAMSFGSGSIEAFKESQRIWVKDQKPALETNLGFVETYRDPHGVRGEWEGFVALVNLERTRAFGKLVDSAEAMIPKLPWGKDFEKDKFLSPDFTSLEVLSFQSSGIPAGINLPNYDDIRQNLGFKNVSLGNVLSAKAPNEPVPFIAEKDLEVYRRCRDAAFEVQVGIHELLGHGTGKLLQETAPGEYNFDVSNPPISPVTNKPISSWYKPGQTWGSVFGAMSSSYEECRAECVAMALSCDFSILQLFGFGDGKEDLANEAGDVLFAGYLQMARAGLVALEFWDPKTQKWGQAHMQARYSILRTFLDAGGEFVKLSYTKDDLSDLEIHLDRSKILTHGRPAVEKYLQKLHVYKSTADFEAGKKLYDDITSVDEWWGTKVREIVLKNKIPRKVFVQGNTILNGDEVTLKEYEPTLEGMIQSFVERNV

[0125] 3.3 Expression vector construction

[0126] Next, the genomic DNA of Aspergillus niger A327 was extracted. The results are as Figure 3, detected by agarose gel electrophoresis, the length of the Aspergillus niger genomic DNA obtained by extraction is greater than 15 kb, and the quality is good, which can be used for the next experiment.

[0127] Using the Aspergillus niger genome extracted above as a template and the specifically designed primers prepared in advance, the candidate genes in Aspergillus niger were amplified. The results are as Figure 4 , with a length of 2265 bp, and the size of the amplified band is correct. Then, using the vector containing the coding sequence as a template, the cDNA sequence of the target gene was amplified. The results are as Figure 5 , the size of the amplified band is correct, with a length of 2121 bp, which can be used for constructing an expression vector.

[0128] PCR amplification was used for vector linearization. The vector PUC19 - acs was stored in our laboratory. The vector contains the amds marker gene, the glaA strong promoter, and the TtrpC terminator. The results are as Figure 6 , and the length of the obtained band is 6960 bp, with the correct length, which can be used for constructing an expression vector.

[0129] The obtained target gene and the linearized vector PUC19 - acs above were ligated by seamless cloning technology to obtain the expression vector PUC19 - acs - AFD. The product was transformed into competent Escherichia coli DH5α cells, inoculated on LB solid medium (containing antibiotics), and cultured inverted at 30 °C to screen for positive clones. A single colony with good growth was picked from them and placed in 10 μL of deionized water to make a bacterial suspension. Then, PCR identification was performed on the colony, and the PCR product was analyzed by 1% agarose gel electrophoresis. The total sequence of the second half of the promoter and the target gene was used as the target sequence for amplification. Using QI - z as the 5' - end primer and AN - R as the 3' - end primer, the length is 2734 bp. The results are as Figure 7 , and the length of the obtained band is correct.

[0130] The PUC19 - acs - AFD plasmid with correct colony PCR verification was verified by double digestion with SalⅠ and KpnⅠ. The lengths of the obtained bands are approximately 6.18 Kb, 1.98 Kb, and 0.92 Kb. The results are as Figure 8 , and the band lengths are in line with expectations, proving that the expression vector was constructed correctly.

[0131] 3.4 Screening of Transformed Strains

[0132] 3.4.1 Primary Screening

[0133] The Aspergillus niger A327 strain activated on the PDA medium slant was eluted with deionized water to prepare a spore suspension, which was then inoculated into the S3Y2 medium and cultured on a shaker at 30 °C and 200 rpm / min for 12 - 16 h. Subsequently, the mycelium was filtered and collected to prepare protoplasts; the expression vector PUC19 - acs - AFD was transformed into the recipient Aspergillus niger A327, and it was cultured upside - down at 30 °C for 2 - 4 days in a selective medium with acetamide as the sole nitrogen source. During this period, the growth status of the colonies was continuously observed.

[0134] According to the growth status and morphology of the colonies, 100 transformants were selected. Each transformant was inoculated into a PDA slant medium and a Czapek slant medium respectively; the selected transformants were made into a bacterial suspension and inoculated into 30 mL of PDB medium at an inoculation amount of 1 mL, and then AFB1 was added to a final mass concentration of 120 ng / mL. It was cultured at 30 °C and 200 rpm / min in a constant - temperature shaker for 96 h, and the residual amount of aflatoxin B1 was measured using an enzyme - linked immunosorbent assay kit. The degradation rate was calculated according to formula (1).

[0135] According to the primary - screening data, among the 100 selected transformants, except for two strains, the degradation ability of the other transformants to aflatoxin B1 was higher than that of the starting strain (CK represents the starting strain). Among them, 53% of the transformants had a degradation rate increased by more than 30 percentage points compared with the starting strain, 40% of the transformants had a degradation rate increased by 15 - 30 percentage points compared with the starting strain, and the transformant with the highest measured degradation rate was No. 15, which was nearly twice as high as that of the starting strain.

[0136] 3.4.2 Re - screening

[0137] Eight transformants with relatively high degradation rates, namely No. 5, No. 15, No. 25, No. 41, No. 61, No. 72, No. 74, and No. 78, were selected. The slants of the PDA slant media of the above eight transformants were eluted to make a bacterial suspension, which was gradient - diluted and spread on plates, and cultured upside - down at 30 °C for 2 - 4 days. Five single colonies were picked from each transformant and cultured on a slant. Then AFB1 was added to a final mass concentration of 120 ng / mL and cultured in PDB medium at 30 °C and 200 rpm / min in a constant - temperature shaker for 96 h for re - screening; the residual amount of aflatoxin B1 was measured using an enzyme - linked immunosorbent assay kit, and the degradation rate was calculated through formula (1); the results are shown in Table 5.

[0138] Table 5 Determination of the re - screening degradation rate of the transformants

[0139]

[0140] Based on the above re-screening data, it can be concluded that the results obtained from the re-screened strains are basically consistent with those of the primary screening; most of them can maintain the same degradation level as in the primary screening. When compared with the CK group, the degradation rates of the strains obtained by single colony isolation of the above eight transformants have all increased to varying degrees. Among them, 37.5% of the strains have a degradation rate increase of more than 30 percentage points compared to the starting strain, 45% of the strains have a degradation rate increase of nearly 20 - 30 percentage points compared to the starting strain, and the remaining 17.5% of the strains have a degradation rate increase of more than 10 percentage points. The strain with the highest degradation rate in the re-screening is strain 15 - 1, with a corresponding degradation rate of 75.98%. This result is consistent with the primary screening result, where strain 15 has the highest degradation rate. The resulting genetically engineered bacterium is named A327 - 15.

[0141] Example 2

[0142] 1 Experimental materials

[0143] Agar powder was purchased from Beijing Bio-Atlas Technology Co., Ltd.; yeast powder was purchased from OXOID; D-sorbitol was purchased from Meilunbio; other common reagents such as sucrose, dextrin, maltose, glucose, peptone, ammonium sulfate, zinc sulfate, ammonium chloride, urea, sodium nitrate, manganese chloride, copper chloride, calcium chloride, magnesium chloride, barium chloride, and disodium hydrogen phosphate were all domestic analytical pure reagents.

[0144] 2 Experimental methods and results

[0145] In this experiment, the carbon source, nitrogen source, metal ions, and their addition amounts in the basic fermentation medium of the Aspergillus niger genetically engineered bacterium A327 - 15 were optimized to explore the effects of different medium components and addition amounts on the degradation of aflatoxin B1 by Aspergillus niger A327 - 15. After selecting the optimal medium components, the addition amounts of the optimal components were determined, so as to determine the optimal fermentation medium for Aspergillus niger to degrade aflatoxin B1; then, the fermentation conditions such as temperature, inoculum size, liquid volume, rotation speed, and initial pH were optimized; finally, the optimal medium components, addition amounts, and optimal fermentation conditions for Aspergillus niger A327 - 15 to degrade aflatoxin B1 were determined, and the specific situation of the degradation of different concentrations of aflatoxin B1 by the strain after fermentation process optimization was studied. Finally, the degradation of aflatoxin B1 by the strain after fermentation process optimization over time was studied; the results are as follows:

[0146] (1) Through single-factor optimization experiments, the optimal medium components were first explored, and then the optimal addition amounts were explored based on this; the results were: the optimal carbon source is sucrose at 19 g / L, the optimal nitrogen source is ammonium chloride at 10 g / L, and the optimal metal ion is potassium chloride at 1.75 g / L.

[0147] (2) Based on the single-factor experiments, a three-factor and three-level response surface Box-Behnken Design experiment was designed. After fermenting the 17 groups of experiments generated by the system, the corresponding degradation rates were measured. The experimental results were regressively fitted to obtain the regression equation Y = 79.59 - 0.3347A + 3.14B + 1.35C + 0.3012AB + 0.3564AC + 2.30BC - 11.30A 2 - 5.33B 2 - 9.82C 2 , this equation shows the relationship between the degradation rate of aflatoxin B1 by Aspergillus niger A327-15 and the three selected factors; for this model, the regression model is extremely significant (P < 0.0001), the model determination coefficient is 0.9796, the adjusted determination coefficient is 0.9533, and the lack-of-fit term is not significant P = 0.7635 (P > 0.05), indicating a high degree of model fitting and relatively high equation reliability; this model can be used for experimental analysis. Using Design-Expert 13 for analysis and prediction, the optimal medium composition and addition amounts are obtained: sucrose 18.98 g / L, ammonium chloride 10.79 g / L, potassium chloride 1.86 g / L, and the predicted degradation rate is 80.16%; fermentative verification of the optimal medium formulation shows that the 84-hour degradation rate of aflatoxin B1 by Aspergillus niger A327-15 is 80.294 ± 0.789%, which is consistent with the predicted value of 80.16%, indicating a relatively high credibility of the prediction results. Compared with the degradation rate of 75.09% at 96 hours before the optimization of the medium composition, the degradation rate is increased to 80.29% at 84 hours, and the degradation time of Aspergillus niger for degrading the same mass concentration of aflatoxin B1 is shortened by 12 hours, and the degradation rate is greatly improved.

[0148] (3) On the basis of optimizing the medium composition and addition amounts, the fermentation conditions of the recombinant Aspergillus niger strain A327-15 were studied, and the optimal fermentation conditions for this strain to degrade aflatoxin B1 were determined: temperature 32 °C, initial pH 6.5, rotation speed 180 rmp / min, inoculation amount 1.5 mL, and liquid loading amount 30 mL; under these conditions, the degradation rate can reach 97.84% after 84 hours of fermentation, which is 17.55% higher than the degradation rate after the optimization of the medium composition and addition amounts.

[0149] Example 3

[0150] Effect of AFB1 mass concentration on the degradation of AFB1 by Aspergillus niger A327-15

[0151] To explore the degradation ability of Aspergillus niger strain A327-15 against high concentrations of aflatoxin B1, in this experiment, aflatoxin B1 with mass concentrations of 120, 240, 480, 720, 960, and 1200 ng / mL was sequentially set. With an inoculation volume of 1.5 mL, it was inoculated into 30 mL of optimized fermentation medium. The pH was natural, and after fermentation at 32 °C and 180 rmp / min for 84 h, the residual amount of aflatoxin B1 was measured, and the degradation rate was calculated using formula (1).

[0152] As Figure 9 shown, when the mass concentration of AFB1 was 120 ng / mL, the highest degradation rate was 97.25%; after the concentration increased, when the mass concentration of AFB1 was 240 - 720 ng / mL, the degradation rate decreased slightly, but the degradation rate remained above 93%; when the AFB1 concentration increased to 960 ng / mL, the degradation rate was 92.19%; when the mass concentration of AFB1 was 1200 ng / mL, the degradation rate was 83.60%; although the degradation rate decreased slightly during the gradual increase of the AFB1 concentration, the overall fluctuation was very small, and the degradation ability of Aspergillus niger strain A327-15 against AFB1 still remained at a high level; from the aspect of the total amount of AFB1 degraded by Aspergillus niger strain A327-15, with the increase in the mass concentration of AFB1 in the medium, the total amount of AFB1 degraded by Aspergillus niger A327-15 was also in a stable upward trend. When the mass concentration of AFB1 was 1200 ng / mL, the total amount of AFB1 degraded was 30.09 μg. In summary, it shows that Aspergillus niger A327-15 also has good degradation ability against high concentrations of AFB1, and Aspergillus niger strain A327-15 has great application potential in the detoxification of severely aflatoxin B1-polluted situations.

[0153] Example 4

[0154] Power curve of Aspergillus niger A327-15 degrading AFB1 after optimizing the fermentation process

[0155] After optimizing the medium components, addition amounts, and culture conditions, the relationship between the degradation characteristics of Aspergillus niger A327-15 against aflatoxin B1 and time was further studied. In this experiment, with an inoculation volume of 1.5 mL, it was inoculated into 30 mL of optimized fermentation medium, and aflatoxin B1 was added to a final mass concentration of 120 ng / mL. The pH was natural, and fermentation was carried out at 32 °C and 180 rmp / min. Sampling was carried out at 24 h, 48 h, 72 h, 78 h, 84 h, 90 h, 96 h, 102 h, and 114 h in sequence, the residual amount of aflatoxin B1 was measured, and the degradation rate was calculated using formula (1).

[0156] As Figure 10As shown, with the passage of time, the degradation rate of AFB1 by Aspergillus niger A327-15 gradually increases. Within 24 hours of the start of fermentation, since Aspergillus niger is still in the growth and reproduction stage, the degradation rate is relatively low; during the fermentation process from 24h to 78h, the degradation rate increases significantly, rising from 2.68% to 92.93%. Within these 54 hours, the aflatoxin B1 degrading enzyme degrades nearly 90% of the AFB1 in the culture medium; during the fermentation process from 78h to 84h, the degradation amplitude slows down, and the degradation rate reaches 97.84% at 84h; after 90h, the degradation rate of AFB1 reaches 100%.

[0157] In summary, in the study of aflatoxin B1 at different mass concentrations, it was found that at concentrations of 120 ng / mL - 960 ng / mL, a degradation rate of about 93% can be maintained, and at a concentration of 1200 ng / mL, a degradation rate of 83.60% can also be maintained, indicating that the Aspergillus niger strain A327-15 also has good degradation ability for high-concentration AFB1.

[0158] Finally, after the optimization of the culture medium, the degradation situation of aflatoxin B1 by recombinant Aspergillus niger A327-15 under the optimized culture medium components, addition amounts, and fermentation conditions was studied. The results showed that the degradation rate of aflatoxin B1 gradually increased with the passage of time. During the period from 24h to 78h, the degradation rate increased significantly, rising from 2.68% to 92.93%. Within these 54 hours, the aflatoxin B1 degrading enzyme degraded nearly 90% of the AFB1 in the culture medium. During the period from 78h to 84h, the degradation amplitude slowed down, and the degradation rate reached 97.84% at 84h; after 90h, the degradation rate of aflatoxin B1 by the aflatoxin B1 degrading enzyme in the culture medium reached 100%.

[0159] The embodiments described above are only used to describe the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene for degrading aflatoxin B1, characterized in that, Its nucleotide sequence is shown in SEQ ID No.

1.

2. An aflatoxin degrading enzyme, characterized in that, Encoded by the gene described in claim 1.

3. A recombinant vector, characterized in that, Containing the gene for degrading aflatoxin B1 described in claim 1.

4. A genetically engineered bacterium, characterized in that, Containing the gene for degrading aflatoxin B1 described in claim 1.

5. The genetically engineered bacterium according to claim 4, wherein The starting strain of the genetically engineered bacterium is Aspergillus niger A327.

6. Use of the gene for degrading aflatoxin B1 described in claim 1, the aflatoxin degrading enzyme described in claim 2, or the recombinant vector described in claim 3 in degrading aflatoxin B1.

7. Use of the genetically engineered bacterium described in claim 4 or 5 in degrading aflatoxin B1.

8. A method for degrading aflatoxin B1, characterized in that, Comprising: Inoculating the genetically engineered bacterium described in claim 4 or 5 into a contaminated substrate containing aflatoxin B1 and culturing.

9. The method according to claim 8, wherein The volume ratio of the inoculation is 5%; the culturing conditions are: temperature 32 °C, pH 6.5, rotation speed 180 rmp / min, and culturing time 84 - 90 h.

10. The method according to claim 1, characterized in that, The mass concentration of aflatoxin B1 in the contaminated substrate containing aflatoxin B1 is 120 - 960 ng / mL.

Images