Aflatoxin B1 degrading enzyme and application thereof

Abstract

The invention discloses an aflatoxin B1 degrading enzyme and application thereof. The degrading enzyme comprises lignin peroxidase, dye decoloring peroxidase and multifunctional peroxidase. According to the invention, an efficient and targeted aflatoxin B1 degrading enzyme screening model is established by adopting a molecular simulation technology, and the problems of high complexity, low screening efficiency and the like commonly existing in a traditional screening method are solved. The engineering strain provided by the invention takes escherichia coli Shuffle T7-B as a host, has a stable degrading enzyme expression characteristic, and ensures the efficient expression of the degrading enzyme. The degradation rate of the degrading enzyme provided by the invention on aflatoxin B1 reaches 84% or above.

Description

(1) Technical field

[0001] The present invention relates to four novel aflatoxin B1 degrading enzymes and applications thereof. (2) Background technology

[0002] Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus, is a significant component of naturally occurring food contaminants, widely contaminating crops such as peanuts, corn, and rice, as well as their products. Aflatoxin B1 is hepatotoxic, nephrotoxic, carcinogenic, and teratogenic, and is classified as a Class I carcinogen by the International Agency for Research on Cancer (IARC). Direct ingestion of contaminated food or indirect ingestion of animal products derived from contaminated feed can lead to liver cancer and other diseases. Strategies for removing aflatoxin primarily include physical, chemical, and biological approaches. While simple to perform, physical methods (such as adsorption and irradiation) do not completely remove the toxin and can affect the nutritional value and sensory quality of food under heating conditions. Chemical methods (such as hydrolysis or redox reactions) can effectively degrade aflatoxin, but they can also result in chemical residues and secondary contamination. In contrast, biological methods (microbial strains and their metabolites) have become a research hotspot in recent years due to their mild reaction conditions, high specificity, high efficiency, and lack of secondary contamination.

[0003] Among biological methods, the method of using microbial enzymes to degrade aflatoxin B1 is considered to be one of the most promising strategies. The molecular structure of aflatoxin B1 has two important active structures with chemical reaction and toxicity characteristics: the C8=C9 double bond structure at the end of the furan ring and the lactone ring structure. The C8=C9 double bond is easily oxidized into AFB1-8,9-epoxide with strong carcinogenic activity, while the lactone ring provides a stable structural feature for aflatoxin B1 and is also a key site for toxicity. Therefore, the degradation mechanism for aflatoxin B1 is to achieve the detoxification effect by opening the C8=C9 double bond or destroying the lactone ring structure. The aflatoxin B1 degrading enzymes currently reported mainly include laccase, peroxidase, F 420-H2-dependent reductases and lactonases. For example, laccase isolated from Pleurotus eryngii hydrolyzes aflatoxin B1 to AFB1-8,9-dihydrodiol by acting on the C8=C9 double bond (Loi M, De Leonardis S, Ciasca B et al., Aflatoxin B1 degradation by Ery4laccase: from in vitro to contaminated corn. Toxins (Basel), 2023). Dye-decolorizing peroxidase from Bacillus subtilis also detoxifies by oxidizing the C8=C9 double bond (Qin X, Su XY, Tu T et al., Enzymatic degradation of multiple major mycotoxins by dye-decolorizing peroxidase from Bacillus subtilis. Toxins, 2021). Laccase isolated from Weizmannia coagulans forms hydrogen bonds and hydrophobic interactions with aflatoxin B1 through asparagine (Asn) and threonine (Thr), and produces aflatoxin Q1 with relatively weak toxicity through C3 hydroxylation (Hao WB, Gu X, Yu X et al., Laccase Lac-W detoxifies aflatoxin B(1)and degrades five other major mycotoxins in the absence of redox mediators. Environmental Pollution, 2023). N-acyl homoserine lactonease isolated from multiple strains of Bacillus detoxifies by acting on the lactone ring of aflatoxin B1, destroying the lactone ring structure (Gonzalez Pereyra ML, Martinez MP, Cavaglieri LR. Presence of aiiA homologue genes encoding for N-Acyl homoserine lactone-degrading enzyme in aflatoxin B(1)-decontaminating Bacillus strains with potential use as feed additives. Food and Chemical Toxicology, 2019).

[0004] While the aforementioned degradative enzymes screened and purified from natural strains demonstrate degradation activity, they suffer from drawbacks such as a wide screening range, cumbersome extraction and purification procedures, and low enzyme yields and activity. Furthermore, some recombinantly expressed enzymes may suffer from low degradation efficiency and instability. For example, patent application CN118256459A, "A Recombinant Enzyme and Its Use in Degrading Aflatoxin B1," discloses a heterologously expressed recombinant multi-copper oxidase, which achieves an optimal degradation rate of only approximately 60%.

[0005] Therefore, it is necessary to find an efficient and targeted screening method for aflatoxin B1 degrading enzymes, explore new aflatoxin B1 degrading enzymes, and construct an efficient expression system to obtain stable new degrading enzymes. (3) Summary of the invention

[0006] The present invention aims to provide a novel aflatoxin B1 degrading enzyme and its application, which solves the problems of low screening efficiency, complicated extraction steps, low enzyme yield and low activity of the degrading enzyme in the prior art.

[0007] The technical solution adopted in the present invention is:

[0008] In a first aspect, the present invention provides an aflatoxin B1 degrading enzyme, wherein the degrading enzyme comprises lignin peroxidase, dye decolorizing peroxidase or multifunctional peroxidase.

[0009] Furthermore, the degrading enzyme is one of the following: a lignin peroxidase (ApeLiP) derived from Agrocybe pediades, the amino acid sequence of which is shown in SEQ ID NO.1; a dye decolorizing peroxidase (KpDyP) derived from Klebsiella pneumoniae, the amino acid sequence of which is shown in SEQ ID NO.2; a multifunctional peroxidase (VPL2) derived from Pleurotus eryngii, the amino acid sequence of which is shown in SEQ ID NO.3; and a lignin peroxidase (TrcLip) derived from Trametopsis cervina, the amino acid sequence of which is shown in SEQ ID NO.4.

[0010] In a second aspect, the present invention provides a method for screening the aflatoxin B1 degrading enzyme, comprising the following steps:

[0011] (1) Select 4-6 known aflatoxin B1-degrading enzymes as screening templates and obtain their 3D structures from the RCSB database (https: / / www.rcsb.org / pdb);

[0012] (2) The degradation enzyme in step (1) was semi-flexibly docked with aflatoxin B1 using AutoDock Vina software to analyze the key amino acid residues in the degradation enzyme that interact with aflatoxin B1, and the regional sequence with more binding sites (about 10–15 amino acids in length) was selected as the target sequence;

[0013] (3) Use the BLAST tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) to perform homology screening in the protein database (pdb database), screen out homologous proteins with high similarity, record the protein sequence, functional annotation and species origin as candidate proteins;

[0014] (4) Molecular docking of the screened candidate proteins with aflatoxin B1 was performed to preliminarily screen candidate enzymes with docking binding energies < -9 kcal / mol;

[0015] (5) The bonding patterns and active amino acid residues of the candidate enzymes were analyzed, focusing mainly on the sites acting on the furan ring and lactone ring of the AFB1 toxic group, and the similarity between the active site sequence and the target sequence was analyzed; finally, proteins with a binding energy lower than -10 kcal / mol and the highest similarity between the active site sequence and the target sequence were selected as AFB1 degrading enzymes.

[0016] In a third aspect, the present invention provides a gene encoding an aflatoxin B1 degrading enzyme. The nucleotide sequences of the gene after optimization of Escherichia coli codon preference and addition of a HIS tag are shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively.

[0017] In a fourth aspect, the present invention provides a recombinant plasmid expressing aflatoxin B1 degrading enzyme, wherein the recombinant plasmid is based on pET-22b(+).

[0018] In a fifth aspect, the present invention provides a recombinant genetically engineered bacterium containing a gene encoding an aflatoxin B1 degrading enzyme, wherein the recombinant genetically engineered bacterium is constructed using Escherichia coli Shuffle T7-B as a host bacterium.

[0019] In a sixth aspect, the present invention provides a use of the aflatoxin B1 degrading enzyme in degrading aflatoxin B1.

[0020] Furthermore, the application is as follows: using aflatoxin B1 as a substrate, using pure enzyme liquid extracted by ultrasonic crushing of wet bacteria after induction culture of recombinant genetically engineered bacteria containing the gene encoding aflatoxin B1 degrading enzyme as a catalyst, and using a pH 4.0-11.0 buffer as a reaction medium to form a reaction system, reacting at 25-60°C in the dark for 0.5-72h (preferably 37°C for 24h) to achieve degradation of aflatoxin B1.

[0021] Furthermore, in the reaction system, the substrate is added to a final concentration of 0.1-10 μg / mL (preferably 1 μg / mL); the amount of pure enzyme solution added is 0.4-0.8 mg / mL (preferably 0.8 mg / mL) based on protein content.

[0022] Furthermore, the buffer solution is a phosphate buffer solution with a pH of 8.0-9.0.

[0023] Furthermore, the reaction system also contains 0.05-0.5M metal ions (preferably 0.1M); the metal ions include Mg 2+ , Ca 2+ , Zn 2+ , Li + , Mn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , K + , preferably K + .

[0024] Furthermore, the pure enzyme solution is prepared as follows:

[0025] (1) Wet bacteria

[0026] The recombinant genetically engineered bacteria containing the gene encoding the aflatoxin B1 degrading enzyme were evenly spread on LB solid medium containing 100 mg / mL ampicillin (AMP) and cultured at 37°C overnight; the colonies were inoculated into LB liquid medium containing 100 mg / mL ampicillin and cultured at 37°C and 250 rpm overnight; the overnight cultured bacterial liquid was inoculated into LB liquid medium containing 100 mg / mL ampicillin at a volume concentration of 1% (v / v), and cultured at 37°C and 250 rpm overnight. When the OD value reached 0.5-0.6, the inducer IPTG was added to a final concentration of 0.2 mM, and expression was induced at 16°C for 24 hours. The wet bacteria were centrifuged at 8000 rpm at 4°C for 20 minutes, and the wet bacteria were collected;

[0027] (2) Crude enzyme solution

[0028] Wet cells were resuspended in 5-10 mL of binding buffer per gram of wet cells. Lysozyme (0.2 mg / mL), DNase (20 μg / mL), 1 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added, and the mixture was mixed at 4°C for 30 min. Cells were disrupted using an ultrasonic cell disruptor at a power of 300 W for 20 min, with a sonication time of 3 s and a pause of 5 s. The lysate was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was obtained as the crude enzyme solution.

[0029] (3) Pure enzyme solution

[0030] A Ni-IDA pre-packed gravity column was used to naturally drain the liquid in the gravity column. The column was balanced with 2 volumes of Binding Buffer and slowly drained. 2 column volumes of crude enzyme solution from step (2) were added, and the crude enzyme solution was stored for 3–5 minutes and then slowly drained. The crude enzyme solution from step (2) was continued to be added until it was added. The total loading volume of crude enzyme solution was 9–11 column volumes. The column was rinsed twice with 2 column volumes of Binding Buffer. 2 volumes of elution buffer were added, and the elution buffer was stored for 8–10 minutes, and then slowly drained. The elution buffer was retained and repeated until all the protein was eluted. The elution buffer containing the target protein was collected as the pure enzyme solution. Binding Buffer composition: 20 mM NaH2PO4·2H2O, 20 mM Na2HPO4·7H2O, 0.5 M NaCl, 10 mM imidazole; Elution Buffer composition: 20 mM NaH2PO4·2H2O, 20 mM Na2HPO4·7H2O, 0.5M NaCl, 250mM imidazole.

[0031] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0032] (1) The present invention uses molecular simulation technology and homology comparison method to establish an efficient and targeted aflatoxin B1 degrading enzyme screening model, which solves the problems of high complexity and low screening efficiency commonly found in traditional screening methods.

[0033] (2) The engineered strain provided by the present invention uses Escherichia coli Shuffle T7-B as a host, has stable degradation enzyme expression characteristics, ensures efficient expression of the degradation enzyme, and increases the yield of the degradation enzyme.

[0034] (3) The degradation enzyme provided by the present invention exhibits excellent degradation activity against aflatoxin B1, with a degradation rate of more than 84%. Under the optimized conditions (40°C, pH, addition of 0.1MK +) reached the optimal degradation rates of 90.06%, 91.55%, 85.09% and 84.33% respectively.

[0035] (4) The novel aflatoxin B1 degrading enzyme provided by the present invention has strong alkali resistance (pH 9.0-11.0), among which the lignin peroxidase of Trametopsis cervina can exert more than 70% degradation activity within 0.5 h, and has the characteristics of high degradation efficiency and strong degradation activity. (IV) Description of the accompanying drawings

[0036] Figure 1 , flow chart of targeted screening of aflatoxin B1-degrading enzymes.

[0037] Figure 2 , Agarose nucleic acid electrophoresis diagram of the recombinant plasmid in different expression strains, A is Shuffle T7-B, B is BL21, and C is Origami B (DE3).

[0038] Figure 3 , SDS-PAGE images of crude enzyme and purified degradation enzyme in different expression systems, A is Shuffle T7-B, B is BL21, and C is Origami B (DE3).

[0039] Figure 4 , High-performance liquid chromatograms after aflatoxin B1 was degraded by different degrading enzymes. The arrow indicates the peak of aflatoxin B1.

[0040] Figure 5 , bar graph of the degradation rate of aflatoxin B1 by homologous proteins; 1 is ScDyp; 2 is MsDyp; 3 is SmPgd; 4 is ElDyp; 5 is KpDyP; 6 is MmFod; 7 is TrcLip; 8 is TtcLr; 9 is ApeLiP; 10 is Fbp1; 11 is VPL2; 12 is SvRmo.

[0041] Figure 6 、 Four Bar graph showing the degradation rates of aflatoxin B1 by various aflatoxin B1-degrading enzymes at different temperatures; A is ApeLip; B is KpDyp; C is TrcLip; and D is VPL2.

[0042] Figure 7 、 Four Bar graph showing the degradation rates of aflatoxin B1 by various aflatoxin B1-degrading enzymes at different pH values; A is ApeLip; B is KpDyp; C is TrcLip; and D is VPL2.

[0043] Figure 8 、 FourBar graph of the degradation rates of aflatoxin B1 by various aflatoxin B1-degrading enzymes under the addition of different metal ions; A is ApeLip; B is KpDyp; C is TrcLip; D is VPL2.

[0044] Figure 9 、 Four The degradation rate curve of aflatoxin B1 by different aflatoxin B1 degrading enzymes at different incubation times. (V) Specific implementation methods

[0045] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified; the materials and reagents used are obtained from commercial sources unless otherwise specified.

[0047] The culture medium and buffer were prepared as follows:

[0048] LB liquid medium: Dissolve 10.0 g of tryptone, 5.0 g of yeast extract powder, and 10.0 g of sodium chloride in 1000 mL of distilled water and sterilize by autoclaving at 121°C for 15 min.

[0049] LB solid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, and 15 g of agar were dissolved in 1000 mL of distilled water and sterilized by autoclaving at 121°C for 15 min.

[0050] Binding Buffer: 20mM NaH2PO4·2H2O, 20mM Na2HPO4·7H2O, 0.5M NaCl, 10mM imidazole.

[0051] Elution Buffer: 20mM NaH2PO4·2H2O, 20mM Na2HPO4·7H2O, 0.5M NaCl, 250mM imidazole.

[0052] Phosphate buffer: 20 mM NaH2PO4·2H2O, 20 mM Na2HPO4·7H2O, 0.5 M NaCl, pH 8.5.

[0053] Example 1: Efficient screening of aflatoxin B1 degrading enzymes based on molecular docking technology

[0054] like Figure 1 As shown, the steps for efficiently screening aflatoxin B1 degrading enzymes are as follows:

[0055] (1) Six known aflatoxin B1-degrading enzymes (Table 1) were selected as screening templates, and their 3D structures were obtained from the RCSB database (https: / / www.rcsb.org / pdb).

[0056] (2) The degradation enzyme was semi-flexibly docked with aflatoxin B1 using AutoDock Vina software. The key amino acid residues in the degradation enzyme that interacted with aflatoxin B1 were analyzed, and the regional sequence with more binding sites (about 10-15 amino acids in length) was selected as the target sequence (Table 1).

[0057] (3) Homology screening was performed in the protein database (pdb database) using the BLAST tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and homologous proteins with high similarity were screened out. The protein sequence, functional annotation, and species origin were recorded as candidate proteins.

[0058] Table 1 Known aflatoxin B1 degrading enzymes and the targeted sequences obtained by screening

[0059]

[0060]

[0061] (4) The selected candidate proteins were molecularly docked with aflatoxin B1. Based on the docking binding energy, the following 12 candidate enzymes with excellent docking binding energy (<-9 kcal / mol) were preliminarily selected:

[0062] 1. Dye-decolorizing peroxidase from Streptomyces coelicolor (ScDyp, PDB id: 4gu7); 2. Dye-decolorizing peroxidase from Mycobacterium smegmatis (MsDyp, PDB id: 7bok); 3. Glycerol dehydrogenase from Sinorhizobium meliloti (SmPgd, PDB id: 3uhj); 4. Dye-decolorizing peroxidase from Enterobacter lignolyticus (ElDyp, PDB id: 5vj0); 5. Dye-decolorizing peroxidase from Klebsiella pneumoniae (KpDyP, PDB id: 6fl2); 6. Iron-oxidizing protein oxidoreductase from Magnetococcus marinus (MmFod, PDB id: id: 6n2n); 7. Lignin peroxidase from Trametopsis cervina (TrcLip, PDB id: 3q3u); 8. Lactate racemase from Thermoanaerobacterium thermosaccharolyticum (TtcLr, PDB id: 4a5g); 9. Lignin peroxidase from Agrocybe pediades (ApeLiP, PDB id: 7oo5); 10. Fructose-1,6-bisphosphatase from Yeast (Fbp1, PDB id: 7ns5); 11. Multifunctional peroxidase from Pleurotus eryngii (VPL2, PDB id: 3fjw); 12. Lactate peroxidase from Streptomyces venezuelae (Lysobacterium spp., PDB id: 3fjw); venezuelae) monooxygenase (SvRmo, PDB id: 5vqb).

[0063] (5) The bonding patterns and active amino acid residues of these 12 candidate enzymes were analyzed, focusing on the sites acting on the furan ring and lactone ring of the AFB1 toxic group, and analyzing the similarity between the active site sequence and the target sequence.

[0064] Finally, the following four proteins with binding energies lower than -10 kcal / mol and active site sequences with the highest similarity to the target site sequences were screened out as AFB1 degrading enzymes: lignin peroxidase (ApeLiP), dye decolorizing peroxidase (KpDyP), multifunctional peroxidase (VPL2) and lignin peroxidase (TrcLip). Although the remaining AFB1 degrading enzymes also had binding energies lower than -8 kcal / mol, they did not have active sites consistent with the target sequence.

[0065] Example 2: Heterologous expression of AFB1 degrading enzyme

[0066] 1. Construction of recombinant plasmid

[0067] Based on the four best AFB1 degrading enzymes screened in Example 1, they were further heterologously expressed. The signal peptide and transmembrane region of the AFB1 degrading enzyme sequence were analyzed, and the coding codons of the AFB1 degrading enzyme were optimized according to the codon preference of Escherichia coli. A His tag was added. The optimized amino acid sequence of lignin peroxidase (ApeLiP) is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the amino acid sequence of dye decolorizing peroxidase (KpDyP) is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; the amino acid sequence of multifunctional peroxidase (VPL2) is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; the amino acid sequence of lignin peroxidase (TrcLip) is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.8.

[0068] SEQ ID NO.1

[0069] MAVTCPTGQTTANEACCVLFPVIDLLQEELFDGECGEEAHAALRLAFHDAIGFSKNGGKGGG

[0070] ADGSILAFHQTETTYAANSGIEDIITAQLPIFQKTNLTAGDFVHLAAAIGTGNCPGSPQLAYSFGR

[0071] PPPVAPAPDGTVPEPTDSVTDILARFSEAGFVTAEVIWLLASHSIAAASKIDTSAPRTPFDSTPALF

[0072] DTQFYLETILNGTLLPGDGGAHTGEVLSPIAGEMRLQSDFAFAQDPRTACLWQEPINDQAFIQG

[0073] KFFAAMKKLQVLGQTGLTDCSDVIPVPASLGPITFPAGFSEADVISACTATPLPSLATIAGKPTI

[0074] PPVPLHHHHHH

[0075] SEQ ID NO.2

[0076] MSQVQSGILPEHCRAAIWIEANLKGDVNALREASKIFVDNVATFQAKFPDAKLGAVVAFGNNV

[0077] WRQLSGGEGADELKDFPVYGGGLAPSTQYDLLIHILSARHEVNFSVAQAALAAFGDAIDVKEEI

[0078] HGFRWVEERDLSGFVAGTENPAGEETRREVAVIKDGVDAGGSYVFVQRWEHNLKQLNRMSV

[0079] PDQEMMIGRTKDANEEIDGDERPVTSHLSRVDLKEDGKGLKIVRQSLPYGTASGTHGLYFCAY

[0080] CARLYNIEQQLLSMFGDTDGKRDAMLRFTKPVTGGYYFAPSLERIQALGPLGMSQVQSGILPEH

[0081] CRAAIWIEANLKGDVNALREASKIFVDNVATFQAKFPDAKLGAVVAFGNNVWRQLSGGEGAD

[0082] ELKDFPVYGGGLAPSTQYDLLIHILSARHEVNFSVAQAALAAFGDAIDVKEEIHGFRWVEERDL

[0083] SGFVAGTENPAGEETRREVAVIKDGVDAGGSYVFVQRWEHNLKQLNRMSVPDQEMMIGRTK

[0084] DANEEIDGDERPVTSHLSRVDLKEDGKGLKIVRQSLPYGTASGTHGLYFCAYCARLYNIEQQLL

[0085] SMFGDTDGKRDAMLRFTKPVTGGYYFAPSLERIQALGHHHHHH

[0086] SEQ ID NO.3

[0087] MAVPLVQKRATCDDGRTTANAACCILFPILDDIQENLFDGAQCGEEVHESLRLTFHDAIGFSPTL

[0088] GGGGADGSIIAFDTIETNFPANAGIDEIVSAQKPFVAKHNISAGDFIQFAGAVGVSNCPGGVRIPF

[0089] FLGRPDAVAASPDHLVPEPFDSVDSILARMGDAGFSPVEVVWLLASHSIAAADKVDPSIPGTPF

[0090] DSTPGVFDSQFFIETQLKGRLFPGTADNKGEAQSPLQGEIRLQSDHLLARDPQTACEWQSMVNN

[0091] QPKIQNRFAATMSKMALLGQDKTKLIDCSDVIPTPPALVGAAHLPAGFSLSDVEQACAATPFPA

[0092] LTADPGPVTSVPPVPGSHHHHHH

[0093] SEQ ID NO.4

[0094] MVPSPLVSCGGGRSVKNAACCAWFPVLDDIQANLFNGGKCEEEAHEAVRLTFHDAVGFSLAA

[0095] QKAGKFGGGGADGSILAFSDIETAFIPNFGLEFTTEGFIPFALAHGVSFGDFVQFAGAVGAANCA

[0096] GGPRLQFLAGRSNISQPSPDGLVPDPTDSADKILARMADIGFSPTEVVHLLASHSIAAQYEVDTD

[0097] VAGSPFDSTPSVFDTQFFVESLLHGTQFTGSGQGGEVMSPIPGEFRLQSDFALSRDPRTACEWQA

[0098] LVNNQQAMVNNFEAVMSRLAVIGQIPSELVDCSDVIPTPPLAKVAQVGSLPPGKSMADVQVAC

[0099] TNGMPFPSLPTSPGPVQTVAPVLGHHHHHH

[0100] SEQ ID NO.5

[0101] catatggcagtgacctgtccgaccggccagaccaccgccaatgaagcctgttgcgttctgtttccggttattgatctgctgcaggaa

[0102] gaactgtttgatggtggcgaatgtggtgaagaagcccatgcagccctgcgtctggcatttcatgatgcaattggctttagcaaaaatg

[0103] gcggtaaaggcggcggtgcagatggcagcattctggcctttcatcagaccgaaaccacctatgccgccaatagtggtattgaagat

[0104] attattaccgcccagctgccgatttttcagaaaaccaatctgaccgccggcgattttgttcatctggccgcagcaattggtaccggca

[0105] attgtccgggcagtccgcagctggcatatagctttggtcgtccgccgccggttgcaccggcacctgatggtaccgttccggaaccg

[0106] accgatagtgtgaccgatattctggcacgttttagcgaagcaggctttgtgaccgcagaagttatttggctgctggccagccatagca

[0107] ttgcagccgcaagcaaaattgataccagcgcaccgcgcaccccgtttgatagcaccccggcactgtttgatacccagttttatctgg

[0108] aaaccattctgaatggtaccctgctgccgggcgatggcggtgcacataccggtgaagttctgagcccgattgcaggtgaaatgcgc

[0109] ctgcagagcgattttgcctttgcacaggaccctcgcaccgcatgcctgtggcaggaaccgattaatgatcaggcattcattcagggt

[0110] aaatttttcgccgccatgaaaaaactgcaggtgctgggtcagaccggtctgaccgattgcagcgatgtgattccggtgccggcaag

[0111] tctgccgggtccgattacctttccggccggttttagcgaagccgatgtgattagtgcatgtaccgccaccccgctgccgagtctggc

[0112] cacaattgcaggtccgaaaccgaccattccgccggtgccgctgcaccatcaccatcaccattaactcgag

[0113] SEQ ID NO.6

[0114] catatgagtcaggttcagagcggtattctgccggaacattgtcgcgcagcaatttggattgaagccaatctgaaaggcgatgtgaat

[0115] gccctgcgtgaagcaagcaaaatttttgttgataatgtggcaaccttccaggccaaatttccggatgcaaaactgggcgccgtggtt

[0116] gcatttggcaataatgtttggcgccagctgagcggtggcgaaggcgctgaatgaactgaaagattttccggtttatggtaaaggtctt

[0117] gccccgagcacccagtatgatctgctgattcatattctgagtgcacgccatgaagtgaattttagcgttgcacaggcagccctggca

[0118] gcctttggtgacgccattgatgtgaaaagaagaaattcatggtttccgctgggttgaagaacgcgatctgagcggctttgttgcaggca

[0119] ccgaaaatccggcaggtgaagaaacccgtcgcgaagtggcagttattaaggatggtgtggatgcaggtggcagttatgtttttgtgc

[0120] agcgttgggaacataatctgaaacagctgaatcgtatgagcgttccggatcaggaaatgatgattggtcgcaccaaagatgcaaat

[0121] gaagaattgatggtgacgaacgcccggtgaccagtcatctgagccgtgtggatctgaaaagaatggtaaaggtttaaaaatcgt

[0122] gcgtcagagtctgccgtatggcaccgcaagtggtacccacggtctgtatttttgcgcctattgcgcacgcctgtataatattgaacag

[0123] cagctgctgagcatgtttggtgacaccgatggcaaacgtgatgccatgctgcgctttaccaaaccggttaccggtggttattatttttgc

[0124] cccgagcctggaacgtattcaggcactgggtccgctgggtatgagccaggttcagagtggtattctgcctgaacattgccgtgccg

[0125] ccatttggattgaggcaaatctgaaaggtgacgttaatgcactgcgtgaagccagtaaaatttttgtggataatgtggcgacctttcag

[0126] gcaaaatttccggacgccaaactgggcgcagttgttgcatttggtaataatgtgtggcgtcagctgagcggcggtgaaggcgccga

[0127] tgaactgaaggattttccggtgtatggcaaaggtctggcgccgagcacccaatatgatctgttaattcatattttaagcgcccgccatg

[0128] aagttaattttagcgtggcccaggcagccttagccgcctttggtgacgcgattgatgttaaagaagaaatccacggttttcgctgggt

[0129] ggaagaacgtgatctgagtggttttgtggcaggtaccgaaaatcctgccggtgaagaaacacgtcgcgaagttgccgttattaagg

[0130] acggtgttgatgccggtggcagctatgtttttgttcagcgttgggagcataatctgaagcagctgaatcgcatgagtgttccggatca

[0131] agaaatgatgatcggtcgcaccaaggatgcaaatgaggaaattgatggcgatgaacgtccggttaccagccatctgagccgcgtt

[0132] gatctgaaagaggatggcaaaggcctgaaaattgttcgtcagagcctgccgtatggtaccgccagcggcacccacggtttatatttt

[0133] tgtgcatattgtgcacgcctgtacaatattgaacaacagctgctgagtatgtttggcgataccgatggtaaacgcgatgccatgttacg

[0134] ttttaccaaacctgttaccggcggttattatttcgcaccgagcctggagcgtattcaggccctgggtcaccatcaccatcaccattaac

[0135] tcgag

[0136] SEQ ID NO.7

[0137] catatggctgtgcctctggttcagaaacgtgccacctgcgatgatggtcgcaccaccgccaatgcagcatgctgcattctgtttccga

[0138] ttctggatgatattcaggaaaatctgtttgatggtgcccagtgtggtgaagaagtgcatgaaagcctgcgcctgacctttcatgatgcc

[0139] attggctttagcccgaccctgggtggtggcggtgccgatggtagtattattgcatttgataccattgaaaccaactttccggcaaatgc

[0140] aggtattgatgaaattgttagcgcccagaaaccgtttgttgccaaacataatattagtgcaggtgactttatccagtttgccggcgcagt

[0141] gggtgttagcaattgcccgggtggtgttcgcattccgtttttcctgggtcgcccggatgcagttgcagcaagcccggatcatctggtg

[0142] ccggaaccgtttgatagtgtggatagtattctggcccgcatgggcgatgcaggttttagcccggttgaagtggtgtggctgctggca

[0143] agccatagcattgcagccgccgataaagttgatccgagtattccgggtaccccgtttgattcaaccccgggtgtgtttgatagtcagtt

[0144] tttcattgaaacccagctgaaaggccgtctgtttccgggcaccgcagataataagggcgaagcacagagtccgctgcagggcgaa

[0145] attcgcctgcagagcgatcatctgctggcccgtgatccgcagaccgcctgcgaatggcagagcatggttaataatcagccgaaaat

[0146] tcagaatcgttttgccgcaaccatgagtaaaatggcactgctgggtcaggataaaaccaaactgattgattgtagcgatgttattccga

[0147] ccccgccggccctggtgggtgcagcacatctgccggccggttttagtctgagtgatgtggaacaggcctgcgcagccaccccgtt

[0148] tccggcactgaccgccgatccgggtccggttaccagcgtgccgccggttccgggtagtcaccatcaccatcaccattaactcgag

[0149] SEQ ID NO.8

[0150] catatggtgcctagtccgctggttagctgcggcggcggccgtagtgttaaaaatgcagcatgctgcgcctggtttccggtgctggat

[0151] gatattcaggcaaatctgtttaatggcggcaaatgcgaagaagaagcccatgaagccgtgcgcctgacctttcatgatgccgtggg

[0152] ctttagtctggcagcacagaaagccggtaaatttggtggcggtggcgccgatggcagcattctggcattttcagatattgaaaccgc

[0153] attcattccgaattttggtctggagtttactaccgaaggttttattccgtttgcactggcccacggtgtgagctttggtgactttgttcagttt

[0154] gccggtgccgtgggcgcagcaaattgtgcaggcggtccgcgcctgcagtttctggccggtcgcagcaatattagccagccgagt

[0155] ccggatggtctggttccggaccctaccgatagcgccgataaaattctggcccgcatggcagatattggttttagcccgaccgaagtt

[0156] gttcatctgctggcaagtcatagcattgcagcacagtatgaagttgataccgatgttgcaggcagtccgtttgatagtaccccgagtgt

[0157] ttttgatacccagtttttcgtggaaagtctgctgcatggcacccagtttaccggtagtggccagggcggtgaagttatgagcccgattc

[0158] cgggtgaatttcgtctgcagagtgattttgcactgagccgtgatccgcgtaccgcatgcgaatggcaggccctggttaataatcagc

[0159] aggcaatggttaataatttcgaagccgttatgagtcgtctggccgtgattggtcagattccgagcgaactggtggattgcagtgatgtt

[0160] attccgaccccgccgctggccaaagttgcccaggttggcagcctgccgccgggtaaaagtatggcagatgtgcaggtggcctgta

[0161] ccaatggtatgccgtttccgagtctgccgaccagtccgggcccggttcagaccgtggcccctgttctgggccaccatcaccatcac

[0162] cattaactcgag

[0163] Shanghai Sangon Biotechnology Co., Ltd. was commissioned to artificially synthesize the optimized genes of each enzyme using the primers described in Table 2 and insert them between the NdeI-XhoI restriction sites of the vector pET-22b(+) to construct a recombinant plasmid.

[0164] Table 2 Recombinant plasmid primers

[0165]

[0166] 2. Heterologous expression in E. coli

[0167] Thaw E. coli Shuffle T7-B competent cells and recombinant plasmid on ice. Add 10 μL of recombinant plasmid (concentration approximately 100 ng / μL) to 100 μL of E. coli competent cells and place on ice for 30 minutes. Heat shock at 42°C for 1 minute, then quickly place on ice for 2 minutes. Add 700 μL of LB liquid medium and incubate at 37°C, 200 rpm for 1 hour.

[0168] The above cultured bacterial liquid was streaked onto LB solid medium plates containing 100 mg / mL ampicillin and cultured overnight at 37°C. A single colony was used as a template and PCR amplification was performed using the primers in Table 2 and the system in Table 3. The PCR product was verified by agarose gel electrophoresis. The results are shown in Table 2. Figure 2 As shown in A, M represents DNA Marker; 0 represents a blank strain without expression; P1-P4 represent the four recombinant plasmids constructed in step 1; A (ApeLip), K (KpDyp), T (TrcLip), and V (VPL2) represent the plasmids extracted from the four expression strains (i.e., PCR amplification products), respectively. According to the plasmid size comparison, the plasmid extracted from the expression strain is consistent with the recombinant plasmid size, proving that the plasmid is successfully expressed in the strain.

[0169] The PCR amplification system is shown in Table 3.

[0170] Table 3 PCR amplification system

[0171]

[0172] The PCR program was as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1.5 min, followed by extension at 72°C for 5 min. PCR product bands were verified by agarose gel electrophoresis.

[0173] 3. Induced expression of recombinant Escherichia coli

[0174] Evenly spread the recombinant E. coli cells verified in step 2 onto LB solid medium containing 100 mg / mL ampicillin and incubate at 37°C overnight. Inoculate the colony onto LB liquid medium containing 100 mg / mL ampicillin and incubate at 37°C, shaking at 250 rpm, overnight. Inoculate the overnight culture into 500 mL of LB liquid medium containing 100 mg / mL ampicillin at a 1% (v / v) inoculum concentration and incubate at 37°C, shaking at 250 rpm, overnight. When the OD value reaches 0.5-0.6, add the inducer IPTG to a final concentration of 0.2 mM. Induce expression at 16°C for 24 hours. Centrifuge at 8000 rpm for 20 minutes at 4°C to collect the wet cells.

[0175] 4. Protein purification using Ni-IDA agarose resin

[0176] (1) Preparation of protein samples:

[0177] Resuspend the wet cells prepared in step 3 in 10 mL of Binding Buffer per gram of wet cells. Add lysozyme to a final concentration of 0.2 mg / mL, 20 μg / mL of DNase, 1 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and mix at 4°C for 30 minutes. Use an ultrasonic cell disruptor to disrupt the cells at a power of 300 W for 20 minutes, with a sonication time of 3 seconds and a pause of 5 seconds. Transfer the lysate to a centrifuge tube and centrifuge at 4°C, 12,000 rpm for 20 minutes. Take the supernatant, which is the crude enzyme solution, for subsequent experiments.

[0178] (2) Ni-IDA prepacked column gravity purification

[0179] Use Ni-IDA pre-loaded gravity column (1mL), drain the liquid in the gravity column naturally, balance the column with 2 volumes of Binding Buffer, and drain slowly. Add 2 column volumes of step (1) crude enzyme solution, keep it for 3-5 minutes, and then drain it slowly. Continue to add step (1) crude enzyme solution until it is added. Add 2 column volumes each time. The total amount of crude enzyme solution loaded is about 10 column volumes. Rinse the column twice with 2 column volumes of Binding Buffer. Add 2 volumes of Elution Buffer, keep it for 8-10 minutes, drain it slowly, retain the eluate, repeat this step until all the protein is eluted, and collect the eluate containing the target protein, which is the pure enzyme solution. Use BCA kit to detect the protein content of the pure enzyme solution for subsequent aflatoxin B1 degradation test.

[0180] 5. SDS-PAGE verification of purified protein

[0181] Prepare the separating gel: 4 mL of ultrapure water, 8 mL of 30% acrylamide solution (ACr-Bis), 7.8 mL of 1.5 M Tris-HCl buffer, 0.2 mL of 10% sodium dodecyl sulfate (SDS) aqueous solution, 0.2 mL of 10% ammonium persulfate (APS) aqueous solution, and 10 μL of tetramethylethylenediamine (TEMED). Mix thoroughly and add to the gel plate. Seal the top 2 cm with isopropyl alcohol. Once the separating gel has solidified, pour off the isopropyl alcohol.

[0182] Prepare stacking gel: 3.8 mL ultrapure water, 1.75 mL 30% Acr-Bis, 1.25 mL Tris-HCl buffer (1 M), 0.1 mL 10% SDS, 0.1 mL 10% APS, and 10 μL TEMED. Mix thoroughly and add to overflow. Insert a 1.5 mm, 15-well comb into the gel plate. Remove the comb after solidification.

[0183] Thoroughly mix the protein sample with 5× denaturing protein loading buffer in a 4:1 ratio and place in a boiling water bath for 3-5 minutes. Allow to cool before loading. After plugging in the power cord, adjust the voltage to 100V. When the protein sample condenses into a straight line, adjust the voltage to 120V. Stop the electrophoresis when the sample line is approximately 2 cm from the bottom. After electrophoresis, remove the gel and stain with Coomassie Brilliant Blue for 1 hour. Then, destain overnight in destaining buffer. Remove the destaining buffer the next day and take photos.

[0184] like Figure 3As shown in Figure A, A, K, T, and V represent four expressed proteins: ApeLip (36 kDa); KpDyp (67.2 kDa); TrcLip (36.5 kDa); and VPL2 (35.2 kDa). "Crude" indicates crude enzyme solution, and "Pure" indicates pure enzyme solution. The molecular weights of the four expressed proteins are consistent, and the purified proteins are free of contaminants.

[0185] Comparative Example 1: Comparison of heterologous expression in E. coli Shuffle T7-B, BL21, and OrigamiB (DE3) as host bacteria

[0186] The host E. coli Shuffle T7-B in Example 2 was replaced with E. coli BL21 and E. coli OrigamiB (DE3), respectively. Other operations were the same, and the results were shown in Table 2. Figure 2 and Figure 3 shown.

[0187] Depend on Figure 2 The agarose gel electrophoresis images shown in B and C show that the four recombinant plasmids can be successfully transformed into BL21, but ApeLip and TrcLip cannot be transformed into Origami B (DE3). Figure 3 As shown in Figures B and C, the protein bands after induction expression in BL21 and Origami B(DE3) are very faint, possibly indicating unsuccessful expression or the formation of inclusion bodies in the precipitate after lysis. The concentration of purified proteins after expression in different systems was assayed using a BCA kit, and the results are shown in Table 4. Therefore, the E. coli Shuffle T7-B competent strain has a higher transformation success rate and expression level than other hosts.

[0188] Table 4 Protein concentrations in different host species

[0189]

[0190]

[0191] Example 3: Detection of AFB1 degradation effect

[0192] The reaction system of the experimental group was 1 mL:0.5 mL of phosphate buffer, 0.5 mL of the pure enzyme solution obtained in Example 2 (the amount of pure enzyme solution added was 0.8 mg / mL based on protein content), and aflatoxin B1 standard was added with a final concentration of 1 μg / mL.

[0193] The system without aflatoxin B1 inoculation served as the control group (CK).

[0194] Each group was reacted at 37°C in the dark for 24 h, and an equal volume of methanol was added to terminate the reaction. The mixture was vortexed thoroughly and filtered through a 0.22 μm filter membrane before the aflatoxin B1 content was determined by high performance liquid chromatography (HPLC).

[0195] The high performance liquid chromatography (HPLC) conditions were as follows: the chromatographic column was a C18 column (5 μm, 250 mm × 4.6 mm); the mobile phase A was methanol, the mobile phase B was pure water, and the volume ratio of phase A to phase B was 40:60; the flow rate was 1 mL / min; the injection volume was 20 μL; the column temperature was 40 °C, the fluorescence detector was used, the excitation wavelength Ex was 360 nm, the emission wavelength Em was 440 nm, and post-column derivatization was used.

[0196]

[0197] Wherein, A0 is the content of aflatoxin B1 in the control group; A1 is the content of aflatoxin B1 in the experimental group.

[0198] like Figure 4 As shown, compared with the control group (CK), the peak of aflatoxin B1 after treatment with the four proteins was significantly reduced, and the degradation rate could reach about 80%.

[0199] Comparative Example 2: Comparison of the degradation effects of four AFB1 degrading enzymes and other homologous proteins

[0200] The reaction system of the experimental group of Example 3 was used to prepare pure enzyme solutions of the eight homologous proteins except the four AFB1 degrading enzymes screened in Example 1 using the method of Example 2. The reactions and detections were performed using the method of Example 3. The results were as follows: Figure 5 As shown in the figure: 1. ScDyp; 2. MsDyp; 3. SmPgd; 4. ElDyp; 5. KpDyP; 6. MmFod; 7. TrcLip; 8. TtcLr; 9. ApeLiP; 10. Fbp1; 11. VPL2; 12. SvRmo. The results showed that the four AFB1-degrading enzymes, ApeLip, KpDyp, TrcLip, and VPL2, not only had binding energies below -10 kcal / mol but also had binding site sequences that were completely identical to the target sequence. In practical applications, they all achieved degradation rates of approximately 80% for aflatoxin B1. While the remaining homologous enzymes also had binding energies below -8 kcal / mol, they lacked active sites that were identical to the target sequence. With the exception of ScDyp and ElDyp, which achieved 40% degradation, the degradation rates of the remaining enzymes were all below 20%.

[0201] Example 4: Effect of incubation conditions on AFB1 degrading enzyme activity

[0202] 1. Effect of temperature on the degradation activity of AFB1 degrading enzyme

[0203] The reaction system was 1 mL:0.5 mL of phosphate buffer, 0.5 mL of the pure enzyme solution obtained in Example 2 (the amount of pure enzyme solution added was 0.8 mg / mL based on protein content), and aflatoxin B1 standard was added with a final concentration of 1 μg / mL.

[0204] The mixture was placed at 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, and 60°C in the dark for 24 h, and an equal volume of methanol was added to terminate the reaction. The mixture was vortexed thoroughly and filtered through a 0.22 μm filter membrane. The aflatoxin B1 content was detected using the method in Example 3.

[0205] like Figure 6 As shown, the degradation rates of the four enzymes for aflatoxin B1 were similar until the temperature reached 45°C, reaching an optimal degradation rate of approximately 83% at 37°C. When the temperature rose to 50°C, the degradation activities of the TrcLip and VPL2 enzymes were inhibited and gradually became completely inactivated as the temperature continued to rise. In contrast, the ApeLip and KpDyp enzymes still maintained a degradation rate of approximately 50% at 50°C, demonstrating a certain degree of heat resistance.

[0206] 2. Effect of pH on the degradation activity of AFB1 degrading enzyme

[0207] The pH of the phosphate buffer was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively. 0.5 mL of the pure enzyme solution obtained in Example 2 (the amount of pure enzyme solution added was 0.8 mg / mL based on protein content) was added to 0.5 mL of the buffer system at different pH values. Aflatoxin B1 was also added to a final concentration of 1 μg / mL. The reaction was incubated at 37°C in the dark for 24 h. An equal volume of methanol was added to terminate the reaction, the mixture was thoroughly vortexed, and the aflatoxin B1 content was determined using the method of Example 3 after filtration through a 0.22 μm filter.

[0208] like Figure 7 As shown in the figure, the degradation effect of the four degradation enzymes under alkaline conditions is stronger than that under acidic and neutral conditions, and the optimal degradation rate is reached at pH 9.0. As the pH value continues to increase, the TrcLip enzyme is least affected and has strong alkaline resistance. Although the degradation activity of the other three enzymes decreases, they can still maintain more than 60%.

[0209] 3. Effects of metal ions on the degradation activity of AFB1 degrading enzyme

[0210] The reaction system consisted of 1 mL:0.5 mL of phosphate buffer, 0.5 mL of the pure enzyme solution obtained in Example 2 (the amount of pure enzyme solution added was 0.8 mg / mL based on protein content), an aflatoxin B1 standard at a final concentration of 1 μg / mL, and a metal ion at a final concentration of 0.1 M. A control group (CK) was used without the addition of metal ions.

[0211] Metal ion: Mg 2+ , Ca 2+ , Zn 2+ , Li + , Mn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , K + .

[0212] The mixture was reacted at 37° C. in the dark for 24 h, and an equal volume of methanol was added to terminate the reaction. The mixture was vortexed thoroughly, and the aflatoxin B1 content was detected using the method of Example 3 after filtering through a 0.22 μm filter membrane.

[0213] like Figure 8 As shown, compared with the CK group, K + The addition of α-aminobutyric acid promoted the degradation activity of the four enzymes, and the degradation rates of ApeLip and KpDyp were more than 90%. 2+ The addition of Li+ slightly improved the degradation activity of the two enzymes, while the addition of Li+ had almost no effect on the degradation activity of the two enzymes.

[0214] 4. Effect of incubation time on the degradation activity of AFB1 degrading enzyme

[0215] The reaction system was 1 mL: 0.5 mL of phosphate buffer (pH 9.0), 0.5 mL of the pure enzyme solution obtained in Example 2 (the amount of pure enzyme solution added was 0.8 mg / mL based on protein content), and aflatoxin B1 standard was added with a final concentration of 1 μg / mL.

[0216] The reaction was carried out at 37°C in the dark for 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. An equal volume of methanol was added to terminate the reaction, the mixture was thoroughly vortexed, and the aflatoxin B1 content was determined using the method of Example 3 after filtration through a 0.22 μm filter membrane.

[0217] like Figure 9As shown, after 0.5 hours, the TrcLip enzyme achieved a degradation rate of approximately 70%, demonstrating high efficiency. The degradation rate then slowly increased, reaching an optimal rate of 85.91% at 72 hours. KpDyp and VPL2 enzymes gradually reached their peak degradation efficiency after 8–10 hours, reaching peak rates of 84.99% and 85.86% at 72 hours, respectively. ApeLip showed a lower initial degradation rate, reaching peak efficiency around 12 hours and 85.88% after 72 hours.

[0218] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. If improvements and modifications are to be made, such improvements and modifications should also be considered as within the scope of protection of the present invention.

Claims

1. A novel aflatoxin B1 degrading enzyme, characterized in that: The degrading enzyme includes lignin peroxidase, dye decolorizing peroxidase or multifunctional peroxidase.

2. The aflatoxin B1 degrading enzyme according to claim 1, wherein The degrading enzyme is one of the following: lignin peroxidase derived from Agrocybe pediades; dye decolorizing peroxidase derived from Klebsiella pneumoniae; multifunctional peroxidase derived from Pleurotus eryngii; lignin peroxidase derived from Trametopsis cervina.

3. The aflatoxin B1 degrading enzyme according to claim 2, wherein The amino acid sequence of the degradation enzyme is one of the following: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.

4.

4. A method for screening an aflatoxin B1-degrading enzyme according to claim 1, characterized in that: The method comprises the following steps: (1) Select 4-6 known aflatoxin B1-degrading enzymes as screening templates and obtain their 3D structures from the RCSB database; (2) Using AutoDock Vina software, the degradation enzyme in step (1) was semi-flexibly docked with aflatoxin B1, and the key amino acid residues in the degradation enzyme that interacted with aflatoxin B1 were analyzed, and the regional sequence with more binding sites was selected as the target sequence; (3) Use the BLAST tool in the NCBI database to perform homology screening in the protein database, screen out homologous proteins with high similarity, record the protein sequence, functional annotation and species origin as candidate proteins; (4) Molecular docking of the screened candidate proteins with aflatoxin B1 was performed to preliminarily screen candidate enzymes with docking binding energies < -9 kcal / mol; (5) Analyze the similarity between the active site sequence and the target sequence of the candidate enzyme acting on the furan ring and lactone ring of the AFB1 toxic group; finally, screen the protein with a binding energy lower than -10kcal / mol and the highest similarity between the active site sequence and the target sequence as the aflatoxin B1 degrading enzyme.

5. A recombinant plasmid expressing the aflatoxin B1 degrading enzyme according to claim 1.

6. A recombinant genetically engineered bacterium containing the gene encoding the aflatoxin B1 degrading enzyme according to claim 1.

7. Use of the aflatoxin B1 degrading enzyme according to claim 1 in degrading aflatoxin B1.

8. The use according to claim 7, characterized in that The application is as follows: aflatoxin B1 is used as a substrate, pure enzyme liquid extracted from wet bacteria after induction culture of recombinant genetically engineered bacteria containing the gene encoding aflatoxin B1 degrading enzyme by ultrasonic crushing is used as a catalyst, a pH 4.0-11.0 buffer solution is used as a reaction medium to form a reaction system, and the reaction is carried out in the dark at 25-60°C for 0.5-72 hours to achieve the degradation of aflatoxin B1.

9. The use according to claim 8, characterized in that In the reaction system, the substrate is added to a final concentration of 0.1-10 μg / mL; the amount of pure enzyme solution added is 0.4-0.8 mg / mL based on protein content.

10. The use according to claim 8, characterized in that The reaction system also contains 0.05-0.5M metal ions; the metal ions include Mg 2+ , Ca 2+ , Zn 2+ , Li + , Mn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , K + .

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