Use of aflatoxin-degrading enzyme, recombinant plasmid and recombinant bacteria in preparation of medicine for degrading aflatoxin

Abstract

This invention provides the use of aflatoxin-degrading enzymes, recombinant plasmids, and recombinant bacteria in the preparation of drugs that degrade aflatoxin, belonging to the field of biotechnology. The aflatoxin-degrading enzymes of this invention are extracted from Bacillus amyloliquefaciens strain 906, and their amino acid sequences are shown in SEQ ID NO.1 or SEQ ID NO.11. The two novel aflatoxin-degrading enzymes extracted from Bacillus amyloliquefaciens strain 906 exhibit excellent degradation effects on aflatoxin B1 and have promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of aflatoxin-degrading enzymes, recombinant plasmids, and recombinant bacteria in the preparation of drugs that degrade aflatoxin. Background Technology

[0002] Aflatoxins are a class of toxins produced by Aspergillus flavus (…). Aspergillus flavus ) and parasitic aspergillus ( Aspergillus parasiticus Secondary metabolites produced by fungi such as aflatoxin B1 (AFB1) are among the most potent and widely distributed natural carcinogens known. AFB1 possesses strong genotoxicity and carcinogenicity, and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). It primarily enters the food chain through contamination of food crops such as corn, peanuts, and cottonseed, posing a serious threat to human and animal health.

[0003] In recent years, due to the impact of global climate change, the contamination range of aflatoxin has expanded from traditional tropical and subtropical regions to temperate regions. The European Food Safety Authority (EFSA) points out that Aspergillus flavus thrives in warm and humid environments, and global warming has made mycotoxins, which were originally prevalent in tropical regions, more common in European crops. As a major agricultural country, China also faces a serious problem of aflatoxin contamination in its grain and oil products, with frequent incidents of AFB1 exceeding the standard, posing a serious threat to food safety and public health.

[0004] Currently, detoxification methods for AFB1 mainly fall into three categories: physical, chemical, and biological methods. Physical detoxification methods include high-temperature treatment, irradiation, and adsorption. While simple to operate, these methods suffer from drawbacks such as high energy consumption, incomplete detoxification, and potential damage to nutrients. Furthermore, adsorption only transfers the toxin rather than truly degrading it. Chemical detoxification methods primarily employ ammoniation and oxidation, which, while highly efficient, easily introduce chemical residues, altering food quality and posing environmental pollution risks, making them difficult to promote in practical production. Biological detoxification utilizes microorganisms or their produced enzymes to degrade AFB1 into low-toxicity or non-toxic products. Due to its high specificity, mild reaction conditions, and lack of secondary pollution, it is considered the most promising AFB1 detoxification technology.

[0005] Enzymatic degradation of AFB1 is a safe, environmentally friendly, and efficient biological detoxification strategy. Studies have shown that the enzymatic degradation of AFB1 mainly involves two major classes of enzymes: hydrolases and oxidoreductases. Its mechanism of action includes reactions such as lactone ring opening, hydroxylation, demethylation, and decarboxylation. By destroying the planar polyaromatic structure and reactive furan lactone ring of AFB1, it loses its DNA-binding ability and mutagenic activity.

[0006] Currently reported enzyme sources with AFB1 degradation activity include fungal, bacterial, and plant-derived enzymes. Fungal enzymes, such as laccase and manganese peroxidase from *Cladosporium chrysogenum* and manganese peroxidase from *Phanerochaete chrysosporium*, can achieve a degradation rate of 95% under acidic conditions. Bacterial enzymes, such as aflatoxin detoxifying enzymes produced by *Bacillus licheniformis* and CotA laccase from *Bacillus subtilis*, exhibit good degradation activity under near-neutral pH and mild temperature conditions. Jiangnan University has screened a strain of *Bacillus amyloliquefaciens* capable of degrading AFB1 in peanut meal. Plant-derived enzymes, such as horseradish peroxidase, enhance their detoxification ability by producing hydroxylated or quinone products.

[0007] However, existing technologies still have significant shortcomings. Most degrading enzymes have an optimal pH that is slightly acidic or require a redox medium, limiting their application in food and feed processing. The specific activity and stability of these enzymes need improvement. Given the serious threat AFB1 poses to food safety and the limitations of existing detoxification technologies, developing novel, efficient, and safe AFB1-degrading enzymes has significant theoretical and practical value. Summary of the Invention

[0008] The purpose of this invention is to provide the use of aflatoxin-degrading enzymes, recombinant plasmids, and recombinant bacteria in the preparation of drugs that degrade aflatoxin.

[0009] This invention provides the use of an aflatoxin-degrading enzyme in the preparation of a drug that degrades aflatoxin, wherein the amino acid sequence of the aflatoxin-degrading enzyme is shown in SEQ ID NO.1 or SEQ ID NO.11.

[0010] Furthermore, the aflatoxin is aflatoxin B1.

[0011] The present invention also provides the use of a recombinant plasmid in the preparation of a drug for degrading aflatoxin, said recombinant plasmid comprising a gene fragment encoding an aflatoxin-degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.11.

[0012] Furthermore, the recombinant plasmid is recombinant pET28a.

[0013] Furthermore, the aflatoxin is aflatoxin B1.

[0014] The present invention also provides the use of a recombinant bacterium in the preparation of a drug for degrading aflatoxin, said recombinant bacterium comprising a gene fragment encoding an aflatoxin-degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.11.

[0015] Furthermore, the recombinant bacteria is recombinant Escherichia coli.

[0016] Furthermore, the aflatoxin is aflatoxin B1.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention extracts two novel aflatoxin-degrading enzymes from Bacillus amyloliquefaciens strain 906. The enzymes extracted in this invention exhibit excellent degradation effects on aflatoxin B1 and have promising application prospects.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 The figure shows the degradation effect of different components (fermentation broth, supernatant and bacterial suspension) on AFB1 after Bacillus amyloliquefaciens fermentation.

[0021] Figure 2 The UV-Vis spectrum and fluorescence characteristics of AFB1 degradation products after degradation by Bacillus amyloliquefaciens 906 supernatant are shown in the figure: (a) UV-Vis spectrum; (b) fluorescence characteristics.

[0022] Figure 3 This is a diagram showing the localization results of AFB1's effective degradative enzymes.

[0023] Figure 4 The figure shows the effect of different treatments on the degradation of the fermentation broth.

[0024] Figure 5 The figure shows the comparison of AFB1 degradation efficiency between acetone-concentrated crude enzyme and the original supernatant.

[0025] Figure 6 The results of absorbance and AFB1 degradation efficiency for each tube in column chromatography are shown in the following figures: (a) Absorbance results; (b) AFB1 degradation efficiency results.

[0026] Figure 7 This is a schematic diagram of the construction of the pet28a-gene recombinant expression vector plasmid.

[0027] Figure 8 For gene sequence PCR fragment amplification products and pet28a- LigWAgarose gel electrophoresis images of positive clones: (a) agarose gel electrophoresis image of the amplified product of the gene sequence PCR fragment; (b) pet28a- LigW Agarose gel electrophoresis image of positive clones; where the marker represents the standard DNA molecular weight.

[0028] Figure 9 for LigW SDS-PAGE electrophoresis image of the protease; where the marker represents the molecular weight of the standard protein, KZ is the negative control with empty plasmid, followed by pet28a- LigW Induced expression and uninduced expression, and their purified enzyme solutions.

[0029] Figure 10 for Lig Ultra-high performance liquid chromatograms of aflatoxin degradation in each group of W enzymes.

[0030] Figure 11 This is an ultra-high performance liquid chromatogram of the enzyme after in vitro expression to degrade aflatoxin.

[0031] Figure 12 for Lig W, frsA and tuF The degradation rate of aflatoxin B1 after in vitro enzyme expression is shown in the figure.

[0032] Figure 13 For gene sequence PCR fragment amplification products and pet28a- pruA Agarose gel electrophoresis images of positive clones: (a) agarose gel electrophoresis image of the amplified product of the gene sequence PCR fragment; (b) pet28a- pruA Agarose gel electrophoresis image of positive clones; where the marker represents the standard DNA molecular weight.

[0033] Figure 14 for pruA SDS-PAGE electrophoresis image of the protease; where the marker represents the molecular weight of the standard protein, KZ is the negative control with empty plasmid, followed by pet28a. -purA Induced and uninduced expression enzyme solutions.

[0034] Figure 15 for pruA Ultra-high performance liquid chromatograms of aflatoxin degradation in each enzyme group.

[0035] Figure 16 This is an ultra-high performance liquid chromatogram used to screen enzymes that degrade aflatoxin B1 after in vitro expression.

[0036] Figure 17 The graph shows the degradation rate of aflatoxin B1 after in vitro expression of the enzyme. Detailed Implementation

[0037] Unless otherwise specified, the chemical reagents used in the specific embodiments are all conventional commercially available reagents, and the technical means used are all conventional means well known to those skilled in the art.

[0038] The aflatoxin-degrading enzyme extracted in this invention is derived from Bacillus amyloliquefaciens strain 906, which was deposited on March 31, 2012, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCCNO: M2012095. This strain is also described in Chinese patent application number 201210128153.5, filed on April 27, 2012.

[0039] Example 1 5-Carboxyvanillic acid decarboxylase ( ligW Discovery and efficacy testing of enzymes In this embodiment, 5-carboxyvanillic acid decarboxylase was extracted from Bacillus amyloliquefaciens strain 906. ligW The amino acid sequence of the enzyme is shown below: MLFRQHFKPVDIHGHLPYSLNFGKNDKTFSDYNRERSERMNLTWFPDPHDVDGDDGEIALMTRWEKELDKYGIDVLNFLTAEESNDRMAAFIQANPARFTGFAYHPLEQKDAYEELKRAVEELGLKGYKLFGPLTDIEFHDPSLKKLWTYLADKRLP VLIHFGLLGRAGGIVSHRNINPLSIYRVAREYADIPFIIPHFGAGYYQELLQLCWSCPNVYIDTSGSNQWMRWMPYRLDLEILFRKTSELIGAERIIFGTDSNGFPRGYVYRYLQEQVRTCREINMREEDIENIFGNNARTLLKIARKEKEVNT (SEQ ID NO.1) This enzyme is an aflatoxin B1 degrading enzyme and has excellent degradation effect on aflatoxin B1.

[0040] The discovery process and efficacy testing of this aflatoxin B1 degrading enzyme are shown below. In addition to obtaining the aflatoxin B1 degrading enzyme according to the following method (…),… ligW Enzymes can also be synthesized artificially based on their sequences.

[0041] 1. The detection method involved in this embodiment In this embodiment, the method for determining aflatoxin content using UPLC is as follows: Add 3 times the volume of dichloromethane to the degraded sample and shake on a shaker for 15 min to ensure complete extraction. After standing vertically to allow for phase separation, remove the lower organic phase into a clean test tube. Repeat the extraction 3 times, combine the collected extracts, and dry them under nitrogen to remove dichloromethane. Add 1 mL of chromatographic grade methanol and sonicate for 5 min to completely reconstitute the extract. Filter the sample through a 0.22 μm organic filter membrane to remove impurities. Detect the aflatoxin content by UPLC. Each treatment is repeated 3 times.

[0042] UPLC conditions: The test was performed using a Waters C18 high performance liquid chromatography column (2.1 × 100 mm, 1.7 μm). The mobile phase (methanol: formic acid aqueous solution (0.1%) = 4:6, V / V) was used for isocratic elution at a flow rate of 0.2 mL / min (0-2 min) and 0.08 mL / min (2-10 min), with a run time of 10 min, a column temperature of 30 °C, and an excitation wavelength of 360 nm and an emission wavelength of 440 nm for the fluorescence detector.

[0043] The degradation rate of aflatoxin B1 (AFB1) was calculated according to Formula I: Y = (X1 - X2) / X1 × 100% Formula I In the formula, X1 is the AFB1 content in the blank group (ng), X2 is the residual AFB1 content in the sample group (ng), and Y is the AFB1 degradation rate (%).

[0044] In this embodiment, the verification methods for fluorescence spectroscopy and ultraviolet spectroscopy are as follows: After 48 h of degradation of the experimental and control samples, three times the volume of dichloromethane was added to the degradation solution for three consecutive extractions. The organic phases were combined and purged with nitrogen until no liquid remained in the tube. The samples were then reconstituted with 1 mL of methanol-water solution (methanol to water volume ratio of 1:1), vortexed for 1–2 min, and filtered through a 0.22 μm organic filter to remove impurities. The samples were then subjected to fluorescence spectroscopy at an excitation wavelength of 365 nm using a fluorescence spectrophotometer, and the ultraviolet absorption wavelength range of 200–800 nm was also scanned.

[0045] 2. Discovery and efficacy testing of aflatoxin B1 degrading enzyme in this embodiment. 2.1 Preparation of Bacillus amyloliquefaciens 906 seed culture Bacillus amyloliquefaciens 906 was taken from a -80℃ freezer, thawed in a 37℃ incubator, and a loopful of bacterial culture was streaked onto an LB agar plate. The plate was then incubated upside down in a 37℃ incubator for 24 h. A single colony was picked and inoculated into 20 mL of LB broth, and cultured overnight in a shaker at 37℃ and 160 r / min to obtain the 906 seed culture.

[0046] 2.2 Determination of the active components of Bacillus amyloliquefaciens 906 in degrading AFB1 Accurately pipette 1 mL of activated Bacillus amyloliquefaciens 906 seed culture and transfer it to 20 mL of fresh LB broth (inoculum size 5%, pH 8.0). Incubate for 24 h at 32℃ and 160 r / min in a shaker. After incubation, take 10 mL of the mixed fermentation broth containing 906 for later use. Centrifuge the remaining 10 mL of fermentation broth at 4℃ and 5000 r / min for 15 min, filter through a sterile membrane, and collect the sterile supernatant for later use. Wash the precipitate from centrifugation three times with sterile ultrapure water, add 10 mL of physiological saline to resuspend the bacterial cells, and prepare a bacterial suspension. 900 μL of fermentation broth, sterile fermentation supernatant, and bacterial suspension were respectively added to 100 μL of aflatoxin B1 (AFB1) standard solution (1 μg / mL) as sample groups. Simultaneously, 900 μL of sterile LB medium (blank control for fermentation broth and sterile fermentation supernatant) or sterile physiological saline (blank control for bacterial suspension) were added to 100 μL of AFB1 standard solution (1 μg / mL) as blank groups. The mixture was incubated at 37℃ and 160 r / min in the dark for 48 h with shaking. The AFB1 degradation rate of each component was determined by UPLC. The AFB1 degradation products after degradation by Bacillus amyloliquefaciens 906 supernatant were also scanned and characterized by UV-Vis and fluorescence spectroscopy.

[0047] UPLC measurement results as follows Figure 1 As shown, the degradation of aflatoxin by Bacillus amyloliquefaciens 906 mainly depends on secondary metabolites secreted into the supernatant during fermentation. Although the bacterial suspension has a partial adsorption effect on the toxin, it is significantly lower than that of the supernatant.

[0048] Characterization results of AFB1 degradation products after degradation by Bacillus amyloliquefaciens 906 supernatant are as follows: Figure 2 As shown. By Figure 2 As shown in (a), compared with the LB blank control, after degradation by Bacillus amyloliquefaciens 906 supernatant, the absorption peak of AFB1 at around 365 nm was significantly reduced, indicating that AFB1 was degraded by the active ingredients in the fermentation supernatant. Figure 2 As shown in (b), after AFB1 is degraded, its fluorescence intensity decreases significantly, indicating that the lactone ring of the key chromophore of AFB1 is broken, and the fluorescence characteristics are weakened. However, the fluorescence intensity of AFB1 does not disappear, indicating that it is not completely degraded or that multiple pathways exist, such as competitive reactions such as furan ring opening and pentanone ring modification. These reactions can retain some conjugated structures, thus leaving some fluorescence.

[0049] 2.3 Subcellular localization of key degradative enzymes Accurately pipette 1 mL of activated Bacillus amyloliquefaciens 906 seed culture and transfer it to 20 mL of fresh LB broth (inoculum size 5%, pH 8.0). Incubate at 32°C and 160 rpm for 24 h in a shaker. After incubation, centrifuge the bacterial suspension at 4°C and 5000 rpm for 15 min. Filter the supernatant obtained by centrifugation through a 0.22 μm filter and store at 4°C; this is the extracellular enzyme. Wash the bacterial pellet three times with PBS buffer (pH 8.0), extract periplasmic enzymes using the osmotic shock method, and store at 4°C. Resuspend the 906 bacterial pellet in 50 mmol / L PBS buffer (pH 8.0) and disrupt the cells using an ultrasonic cell disruptor (40% power, 5 s on, 5 s off, 20 min total). Centrifuge the lysate at 10000 rpm for 20 min; the supernatant is the intracellular enzyme. Determine the concentration of each protein component using the Bradford assay kit and adjust the concentrations accordingly. 900 μL of extracellular enzyme, intracellular enzyme, and periplasmase were mixed with 100 μL of AFB1 (1 μg / mL) solution to form sample groups. At the same time, 100 μL of AFB1 (1 μg / mL) was added to 900 μL of sterile LB medium (blank control for extracellular enzyme) or sterile PBS buffer (blank control for periplasmase and intracellular enzyme) to form blank groups. The mixtures were incubated at 37℃ and 160 r / min in the dark for 48 h. The degradation rate of AFB1 in each component was determined by UPLC.

[0050] The results are as follows Figure 3 As shown, the extracellular enzymes of Bacillus amyloliquefaciens 906 exhibit the highest degradation activity for AFB1, followed by intracellular enzymes, while periplasmic enzymes show the least catalytic degradation activity for AFB1. Therefore, the effective degradation components for AFB1 by Bacillus amyloliquefaciens 906 can be secreted extracellularly.

[0051] 2.4 Study on the physicochemical properties of the active components for AFB1 degradation To determine that the degradation of AFB1 by Bacillus amyloliquefaciens 906 fermentation supernatant was due to enzymatic hydrolysis, the following treatments were performed: (1) the fermentation broth was heated in an 80℃ water bath for 10 min and then allowed to return to room temperature; (2) proteinase K (final concentration 1 mg / mL) was added to the fermentation broth; (3) proteinase K (final concentration 1 mg / mL) and SDS (concentration 1%) were added to the fermentation broth; (4) EDTA (final concentration 1 mg / mL) was added to the fermentation broth, and the mixture was reacted at 55℃ for 1 h. Normal fermentation broth was used as the untreated positive control, and LB broth medium was used as the negative control (blank group). AFB1 (final concentration 100 ng / mL) was added to each of the three groups, and three parallel reactions were set up. The mixture was reacted at 37℃ and 160 r / min in the dark for 48 h. After degradation, the degradation rate of AFB1 was detected by UPLC.

[0052] The results are as follows Figure 4 As shown, the aseptic fermentation broth of Bacillus amyloliquefaciens 906 was subjected to heating, proteinase K, SDS and EDTA treatments. It was found that the degradation activity of the fermentation broth decreased to varying degrees after treatment, indicating that the main degradative substances in the aseptic fermentation broth are enzymes, and that these enzymes are somewhat dependent on metal ions.

[0053] 2.5 Isolation and purification of extracellular enzymes The fermentation broth of *Bacillus amyloliquefaciens* 906, cultured for 24 h under optimal conditions (32°C, 5% inoculum, pH 8.0), was centrifuged at 8000 r / min for 20 min at 4°C and filtered through a 0.22 μm filter. Two volumes of pre-chilled acetone were slowly added to the supernatant while stirring gently for 60 min, followed by incubation at 4°C overnight. The sample was then centrifuged at 8000 r / min for 15 min at 4°C, the supernatant was discarded, and the protein precipitate was redissolved in sterile 50 mM Tris-HCl buffer (pH 7.4) to obtain a concentrated crude enzyme solution. The concentration was determined using a Bradford assay kit and adjusted to match the concentration of the *Bacillus amyloliquefaciens* 906 supernatant. UPLC was used to determine its aflatoxin degradation activity, with either LB broth (a blank control of the fermentation supernatant) or Tris-HCl buffer (pH 7.4) as blank controls.

[0054] The results are as follows Figure 5 As shown, the protein sample reconstituted after acetone precipitation exhibited a significantly lower degradation rate of AFB1 compared to the supernatant at the same protein concentration, indicating that the organic reagent treatment resulted in the loss of some protease activity. Considering the concentration factor (approximately 10-fold), acetone precipitation significantly reduced the sample volume and increased the total protein content per unit volume, thus meeting the requirements for sample concentration and volume in subsequent chromatographic purification.

[0055] 2.6 Sephadex G-100 gel chromatography The dry gel particles were fully swollen in 10 times the amount of eluent. After swelling, the fine particles were discarded, and the column was packed under vacuum. The outlet was closed, and the gel homogenate was poured into the chromatographic column (16 mm × 60 cm) through a glass rod while stirring. After the gel had settled to about 1 cm - 2 cm, the column outlet was opened to allow water to flow out, while the gel suspension was slowly added continuously. When the deposited gel surface rose to 2 cm - 3 cm above the top, the outlet was closed.

[0056] Equilibrate with 3-5 column volumes of eluent, compact the gel and keep the bed surface moist. After equilibration, open the outlet to allow eluent to flow out until it just exposes the bed surface, then immediately close the outlet. Use a dropper to slowly flow the sample (the crude enzyme solution precipitated with acetone obtained in step 2.5) along the inner wall of the column to the bed surface, then open the outlet to allow the sample to enter the bed until the bed surface is exposed again. Immediately and slowly add 1-2 sample volumes of eluent (50 mM Tris-HCl buffer, pH 7.4) to rinse the inner wall. Connect the fraction collector and adjust the flow rate to start elution. Elution flow rate is 0.5 mL / min, collecting one tube every 3 min. Finally, detect the UV absorbance of the eluted fraction at 280 nm and plot the elution curve. Determine the AFB1 degradation effect of each collected tube, retaining the active component.

[0057] Figure 6 The graph shows the absorbance and AFB1 degradation efficiency of each tube in the column chromatography. The crude enzyme solution extracted with acetone was further separated and purified. After separation by Sephadex G-100 column chromatography, 40 solutions were obtained. At 280 nm, three main peaks were observed (e.g., ...). Figure 6 As shown in (a)). A BFB1 degradation experiments were conducted on the separated solutions from all tubes, and the ninth tube showed the highest degradation efficiency, with tubes 7-11 also showing effectiveness (as shown in [reference]). Figure 6 As shown in (b) in the figure), the 7-11 tubes are combined to obtain a combined solution for later use.

[0058] 2.7 Ultrafiltration Prepare an ultrafiltration tube with a capacity of 3 KD (Vmax 15 mL). Combine the effective enzyme solution extracted by organic reagents and separated by column chromatography (the combined solution of tubes 7-11 obtained in step 2.6) and add 15 mL to the ultrafiltration tube. Centrifuge at 4℃ and 4000 xg for 10 min to obtain a concentrated solution with a final volume of approximately 1 mL.

[0059] 2.8 SDS-PAGE Analysis Take 15 μL of the crude enzyme solution obtained through ultrafiltration and concentration (the concentrate obtained in step 2.7), add 5 μL of 4× protein loading buffer, mix well, and incubate at 95℃ for 5 min. Immediately after heat treatment, place on ice for 5 min. Load an appropriate amount of sample onto a 4%–12% protein gel, ensuring the loading volume is consistent with the total protein content. Set the voltage to 150 V and incubate for 60 min. After electrophoresis, remove the gel, stain with Coomassie Brilliant Blue for 30 min, destain, and observe.

[0060] 2.9 Mass Spectrometry Identification of Purified Enzymes Different individual bands separated by SDS-PAGE were cut off with a scalpel and placed in 1.5 mL centrifuge tubes. A blank gel was also cut off as a control. The gel blocks were destained with 500 μL of 30% acetonitrile / 100 mmol / L NH4HCO3, allowed to stand for 30 min, the destaining solution was discarded, and the gel blocks were washed repeatedly with 500 μL of water until transparent. The supernatant was discarded, and the gel blocks were lyophilized. After lyophilization, 90 μL of 100 mmol / L NH4HCO3 and 10 μL of DTT were added to the centrifuge tubes, and the mixture was incubated at 56°C for 30 min, after which the supernatant was discarded. 70 μL of 100 mmol / L NHHCO3 and 30 μL of 200 mmol / L IAA were added to each tube, and the mixture was reacted in the dark for 20 min. The supernatant was discarded, 100 mmol / L NHHCO3 was added, and the mixture was incubated at room temperature for 15 min to reduce the protein. After removing the supernatant, add 100 μL of 100% acetonitrile to the centrifuge tube, and after 5 min, discard the supernatant and lyophilize. Add 5 μL of 2.5–10 ng / μL trypsin solution to the lyophilized sample and react at 37°C for 20 h. Transfer the enzyme digest to a new centrifuge tube. Add 100 μL of extraction buffer (60% acetonitrile / 0.1% TFA), sonicate for 15 min, combine the extract with the enzyme digest, lyophilize, and remove. Add 60 μL of 0.1% TFA aqueous solution to reconstitute, then filter through a 0.22 μm filter tube for later use.

[0061] Separation by capillary liquid chromatography: Solution A is an aqueous solution of 0.1% formic acid, and Solution B is an aqueous solution of 0.1% formic acid in acetonitrile (acetonitrile content 84%). After equilibration of the column with 95% solution A, the sample is loaded onto the trap column via an autosampler. A linear chromatographic gradient is used for solution B: from 0 to 50 min, the concentration gradient of solution B increases from 4% to 50%; from 50 to 54 min, the concentration gradient increases from 50% to 100%; from 54 to 60 min, solution B is maintained at 100%. Quadrupole orbital trap mass spectrometry detection: The separated sample is fully scanned using a quadrupole orbital trap mass spectrometer (Q-Exactive), and spectra of peptides and fragment mass-charge ratios of peptides are acquired (primary and secondary mass spectrometry).

[0062] The raw mass spectrometry test files were searched in the Uniprot database for Bacillus amyloliquefaciens using Mascot 2.2 software to obtain the characteristic amino acid sequences. The enzyme protein sequences were analyzed using the BLAST program in the GenBank database of the National Center for Biotechnology Information to find sequences related to strains with high homology to the enzyme sequences. Multiple alignments of these amino acid sequences were performed using DNAMAN software and the CLUSTALW program (ftp: / / ftp-igbmc.u-strasbg.fr / pub / ClustalW / ), and the alignment results were viewed on ESPript3.

[0063] The solutions from tubes 7-11 were combined and ultrafiltered. After the solution volume was concentrated, SDS-PAGE was performed. Strips with clearly defined bands were cut and subjected to LC-MS / MS mass spectrometry. The results were compared with database data and, combined with comparison with the strain's whole genome data, revealed serine aminopeptidase (…). frsA Translational elongation factor enzyme (CEE) tuF), 5-Carboxyvanillate decarboxylase ( ligW ).in ligW It is a novel enzyme that can degrade aflatoxin. It belongs to the amide hydrolase family of proteins, but no studies have yet found that it can degrade aflatoxin. tuF It is an enzyme with a known amino acid sequence, and its uniprot number is A7Z0N5. ligW The amino acid sequence is shown in SEQ ID NO.1. frsA The amino acid sequence is as follows: MILIDHQTVSGIPFLHIVKEENRDRPAPLVFFIHGFTSAKEHNLHFAYLLAEKGFRAVLPEALYHGERAEQLSAEELAVHFWDIVLNEIEELDVLKKDFEGRGLIEDGRIGLAGTSMGGITTFGAMAAY DWVKAGVSLMGSPNYTAFFQQQIDHIQKQDIDIDVTKEQVDELFARLKPFDLSLEPEKLRSRPLLFWHGVQDKVVPYAPTRGFYETIKPHYSSRPDHLQFLKDERADHKVPRYAVLETVAWFDKHL (SEQ ID NO.2) The selected enzymes were then expressed in large quantities in vitro using biosynthesis methods, which facilitated the determination of their degradation effect on AFB1.

[0064] 2.10 Design of PCR amplification primers Two suitable restriction enzyme sites are selected at the multiple cloning site of the pET28a vector. These sites are then ligated to the restriction enzyme ends of the vector and the target gene fragment, thus constructing a recombinant expression vector containing the target gene. For details, see [link to documentation]. Figure 7 .

[0065] according to LigThe amino acid sequence of the W enzyme (SEQ ID NO.1) was used to design and screen a pair of PCR primers for amplifying the full-length sequence of the target gene using SnapGene and Oligo software, and corresponding restriction sites were added. The upstream primer was 5'-cgcGAATTCCTTTTCAGGCAGCATTTTAA-3' (SEQ ID NO.3), and the downstream primer was 5'-acgcGTCGACCGTATTTACTTCCTTTTCTTT-3' (SEQ ID NO.4). Other enzymes were designed using the same method (…). frsA , tuF The upstream and downstream primers are shown in Table 1.

[0066] Table 1. Upstream and downstream primers for each enzyme 2.11 PCR amplification of the target gene fragment After activating Bacillus amyloliquefaciens 906, the bacterial cells were washed by centrifugation, and the genome of Bacillus amyloliquefaciens 906 was extracted using a DNA extraction kit according to the instructions. The target gene was amplified using upstream and downstream primers of the corresponding enzyme gene and high-fidelity enzyme (PCR system is shown in Table 2).

[0067] The PCR conditions were as follows: 95℃ pre-deformation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ extension for 10 min, and incubation at 4℃.

[0068] Table 2. PCR reaction system 2.12 Ligation of the target gene DNA fragment to the vector by Lig Taking W as an example, Lig The W gene fragment was purified and recovered using a purification kit, and the fragment concentration was determined. The purified target gene fragment was then subjected to double enzyme digestion with plasmid pET-28a, according to the reaction system prepared in Table 3 below. The mixture was incubated at 37°C for 20 min in a PCR instrument.

[0069] Table 3. Enzyme digestion systems for expression vectors and purified PCR fragments The bands were detected by 1% agarose gel electrophoresis, and the enzyme digestion products were purified and recovered using a product purification kit. The concentrations of the target gene fragment and plasmid pET-28a after enzyme digestion were determined by a micro spectrophotometer.

[0070] The target gene fragment purified by enzyme digestion was ligated with the plasmid pET-28a fragment. After preparing the reaction system according to Table 4, it was reacted at 22℃ for 20 min, then at 4℃ for 5 min, and stored in a refrigerator for later use.

[0071] Table 4. Connection Reaction System Using the genome of Bacillus amyloliquefaciens 906 as a template, aflatoxin-degrading enzymes ( LigW The coding gene for the gene was amplified by PCR. The PCR products were analyzed by 1% agarose gel electrophoresis. The band size of the coding gene was 943 bp (e.g., ...). Figure 8 As shown in (a)), the band size of the positive clone was 1181 bp (as shown in [reference]). Figure 8 As shown in (b) in the figure, the size is the same as the expected target gene fragment, indicating that the vector was successfully constructed.

[0072] 2.13 Recombinant plasmid pET28a- Lig W was transformed into Escherichia coli DH5α Take a 100 μL vial of *E. coli* DH5α Competent Cells from a -80℃ freezer, thaw on ice for 10 min, and gently mix. Add 10 μL of the mixed ligation system to 100 μL of competent cells, gently aspirate and mix, and incubate on ice for 30 min. Heat shock at 42℃ for 60 s, then incubate on ice for 2-3 min. Next, in a laminar flow hood, add 1 mL of antibiotic-free LB broth and incubate at 37℃ with shaking at 160 rpm for 1 h. Centrifuge the bacterial culture at room temperature at 6000 rpm for 5 min. In a laminar flow hood, discard 700 μL of supernatant, thoroughly mix the remaining bacterial cells and liquid by pipetting, and spread 100 μL onto a TSA plate containing Kana (final concentration 50 μg / mL) resistance, incubate upside down at 30℃ for 24 h.

[0073] 2.14 Recombinant plasmid pET28a- Lig Screening and identification of W Single colonies from the plating plate were picked and directly added to the PCR reaction system. YZ-F and YZ-R were used as upstream and downstream primers, respectively (YZ-F sequence: 5'-GACTCACTATAGGGGAATTGT-3' (SEQ ID NO. 9); YZ-R sequence: 5'-CCCTCAAGACCCGTTTAGA-3' (SEQ ID NO. 10)). Positive transformants successfully ligated into the vector pET28a were screened by PCR and 1% agarose gel electrophoresis. PCR conditions were as follows: 95℃ pre-deformation for 20 min; 95℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 1 min 15 s, for a total of 32 cycles; 72℃ extension for 10 min; and incubation at 4℃.

[0074] After successful sequencing, the positive transformants were transferred into 20 mL of antibiotic-containing LB broth for amplification culture to obtain the ligation vector pET28a- Lig W.

[0075] Table 5. Colony PCR Reaction System 2.15 Recombinant plasmid pET-28a- Lig W was transformed into Escherichia coli BL21(DE3). The plasmid pET28a- was extracted from the cultured positive clone of E. coli using a plasmid extraction kit. Lig W, and the extracted plasmid was transformed into BL21(DE3) competent cells (total amount not exceeding 100 ng) using the hot-shock method. After activation culture, the bacteria were inoculated onto Kana-resistant plates, and colony PCR was performed using verification primers. The successfully transformed bacteria were inoculated into 20 mL of LB broth containing Kana resistance for expansion culture and stored at -80℃ for later use.

[0076] 2.16 Induction and lysis of bacterial culture Successfully validated positive clones were selected and co-inoculated with *E. coli* (KZ) containing the PET28A empty vector plasmid into LB broth (50 μg / mL kanamycin resistant). The mixture was incubated overnight at 37°C in a shaker for activation. The activated bacterial solution was then transferred at a ratio of 1% to LB broth containing kanamycin resistant (50 μg / mL) and cultured at 37°C and 160 r / min until OD500. 600nmWhen the concentration was 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was then incubated in a shaker at 160 r / min and 20℃ for 12 h. After incubation, the bacterial suspension was centrifuged at 5000 r / min for 15 min at 4℃ to obtain a bacterial pellet. The pellet was then resuspended in 5 mL of pre-chilled Tris-HCl (pH 7.4), mixed thoroughly by pipetting, and centrifuged again for 15 min. This step was repeated twice. The obtained bacterial pellet was resuspended in pre-chilled lysis buffer such as Tris-HCl (pH 8.0, with the option to add the protease inhibitor PMSF), and sonicated at 20% power for 5 s with a 5 s interval, for a total of 20 min. Cell lysis was performed on ice. The crude enzyme mixture (i.e., the lysis buffer) was centrifuged at 10000 r / min for 20 min at 4℃, and the supernatant was collected for subsequent analysis. This supernatant is the lysate. LigW Crude enzyme solution. The concentration of crude enzyme was determined according to the instructions of the Bradford Protein Assay Kit, and the concentration was adjusted to be consistent with Tris-HCl.

[0077] 2.17 SDS detection of enzyme expression Take 15 μL of crude enzyme solution (total amount 50-100 μg), add 5 μL of 4× protein loading buffer, mix well, and incubate at 95℃ for 5 min. Immediately after warm compress, incubate on ice for 5 min. Load an appropriate amount of sample onto a 10% protein gel, ensuring the loading volume is consistent with the total protein amount. Set the voltage to 80 V for 20 min. When the sample reaches the separating gel, change the voltage to 120 V for 60 min. After electrophoresis, remove the gel, stain with Coomassie Brilliant Blue for 30 min, destain, and observe and photograph.

[0078] The constructed recombinant expression vector pet28a- Lig W was transformed into E. coli BL21(DE3), such as Figure 9 As shown, the target protein band appeared after induction with 0.4 mM IPTG. However, the negative control bacteria transformed with an empty plasmid containing the target gene, and the recombinant vector cultured under the same conditions without an inducer, did not express the target protein. Therefore, the protein vector was successfully constructed and expressed. Purification using a Ni column yielded... Lig Purification solution of W protein.

[0079] 2.18 Determination of Aflatoxin Degradation Efficiency The sample obtained by pyrolysis using method 2.16 LigAdd 900 μL of enzyme solution to 100 μL of AFB1 standard solution (1 μg / mL), and use high-temperature inactivated enzyme (treated at 121℃ for 10 min) as a negative control and Tris-HCl as a blank group. Incubate at 37℃ and 160 r / min in the dark for 48 h with shaking. UPLC is used to determine the AFB1 degradation rate of each component.

[0080] Determined by ultra-high performance liquid chromatography Lig The degradation efficiency of W for aflatoxin was as follows: Figure 10 As shown: the crude enzyme solution was mixed with AFB1 for degradation, with Tris-HCl as the blank group, and the enzyme was inactivated by high temperature. Lig W proteinase solution served as a negative control, and it was found that... Lig The degradation efficiency of W enzyme for aflatoxin can reach 62%.

[0081] Other enzymes were evaluated using the same method described above. frsA , tuF The degradation effect of ) on aflatoxin (AFB1) was shown in the following results. Figure 11 and Figure 12 As shown: You can see ligW It exhibits excellent degradation effect on AFB1, and frsA and tuF It has almost no degradation effect on AFB1.

[0082] Example 2 1-Pyrrololine-5-carboxylic acid dehydrogenase ( pruA The discovery of enzymes In this embodiment, 1-pyrrolidone-5-carboxylic acid dehydrogenase was extracted from Bacillus amyloliquefaciens strain 906. pruA The amino acid sequence of the enzyme is shown below: MTTPYKHEPFTNFQDQSNVEEFKKALATVNEYLGKDYPLVINGEKVETEAKIVSINPADKEEVVGKVSKASQEHAEQAIEAAAKAFEEWRYTSPEERAAVLFRAAAKVRRRKHEFSALLVKEAGKPWNE ADADTAEAIDFMEYYARQMVELAKGKPVNSREGEKNQYVYTPTGVTVVIPPWNFLFAIMAGTTVAPIVTGNTVVLKPASATPVIAAKFVEVLEESGLPKGVVNFVPGSGAEVGDYLVDHPKTSIITFTGS REVGTRIFERAAKVQPGQQHLKRVIAEMGGKDTVVVDEDADIELAAQSIFTSAFGFAGQKCSAGSRAVVHEKVYDQVLERVIEITESKVTASPDSADVYMGPVIDQGSYDKIMSYIEIGKEEGRLVSGGT GDDSKGYFIKPTIFADLDPKARLMQEEIFGPVVAFSKVSNFDEALEVANNTEYGLTGAVITNNRKHIERAKQEFHVGNLYFNRNCTGAIVGYHPFGGFKMSGTDSKAGGPDYLALHMQAKTISEMF (SEQ IDNO.11) This enzyme is an aflatoxin B1 degrading enzyme and has excellent degradation effect on aflatoxin B1.

[0083] The discovery process and efficacy testing of this aflatoxin B1 degrading enzyme are shown below. In addition to obtaining the aflatoxin B1 degrading enzyme according to the following method (…),… pruA Enzymes can also be synthesized artificially based on their sequences.

[0084] 1. The detection method involved in this embodiment In this embodiment, the method for determining aflatoxin content using UPLC is as follows: Add 3 times the volume of dichloromethane to the degraded sample and shake on a shaker for 15 min to ensure complete extraction. After standing vertically to allow for phase separation, remove the lower organic phase into a clean test tube. Repeat the extraction 3 times, combine the collected extracts, and dry them under nitrogen to remove dichloromethane. Add 1 mL of chromatographic grade methanol and sonicate for 5 min to completely reconstitute the extract. Filter the sample through a 0.22 μm organic filter membrane to remove impurities. Detect the aflatoxin content by UPLC. Each treatment is repeated 3 times.

[0085] UPLC conditions: The test was performed using a Waters C18 high performance liquid chromatography column (2.1 × 100 mm, 1.7 μm). The mobile phase (methanol: formic acid aqueous solution (0.1%) = 4:6, V / V) was used for isocratic elution at a flow rate of 0.2 mL / min (0-2 min) and 0.08 mL / min (2-10 min), with a run time of 10 min, a column temperature of 30 °C, and an excitation wavelength of 360 nm and an emission wavelength of 440 nm for the fluorescence detector.

[0086] The degradation rate of aflatoxin B1 (AFB1) was calculated according to Formula I: Y = (X1 - X2) / X1 × 100% Formula I In the formula, X1 is the AFB1 content in the blank group (ng), X2 is the residual AFB1 content in the sample group (ng), and Y is the AFB1 degradation rate (%).

[0087] In this embodiment, the verification methods for fluorescence spectroscopy and ultraviolet spectroscopy are as follows: After 48 h of degradation of the experimental and control samples, three times the volume of dichloromethane was added to the degradation solution for three consecutive extractions. The organic phases were combined and purged with nitrogen until no liquid remained in the tube. The samples were then reconstituted with 1 mL of methanol-water solution (methanol to water volume ratio of 1:1), vortexed for 1–2 min, and filtered through a 0.22 μm organic filter to remove impurities. The samples were then subjected to fluorescence spectroscopy at an excitation wavelength of 365 nm using a fluorescence spectrophotometer, and the ultraviolet absorption wavelength range of 200–800 nm was also scanned.

[0088] 2. Discovery and efficacy testing of aflatoxin B1 degrading enzyme in this embodiment. 2.1 Activation of microbial strains Bacillus amyloliquefaciens 906 was taken from a -80℃ freezer, thawed in a 37℃ incubator, and a loopful of bacterial culture was streaked onto an LB agar plate and incubated upside down in a 37℃ incubator for 24 h. A single colony was picked and inoculated into 20 mL of LB broth and cultured overnight in a shaker at 37℃ and 180 r / min to obtain the strain seed culture.

[0089] 2.2 Sample Preparation The seed culture of activated Bacillus amyloliquefaciens 906 was transferred at a 5% inoculum to 100 mL of LB broth containing AFB1 (100 ng / mL) (experimental group) and LB broth without AFB1 (control group), and cultured at 37℃ and 160 r / min for 24 h and 48 h, respectively. After culture, the bacterial cells were collected by centrifugation at 4000 r / min, the supernatant was removed, and the bacterial pellet was washed 2-3 times with PBS solution. Each group was performed in triplicate.

[0090] 2.3 RNA extraction and detection After culturing each sample group, the supernatant was removed by filtration and centrifugation. The bacterial precipitate was washed 2-3 times with PBS buffer. All samples were treated with lysozyme, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). The purity (OD260 / 280 and OD260 / 230) and concentration of total RNA were determined by NanoDrop. RNA integrity, including RNA integrity value and 23S / 16S ratio, was accurately determined using an Agilent 2100. Finally, quantification and banding were performed using 1% agarose gel electrophoresis. 1 μg of total RNA samples with an RNA integrity value higher than 6.5 was prepared for subsequent library preparation.

[0091] 2.4 Library preparation, quality control, and Illumina HiSeq sequencing After total RNA testing was passed, rRNA was removed from the total RNA, and then the ribosome-free RNA was fragmented and reverse transcribed. A library was constructed using the Trussq™ RNA sample prep Kit, but dUTP was used instead of dTTP in the dNTPs reagent for synthesizing the second strand of cDNA, ensuring that the second strand of cDNA contained A / U / C / G bases. Before PCR amplification, the second strand of cDNA was digested with UNG enzyme, ensuring that the library contained only the first strand of cDNA. After library construction, preliminary quantification was performed using Qubit 2.0, and the insertion size of the library was detected using an Agilent 2100. Once the expected value was met, the effective concentration of the library was accurately quantified by qPCR. After passing the library test, it was added to the Illumina HiSeq instrument according to the manufacturer's instructions. Sequencing was performed using 2×150 bp paired-end sequencing. Image analysis and base identification were performed using the HiSeq control software (HCS), OLB, and GAPipeline-1.6 on the instrument.

[0092] Cutadapt was used to filter low-quality data from the raw sequencing data, removing reads with adapter sequences, 5' or 3' end quality values ​​below a certain threshold, N ratios greater than 10%, Q ≤ 10 bases comprising more than 50% of the entire read, and reads shorter than 75 bp after trimming, resulting in high-quality (Clean Reads). CPC2 was used to assess the coding potential of the unigene sequences. Subsequently, the discovered coding unigenes were compared with the NCBI Non-Redundant Protein Database (Nr) using the BLAST program to obtain homologous sequence information (Altschul et al 1997). Further annotation information from the GO, COG / KOG, KEGG, and Pfam (protein family) databases was obtained to comprehensively analyze the potential functions of these new genes.

[0093] To ensure that the number of fragments accurately reflects transcript expression levels, the number of sequencing reads and transcript lengths in the samples need to be normalized. The software RSeQC resamples the total aligned reads, and the HTSeq software evaluates FPKMs (Fragments Per Kilobase of Transcript Sequence Per Millions Basepairs Sequenced) at the current sequencing depth, using relative error rate to measure and assess the accuracy of FPKMs. Correlation checks and PCA analysis are also performed on the samples. A gene's expression level is directly reflected in its abundance; higher gene abundance indicates higher gene expression levels.

[0094] Differential gene expression analysis was performed using DESeq2 (V1.6.3) from the Bioconductor software package. This model is based on a negative binomial distribution model, and the significance of the original hypothesis test was evaluated using the Benjamini correction method. p The values ​​were corrected. The test results were screened according to the significance criteria (|log2(FoldChange)|>2 and padj<0.05), and the number of differentially expressed genes upregulated or downregulated was counted.

[0095] The transcriptome results (with AFB1 added vs. without addition) are shown in Table 6.

[0096] Table 6. Basic Information on Screened Genes Gene pruA The encoded amino acid sequence is shown in SEQ ID NO.11. AcoA The uniprot number is A0A410L080. licHThe uniprot number is A0AAP3YEE6. Hutl The uniprot number is A0A411A993 pstA The amino acid sequence is as follows: MNRKITDKLATGVFGLCAAVIAAILAGLFLYILIHGVSEISLRFLTSKSSAIASGGGIRDQLFNSFYILFITMLITIPLGVGGGVFMAEYAPQNKITDFIRTCIEVLSSLPSIVIGMFGMLMFVNLTGWGYTIIGGALALTVFNLPVMV RVTEGALTAVPKELKEASLALGVSRWHTVKTVLIPSAIPSILTGAILASGRVFGEAAALLFTAGLTTPRLNFTDLNPFSESSPFNIFRPAETLAVHIWSVNTQGIIPDAEAIANGGSAVLVISVLLFNLSARWLGSVIYKKLTAN (SEQ ID NO.12) 2.5 Design of PCR amplification primers according to pruA The amino acid sequence of the enzyme (SEQ ID NO. 11) was used to design and screen a pair of PCR primers for amplifying the full-length sequence of the target gene using SnapGene and Oligo software, and corresponding restriction sites were added. The upstream primer was 5'-cgcggatccACACCTTATAAACACGAACC-3' (SEQ ID NO. 13), and the downstream primer was 5'-acgcgtcgacCATTTCACTGATTGTTTTCG-3' (SEQ ID NO. 14). Other enzymes were designed using the same method (…). AcoA , pstA , Hutl , licH The upstream and downstream primers are shown in Table 7.

[0097] Table 7. Upstream and downstream primers for each enzyme 2.6 PCR amplification of the target gene fragment After activating Bacillus amyloliquefaciens 906, the bacterial cells were washed by centrifugation, and the genome of Bacillus amyloliquefaciens 906 was extracted using a DNA extraction kit according to the instructions. The target gene was amplified using upstream and downstream primers of the corresponding enzyme gene and high-fidelity enzyme (PCR system is shown in Table 8).

[0098] PCR conditions were as follows: 95℃ pre-deformation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min 30 s, for a total of 35 cycles; 72℃ extension for 10 min, followed by incubation at 4℃. Table 8. PCR reaction system 2.7 Ligation of the target gene DNA fragment to the vector by pruA For example, pruA After the gene fragment was purified and recovered using a purification kit, the concentration of the DNA fragment was determined. The purified and recovered target gene DNA fragment and plasmid pET-28a were first subjected to double enzyme digestion reactions, and the reaction system was prepared according to Table 9 below. The reaction was incubated in a PCR instrument at 30℃ for 20 min, and then at 37℃ for 20 min.

[0099] Table 9. Enzyme digestion systems for expression vectors and purified PCR fragments The bands were detected by 1% agarose gel electrophoresis. The enzyme digestion products were purified and recovered using a product purification kit. The enzyme digestion products were measured by a micro spectrophotometer. pruA Concentrations of gene fragments and plasmid pET-28a.

[0100] The target gene fragment purified by enzyme digestion was ligated with the plasmid pET-28a fragment. After preparing the reaction system according to Table 10, it was reacted at 22℃ for 20 min, then at 4℃ for 5 min, and stored in a refrigerator for later use.

[0101] Table 10. Connection Reaction System Using the genome of Bacillus amyloliquefaciens 906 as a template, aflatoxin-degrading enzymes ( pruA The coding gene for the gene was amplified by PCR. The PCR products were analyzed by 1% agarose gel electrophoresis. The band size of the coding gene was 1549 bp (e.g., ...). Figure 13 As shown in (a), the band size of the positive clone was 1876 bp (as shown in the middle). Figure 13 As shown in (b), the size is the same as the expected target gene fragment, indicating that the vector was successfully constructed.

[0102] 2.8 Recombinant plasmid pET28a- pruA Transformed into Escherichia coli DH5α Take a 100 μL vial of *E. coli* DH5α Competent Cells from a -80℃ freezer, thaw on ice for 10 min, and gently mix. Add 10 μL of the ligation system to 100 μL of competent cells, gently mix by pipetting, and incubate on ice for 30 min. Heat shock at 42℃ for 60 s, then incubate on ice for 2-3 min. Next, in a laminar flow hood, add 1 mL of antibiotic-free LB broth and incubate at 37℃ with shaking at 160 rpm for 1 h. Centrifuge the culture at room temperature at 6000 rpm for 5 min. In a laminar flow hood, discard 700 μL of the supernatant, thoroughly mix the remaining cells and liquid by pipetting, and spread 100 μL onto a TSA plate containing Kana (final concentration 50 μg / mL) resistance, incubate upside down at 30℃ for 24 h.

[0103] 2.9 Recombinant plasmid pET28a- pruA Screening and identification Single colonies from the plating plate were picked and directly added to the PCR reaction system. YZ-F and YZ-R were used as upstream and downstream primers, respectively (the sequences of YZ-F and YZ-R are the same as in Example 1; the sequence of YZ-F is shown in SEQ ID NO. 9, and the sequence of YZ-R is shown in SEQ ID NO. 10). Positive transformants successfully ligated into the vector pET28a were screened by PCR and 1% agarose gel electrophoresis. PCR conditions were: 95℃ pre-deformation for 20 min; 95℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 1 min 15 s, for a total of 32 cycles; 72℃ extension for 10 min, and incubation at 4℃.

[0104] After successful sequencing, the positive transformants were transferred into 20 mL of antibiotic-containing LB broth for amplification culture to obtain the ligation vector pET28a- pruA .

[0105] Table 11. Colony PCR Reaction System 2.10 Recombinant plasmid pET28a- pruA Transformed into Escherichia coli BL21(DE3) The plasmid pET28a- was extracted from the cultured positive clone of E. coli using a plasmid extraction kit. pruA The extracted plasmids were transformed into BL21(DE3) competent cells (total amount not exceeding 100 ng) using the hot-shock method. After activation culture, the bacteria were inoculated onto Kana-resistant plates, and colony PCR was performed using verification primers. Successfully transformed bacteria were inoculated into 20 mL of LB broth containing Kana resistance for expansion culture and stored at -80℃ for later use.

[0106] 2.11 Induction and lysis of bacterial culture Successfully validated positive clones and *E. coli* (KZ) containing the PET28A empty vector plasmid were inoculated into LB broth (50 μg / mL kanamycin resistant) and incubated overnight at 37°C in a shaker for activation. The activated bacterial culture was then transferred at a ratio of 1% to LB broth containing kanamycin resistant bacteria (50 μg / mL) and incubated at 37°C and 160 r / min until OD500. 600nm When the concentration was 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was then incubated in a shaker at 160 r / min and 20℃ for 12 h. After incubation, the bacterial suspension was centrifuged at 5000 r / min for 15 min at 4℃ to obtain a bacterial pellet. The pellet was then resuspended in 5 mL of pre-chilled Tris-HCl (pH 7.4), mixed thoroughly by pipetting, and centrifuged again for 15 min. This step was repeated twice. The obtained bacterial pellet was resuspended in pre-chilled lysis buffer such as Tris-HCl (pH 8.0, with the option to add the protease inhibitor PMSF), and sonicated at 20% power for 5 s with a 5 s interval, for a total of 20 min. Cell lysis was performed on ice. The crude enzyme (i.e., the lysis buffer) was centrifuged at 10000 r / min for 20 min at 4℃, and the supernatant was collected for subsequent analysis. This supernatant is the lysate. pruA The crude enzyme solution was prepared. The concentration of the crude enzyme was determined according to the instructions of the Bradford Protein Assay Kit, and the concentration was adjusted to be consistent with Tris-HCl.

[0107] 2.12 SDS detection of enzyme expression Take 15 μL of crude enzyme solution (total amount 50-100 μg), add 5 μL of 4× protein loading buffer, mix well, and incubate at 95℃ for 5 min. Immediately after warm compress, incubate on ice for 5 min. Load an appropriate amount of sample onto a 10% protein gel, ensuring the loading volume is consistent with the total protein amount. Set the voltage to 80 V for 20 min. When the sample reaches the separating gel, change the voltage to 120 V for 60 min. After electrophoresis, remove the gel, stain with Coomassie Brilliant Blue for 30 min, destain, and observe and photograph.

[0108] The constructed recombinant expression vector pet28a- pruA Transformed into E. coli BL21(DE3), such as Figure 14 As shown, the target protein band appeared after induction with 0.4 mM IPTG. However, the negative control bacteria transformed with an empty plasmid containing the target gene, and the recombinant vector cultured under the same conditions without an inducer, did not express the target protein. Therefore, the protein vector was successfully constructed and expressed.

[0109] 2.13 Determination of aflatoxin degradation effect Take the product obtained by pyrolysis according to method 2.11 pruA Add 900 μL of enzyme solution to 100 μL of AFB1 standard solution (1 μg / mL), and use high-temperature inactivated enzyme (treated at 121℃ for 10 min) as a negative control and sterile Tris-HCl buffer as a blank group. Incubate at 37℃ and 160 r / min in the dark for 48 h with shaking. UPLC was used to determine the AFB1 degradation rate of each component.

[0110] Determined by ultra-high performance liquid chromatography pruA The degradation efficiency of aflatoxin was as follows: Figure 15 As shown: The crude enzyme solution was mixed with AFB1 for degradation, with Tris-HCl as a blank control, and the enzyme was inactivated by high temperature. pruA The protease solution served as a negative control, and it was found that... pruA The enzyme can degrade aflatoxin up to 70% efficiently.

[0111] Other enzymes were evaluated using the same method described above. hutL , licH , AcoA and pstA The degradation effect of ) on aflatoxin (AFB1) was shown in the following results. Figure 16 and Figure 17 As shown: You can see pruA The enzyme showed the best degradation effect on aflatoxin, significantly better than other enzymes.

[0112] In summary, this invention has extracted two novel aflatoxin-degrading enzymes from Bacillus amyloliquefaciens strain 906. The enzymes extracted in this invention exhibit excellent degradation effects on aflatoxin B1 and have promising application prospects.

Claims

1. The use of an aflatoxin-degrading enzyme in the preparation of aflatoxin-degrading drugs, characterized in that: The amino acid sequence of the aflatoxin-degrading enzyme is shown in SEQ ID NO.1 or SEQ ID NO.

11.

2. The use according to claim 1, characterized in that: The aflatoxin in question is aflatoxin B1.

3. The use of a recombinant plasmid in the preparation of a drug that degrades aflatoxin, characterized in that: The recombinant plasmid contains a gene fragment encoding an aflatoxin-degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.

11.

4. The use according to claim 3, characterized in that: The recombinant plasmid is recombinant pET28a.

5. The use according to claim 3 or 4, characterized in that: The aflatoxin in question is aflatoxin B1.

6. The use of a recombinant bacterium in the preparation of a drug that degrades aflatoxin, characterized in that: The recombinant bacteria contain a gene fragment encoding an aflatoxin-degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.

11.

7. The use according to claim 6, characterized in that: The recombinant bacteria is recombinant Escherichia coli.

8. The use according to claim 6 or 7, characterized in that: The aflatoxin in question is aflatoxin B1.

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