Laccase HcCotA molecule capable of efficiently degrading aflatoxin B1 and application of laccase HcCotA molecule

Abstract

The invention discloses a laccase HcCotA molecule capable of efficiently degrading aflatoxin B1 and application thereof, and relates to the technical field of microorganisms and microorganism application, and the laccase HcCotA molecule is technically characterized in that HcCotA has excellent thermal stability and pH stability and also has tolerance to pepsin and trypsin, so that the HcCotA keeps enzyme activity in a complex biological environment, and the aflatoxin B1 can be efficiently degraded. The practical application range of the laccase in the field of grain safety guarantee is expanded, the laccase has great economic value and wide market prospect, and key technical support is provided for green and safe development of grain processing and storage industries.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and their applications, and particularly to the molecule of laccase HcCotA with high efficiency in degrading aflatoxin B1 and its applications. Background Art

[0002] Aflatoxins (AFT) are derivatives of dihydrofuranocoumarin, mainly produced by Aspergillus flavus and Aspergillus parasiticus. Common aflatoxin compounds include aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among the four aflatoxins, AFB1 has the highest toxicity and contamination rate in food grains and animal feeds, and has strong carcinogenicity, teratogenicity, and mutagenicity. The International Agency for Research on Cancer (IARC) of the World Health Organization has also classified AFB1 as a class I carcinogen. Research shows that 5%-28% of liver cancers may be related to the daily intake of AFB1. AFB1 is a pathogenic factor that causes growth inhibition, immune system disorders, and malnutrition in animals, and is an important obstacle to the immune system, cell metabolism, growth, and development of humans and animals.

[0003] Some studies have shown that Ery4 laccase can degrade 90% of AFB1 in feed raw materials through a mediator. However, there are some problems with the existing mediator systems, such as high cost, which increases the usage cost of the enzyme; poor mediator stability, and the generation of some toxic metabolic by-products, etc. These problems may affect the stability of the enzyme, resulting in a decrease in enzyme activity, which may limit the rate of electron transfer between the laccase and the recalcitrant substrate by the mediator small molecule, further affecting the progress of the oxidation reaction. Laccase CotA can catalyze AFB1 into less toxic AFQ1 without the assistance of a mediator, but it has disadvantages such as poor thermal stability, narrow pH range, and low substrate adaptability of the binding pocket, which severely limits the detoxification application of CotA to AFB1 in actual contaminated feeds and their raw materials.

[0004] Therefore, the present invention aims to provide the molecule of laccase HcCotA with high efficiency in degrading aflatoxin B1 and its applications to solve the above problems. Summary of the Invention

[0005] The object of the present invention is to solve the above problems, and to provide a molecule of laccase HcCotA that can efficiently degrade aflatoxin B1 and its application. HcCotA has excellent thermal stability and pH stability, and also has tolerance to pepsin and trypsin, enabling it to maintain enzyme activity in a complex biological environment, expanding the practical application scope of laccase in the field of food safety assurance, having great economic value and broad market prospects, and providing key technical support for the green and safe development of the food processing and storage industries.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a molecule of laccase HcCotA that can efficiently degrade aflatoxin B1 and its application, and the amino acid sequence of the laccase HcCotA is as follows:

[0008] MGSPNLEKFVDRLPLAEKIRPVREEGGIAYYEVTMEEFWQKLHRDLRPTRLWGYNRSFPGPLFDVPHGKKIRVKWTNHLPQRHFLPMDTTILDEMGTDFPEVRTVVHLHGGETEPDSDGYPEAWFTRDFNKTGPDFKKEVYEYTNSQRPATLWYHDHAIGITRLNVYAGLAGMYIIRDPKEKAFHLPSGKYEIPLLLTDRTFNNDGSLFYPRQPQNPGPETPDPSVVPFFLGDTILVNGKVWPYLEVEPRKYRFRIVNASNTRAYRLYLDSGQAFYQIGTDGGLLRRPVQVENLALEPAERADLILDFSEYAGQTILLKNDLGPNADPADQTGDVMQFRVVLPVAGEDTSRIPRSLSSIPVPSSHNVSAIRHLKLTGATDSYGRPLLLLDKKRWMDPVTETPRLGTTEIWSLANTTAFTHPIHIHLIQFQILDRRPFDLDLYNETGQIVYTGPATPPEPNERGFKDTVAAPGGQITRVMMRFSPYAGDYVWHCHILEHEDYDMMRPFQVIDPDLPASDGPLLDLEHHHHHH;

[0009] The gene sequence of the HcCotA is as follows:

[0010]

[0011] The HcCotA is tolerant to pepsin and trypsin.

[0012] The HcCotA can degrade aflatoxin B1, and the degradation rate of aflatoxin B1 by the HcCotA can be increased in an alkaline environment; the degradation rate of aflatoxin B1 by the HcCotA can be increased at a high temperature of 80°C.

[0013] The HcCotA mutant L386A can be constructed by genetic engineering technology, and the mutant L386A of the HcCotA can degrade aflatoxin B1 in corn flour and peanut oil.

[0014] The amino acid sequence of the HcCotA mutant L386A:

[0015] MGSPNLEKFVDRLPLAEKIRPVREEGGIAYYEVTMEEFWQKLHRDLRPTRLWGYNRSFPGPLFDVPHGKKIRVKWTNHLPQRHFLPMDTTILDEMGTDFPEVRTVVHLHGGETEPDSDGYPEAWFTRDFNKTGPDFKKEVYEYTNSQRPATLWYHDHAIGITRLNVYAGLAGMYIIRDPKEKAFHLPSGKYEIPLLLTDRTFNNDGSLFYPRQPQNPGPETPDPSVVPFFLGDTILVNGKVWPYLEVEPRKYRFRIVNASNTRAYRLYLDSGQAFYQIGTDGGLLRRPVQVENLALEPAERADLILDFSEYAGQTILLKNDLGPNADPADQTGDVMQFRVVLPVAGEDTSRIPRSLSSIPVPSSHNVSAIRHLKLTGATDSYGRPLALLDKKRWMDPVTETPRLGTTEIWSLANTTAFTHPIHIHLIQFQILDRRPFDLDLYNETGQIVYTGPATPPEPNERGFKDTVAAPGGQITRVMMRFSPYAGDYVWHCHILEHEDYDMMRPFQVIDPDLPASDGPLLDLEHHHHHH;

[0016] The gene sequence of the HcCotA mutant L386A:

[0017]

[0018] Advantages of this solution compared with the prior art:

[0019] In the present invention, the laccase HcCotA is cloned from Heyndrickxia coagulans, which has excellent thermal stability and pH stability, and also has tolerance to pepsin and trypsin, enabling it to maintain enzyme activity in a complex biological environment; the degradation rate of HcCotA for 2000 μg / L of AFB1 reaches 98.3% within 8 h. At the same time, the mutant L386A of HcCotA can degrade AFB1 in the corn flour and peanut oil system, effectively solving the problem of AFB1 contamination in grains such as corn and peanuts and their processed products, greatly expanding the practical application scope of laccase in the field of food safety assurance, having great economic value and broad market prospects, and providing key technical support for the green and safe development of the grain processing and storage industry. Brief Description of the Drawings

[0020] Figure 1 It is a schematic diagram of SDS-PAGE and ultraviolet-visible light scanning for purifying HcCotA in the embodiment of the present invention. Among them, A is the SDS-PAGE for purifying HcCotA, lane M: protein marker; lane 1: purified HcCotA; B is the full-wavelength scanning diagram of HcCotA. The purified HcCotA is placed in 0.02 mol / L phosphate buffer (pH 7.4), and the ultraviolet-visible absorption spectrum is recorded in the range of 300 - 800 nm.

[0021] Figure 2 It is a pH diagram of HcCotA degrading AFB1 in the embodiment of the present invention.

[0022] Figure 3 It is a temperature diagram of HcCotA degrading AFB1 in the embodiment of the present invention.

[0023] Figure 4 It is a schematic diagram showing the influence of HcCotA enzyme concentration on AFB1 degradation in the embodiment of the present invention.

[0024] Figure 5 It is a schematic diagram of enzyme kinetics of HcCotA using AFB1 as a substrate in the embodiment of the present invention.

[0025] Figure 6 It is a schematic diagram showing the enzymatic hydrolysis tolerance of HcCotA to pepsin and the enzymatic hydrolysis resistance characteristics to trypsin in the embodiment of the present invention.

[0026] Figure 7 It is a binding model diagram of AFB1 and the molecular surface of laccase HcCotA (the loop is the key amino acid residue) in the embodiment of the present invention.

[0027] Figure 8 In A, it is the analysis of the conserved sequence of HcCotA. The red triangle represents the copper ion binding site, the black triangle represents the predicted AFB1 binding site, the green represents the non-conserved sequence, and the magenta represents the conserved sequence; in B, it is the molecular docking of HcCotA and AFB1 (the white ring around the pocket is the key amino acid residue).

[0028] Figure 9 It is the kinetic schematic diagram of L386A using AFB1 as a substrate in the embodiment of the present invention;

[0029] Figure 10 It is the schematic diagram of the degradation effect of L386A and WT on AFB1 in corn flour in the embodiment of the present invention;

[0030] Figure 11 It is the influence of L386A and WT on the degradation of AFB1 in peanut oil in the embodiment of the present invention. Detailed implementation manners

[0031] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0032] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0033] Example 1: Reagents

[0034] (1) Liquid LB: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder, and made up to 1 L with deionized water. Solid LB: 25 g of agar powder was added to 1 L of liquid LB. Sterilize at 121 °C for 20 min and reserve for use.

[0035] (2) Liquid fermentation medium: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder, 20 g of glucose. Made up to 1 L with deionized water. Solid fermentation medium: 25 g of agar powder was added to 1 L of liquid fermentation medium. Sterilize at 121 °C for 20 min and reserve for use after sterilization.

[0036] (3) Glycine buffer: 3.0 g of tris(hydroxymethyl)aminomethane, 1.0 g of sodium dodecyl sulfate, 14.4 g of glycine, made up to 1 L with deionized water, and stored at room temperature.

[0037] (4) Coomassie Brilliant Blue R-250 Staining Solution: 1.0 g of Coomassie Brilliant Blue R-250, 450 mL of methanol, 100 mL of glacial acetic acid, 450 mL of deionized water, stored at room temperature in a brown bottle.

[0038] (5) Coomassie Brilliant Blue G-250 Staining Solution: 0.1 g of Coomassie Brilliant Blue G-250, 50 mL of 95% ethanol, 120 mL of 85% phosphoric acid, made up to 1 L with deionized water, stored in the dark at 4°C in a refrigerator.

[0039] (6) Coomassie Staining Decolorizing Solution: 100 mL of glacial acetic acid, 200 mL of methanol, made up to 1 L with deionized water, stored at room temperature in the dark.

[0040] (7) 50 mmol / L Citric Acid-Sodium Citrate Buffer (pH 3.0 - 5.0): Prepare stock solution A by dissolving 2.1 g of citric acid in 100 mL of deionized water. Prepare stock solution B by dissolving 2.94 g of sodium citrate in 100 mL of deionized water. For pH 3.0 buffer, add 93.0 mL of stock solution A and 7.0 mL of stock solution B. For pH 4.0 buffer, add 65.5 mL of stock solution A and 34.5 mL of stock solution B. For pH 5.0 buffer, add 41.0 mL of stock solution A and 59.0 mL of stock solution B.

[0041] (8) 50 mmol / L Phosphate Buffer (pH 6.0 - 8.0): Dissolve 0.78 g of sodium dihydrogen phosphate in 100 mL of deionized water to prepare stock solution A, and dissolve 1.795 g of disodium hydrogen phosphate in 100 mL of deionized water to prepare stock solution B. For pH 6.0 buffer, add 87.7 mL of stock solution A and 12.3 mL of stock solution B. For pH 5.0 buffer, add 39.0 mL of stock solution A and 61.0 mL of stock solution B. For pH 8.0 buffer, add 5.3 mL of stock solution A and 94.7 mL of stock solution B.

[0042] (9) 50 mmol / L Tris-HCl Buffer (pH 8.9): Dissolve 1.211 g of Tris in 100 mL of deionized water to prepare stock solution A, and dissolve 1 mL of concentrated hydrochloric acid in 119 mL of deionized water to prepare stock solution B. Add 50.0 mL of stock solution A and 7.0 mL of stock solution B, and make up to 100 mL with deionized water.

[0043] (10) 1% Agarose Nucleic Acid Gel: Add 0.5 g of agarose to 50 mL of 1×TAE buffer, heat for 2 min until completely melted, add 5 μL of 10000×ExRed nucleic acid dye, pour into the gel tank, insert the comb, and wait for it to solidify.

[0044] (11) 10% Protein Gel: Add 2.7 mL each of separating gel buffer and separating gel solution, add 55 μL of modified ammonium persulfate solution, mix well, and inject into the gel-casting glass plate; Add 0.75 mL each of stacking gel buffer and stacking gel solution, add 15 μL of modified ammonium persulfate solution, mix well, inject into the gel-casting glass plate, and insert the comb teeth.

[0045] Example 2: Method

[0046] 1. Cloning of HcCotA

[0047] The glycerol bacteria of H. coagulans stored at -80 °C were streaked in three zones on the solid fermentation medium and cultured inverted at 40 °C for 24 h until single colonies grew on the plate. Pick a single colony and inoculate it into 5 mL of liquid fermentation medium, and culture it at 40 °C at 200 rpm for 24 h. Repeat the above operations twice.

[0048] Extract the genomic DNA of H. coagulans. Using the genomic DNA of H. coagulans as the DNA template, amplify the target gene with reference to the upstream and downstream primers with restriction enzyme sites added in Table 1. The amplified PCR product was detected by 1% agarose gel and recovered and purified by the micro-column concentrated DNA gel recovery kit of Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0049] Table 1 DNA Template Reference Table

[0050]

[0051] Use the micro-column concentrated DNA gel recovery kit to recover the digested HcCotA gene fragment and pET28a vector, and perform ligation by reacting with T4 DNA ligase at 25 °C for 30 min. Transform the ligated product into Escherichia coli competent cell DH5α, ice-bath for 30 min, heat-shock at 42 °C for 90 s, and ice-bath again for 5 min. Add 900 μL of liquid LB, resuscitate at 37 °C and 220 rpm in a shaker for 1 h, centrifuge at 12000 rpm for 5 min, discard most of the supernatant, gently pipette the precipitate to mix it evenly and spread it evenly on a solid LB plate containing 50 μg / mL kanamycin (Kana), and culture it inverted at 37 °C overnight.

[0052] Pick the monoclonal on the plate and pipette it repeatedly in ddH2O. Take the bacterial solution as the template for colony PCR for PCR amplification and verify it by 1% agarose gel electrophoresis. Pick the positive clone and expand it in a 10 mL liquid LB test tube containing Kana for 8 h, and extract the plasmid. Send part of the plasmid for sequencing, and the sequencing primers are the universal primers T7 and T7ter of pET28a. Use the SnapGene software to align the sequencing results with the target gene to judge whether the plasmid is successfully constructed.

[0053] 2. Heterologous expression and purification of HcCotA

[0054] Transform pET28a-HcCotA into Escherichia coli BL21(DE3) and incubate it upside down overnight at 37°C. Pick a single colony and inoculate it into 10 mL of sterile LB containing 50 μg / mL of Kana final concentration, and shake it at 37°C for 12 h. Inoculate the bacterial solution into liquid LB containing 50 μg / mL of Kana at a ratio of 1:100, and shake it at 37°C until the OD600nm reaches 0.6 - 0.8. Then add copper sulfate (CuSO4) with a final concentration of 0.5 mmol / L and IPTG with a final concentration of 0.5 mmol / L, and culture it at 180 rpm at 16°C for 6 h. Centrifuge the bacteria at 6000 rpm for 10 min to collect the cells, and resuspend the cells in 50 mL of buffer (20 mmol / L phosphate buffer, pH 7.4). Break the cells using a low-temperature ultra-high pressure cell disruptor, and centrifuge at 12000 rpm at 4°C for 30 min. The supernatant is the crude enzyme solution. Elute the target protein through a nickel ion affinity chromatography column, and concentrate it using an ultrafiltration tube with a cut-off value of 30 kDa (Millipore, USA).

[0055] 3. Determination of the enzymatic properties of HcCotA

[0056] (1) Determination of the enzyme activity of HcCotA

[0057] The ABTS oxidation method was used to determine the activity. Laccase can oxidize ABTS to generate ABTS + radicals, and the product shows a dark green color and has the maximum absorbance at OD420nm. The total reaction system is 200 μL. In 50 mM sodium citrate buffer pH 4.0, it contains 1 mM ABTS and an appropriate amount of enzyme solution. After reacting at 70°C for 10 min, terminate the reaction on ice, and measure the absorbance at 420 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, USA).

[0058] (2) Optimal reaction conditions of HcCotA

[0059] Under buffer conditions such as citrate-sodium citrate buffer (pH 3.0 - 6.0), Tris-HCl buffer (pH 7.0 - 9.0), etc., using ABTS as the substrate, measure the enzyme activity of HcCotA at 70°C, and measure the absorbance of ABTS and DMP at 420 nm and 469 nm respectively. The pH at the highest enzyme activity obtained is the optimal pH of HcCotA.

[0060] Under the condition of 50 mmol / L citric acid-sodium citrate buffer (pH 4.0), using ABTS as the substrate, the enzyme activity of HcCotA was measured at 50 °C - 90 °C, and the temperature at the highest enzyme activity was taken as the optimum temperature of HcCotA.

[0061] (3) pH stability and thermal stability of HcCotA

[0062] HcCotA was incubated at 4 °C for a certain time under different pH conditions such as 50 mmol / L citric acid-sodium citrate buffer (pH 3.0 - 4.0), 50 mmol / L phosphate buffer (pH 7.0), and 50 mmol / L Tris-HCL buffer (pH 8.9). Then, using ABTS as the substrate, the residual enzyme activity of HcCotA was measured at 70 °C, with the enzyme activity of untreated HcCotA taken as 100%.

[0063] HcCotA was incubated at 50 °C, 70 °C, and 80 °C for a certain time in 20 mmol / L phosphate buffer (pH 7.4). Then, using ABTS as the substrate, the residual enzyme activity of HcCotA was measured at 70 °C, with the enzyme activity of untreated HcCotA taken as 100%.

[0064] (4) Determination of enzyme kinetic parameters of HcCotA

[0065] Under the condition of 50 mmol / L citric acid-sodium citrate buffer (pH 4.0), the enzyme activity of HcCotA towards different concentrations of ABTS was measured at 70 °C, and Km and kcat were calculated and analyzed by the Miachelis-Menten equation. m and k cat .

[0066] Under the condition of 50 mmol / L phosphate buffer (pH 6.0), the enzyme activity of HcCotA towards different concentrations of DMP was measured at 70 °C, and Km and kcat were calculated and analyzed by the Miachelis-Menten equation. m and k cat .

[0067] 4. Detection of AFB1 by fluorescence-high performance liquid chromatography and ELISA kit

[0068] Determination of AFB1 by fluorescence - high performance liquid chromatography: C18 reverse phase column (5μm, 6×100mm), photochemical derivatizer (Aura Industries, Staten Island, NY USA) and fluorescence detector (Shimadzu RF - 20A, Shimadzu, Tokyo, Japan). The sample was filtered using a 0.22μm filter. The excitation wavelength and emission wavelength for detecting AFB1 were 360nm and 440nm respectively. The injection volume was 10μL. The mobile phase was methanol and water (45:55, v / v), and the flow rate was 0.8mL / min. A linear relationship was established by analyzing 7 AFB1 standard solutions in the range of 0, 0.001, 0.01, 0.1, 0.5, 1.0 and 2.0μg / mL.

[0069] Detection of AFB1 by ELISA kit: Enough well strips for standard and sample detection were inserted into the microplate holder. Record the positions of the standards and samples. Add 50μL of the prepared sample solution to the corresponding wells. Add 50μL of the enzyme conjugate solution to each well. Add 50μL of the aflatoxin antibody solution to each well, mix well, and incubate at room temperature (20℃ - 25℃) for 30 minutes. Pour out the liquid in the wells, invert the microplate holder and tap it on the absorbent paper (tap 3 times per round) to ensure complete removal of the liquid in the wells. Add 250μL of the washing buffer, and pour out the liquid in the wells again. The above operations were repeated twice. Add 100μL of the substrate / chromogenic agent to each well, mix well and incubate in the dark at room temperature (20℃ - 25℃) for 15min. Add 100μL of the reaction termination solution to each well and mix well. Measure the absorbance at 450nm within 15min after adding the reaction termination solution.

[0070] 5. Degradation of AFB1 by HcCotA

[0071] (1) Optimal pH for HcCotA to degrade AFB1

[0072] In 50mmol / L citric acid - sodium citrate buffer (pH3.0 - 5.0), 50mmol / L phosphate buffer (pH6.0 - 8.0), 50mmol / L Tris - HCL buffer (pH8.9), add HcCotA with a concentration of 0.02mg / mL and AFB1 with a concentration of 2000μg / L. The control group was adding the same concentration of AFB1 without enzyme in the corresponding pH buffer. React at 37℃ for 2h and terminate the reaction by boiling for 10min. Determine the degradation rate of AFB1, and calculate the degradation rate using DR=(1 - EG / CG)×100%, where DR is the degradation rate; EG and CG are the AFB1 concentrations of the experimental group and the control group respectively. The pH corresponding to the highest degradation rate is the optimal pH for HcCotA to degrade AFB1.

[0073] (2) Optimal temperature for HcCotA to degrade AFB1

[0074] Under the condition of 50 mmol / L phosphate buffer (pH 8.0), add HcCotA with a concentration of 0.02 mg / mL and AFB1 with a concentration of 2000 μg / L, react at 50 °C - 90 °C for 2 h, and terminate the reaction by boiling for 10 min. The degradation rate of AFB1 by HcCotA is determined by an ELISA kit, and the temperature corresponding to the highest degradation rate is the optimal temperature for HcCotA to degrade AFB1.

[0075] (3) Enzyme kinetics of HcCotA with AFB1 as the substrate

[0076] The kinetic determination of HcCotA with AFB1 as the substrate is carried out under the condition of 50 mmol / L phosphate buffer (pH 8.0). HcCotA with a concentration of 0.02 mg / mL is incubated with different concentrations of AFB1 at 80 °C for 20 min, and K m and k cat .

[0077] (4) Degradation effect of HcCotA on AFB1

[0078] Under the condition of 50 mmol / L phosphate buffer (pH 8.0), add AFB1 with a concentration of 2000 μg / L and the corresponding concentration of HcCotA, incubate at 50 °C, the total system is 200 μL, and the control group is set without adding enzyme, incubate for the same time, and terminate the reaction on ice after the reaction. Take 10 μL and add 900 μL of 70% methanol, shake for 10 min, then mix evenly with deionized water at a ratio of 1:1, centrifuge at 7000 rpm for 5 min, and then determine the AFB1 degradation efficiency with an ELSA kit.

[0079] 6. Tolerance of HcCotA to pepsin and trypsin

[0080] Under the condition of 50 mmol / L phosphate buffer (pH 8.0), add HcCotA with a concentration of 0.2 mg / mL and pepsin solution with a concentration of 0.025 mg / mL and pH 4, incubate at 37 °C for 6 h, 12 h, 24 h, and detect by SDS-PAGE.

[0081] Under the condition of 50 mmol / L phosphate buffer (pH 8.0), add HcCotA with a concentration of 0.2 mg / mL and trypsin solution with a concentration of 0.025 mg / mL and pH 8, incubate at 37 °C for 6 h, 12 h, 24 h, and detect by SDS-PAGE.

[0082] 7. Molecular Docking of HcCotA and AFB1

[0083] The tertiary structure of laccase HcCotA was analyzed using PyMol. The substrate molecule AFB1 was docked into the HcCotA molecule using the software Auto Dock Tools 1.5.6 to predict the key amino acid residues.

[0084] 8. Construction of the HcCotA-L386 Mutant

[0085] Using pET28a-HcCotA as a template, according to the upstream and downstream primers of the mutants in Table 2, a PCR reaction system was configured. DpnⅠ restriction endonuclease was added to the PCR product and digested at 37 °C for 4 h. The digested product was transformed into Escherichia coli competent cells DH5α, and monoclonal colonies were picked and cultured in a shaker. Then the plasmids were extracted and sequenced to verify whether the mutants were successfully constructed.

[0086] Table 2 Mutant Sequence Table

[0087]

[0088]

[0089]

[0090] 9. Identification of the HcCotA Mutant with High-efficiency Degradation of AFB1

[0091] 0.02 mg / mL of the enzyme and 2000 μg / L of AFB1 were added to 50 mmol / L phosphate buffer (pH 8.0) and incubated at 45 °C for 2 h. The control group was treated under the same conditions without adding the enzyme. The reaction was terminated on ice, and the AFB1 concentration was measured using an ELISA kit.

[0092] 10. Determination of the Enzyme Kinetic Parameters of L386A

[0093] The enzyme kinetics in HcCotA was referred for determination.

[0094] 11. Detoxification Effect of L386A on AFB1 in Corn Flour

[0095] The corn flour was treated with 50 mmol / L phosphate buffer with a humidity of 250 mg / mL, 0.02 mg / mL enzyme was added, the AFB1 concentration was 100 μg / L, the treatment temperature was 70 °C, and the reaction was carried out at 1500 rpm for 2 h. After the reaction, 200 μL of the sample, 600 μL of methanol and water extractant (45:55, v / v) were added to a 1.5 mL centrifuge tube and vortexed for 5 min. The sample was placed at -20 °C for 2 h to precipitate proteins. Then it was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm nylon microfiltration membrane and collected in a sample bottle for analysis. It should be noted that during the extraction process, two grinding beads need to be added to shake and break the particle agglomerates in the system, thereby improving the extraction efficiency of AFB1.

[0096] 12. Detoxification effect of L386A on AFB1 in peanut oil

[0097] In the degradation system, the concentration of L386A was 0.05 mg / mL, the AFB1 concentration was 100 μg / L, and the corresponding volume of 50 mmol / L phosphate buffer (pH 8.0). The effect of the buffer volume ratio on the degradation of AFB1 was studied. The buffer volumes were 0%, 5%, 10%, 15% and 20% (v / v) respectively. The control group was treated under the same conditions without adding enzyme. After the reaction, an equal volume of 75% methanol was added, and AFB1 in peanut oil was extracted according to the national standard (GB5009.96 - 2016). 200 μL of peanut oil sample and 600 μL of methanol and water extractant (45:55, v / v) were added to a 1.5 mL centrifuge tube. It was placed at -20 °C for 2 h to precipitate proteins, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm nylon microfiltration membrane and collected in a sample bottle for analysis.

[0098] AFB1 with a final concentration of 100 μg / mL and 500 μg / mL was added to peanut oil respectively, 0.05 mg / mL of enzyme and 15% (pH 8.0) phosphate buffer were added respectively, and the reaction was carried out at 70 °C for 5 h at 1500 rpm.

[0099] Example 3: Conclusion

[0100] 1. Expression and purification of HcCotA

[0101] pET28a - HcCotA was expressed in Escherichia coli BL21(DE3) cells and purified by Ni column. As Figure 1As shown in Figure A, the recombinant protein HcCotA was successfully expressed in Escherichia coli BL21(DE3) strain. After one-step purification by nickel ion affinity chromatography, the purity could reach over 90%, which could be used for subsequent property exploration. The SDS-PAGE of HcCotA showed that the molecular weight of the protein was 60 kDa, which was consistent with the predicted molecular weight of HcCotA with 6 His tags at the C-terminus. As Figure 1 shown in Figure B, the ultraviolet-visible spectrum of HcCotA had an obvious absorption peak at 610 nm, possessing the characteristics of a typical blue laccase.

[0102] 2. Optimal conditions for HcCotA to degrade AFB1

[0103] (1) Optimal pH for HcCotA to degrade AFB1

[0104] pH is a crucial factor in the degradation of AFB1 by HcCotA. Studies have shown that the concentration of AFB1 is 2000 μg / L, the concentration of laccase HcCotA is 0.05 mg / mL, at 37 °C, and incubated for 2 h under the conditions of pH 5.0 - 9.0. The degradation effect is as Figure 2 shown. The degradation of AFB1 proceeds rapidly in an alkaline environment. The highest degradation rate of AFB1 by HcCotA is 60.6% at pH 8.0; the degradation ability decreases sharply under acidic and neutral conditions. When pH is 5.0 - 7.0, the degradation rate of AFB1 is between 6.2% and 12.6%.

[0105] (2) Optimal temperature for HcCotA to degrade AFB1

[0106] The effect of temperature on the degradation of AFB1 catalyzed by HcCotA is shown in Figure 3 Figure. It can be seen from the figure that the optimal temperature for HcCotA to degrade AFB1 is 80 °C. When the reaction temperature increases from 50 °C to 80 °C, the degradation efficiency of AFB1 by it increases from 43.5% to 97.5%; when the reaction temperature increases from 80 °C to 90 °C, the degradation efficiency of AFB1 by HcCotA decreases to 20%. Therefore, HcCotA has a high degradation ability for AFB1 under appropriate high-temperature conditions. Due to the high-temperature tolerance of HcCotA, during the high-temperature degreasing process of peanut oil, it can directly play a degradation role without cooling, saving industrial processing costs.

[0107] 3. Degradation effect of HcCotA on AFB1

[0108] Studies have shown that the degradation rates of 0.02 mg / mL and 0.04 mg / mL of HcCotA on 2000 μg / L of AFB1 reach 90.9% and 98.3% respectively in 8 h ( Figure 4)。In the study by Sun et al., BlCotA at a concentration of 0.04 mg / mL required up to 24 h of catalysis to degrade 1000 μg / L of AFB1 to achieve a degradation efficiency of 90.3%. The concentration of AFB1 in this system was much lower than that used in the current study, and HcCotA was far superior to BlCotA in terms of the degradation rate and efficiency of AFB1. Therefore, HcCotA demonstrated the ability to rapidly and efficiently degrade AFB1.

[0109] The kinetic parameters of HcCotA for AFB1, such as Figure 5 , K m , k cat and k cat / K m were 25.67 μmol*L -1 , 2.10 min -1 and 0.08 min -1 *μmol*L -1 . The K m of laccase BlCotA from Bacillus licheniformis for AFB1 as the substrate was 60.62 μmol / L. In contrast, the K m of HcCotA for AFB1 was lower and the affinity was higher. Therefore, its oxidation efficiency for the substrate was also higher than that of BlCotA.

[0110] 4. Tolerance of HcCotA to pepsin and trypsin

[0111] The results, as Figure 6 shown, indicated that the major proteases in the gastrointestinal tract hardly damaged the structural integrity of HcCotA.

[0112] 7. Interaction between HcCotA and AFB1

[0113] The structural model of the HcCotA-AFB1 complex obtained by molecular docking guided the study of the interaction between HcCotA and AFB1 to a certain extent. As Figure 7 shown, when L386A bound to AFB1, there were electrostatic interactions, hydrogen bond interactions, hydrophobic interactions, etc. between them. Among them, the cyclopentenone structure at the toxic site 3 of AFB1 was close to the T1 copper ion binding sites H419 and H497, and F228, L386, L230, etc. mainly stabilized AFB1 through π-π and hydrophobic interactions.

[0114] 8. Analysis of the conserved sequence of HcCotA

[0115] Based on the conservative sequence analysis of HcCotA, it was found that the key amino acid sites of HcCotA and AFB1 are non-conservative sequences. Based on the molecular docking results of HcCotA and AFB1, candidate amino acid residues L321, L386, F417, etc. that interact with AFB1 will be mutated at these sites.

[0116] 9. Identification of HcCotA Mutants with High Efficiency in Degrading AFB1

[0117] To effectively screen candidate mutants, the initially predicted mutants were expressed and purified, and the degradation of AFB1 was studied. The degradation efficiencies of the mutants and the wild type were compared to screen for highly active mutant enzymes. The screening of mutants at the F417 site is shown in Table 3. The degradation activities of mutants F417M, F417I, F417K, F417N, F417P, F417R, F417G and WT against AFB1 were almost the same, and had almost no effect on the degradation activity of AFB1. The activities of F417A, F417H, F417Y were increased by 27.4%, 25.4% and 27.4% respectively compared to WT. However, the degradation activities of F417E, F417C, F417L, F417W against AFB1 decreased significantly, which may be due to the changes in protein structure caused by these mutations, thus affecting their degradation activities.

[0118] Table 3 Screening of Mutants at the F418 Site

[0119]

[0120]

[0121] Explore the effects of sites such as F228, N324, and T418 on the degradation of AFB1. The results are shown in Table 4. The degradation activities of 13 mutants such as F228W and N324A against AFB1 had no significant effect compared to WT. The current model may not be able to accurately predict the effects of key amino acid sites on activity and cannot further guide the adaptation modification of the substrate pocket for AFB1.

[0122] Table 4 Screening of the HcCotA Mutant Library

[0123]

[0124] The large side chain of L386 has a certain steric hindrance for AFB1 to approach H497 further. The degradation rate of AFB1 by the mutant L386A with a small side chain amino acid residue at position 386 was significantly increased, while the activity of L386Y in degrading AFB1 decreased significantly (Table 5). The molecular docking results showed that in the L386A mutant, H497 was closer to the cyclopentenone double bond of AFB1, and the electron transfer efficiency was more efficient.

[0125] Table 5 Effects of L386 and L321Y on activity

[0126]

[0127] Studies on the L321 site have shown that mutating it to L321Y can significantly enhance the degradation activity by 30.1%. This may be because the L321Y mutant can further contract the HcCotA substrate pocket, increasing the adaptability to AFB1. However, the degradation efficiency of the L321Y / L386A double mutant for AFB1 has decreased significantly, and the current molecular docking model may not be able to explain this result. It is speculated that L321Y / L386A may have an adverse effect on the structure or functional region of the enzyme, resulting in a decrease in the degradation rate.

[0128] 10. Enzyme kinetics of L386A catalyzing AFB1 degradation

[0129] The Km and kcat of L386A for AFB1 m and kcat cat are 13.19 μmol·L -1 , 41.27 min -1 , respectively. The degradation activity kcat / Km cat / Km m = 3.12 min -1 ·μmol·L -1 . The kcat / Km of L386A for AFB1 cat / Km m is nearly 40 times higher than that of WT, with higher substrate affinity, more efficient electron transfer efficiency, and a significantly increased turnover number for oxidizing AFB1.

[0130] 11. Removal of AFB1 contamination in corn flour by HcCotA and L386A

[0131] The effects of complex components such as the moisture content and treatment temperature of real sample contaminated environments, such as corn flour and its processed products, on the degradation of AFB1 by related enzymes are not clear. To explore the detoxification effect of L386A in AFB1-contaminated corn flour ( Figure 8 ), in corn flour with a certain humidity, the degradation rate of L386A for 100 μg / L AFB1 at 0.5 h was 74.7%, which was 11.1% higher than that of WT. The degradation rate of L386A for 500 μg / L AFB1 at 0.5 h was 74.4%, and the degradation rate of AFB1 at 2 h was as high as 98.3%, which was significantly higher than that of WT. In this study, the detoxification effect of L386A was evaluated in the real environment of AFB1-contaminated corn flour, and a technical system for purifying AFB1 contamination in grains was constructed to provide technical support for achieving high-efficiency removal of AFB1 in feed.

[0132] 12. Removal of AFB1 contamination in peanut oil by L386A

[0133] Agricultural products such as peanuts and soybeans are vulnerable to mycotoxins such as AFB1, which will ultimately spread to the extracted oil. In this study, the contaminated peanut oil under simulated natural conditions was as follows Figure 9 . It was found that the degradation rate of L386A to 100 μg / L AFB1 was 94.0% at 3 h, which was significantly increased by 20.2% compared with the wild type (WT). Therefore, L386A has the advantages of high efficiency and strong specificity, and is more suitable for the detoxification of AFB1 in peanut oil than WT. L386A can adapt to the industrial application of high-temperature degumming of crude oil, reduce the cost of AFB1 removal, ensure the food security of our country, and promote the high-quality development of grain industries such as corn and peanuts.

[0134] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

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