Bacterial laccase Lac_P.E., encoding gene, its application and feed additive

Abstract

The present invention relates to a bacterial laccase Lac_P.E., its encoding gene, its application and a feed additive, belonging to the technical field of enzyme engineering. In the present invention, an expression strain of Lac_P.E. is constructed, and the bacterial laccase Lac_P.E. is obtained after induced expression and purification. Through high performance liquid chromatography detection and analysis, it is found that the laccase Lac_P.E. can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator, and can be widely used in the field of feed mycotoxin detoxification.

Description

Technical Field

[0001] The present invention relates to a bacterial laccase Lac_P.E., its encoding gene, its application and a feed additive, belonging to the technical field of enzyme engineering. Background Art

[0002] Mycotoxins are secondary metabolites of molds, which are carcinogenic, mutagenic and teratogenic, and can cause death of humans and animals. There are more than 500 kinds of mycotoxins, and the six mycotoxins that are mainly focused on and monitored in various countries and regions are aflatoxin (AF), zearalenone (ZEN), ochratoxin A (OTA), fumonisin (FB), vomitoxin (DON), and T-2 toxin. Grain crops are easily infected by toxin-producing molds in the field. If the environmental temperature and humidity are suitable, the molds will continue to grow and reproduce during the processes of processing, transportation and storage, and the content of mycotoxins will continue to increase.

[0003] The methods for degrading mycotoxins mainly include physical method, chemical method and biological method. The physical method has unstable effect and is easy to desorb, and at the same time, there is a phenomenon of non-specific adsorption of nutrients such as vitamins, mineral elements and amino acids. The chemical method has high treatment cost, and chemical residues and introduction of new harmful components affect the safety of feed. The biological method includes microbial method and enzymatic method, which have the advantages of safety, high efficiency and environmental friendliness, and is an important and effective method for mycotoxin detoxification. Since microorganisms contain harmful components such as virulence factors, and the essence of microbial detoxification is to degrade mycotoxins through enzymes produced by microorganisms, enzymatic detoxification is considered to be the best method for mycotoxin detoxification.

[0004] Enzymatic detoxification refers to the degradation enzyme converting the toxin into a low-toxic or non-toxic product through an enzymatic reaction, mainly including oxidases (such as laccase, manganese peroxidase) and hydrolases (such as esterase), etc. Peroxidase needs hydrogen peroxide as a substrate in the process of catalytic degradation of aflatoxin or zearalenone, and laccase catalytic degradation of aflatoxin or zearalenone requires the participation of a mediator. Therefore, the effectiveness and safety of peroxidase and laccase-mediator system for mycotoxin detoxification in feed and food need to be further evaluated.

[0005] At present, most of the degradation enzymes reported at home and abroad can only specifically degrade a single type of mycotoxin and cannot effectively degrade toxins of different structural types, which are greatly limited in practical applications. Therefore, finding and developing new broad-spectrum enzymes for simultaneous degradation of multiple mycotoxins is the key to enzymatic detoxification technology. Summary of the Invention

[0006] The present invention provides a protein encoding gene Lac_P.E. of a bacterial laccase Lac_P.E., and this gene can encode the bacterial laccase Lac_P.E.

[0007] The present invention provides a bacterial laccase Lac_P.E., which can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator.

[0008] The present invention provides the application of the bacterial laccase Lac_P.E., which can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator, and can be widely used for detoxifying mycotoxins in grains, foods, feeds and their processing by-products.

[0009] The present invention also provides a feed additive, which can effectively degrade mycotoxins in moldy feeds.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] A bacterial laccase Lac_P.E. encoding gene Lac_P.E. gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] The Lac_P.E. gene encodes a bacterial laccase Lac_P.E., which can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator, and has important significance for the research in the field of mycotoxin detoxification.

[0013] A bacterial laccase Lac_P.E., the amino acid sequence of which is shown in SEQ ID NO.2.

[0014] This bacterial laccase can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator, and can be used in the field of mycotoxin detoxification.

[0015] The application of the bacterial laccase Lac_P.E. can be applied in the field of mycotoxin detoxification.

[0016] This bacterial laccase can degrade aflatoxin, zearalenone, vomitoxin, ochratoxin, T-2 toxin and fumonisin simultaneously without relying on any mediator, and can be applied in the field of mycotoxin detoxification such as grains, foods, feeds, etc.

[0017] Preferably, the mycotoxin is aflatoxin B 1 (AFB 1 ), zearalenone (ZEN), vomitoxin (DON), ochratoxin (OTA), T-2 toxin (T-2) and fumonisin (FB 1 ) or a combination of two or more of them.

[0018] Further preferably, the degradation of the mycotoxin by bacterial laccase Lac_P.E. is carried out under mediator-free conditions.

[0019] Further preferably, the pH for the degradation of aflatoxin B by bacterial laccase Lac_P.E. 1 is 4 - 12, and the reaction temperature is 20 - 60 °C.

[0020] Further preferably, the pH for the degradation of aflatoxin B by bacterial laccase Lac_P.E. 1 is 9, and the reaction temperature is 30 °C.

[0021] A feed additive comprising bacterial laccase Lac_P.E.

[0022] This feed additive can effectively degrade mycotoxins in moldy feed. Brief Description of the Drawings

[0023] Figure 1 It is the SDS-PAGE diagram of the purified expression product of recombinant plasmid pET-21a-Lac_P.E. in Example 2 of the present invention; wherein lane 1 is the purified recombinant Lac_P.E., and lane M is the protein molecular weight standard (250, 150, 100, 70, 50, 40, 35, 20 kDa);

[0024] Figure 2 It is the HPLC analysis result of the degradation of AFB by recombinant Lac_P.E. in Example 3 of the present invention 1 (A is the AFB 1 blank control group, B is the treatment group with Lac_P.E. added);

[0025] Figure 3 It is the HPLC analysis result of the degradation of ZEN by recombinant Lac_P.E. in Example 3 of the present invention (A is the ZEN blank control group, B is the treatment group with Lac_P.E. added);

[0026] Figure 4 It is the HPLC analysis result of the degradation of DON by recombinant Lac_P.E. protein in Example 3 of the present invention (A is the DON blank control group, B is the treatment group with Lac_P.E. added);

[0027] Figure 5 It is the HPLC analysis result of the degradation of OTA by recombinant Lac_P.E. in Example 3 of the present invention (A is the OTA blank control group, B is the treatment group with Lac_P.E. added);

[0028] Figure 6HPLC analysis results of the degradation of T-2 by recombinant Lac_P.E. in Example 3 of the present invention (A is the T-2 blank control group, and B is the group treated with Lac_P.E.);

[0029] Figure 7 HPLC analysis results of the degradation of FB by recombinant Lac_P.E. in Example 3 of the present invention 1 (A is the FB 1 blank control group, and B is the group treated with Lac_P.E.);

[0030] Figure 8 Degradation of AFB by Lac_P.E. under different pH conditions in Example 4 of the present invention 1 ;

[0031] Figure 9 Degradation of AFB by Lac_P.E. under different temperature conditions in Example 4 of the present invention 1 ;

[0032] Figure 10 Degradation of AFB by Lac_P.E. under different enzyme amount conditions in Example 4 of the present invention 1 ;

[0033] Figure 11 Effect of Lac_P.E. on the simultaneous degradation of AFB 1 , ZEN, DON, OTA, T-2 and FB 1 in Example 5 of the present invention. Detailed implementation manners

[0034] The present invention will be further described in detail below with reference to specific examples. Unless otherwise specified, the equipment and reagents used in each example can be obtained commercially, and the technical means used in the examples are conventional means used by those skilled in the art.

[0035] Some of the biological materials, experimental reagents, etc. involved in the following examples are briefly introduced as follows:

[0036] Biological materials:

[0037] Vector: Escherichia coli expression vector pET-21a, purchased from Invitrogen.

[0038] Strain: BL21(DE3) chemically competent cells, purchased from Invitrogen.

[0039] Experimental reagents: Aflatoxin B 1 , zearalenone, deoxynivalenol, ochratoxin, T-2 toxin and fumonisin standards, purchased from sigma; other reagents are domestic analytical pure.

[0040] Example 1 Bacterial laccase Lac_P.E. encoding gene Lac_P.E. gene

[0041] The nucleotide sequence of the bacterial laccase Lac_P.E. encoding gene Lac_P.E. gene in this example is shown in SEQ ID NO.1.

[0042] Example 2 Bacterial laccase Lac_P.E.

[0043] The amino acid sequence of the bacterial laccase Lac_P.E. in this example is shown in SEQ ID NO.2. The specific description of preparing the bacterial laccase Lac_P.E. is as follows:

[0044] 1. Construction of Lac_P.E. expression strain

[0045] 1.1 Construction of Lac_P.E. expression vector

[0046] The Lac_P.E. encoding gene from Priestia was synthesized by Anhui General Biosystems Co., Ltd. and ligated between the BamHI and XhoI restriction sites of pET-21a. The Lac_P.E. encoding gene from Priestia was codon-optimized to be more conducive to expression in Escherichia coli; site-directed mutations were made on individual amino acids (Q212R, D320N) to make the catalytic activity of the encoded bacterial laccase Lac_P.E. protein higher.

[0047] 1.2 Construction of Lac_P.E. expression strain

[0048] Transform Escherichia coli competent cell BL21(DE3), screen on an Amp-resistant plate, pick positive transformants, extract the transformed plasmid, and perform double digestion verification and sequencing to determine the correct recombinant strain BL21(DE3) / pET-21a-Lac_P.E.

[0049] 2. Induced expression and purification of Lac_P.E. in Escherichia coli

[0050] 2.1 Induced expression of Lac_P.E.

[0051] Inoculate BL21(DE3) / pET-21a-Lac_P.E. into 50 mL of LB culture medium, culture with shaking at 37 °C and 180 rpm for 12 h, then transfer it to 400 mL of LB medium at a ratio of 1%, culture with shaking at 37 °C and 180 rpm for about 3 h (OD600 ≈ 0.6), add IPTG with a final concentration of 1 mM and CuSO 4 , and induce at 16 °C for 16 h.

[0052] 2.2 Purification of Lac_P.E.

[0053] Collect the fermentation broth, centrifuge at 12,000 rpm for 30 min at 4°C, and discard the supernatant; resuspend the cells with phosphate buffer (pH 7.0), centrifuge at 12,000 rpm for 30 min at 4°C, discard the supernatant, and repeat the cell washing three times. Then resuspend the cells in phosphate buffer-0.5 mM CuSO 4 (20 mM, pH 7.0), sonicate, centrifuge at 12,000 rpm for 10 min at 4°C, collect the supernatant and filter. Since the C-terminus of the expressed Lac_P.E. carries a histidine tag (6×His), a nickel ion affinity chromatography column (Ni2+-NTA) is used to purify the recombinant protein. The procedures of equilibration, sample loading, elution, etc. refer to the Qiagen user manual. The purified protein is ultrafiltered using a cut-off tube (10 kDa) to remove the imidazole contained therein, and the purification result of the target protein is detected by SDS-PAGE electrophoresis. The results are as Figure 1 shown. Lane 1 is the expression product, indicating that the molecular weight of the protein expressed by the recombinant strain is approximately 63 kDa, which is consistent with the theoretical molecular weight.

[0054] Example 3 Application of Bacterial Laccase Lac_P.E. in Mycotoxin Detoxification

[0055] For the application of the bacterial laccase Lac_P.E. in mycotoxin detoxification in this example, the bacterial laccase Lac_P.E. is used to degrade aflatoxin B 1 , zearalenone, vomitoxin, ochratoxin, T2 toxin, fumonisin respectively under mediator-free conditions, and its degradation activity is detected by high performance liquid chromatography. The specific steps are as follows:

[0056] 1. Detection of the degradation activity of Lac_P.E. against aflatoxin B 1

[0057] Dissolve aflatoxin B 1 in methanol to prepare a stock solution of 100 μg / mL. Conduct experiments according to the following 1000 μL reaction system: 980 μL sodium phosphate buffer (0.1 M, pH 9.0), 10 μL Lac_P.E. protein (final enzyme activity 3 U / mL), 10 μL aflatoxin B 1 solution. Use the system without adding Lac_P.E. as a control. After the reaction is carried out at 30°C for 24 h, add 3 mL of methanol to terminate the reaction, and detect by high performance liquid chromatography. Analyze whether Lac_P.E. has degradation activity against AFB 1 according to the change in the AFB 1 concentration.

[0058] Detection of AFB by high performance liquid chromatography1 The chromatographic conditions are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm); mobile phase: methanol-water (50:50); flow rate: 1 mL / min; pump pressure: 162 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 360 nm, λ em = 440 nm; acquisition time: 10 min.

[0059] Under the above chromatographic conditions, as Figure 2 shown, the control group sample had a strong absorption peak at the retention time RT = 8.246 min, while a smaller AFB 1 target peak was detected in the group added with Lac_P.E. By calculating the absorption peak area, 73% of the AFB 1 molecules had been degraded, indicating that recombinant Lac_P.E. had the activity to degrade aflatoxin B 1 .

[0060] 2. Detection of the degradation activity of Lac_P.E. against zearalenone

[0061] Zearalenone was dissolved in acetonitrile to prepare a stock solution of 100 μg / mL, and the following 1000 μL reaction system was used for the experiment: 950 μL of sodium phosphate buffer (0.1 M, pH 9.0), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 40 μL of zearalenone solution. The system without adding Lac_P.E. was used as the control. After the reaction was carried out at 30 °C for 24 h, 3 mL of methanol was added to terminate the reaction, and high performance liquid chromatography was used for detection. According to the change of ZEN concentration, the degradation activity of Lac_P.E. against ZEN was analyzed.

[0062] The chromatographic conditions for detecting ZEN by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×250 mm, 5 μm); mobile phase: acetonitrile-water-methanol (46:46:8); flow rate: 1 mL / min; pump pressure: 145 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 274 nm, λ em = 440 nm; acquisition time: 12 min.

[0063] Under the above chromatographic conditions, as Figure 3 shown, the control group sample had a strong absorption peak at the retention time RT = 9.869 min, while a smaller ZEN target peak was detected in the group added with Lac_P.E. By calculating the absorption peak area, 60% of the ZEN molecules had been degraded, indicating that recombinant Lac_P.E. had the activity to degrade zearalenone.

[0064] 3. Detection of the degradation activity of Lac_P.E. against vomitoxin

[0065] Dissolve vomitoxin in methanol to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 910 μL of sodium phosphate buffer (0.1 M, pH 9.0), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 80 μL of vomitoxin solution. Use the system without adding Lac_P.E. as a control. After the reaction is carried out at 30 °C for 24 h, add 3 mL of methanol to terminate the reaction, and detect it by high-performance liquid chromatography. Analyze whether Lac_P.E. has degradation activity against DON based on the change in DON concentration.

[0066] The chromatographic conditions for detecting DON by high-performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×250 mm, 5 μm); mobile phase: acetonitrile-water (10:90); flow rate: 0.8 mL / min; pump pressure: 150 bar; injection volume: 10 μL; detection wavelength of ultraviolet detector: 218 nm; collection time: 15 min.

[0067] Under the above chromatographic conditions, as Figure 4 shown, the control group sample has a strong absorption peak at the retention time RT = 12.556 min, while a smaller DON target peak is detected in the group added with Lac_P.E. After calculating the absorption peak area, 34% of the DON molecules have been degraded, indicating that the recombinant Lac_P.E. has the activity of degrading vomitoxin.

[0068] 4. Detection of the degradation activity of Lac_P.E. against ochratoxin

[0069] Dissolve ochratoxin in methanol to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 980 μL of sodium phosphate buffer (0.1 M, pH 8.0), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 10 μL of OTA solution. Use the system without adding Lac_P.E. as a control. After the reaction is carried out at 30 °C for 24 h, add 3 mL of methanol to terminate the reaction, and detect it by high-performance liquid chromatography. Analyze whether Lac_P.E. has degradation activity against OTA based on the change in OTA concentration.

[0070] The chromatographic conditions for detecting OTA by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: acetonitrile-water-glacial acetic acid (48:51:1); flow rate: 1mL / min; pump pressure: 125bar; injection volume: 10μL; fluorescence detector detection wavelength: λ ex = 333nm, λ em = 460nm; acquisition time: 20min.

[0071] Under the above chromatographic conditions, as Figure 5 shown, the control group sample had a strong absorption peak at the retention time RT = 17.232min, while a smaller OTA target peak was detected in the group added with Lac_P.E. By calculating the absorption peak area, 12% of the OTA molecules had been degraded, indicating that recombinant Lac_P.E. has the activity of degrading ochratoxin.

[0072] 5. Detection of the degradation activity of Lac_P.E. against T-2 toxin

[0073] Dissolve T-2 toxin in methanol to prepare a stock solution of 1000μg / mL, and conduct experiments according to the following 1000μL reaction system: 950μL sodium phosphate buffer (0.1M, pH 9.0), 10μL Lac_P.E. (final enzyme activity 3U / mL), 40μL T-2 toxin solution. The system without adding Lac_P.E. was used as a control. After the reaction was carried out at 30°C for 24h, 3mL of methanol was added to terminate the reaction, and high performance liquid chromatography was used for detection. Whether Lac_P.E. has the degradation activity against T-2 toxin was analyzed according to the change of T-2 toxin concentration.

[0074] The chromatographic conditions for detecting T-2 by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: acetonitrile-water (50:50); flow rate: 1mL / min; pump pressure: 125bar; injection volume: 10μL; ultraviolet detector detection wavelength: 208nm; acquisition time: 10min.

[0075] Under the above chromatographic conditions, as Figure 6 shown, the control group sample had a strong absorption peak at the retention time RT = 5.983min, while a smaller T-2 toxin target peak was detected in the group added with Lac_P.E. By calculating the absorption peak area, 19% of the T-2 toxin had been degraded, indicating that recombinant Lac_P.E. has the activity of degrading T-2 toxin.

[0076] 6. Detection of the degradation activity of Lac_P.E. against fumonisin

[0077] Dissolve fumonisin in methanol to prepare a stock solution of 1000 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 970 μL of sodium phosphate buffer (0.1 M, pH 9.0), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 20 μL of FB 1 solution. Use the system without adding Lac_P.E. as a control. After the reaction is carried out at 30 °C for 24 h, add 3 mL of methanol to terminate the reaction, and detect it by high-performance liquid chromatography. According to the change in the FB 1 concentration, analyze whether Lac_P.E. has degradation activity on FB 1 .

[0078] The chromatographic conditions for detecting FB 1 by high-performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm); mobile phase: methanol: 0.1 M sodium dihydrogen phosphate (pH 3.3) (77:23); flow rate: 0.6 mL / min; pump pressure: 125 bar; injection volume: 10 μL; fluorescence detector detection wavelength: λ ex = 333 nm, λ em = 440 nm; collection time: 15 min.

[0079] Under the above chromatographic conditions, as Figure 7 shown, the control group sample has a strong absorption peak at the retention time RT = 8.018 min, while a smaller FB 1 target peak is detected in the group added with Lac_P.E. Calculated by the absorption peak area, 18% of FB 1 has been degraded, indicating that recombinant Lac_P.E. has the activity of degrading FB 1 .

[0080] In summary, bacterial laccase Lac_P.E. can degrade aflatoxin B 1 (AFB 1 ), zearalenone (ZEN), deoxynivalenol (DON), ochratoxin (OTA), T-2 toxin (T-2), and fumonisin (FB1), and it is a broad-spectrum enzyme.

[0081] Example 4 Effects of reaction conditions and enzyme amount on the degradation activity of Lac_P.E. on aflatoxin B 1

[0082] In enzymatic reactions, the pH and reaction temperature of the reaction system have a great influence on the enzyme activity. In this example, the pH and reaction temperature of the reaction system are changed respectively to detect the degradation of aflatoxin B by Lac_P.E. 1 ​After determining the changes in activity and identifying the most suitable pH and reaction temperature, the addition amount of bacterial laccase Lac_P.E. was optimized accordingly. The specific steps are as follows:

[0083] 1. Effect of pH on the degradation of aflatoxin B by Lac_P.E. 1 Activity

[0084] Test the activity of Lac_P.E. in degrading AFB 1 using the reaction system described in step 1 of Example 3: 980 μL of buffer (0.1 M Na 2 HPO 4 -citrate buffer, pH 4 - 7; 0.1 M Tris-HCl buffer, pH 8 - 9; 0.1 M glycine-NaOH buffer, pH 10 - 12), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 10 μL of aflatoxin B 1 solution. After the reaction was carried out at 30 °C for 24 h, 3 mL of methanol was added to terminate the reaction, and the residual AFB 1 content in the system was detected using the method described in step 1 of Example 3. The results are as Figure 8 shown. Under alkaline conditions, the activity of Lac_P.E. in degrading AFB 1 is relatively high, and the optimal pH is 9.0.

[0085] 2. Effect of temperature on the degradation of aflatoxin B by Lac_P.E. 1 Activity

[0086] To test the activity of Lac_P.E. in degrading AFB 1 at different temperature conditions, use the reaction system described in step 1 of Example 3: 980 μL of buffer (0.1 M sodium phosphate buffer, pH 9.0), 10 μL of Lac_P.E. (final enzyme activity 3 U / mL), and 10 μL of aflatoxin B 1 solution. After the reaction was carried out at different temperatures (20 °C, 30 °C, 40 °C, 60 °C) for 24 h, 3 mL of methanol was added to terminate the reaction. The residual AFB 1 content in the system was detected using the method described in step 1 of Example 3. The results are as Figure 9 shown. The optimal temperature for Lac_P.E. to degrade AFB 1 is 30 °C.

[0087] 3. Effect of enzyme amount on the degradation of aflatoxin B by Lac_P.E. 1 Activity

[0088] To test the activity of Lac_P.E. in degrading AFB 1Activity was determined using the reaction system described in Step 1 of Example 3: The final concentration of Lac_P.E. was 0.3, 1, 3, or 10 U / mL, and 10 μL of aflatoxin B 1 was added. The solution was made up to 1000 μL with Tris-HCl buffer (0.1 M, pH 9.0). A system without Lac_P.E. was used as a control. After reacting at 30 °C for 24 h, 3 mL of methanol was added to terminate the reaction. The residual AFB 1 content in the system was detected using the method described in Step 1 of Example 3. The results are shown in Figure 10 Figure, and the efficiency of Lac_P.E. in degrading AFB 1 increased with increasing Lac_P.E. concentration, but the increasing trend gradually slowed down. Considering economic factors, the optimal enzyme amount was 1 U / mL.

[0089] Example 5 Effect of Lac_P.E. on the simultaneous degradation of AFB 1 , ZEN, DON, OTA, T-2 toxin, and FB 1 Separate stock solutions of AFB

[0090] , DON, OTA, T-2 toxin, and FB 1 were dissolved in methanol to concentrations of 100 μg / mL, 800 μg / mL, 100 μg / mL, 4000 μg / mL, and 2000 μg / mL, respectively, and ZEN was dissolved in acetonitrile to a concentration of 400 μg / mL. The following 1000 μL reaction system was used for the experiment: 930 μL of sodium phosphate buffer (0.1 M, pH 9.0), 10 μL of Lac_P.E. protein (final enzyme activity 18 U / mL), and 10 μL of AFB 1 solution, ZEN solution, DON solution, OTA solution, T-2 toxin solution, and FB 1 solution were added respectively. A system without Lac_P.E. was used as a control. After reacting at 30 °C for 24 h, 3 mL of methanol was added to terminate the reaction. High-performance liquid chromatography was used for detection, and the changes in the concentrations of AFB 1 , DON, OTA, T-2 toxin, and FB 1 were used to analyze whether Lac_P.E. had simultaneous degradation activity against AFB 1 , DON, OTA, T-2 toxin, and FB 1 , DON, OTA, T-2 toxin, and FB 1 .

[0091] High-performance liquid chromatography for the detection of AFB 1The chromatographic conditions are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: methanol-water (50:50); flow rate: 1 mL / min; pump pressure: 162 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 360 nm, λ em = 440 nm; collection time: 10 min.

[0092] The chromatographic conditions for the determination of ZEN by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×250mm, 5μm); mobile phase: acetonitrile-water-methanol (46:46:8); flow rate: 1 mL / min; pump pressure: 145 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 274 nm, λ em = 440 nm; collection time: 12 min.

[0093] The chromatographic conditions for the determination of DON by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×250mm, 5μm); mobile phase: acetonitrile-water (10:90); flow rate: 0.8 mL / min; pump pressure: 150 bar; injection volume: 10 μL; detection wavelength of ultraviolet detector: 218 nm; collection time: 15 min.

[0094] The chromatographic conditions for the determination of OTA by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: acetonitrile-water-glacial acetic acid (48:51:1); flow rate: 1 mL / min; pump pressure: 125 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 333 nm, λ em = 460 nm; collection time: 20 min.

[0095] The chromatographic conditions for the determination of T-2 by high performance liquid chromatography are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: acetonitrile-water (50:50); flow rate: 1 mL / min; pump pressure: 125 bar; injection volume: 10 μL; detection wavelength of ultraviolet detector: 208 nm; collection time: 10 min.

[0096] High performance liquid chromatography for the detection of FB 1The chromatographic conditions were as follows: Chromatographic column: Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm); Mobile phase: methanol: 0.1 M sodium dihydrogen phosphate (pH 3.3) (77:23); Flow rate: 0.6 mL / min; Pump pressure: 125 bar; Injection volume: 10 μL; Detection wavelengths for fluorescence detector: λ ex = 333 nm, λ em = 440 nm; Sampling time: 15 min.

[0097] In summary, bacterial laccase Lac_P.E. can simultaneously degrade aflatoxin B 1 (AFB 1 ), zearalenone (ZEN), deoxynivalenol (DON), ochratoxin (OTA), T-2 toxin (T-2), and fumonisin (FB 1 ). Compared with the degradation of a single toxin alone, the degradation rate has a relatively small difference, and the results are as Figure 11 shown.

[0098] Example 6 A feed additive containing bacterial laccase Lac_P.E.

[0099] The feed additive containing bacterial laccase Lac_P.E. in this example is a liquid preparation. Bacterial laccase Lac_P.E. is dissolved in phosphate buffer buffer-0.5 mM CuSO 4 (20 mM, pH 7.0) at a concentration of 10 U / mL to obtain the additive, which is stored at 4 °C or room temperature.

[0100] Experimental Example 1 Inhibition of Aspergillus flavus growth under storage conditions by the feed additive

[0101] The feed additive used in this experimental example was prepared in Example 6.

[0102] Preparation of Aspergillus flavus spore suspension: The Aspergillus flavus strain isolated from moldy corn was inoculated on a PDA test tube slant and cultured at 30 °C for 7 d. The slant was rinsed with 0.9% physiological saline to prepare the spore suspension. The spore concentration was adjusted to 10 5 CFU / mL and stored at 4 °C for later use.

[0103] The water content of corn was adjusted to 28% by the seed soaking method. It was soaked in 1% sodium hypochlorite solution for 2 min for disinfection, then rinsed 3 times with sterile water and placed in tissue culture bottles, 50 g of corn per bottle. Under sterile conditions, 1 mL of Aspergillus flavus spore suspension with a concentration of 10 5 CFU / mL was added to each respectively. 1 mL of the additive was added to the treatment group, and 1 mL of sterile water was added to the control group. After shaking well, it was cultured at 30 °C for 7 d, and the moldy situation of each group of corn was observed.

[0104] Accurately weigh 5 g of corn samples and add them to an Erlenmeyer flask containing 45 mL of sterile water. Mix well, count with a hemocytometer, calculate the concentration of Aspergillus flavus spores, and calculate the inhibition rate. Inhibition rate (%) = (1 - concentration of Aspergillus flavus spores in the control group / concentration of Aspergillus flavus spores in the treatment group) × 100%.

[0105] After calculation, the inhibition rate of the additive against Aspergillus flavus was 89.13%.

[0106] Experimental Example 2 Treatment of AFB in Naturally Moldy Corn by Feed Additive 1

[0107] Put the naturally moldy corn into tissue culture flasks, 50 g of corn in each flask. Add 1 mL of the additive as the treatment group, and add 1 mL of sterile water to the control group. Shake well and incubate at a constant temperature of 30 °C for 24 h. Detect AFB in the corn by HPLC method 1 .

[0108] High-performance liquid chromatography detection of AFB 1 The chromatographic conditions for the detection of AFB are as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6 × 150 mm, 5 μm); mobile phase: methanol-water (50:50); flow rate: 1 mL / min; pump pressure: 162 bar; injection volume: 10 μL; detection wavelength of fluorescence detector: λ ex = 360 nm, λ em = 440 nm; acquisition time: 10 min.

[0109] After determination, the degradation rate of AFB 1 was 71.28%.

Claims

1. A bacterial laccase Lac_P.E. encoding gene Lac_ P.E. gene It is characterized in that: Its nucleotide sequence is as shown in SEQ ID NO.

1.

2. A bacterial laccase Lac_P.E . , It is characterized in that: Its amino acid sequence is as shown in SEQ ID NO.

2.

3. The application of the bacterial laccase Lac_P.E. according to claim 2, It is characterized in that: Application in mycotoxin detoxification; the mycotoxin is aflatoxin B 1 , zearalenone, deoxynivalenol, ochratoxin, T-2 toxin, fumonisin, or a combination of two or more thereof.

4. The application of the bacterial laccase Lac_P.E. according to claim 3, It is characterized in that: The degradation of the mycotoxin by the bacterial laccase Lac_P.E. is carried out under mediator-free conditions.

5. The application of the bacterial laccase Lac_P.E. according to claim 4, It is characterized in that: Degradation of Aflatoxin B by Bacterial Laccase Lac_P.E. 1 The pH is 4 - 12 and the reaction temperature is 20 - 60 °C.

6. The application of the bacterial laccase Lac_P.E. according to claim 5, It is characterized in that: Degradation of Aflatoxin B by Bacterial Laccase Lac_P.E. 1 The pH is 9 and the reaction temperature is 30 °C.

7. A feed additive comprising the bacterial laccase Lac_P.E. according to claim 2.

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