A mycotoxin degrader and its application

Abstract

The present invention relates to a mycotoxin degrading agent and its application, belonging to the field of agricultural biology. The present invention provides a highly efficient mycotoxin degrading agent that can be used for mycotoxin degradation. The mycotoxin degrading agent of the present invention comprises bacterial laccase Lac_P.E. and an artificial mediator, and can efficiently degrade mycotoxins, including aflatoxin B1 and zearalenone, and is widely used in the field of mycotoxin detoxification.

Description

Technical Field

[0001] The present invention relates to a mycotoxin degrading agent and its application, belonging to the field of agricultural biology. Background Art

[0002] Mycotoxins are secondary metabolites of fungi. Due to factors such as climate and storage conditions, feeds such as corn are extremely vulnerable to contamination by various mycotoxins. Therefore, mycotoxin contamination is widespread in agricultural products, feed raw materials, and animal feeds. There are more than 500 kinds of mycotoxins. The mycotoxins that are mainly focused on and monitored in various countries and regions are mainly aflatoxin (AF), zearalenone (ZEN), ochratoxin A (OTA), fumonisin (FB), deoxynivalenol (DON), T-2 toxin, etc. Mycotoxins are carcinogenic, mutagenic, and teratogenic, can cause death of humans and animals, and the probability of coexistence of multiple mycotoxins is high, with synergistic toxicity.

[0003] The methods for degrading mycotoxins mainly include physical methods, chemical methods, and biological methods. The physical method has unstable effects, is prone to desorption, 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 costs, and chemical residues and the introduction of new harmful components affect the safety of feeds. Biological methods include microbial methods and enzymatic methods, which have the advantages of safety, high efficiency, and environmental friendliness, and are important and effective methods 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. Enzymatic detoxification refers to the degradation enzyme converting the toxin into a low-toxic or non-toxic product through an enzymatic reaction. The degradation enzymes mainly include oxidases (such as laccase, manganese peroxidase) and hydrolases (such as esterase), etc.

[0004] Most of the reported degradation enzymes can only degrade a single type of mycotoxin. Laccase has no strict selectivity for substrates, and there are as many as 300 kinds of substrate types, and it has excellent catalytic characteristics and advantages such as green environmental protection (the by-product is only water and no toxic intermediates), and is an ideal industrial enzyme. However, the degradation rate of existing laccase for mycotoxins is generally low.

[0005] Laccase can degrade various mycotoxins with different structures in the presence of a mediator. A mediator is a type of small molecule phenolic compound that acts as an electron transfer intermediate. It is itself a substrate of laccase and can form a highly active and stable intermediate under the action of laccase and participate in the indirect oxidation of the substrate by laccase. Through the laccase-mediator system, laccase has a wider substrate range and a higher degradation rate.

[0006] It is reported in the literature that when using the laccase-mediator system for dye decolorization, the effect of the mediator depends on the type of dye to be treated. For example, white rot fungus laccase can effectively degrade acid red using HBT as a mediator. When using Trametes versicolor laccase for pesticide degradation, different pesticide substrates also showed selectivity for the mediator. For example, when degrading pyrimethanil and isoproturon, the best mediator is purpuric acid, and acetylsyringone and HBT are the best mediators for degrading chlorothalonil and norflurazon. Thus, it can be seen that different mycotoxins also have selectivity for the mediator, and the appropriate mediator for laccase varies significantly due to the differences in the nature of its degradation substrates. Therefore, finding an efficient mediator to improve the degradation rate of laccase for mycotoxins is the key to the enzymatic detoxification technology. Summary of the Invention

[0007] The present invention provides a mycotoxin degrading agent, which can efficiently degrade mycotoxins and is of great significance for the research in the field of mycotoxin detoxification.

[0008] The present invention also provides the application of the mycotoxin degrading agent. The mycotoxin degrading agent can efficiently degrade mycotoxins and can be widely used for mycotoxin detoxification in grains, foods, feeds and their processing by-products.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A mycotoxin degrading agent, comprising bacterial laccase Lac_P.E. and an artificial mediator, wherein the artificial mediator is 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, and the amino acid sequence of the bacterial laccase Lac_P.E. is as shown in SED IDNO.2.

[0011] This mycotoxin degrading agent can efficiently degrade mycotoxins and is of great significance for the research in the field of mycotoxin detoxification.

[0012] Preferably, the nucleotide sequence of the bacterial laccase Lac_P.E. is as shown in SED ID NO.1.

[0013] Preferably, in the system for degrading mycotoxins, the mycotoxin degrading agent is in a liquid dosage form, wherein the final enzyme activity of the bacterial laccase Lac_P.E. is 1 U / mL, and the dosage of the mediator is 1 mM.

[0014] More preferably, the pH of the liquid dosage form is 8.0.

[0015] More preferably, the mycotoxin is aflatoxin B 1 and / or zearalenone.

[0016] The application of the mycotoxin degrading agent can be applied to the field of mycotoxin detoxification.

[0017] The mycotoxin degrading agent can efficiently degrade mycotoxins and can be widely used for mycotoxin detoxification in grains, food, feeds and their processing by-products.

[0018] Preferably, the mycotoxin is aflatoxin B 1 and / or zearalenone.

[0019] More preferably, the degradation of the mycotoxin is carried out in an environment with a pH of 8. Description of the Drawings

[0020] Figure 1 Degradation effect of the laccase-ABTS system derived from recombinant Priestia sp. in Example 2 of the present invention on aflatoxin B 1 and zearalenone;

[0021] Figure 2 HPLC analysis result of the degradation of aflatoxin B by the laccase-ABTS system derived from recombinant Priestia sp. in Example 2 of the present invention 1 ;

[0022] Figure 3 HPLC analysis result of the degradation of zearalenone by the laccase-ABTS system derived from recombinant Priestia sp. in Example 2 of the present invention;

[0023] Figure 4 HPLC analysis result of the degradation of aflatoxin B by the laccase-HBT system derived from recombinant Priestia sp. in Comparative Example 1 of the present invention 1 ;

[0024] Figure 5 HPLC analysis result of the degradation of zearalenone by the laccase-HBT system derived from recombinant Priestia sp. in Comparative Example 1 of the present invention;

[0025] Figure 6 HPLC analysis result of the degradation of aflatoxin B by the laccase-2,6-DMP system derived from recombinant Priestia sp. in Comparative Example 2 of the present invention 1 ;

[0026] Figure 7 HPLC analysis result of the degradation of zearalenone by the laccase-2,6-DMP system derived from recombinant Priestia sp. in Comparative Example 2 of the present invention. Detailed Embodiments

[0027] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and comparative example can be obtained commercially.

[0028] A brief introduction to some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following examples and test examples is as follows:

[0029] Biological materials:

[0030] Strain: An engineered Escherichia coli strain expressing laccase Lac_P.E. derived from Priestia, constructed in our laboratory.

[0031] Experimental reagents: Aflatoxin B 1 and zearalenone, purchased from Sigma; other reagents such as ABTS are of domestic analytical purity.

[0032] Example 1 A mycotoxin degrading agent

[0033] The mycotoxin degrading agent in this example comprises bacterial laccase Lac_P.E. and an artificial mediator. The amino acid sequence of laccase Lac_P.E. is shown in SED ID NO.2. The preparation method of bacterial laccase Lac_P.E. is as follows:

[0034] Take the engineered Escherichia coli strain BL21(DE3) / Lac_P.E. containing the recombinant plasmid, inoculate it into 50 mL of LB culture medium, and culture it with shaking at 37 °C and 180 rpm for 12 h. Then transfer it to 400 mL of LB culture medium at a ratio of 1% and culture it 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 it at 16 °C for 16 h, then centrifuge to collect the bacterial cells. Resuspend the bacterial cells in phosphate buffer - 0.5 mM CuSO 4 (20 mM, pH 7.0). Lyse the bacterial cells by ultrasonic disruption. Centrifuge to remove the disrupted cell debris, and purify it using a Ni affinity chromatography column. The buffer is 20 mM PB, 0.5 M NaCl, 20 mM imidazole, pH 7.4, and collect the eluted fraction with electrophoretic purity.

[0035] Example 2 Application of the mycotoxin degrading agent in mycotoxin detoxification

[0036] The application of the mycotoxin degrading agent in this example in mycotoxin detoxification. The mycotoxin degrading agent contains bacterial laccase Lac_P.E. and an artificial mediator. Compared with bacterial laccase Lac_P.E., when ABTS is added to the degradation system, the degradation rates of aflatoxin B 1 and zearalenone are significantly increased. The specific implementation steps are as follows:

[0037] 1. Degradation of aflatoxin B by the Lac_P.E.-ABTS system 1

[0038] Dissolve aflatoxin B 1 in methanol to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 970 μL of Tris-HCl (0.1 M, pH 8.0), 10 μL of aflatoxin B 1 solution (100 μg / mL), 10 μL of ABTS solution (100 mM), and 10 μL of Lac_P.E. (final enzyme activity 1 U / mL). Use the system without adding laccase Lac_P.E. as a control, and set 3 replicates for the reaction system. After the reaction is carried out at 30 °C for 24 h, add three volumes of methanol to terminate the reaction, and analyze the degradation rate of aflatoxin B 1 by high-performance liquid chromatography. The liquid chromatography is an Agilent 1290 HPLC high-performance liquid chromatography analysis system, the chromatographic column is Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm), the mobile phase: methanol-water (50:50); the flow rate: 1 mL / min; the injection volume: 10 μL; the detection wavelength of the fluorescence detector: λ ex = 360 nm, λ em = 440 nm.

[0039] The results are as shown in Figure 1 and Figure 2 . Part of aflatoxin B 1 has been degraded. When using ABTS as a mediator, the degradation rate is 100%, while when ABTS is not added to the system, the degradation rate is 16%. It can be seen that when ABTS is added to the degradation system, complete degradation of aflatoxin B 1 is achieved.

[0040] 2. Degradation of zearalenone by the Lac_P.E.-ABTS system

[0041] 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: 940 μL Tris-HCl (0.1 M, pH 8.0), 40 μL zearalenone solution, 10 μL ABTS solution (100 mM), and 10 μL Lac_P.E. (final enzyme activity 1 U / mL). The system without adding laccase Lac_P.E. was used as a control, and 3 replicates were set for the reaction system. After the reaction was carried out at 30 °C for 24 h, three volumes of methanol were added to terminate the reaction, and the degradation rate of zearalenone was analyzed by high performance liquid chromatography. The liquid chromatography was an Agilent 1290 HPLC high performance liquid chromatography analysis system, the chromatographic column was Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm), the mobile phase: acetonitrile-water-methanol (46:46:8); flow rate: 1 mL / min; fluorescence detector detection wavelength: λ ex = 274 nm, λ em = 440 nm.

[0042] The results are as Figure 1 、 Figure 3 shown. Part of the zearalenone has been degraded. When ABTS is used as a mediator, the degradation rate is 100%, while when ABTS is not added to the system, the degradation rate is only 30%. It can be seen from this that when ABTS is added to the degradation system, the complete degradation of zearalenone is achieved.

[0043] From the above results, it can be seen that when ABTS is added to the degradation system of bacterial laccase Lac_P.E., the degradation rate of aflatoxin B 1 、zearalenone by bacterial laccase Lac_P.E. can be significantly improved, and the degradation efficiency can reach 100%.

[0044] Different mycotoxins have selectivity for mediators, and the appropriate mediators for laccase vary significantly due to the differences in the properties of their degradation substrates. In the present invention, a Lac_P.E.-HBT system and a Lac_P.E.-2,6-DMP system were also constructed for common artificial mediators, and the degradation rates of mycotoxins in their systems were detected. The specific operation instructions are as follows.

[0045] Comparative Example 1 Degradation effect of Lac_P.E.-HBT (1-hydroxybenzotriazole) system on mycotoxins

[0046] 1. Degradation of aflatoxin B by Lac_P.E.-HBT system 1

[0047] Aflatoxin B 1Dissolve it in methanol to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 970 μL Tris-HCl (0.1 M, pH 8.0), 10 μL aflatoxin B 1 solution (100 μg / mL), 10 μL HBT solution (100 mM), 10 μL Lac_P.E. (final enzyme activity 1 U / mL). Use the system without adding laccase Lac_P.E. as a control, and set 3 replicates for the reaction system. After the reaction is carried out at 30 °C for 24 h, add three volumes of methanol to terminate the reaction, and analyze the degradation rate of aflatoxin B 1 by high performance liquid chromatography. The liquid chromatography is an Agilent 1290 HPLC high performance liquid chromatography analysis system, the chromatographic column is Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm), mobile phase: methanol-water (50:50); flow rate: 1 mL / min; injection volume: 10 μL; fluorescence detector detection wavelength: λ ex = 360 nm, λ em = 440 nm.

[0048] The results are as Figure 4 shown. Part of aflatoxin B 1 has been degraded. When using HBT as a mediator, the degradation rate is 25%, while when HBT is not added to the system, the degradation rate is 16%. It can be seen that when HBT is added to the degradation system, the degradation rate of laccase Lac_P.E. to aflatoxin B 1 is increased, but the increase amplitude is very small.

[0049] 2. Degradation of zearalenone by Lac_P.E.-HBT system

[0050] Dissolve zearalenone in acetonitrile to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 940 μL Tris-HCl (0.1 M, pH 8.0), 40 μL zearalenone solution, 10 μL HBT solution (100 mM), 10 μL Lac_P.E. (final enzyme activity 1 U / mL). Use the system without adding laccase Lac_P.E. as a control, and set 3 replicates for the reaction system. After the reaction is carried out at 30 °C for 24 h, add three volumes of methanol to terminate the reaction, and analyze the degradation rate of zearalenone by high performance liquid chromatography. The liquid chromatography is an Agilent 1290 HPLC high performance liquid chromatography analysis system, the chromatographic column is Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm), mobile phase: acetonitrile-water-methanol (46:46:8); flow rate: 1 mL / min; fluorescence detector detection wavelength: λ ex= 274 nm, λ em = 440 nm.

[0051] The results are as Figure 5 shown. A part of zearalenone has been degraded. When HBT is used as a mediator, the degradation rate is 24%, while when HBT is not added to the system, the degradation rate is 30%. It can be seen from this that when HBT is added to the degradation system, the degradation rate of zearalenone by laccase Lac_P.E. is reduced.

[0052] Generally speaking, when HBT is added to the degradation system of bacterial laccase Lac_P.E., the degradation rate of aflatoxin B 1 by laccase Lac_P.E. is improved to a certain extent, but the degradation rate of zearalenone is not increased and shows a downward trend, indicating that the effect of HBT as a mediator of laccase Lac_P.E. is not as good as ABTS.

[0053] Comparative Example 2 Degradation effect of Lac_P.E.-2,6-DMP (2,6-dimethylphenol) system on mycotoxins

[0054] 1. Degradation of aflatoxin B by Lac_P.E.-2,6-DMP system 1

[0055] Dissolve aflatoxin B 1 in methanol to prepare a stock solution of 100 μg / mL, and conduct experiments according to the following 1000 μL reaction system: 970 μL Tris-HCl (0.1 M, pH 8.0), 10 μL aflatoxin B 1 solution (100 μg / mL), 10 μL 2,6-DMP solution (100 mM), 10 μL Lac_P.E. (final enzyme activity 1 U / mL). Use the system without adding laccase Lac_P.E. as a control, and set 3 replicates for the reaction system. After the reaction is carried out at 30 °C for 24 h, add three volumes of methanol to terminate the reaction, and analyze the degradation rate of aflatoxin B 1 by high performance liquid chromatography. The liquid chromatography is an Agilent 1290 HPLC high performance liquid chromatography analysis system, the chromatographic column is Waters-Acchrom Tnature C18 (4.6×150 mm, 5 μm), mobile phase: methanol-water (50:50); flow rate: 1 mL / min; injection volume: 10 μL; fluorescence detector detection wavelength: λ ex = 360 nm, λ em = 440 nm.

[0056] The results are as Figure 6 shown. A part of aflatoxin B 1has been degraded. When 2,6-DMP is used as a mediator, the degradation rate is 76%, while when 2,6-DMP is not added to the system, the degradation rate is 16%. It can be seen from this that when 2,6-DMP is added to the degradation system, the degradation rate of aflatoxin B by laccase Lac_P.E. is increased. 1 of.

[0057] 2. Degradation of zearalenone by the Lac_P.E.-2,6-DMP system

[0058] Zearalenone was dissolved in acetonitrile to prepare a stock solution of 100 μg / mL. The following 1000 μL reaction system was used for the experiment: 940 μL Tris-HCl (0.1 M, pH 8.0), 40 μL zearalenone solution, 10 μL 2,6-DMP solution (100 mM), and 10 μL Lac_P.E. (final enzyme activity 1 U / mL). A system without laccase Lac_P.E. was used as a control, and the reaction system was set with 3 replicates. After the reaction was carried out at 30 °C for 24 h, three volumes of methanol were added to terminate the reaction, and the degradation rate of zearalenone was analyzed by high performance liquid chromatography. The liquid chromatography was an Agilent 1290 HPLC high performance liquid chromatography analysis system, the chromatographic column was Waters-Acchrom Tnature C18 (4.6 × 150 mm, 5 μm), the mobile phase: acetonitrile-water-methanol (46:46:8); the flow rate: 1 mL / min; the detection wavelength of the fluorescence detector: λ ex = 274 nm, λ em = 440 nm.

[0059] The results are as Figure 7 shown. Part of the zearalenone has been degraded. When 2,6-DMP is used as a mediator, the degradation rate is 23%, while when 2,6-DMP is not added to the system, the degradation rate is 30%. It can be seen from this that when 2,6-DMP is added to the degradation system, the degradation rate of zearalenone by laccase Lac_P.E. is decreased.

[0060] Generally speaking, when 2,6-DMP is added to the degradation system of bacterial laccase Lac_P.E., the degradation rate of aflatoxin B by laccase Lac_P.E. 1 is increased, but the degradation rate of zearalenone is not increased and shows a downward trend, indicating that the effect of 2,6-DMP as a mediator of laccase Lac_P.E. is not as good as ABTS.

[0061] To sum up, the effects of HBT and 2,6-DMP as mediators of laccase Lac_P.E. on the degradation of aflatoxin B 1 and zearalenone are not as good as ABTS as a mediator of laccase Lac_P.E.

Claims

1. A mycotoxin degrading agent, characterized in that: The mycotoxin degrading agent described above includes bacterial laccase Lac_ P.E. and an artificial mediator; the artificial mediator is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); the amino acid sequence of the bacterial laccase Lac_ P.E. is as shown in SED ID NO.

2.

2. The mycotoxin degrading agent according to claim 1, characterized in that: The described bacterial laccase Lac_ P.E. Nucleotide The sequence is as shown in SED ID NO.

1.

3. The mycotoxin degrading agent according to claim 1, characterized in that: The mycotoxin degrading agent described is in liquid dosage form, in which the final enzyme activity of bacterial laccase Lac_ P.E. is 1 U / mL, and the dosage of the mediator is 1 mM.

4. The mycotoxin degrading agent according to claim 3, characterized in that: The pH of the liquid dosage form is 8.

0.

5. The mycotoxin degrading agent according to claim 3, characterized in that: The mycotoxin is aflatoxin B 1 and / or zearalenone.

6. The application of the mycotoxin degrading agent according to claim 1, characterized in that: Application in mycotoxin detoxification; the mycotoxin is aflatoxin B 1 and / or zearalenone.

7. The application of the mycotoxin degrading agent according to claim 6, characterized in that: The degradation of the mycotoxin is carried out in an environment with a pH of 8.

Images