A method for increasing the rate of degradation of vomitoxin in feed

Abstract

The application relates to the field of agricultural biotechnology, in particular to a method for improving the degradation rate of vomitoxin in feed. The method comprises the steps of degrading vomitoxin by using glutathione, manganese peroxidase and single-chain antibody fusion enzyme. The fusion enzyme of the application can efficiently degrade mycotoxins, has low cost and wide application range, and can be widely used in the field of feed toxin-degrading enzymes; the addition of glutathione in the reaction can effectively improve the effect of the enzyme in degrading mycotoxins in the feed.

Description

Technical Field

[0001] The present application relates to the field of agricultural biotechnology, and in particular to a method for improving the degradation rate of vomitoxin in feed. Background Art

[0002] Deoxynivalenol (DON) is a type B trichothecene toxin produced as a secondary metabolite by Fusarium graminearum, Fusarium oxysporum, and Fusarium nivale. It is widely toxic to humans and animals, causing significant harm. Furthermore, DON is a major mycotoxin contaminating crops and feed such as wheat, barley, and corn.

[0003] Manganese peroxidase is a heme-containing fungal enzyme that converts Mn 2+ Oxidation to Mn 3+ , Mn generated during the reaction 3+ It is not stable and can chelate with carboxylic acid to form Mn 3+ -Organic acid chelates, which have a higher redox potential and can participate in the degradation of vomitoxin in pure solution.

[0004] Glutathione (GSH) is the most abundant non-protein thiol species in biological systems. It is an important endogenous antioxidant that plays a crucial role in defending against toxins and maintaining redox homeostasis in organisms. Currently, there is no technical guidance on the use of glutathione to enhance the degradation of mycotoxins in food and feed. Summary of the Invention

[0005] The object of the present invention is to provide a method for improving the degradation rate of vomitoxin in feed.

[0006] The method for improving the degradation rate of vomitoxin in feed according to the present invention comprises the step of using a mixture of glutathione and a fusion enzyme to degrade vomitoxin, wherein the fusion enzyme is composed of manganese peroxidase and a single-chain antibody.

[0007] According to the method for improving the degradation rate of vomitoxin in feed of the present invention, the manganese peroxidase is CsMnP derived from Ceriporiopsis subvermispora, whose amino acid sequence is shown in SEQ ID No: 2, the accession number of the single-chain antibody is AAN75452, whose amino acid sequence is shown in SEQ ID No: 1, CsMnP is upstream, ScFv is downstream, and the two are connected by GGGGS.

[0008] According to the method for improving the degradation rate of vomitoxin in feed of the present invention, the feed is a wheat soybean meal diet and a corn soybean meal diet.

[0009] According to the technical solution of the present application, the introduction of glutathione (GSH) generates a new free radical capable of degrading DON in the presence of feed. However, manganese peroxidase itself is also a substrate for the new free radical. In this case, the degradation of DON catalyzed by manganese peroxidase involves a complex interaction between DON, lignin, and GSH. The presence of lignin and vomitoxin prevents the new free radical from attacking the manganese peroxidase.

[0010] The fusion enzyme of the present invention can efficiently degrade mycotoxins, has low cost, and a wide range of applications, and can be widely used in the field of feed toxin degrading enzymes; adding glutathione to the reaction can effectively enhance the effect of the enzyme in degrading mycotoxins in feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 HPLC analysis results showing the degradation of pure DON by the fusion enzyme;

[0012] Figure 2 HPLC analysis results showing the degradation of pure DON by the fusion enzyme after addition of glutathione;

[0013] Figure 3 HPLC analysis results showing the degradation of DON by the fusion enzyme in a wheat-soybean meal diet;

[0014] Figure 4 HPLC analysis results showing the degradation of DON by the fusion enzyme in a corn-soybean meal diet;

[0015] Figure 5 HPLC analysis results showing the degradation of DON by the fusion enzyme in a wheat-soybean meal diet after glutathione supplementation;

[0016] Figure 6 HPLC analysis showing the degradation of DON by the fusion enzyme in a corn-soybean meal diet after glutathione supplementation. DETAILED DESCRIPTION

[0017] Test materials and reagents

[0018] 1. Strain: An engineered E. coli strain producing CsMnP and single-chain antibody fusion enzyme derived from Ceriporiopsis subvermispora;

[0019] 2. Biochemical reagents: vomitoxin; chromatographic grade acetonitrile; glutathione;

[0020] 3. Culture medium: Escherichia coli broth (LB) (1% peptone, 0.5% yeast extract, 1% sodium chloride);

[0021] Example 1 Preparation of recombinant fusion enzyme CsMnP-ScFv

[0022] The CsMnP and ScFv gene sequences from Ceriporiopsis subvermispora were synthesized and the CsMnP gene sequence and the ScFv gene sequence were connected by the base sequence 5'GGCGGCGGTGGTAGC3'(encoding GGGGS). The BL21 / CsMnP-ScFv containing the recombinant plasmid was inoculated into 50 mL LB medium added with 50 μg / mL ampicillin and cultured at 37°C, 220 rpm in a shaker overnight; 2% of the bacterial liquid was transferred into 300 mL LB liquid medium added with ampicillin and the bacterial liquid was cultured at 37°C, 200 rpm in a shaker for about 3 h to OD 600 ≈0.6-0.8; IPTG was added to the culture medium to a final concentration of 1 mM, and the expression of the recombinant fusion enzyme protein was induced by continuing to culture at 37°C for 4 h; the bacterial liquid was centrifuged at 12,000 rpm for 2 min to collect the bacterial cells, which were stored at -20°C for standby.

[0023] To purify the recombinant protein, the induced E. coli culture was resuspended in 20 mL buffer containing 50 mM Tris-HCl (including 10 mM EDTA and 5 mM DTT, pH 8.0). Lysozyme was added to a final concentration of 2 mg / mL, and incubated on ice for 1 h. Then 20 μL DNase I was added in the mixed system, and after incubation on ice for 30 min, the supernatant was removed by centrifugation at 12,000 rpm for 30 min at 4°C. The insoluble part was washed twice with 20 mM Tris-HCl buffer (including 1 mM EDTA, 5 mM DTT, 2 M urea and 1% Triton-100, pH 8.0), transferred to 5 mM Tris-HCl (including 8 M urea, 1 mM EDTA, 1 mM DTT, 10% glycerol, pH 8.0), and placed on 4°C upside down until completely dissolved. The recombinant enzyme was purified by immobilized affinity chromatography with nickel-coated magnetic beads. The purified protein was dialyzed in a refolding buffer containing 50 mM Tris-HCl (pH 9.5), 0.6 M urea, 0.5 mM GSSG, 0.1 mM DTT, 5 mM CaCl2 and 10% glycerol for 24 h, then 5 μM of hemin solution was added, and incubated at 4°C for 12 h to obtain the functional recombinant enzyme CsMnP-ScFv.

[0024] Example 2 Degradation of vomitoxin by the fusion enzyme

[0025] Vomitoxin was dissolved in methanol to prepare a 1 g / L stock solution, and the following reaction system was used: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L vomitoxin, 0.1 U / mL of CsMnP-ScFv. The system without the addition of the fusion enzyme was used as a control, and the reaction system was set in triplicate. The reaction was carried out at 30°C, and after 24 h, the reaction was terminated by adding three volumes of methanol, and the degradation rate of vomitoxin was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography was a Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, the chromatographic separation column was Zorbax SB-C18 (4.6 x 250 mm, 5 μm), 10% acetonitrile was used as the mobile phase, and vomitoxin was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 1 Figure 1, and the degradation rate of vomitoxin was 37.7%.

[0026] Example 3 Degradation of vomitoxin by the fusion enzyme after the addition of glutathione

[0027] Vomitoxin was dissolved in methanol to prepare a 1 g / L stock solution, and the following reaction system was used: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L vomitoxin, 5 mM glutathione, 0.1 U / mL of CsMnP-ScFv. The system without the addition of the fusion enzyme was used as a control, and the reaction system was set in triplicate. The reaction was carried out at 30°C, and after 24 h, the reaction was terminated by adding three volumes of methanol, and the degradation rate of vomitoxin was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography was a Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, the chromatographic separation column was Zorbax SB-C18 (4.6 x 250 mm, 5 μm), 10% acetonitrile was used as the mobile phase, and vomitoxin was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 2 Figure 2, and the degradation rate of vomitoxin was 81.4%.

[0028] Example 4 Degradation of vomitoxin in feed by the fusion enzyme

[0029] The vomitoxin was dissolved in methanol to prepare a 1 g / L stock solution, and the following reaction system was used: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L vomitoxin, 0.5 U / mL of CsMnP-ScFv, 16 mg / mL of wheat soybean meal or 16 mg / mL of corn soybean meal. The system without the addition of the fusion enzyme was used as a control, and the reaction system was set in triplicate. The reaction was carried out at 30°C, and after 96 h, three volumes of methanol were added to terminate the reaction, and the degradation rate of vomitoxin was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography was a Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, the chromatographic separation column was Zorbax SB-C18 (4.6 x 250 mm, 5 μm), 10% acetonitrile was used as the mobile phase, and the vomitoxin was monitored under ultraviolet light at a wavelength of 218 nm.

[0030] The results are shown in Table 1, and the degradation rate of vomitoxin in the wheat soybean meal was 11.4%. The results are shown in Table 2, and the degradation rate of vomitoxin in the corn soybean meal was 21.6%. It can be seen that the presence of the feed seriously affected the degradation of vomitoxin. Figure 3 Figure 4 The results are shown in Table 1, and the degradation rate of vomitoxin in the wheat soybean meal was 11.4%. The results are shown in Table 2, and the degradation rate of vomitoxin in the corn soybean meal was 21.6%. It can be seen that the presence of the feed seriously affected the degradation of vomitoxin.

[0031] Example 5 Degradation of vomitoxin in feed by the fusion enzyme after the addition of glutathione

[0032] The vomitoxin was dissolved in methanol to prepare a 1 g / L stock solution, and the following reaction system was used: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L vomitoxin, 0.5 U / mL of CsMnP-ScFv, 16 mg / mL of wheat soybean meal or 16 mg / mL of corn soybean meal, and 20 mM GSH. The system without the addition of the fusion enzyme was used as a control, and the reaction system was set in triplicate. The reaction was carried out at 30°C, and after 96 h, three volumes of methanol were added to terminate the reaction, and the degradation rate of vomitoxin was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography was a Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, the chromatographic separation column was Zorbax SB-C18 (4.6 x 250 mm, 5 μm), 10% acetonitrile was used as the mobile phase, and the vomitoxin was monitored under ultraviolet light at a wavelength of 218 nm.

[0033] The results are shown in Table 3, and the degradation rate of vomitoxin in the wheat soybean meal was 82.7%. The results are shown in Table 4, and the degradation rate of vomitoxin in the corn soybean meal was 57.2%. The presence of glutathione increased the degradation rate of vomitoxin by 7.3 times in the wheat soybean meal and by 2.6 times in the corn soybean meal. Figure 5 Figure 6 The results are shown in Table 3, and the degradation rate of vomitoxin in the wheat soybean meal was 82.7%. The results are shown in Table 4, and the degradation rate of vomitoxin in the corn soybean meal was 57.2%. The presence of glutathione increased the degradation rate of vomitoxin by 7.3 times in the wheat soybean meal and by 2.6 times in the corn soybean meal.

[0034] ​​The above examples are only for understanding the technical solutions of the present application and do not limit the protection scope of the present application.

Claims

1. A method for increasing the degradation rate of vomitoxin in feed, characterized in that: The method includes the step of degrading vomitoxin using a mixture of glutathione and a fusion enzyme, wherein the fusion enzyme consists of a manganese peroxidase and a single-chain antibody, the manganese peroxidase is upstream, the single-chain antibody is downstream, and the two are linked by GGGGS, the amino acid sequence of the manganese peroxidase is shown in SEQ ID No: 2, and the amino acid sequence of the single-chain antibody is shown in SEQ ID No:

1.

2. The method for improving the degradation rate of vomitoxin in feed according to claim 1, characterized in that: The glutathione is reduced glutathione.

3. The method for improving the degradation rate of vomitoxin in feed according to claim 1, characterized in that: The feed is a wheat soybean meal diet or a corn soybean meal diet.

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