A method of targeted degradation of vomitoxin in feed and a fusion enzyme

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a method for targeted degradation of vomitoxin in feed and a fusion enzyme. The application fuses and expresses a DON degrading enzyme, i.e. a manganese peroxidase, with a single-chain antibody capable of specifically recognizing DON, and the obtained fusion enzyme can efficiently target and degrade vomitoxin in feed, can efficiently degrade mycotoxins, has low cost, and has wide application range, and can be widely used in the field of feed toxin degrading enzymes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for targeted degradation of vomitoxin in feed and a fusion enzyme. BACKGROUND

[0002] Fungal toxins are a class of more than 500 secondary fungal metabolites with different structures, which pose a threat to human and animal health. They mainly exist in food and feed, among which vomitoxin (DON) is one of the main fungal toxins, which is produced by fusarium and can cause severe gastrointestinal inflammation and immunosuppression.

[0003] Single-chain antibodies are small molecules composed of antibody heavy chain variable regions and light chain variable regions connected by a peptide chain, and are the smallest functional structural units with antibody activity. They have the characteristics of small molecular weight, strong penetration, high specificity, and have been widely used in drug targeting and immunobiological therapy in many medical fields.

[0004] Research has found that vomitoxin can be detoxified by physical, chemical, biological and other methods. However, physical and chemical methods have some disadvantages, such as destroying nutrients and introducing unsafe chemical components. At present, the research on vomitoxin-degrading bacteria at home and abroad has achieved certain results, such as Bacillus subtilis (Bacillus subtilis) ASAG 216, Nocardioides sp. (Nocardioides sp.) ZHH-013, etc. Due to the difficulty in expressing and separating and identifying in engineering bacteria, the number of DON-degrading enzymes reported so far is very small. In addition, some degradation enzymes also require complex and expensive auxiliary molecules. Even for the manganese peroxidase that can efficiently degrade vomitoxin, it is more challenging, and this type of degradation enzyme cannot degrade DON under the real situation of vomitoxin existing in food and feed. SUMMARY

[0005] The purpose of the present application is to provide a method for targeted degradation of vomitoxin in feed.

[0006] The purpose of the present application is to provide a method for targeted degradation of vomitoxin in feed.

[0007] The method for targeted degradation of vomitoxin in feed according to the present application comprises the following steps:

[0008] The manganese peroxidase and the single-chain antibody are fused to obtain a fusion enzyme;

[0009] The above-mentioned fusion enzyme is used to degrade vomitoxin in feed.

[0010] The method for targeted degradation of vomitoxin in feed according to the present application, wherein the feed is wheat soybean meal and corn soybean meal.

[0011] The fusion enzyme for targeted degradation of vomitoxin in feed according to the present invention comprises manganese peroxidase and single-chain antibody, wherein the manganese peroxidase is at the upstream and the single-chain antibody is at the downstream, and the two are connected by GGGGS.

[0012] According to the fusion enzyme for targeted degradation of vomitoxin in feed of the present invention, the manganese peroxidase provided is CsMnP derived from Ceriporiopsis subvermispora, and its amino acid sequence is shown in SEQ ID No: 2.

[0013] According to the fusion enzyme for targeted degradation of vomitoxin in feed of the present invention, the accession number of the single-chain antibody is AAN75452, and the amino acid sequence thereof is shown in SEQ ID No: 1.

[0014] The present invention fuses DON-degrading enzyme, manganese peroxidase, with a single-chain antibody that specifically recognizes DON to express the resulting fusion enzyme, which can efficiently target and degrade vomitoxin in feed. Manganese peroxidase catalyzes DON degradation usually in an indirect manner, involving Mn 3+ The single-chain antibody used in this application can target and recognize DON, thereby increasing the local concentration of enzyme to DON. 2+ During oxidation, the newly formed Mn 3+ The fusion enzyme is characterized by its ability to efficiently degrade mycotoxins, is low-cost, and has a wide range of applications, making it suitable for use in the field of feed toxin-degrading enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 HPLC analysis results showing the degradation of pure DON by manganese peroxidase;

[0016] Figure 2 HPLC analysis results showing the degradation of pure DON by the fusion enzyme;

[0017] Figure 3 HPLC analysis results showing the degradation of DON by manganese peroxidase in wheat soybean meal;

[0018] Figure 4 HPLC analysis results showing the degradation of DON by manganese peroxidase in corn and soybean meal;

[0019] Figure 5 HPLC analysis results showing the degradation of DON by the fusion enzyme in wheat soybean meal;

[0020] Figure 6 HPLC analysis results showing the degradation of DON by the fusion enzyme in corn and soybean meal. DETAILED DESCRIPTION

[0021] Test materials and reagents

[0022] 1. Strain: An engineered E. coli strain producing CsMnP and single-chain antibody fusion enzyme derived from Ceriporiopsis subvermispora;

[0023] 2. Biochemical reagents: vomitoxin, chromatographic grade acetonitrile;

[0024] 3. Culture medium: Escherichia coli LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride).

[0025] Example 1 Preparation of recombinant fusion enzyme CsMnP-ScFv

[0026] CsMnP and ScFv gene sequences from Ceriporiopsis subvermispora were synthesized and linked to the ScFv gene sequence via the base sequence 5'GGCGGCGGTGGTAGC3' (encoding GGGGS). The BL21 / CsMnP-ScFv plasmid containing the recombinant plasmid was inoculated into 50 mL of LB medium supplemented with 50 μg / mL ampicillin and cultured overnight at 37°C in a shaker at 220 rpm. A 2% bacterial suspension was transferred to 300 mL of LB liquid medium supplemented with ampicillin and cultured at 37°C in a shaker at 200 rpm for approximately 3 hours until the OD reached 0. 600 ≈0.6-0.8; add IPTG to a final concentration of 1 mM to the culture medium and continue culturing at 37°C for 4 h to induce the expression of the recombinant fusion enzyme protein; centrifuge the bacterial solution at 12,000 rpm for 2 min to collect the bacteria and store at -20°C for later use.

[0027] To purify the recombinant protein, the induced E. coli culture was resuspended in 20 mL of 50 mM Tris-HCl buffer (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 hour. Then, 20 μL of DNase I was added to the mixture. After incubation on ice for 30 minutes, the mixture was centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant was removed. The insoluble fraction was washed twice with 20 mM Tris-HCl buffer (1 mM EDTA, 5 mM DTT, 2 M urea, and 1% Triton-100, pH 8.0) and transferred to 5 mM Tris-HCl (8 M urea, 1 mM EDTA, 1 mM DTT, 10% glycerol, pH 8.0) and incubated end over end at 4°C until completely dissolved. The recombinant enzyme was purified by immobilized affinity chromatography using nickel-coated magnetic beads. The purified protein was dialyzed for 24 h in a refolding buffer containing 50 mM Tris-HCl (pH 9.5), 0.6 M urea, 0.5 mM oxidized glutathione, 0.1 mM DTT, 5 mM CaCl2, and 10% glycerol. Then, a 5 μM heme solution was added and incubated at 4°C for 12 h to obtain the functional recombinant enzyme CsMnP-ScFv.

[0028] Example 2 Degradation of DON by Manganese Peroxidase

[0029] Deoxynivalenol was dissolved in methanol to prepare a 1 g / L stock solution, and the reaction system was as follows: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L deoxynivalenol, and 0.1 U / mL CsMnP. The system without manganese peroxidase was used as a control, and the reaction system was repeated three times. The reaction was carried out at 30°C, and after 24 hours, three volumes of methanol were added to terminate the reaction. The degradation rate of deoxynivalenol was analyzed by high-performance liquid chromatography (HPLC). The liquid chromatography was performed on a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, with a chromatographic separation column of Zorbax SB-C18 (4.6×250 mm, 5 μm), 10% acetonitrile as the mobile phase, and deoxynivalenol was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 2. Figure 1 As shown in the figure, the degradation rate of DON was 41.5%.

[0030] Example 3 Degradation of DON by Fusion Enzyme

[0031] Deoxynivalenol was dissolved in methanol to prepare a 1 g / L stock solution, and the reaction system was as follows: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L deoxynivalenol, and 0.1 U / mL CsMnP-ScFv. The system without the addition of fusion enzyme was used as a control, and the reaction system was repeated three times. The reaction was carried out at 30°C, and after 24 hours, three volumes of methanol were added to terminate the reaction. The degradation rate of deoxynivalenol was analyzed by high-performance liquid chromatography (HPLC). The liquid chromatography was performed on a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, with a chromatographic separation column of Zorbax SB-C18 (4.6×250 mm, 5 μm), 10% acetonitrile as the mobile phase, and deoxynivalenol was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 2. Figure 2 As shown in the figure, the degradation rate of vomitoxin was 37.7%.

[0032] Example 4 Degradation of vomitoxin in feed by manganese peroxidase

[0033] Deoxynivalenol was dissolved in methanol to prepare a 1 g / L stock solution, and the reaction system was as follows: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L deoxynivalenol, 0.1 U / mL CsMnP, 16 mg / mL wheat soybean meal or 16 mg / mL corn soybean meal. The system without manganese peroxidase was used as a control, and the reaction system was repeated three times. The reaction was carried out at 30°C and terminated after 24 h by adding three volumes of methanol. The degradation rate of deoxynivalenol was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography was performed on a Shimadzu Nexera UHPLC high performance liquid chromatography system, with a chromatographic separation column of ZorbaxSB-C18 (4.6×250 mm, 5 μm), 10% acetonitrile as the mobile phase, and deoxynivalenol was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 2. Figure 3 As shown in Figure 2, the degradation rate of DON in the presence of wheat soybean meal was 3.7%. Figure 4 As shown in the figure, the degradation rate of DON in the presence of corn soybean meal was 5.1%.

[0034] Example 5 Degradation of vomitoxin in feed by fusion enzyme

[0035] Deoxynivalenol was dissolved in methanol to prepare a 1 g / L stock solution. The reaction system was as follows: 50 mM malonate buffer, 1 mM MnSO4, 0.1 mM H2O2, 50 mg / L deoxynivalenol, 0.1 U / mL CsMnP-ScFv, 16 mg / mL wheat soybean meal or 16 mg / mL corn soybean meal. The system without the addition of fusion enzyme was used as a control, and the reaction system was repeated three times. The reaction was carried out at 30°C and terminated after 24 h by adding three volumes of methanol. The degradation rate of deoxynivalenol was analyzed by high-performance liquid chromatography (HPLC). The liquid chromatography was performed on a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, with a chromatographic separation column of ZorbaxSB-C18 (4.6×250 mm, 5 μm). The mobile phase was 10% acetonitrile, and deoxynivalenol was monitored under ultraviolet light at a wavelength of 218 nm. The results are shown in Figure 2. Figure 5 As shown in Figure 2, the degradation rate of DON in the presence of wheat soybean meal was 12.4%. Figure 6 As shown in the figure, the degradation rate of DON was 20.8% in the presence of corn soybean meal.

[0036] From the above data, we can see that the fusion enzyme has a great influence on the 2+ The specific activity of fusion enzyme is lower than that of manganese peroxidase, so in the absence of any interference, it is not as good as manganese peroxidase in degrading vomitoxin, but in the presence of feed, the effect of fusion enzyme is highlighted.

[0037] The above embodiments are only used to understand the technical solutions of the present invention and do not limit the scope of protection of this application.

Claims

1. A method for targeted degradation of vomitoxin in feed, characterized in that: The method comprises the following steps: Manganese peroxidase was fused to a single-chain antibody to obtain a fusion enzyme; Using the fusion enzyme to degrade vomitoxin in feed, The amino acid sequence of the manganese peroxidase is shown in SEQ ID No: 2, the amino acid sequence of the single-chain antibody is shown in SEQ ID No: 1, the manganese peroxidase is upstream, the single-chain antibody is downstream, and the two are connected with GGGGS.

2. The method for targeted degradation of vomitoxin in feed according to claim 1, characterized in that: The feed is wheat soybean meal or corn soybean meal.

3. A fusion enzyme for targeted degradation of vomitoxin in feed, characterized in that: The fusion enzyme is formed by the fusion of manganese peroxidase and a single-chain antibody, wherein the manganese peroxidase is upstream and the single-chain antibody is downstream, and the two are connected 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.

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