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Vomitoxin degrading enzyme DDH and application of DDH in detoxification of Trichothecenes

A technology of trichothecenes, vomitoxin, applied in the field of agricultural biology

Active Publication Date: 2020-07-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no other sources of enzymes that can degrade DON.

Method used

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  • Vomitoxin degrading enzyme DDH and application of DDH in detoxification of Trichothecenes
  • Vomitoxin degrading enzyme DDH and application of DDH in detoxification of Trichothecenes
  • Vomitoxin degrading enzyme DDH and application of DDH in detoxification of Trichothecenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Acquisition and expression of deoxynivalenol-degrading enzyme DDH protein

[0064] The DDH protein coding gene was amplified with the genomic DNA of M. halotolerant ANSP101 as the amplification template, and the recombinant expression vector containing the DDH protein coding gene sequence and its engineering bacteria were constructed to express the DDH protein. Specific steps are as follows:

[0065] 1. Cloning of DDH gene encoding deoxynivalenolase

[0066] 1.1 Extract the genomic DNA of M. halotolerant according to the following steps:

[0067] Take 50 μL of the preserved bacteria solution from the glycerol tube, inoculate and streak on the LB solid plate, and culture at 37°C for 12 hours.

[0068] Pick a single colony from the cultured plate and inoculate it in 5 mL liquid LB medium, culture at 180 r / min, 37°C for 12 h.

[0069] Aliquot the bacterial solution into sterilized 1.5mL microcentrifuge tubes, centrifuge at 12000r / min for 1min to collect the ba...

Embodiment 2

[0103] Example 2 Effect of temperature on activity of deoxynivalenol-degrading enzyme DDH degrading DON

[0104] Dissolve solid vomitoxin in acetonitrile to make a 500 μg / mL mother solution, and dissolve the prosthetic group PMS (phenazine methyl sulfate) in ultrapure water to make a 5 mM mother solution. The experiment is carried out according to the following 500 μL reaction system: 350 μL glycine-hydrogen Sodium oxide buffer (0.05M, pH9.0), 50 μL DDH protein (23.5 μg), 50 μL prosthetic PMS solution, 50 μL DON solution. The system without adding DDH protein was used as a control. The reaction was carried out at different temperatures (20, 25, 30, 35, 40, 45°C) for 1 h, then 500 μL of methanol was added to terminate the reaction, centrifuged at 12,000 rpm for 1 min, and the supernatant was filtered with a Millex-GV filter membrane (0.22 μm). The residual DON content in the system was detected by liquid chromatography.

[0105] The chromatographic conditions for high perform...

Embodiment 3

[0108] Example 3 Effect of pH on Degradation DON Activity of Deoxynivalenol-degrading Enzyme DDH

[0109] In order to test the effect of the deoxynivalenol degrading enzyme DDH on the DON degradation activity under different pH conditions, the reaction system used in case implementation 2 was adopted: 350 μL buffer (0.05M sodium phosphate buffer, pH6-8; 0.05M glycine-NaOH buffer, pH9-11), 50 μL DDH protein (23.5ug), 50 μL prosthetic PMS solution, 50 μL DON solution. After the reaction was carried out at 35°C for 1 hour, 500 μL of methanol was added to terminate the reaction, centrifuged at 12,000 rpm for 1 minute, and the supernatant was filtered through a Millex-GV filter membrane (0.22 μm), and the residual DON content in the system was detected by the method described in Case Example 2.

[0110] The result is as image 3 It was shown that the optimum pH for degrading DON by the deoxynivalenol-degrading enzyme DDH was 9.0.

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Abstract

The invention belongs to the field of agricultural biotechnology, and in particular relates to a vomitoxin degrading enzyme DDH, and a coding gene and application of DDH. The vomitoxin degrading enzyme DDH involved in the present invention has an amino acid sequence as shown in SEQ ID NO. 1. The invention also discloses the gene encoding the above vomitoxin degrading enzyme DDH, and the DNA sequence of the gene is shown in SEQ ID NO. 2 or SEQ ID NO. 3. The invention further discloses a preparation method of the above vomitoxin degrading enzyme DDH, the application of DDH in detoxification of Trichothecenes, and the preparation method and application of DDH as a biodegradation agent of Trichothecenes. The vomitoxin degrading enzyme DDH of the invention has broad application prospects in thefield of the detoxification of Trichothecenes.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a deoxynivalenol degrading enzyme DDH and its coding gene and application, especially the application in the detoxification of trichothecene toxins. Background technique [0002] Trichothecene mycotoxins (Trichothecene mycotoxins) are a large class of mycotoxins produced by Fusarium, mainly including vomitoxin (also known as deoxynivalenol, Deoxynivalenol, DON), T-2 toxin, nivalenol Nivalenol (NIV), Fusarenon X (FUS), etc. Such toxins are easy to contaminate crops such as wheat, barley, and corn, and are often detected in these grains and animal feed, and some of them exceed the standard. This type of toxin can cause vomiting, food refusal, damage to digestive tract mucosa, decline in production performance and immune function in animals, and cause serious economic losses to livestock farmers. At present, there are two main methods for mycotoxin detoxificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21A23L5/20A01G7/06
CPCC12N9/0006C12Y101/01021C12N15/70A23L5/25A01G7/06Y02P60/87
Inventor 赵丽红马秋刚秦晓娟郭永鹏计成
Owner CHINA AGRI UNIV
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