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Dye decolorizing peroxidase BsDyP and application of dye decolorizing peroxidase BsDyP in mycotoxin detoxification

A peroxidase and mycotoxin technology, applied in the field of agricultural biology, can solve the problems of unclear toxicity, limiting the research and application of mycotoxin-degrading enzymes, etc.

Active Publication Date: 2020-04-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The enzymes reported to degrade zearalenone include the lactone hydrolase ZHD101 from Gliocladium rubrum and the zearalenone degrading enzyme ZENdease-N2 from the fungus Vitis vinifera; Aspergillus toxin enzymes include aflatoxin oxidase ADTZ from Armillaria pseudoarmilla, F420H2-dependent reductase FDR from mycobacteria, and fungal laccase from white-rot fungi, but the degradation mechanism of these enzymes and The toxicity of degradation products is not very clear, which limits the research and application of mycotoxin-degrading enzymes

Method used

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  • Dye decolorizing peroxidase BsDyP and application of dye decolorizing peroxidase BsDyP in mycotoxin detoxification
  • Dye decolorizing peroxidase BsDyP and application of dye decolorizing peroxidase BsDyP in mycotoxin detoxification
  • Dye decolorizing peroxidase BsDyP and application of dye decolorizing peroxidase BsDyP in mycotoxin detoxification

Examples

Experimental program
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Effect test

Embodiment 1

[0064] The acquisition and expression of embodiment 1 dye decolorization peroxidase BsDyP

[0065] Using Bacillus subtilis 168 genomic DNA as the amplification template, amplify the coding gene of dye decolorization peroxidase BsDyP, then construct the recombinant expression vector containing the BsDyP coding gene sequence and its engineering bacteria, and express the dye decolorization peroxidase BsDyP . Specific steps are as follows:

[0066] 1. Cloning of the gene encoding dye decolorization peroxidase BsDyP

[0067] 1.1 Extract Bacillus subtilis genomic DNA according to the following steps

[0068] (1) Inoculate and streak on LB solid plates from glycerol tubes, and culture at 37°C for 12 hours.

[0069] (2) 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 hours.

[0070] (3) Aliquot the bacterial solution into sterilized 1.5mL microcentrifuge tubes, centrifuge at 12000r / min for 1min to collect th...

Embodiment 2

[0098] Example 2 Recombinant dye decolorization peroxidase rBsDyP degrades zearalenone under different temperature conditions

[0099] Zearalenone (ZEN) was dissolved in methanol to form a 1000 μg / mL stock solution, and the degradation reaction was carried out in 1 mL of sodium phosphate buffer (0.1 M, pH 8.0). The rBsDyP protein concentration obtained in Example 1 was is 100μg / mL, ZEN concentration is 10μg / mL, H in the reaction system 2 o 2 The content was 100 μM, and the system without adding rBsDyP protein was used as a control; reacted at 27, 32, 37, 42, 47, 52 and 57°C for 1 hour, and added 1 mL of methanol to terminate the reaction, and detected the ZEN content by high performance liquid chromatography.

[0100] The chromatographic conditions for HPLC detection of ZEN are: chromatographic column: Agilent C18 chromatographic column, 4.6mm×150mm×5μm; mobile phase: acetonitrile-water-methanol (46:46:8); flow rate: 1mL / min; pump pressure : 45 bar; injection volume: 20 μL; ...

Embodiment 3

[0102] Example 3 Recombinant dye decolorization peroxide rBsDyP degrades zearalenone under different pH conditions

[0103] In order to test the activity of rBsDyP prepared in Example 1 to degrade ZEN under different pH conditions, in 1 mL of buffers with different pH values ​​(pH4-6, 0.1M sodium citrate buffer; pH7-8, 0.1M sodium phosphate buffer; pH9-10, 0.1M glycine-NaOH buffer) for degradation reaction, H in the reaction system 2 o 2 The concentration of rBsDyP protein prepared in Example 1 was 100 μg / mL, and the concentration of ZEN was 10 μg / mL; the system without adding rBsDyP protein was used as a control; reacted at 42°C for 1 hour, and added 1 mL of methanol to terminate the reaction. The content of ZEN was detected by liquid chromatography.

[0104] The result is as image 3 As shown, the optimum pH for the degradation of ZEN by rBsDyP protein is 8, and the degradation rate of rBsDyP prepared in Example 1 and ZEN (10 μg / mL) at 42°C for 1 hour under the optimal pH...

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Abstract

The invention belongs to the technical field of agricultural biology, and specifically relates to a dye decolorizing peroxidase BsDyP as well as an encoding genes and application thereof; and the dyedecolorizing peroxidase BsDyP disclosed by the invention has an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention further discloses a gene encoding the dye decolorizing peroxidase BsDyP; and the gene encoding the dye decolorizing peroxidase BsDyP has a DNA sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4. The invention still further discloses a preparation method of the dye decolorizing peroxidase BsDyP as well as application of the dye decolorizing peroxidase BsDyP in mycotoxin detoxification, and a preparation method of a mycotoxin biodegradation agent by using the dye decolorizing peroxidase BsDyP as well as application of the prepared mycotoxin biodegradation agent. Thus, the dye decolorizing peroxidase BsDyP disclosed by the invention has great application prospect in the field of mycotoxin detoxification.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to dye decolorization peroxidase BsDyP and its coding gene and application, especially the application in the detoxification of mycotoxins. Background technique [0002] Mycotoxins are secondary metabolites of fungi, which have carcinogenic, genotoxic, reproductive toxic, neurotoxic and immunosuppressive effects, and are extremely harmful to humans and various livestock and poultry. At present, there are more than 300 kinds of mycotoxins that have been isolated and identified, among which the most polluting and toxic, the most studied are aflatoxin (Aflatoxin, AFT), zearalenone (Zearalenone, ZEN), and trichothecenes. Trichothecene mycotoxins (including vomitoxin and T-2 toxin, etc.), ochratoxin (Ochratoxin) and fumonisin (Fumonisin), etc. At present, there are two main methods for mycotoxin detoxification in the food and feed industry. One is physical adsorption...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N9/04C12N15/53A23L5/20
CPCC12N9/0065C12N9/0006A23L5/25C12Y111/01C12Y101/03004
Inventor 赵丽红郭永鹏马秋刚计成唐彧
Owner CHINA AGRI UNIV
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