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A kind of zearalenone degrading enzyme and its coding gene and application

A technology of zearalenone and zearalenol, which is applied in the field of zearalenone degrading enzymes and their coding genes and applications, can solve the problems of insufficient enzyme research and achieve good stability

Inactive Publication Date: 2020-10-16
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many studies on the enzymes related to the degradation of zearalenone in the screened microorganisms

Method used

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  • A kind of zearalenone degrading enzyme and its coding gene and application
  • A kind of zearalenone degrading enzyme and its coding gene and application
  • A kind of zearalenone degrading enzyme and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, preparation and purification of protein and gene

[0046] 1. Artificial synthesis of gene sequence

[0047] The nucleotide sequence shown in SEQ ID NO.2 was entrusted to Wuhan Jinkairui Bioengineering Co., Ltd. to artificially synthesize the gene according to the conventional technology in the field, and the gene was inserted into the plasmid vector pUC57, and stored for future use.

[0048] 2. Gene sequence amplification

[0049] According to the nucleotide sequence shown in SEQ ID NO.2, the primer pair is designed as follows:

[0050] Forward primer: 5′-CGC GGATCC ATGGCCGCTACACGTACACGAGG-3', as shown in SEQ ID NO.3;

[0051] Reverse primer: 5′-CCG CTCGAG CTATTTCAAATACTTCCGACTCG-3', as shown in SEQ ID NO.4;

[0052] The underlined part of the forward primer is the restriction site of BamHI, and the underlined part of the reverse primer is the restriction site of XhoI.

[0053] PCR reaction system:

[0054] 10× buffer 5μL dNTP 4...

Embodiment 2

[0071] Example 2. Verification of protein function using zearalenone as a substrate

[0072] The enzyme activity unit is defined as the amount of enzyme required to degrade 1 μg of the substrate zearalenone within 1 min as an enzyme activity unit U.

[0073] (1) Optimum temperature

[0074] The Zhd518 pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer solution of pH 8.0, and the enzyme activity was measured with the diluted enzyme solution. The diluted enzyme solution was recorded as the diluted enzyme solution.

[0075] The composition of solution A: consists of 50 mM, pH 8.0 Tris-HCl buffer solution and zearalenone solution; the final concentration of the substrate zearalenone in the reaction system 0.5 mL is 20.0 μg / ml.

[0076] Experimental group: The reaction system for activity determination is 0.5mL, diluted with 0.45mL solution A and 0.05mL enzyme solution; the pH value of the reaction system is 8.0; mL of chromatographic grade methan...

Embodiment 3

[0113] Example 3. Verification of protein function using β-zearalenol as a substrate

[0114] The enzyme activity unit is defined as the amount of enzyme required to degrade 1 μg of the substrate zearalenone within 1 min as an enzyme activity unit U.

[0115] The diluted enzyme solution described below is obtained by diluting the Zhd518 pure enzyme solution in Step 5 of Example 1 with 50 mM Tris-HCl buffer solution.

[0116] Experimental group: β-zearalenol was used as the substrate (the final concentration of the substrate in the reaction system was 20.0 μg / ml), the activity assay reaction system was 0.5mL, and 0.45mL substrate solution and 0.05mL diluted enzyme solution; the pH value of the reaction system was 8.0; after the reaction system was reacted at an optimum temperature of 40° C. for 10 min, 0.5 mL of chromatographic grade methanol was used to terminate the reaction, and after cooling, the amount of degradation of the substrate was measured using high performance liq...

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Abstract

The invention relates to zearalenone degradation enzyme and coding gene and application thereof. The degradation enzyme has an amino acid sequence indicated in SEQ ID NO. 1 or the degradation enzyme is an conservative variant obtained from conservative mutation by missing, substituting, inserting or / and adding one to several amino acids. The zearalenone degradation enzyme has the advantages of high enzyme activity, good pH tolerance and the like and can be widely applied to enzymolysis of zearalenone and several derivatives thereof, and substrate specificity for zearalenone and beta-zearalanel is especially good.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a zearalenone degrading enzyme, its coding gene and its application. Background technique [0002] Zearalenone, first isolated from corn, is a non-steroidal estrogenic mycotoxin that can be produced by many Fusarium species, both before and after harvest. Zearalenone is consistently found in many crop and cereal by-products including corn, barley, wheat, etc., especially in environments that are suitable for fungal growth. [0003] There are many derivatives of zearalenone, such as zearalenol, which will enter the food chain through contaminated crops and accumulate in human and animal bodies, causing damage to organisms. The chemical structure of zearalenone and its derivatives is similar to natural estrogen, so they can competitively bind to estrogen receptors, causing external and internal genital changes and reproductive disorders, leading to hyperoestrogenism and in...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/11
CPCC12N9/18
Inventor 张桂敏王美星尹李峰周玉玲马延和
Owner HUBEI UNIV
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