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Zeranol decomposing enzyme and coding gene and application thereof

A technology of erythralenone and coding genes, which is applied in hydrolytic enzymes, genetic engineering, plant genetic improvement, etc., can solve the problems of nutrient loss, chemical reagent residue, toxin degradation effect is not obvious, etc., to achieve the prevention and treatment of mycotoxin poisoning Effect

Inactive Publication Date: 2017-08-11
广州和仕康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The toxin degradation effect of the physical method is not obvious. Although the effect of the chemical method is better than the physical method, it may cause the loss of nutrients or the residue of chemical reagents

Method used

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  • Zeranol decomposing enzyme and coding gene and application thereof
  • Zeranol decomposing enzyme and coding gene and application thereof
  • Zeranol decomposing enzyme and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Acquisition of zearalenone decomposing enzyme gene (ZEN-jlm)

[0021] The inventors used TaKaRa Smart TM The cDNA library construction kit was used to construct the cDNA library of Gliocladium roseum. Randomly picked clones were sequenced, and the measured sequence was compared with the sequence in the international gene bank (GenBank) by BLAST to obtain the red cDNA of enonelolytic enzyme (ZEN-jlm).

[0022] 1. Extraction of total RNA from Gliocladium roseum

[0023] Clonospora rosea was cultured in liquid PDA medium (200 rpm, 30° C., 48 h). After the culture was completed, the precipitate was collected by centrifugation at 12000 r / min for 10 min. Wash with 75% ethanol solution prepared with DEPC treated water. Dry at room temperature, dissolve and precipitate RNA in RNase-free water, and two clear bands of 28S and l8S can be seen by 1% agarose gel electrophoresis; 260 / OD 280 The ratio identifies the purity of the total RNA, and the ratio is 1.95; stor...

Embodiment 2

[0061] Example 2: Recombinant expression of zearalenone decomposing enzyme in Bacillus subtilis

[0062] 1. According to SEQ ID NO: 1, design primers as follows:

[0063] P1: 5'-GTGGGGTTCAAAGAAAGGCCTG-3' (SEQ ID NO: 3);

[0064] P2: 5'-CCCAAGCTT CTAGAACTTGCCAAGGAACTTGGC-3' (introducing a HindIII restriction site) (SEQ ID NO: 4).

[0065] Using the pET30a-ZENjlm plasmid as a template to amplify the ZEN-jlm gene, the PCR system is as follows:

[0066]

[0067]

[0068] Reaction program: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min. 1% agarose gel electrophoresis to detect PCR products.

[0069] 2. Using the Bacillus subtilis genomic DNA as a template, set primers to amplify P 43 Promoter sequence, EcoRI restriction site was introduced into primer P3, and the designed primers were as follows:

[0070] P3: 5'-CGGAATTCCTTGTAGAGCTCAGCATTATTG-3' (SEQ ID NO: 5);...

Embodiment 3

[0106] Example 3: Separation and purification of recombinant erythralenone decomposing enzyme

[0107] Preparation of seed solution: Inoculate the recombinant strain WB800-ZEN-jlm into LB medium, fill a 250mL Erlenmeyer flask with a liquid volume of 50mL, then place it in a constant temperature shaking shaker at 37°C, 200r / min, and cultivate for 24h.

[0108] Preparation and separation and purification of fermentation supernatant: 14L LB medium was placed in a 20L fermentation hall, and after autoclaving, 5% (v / v) inoculum was used to inoculate the shake flask seed solution. During the fermentation, the dissolved oxygen content was 60%, the magnetic stirring speed was 200r / min, and the fermentation was carried out at 37°C for 24 hours. After the fermentation was completed, the fermentation broth was centrifuged at 5000r / min for 10min in a refrigerated centrifuge at 4°C. The supernatant was used for protein precipitation. With 40% saturation (NH 4 ) 2 SO 4 Solution, 4°C, 5h...

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Abstract

The invention provides a zeranol decomposing enzyme and a coding gene thereof. An amino acid sequence of the zeranol decomposing enzyme is shown as in SEQ ID NO: 2, or the zeranol decomposing enzyme is a protein derivative, with a zeranol decomposing function, obtained by modification of the amino acid sequence shown as in SEQ ID NO: 2. The enzyme is capable of completely decomposing zeranol. The recombinant zeranol decomposing enzyme described herein is capable of decomposing fungaltoxin zeranol in feed and has an effective function in prevention and treatment of swine mycotoxicosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an erythralenone decomposing enzyme and its coding gene, as well as the application of the decomposing enzyme. Background technique [0002] Zearalenone (ZEN) is a non-steroidal mycotoxin produced by Fusarium graminearum, first introduced in 1962 [0003] Years were isolated from moldy corn by Stob et al. In 1966, Urry et al. determined the chemical structure of the substance by means of classical chemistry, nuclear magnetic resonance and mass spectrometry, and called it F-2 toxin. Its chemical name is: 6-(10-hydroxy-6-oxyl-undecenyl)-β-lenacarboxylate. [0004] ZEN is a type of mycotoxin with the widest pollution range in the world, including wheat, corn, sorghum, oats and other grains and their products. Among them, the detection rate of ZEN in corn is extremely high, seriously affecting food safety. ZEN has a strong estrogen effect, and its main action site is the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/75C12N1/21A23K20/189C12R1/125
CPCC12N9/18
Inventor 汪猜皮灿辉刘耿霞
Owner 广州和仕康生物技术有限公司
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