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Mycoenone hydrolase zen214 and its coding gene and application

A technology of ZEN214 and hydrolase, applied in the field of feed enzymes

Active Publication Date: 2021-02-23
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection method of ZEN has been relatively perfect, but the problems related to the conversion and degradation of ZEN are imminent but still unresolved.

Method used

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  • Mycoenone hydrolase zen214 and its coding gene and application
  • Mycoenone hydrolase zen214 and its coding gene and application
  • Mycoenone hydrolase zen214 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of recombinant erythralenone hydrolase ZEN214 expression vector

[0035] Using the overlap-PCR method, the expression vector pet30a was connected with the zearalenone hydrolase coding gene zen214 by two-step PCR method to obtain the recombinant plasmid pet30a-zen214 and transform Escherichia coli BL21 to obtain the recombinant Escherichia coli strain BL21 / zen214.

[0036] The primers used in the first step of PCR are as follows:

[0037] 214-30a F:5'-TATGCACCATCATCATCATCATATGGTTTTCGAAAGATTG-3'

[0038] 214-30a R: 5'-AGTGGTGGTGGTGGTGGTGTTAAGGCAAGTGAGATCTAGT-3'

[0039] 30a-214F:5'-GCCTTAACACCACCACCACCACCACTGAGATCCG-3'

[0040] 30a-214R:5'-TCGAAAACCATATGATGATGATGATGGTGCATAT-3'

[0041] Among them, 214-30a F and 214-30a R are used to amplify the fragment of zen214, and 30a-214F and 30a-214R are used to amplify the vector fragment of pet30a.

[0042] The PCR reaction system is as follows:

[0043]

[0044] The PCR reaction conditions are: 95°C f...

Embodiment 2

[0051] Example 2 Activity analysis of recombinant erythralenone hydrolase

[0052] The codon-optimized erythralenone hydrolase ZEN214 has a protein expression level that is about 2.2 times higher than that before optimization, which can significantly reduce production costs. The results of SDS-PAGE analysis of the expressed and purified protein are as follows figure 1 Shown, zearalenone hydrolase degrades zearalenone substrate HPLC analysis as figure 2 as shown,

[0053] The reaction system is:

[0054] 800ul of disodium hydrogen phosphate citrate buffer solution with pH6.0, plus 100ul of 0.5g / L zearalenone toxin (purchased from sigma company) dissolved in DMSO, and then add 100ul of enzyme solution properly diluted, 37 Incubate at 100°C for one hour, then add 2 times the volume of DMSO to terminate the reaction, and shake vigorously at the same time, draw part of the sample through a microporous membrane, and load it for high-performance liquid phase analysis (HPLC). The c...

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Abstract

The invention relates to the field of feed enzymes, in particular to zearalenone hydrolase ZEN214 and its coding gene and application. Its amino acid sequence is shown in SEQ ID NO.1. The enzyme can hydrolyze zearalenone and remove toxins. The application of moldy feed and feed raw materials has broad development prospects.

Description

technical field [0001] The invention relates to the field of feed enzymes, in particular to mycoenone hydrolase ZEN214 and its coding gene and application. Background technique [0002] Zearalenone (Zearalenone, ZEN) is the most widely distributed mycotoxin produced by Fusarium in the world. It occurs in grains and agricultural by-products in Asia, Europe and America. ZEN has estrogen-like properties, also known as F-2 toxin, its chemical name is 6-(10-hydroxy-6-oxyl-undecenyl) β-leucanolide, its molecular formula is C18H22O5, and its molecular mass is It is 318, the melting point is 165°C, it is relatively stable to heat, and it will not be degraded when heated at 120°C for 4 hours. ZEN has strong reproductive toxicity, can produce competitive binding with estrogen receptor and then exhibit estrogenic activity. After the combination of estrogen receptor and ZEN, the configuration of the receptor changes, and it transfers to the nucleus to further combine with chromatin to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12N15/70
Inventor 姚斌柏映国杨虹罗会颖陈家明涂涛孟昆王亚茹黄火清苏小运王苑马锐师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI