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Zearalenone hydrolytic enzyme and coding gene and application thereof

A technology of zearalenone and coding genes, applied in the field of feed enzymes, can solve problems such as loss of nutrients, residual chemical reagents, and ineffective degradation of toxins

Inactive Publication Date: 2020-07-10
BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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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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  • Zearalenone hydrolytic enzyme and coding gene and application thereof
  • Zearalenone hydrolytic enzyme and coding gene and application thereof
  • Zearalenone hydrolytic enzyme and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Construction of recombinant erythralenone hydrolase ZENWJ expression vector

[0035] The optimized gene sequence was directly connected to the expression vector pHT43 and the zearalenone hydrolase coding gene zenwj by Gene Synthesis Company to obtain the recombinant plasmid pHT43-zenwj and transform it into Bacillus subtilis WB800N to obtain the recombinant Bacillus subtilis strain WB800N / zenwj.

[0036] After the sequencing was correct, the recombinant plasmid pHT43-zenwj was transformed into Bacillus subtilis competent WB800N for induced expression. Pick 3-5 positive transformants and inoculate them into 30ml 2xYT liquid medium containing 100μg / ml kanamycin, culture at 37°C and 250rpm for 6 hours, this is pre-culture; transfer this pre-culture to 300ml containing In 2xYT liquid medium with 100 μg / ml kanamycin, culture at 37°C and 250 rpm for 2-3 hours, the OD600 is about 0.5. 1M IPTG stock solution was added to the culture to make the final concentration...

Embodiment 2

[0037] Example 2 Activity detection and analysis of recombinant erythralenone hydrolase

[0038] ZENWJ of codon-optimized erythralenone hydrolase, induced by IPTG, expressed and purified, the protein SDS-PAGE analysis results are as follows figure 1 As shown, the analysis of the degradation of different concentrations of zearalenone by zearalenone hydrolase is as follows figure 2 as shown,

[0039] The reaction system is:

[0040] 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 control is an appropriately diluted enzyme solution in a boiling water bath...

Embodiment 3

[0042] Example 3 Application experiment of zearalenone hydrolase ZENWJ

[0043]Corn dregs contaminated with ZEN were collected from the market, and the ZEN content was detected to be 1225 μg / kg. The Bacillus subtilis fermentation supernatant (enzyme liquid) was diluted 2, 5, and 10 times with buffer solution. According to the 1:1 ratio of fermentation liquid and corn dregs (v:w), mix evenly (that is, the enzyme liquid addition amount is 0.1, 0.2, 0.5mL / g), and react at 37°C for 0, 3, 6, 12, 24h, without Enzyme solution was added as a control. ZEN in corn dregs was extracted according to GB / T 23504-2009, the residual amount of ZEN was detected by HPLC, and the degradation rate of ZEN in corn dregs was calculated.

[0044] Such as image 3 As shown, with the extension of the degradation time, the ZEN content in the sample gradually decreased, the degradation rate was faster in the first 6 hours of the reaction, and the degradation rate tended to be gentle after 6 hours. The ...

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Abstract

According to the invention, the amino acid sequence of a zearalenone detoxification enzyme is optimized, and the 10 amino acid residues (10 amino acid differences exists with zhd 101 detoxification enzyme) of the detoxification enzyme are changed, so that the optimum reaction pH of the reassembled zearalenone detoxification enzyme is reduced from 8.5 to 6.5, and is very consistent with the pH of the intestinal tract of chicken and pigs; and when the pH is as low as 3.0, the detoxification enzyme activity remains 40% or above, so that the detoxification efficiency of the zearalenone detoxification enzyme as a feed additive in practical application is greatly improved. The invention also performs codon optimization on the zearalenone detoxification enzyme gene and expresses the gene in bacillus subtilis.

Description

technical field [0001] The invention relates to the field of feed enzymes, in particular to zearalenone hydrolase ZENWJ and its coding gene and application. Background technique [0002] Zearalenone (ZEN) is a non-steroidal mycotoxin produced by Fusarium graminearum, which was first isolated from moldy corn by Stob et al. in 1962. In 1966, Urry et al. determined the chemical structure of the substance by means of classical chemistry, nuclear magnetic resonance and pristine analysis, and called it F-2 toxin. Its chemical name is: 6-(10-hydroxyl-6-oxyl-undecenyl)-β-threonolactone, also known as F-2 toxin, its chemical name is 6-(10-hydroxyl- 6-Oxy-Undecenyl) β-Raycarboxylate, Molecular Formula C 18 h 22 o 5 , the molecular weight 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. [0003] According to the estimates of the Food and Agriculture Organization of the United Nations (FAO), about 25%...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/75C12N1/21A23L5/20C12R1/125
CPCC12N9/18C12N15/75A23L5/25
Inventor 褚华硕于海娜姚宏明宫瑞雪黄玉岚高长斌刘延亭
Owner BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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