Detoxication engineering bacteria, and preparation method and application thereof

A technology of engineering bacteria and detoxification esters, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of excessive pesticide residues, export losses of agricultural products, export scale restrictions, etc., and achieve a widening of the degradation spectrum , Solve the pollution problem and eliminate the effect of loss

Inactive Publication Date: 2013-01-02
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of standardized management of the use of pesticides in my country, pesticide residues in agricultural products generally exceed the standard, resulting in heavy losses in the export of agricultural products in my country. Traditional export products, such as fruits, tea and Chinese herbal medicines, have repeatedly returned in large quantities, and the export scale has been severely restricted.

Method used

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  • Detoxication engineering bacteria, and preparation method and application thereof
  • Detoxication engineering bacteria, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the construction of engineering bacteria

[0040] 1. Construction of recombinant plasmid (pETDuet-E4-mpd)

[0041] 1. Preparation of hydrolase gene

[0042] The DNA shown in sequence 4 is artificially synthesized, the 11th to 901st nucleotides from the 5' end of sequence 4 are the hydrolase (mpd) gene, the upstream is the BamHI site, and the downstream is the HindIII site.

[0043] 2. Preparation of detoxification esterase gene

[0044] The DNA shown in the artificially synthesized sequence 2, sequence 2 from the 8th to the 1666th nucleotide at the 5' end is the detoxification esterase (E4) gene, its upstream is the BglII site, and its downstream is the XhoI site.

[0045] 3. Construction of recombinant plasmid (pETDuet-E4-mpd)

[0046] ① Digest the hydrolase gene in step 1 with restriction endonucleases BamHI and HindIII.

[0047] ② Digest plasmid pETDuet-1 with restriction endonucleases BamHI and HindIII.

[0048]③ Ligate the digested product of ste...

Embodiment 2

[0082] The crude enzyme solution of embodiment 2, Pet30-mpd bacterial strain is to the degradation of carbaryl and malathion

[0083] 1. Degradation of carbaryl by crude enzyme solution of Pet30-mpd

[0084] The degradation of carbaryl was determined according to the method of Hayatsu and Nagata (1993), specifically as follows:

[0085] Buffer: 10mM KH 2 PO 4 / KOH buffer; pH 7.5.

[0086] Chromogenic reagent (DBLS): Dissolve 4.5mg of Fast Blue B salt in 4.5ml of water, and then add 10.5ml of 5% SDS solution.

[0087] Reaction system (20ml): add Carbaryl and the crude enzyme solution of the Pet30-mpd bacterial strain that embodiment 1 prepares in buffer solution, contain 130 μ M Carbaryl and 5ml crude enzyme solution (107U ml -1 ).

[0088] Control system (20ml): replace the crude enzyme solution with deionized water, and the others are the same as the reaction system.

[0089] The reaction temperature was 37°C, and 2ml samples were taken every 30 minutes. Add 0.5ml fres...

Embodiment 3

[0098] Embodiment 3, the detoxification experiment of chicken chlorpyrifos poisoning

[0099] Adult aircraft carrier chickens (Gallus gallus) (12 months old, weighing about 1.5 kg) were purchased from the Experimental Chicken Farm of Beijing Agricultural University. After the chickens were bought back, they were fed individually, and the experiment was started after at least one week of adaptation to the environment. Free access to food and water, during the experiment, the temperature of the chicken house was controlled at about 20°C, and the light was about 10h per day.

[0100] The experimental chickens were divided into seven groups, 10 in each group:

[0101]A group of tests: give chlorpyrifos (in empty capsules, the dose is 170mg / kg, orally) and Pet30-mpd (engineering bacteria) (0.5g wet thalline is packed in empty capsules, orally) at the same time;

[0102] Experimental group two: first give chlorpyrifos (put into empty capsules, the dose is 170mg / kg, take orally), t...

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Abstract

The invention discloses detoxication engineering bacteria, and a preparation method and application thereof. The invention provides a recombinant plasmid which is obtained by introducing coded genes of detoxication esterase and coded genes of hydrolase into an enzyme cutting site in plasmid pETDuet-1. The engineering bacteria of the invention can be obtained by introducing the recombinant plasmidinto host bacteria. The engineering bacteria comprise the coded genes of the detoxication esterase and the hydrolase and can realize inducible expression of the detoxication esterase and the hydrolase. Crude enzyme solution obtained by fermentation of the engineering bacteria can also be protected. The engineering bacteria and the crude enzyme solution thereof can simultaneously degrade organic acid esters such as malathion, parathion, high-efficiency cypermethrin, acetofenate and the like and organophosphorus pesticides, and can be used for detoxication of organophosphorus poisoned warm-blooded animals. The invention provides a new way for biological pollution treatment of toxic and harmful compounds such as pesticides remained in water sources, soil and the like by using natural resources of eukaryote and prokaryote.

Description

technical field [0001] The invention relates to a detoxification engineering bacterium and its preparation method and application. Background technique [0002] Modern agricultural production has played a huge role in satisfying people's desire for high food production, and was once regarded as a panacea for third world countries to get rid of poverty and hunger. Crop diseases and insect pests are becoming more and more rampant, agricultural resources are wasting, and the agricultural environment is degrading. Faced with the food crisis in Africa, people gradually have a kind of confusion and doubts while rejoicing, and begin to re-examine the concept of modern agriculture. Re-understand the connotation of agricultural resources and environment, and rethink the direction of agricultural research and decision-making. [0003] The application of insecticides (ie pesticides) is a double-edged sword. On the one hand, pests eat a large number of crops. For example, in India, al...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/55C12N9/16C12N9/14C12N1/21A62D3/02A61K38/46A61P39/02C12R1/19A62D101/04
Inventor 乔传令杨超兰文升
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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