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Complex enzyme preparation for eliminating residual pesticides and application thereof

A compound enzyme preparation and residual pesticide technology, which is applied to the compound enzyme preparation of residual pesticides in tea leaves and melons and fruits, eliminates the field of vegetables, can solve the problems of unacceptable vegetables and fruits, and achieves excellent removal of pesticide residues, wide degradation spectrum, and responsive The effect of mild conditions

Active Publication Date: 2008-10-29
澳联健康科技(江苏)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if there is no impact, it is estimated that the vegetables and fruits treated in this way will be difficult to be accepted by people

Method used

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  • Complex enzyme preparation for eliminating residual pesticides and application thereof
  • Complex enzyme preparation for eliminating residual pesticides and application thereof
  • Complex enzyme preparation for eliminating residual pesticides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0053] Preparation of Pesticide Degrading Enzyme

[0054] The pesticide-degrading enzymes derived from microorganisms can be obtained by cultivating microorganisms that produce the enzymes, or can be prepared by genetic engineering methods. For example, the recombinant protein can be produced by cloning the gene encoding the pesticide degrading enzyme in a prokaryotic expression vector such as an Escherichia coli expression vector or a eukaryotic expression vector such as a yeast expression vector, which is well known to those skilled in the art.

[0055] Enzymes that eliminate pesticide residues in vitro have much lower purity requirements than protein pharmaceuticals. In order to simplify the purification process and reduce costs, tags for easy purification can be added to the N-terminal or C-terminal of the protein, such as FLAG, 6×His, etc., among which 6×His is more mature and effective. As a preference, the present invention chooses to introduce a 6×His tag at the N-ter...

Embodiment 1

[0059] Embodiment 1, the preparation of opd gene product

[0060] (1) Expression and purification

[0061] A. According to the gene sequence published by genbank accession number M20392, an Nde I site was introduced at the 5' end, and an Eco RI site was added after the stop codon to design the opd expression gene;

[0062] B, the gene is cloned between the Nde I and Eco RI sites of the pET30a (+) plasmid, and the expression plasmid is constructed;

[0063] C. Use CaCl 2 Transform the expression plasmid into Escherichia coli BL21 (DE3) strain, select Kanna-resistant clones, and obtain engineering bacteria;

[0064] D. Inoculate the engineered bacteria in step (2) in LB medium containing 50 μg / ml kanamycin, culture overnight at 37° C. on a shaker. Transfer to fresh LB medium containing kanamycin at a ratio of 1:100, continue culturing until OD600≈0.8, add IPTG to a concentration of 0.5mmol / L to induce expression for 4 hours, collect the bacteria by centrifugation, and discard...

Embodiment 2

[0074] Embodiment 2, the preparation of cehA gene product

[0075] According to the gene sequence published by genbank accession number: Ab069723, an Nde I site was introduced at the 5' end, and an Eco RI site was added after the stop codon to design the cehA expression gene;

[0076] The cehA gene expression product was prepared and characterized according to the same method as in Example 1, and a total of 67 ml of target protein solution was obtained; the purity was 85.4%, and the concentration was 2.7 mg / ml.

[0077] Measure the activity as follows: the basic reaction system is 1ml of PB buffer (50mmol / L, pH7.5) containing 2mmol / L of the corresponding substrate carbaryl, add about 5 μg of enzyme, react at room temperature for 10 minutes, and stop with dilute sulfuric acid reaction. A control group was also established. After the reaction, the carbamate peak areas of the control group and the test group were analyzed by gas chromatography according to NY / T 761-2004, and th...

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Abstract

The purpose of the invention is to provide a compound enzymatic preparation which has a wide degrading spectrum and is capable of clearing residual pesticide on common vegetables and fruits, and decomposes the pesticide remained on the vegetables and the fruits into a nonpoisonous or low-toxic outcome and reduces the poisoning effect of the pesticide to a man and a non-target organism as well as the pollution to the environment. The invention also provides the application of the compound enzymatic preparation. The compound enzymatic preparation clearing the residual pesticide of the invention comprises organophosphorus hydrolase, carbamate hydrolase and pyrethroid hydrolase. Preferably, the three enzymes are derived from a gene expression of a microorganism. Compared with the traditional method, the method has the advantages of simplicity, safety and moderate required reaction condition, and can not influence the taste and the nutrition of the vegetables and the fruits; the compound enzymatic preparation is extremely easy to be cleaned and really clears the residual pesticide at the same time.

Description

technical field [0001] The invention relates to a compound enzyme preparation, in particular to a compound enzyme preparation for eliminating residual pesticides in vegetables, tea leaves and fruits and its application. Background technique [0002] my country needs to feed 20% of the world's population with 7% of the world's land, so it is very dependent on pesticides. Especially in order to reduce the losses caused by diseases and insect pests, a large amount of insecticides are used. But on the one hand, pesticides inhibit the occurrence of pests and diseases; on the other hand, it also brings great ecological and social problems. [0003] Most pesticides are toxic and lack selectivity, often causing direct poisoning to non-target organisms. In addition, because pesticides are difficult to degrade, some of them remain in crops, pastures, soil and water bodies, and are transferred and enriched along the food chain, so that humans at the highest position in the food chain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A62D3/02A62D101/04A62D101/20
Inventor 苏勇杨柏成
Owner 澳联健康科技(江苏)有限公司
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