Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof

A technology of organophosphorus pesticides and coding genes, which is applied in the field of organophosphorus pesticides degrading enzymes, can solve the problems of no achievements and achieve the effects of great application value, high degradation efficiency, and not easy to inactivate

Active Publication Date: 2014-07-23
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with the organophosphorus pesticide degrading enzyme OPH, the genetic modification of the organophosphorus pesticide degrading enzyme MPH has not achieved such remarkable results

Method used

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  • Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof
  • Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof
  • Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Expression of the organophosphorus pesticide degrading enzyme of the present invention in E. coli (1) PCR amplification: PCR amplification of the present invention with upstream primer 5'-ATTCATATGGCCGCACCGCAGGTG-3' and downstream primer 5'-TAACTCGAGCTTGGGGTTGACGACCG-3' The organophosphorus pesticide degrading enzyme gene.

[0026] PCR (50μL system) reaction conditions: 50ng of the organophosphorus pesticide degrading enzyme gene of the present invention (SEQ ID No.1) as a template, 0.3μM each of the upstream primer and downstream primer, 200μM each of dNTPs, 5U Taq DNA polymerase, 5μL PCR buffer Solution, 30 cycles, denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and the last cycle for extension at 72°C for 10 min.

[0027] The composition of PCR buffer is: 100mM Tris-HCl pH8.3, 500mM KCl and 15mM MgCl 2 , The solvent is deionized water.

[0028] (2) Expression vector construction: The above...

Embodiment 2

[0074] Example 2: Determination of the degradation specific activity of the organophosphorus pesticide of the present invention

[0075] Using methyl parathion and chlorpyrifos as substrates, respectively, the specific activities of the organophosphorus pesticide degrading enzyme and the wild-type enzyme of the present invention are measured.

[0076] The production method of the wild-type enzyme is as follows: the organophosphorus pesticide degrading enzyme gene mpd (GenBank accession number: JQ686087) is used as a PCR template, and the other operation steps are completely performed in accordance with the method of Example 1.

[0077] With methyl parathion as the substrate, the specific activity determination method is as follows:

[0078] (1) Preparation of p-nitrophenol standard solution: use 20μM Tris-HCl (pH8.0) buffer as a solvent to prepare p-nitrophenol with concentrations of 10μM, 20μM, 30μM, 40μM, 50μM, 60μM. Phenol standard solution.

[0079] (2) Drawing the standard curve o...

Embodiment 3

[0108] Example 3: Determination of the thermal stability of the organophosphorus pesticide degrading enzyme of the present invention

[0109] The purified organophosphorus pesticide degrading enzyme and wild-type enzyme of the present invention with a concentration of 100 μg / ml obtained in Example 1 were heated at 40°C, 42.5°C, 45°C, 47.5°C, 50°C, 52.5°C, 55°C, respectively After heat treatment at 57.5°C and 60°C for 10 minutes, immediately place it on ice and keep for 30 minutes. Use chlorpyrifos as a substrate to determine enzyme activity and analyze thermal stability.

[0110] The production method of the wild-type enzyme is as follows: the organophosphorus pesticide degrading enzyme gene mpd (GenBank accession number: JQ686087) is used as a PCR template, and the other operation steps are completely carried out in accordance with the method of Example 1.

[0111] The determination method of enzyme activity is as follows:

[0112] (1) Preparation of chlorpyrifos standard solution: u...

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Abstract

The invention relates to an organophosphorus pesticide degrading enzyme obtained through gene engineering in a transformation mode. The organophosphorus pesticide degrading enzyme transformed through random mutation is generated through replacing three amino acid locuses of organophosphorus pesticide degrading enzyme derived from pseudomonas stutzeri. The amino acid replacing locuses replace a seventh locus, a one hundred and twenty-fourth locus and a two hundred and forty-sixth locus. The organophosphorus pesticide degrading enzyme not only is excellent in activity degrading of chlorpyrifos parathion-methyl, but also has excellent heat stability.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and relates to an organophosphorus pesticide degradation enzyme modified through genetic engineering (random mutation modification). Background technique [0002] Pesticide residues seriously harm the ecological environment and the health of the people. This is a serious problem that all countries need to face. Research has found that groundwater, crops and soil in different areas are all polluted by pesticide residues. my country is a large agricultural country, and the amount of pesticides used every year is huge, and the problem of pesticide residues that comes with it has become increasingly serious. Relevant data show that pesticide residues in agricultural products in my country are exceedingly serious, and the daily intake of various pesticides in the daily diet of residents is dozens of times higher than in developed countries. In recent years, pesticide residue removal technology has beco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52
Inventor 谢建飞张惠文石元亮卢宗云徐明恺张成刚
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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