Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof

A technology of organophosphorus pesticides and random mutation, which is applied in the field of genetic engineering, can solve problems such as no results, and achieve the effects of not easy inactivation, high degradation efficiency, excellent enzyme activity and thermal stability

Active Publication Date: 2013-06-12
SHENYANG INST OF APPLIED 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 by random mutation and encoding gene thereof
  • Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof
  • Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the expression of organophosphorus pesticide degrading enzyme of the present invention in Escherichia coli

[0028] (1) PCR amplification: use the upstream primer 5'-ATTCATATGGCCGCACCGCAGGTG-3' and the downstream primer 5'-TAACTCGAGCTTGGGGTTGACGACCG-3' to PCR amplify the organophosphorus pesticide degrading enzyme gene of the present invention.

[0029] The reaction conditions for PCR (50 μL system) are: 50ng of the organophosphorus pesticide degrading enzyme gene (SEQ ID No.1) of the present invention as a template, 0.3 μM each of the upstream primer and downstream primer, 200 μM each of dNTPs, 5U Taq DNA polymerase, 5 μL 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 of extension at 72°C for 10 min. The composition of PCR buffer is: 100mM Tris-HCl pH8.3, 500mM KCl and 15mMMgCl 2 , the solvent is deionized water.

[0030] (2) Con...

Embodiment 2

[0076] Embodiment 2: the mensuration of organophosphorus pesticide degradation specific activity of the present invention

[0077] Using methyl parathion and chlorpyrifos as substrates respectively, the specific activity of the organophosphorus pesticide degrading enzyme of the present invention and the wild-type enzyme is determined.

[0078] 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 other operating steps are completely carried out according to the method in Example 1.

[0079] With methyl parathion as substrate, the assay method of specific activity is as follows:

[0080] (1) Preparation of p-nitrophenol standard solution: using 20 μM Tris-HCl (pH 8.0) buffer as solvent, prepare p-nitrophenol concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively. Phenol standard solution.

[0081] (2) Drawing of p-nitrophenol...

Embodiment 3

[0109] Embodiment 3: Determination of thermal stability of organophosphorus pesticide degrading enzyme of the present invention

[0110] The purified organophosphorus pesticide-degrading enzyme and the wild-type enzyme obtained in Example 1 at a concentration of 100 μg / ml were respectively incubated at 40°C, 42.5°C, 45°C, 47.5°C, 50°C, 52.5°C, and 55°C , 57.5°C, 60°C heat treatment for 10min, immediately place it on ice for 30min, use chlorpyrifos as substrate, measure enzyme activity and analyze thermal stability.

[0111] 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 other operating steps are completely carried out according to the method in Example 1.

[0112] The assay method of enzyme activity is as follows:

[0113] (1) Preparation of chlorpyrifos standard solution: using 20 μM Tris-HCl (pH 8.0) buffer solution containing 0.1% (volu...

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Abstract

The invention relates to an organophosphorus pesticide degrading enzyme obtained by genetic engineering transformation. The organophosphorus pesticide degrading enzyme transformed by random mutation is generated by replacing an amino acid site of the organophosphorus pesticide degrading enzyme from pseudomonas stutzeri, and the replacing amino acid site is located at the 71st site. The organophosphorus pesticide degrading enzyme is excellent in degrading activity of chlorpyrifos and methyl to ethion and excellent in heat stability.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to an organophosphorus pesticide degrading enzyme transformed by genetic engineering. Background technique [0002] Pesticide residues seriously endanger the ecological environment and the health of the people. It is a serious problem that all countries need to face. Studies have found that groundwater, crops and soil in different regions have been polluted by pesticide residues. my country is a large agricultural country, and the annual use of pesticides is huge, and the problem of pesticide residues is also becoming more and more serious. Relevant data show that pesticide residues in agricultural products in my country exceed the standard seriously, and the intake of various pesticides in the daily diet of residents is dozens of times higher than that in developed countries. In recent years, the removal technology of pesticide residues has become the focus of research, and it is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12R1/38
Inventor 张惠文谢建飞徐明恺张成刚石元亮卢宗云
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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