Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof

An organophosphorus pesticide and random mutation technology, applied in the field of genetic engineering, can solve problems such as failure to obtain results, and achieve the effects of great application value, improved enzyme activity and thermal stability, and less inactivation

Active Publication Date: 2013-06-12
辽宁中科生物工程股份有限公司 +1
View PDF2 Cites 5 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof
  • Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof
  • Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the expression of organophosphorus pesticide degrading enzyme of the present invention in Escherichia coli

[0027] (1) PCR amplification: use the upstream primer 5'-ATTCATATGGCCGCACCGCAGG TG-3' and the downstream primer 5'-TAACTCGAGCTTGGGGTTGACGACCG-3' to PCR amplify the organophosphorus pesticide degrading enzyme gene of the present invention.

[0028] 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.

[0029] The composition of PCR buffer is: 100mM Tris-HCl pH8.3, 500mM KCl and 15mMMgCl 2 , the solvent is deionized water.

[003...

Embodiment 2

[0041] Embodiment 2: the mensuration of organophosphorus pesticide degradation specific activity of the present invention

[0042] 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.

[0043] 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.

[0044] With methyl parathion as substrate, the assay method of specific activity is as follows:

[0045] (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.

[0046] (2) Drawing of p-nitrophenol...

Embodiment 3

[0075] Embodiment 3: Determination of thermal stability of organophosphorus pesticide degrading enzyme of the present invention

[0076] 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.

[0077] 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.

[0078] The assay method of enzyme activity is as follows:

[0079] (1) Preparation of chlorpyrifos standard solution: using 20 μM Tris-HCl (pH 8.0) buffer solution containing 0.1% (volume...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an organophosphorus pesticide degradation enzyme obtained through genetic engineering reconstruction. A random mutation reconstructed organophosphorus pesticide degradation enzyme is generated by the fact that three amino acid loci of an organophosphorus pesticide degradation enzyme are derived from pseudomonas stutzeri. The replaced amino acid loci are a 31st locus, a 124th locus and a 246th locus. The organophosphorus pesticide degradation enzyme is good in degradation activity for chlorpyrifos and parathion-methyl, and also has good 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12R1/38
Inventor 谢建飞张惠文石元亮卢宗云
Owner 辽宁中科生物工程股份有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap