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Application of esterase gene est816 and recombinant esterase thereof in degrading pyrethroid pesticides

A technology of pyrethroids and est816, applied in the field of genetic engineering, achieves the effect of good stability and broad application prospects

Inactive Publication Date: 2017-08-18
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there are few reports on pyrethroid degrading enzymes, therefore, it is of great significance to study and explore biocatalysts pyrethroid degrading enzymes with excellent performance.

Method used

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  • Application of esterase gene est816 and recombinant esterase thereof in degrading pyrethroid pesticides
  • Application of esterase gene est816 and recombinant esterase thereof in degrading pyrethroid pesticides
  • Application of esterase gene est816 and recombinant esterase thereof in degrading pyrethroid pesticides

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Cloning and Escherichia coli expression of embodiment 1 esterase gene est816

[0062] 1. Cloning of gene fragments

[0063] (1) according to the gene sequence design primer of esterase gene est816 as follows:

[0064] est816-fw: 5'-CGC ATGCCGCATGTAGAGAACGA-3' (the bold and underlined part is the BamHI restriction site);

[0065] est816-rv: 5'-CCG TCAGGACACCAATGAAGCTTCTCGA-3' (the part in bold and underlined is the EcoRI restriction site).

[0066] (2) PCR amplification

[0067] Using the plasmid pUC118-A (the plasmid obtained by cloning the est816 gene sequence based on the plasmid pUC118) as a template, and est816-fw and est816-rv as primers, the est816 gene was amplified by PCR. The system is as follows:

[0068]

[0069] The PCR reaction conditions are as follows:

[0070] The first stage: pre-denaturation at 98°C for 3 minutes;

[0071] The second stage: denaturation at 98°C for 10 sec, annealing at 64°C for 15 sec, extension at 72°C for 1 min, a total o...

Embodiment 2

[0080] Cloning and Pichia pastoris expression of embodiment 2 esterase gene est816

[0081] 1. Cloning of gene fragments

[0082] (1) according to the gene sequence design primer of esterase gene est816 as follows:

[0083] EcoRIfw: 5'-CCG ATGCCGCATGTAGAGAACGA-3' (the bold and underlined part is the EcoRI restriction site);

[0084] KpnIrv: 5'-CGG GGACACCAATGAAGCTTCTCGA-3' (the bold and underlined part is the KpnI restriction site).

[0085] (2) PCR amplification

[0086] The plasmid pET-28a(+)-est816 was used as a template, and EcoRIfw and KpnIrv were used as primers to perform PCR amplification of the est816 fragment. The system is as follows:

[0087]

[0088] The PCR reaction conditions are as follows:

[0089] The first stage: pre-denaturation at 98°C for 3 minutes;

[0090] The second stage: denaturation at 98°C for 10 sec, annealing at 64°C for 15 sec, extension at 72°C for 1 min, a total of 30 cycles;

[0091] The third stage: extension at 72°C for 10 minu...

Embodiment 3

[0105] The enzymatic property research of embodiment 3 recombinant esterase Est816

[0106] 1. Optimum reaction temperature and thermal stability of recombinant esterase

[0107] The enzyme solution of the recombinant esterase is subjected to an enzymatic reaction at 20-80°C, the reaction system is 405 μl, and its composition is 400 μl of potassium phosphate solution (pH6.8) containing 40 μM p-nitrophenyl acetate and 5 μl of enzyme solution . The reaction system was reacted at different temperatures for 5 minutes, and then the absorbance of p-nitrophenol released during the process was measured at a wavelength of 405 nm, and a blank control without enzyme solution was made at the same time. The enzyme activity was measured to obtain the optimum reaction temperature (the highest enzyme activity was recorded as 100%).

[0108] The test results are attached image 3 shown. In 50mM potassium phosphate buffer (pH 6.8), the recombinant AHLs-lactonase enzyme solution was placed a...

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Abstract

The invention discloses an application of an esterase gene est816 and a recombinant esterase thereof in degrading pyrethroid pesticides. Specifically, the invention relates to heterologous expression of esterase gene est816 and a recombinant esterase thereof in degrading pyrethroid pesticides. A recombinant esterase Est816 is constructed via both an Escherichia coli expression system and a Pichia pastoris expression system, has efficient soluble expression in two expression systems, and has good thermal stability and acid and alkali resistance. The recombinant esterase est816 has a strong degradation effect on pyrethroid pesticides (such as cyhalothrin, cypermethrin, fenvalerate, deltamethrin, and so on), and the degradation rate is up to 90% or higher. Therefore, the esterase gene est816 and the recombinant esterase Est816 have broad application prospects in removing residual pyrethroid pesticides.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, it relates to the heterologous expression of esterase gene est816 and the application of its recombinant esterase in degrading pyrethroid pesticides. Background technique [0002] At present, pyrethroid pesticides play a huge role in the prevention and control of agricultural, forestry and sanitation diseases, and their usage ranks second in the pesticide market, and is still growing. Absorption through the respiratory tract and skin can cause poisoning and have a certain impact on human health. Moreover, with the improvement of people's living standards and the enhancement of environmental awareness, the environmental and food pollution problems caused by the large, frequent and irregular use of pyrethroids have attracted more and more attention. Therefore, in the situation that the natural decomposition ability cannot meet the safety needs of human beings, it ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/81C12N1/21C12N1/19C12N9/18A62D3/02C12R1/19C12R1/84A62D101/04A62D101/49
CPCA62D3/02C12N9/18C12N15/70C12N15/815A62D2101/04A62D2101/49C12N2800/101C12N2800/102
Inventor 刘玉焕范新炯
Owner SUN YAT SEN UNIV
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