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Organophosphorus pesticide degrading enzyme gene and application thereof

An organophosphorus pesticide and enzyme-degrading technology, which is applied in the fields of application, hydrolase, and genetic engineering, can solve problems such as the impact of enzyme activity and food safety hazards, and achieve the goal of improving enzyme activity, maintaining activity and stability, and great application value Effect

Inactive Publication Date: 2014-01-22
SHANGHAI ACAD OF AGRI SCI
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Problems solved by technology

[0005] The organophosphate degrading enzyme (OpdA) expressed in Escherichia coli has food safety hazards in its application; at the same time, the recombinant protein is produced in the form of inclusion bodies in the cytoplasm of Escherichia coli, and the purification and renaturation process of inclusion bodies will affect the enzyme activity. have a certain negative impact

Method used

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  • Organophosphorus pesticide degrading enzyme gene and application thereof
  • Organophosphorus pesticide degrading enzyme gene and application thereof
  • Organophosphorus pesticide degrading enzyme gene and application thereof

Examples

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Embodiment 1

[0026] 1) Obtaining the sequence of organophosphorus pesticide degrading enzyme gene (opdA)

[0027] The complete gene sequence of the organophosphorus degrading enzyme gene opdA of Agrobacterium radiobacterium was obtained from the NCBI database (NCBI accession number: ACCESSION: AY043245), and synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (the sequence shown in SEQ ID NO: 3). shown), and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 4. The forward primer opdA-F: ​​5'-AAAGGATCCGCAAACGAGAAGAGATGCAC-3' (as shown in SEQ ID NO: 5) and the reverse primer opdA-R: 5'-AAACTCGAGTCGTTCGGTATCTTGACG-3' (as shown in SEQ ID NO: 6) were then used ) was amplified by PCR to obtain the amplified product of the opdA gene and recovered by electrophoresis. The PCR product and pUC57 vector were recovered by double-enzyme digestion with restriction enzymes BamH I and Xho I, respectively, and the fragments cut by the two were ligated and transformed into Escheric...

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Abstract

The invention discloses an organophosphorus pesticide degrading enzyme gene and application thereof. The gene is obtained through site-specific mutagenesis on three amino acid coding loci, the 253rd locus, the 256th locus and the 271st, on the organophosphorus degrading enzyme gene. The nucleotide sequence with mutated organophosphorus pesticide degrading enzyme gene is connected to a pHT43 vector, then the expression vector is transferred into Bacillus subtilis host cells for expression, so as to isolate expression protein with organophosphorus pesticide degrading activity. The organophosphorus pesticide degrading enzyme expressed by mutant gene provided by the invention has obviously improved affinity and sensitivity with a substrate ethyl-parathion, and obviously improved enzyme activity; the invention can be used for degradation and elimination of organic phosphorus pesticide residues in agricultural products and opens up a novel path for the engineering enzymatic degradation of organophosphorus pesticide.

Description

technical field [0001] The invention belongs to the field of genetic bioengineering, in particular to a mutant organophosphorus pesticide degrading enzyme gene and its application. Background technique [0002] With the development of science and the progress of society, more and more compounds have entered the production and living environment of human beings. At the same time, many synthetic pollutants that are difficult to be degraded and transformed by the natural environment enter nature, and pesticides are one of them. [0003] The pesticides currently used are mainly pesticides, accounting for 70% of the total consumption; among which organophosphorus pesticides account for more than 70% of the total pesticide consumption; and the output of highly toxic varieties of organophosphorus pesticides also accounts for the output of organophosphorus pesticides This is the so-called three "70%" of unreasonable structure. There are many kinds of organophosphorus pesticides, a...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/63C12N15/55A62D3/02A62D101/04A62D101/26A62D101/28
CPCC12N9/16C12N15/75
Inventor 王金斌唐雪明李文蒋玮何建华吴潇宿学峰任芳芳
Owner SHANGHAI ACAD OF AGRI SCI
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