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Mutant of organophosphorus hydrolase, expression vector, recombinant bacterium and application of recombinant bacterium

An expression vector and mutant technology, applied in the fields of genetic engineering and enzymology, can solve the problems of inability to meet the requirements of fast and efficient, fast degradation of organophosphorus pesticides, narrow applicable environment, insufficient secretion and expression of enzymes, etc.

Inactive Publication Date: 2020-03-06
KARAMAY SANDA NEW TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a biocatalyst, the specificity and efficiency of biological enzymes are unmatched by other catalysts. It can specifically speed up a series of biochemical reactions. However, due to its own nature, wild bacteria OPH has low activity in degrading organophosphorus pesticides. , The secretion and expression of enzymes are insufficient, and the applicable environment is narrow, which cannot meet the needs of fast, efficient and rapid degradation of organophosphorus pesticides in various situations

Method used

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  • Mutant of organophosphorus hydrolase, expression vector, recombinant bacterium and application of recombinant bacterium
  • Mutant of organophosphorus hydrolase, expression vector, recombinant bacterium and application of recombinant bacterium
  • Mutant of organophosphorus hydrolase, expression vector, recombinant bacterium and application of recombinant bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Organophosphate hydrolase point mutations and secreted expression

[0043] (1) Point mutation key gene

[0044] Based on the data structure of Fs OPH (obtained from the separation and purification of broad-spectrum organophosphate degrading bacteria (Flavobacterium sp. Mutations change the base pairs at key positions to change the protein sequence and three-dimensional structure, and obtain organophosphate hydrolase with higher hydrolytic activity. The OPH gene fragment containing K185R (the lysine at the 185th position is mutated to arginine) and I274N (the isoleucine at the 274th position is mutated to asparagine) mutation sites was obtained by overlapping PCR technology.

[0045] (2) Design expression system

[0046] Bacillus subtilis is selected as the secretory expression bacteria. Bacillus subtilis is a new expression bacteria. At present, there are few domestic studies. It has a strong protein secretion ability and can directly secrete foreign proteins into the...

Embodiment 2

[0066] Activity Determination of Recombinant Organophosphate Hydrolase

[0067] The recombinant strain was inoculated into 30 mL of TB expression medium, and cultured with constant temperature and shaking for 72 h under the same conditions. The fermentation broth was centrifuged, and the supernatant was taken as a crude enzyme solution, and the molecular weight of the enzyme produced by the recombinant bacteria was identified by SDS-PAGE protein electrophoresis, the protein concentration was determined by Bradford, and the ability of the produced enzyme to degrade paraoxon was detected by the p-nitrophenol method. Test the hydrolysis ability of genetically engineered recombinant strains.

[0068] SDS-PAGE protein electrophoresis: Take 40 μL of fermentation supernatant, add 10 μL of 5×SDS-PAGE loading buffer containing PMSF to it, let it stand at room temperature for 30 minutes, then bathe in boiling water for 3-5 minutes. Use 5% stacking gel and 12% separating gel for protein...

Embodiment 3

[0077] The isoleucine at position 274 of the organophosphate hydrolase is mutated to asparagine (I274N). The point mutation and secreted expression of the organophosphate hydrolase and the activity determination of the recombinant organophosphate hydrolase are the same as in Examples 1 and 2.

[0078] The result is: I274N can increase the hydrolysis rate of paraoxon from 0.014mmol / min.mg to 0.395mmol / min.mg.

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Abstract

The invention provides a mutant of an organophosphorus hydrolase, an expression vector, a recombinant bacterium and an application of the recombinant bacterium, and belongs to the technical field of genetic engineering and enzymology. According to the mutant, isoleucine at a 274th position of organophosphorus hydrolase mutates into asparagin. Compared with organophosphorus hydrolase, the providedmutant of the organophosphorus hydrolase improves the hydrolysis rate of paraoxon compounds.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzymology, and in particular relates to a mutant, an expression vector, a recombinant bacterium and an application of an organophosphate hydrolase. Background technique [0002] Organophosphate hydrolase (Organophosphorus hydrolase, OPH) is widely used in the biodegradation of organophosphorus pesticide residues, which is of great significance to the restoration of environmental pollution and the protection of human health. Organophosphate hydrolase OPH is a typical phosphotriesterase, which dissociates various phosphate bonds (P-O bond, P-CN bond, P-F bond and P- S bond), thus hydrolyzing many organophosphate triesters, thioesters, and fluorophosphoric esters. There are three hydrophobic pockets in the active center of OPH enzyme, which respectively interact with the three substituents of the substrate molecule to affect the release rate of the leaving group and determine the s...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/70C12N1/21A62D3/02C12R1/19A62D101/04A62D101/26A62D101/28
CPCA62D3/02A62D2101/04A62D2101/26A62D2101/28C12N9/16C12N15/70C12Y301/08001
Inventor 黄保军贾剑平何蕙利谢建兵耿耀民吴阳高则彬何菲
Owner KARAMAY SANDA NEW TECH
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