Recombinant Bacillus subtilis heterologously expressing phosphotriesterase

A Bacillus subtilis, phosphotriesterase technology, applied in the field of genetic engineering, can solve problems such as large consumption, environmental pollution, heavy logistics burden, etc., and achieve the effects of being beneficial to large-scale production, expanding application scope, and simplifying post-processing technology.

Pending Publication Date: 2019-01-11
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the decontamination of organophosphorus nerve agents is mainly based on chemical decontamination agents based on chlorination and oxidation. , serious environmental pollution, large consumption and heavy logistical burden, etc.

Method used

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  • Recombinant Bacillus subtilis heterologously expressing phosphotriesterase
  • Recombinant Bacillus subtilis heterologously expressing phosphotriesterase
  • Recombinant Bacillus subtilis heterologously expressing phosphotriesterase

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Embodiment 1

[0058] A recombinant bacillus subtilis expressing phosphotriesterase heterologously, comprising the following process:

[0059] (1) Acquisition of phosphotriesterase

[0060] Autodck software was used to simulate the docking of the enzyme source and the organophosphorus compound and calculate the binding energy, carry out reverse virtual screening, obtain the binding model of the enzyme source and the organophosphorus compound, find the enzyme source with the smallest binding energy and determine it as the enzyme source. Name it ob3. It was sent to Shanghai Sangon Bioengineering Co., Ltd. for optimized synthesis according to the preferred codons of Bacillus subtilis to obtain an optimized phosphotriesterase gene. The gene sequence is SEQ ID NO.1.

[0061] (2) Construction of recombinant plasmid pHY-ZL-ob3

[0062] Using pHY-PLK300 as the backbone, insert the promoter BSxyl, terminator Ter, and signal peptide elements in sequence at the multiple cloning site to obtain the pla...

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Abstract

The invention discloses a recombinant Bacillus subtilis heterologously expressing phosphotriesterase. The preparation method of the recombinant Bacillus subtilis comprises the following steps: (1) determining the gene coding of the phosphotriesterase; (2) inserting the gene determined in step (1) into pHY-ZL, obtaining recombinant expression vector; and (3) transforming the recombinant expressionvector into the host Bacillus subtilis and expressing the phosphatriesterase gene in the host strain. As the recombinant Bacillus subtilis can solubly secrete and express the phosphatriesterase, the enzyme can hydrolyze a plurality of organic phosphorus compound, the restriction of the specificity of the organic phosphorus hydrolase substrate is solved, and the degradation rate of the organic phosphorus compound is greatly improved; and the recombinant Bacillus subtilis can solubly secrete and express the phosphatriesterase. At the same time, the obtained recombinase is secreted by Bacillus subtilis of food safety bacteria (GRAS), and the application field of the recombinase is enlarged.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for integrating a phosphotriesterase gene into a Bacillus subtilis gene to make a recombinant Bacillus subtilis. Background technique [0002] Phosphotriesterases (PTE, EC3.1.8.1) derived from microorganisms can extensively hydrolyze organophosphorus compounds, break the phosphoester bonds and detoxify them, which has the advantages of environmental protection and in-situ detoxification, and provides a new way for bioremediation. Phosphotriesterases belong to the amidohydrolase superfamily, which catalyzes the hydrolysis of compounds such as amides, esters, sugars, and organophosphates; the active site has a mononuclear or binuclear metal center. The metal center has dual functions: polarized water molecules enhance nucleophilic attack; and enhance the breaking of the bond of the dissociated group of the substrate molecule. [0003] In recent years, there ha...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N1/21C12N15/55C12N15/75C12N15/65C12N9/16C12R1/125
CPCC12N9/16C12N15/65C12N15/75C12Y301/08001
Inventor张梁杨贞妮郭旋钟近艺郑禾辛瑜李由然石贵阳
OwnerJIANGNAN UNIV