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Organic phosphorus hydrolase mutant and application thereof

A hydrolase and mutant technology, which is applied in the fields of genetic engineering and biological enzymology, can solve the problems such as the inability to meet the requirements of fast, efficient and fast degradation of organophosphorus compounds.

Inactive Publication Date: 2021-06-08
中国人民解放军陆军防化学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitation of its own properties, the activity of wild bacteria OPH to degrade organophosphorus compounds cannot meet the needs of fast, efficient and rapid degradation of organophosphorus compounds in various situations.

Method used

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  • Organic phosphorus hydrolase mutant and application thereof

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Experimental program
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Effect test

Embodiment 1

[0017] Organophosphate hydrolase point mutations and secreted expression

[0018] (1) Point mutation key gene

[0019] Based on the data structure of the organophosphate hydrolase OPH found in the Sphingobium fuliginis (ATCC 27551) strain, and according to its hydrolysis mechanism, the impact of protein structure changes on the enzymatic hydrolysis performance was analyzed, and point mutations changed the base pairs at key positions to change According to the protein sequence and three-dimensional structure, an organophosphate hydrolase with higher hydrolysis activity was obtained. The amino acid sequence of the designed organophosphate hydrolase mutant is shown in SEQ NO.1. The OPH gene fragment containing the H257Y and L303T mutation sites was obtained by conventional overlapping PCR technology in the art, and sequenced. The nucleotide sequence encoding the organophosphate hydrolase mutant gene is shown in SEQ NO.2.

[0020] Amino acid sequence SEQ NO.1 is:

[0021] MQTRRV...

Embodiment 2

[0051] According to the method described in Example 1, the organophosphate hydrolase mutant was mutated from histidine at position 257 to tyrosine (H257Y), which was designated as OPH-Y.

Embodiment 3

[0053] According to the method described in Example 1, the organophosphorus hydrolase mutant was mutated from leucine at position 303 to threonine (L303T), which was designated as OPH-T.

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Abstract

The invention relates to an organophosphorus hydrolase mutant and application thereof, and belongs to the technical field of genetic engineering and bio-enzymology. The mutant is characterized in that histidine at the 257th site of organophosphorus hydrolase is mutated into tyrosine, leucine at the 303rd site of the organophosphorus hydrolase is mutated into threonine, and the amino acid sequence of the organophosphorus hydrolase mutant is shown as SEQNO.1. The mutant effectively enhances the activity of the organophosphorus hydrolase and the ability of hydrolyzing a substrate, and when the mutant is used for degrading paraoxon compounds, the hydrolysis rate of the paraoxon compounds is effectively increased.

Description

technical field [0001] The invention relates to an organophosphorus hydrolase mutant and its application, belonging to the technical fields of genetic engineering and biological enzymology. Background technique [0002] As biological catalysts, biological enzymes can specifically accelerate a series of biochemical reactions. Among them, organophosphorus hydrolase (Organophosphorus hydrolase, OPH) is used to biodegrade organophosphorus poisons, which is of great significance to the decontamination of neurotoxins, the protection of human health and the protection of the ecological environment. Organophosphorus hydrolase (OPH) is a typical phosphotriesterase, which dissociates various phosphate bonds (P-O bonds, P-CN bonds, P-F bonds and P-S bonds) of group ester bonds by hydrolyzing phosphorus atoms and electrophilic atoms, Thereby hydrolyzing many organophosphate triesters, thioesters, and fluorophosphoric esters. [0003] Organophosphorus poisons such as sarin (GB, sarin) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55A62D3/02A62D101/28A62D101/26
CPCC12N9/16A62D3/02C12Y301/08001A62D2101/28A62D2101/26
Inventor 刘敏顾恰敏赵传欣
Owner 中国人民解放军陆军防化学院
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