Enzymatic performance improved methyl parathion hydrolase mutant and application thereof

A technology of methyl parathion and mutants, applied in the field of genes encoding methyl parathion degrading enzyme mutants, mutants of organophosphate degrading enzymes, and methyl parathion hydrolase mutants, to improve enzyme The effects of chemical properties, widening of application fields and efficiency of action

Active Publication Date: 2017-09-22
北京森根比亚生物工程技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with OPH (18.Mulbry., W., et al., Identification of a Plasmid-Borne ParathionHydrolase Gene from Flavobacterium sp.by Southern Hybridization with opd from Pseudomonas diminuta.Applied and environmental microbiology, 1986.51(5):p.926 -930; C.STEVEN McDANIEL, LINDA L.HARPER, and J.R.WILD*, Cloning and Sequencing of a Plasmid-Borne Gene(opd) Encoding a Phosphotriesterase.Journal ofbacteriology 1988.170(5):p.2306-2311.), MPH( Chu Xiaona., et

Method used

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  • Enzymatic performance improved methyl parathion hydrolase mutant and application thereof
  • Enzymatic performance improved methyl parathion hydrolase mutant and application thereof
  • Enzymatic performance improved methyl parathion hydrolase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Molecular transformation, mutant gene cloning and protein expression of methyl parathion degrading enzyme

[0035] 1. Test method

[0036] 1.1 Molecular simulation technology to determine the site to be mutated

[0037] 1.1.1 The I-tassel online program simulates the OPHC2 protein structure, and determines the candidate sites according to the structural characteristics of amino acid residues, hydrophobicity, isoelectric point, B-factor, and secondary structure;

[0038] 1.1.2 Use the Discovery studio software bulid mutant program to simulate the structure of the candidate point mutation site, and compare and analyze the tertiary structure force of the site before and after the mutation, and select the amino acid site with a more stable secondary structure after the mutation as a mutation target.

[0039] 2.1 Mutant gene cloning, expression and protein induction

[0040] 2.1.1 Design primers, add point mutation bases to the primer sequence, use the OPHC2 wil...

experiment example 2

[0061] Test of experimental example 2 mutant enzymatic properties

[0062] The relative enzyme activity is plotted, the pH curve, temperature curve and the measurement results of the influence of salt solution on the enzyme activity are shown in Figure 6-9 . The optimum action temperature of wild-type OPHC2 is 65°C, and the optimum action pH is 9.0. The pH curve and temperature response trend of the two mutant enzymes are consistent with wild-type OPHC2, but V117HQ119E shows good high temperature stability. When the temperature is higher than 65°C, the enzyme activity retention ability of the modified protein is significantly higher than that of the wild type. As the temperature increases, the advantage of heat resistance of the modified protein is more obvious. Wild type 13.5±0.7%, 80 ℃ higher than wild type 16±1.4% ( Figure 4 B).

[0063] At the same time, the mutant enzyme was subjected to time-gradient treatment at 65°C and 75°C, and after incubation at high temperatu...

experiment example 3

[0067] Experimental example 3 Stability detection experiment of mutant F263 in other surfactants

[0068] In view of the excellent stability of F263W in SDS, other surfactants were also selected to test the tolerance of F263W, and the treatment method was referred to SDS. The types of surfactants to choose from are as follows:

[0069] (1) Benzalkonium bromide, a cationic surfactant, is used in fields such as sterilization, disinfection, and algae killing;

[0070] (2) Apple amino acid foaming agent, compound amino acid surfactant, contains active substances extracted from apples, and is used in the fields of daily chemical and cleaning;

[0071] (3) Potassium cocoyl glycinate, an amino acid-type surfactant, used in the fields of daily chemicals and cleaning;

[0072] (4) Sodium lauroyl glutamate, an amino acid-type surfactant, used in the fields of daily chemicals and care;

[0073] (5) Firefighting foam Ⅰ: Composite foam fire extinguishing agent, the ingredients are fluor...

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Abstract

The invention discloses an enzymatic performance improved methyl parathion hydrolase mutant and application thereof. Wild-type enzymatic performance improved methyl parathion hydrolase is targetedly modified, mutation in vitro and expression to OPHC2 are performed by combining with a protein structure calculation and stimulation technology and a molecular biology technology, and kinetic parameters and the stability of protease are detected by applying biochemical means, so that a mutant V117HQ119E with remarkably improved thermal stability and substrate affinity and a mutant F263W with remarkably improved catalytic efficiency and improved stability in a surfactant are obtained through screening, wherein amino acid sequences of the mutant V117HQ119E and the mutant F263W are shown as SEQ ID No. 2 and SEQ ID No. 4 respectively. The enzymatic performance improved methyl parathion hydrolase mutant remarkably improves the enzymatic performance of the OPHC2, widens the application field and the action efficiency of the OPHC2 and has a wide application prospect on the aspects of degradation of an organophosphorus pesticide, organism detoxication, washing and disinfection of a nerve agent and the like.

Description

technical field [0001] The present invention relates to mutants of organophosphate degrading enzymes, especially methyl parathion hydrolase mutants with improved catalytic performance or stability. The present invention further relates to genes encoding methyl parathion degrading enzyme mutants, belonging to organophosphate Degrading enzyme mutants and their fields of application. Background technique [0002] Organophosphorus compounds are widely used in the fields of pesticides, nerve agents, and flame retardants. They are characterized by strong toxicity, high destructiveness, and refractory degradation, which have brought huge safety hazards to humans and society (Bajgar, J., Complex view onpoisoning with nerve agents and organophosphates.Acta Medical, 2005.48(1):p.3-21.), the development of efficient and environmentally friendly organic phosphorus degradation system is one of the research hotspots in recent decades (Balali-Mood., M .and H. Saber, Recent advances in the...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55A62D3/02A62D101/04A62D101/28
CPCA62D3/02A62D2101/04A62D2101/28C12N9/16C12Y301/08001
Inventor 龚晓洁李立公李光雷
Owner 北京森根比亚生物工程技术有限公司
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