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High-activity methyl parathion hydrolase mutant and use thereof

A methyl parathion, mutant technology, applied in the biological field, can solve problems such as poisoning, and achieve the effects of high degradation rate, high catalytic activity and performance

Inactive Publication Date: 2015-06-24
BIOTRAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although such pesticides have been banned or restricted in many developed countries, and have been included in the PIC (Prior Informed Consent) Convention, they are still produced and used in large quantities in my country, and poisoning incidents occur from time to time

Method used

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  • High-activity methyl parathion hydrolase mutant and use thereof
  • High-activity methyl parathion hydrolase mutant and use thereof
  • High-activity methyl parathion hydrolase mutant and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Error-prone PCR ( error-prone PCR ) and mutant screening:

[0036] Directed evolution experiments were performed using the parental plasmid as a template. Error-prone PCR primers were designed using Primer prime software. PCR system 50ul.

[0037] Primer sequence (the underlined part is restriction site BamH I and Xho I):

[0038] Upstream Primer-5'-ACTAT GGATCC GGCCGCACCGCAGG-3'

[0039] Downstream Primer-5'-TCCC CTCGAG CTTGGGGTTGAC-3'

[0040] Error-PCR reaction system:

[0041]

[0042] PCR conditions:

[0043]

[0044] After the PCR product was purified, it was ligated into the pET21b plasmid vector after double digestion, and transformed into BL21(DE3) competent cells. After the library strains were grown on the LB+Amp resistant plate for 10 hours, a single clone was transferred in parallel to two LB+Amp resistant solid plates in sequence, and cultured at 37°C for 12 hours, and then the Pour a layer of pre-cooled screening plate medium on one o...

Embodiment 2

[0047] Site-directed mutagenesis based on structure-function analysis design:

[0048] Based on the structure-function relationship, the analysis found that the area where the negative charge is concentrated is the active site, including two Zn ion binding sites and the substrate binding site. The negative charge is conducive to the occurrence of nucleophilic reaction in the hydrolysis reaction. Therefore, site-directed mutagenesis was designed to achieve an increase in the affinity of the methyl parathion hydrolase mutant to the substrate by enhancing the interaction of the substrate molecule with the phenyl ring and phosphate ends of the methyl parathion hydrolase protein, thereby enhancing the mutation The purpose of high body activity. And in order to facilitate site-directed derivation of PEG, a group of cysteine ​​mutants were designed, and point mutations were carried out using the full-form Fast Mutagenesis System kit. The mutants and corresponding primers are as fo...

Embodiment 3

[0052] Fermentation and purification of methyl parathion hydrolase mutants:

[0053] The mutants that passed the screening were fermented and purified. Add antibiotics and trace elements at the beginning of fermentation, let it grow naturally at 37°C, use a lower initial medium concentration, so that the growth rate can be carried out according to the expected ratio during the fermentation process, the growth rate is natural during the initial culture, and the wet weight reaches 50g / L Then start to add ammonia water, the flow acceleration rate is determined based on the specific growth rate = 0.1, and no longer add yeast extract. Add trace elements into the sugar solution and add them together with the feed. When the wet weight is between 50g / L and 70g / L, induce at 30°C. Induced by low concentration IPTG (0.1mM), the specific growth rate was controlled at 0.05 at the initial stage of induction. When the wet weight > 120, the rate of ammonia supplementation will not be cha...

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PUM

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Abstract

The invention discloses high-activity methyl parathion hydrolase and a use thereof. Compared with the existing methyl parathion hydrolase, the recombinant methyl parathion hydrolase obtained by site directed mutagenesis design and screening has substantially improved activity, and has a good capability of degrading organophosphorus pesticide such as methyl parathion. A fruit and vegetable pesticide cleaning agent prepared from the methyl parathion hydrolase has effects better than those of the same kind products on the market. The high-activity methyl parathion hydrolase can be used for removing pesticide on fruits and vegetables, tea and traditional Chinese medicine raw materials in processing, can be used in soil restoration and has a wide application prospect. The invention also discloses a preparation method of the recombinant methyl parathion hydrolase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly active methyl parathion hydrolase and its application. Compared with the existing methyl parathion hydrolase, the activity of the recombinant methyl parathion hydrolase obtained through site-directed mutagenesis design and screening is significantly improved, and it has a stronger ability to degrade organophosphorus pesticides such as methyl parathion. Ability, the effect of the fruit and vegetable pesticide cleaning agent developed on the basis of it is better than similar products on the market. In addition, the enzyme can also be used in the removal of pesticides in the processing of fruits and vegetables, tea, and raw materials of traditional Chinese medicine, as well as in soil restoration, and has broad application prospects. The invention also discloses a preparation method of the above-mentioned recombinant methyl parathion hydrolase. technical backgroun...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N1/21C11D3/386A23L1/015B09C1/10A23L5/20
CPCC12N9/16B09C1/10C11D3/386C12Y301/08001
Inventor 郑春杨王磊鲍蓬张先恩张治平王殿冰王琳
Owner BIOTRAND
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