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Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant

An organophosphorus and hydrolase technology, applied in the field of bioengineering, can solve problems such as poor performance of malathion, and achieve the effects of high organophosphorus hydrolysis activity and thermal stability, good practical application value, and good thermal stability.

Active Publication Date: 2016-01-20
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] In order to overcome the above-mentioned problem of poor performance of the known organophosphorus hydrolase on the commonly used organophosphorus pesticide malathion, the present invention carries out gene mutation modification on the known organophosphorus hydrolase to improve the activity of the organophosphorus hydrolase while retaining good Thermostability, and then provide an engineered organophosphate hydrolase, its coding gene, recombinant expression plasmid containing the gene, recombinant expression transformant and its preparation method, as well as the application technology of the mutant protein in degrading organophosphorus pesticides

Method used

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  • Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant
  • Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant
  • Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Site-directed Saturation Mutagenesis of Organophosphate Hydrolases

[0056] In this example, the method of active site cassette mutation (CASTing) is used to transform organophosphate hydrolase, and the plasmid pET28a-PoOPH preserved by the inventor is selected. M2 , Carry out CASTing mutations to transform protein active pocket amino acids. According to the protein structure information, select Val55, Leu57, Leu62, Thr87, Val89, Phe111, Leu115, Trp172, Met188, Ile250, Trp263, Phe265 for site-directed saturation mutation.

[0057] Design primers for site-directed saturation mutagenesis, as shown in Table 1.

[0058] Table 1. List of primers for site-directed saturation mutagenesis

[0059]

[0060]

[0061] Among them, N represents any one of the four bases A, C, G, and T, M represents any one of the two bases T and G, and K represents any one of the two bases A and C , D represents any one of the three bases A, G, and T, and H represents any one of the three b...

Embodiment 2

[0073] Site-directed Saturation Mutagenesis of Organophosphate Hydrolases

[0074] The present embodiment adopts error-prone PCR (epPCR) technology to the mutant PoOPH that CASTing obtains M5 Carry out random mutation of the whole sequence to transform organophosphate hydrolase.

[0075] Design gene cloning primers as follows:

[0076] PoOPH-F: 5'-GAATTC GAGCTC ATGCGTCTTTTTCTCG-3'

[0077] PoOPH-R: 5'-CCC AAGCTT GGCGGTCGCTACGGA-3'

[0078] Wherein, the underlined part of the upstream primer PoOPH-F is the SacI restriction site, and the underlined part of the downstream primer PoOPH-R is the HindIII restriction site.

[0079] with PoOPH M5 Gene sequence of pET28a-PoOPH M5 The plasmid was used as a template for PCR amplification. The PCR system is: template 25ng, upstream and downstream primers (10pmol / μl) 1μl each, 2×TaqMix 12.5μl, 150μM nCl 2 , ddH 2 O to make up to 25 μl. The PCR amplification program is: (1) 95°C for 2min; (2) 94°C for 30sec; (3) 55°C for 30sec; ...

Embodiment 3

[0087] DNA Recombination Transformation of Organophosphate Hydrolase

[0088] Using DNA rearrangement technology, the dominant mutants obtained in the error-prone PCR mutation library are randomly combined to merge beneficial mutations. All the dominant mutant genes obtained in the above-mentioned error-prone PCR mutation library were mixed as a template for gene cloning, and the cloning primers were the same as error-prone PCR. The PCR reaction system is: mixed recombinant plasmid template 25ng, 10×Buffer 2.5μl, dNTPs (2mM) 2.5μl, a pair of cloning primers (10pmol / μl) 0.75μl each, KOD-plus-enzyme 0.5U, distilled water to make up to 25μl. The PCR amplification program is: (1) 2 min at 94°C; (2) 10 sec at 98°C; (3) 30 sec at 55°C; (4) 1 min at 68°C; steps (2) to (4) are performed for 25 cycles. Use an agarose gel DNA recovery kit to recover the only target band, concentrate the resulting mixed mutant gene to 200ng / μl, and use DNaseI to perform random cutting. The enzyme cuttin...

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Abstract

The invention relates to engineering organophosphorus hydrolase, nucleic acid, a mutant and an application of the engineering organophosphorus hydrolase and the mutant and belongs to the technical field of bioengineering. The engineering organophosphorus hydrolase is an organophosphorus hydrolase mutant constituted by new amino acid sequences formed after amino acid substitution of one or more of amino acid residues on the 55th site, the 57th site, the 83th site, the 114th site, the 137th site, the 188th site or the 262th site of an amino acid sequence shown as SEQ ID No.1, and the activity of the engineering organophosphorus hydrolase is improved. Compared with the prior art, the organophosphorus hydrolase mutant has the advantages that the malathion catalytic activity is improved by 36.7 times, the mutant keeps excellent thermal stability of a female parent, and the half-life period at 50 DEG C reaches 65 h. The organophosphorus hydrolase mutant can efficiently degrade common organophosphorus pesticides such as malathion and the like, has excellent thermal stability and has very good application prospect in the field of degradation of the organophosphorus pesticides.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an organophosphorus hydrolase, a mutant and its coding gene, and the application of the organophosphate hydrolase and its mutant in the degradation of organophosphorus pesticides. Background technique [0002] my country is a big country in the production and use of pesticides. There are more than 2,000 pesticide manufacturers nationwide, producing more than 200 types of pesticides. Organophosphorus pesticides have the characteristics of low price, high efficiency, and broad spectrum. They are widely produced and applied insecticides, accounting for 38% of the world's insecticide market. In 2010, the total domestic pesticide output was 2.2622 million tons, of which organophosphorus pesticide products accounted for 80% of the total. In my country, the annual use of pesticides is as high as 800,000 to 1,000,000 tons, ranking first in the world. Although organo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63B09C1/10
CPCB09C1/10C12N9/16
Inventor 许建和罗晓晶潘江
Owner EAST CHINA UNIV OF SCI & TECH
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