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Organic phospholipid hydrolase, gene, recombinant expression transformant and application thereof

A technology of organophosphorus ester and hydrolase, which is applied in the field of bioengineering, can solve the problems of low organophosphorus ester hydrolase activity and poor thermal stability, and achieve excellent thermal stability, high thermal stability, and high organophosphorus hydrolysis active effect

Inactive Publication Date: 2014-07-16
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to provide a kind of organophosphorus ester hydrolase, gene, recombinant expression transformant and application thereof in view of the reported problems of low activity and poor thermal stability of organophosphorus ester hydrolase

Method used

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  • Organic phospholipid hydrolase, gene, recombinant expression transformant and application thereof
  • Organic phospholipid hydrolase, gene, recombinant expression transformant and application thereof
  • Organic phospholipid hydrolase, gene, recombinant expression transformant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Screening and Gene Cloning of Organophospholipid Hydrolase PoOPH

[0042] Select the strains preserved by our research group, inoculate them into the improved inorganic salt medium, add methyl parathion with a final concentration of 100ppm, cultivate them at 30°C and 200rpm for 1-2 weeks, pass them three times, and use TLC Analysis and high performance liquid chromatography to identify the degradation effect of strains on methyl parathion.

[0043] The composition of the improved inorganic salt medium is: 1g / L NaCl, 1g / L NH 4 NO 3 , 1.5g / L K 2 HPO 4 , 0.5g / L KH 2 PO 4 , 0.2g / L MgSO 4 ·7H 2 O, 1g / L yeast extract.

[0044] The cultured bacteria solution was extracted three times with chloroform-ether (1:1, v / v). After evaporating the extractant at room temperature, it was dissolved in methanol and used for thin-layer chromatography and high-performance liquid chromatography analysis.

[0045] The thin-layer chromatography developing solvent is n-hexane:chloroform...

Embodiment 2

[0055] Preparation and activity screening of site-directed saturation mutants of PoOPH

[0056] The saturation mutants at positions 250 and 263 of the PoOPH gene were constructed respectively.

[0057] The PCR primers were designed as follows:

[0058] H250X-F: 5′-ATGGGGCGACATTCTG NNK AACCACGCCGTGCAG-3′

[0059] H250X-R: 5′-CTGCACGGCGTGGTT MNN CAGAATGTCGCCCCAT-3′

[0060] I263X-F: 5′-CCAAGCCTGAAGTGGTC NNK GAGTTCGATGTCGACA-3′

[0061] I263X-R: 5′-TGTCGACATCGAACTC MNN GACCACTTCAGGCTTGG-3′

[0062] The underlined part is the degenerate codon of the mutation site. Wherein, 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.

[0063] Using the recombinant expression plasmid containing the pooph gene obtained in Example 1 as a template, PCR site-directed mutagenesis was performed on the whole plasmid.

[0064] The above-mentioned complete plasmid PCR site-directed mutag...

Embodiment 3

[0073] Purification of PoOPH and its mutants and determination of pure enzyme activity

[0074] Use Ni affinity chromatography column (GE company HisTrap TM FF column, 5mL) to purify PoOPH and its mutant proteins PoOPH(H250I), PoOPH(I263W) and PoOPH(H250I / I263W), the specific method is as follows:

[0075] (1) Equilibrate the Ni chromatography column with Tris-HCl buffer (20mM, pH8.0, containing 0.5M NaCl and 20mM imidazole) of 5 times the bed volume;

[0076] (2) The crude enzyme liquid obtained in Example 2 is passed through the Ni chromatography column with a flow rate of 1ml / min;

[0077] (3) Use 5 times the bed volume of 20mM pH8.0 Tris-HCl buffer (containing 0.5M NaCl and 20mM imidazole) to elute non-specifically bound foreign proteins;

[0078] (4) Elute the specifically bound target protein with 5 times the bed volume of 20 mM pH 8.0 Tris-HCl buffer (containing 0.5 M NaCl and 0.5 M imidazole).

[0079] The purified proteins PoOPH, PoOPH(H250I), PoOPH(I263W) and PoO...

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Abstract

The invention relates to organic phospholipid hydrolase, a mutant, an encoding gene and an application thereof in degradation of organophosphorus pesticides. The organic phospholipid hydrolase PoOPH is originated from edible oil pseudomonadaceae, the mutant of which is a protein which is derived from the protein shown as an amino acid sequence SEQ ID No.1 in a sequence table and has the activity of the organic phospholipid hydrolase, wherein the 250th and the 263rd amino acids are substituted by a single site or double sites. Compared with a parent, the catalytic activities of the PoOPH mutant to parathion-methyl and paraoxon-ethyl are respectively improved by 5680 and 37.5 folds. Meanwhile, the PoOPH mutant maintains the excellent heat stability of the parent. The PoOPH mutant provided by the invention has two attributes at the same time: high vitality and excellent heat stability and has a good application prospect in the field of degrading organophosphorus pesticides.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an organophosphorus ester hydrolase, a gene, a recombinant expression transformant and an application thereof. Background technique [0002] Organophosphorus pesticides have the characteristics of low price, high efficiency, and broad spectrum. They are widely produced and applied pesticides and insecticides, accounting for 38% of the world's insecticide market share. In 2010, the total domestic pesticide output was 2.2622 million tons, of which organophosphorus pesticide products accounted for 80% of the total. The widespread use of organophosphorus pesticides has effectively prevented crop pests and diseases and significantly increased crop yields, but long-term and large-scale use has brought many negative effects on food safety and the ecological environment. On the one hand, domestic vegetable pesticide residue detection incidents occur from time to time,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21A62D3/02A62D101/04A62D101/28
CPCC12N9/16
Inventor 许建和罗晓晶潘江张志钧
Owner EAST CHINA UNIV OF SCI & TECH
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