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Novel esterase and application thereof

A new type of esterase technology, applied in the field of genetic engineering, to achieve the effect of broad application prospects

Inactive Publication Date: 2012-06-20
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no research report on the discovery of new broad-spectrum and high-efficiency degradation pyrethroid pesticide-degrading enzymes from seabed mud by using metagenomic technology at home and abroad.

Method used

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  • Novel esterase and application thereof
  • Novel esterase and application thereof
  • Novel esterase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0036] 1. Extraction of total DNA:

[0037] Weigh 6g sample, add 13.5mL DNA extraction buffer (0.1M Tris, 0.1M EDTA-Na, 0.1M Na 3 PO 4 , 1.5M NaCl, 1% CTAB, pH 8.0), shake vigorously for 3-5min, add 200μL lysozyme (100mg / ml), invert repeatedly 5-6 times, 37℃ water bath for 30min, add 1.5ml 20% SDS, 65 ℃ water bath for 1 h (during this period, turn it upside down several times every 15 min), centrifuge at 8000 r / min for 5 min, take the supernatant, extract twice with an equal volume of chloroform, centrifuge at 16000 r / min for 10 min, take the supernatant, add 0.6 times the volume of iso Propanol, place at room temperature for 2 hours, centrifuge at 20,000r / min for 20min, discard the supernatant, add 5mL of pre-cooled 70% ethanol to the pellet, centrifuge at 20,000r / min for 10min, collect the DNA precipitate, air-dry, and dissolve with an appropriate amount ...

Embodiment 2

[0060] Research on the enzymatic properties of embodiment 2 recombinant esterase F816

[0061] Weigh 11 mg of p-nitrophenol acetate, dissolve it completely with 1 mL of methanol, then pipette 0.4 mL of p-nitrophenol acetate solution into 9.6 mL of 0.05M Tris-HCl (pH=6.8) buffer solution, and take 100 μL of the enzyme solution to be tested was added to 2 mL of p-nitrophenol acetate solution, and incubated for 5 minutes. At the same time, the inactivated crude enzyme solution was used as a control, and the light absorption value was measured at 405 nm. The greater the absorption value, the higher the enzyme activity.

[0062] 1. Optimum reaction temperature and thermal stability of recombinant esterase F816

[0063] After the crude enzyme solution of recombinant esterase F816 is subjected to enzymatic reaction at 20-80° C., its enzyme activity is measured according to the above method to obtain its optimum reaction temperature (recorded as 100% when the enzyme activity is the hi...

Embodiment 3

[0070] Degradability determination of embodiment 3 recombinant esterase F816 pyrethroid pesticides

[0071] In this study, cyhalothrin, cypermethrin, fenvalerate and deltamethrin were selected as the degradation objects, and GC-2010 gas chromatography (Shimadzu Corporation, Japan) was used for quantitative analysis to determine the effect of recombinant esterase F816 on pyrethroids. Degradability of pesticides.

[0072] 1. Sample processing

[0073] Add 0.3mL of crude enzyme solution to 1mL of cyhalothrin, cypermethrin, fenvalerate and deltamethrin with a concentration of 4mg / L, and add 0.3mL of crude enzyme solution to 1mL of pH 6.8 0.1 M potassium phosphate buffer, add 0.1M potassium phosphate buffer with pH 6.8 to a final volume of 4 mL, react at 37°C for 60 min, add n-hexane to the sample to be tested at a ratio of 1:2 (v / v), Shake on a constant temperature shaker for 20min (200r / min), absorb the supernatant and dilute to 1mL, accurately absorb 1μL and perform quantitati...

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Abstract

The invention discloses deoxyribonucleic acid (DNA) of novel esterase. The nucleotide sequence of the DNA is shown as SEQ ID NO.1, and the amino acid sequence of the DNA is shown as SEQ ID NO.3. The invention also discloses an expression vector which contains the DNA of the novel esterase, recombinant esterase and the application of the recombinant esterase to the degradation of pyrethroid pesticide residues. The novel esterase and the recombinant esterase have efficient soluble expression in a bacillus coli expression system, and the recombinant esterase has efficient degrading effects on cyhalothrin, cypermethrin, fenvalerate, deltamethrin and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a novel esterase extracted from seabed mud and its expression vector, a recombinant esterase and the application of the recombinant esterase in degrading pyrethroid pesticide residues. Background technique [0002] Pyrethroid pesticides are a class of biomimetic insecticides artificially synthesized according to the structure of natural pyrethrins after organochlorine, organophosphorus and carbamate pesticides, and their appearance represents the era of ultra-efficient insecticides in the insecticide industry . The insecticidal mechanism of pyrethroid pesticides is mainly to affect the nervous system of insects. The insecticidal activity is strong, and the insecticidal efficacy is generally more than 100 times greater than that of organophosphorus insecticides. They also have good quick-acting properties and low dosage and low residue. It is widely used in various ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/10C12N15/63C12N15/70A62D3/02A62D101/04A62D101/28
Inventor 刘玉焕梁卫驱刘孝龙罗华建范新炯胡珊罗诗刘远星
Owner SUN YAT SEN UNIV
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