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Burkholderia jiangsuensis strain, organophosphorus ester hydrolase and gene of organophosphorus ester hydrolase, and application of organophosphorus ester hydrolase in degradation of organophosphorus pesticide

A technology of organophosphorus pesticides and organophosphorus esters, which is applied in the field of bioengineering technology in the Ming Dynasty, and can solve the problems of diversity and novelty of degrading strains to be enriched

Inactive Publication Date: 2014-07-30
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhou Ningyi and others from Wuhan Institute of Virology cloned the mpd gene from Pseudomonas sp.WBC-3, and obtained the MPH enzyme. The specific activity of its crude enzyme solution to catalyze methyl parathion was 12.7 U / mg (Biochem Biophys Res Commun., 334:1107-1114), the pure enzyme solution is 50.5U / mg (Research in Microbiology.,158:143-149), the catalytic activity of the recombinant organophospholipid hydrolase reported in the current research needs to be improved, and the diversity of the degrading strains And the novelty also needs to be enriched

Method used

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  • Burkholderia jiangsuensis strain, organophosphorus ester hydrolase and gene of organophosphorus ester hydrolase, and application of organophosphorus ester hydrolase in degradation of organophosphorus pesticide
  • Burkholderia jiangsuensis strain, organophosphorus ester hydrolase and gene of organophosphorus ester hydrolase, and application of organophosphorus ester hydrolase in degradation of organophosphorus pesticide
  • Burkholderia jiangsuensis strain, organophosphorus ester hydrolase and gene of organophosphorus ester hydrolase, and application of organophosphorus ester hydrolase in degradation of organophosphorus pesticide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Shake flask culture of embodiment 1 Burkholderia jiangsuensis new bacterial strain (Burkholderia jiangsuensis CGMCC9028)

[0064] Fill 50mL medium into a 250mL shaker flask, and its medium composition is as follows: Glucose 5g l -1 , yeast extract 5g·l -1 , NaCl1g l -1 , KH 2 PO 4 1 g l -1 , MgSO 4 0.2g·l -1 , CaCl 2 0.1g·l -1 , pH7.0, prepared with tap water, sterilized at 121°C for 20min. The strain CGMCC9028 was inoculated into shake flasks, pre-cultivated at 30°C and 180rpm for 12 hours as a seed solution, and the seed solution was inoculated into a 500mL shake flask containing 100mL of the same medium at an inoculation amount of 1% (v / v). , cultivated at 30° C. and 180 rpm for 24 hours, and centrifuged at 9000 rpm to obtain wet cells containing organophosphate hydrolase.

Embodiment 2

[0065] The fermentation culture of embodiment 2 Burkholderia new bacterial strain (Burkholderia jiangsuensis CGMCC9028)

[0066] Seed culture is the same as in Example 1. Load 3L medium into a 5L fermenter, its composition is as follows: glucose 60g, peptone 30g, yeast extract 30g, KH 2 PO 4 3g, MgSO 4 1.5g, NaCl3.0g, tap water 3L, pH7.0. After the culture medium is sterilized, insert 200ml of the seed liquid pre-cultivated for 12h, and ferment at 30°C, 400rpm and 1vvm aeration conditions, during which, add glycerol (8mL / h) at a constant speed for 4-12h, and add bubbles dropwise if foaming occurs. Foam (1% aqueous solution, w / v, purchased from Shanghai Feida Trading Co., Ltd.) for defoaming. After 12 hours of fermentation, put the tank. After the cultivation, it was centrifuged at 9,000rpm to obtain the wet bacterium containing organophosphate hydrolase.

Embodiment 3

[0067] Cloning of Example 3 Organophospholipid Hydrolase Gene Bjmpd

[0068] Synthetic primers are as follows:

[0069] mpdA: 5'-GAATT CATATG GCCGCACCGCAGGTGCGCACCTCG-3'

[0070] mpdB: 5'-GAATT CTCGAG CTTGGGGTTGACGACCG-3'

[0071] Wherein, mpdA primer (its sequence is as shown in SEQ ID No.3 in the sequence listing) underlined part is the Nde I restriction site, mpdB primer (its sequence is as shown in SEQ ID No.4 in the sequence listing) underlined part As the Xho I restriction site, the genomic DNA of Burkholderia jiangsuensis CGMCC9028 was used as a template, and the DNA was amplified by polymerase chain reaction (PCR). 20μl PCR reaction system: dd H 2 O7 μl, genome 1 μl, mpdA 1 μl, mpdB 1 μl, 2×Taq Mix 10 μl. After mixing evenly, put it into the PCR machine and carry out the reaction according to the following procedures: (1) 95°C, pre-denaturation for 5 minutes; (2) 94°C, denaturation for 1 minute; (3) 61°C, annealing for 40s; (4) 72°C extension 1min30s; (2)-(4) 3...

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Abstract

The invention discloses a strain ECU1088, which is identified as burkholderia jiangsuensis. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.9028. The invention also discloses an organophosphorus ester hydrolase and the gene of the organophosphorus ester hydrolase expressed by the strain, a recombinant expression vector containing the gene of the organophosphorus ester hydrolase, a recombinant expression transformant, a recombinase of the strain and a preparation method of the recombinase, and a method for degrading an organophosphorus pesticide through the organophosphorus ester hydrolase or recombinant organophosphorus ester hydrolase. The burkholderia jiangsuensis strain and the recombinant organophosphorus ester hydrolase thereof has remarkable advantages of being good in catalyzing effect, mild in reaction conditions and environment-friendly in degrading the organophosphorus pesticide.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a new strain of Burkholderia jiangsuensis ECU1088, the preservation number of which is CGMCC9028, the organophosphorus ester hydrolase and its gene expressed by the new Burkholderia strain, and the gene containing the gene The recombinant expression vector, the recombinant expression transformant, and the preparation method of the recombinant enzyme also relate to the application of the organophosphorus ester hydrolase or the recombinant enzyme as a catalyst to degrade the organophosphorus pesticide. Background technique [0002] Organophosphorus pesticides are inhibitors of acetylcholinesterase, which are highly toxic to the human body and threaten human health. Pesticide residues are an inevitable phenomenon after large-scale application of pesticides. If the maximum residue limit is exceeded, it will have adverse effects on humans and animals, or cause poiso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/16C12N15/55C12N15/70B09C1/10C12R1/01
Inventor 许建和柳絮云李春秀罗晓晶潘江
Owner EAST CHINA UNIV OF SCI & TECH
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