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Pyrethroid degrading enzyme, coding gene of pyrethroid degrading enzyme and recombination strain and application of coding gene

A technology of pyrethroids and pyrethroids, which is applied in the field of pyrethroid degrading enzymes, can solve the problems of complex preparation process, high risk of E. coli endotoxin, and few types of pyrethroid pesticides, and achieve good fermentation stability, Simple preparation process and obvious degradation effect

Pending Publication Date: 2020-05-08
WUHAN SUNHY BIOLOGICAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If it is used in the fields related to the cleaning of edible agricultural products, the risk of the pyrethroid-degrading enzyme from the above-mentioned sources bringing in E. coli endotoxin is relatively high, the preparation process is complicated, and the yield of the pyrethroid-degrading enzyme is not mentioned.
[0005] It can be seen that the current enzymatic removal technology for pyrethroid pesticides still has a relatively high risk of using the source strain of pyrethroid degrading enzyme, complex preparation process, unknown yield of pyrethroid degrading enzyme, and types of pyrethroid pesticides that can be significantly degraded minor defects

Method used

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  • Pyrethroid degrading enzyme, coding gene of pyrethroid degrading enzyme and recombination strain and application of coding gene
  • Pyrethroid degrading enzyme, coding gene of pyrethroid degrading enzyme and recombination strain and application of coding gene
  • Pyrethroid degrading enzyme, coding gene of pyrethroid degrading enzyme and recombination strain and application of coding gene

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Embodiment 1

[0024] The embodiment of the present invention provides a pyrethroid degrading enzyme coding gene YLL2-C244A, the nucleotide sequence of the coding gene YLL2-C244A is shown in SEQ ID No.1.

[0025] The present invention also provides a recombinant strain YLL2-X33 containing the above-mentioned pyrethroid degrading enzyme coding gene YLL2-C244A; its construction method is as follows: use Pichia pastoris universal signal peptide α-factor as a guide, and use pPICZaA as an expression vector , by screening a series of lipases, the YLL2-C244A mutant secreted expression vector was constructed and transformed into Pichia pastoris X33, and the multi-copy recombinant strain YLL2-X33 was obtained by screening with the antibiotic bleomycin Zeocin. The Pichia strains used in the recombinant Pichia pastoris YLL2-X33 in this example are stable and safe, the fermentation process is simple, the fermentation titer is high, and it can obviously degrade various pyrethroid pesticides.

[0026] The...

Embodiment 2

[0027] Example 2 Preparation method of pyrethroid degrading enzyme enzyme liquid

[0028] Shake flask culture of seeds: get the recombinant strain YLL2-X33 containing the coding gene YLL2-C244A shown in SEQ ID No.1 to activate the recombinant strain YLL2-X33, and continue to use YPD medium to shake the flask at 28°C and 150rpm for 21h ,spare;

[0029] Fermentation culture: Take a fermenter and use BSM medium. The base material formula is dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, potassium sulfate, calcium sulfate, potassium hydroxide, glycerin, PTM1, biotin, etc., and tap water; 120°C Sterilize for 30 minutes, cool and insert the strain into the shake flask to ferment for 110 hours under the conditions of temperature control 28°C and pH 4.8. Through feeding, controlling the ventilation rate and rotating speed, the dissolved oxygen in the fermenter is always controlled to be greater than 20%; during this period, the velocity of the 2# feed contai...

Embodiment 3

[0032] Example 3 Enzymatic properties of the pyrethroid degrading enzyme

[0033] 1. Enzyme activity of the pyrethroid degrading enzyme

[0034] 1 gram of pyrethroid degrading enzyme powder (or 1 mL of pyrethroid degrading enzyme solution), hydrolyzes p-nitrophenyl palmitate for 1 min in 25 °C, pH7.6 buffer solution to produce 1 umol of p-nitrophenol, which is 1 A unit of enzyme activity expressed in u / g (or u / mL).

[0035]Enzyme activity detection uses a self-built method for measuring the production rate of p-nitrophenyl palmitate (referred to as "pNPP") to p-nitrophenol. The specific method is as follows:

[0036] Prepare a 10mL glass test tube, draw 5900mL 0.05mmol / L pH7.6 Tris-hydrochloric acid buffer solution, 75mL10mmol / L p-nitrophenyl palmitate isopropanol solution, mix well and bathe in a water bath at 25°C for 3min, then add 120uL permethrin Immediately after degrading the enzyme sample water dilution solution, start timing from 0s and mix evenly. Use the blank sam...

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Abstract

The invention belongs to the technical field of bioengineering, and specifically relates to a coding gene of a pyrethroid degrading enzyme. The nucleotide sequence of the coding gene YLL2-C244A is shown in SEQ ID No. 1. The invention also provides a recombinant strain YLL2-X33 containing the coding gene YLL2-C244A of the pyrethroid degrading enzyme and the pyrethroid degrading enzyme which is expressed by the recombinant strain YLL2-X33, and also provides application of the coding gene YLL2-C244A in preparation of the pyrethroid pesticide degrading enzyme, application of the pyrethroid degrading enzyme in degradation of pyrethroid pesticide and application of the recombinant strain YLL2-X33 in preparation of a pyrethroid degrading enzyme solution, wherein preparation of the pyrethroid degrading enzyme solution comprises the steps: performing shake-flask culture on the recombinant strain YLL2-X33, performing fermentation culture, performing filtration, and conducting antiseptic treatment so as to obtain the pyrethroid degrading enzyme solution. The pyrethroid degrading enzyme has a wide range of esbiothrin for degradation, and has an obvious degradation effect, and the average degradation rate of common pyrethroid which is remained on vegetables and fruits can reach 85% or above, and the pyrethroid degrading enzyme can be applied to degradation of pyrethroid pesticide residues in food or the environment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a pyrethroid degrading enzyme, its coding gene, recombinant strain and application. Background technique [0002] Pyrethroids were developed in the 1970s and can be used to prevent and control tea trees, vegetables, fruit trees, flowers, trees, and sanitary pests. This kind of pesticide generally has the characteristics of stable environment and slow degradation speed, which has a certain impact on the ecological environment; it has cumulative toxicity to humans, and long-term low-dose exposure can cause chronic diseases; it has moderate neurotoxicity and immune system toxicity to mammals. Cardiovascular toxicity and genotoxicity, some varieties even have teratogenic, carcinogenic and mutagenic effects. [0003] Common methods for reducing pesticide residues in edible agricultural products include cleaning, peeling, cooking, storage, and ozone methods, which ha...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/81C12N1/19A23L5/20C12R1/84
CPCC12N9/20C12Y301/01003C12N15/815A23L5/25Y02A50/30
Inventor 吴启涛詹志春周樱曾世超顾爱玲段垒杨溢民
Owner WUHAN SUNHY BIOLOGICAL
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