Organophosphorus degradating enzyme and coding gene

A technology for organophosphorus pesticides and enzyme degradation, applied in the fields of enzymes, biochemical equipment and methods, bacteria, etc.

Inactive Publication Date: 2006-09-13
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, organophosphorus pesticides have the function of inhibiting human acetylcholinesterase, and have different degrees of toxicity to humans.

Method used

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  • Organophosphorus degradating enzyme and coding gene
  • Organophosphorus degradating enzyme and coding gene
  • Organophosphorus degradating enzyme and coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This example illustrates the screening process of natural strain C2-1 that can degrade a variety of organophosphorus pesticides. The main steps are as follows:

[0037] 1. Take 0.5 g of soil sample in 100mL Burk inorganic salt liquid medium, shake culture overnight at 32°C.

[0038] 2. Bacterial liquid is gradually diluted with sterile water, the dilution factor is 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , Respectively spread on the Burk inorganic salt solid medium plate supplemented with 0.2mg / mL dichlorvos (pure product) and 0.2mg / mL methyl parathion (pure product), cultured in a 32 ℃ incubator, pick A single colony was taken and purified by streaking on the plate.

[0039] 3. The isolated single colony strains were respectively inoculated to the organophosphorus pesticides methyl parathion (0.05%), parathion (0.05%), methamidophos (0.05%) and omethoate (0.05%). , Phoxim (0.05%) (both v / v) (the above-mentioned pesticides were purchased from China National Pesticide Quality Sup...

Embodiment 2

[0042] This experiment illustrates the purification process of OPHC2, the main steps of which are as follows:

[0043] 1. Extraction of Pesticide Degrading Enzyme (OPHC2) Crude Enzyme Solution: Use 1000mL LB complete medium to culture C2-1 cells in large quantities, culture with shaking at 32℃ overnight, centrifuge at 5000rpm for 10 minutes, discard the supernatant, and suspend the bacterial weight in In 100mL 50mmol / L Tris-Cl (pH8.0) buffer containing 0.1mmol / L protease inhibitor PMSF, crush the cells with an ultrasonic disruptor (Branson, USA), centrifuge at 12,000 rpm for 20 minutes, collect the supernatant, and saturate it with The ammonium sulfate with a degree of 20%-90% precipitates the protein. The precipitate was resuspended in 20mL of 50mmol / L, pH8.0 Tris-HCl buffer, and dialyzed for desalting and concentration to obtain OPHC2 crude enzyme solution.

[0044] 2. Separation and purification of OPHC2: Separate the protein components in the crude enzyme solution by polyacryl...

Embodiment 3

[0046] This example illustrates the research on the enzymatic properties of the pesticide-degrading enzyme OPHC2. The main steps are as follows:

[0047] 1. Determination method of organophosphate degrading enzyme enzyme activity

[0048] The organophosphorus degrading enzyme can equally decompose methyl parathion into diethyl phosphorothioate and yellow p-nitrophenol. The activity of the enzyme can be determined by measuring the amount of the product p-nitrophenol.

[0049] The determination of the content of p-nitrophenol and the preparation of the standard curve are as follows: weigh 0.08346 g of p-nitrophenol, first dissolve it with a small amount of 95% ethanol, and then dilute to 100 mL with water, with a concentration of 6 mmol / L. Add different amounts of p-nitrophenol solution and 50mmol / L Tris-Cl (pH8.0) buffer solution according to the following table, the total volume is 1mL, then add 1mL 10% trichloroacetic acid to each tube, and then add 1mL 10% Na 2 CO 3 The total vo...

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Abstract

The invention provides a bacteria strain having a high efficiency broad spectrum degradation ability for an organophosphorus pesticide, the strain belongs to Pseudomonas pseudoalcaligenes, and is named as C2-1, the invention also provides a seperation process, cultivation conditions, and a degradation characteristic of the strain, the strain can be grew on an inorganic salt culture medium by using the pesticide. Also provided is seperation and purification of an organophosphorus pesticide degradation enzyme excreted by the strain, and study on enzyme properties, and seperation and clone of genes coding the enzyme. The invention provides a good gene source for cloning organophosphorus pesticide degradation enzyme genes by using a gene project means, finally realizes an object of producing the organophosphorus pesticide degradation enzyme.

Description

Technical field [0001] The present invention relates to the isolation of a bacterial strain with high-efficiency and broad-spectrum degradation ability for organophosphorus pesticides-C2-1, and to the study of organophosphorus pesticide degrading enzyme secreted by this strain and its enzymatic properties, as well as the enzyme encoding this enzyme Gene cloning. Background technique [0002] Organophosphorus pesticides are the main category of pesticides, accounting for 80% of the world’s total pesticides [1] , Is essential for agricultural production. However, organophosphorus pesticides have the function of inhibiting human acetylcholinesterase and have varying degrees of toxicity to humans. With the improvement of people's quality of life and the strengthening of environmental protection awareness, the residual toxicity of organophosphorus pesticides has attracted more and more attention. [0003] Sethunarhan and Yoshida since 1973 [2] Since the first Flavobacterium with diazi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/20
Inventor 伍宁丰姚斌史秀云范云六邓敏捷
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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