Method for preparing methyl parathion degradation bacterium and enzyme preparation thereof

A technology of methyl parathion and degrading bacteria, applied in the high biological field, can solve problems such as products that are not cost-effective, and achieve the effects of thorough removal of pesticide residues, protection of the ecological environment, and protection of people's lives and health

Inactive Publication Date: 2008-04-30
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

There are also relevant patents on the treatment of field organophosphorus pesticide pollution by microbial agents, but there are no mature reports on the removal of pesticide residues by preparing enzyme preparations, especially household detergents with enzyme preparations for removing pesticide residues have not been available with more cost-effective products.

Method used

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  • Method for preparing methyl parathion degradation bacterium and enzyme preparation thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The surface soil of soybean fields that had been treated with methyl parathion for 10 consecutive years at the Shenyang Ecological Station of the Chinese Academy of Sciences was collected. Get 10g soil, add 90ml distilled water, make soil suspension, insert in the inorganic salt culture medium of methyl parathion only carbon source (methyl parathion concentration is 100mg / L) with 10% volume ratio, inorganic salt The formula of culture medium is (both mass ratio): NH 4 NO 3 0.2%, KH 2 PO 4 0.3%, K 2 HPO 4 0.15%, CaCl 2 0.001%, MgSO 4 ·7H 2 O0.2%, FeSO 4 ·7H 2 O0.001%, the balance is water, pH7.0-7.2, 30 ° C, 200rpm enrichment culture for 5 days, use the enrichment culture solution for the first time to insert in the corresponding medium in a volume ratio of 10%, and simultaneously The concentration of methyl parathion is increased to 200mg / L, cultivated under the same conditions, and the second enrichment culture solution is also inserted into the corresponding...

Embodiment 2

[0034] Get in Example 1 or the slant of P. shigella-like P. shigella with Negative Gram staining in the market and inoculate in 200ml LB medium, by mass percentage, LB medium formula is as follows: yeast extract 0.5%, NaCl1%, peptone 1%, the balance is water; constant temperature shaking culture to logarithmic phase, to obtain seed liquid, ready to inoculate a small fermenter. Fermentor 10L, feeding capacity is 8L, and medium formula is (both mass ratio): glucose 0.8%, (NH 4 ) 2 SO 4 1%, K 2 HPO 4 0.2%, MgSO 4 0.05%, NaCl0.01%, CaCO 3 0.3%, yeast extract 0.02%, the balance is water, pH7.2-7.5. After the feeding is completed, 121°C high-pressure damp heat sterilization, after cooling to 35°C, the above-mentioned cultivated shake flask seed liquid is transferred to a fermenter according to the 10% volume inoculation amount, and cultivated to the logarithmic growth phase; during the cultivation of the fermenter, The stirring speed is 220 rpm, the ventilation rate of steril...

Embodiment 3

[0036] 3000g of the fermented liquid cultivated in the above-mentioned Example 2 was centrifuged for 10 minutes to collect the thalline, and the precipitate was suspended in PBS (40mmol / L, pH7.0) buffer solution. The preparation method of PBS buffer solution was as follows: dissolve 8gNaCl in 800ml distilled water, 0.2gKCl, 1.44gNa 2 HPO 4 , 0.24gKH 2 PO 4 , adjust the pH to 7.4 with HCl, add water to make up to 1L. The bacterial cells were disrupted mechanically, centrifuged at 12000 g for 10 minutes, and the supernatant was collected. 20%~80% mass concentration (NH 4 ) 2 SO 4 Fractional precipitation, collecting 80% mass concentration (NH 4 ) 2 SO 4 Precipitation, dissolved in HEPES buffer solution (20mmol / L, pH7.5), and dialyzed to the above buffer solution for 16-18 hours (dialysis with distilled water or deionized water), to remove ammonium sulfate, to obtain crude enzyme solution, the recovery was 97.8% are bottled and packaged as liquid enzyme preparations.

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Abstract

The invention relates to methyl parathion degradation fungus and the preparing method of the enzyme preparation thereof, and belongs to the biological high technical field. The methyl parathion degradation fungus strain is plesiomus shigelloides, and the production process of liquid enzyme preparation adopts the steps of slant culture, seed liquid shaking, seed fermenter, fermenter, bacterial collection, mechanical cell crushing, clear liquid collection, (NH4)2SO4 fractional precipitation, HEPES buffer solution suspension, dialysis, and crude enzymes production. Enzyme dry powder preparation which is easy to be stored or transported can also be further refined, and processing steps added on the basis of the crude enzymes production are as follows: DEAE-Sephadex-A50 anion column chromatography, CM Sepharose Fast Flow cation column chromatography, dialysis, cryodesiccation and enzyme dry powder production. The direct application of the liquid crude enzymes preparation can ensure the organic phosphorus pesticide residue quantity in agricultural crops to be reduced by more than 90 percent, and the residual on the surface of garden spgarden stuff washed to be reduced by about 98 percent; after the enzyme dry powder preparation is diluted according to a specific proportion, the pesticide residue quantity can be reduced by more than 95 percent. The enzyme preparation product resolves the problem that the pesticide residue is over-standard in the agricultural production.

Description

technical field [0001] The invention relates to a methyl parathion-degrading bacterium and a preparation method of an enzyme preparation thereof, which belongs to the field of biological high technology, uses microorganism technology to degrade chemical pesticides, and is suitable for the production and deep processing of green and pollution-free agricultural products in modern agricultural production. Household cleaning of pesticide residues in vegetables and fruits. Background technique [0002] The use of chemical pesticides such as 666 and DDT has greatly promoted the development of modern agriculture, made an important contribution to the improvement of the output and quality of agricultural products, and greatly improved the labor efficiency and mechanization degree of agricultural production. However, these agrochemicals that are artificially synthesized and released into the environment have brought huge potential risks to human health and the ecological environment....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20
Inventor 徐慧李慧张颖韩斯琴史荣久倪志龙杨伟超张忠泽陈冠雄
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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