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Immobilized 3-phenoxy benzoic acid degrading enzyme and its preparation method

A phenoxybenzoic acid, immobilized enzyme technology, applied in biochemical equipment and methods, microorganism-based methods, immobilized on/in organic carriers, etc., can solve the problem of immobilization of 3-PBA degrading enzymes not seen Enzyme research and other issues, to achieve the effect of easy industrial production and preparation, good stability and high degradation rate

Inactive Publication Date: 2014-03-19
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been relatively few studies on the microbial degradation of 3-PBA, and mainly on the screening and characteristics of degrading bacteria, while there have been fewer studies on 3-PBA degrading enzymes. Some scholars have isolated multiple strains that can degrade 3-PBA, such as Ochrobactrum lupini, Streptomyces aureus, Bacillus sp., Stenotrophomonas sp., studied the degradation characteristics and degradation pathways of the degrading strains, but did not study the 3-PBA degrading enzyme, There is also no research on immobilized enzymes for 3-PBA degrading enzymes

Method used

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  • Immobilized 3-phenoxy benzoic acid degrading enzyme and its preparation method
  • Immobilized 3-phenoxy benzoic acid degrading enzyme and its preparation method
  • Immobilized 3-phenoxy benzoic acid degrading enzyme and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of immobilized 3-phenoxybenzoic acid degrading enzyme beads and enzyme activity assay method.

[0023] A single colony of SC-1 was picked from the plate, streaked on the LB slant medium, and incubated at 30°C for 24 hours. Use sterile saline to elute the SC-1 bacterial lawn from the slant culture medium and mix it to make a bacterial suspension, inoculate 1.0mL of the SC-1 bacterial suspension (the bacterial concentration is about 10 8 cfu / mL) in 30 mL liquid fermentation medium, 30 ° C, 180 r / min constant temperature shaking culture for 24 h to obtain seed liquid. Inoculate the seed solution at 3% (v / v) into a 10L fermenter (fermentation medium: 1.5g ammonium sulfate; 0.5g magnesium sulfate; 0.5g sodium chloride; 0.5g potassium dihydrogen phosphate; 1.5g dipotassium hydrogen phosphate ; glucose 5.0g; yeast extract powder 5.0g; water 1000mL; adjust the pH value to 7.0; add 3-PBA to 100μg / mL, and sterilize at 121°C for 15min.), the filling coeffic...

Embodiment 2

[0029] Example 2: Determination of the optimal reaction temperature and thermal stability of the immobilized enzyme degrading 3-phenoxybenzoic acid.

[0030] Weigh 0.5 g of immobilized enzyme beads, add 0.5 mL of substrate buffer containing 20 μg / mL 3-phenoxybenzoic acid, the buffer is 0.02 mol / LTris-HCl buffer at pH 7.0, and mix well. They were respectively placed at 4°C, 15°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, and 70°C for 30 minutes with shaking. The reaction was terminated with acetonitrile, the residual amount of 3-phenoxybenzoic acid was detected, and the optimum reaction temperature of the immobilized enzyme beads was determined by the relative enzyme activity. After the immobilized enzyme beads were incubated in a water bath at different temperatures for 1 h, the substrate buffer was added and shaken at the optimum reaction temperature for 30 min to determine the residual enzyme activity and investigate the thermal stability of the immobilized enzyme beads.

...

Embodiment 3

[0032] Example 3: Determination of the optimal reaction pH value and pH stability of immobilized enzyme degrading 3-phenoxybenzoic acid.

[0033] Weigh 0.5 g of immobilized enzyme beads and add 0.5 mL of 20 μg / mL 3 -Phenoxybenzoic acid substrate buffer solution, reacted at 30°C for 30 min, detected the residual amount of 3-phenoxybenzoic acid, and determined the optimal reaction pH of the immobilized enzyme beads from the relative enzyme activity. The immobilized enzyme beads were added to buffer solutions with different pH, and stood in a refrigerator at 4°C for 1 hour. Remove the buffer by filtration, wash and filter with normal saline, repeat once, blot the water on the surface of the immobilized enzyme, measure the residual enzyme activity at the optimum pH at 30°C, and investigate the pH stability of the immobilized enzyme beads.

[0034] The result is as image 3 The optimal reaction pH and pH stability curve of the immobilized 3-phenoxybenzoate degrading enzyme shown,...

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Abstract

Belonging to the immobilized enzyme field, the invention discloses a preparation method for an immobilized 3-phenoxy benzoic acid degrading enzyme. The method includes the steps of: (1) centrifuging a Sphingomonas sp. SC-1 fermentation liquid, collecting thalli, mixing the thalli with a Tris-HCl buffer solution evenly, then conducting ultrasonic crushing under ice water bath, performing centrifugation to remove intracellular enzyme, adding the Tris-HCl buffer solution to cell debris deposits to a volume before crushing to make an enzyme solution; and (2) mixing the enzyme solution with a sodium alginate solution in certain proportion, dripping the mixed solution in a calcium chloride solution uniformly to perform embedding immobilization. The immobilized 3-phenoxy benzoic acid degrading enzyme prepared by the invention has high enzyme activity under a wide temperature range (4DEG C-40DEG C) and a wide pH range (5.0-9.0), and has strong continuous reaction performance. The preparation method is simple, and the production cost is low.

Description

technical field [0001] The invention relates to the field of immobilized enzymes, specifically a sphingomonas ( Sphingomonas sp.) A method for preparing 3-phenoxybenzoate-degrading enzymes in SC-1 cells into immobilized enzymes under calcium alginate embedding. Background technique [0002] Pyrethroid pesticides are the second largest class of insecticides. At present, the application amount of pyrethroid pesticides in the world accounts for about 20% of the total amount of pesticides, and the application area accounts for 25% of the total application area of ​​insecticides. . This type of pesticide is stable to light and heat, and is difficult to degrade rapidly under natural conditions. Long-term exposure may cause teratogenic, carcinogenic, and mutagenic hazards. 3-Phenoxybenzoic acid (3-PBA) is one of the decomposition products of most pyrethroid pesticides. Compared with pyrethroid pesticides, the migration rate in the environment is faster, and 3-PBA is in the soil ...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12R1/01
Inventor 不公告发明人
Owner SICHUAN AGRI UNIV
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