Application of chymotrypsin as degradation agent for organic pesticide
A technology for chymotrypsin and pesticides, which is applied in the application field of chymotrypsin as a degrading agent for organic pesticides, and can solve the problems of insufficient indication and the like
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Embodiment 1
[0027] Example 1 Degradation of deltamethrin by Culex chymotrypsin (NYD-Ch)
[0028] 1) Using gene recombination method, construct NYD-Ch prokaryotic expression vector and induce expression
[0029] Primers were designed according to the full-length sequence of the Culex chymotrypsin gene (see GENBANK accession number: AF468495):
[0030] Upstream primer L: 5′-CGGAATTCAACATGGCATCGAAACTGACGGT-3′
[0031] Downstream primer R: 5′-ACTAGCGGCCGCTTAAGCATCCTGACGCAGCTG-3′
[0032] Introduce the EcoR I restriction site (GAATTC) into the upstream primer, and introduce the protective base CG upstream of the restriction site; introduce the Not I restriction site (GCGGCCGC) into the downstream primer, and introduce the protective base outside it Based on ACTA, so as to ensure the digestion efficiency of the endonuclease.
[0033] The full-length sequence of NYD-Ch (813bp) was amplified by PCR method, and the prokaryotic cloning vector pGEM-T / NYD-Ch was constructed. Subcloned into expres...
Embodiment 2
[0045] Example 2 Toxicity determination of metabolites after degradation of deltamethrin by NYD-Ch to fourth-instar larvae of different strains of Culex palustris
[0046] The experiment was divided into sensitive and resistant groups, and the final volume was 300ml.
[0047] Sensitive group determination:
[0048] Group A:
[0049] Toxicity determination of deltamethrin to fourth instar larvae of sensitive strain Culex palustris
[0050] Deltamethrin (ppm) 0 0.001 0.005 0.01 0.05 0.1 0.5
[0051] Fourth instar larva (bar) 50 50 50 50 50 50 50
[0052] After 24 hours of action, the number of dead larvae was recorded
[0053] Group B
[0054] Toxicity determination of metabolites of deltamethrin treated with NYD-Ch to sensitive 4th instar larvae of Culex palustris
[0055] Deltamethrin (ppm) 0 0.001 0.005 0.01 0.05 0.1 0.5
[0056] NYD-Ch(mg / ml) 1 1 1 1 1 1 1
[0057] After 24 hours at 28°C, add the fourth instar larvae
[0058] Fourth instar larva (bar) 50 50 50 50 5...
Embodiment 3
[0098] Example 3 Degradation of deltamethrin by bovine-derived chymotrypsin of the present invention
[0099] In the four samples, 5, 10, 50, 100 U of bovine-derived chymotrypsin (product of Ameresco) and 5 ug of deltamethrin were respectively added to 1 ml with water, and incubated at 29° C. for 24 hours. Add 1ml of acetone to the sample solution, and vortex for 30s. Add 2ml of cyclohexane to mix well, extract, centrifuge at 2,000rpm / min for 10min, absorb the supernatant; after repeated extraction with cyclohexane twice, combine all the supernatants, and dry the solvent with nitrogen in a 60°C water bath. Infiltrate and dissolve the residue with 0.2ml cyclohexane, and measure the content of deltamethrin by chromatography with 0.6ul injection volume. It is found that the degradation rate of deltamethrin has a good correlation with the enzyme concentration in the range of 5-100U . GC / MS analysis showed that the expressed protein NYD-Ch could degrade deltamethrin into Br2CA an...
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