Amidase gene dimtH for degrading dimethoate, chlorpropham and propanil amide, and encoded protein and application thereof
A technology of amidase and amidase, which is applied in the fields of application, hydrolase, genetic engineering, etc., and can solve the problems of unreported degradation genes
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Embodiment 1
[0041] (1) Isolation of amide pesticide degrading bacteria Dimd-2 (CCTCC M 2011234)
[0042] The enrichment matrix used to enrich amide pesticide-degrading strains was taken from the activated sludge of the pesticide wastewater biochemical treatment tank of Yangzhou Pesticide Factory. Take 5.0 g of activated sludge and add it to 100 ml of basic salt medium, and add 50 mg L -1 Hexaflumuron, 30°C, 180r·min -1 Cultivate for 7 days, transfer to the same medium with 5% inoculum size, transfer continuously for 3 times, dilute the enrichment solution in gradient, take 10 -4 ~10 -7 Each 0.1mL of the diluted enrichment solution was applied to the 100mg·L -1 On the solid medium plate of hexaflumuron, after culturing at 30°C for 4 days, pick the grown single colony and inoculate it in a medium containing 100mg·L -1 In the culture medium of various amide pesticides (hexaflumuron, dimethoate, propanil and chlorphenamine), 30°C, 180r·min -1 Cultivate on a shaking table for 5 days to ver...
Embodiment 2
[0049] Cloning of embodiment 2 amidase gene dimtH
[0050] (1) Extraction of bacterial genome total DNA
[0051] Strain Dimd-2 (CCTCC M 2011234) was mass-cultured in LB medium, and the high-purity, large-fragment Dimd-2 genomic total DNA was extracted by CTAB method, dissolved in TE buffer (pH8.0), placed in Preserve at -20°C. For specific methods, refer to the "Refined Molecular Biology Experiment Guide" edited by F. Osper et al.
[0052] (2) pUC118(BamHI) was purchased from Bao Biological Engineering (Dalian) Co., Ltd.
[0053] (3) Digestion of total DNA The total DNA of Paracoccus sp.Dimd-2 was partially digested with Sau3AI.
[0054] (4) Recovery of DNA
[0055] The digested total DNA was purified by electrophoresis (TAE buffer) and recovered using axygen biosciences (China) recovery kit. The recovered DNA was dissolved in 10 mmol / L Tris·HCl (pH8.0) and placed in- Store at 20°C.
[0056] (5) enzyme-linked
[0057] Establish the following reaction system:
[0058] ...
Embodiment 3
[0064] Example 3 High-efficiency expression of amidase gene dimtH in BL21 (pET-29a (+)) (see strategy diagram figure 2 )
[0065] (1) PCR amplification of amidase gene dimtH
[0066] With forward primer: 5’-TCTGGA CATATG ATACCGAGACTGACCAACG-3' (SEQ ID NO.3) and reverse primer: 5'-TCTGGA GAATTC GCCTTCCATAAGAGCGCCGATAGC-3' (SEQ ID NO.4) was used as a primer to amplify the amidase gene dimtH sequence from the total DNA of Paracoccus sp.Dimd-2 by PCR.
[0067] Amplification system:
[0068]
[0069] PCR amplification program:
[0070] a. Denaturation at 95°C for 3 minutes;
[0071] b. Denaturation at 95°C for 1.5min, annealing at 53°C for 0.5min, extension at 72°C for 1.5min, and 25 cycles;
[0072] c. Extend at 72°C for 10 minutes and cool to room temperature.
[0073] (2) The PCR product was double-digested with NdeI and EcoRI.
[0075]
[0076] In a water bath at 37°C, react for more than 3 hours. The digested products were rec...
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