Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof
A technology of organophosphorus pesticides and random mutation, which is applied in the field of genetic engineering, can solve problems such as no results, and achieve the effects of not easy inactivation, high degradation efficiency, excellent enzyme activity and thermal stability
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Embodiment 1
[0027] Embodiment 1: the expression of organophosphorus pesticide degrading enzyme of the present invention in Escherichia coli
[0028] (1) PCR amplification: use the upstream primer 5'-ATTCATATGGCCGCACCGCAGGTG-3' and the downstream primer 5'-TAACTCGAGCTTGGGGTTGACGACCG-3' to PCR amplify the organophosphorus pesticide degrading enzyme gene of the present invention.
[0029] The reaction conditions for PCR (50 μL system) are: 50ng of the organophosphorus pesticide degrading enzyme gene (SEQ ID No.1) of the present invention as a template, 0.3 μM each of the upstream primer and downstream primer, 200 μM each of dNTPs, 5U Taq DNA polymerase, 5 μL PCR buffer solution, 30 cycles, denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and the last cycle of extension at 72°C for 10 min. The composition of PCR buffer is: 100mM Tris-HCl pH8.3, 500mM KCl and 15mMMgCl 2 , the solvent is deionized water.
[0030] (2) Con...
Embodiment 2
[0076] Embodiment 2: the mensuration of organophosphorus pesticide degradation specific activity of the present invention
[0077] Using methyl parathion and chlorpyrifos as substrates respectively, the specific activity of the organophosphorus pesticide degrading enzyme of the present invention and the wild-type enzyme is determined.
[0078] The production method of the wild-type enzyme is as follows: the organophosphorus pesticide degrading enzyme gene mpd (GenBank accession number: JQ686087) is used as a PCR template, and other operating steps are completely carried out according to the method in Example 1.
[0079] With methyl parathion as substrate, the assay method of specific activity is as follows:
[0080] (1) Preparation of p-nitrophenol standard solution: using 20 μM Tris-HCl (pH 8.0) buffer as solvent, prepare p-nitrophenol concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively. Phenol standard solution.
[0081] (2) Drawing of p-nitrophenol...
Embodiment 3
[0109] Embodiment 3: Determination of thermal stability of organophosphorus pesticide degrading enzyme of the present invention
[0110] The purified organophosphorus pesticide-degrading enzyme and the wild-type enzyme obtained in Example 1 at a concentration of 100 μg / ml were respectively incubated at 40°C, 42.5°C, 45°C, 47.5°C, 50°C, 52.5°C, and 55°C , 57.5°C, 60°C heat treatment for 10min, immediately place it on ice for 30min, use chlorpyrifos as substrate, measure enzyme activity and analyze thermal stability.
[0111] The production method of the wild-type enzyme is as follows: the organophosphorus pesticide degrading enzyme gene mpd (GenBank accession number: JQ686087) is used as a PCR template, and other operating steps are completely carried out according to the method in Example 1.
[0112] The assay method of enzyme activity is as follows:
[0113] (1) Preparation of chlorpyrifos standard solution: using 20 μM Tris-HCl (pH 8.0) buffer solution containing 0.1% (volu...
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