High-activity methyl parathion hydrolase mutant and use thereof
A methyl parathion, mutant technology, applied in the biological field, can solve problems such as poisoning, and achieve the effects of high degradation rate, high catalytic activity and performance
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Embodiment 1
[0035] Error-prone PCR ( error-prone PCR ) and mutant screening:
[0036] Directed evolution experiments were performed using the parental plasmid as a template. Error-prone PCR primers were designed using Primer prime software. PCR system 50ul.
[0037] Primer sequence (the underlined part is restriction site BamH I and Xho I):
[0038] Upstream Primer-5'-ACTAT GGATCC GGCCGCACCGCAGG-3'
[0039] Downstream Primer-5'-TCCC CTCGAG CTTGGGGTTGAC-3'
[0040] Error-PCR reaction system:
[0041]
[0042] PCR conditions:
[0043]
[0044] After the PCR product was purified, it was ligated into the pET21b plasmid vector after double digestion, and transformed into BL21(DE3) competent cells. After the library strains were grown on the LB+Amp resistant plate for 10 hours, a single clone was transferred in parallel to two LB+Amp resistant solid plates in sequence, and cultured at 37°C for 12 hours, and then the Pour a layer of pre-cooled screening plate medium on one o...
Embodiment 2
[0047] Site-directed mutagenesis based on structure-function analysis design:
[0048] Based on the structure-function relationship, the analysis found that the area where the negative charge is concentrated is the active site, including two Zn ion binding sites and the substrate binding site. The negative charge is conducive to the occurrence of nucleophilic reaction in the hydrolysis reaction. Therefore, site-directed mutagenesis was designed to achieve an increase in the affinity of the methyl parathion hydrolase mutant to the substrate by enhancing the interaction of the substrate molecule with the phenyl ring and phosphate ends of the methyl parathion hydrolase protein, thereby enhancing the mutation The purpose of high body activity. And in order to facilitate site-directed derivation of PEG, a group of cysteine mutants were designed, and point mutations were carried out using the full-form Fast Mutagenesis System kit. The mutants and corresponding primers are as fo...
Embodiment 3
[0052] Fermentation and purification of methyl parathion hydrolase mutants:
[0053] The mutants that passed the screening were fermented and purified. Add antibiotics and trace elements at the beginning of fermentation, let it grow naturally at 37°C, use a lower initial medium concentration, so that the growth rate can be carried out according to the expected ratio during the fermentation process, the growth rate is natural during the initial culture, and the wet weight reaches 50g / L Then start to add ammonia water, the flow acceleration rate is determined based on the specific growth rate = 0.1, and no longer add yeast extract. Add trace elements into the sugar solution and add them together with the feed. When the wet weight is between 50g / L and 70g / L, induce at 30°C. Induced by low concentration IPTG (0.1mM), the specific growth rate was controlled at 0.05 at the initial stage of induction. When the wet weight > 120, the rate of ammonia supplementation will not be cha...
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