Method utilizing oxygenase to oxidize amidogen
An amino oxidation and oxygenase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, can solve the problems of difficult control of the synthesis process, high risk factor, and high cost. Achieve the effects of safe and green reaction, simple production process and mild reaction process
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[0025] Example 1: Construction of E. coli engineered bacteria expressing nitrogen oxygenase gene
[0026] The 3 types are derived from Streptomycesthioluteus Nitrogen oxygenase gene aurF From Streptomycesvenezuelae Nitrogen oxygenase gene cmlI And from Pseudomonasfluorescens Nitrogen oxygenase gene prnD NdeI and XohI restriction sites are added at both ends, and the gene fragments are inserted into the corresponding restriction sites of expression vector pET22b through double restriction digestion and ligation, and placed under the control of the T7 promoter to construct the expression of AurF, CmlI, PrnD recombinant plasmid pET22b-AurF, pET22b-CmlI, pET22b-PrnD. Transform pET22b-AurF, pET22b-CmlI, and pET22b-PrnD into E. coli BL21 (DE3), respectively, to obtain engineered bacteria E. coli BL21 (DE3) / pET22b-AurF, E. coli BL21 (DE3) / pET22b-AurF, E. coliBL21(DE3) / pET22b-CmlI, E.coliBL21(DE3) / pET22b-PrnD.
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[0027] Example 2: Fermentation of E. coli engineered bacteria to produce enzyme
[0028] Pick engineering bacteria separately E.coli BL21(DE3) / pET22b-AurF, E.coli BL21(DE3) / pET22b-CmlI, E.coli BL21(DE3) / pET22b-PrnD to LB liquid medium containing 100μg / mL ampicillin. After incubating overnight at 37℃ and 200rpm, inoculate 1% (v / v) inoculum into LB liquid medium (containing 100μg / mL). mL ampicillin), culture at 37°C and 200rpm until the OD600 is 0.6~0.8 (about 3h), add the inducer IPTG, and induce expression for 6 hours. Centrifuge the bacterial solution at 8000rpm and 4°C for 5min, discard the supernatant, and precipitate for later use.
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[0029] Example 3: Preparation of E. coli crude enzyme solution expressing nitrogen oxygenase
[0030] Take the bacterial pellet in Example 2 and wash it twice with potassium phosphate buffer (100mmol / L, pH7.2). The washed pellet is suspended in potassium phosphate buffer, and the cells are treated with ultrasound (130W, ultrasound for 2s, intermittent 2s, 10min in total), the broken liquid is at 8000rpm, 4 ℃ Centrifuge for 20 minutes and take the supernatant to obtain the crude enzyme solution.
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