A high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria
A technology of genetically engineered bacteria and high-density fermentation, which is applied in the field of high-density fermentation of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria, can solve the problems of large equipment investment, low efficiency and high cost, and achieves simplified fermentation equipment and improved production. Efficiency, the effect of reducing the amount of three wastes
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Embodiment 1
[0038] The mensuration of embodiment 1 bacterial classification and enzymatic activity
[0039] (1) Strain construction
[0040] The strain used in this example is: E. coli BL21(DE3) / pET-30a(+)-NHaseP, a genetically engineered strain expressing nitrile hydratase derived from Bordetella petrii DSM12804. For the specific method of genetically engineered bacteria construction, please refer to the invention patent application document with the application publication number CN104498466A entitled "Nitrile Hydratase and Its Application". The base sequence of the nitrile hydratase gene in the genetically engineered bacteria is shown in SEQ ID NO.1.
[0041] Genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-pENHase-1229 expressing nitrile hydratase derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A), the base of nitrile hydratase gene in genetically engineered bacteria The base sequence is shown in SEQ ID NO.2. The construction method of gen...
Embodiment 2
[0062] 10L fermentation of embodiment 2 genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseP
[0063] (1) Strain activation: use an inoculation loop to take the genetically engineered bacteria E.coli BL21(DE3) / Streak the pET-30a(+)-NHaseP strain on the surface of LB-Kan-agar solid medium (plate or eggplant bottle), place the plate upside down in a constant temperature incubator at 37°C, and cultivate for 12 hours;
[0064] (2) Seed cultivation: re-inoculate the activated strains into 40mL of primary seed culture medium, shake and culture at 37°C for 12 hours to obtain primary seed liquid; then, transfer the primary seed liquid into 400mL of secondary seed culture medium with shaking at 35°C for 4 hours to obtain a secondary seed liquid;
[0065] Among them, the primary seed medium is LB medium, 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 5M sodium hydroxide solution to adjust the pH value to 7.0, sterilized at 121°C for 20min, and inoculated Befo...
Embodiment 3
[0076] 15L fermentation of embodiment 3 genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-pENHase-1229
[0077] (1) Strain activation: use an inoculation loop to take the genetically engineered strain E.coli BL21 (DE3) expressing nitrile hydratase derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A) preserved in a -80°C glycerol tube in the strain tube / pET-30a(+)-pENHase-1229 strains were drawn on the surface of LB-Kan-agar solid medium (plate or eggplant bottle), and the plate was placed upside down in a constant temperature incubator at 37°C for 12 hours;
[0078] (2) Seed cultivation: re-inoculate the activated strains into 50 mL of primary seed culture medium, shake and culture at 37°C for 12 hours to obtain primary seed liquid; then, transfer the primary seed liquid into 500 mL of secondary seed culture medium with shaking at 35°C for 4 hours to obtain a secondary seed liquid;
[0079] Among them, the primary seed medium is LB med...
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