Method for preparing genetically engineered bacteria for efficiently compounding pantothenic acid and application thereof
A technology of genetically engineered bacteria and pantothenic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of low enzyme activity, substrate toxicity, high production cost, etc., achieve high yield, short fermentation time, highly active effect
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[0017] Example 1
[0018] Construction of genetically engineered bacteria
[0019] They were selected from Escherichia coli BL21 (DE3), Corynebacterium glutamicum ATCC 13032, Bacillus subtilis subsp.subtilis str.168, and Bacillus cereus E33L. Enterobacter cloacae EcWSU1, Bacillus thuringiensis BMB171, and E. coli high-efficiency expression system BL21(DE3) / PET30 were used to construct genetically engineered bacteria.
[0020] Using E.coli BL21(DE3) panc gene (sequence table 1) as template, PCR amplification was performed with primers E-panc-for and E-panc-rev to obtain the fragment panC E , The PCR product panC E The fragment was recovered by 1% agarose electrophoresis, and after double digestion with restriction enzymes BamH 1 and Xho 1, it was ligated to the same digested vector pET30 to obtain the recombinant plasmid pET30-panC E , Transform E. coli DH5α competent cells, screen positive recombinants by colony PCR, double digestion with restriction enzymes BamH 1 and Xho1 to identi...
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[0029] Example 2
[0030] Enzyme activity detection of genetically engineered strains
[0031] Pick E.coli BL21(DE3) / pET30-panC separately from the plate E , E.coli BL21(DE3) / pET30-panC C , E.coli BL21(DE3) / pET30-panC b , E.coli BL21(DE3) / pET30-panC bc , E.coli BL21(DE3) / pET30-panC ec And E.coli BL21(DE3) / pET30-panC bt A single colony was inoculated into 5 mL of LB medium (containing 50 μg / ml Kan) and cultured overnight at 37°C. Transfer the above culture medium 1:100 to 20 mL of LB medium (containing 50 μg / ml Kan), culture at 37°C, and when the OD reaches 0.4-0.6, add 0.2mM IPTG and culture at 30°C for 16 hours. Collect 10OD bacteria, use 1ml buffer (100mM Hepes, 20mM MgCl 2 .6H 2 O, 1mM EDTA, pH8.0) suspended, then ultrasonically disrupted the bacterial cells, centrifuged and diluted the supernatant 10 times. Take 5μL of diluted liquid and add 1.1mL reaction buffer (25mMβ-alanine, 25mM DL-pantoic acid, 4.5mM ATP, 10mM MgCl 2 , 15mM KCl). At 37°C, react for 20 minutes, centr...
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[0037] Example 3
[0038] High density fermentation
[0039] (1) Pick the engineered strain E.coli BL21(DE3) / PET30-panC containing the pantothenate synthase of Corynebacterium glutamicum from the plate C A single colony was inoculated in 10mL of LB liquid medium ((yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L)) and cultured overnight at 37°C;
[0040] (2) Take 3mL and transfer to 100mL high-density fermentation medium (glucose 20g / L, (NH 4 ) 2 SO 4 9g / L, Na 2 CO 3 2g / L, KH 2 PO 4 6.67g / L, (NH 4 ) 2 HPO 4 4g / L, MgSO 4 ·7H 2 O 0.8g / L, citric acid 0.8g / L, NaHCO 3 2g / L, ion stock solution 5mL, pH 7.0), 50μg / ml Kanamycin (Kan), incubate at 37°C for 12h;
[0041] (3) Take 60ml from the 100ml medium in the second step, transfer it to a 1L fermentor containing 600ml of fermentation medium at 1:10, and cultivate with a dissolved oxygen control of 15% and cultivate at 30℃; the 8th hour of fermentation Add feed (glucose, 650g / L); at the 9th hour of fermentation, add IPTG (final con...
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