Gene engineering bacterium for producing glucosamine and application thereof
A technology of genetically engineered bacteria and glucosamine, applied in bacteria, microorganism-based methods, microorganisms, etc., can solve the problems of low conversion efficiency of chitin hydrolysis method, not yet reached industrialized production, low fermentation yield, etc., and achieves low production cost, The effect of high production intensity and short fermentation time
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Embodiment 1
[0015] Example 1: E. coli K-12- glmS build method
[0016] Design primers:
[0017] Upper primer: 5'-C GA GCT C AT GTG TGG AAT TGT TGG C-3' (underlined sequences indicate restriction endonuclease recognition sites Sac I)
[0018] Lower primer: 5'-CCC AAG CTT TTA CTC AAC CGT AAC CGA-3' (underlined sequence indicates restriction enzyme site Hind III)
[0019] glucosamine synthase gene glmS is to use E. coli BL21(DE3) genome (GenBank No. CP001509.3) was used as a template for PCR. restriction endonuclease Sac I and Hind Ⅲ pair glmS The gene fragment and pET-28a(+) were digested, and ligated glmS Insert the gene fragment into the plasmid pET-28a(+) Sac I and Hind Between the Ⅲ sites, the recombinant plasmid pET-28a(+)- glmS . The recombinant plasmid pET-28a(+)- glmS convert E. coli K-12 (ATCC No: 25947), obtained recombinant Escherichia coli E. coli K-12- glmS .
Embodiment 2
[0020] Embodiment 2: the detection method of glucosamine;
[0021] Glucosamine standard curve drawing: Accurately weigh 0.0100 g of glucosamine, add 20.0 ml of distilled water to prepare a 0.5000 g / l glucosamine solution, and then dilute to concentrations of 0.2500 g / l, 0.1250 g / l, and 0.0625 g / l , 0.0313 g / l solution. Take 0.5 ml of glucosamine solution of corresponding concentration in a glass stoppered test tube, add 1.0 ml of acetylacetone reagent, treat in 90°C water bath for 1 hour, cool to room temperature, slowly add 10.0 mL of 96% (v / v) ethanol (do not stir), Then add 1.0 ml of DMAB reagent and mix well. Because the reaction system changes from alkaline to acidic, attention should be paid to the generation of a large amount of carbon dioxide gas. After mixing, it was left at room temperature for 1 hour to be stable, and the color was measured at 530nm. Draw the standard curve with the sample concentration as the abscissa and the OD value as the ordinate.
[0022] ...
Embodiment 3
[0023] Example 3: E. coli K-12- glmS fermentation method
[0024] Escherichia coli cultured with LB slant medium E. coli K-12- glmS After culturing for 12 h, use an inoculation loop to take 1 loop and insert it into a 250 mL Erlenmeyer flask containing 20 mL of seed medium for seed cultivation. The seed medium is: Seed medium: 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol, kanamycin is added at 50 μg / ml, and the pH is natural. The incubation time was 12 h, the temperature was 37°C, and the shaking speed was 200 rpm.
[0025] Insert 5% of the inoculum into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium for fermentation. Fermentation medium: 12 g of peptone, 24 g of yeast extract, 10 g of glucose, and kanamycin was added at 50 μg / ml at the same time, and the pH was natural. The incubation time was 12 h, the temperature was 37°C, the shaker speed was 200 rpm, and the OD 600 When it reached 0.8, lactose was added at a concentration of 10...
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