A construction method of recombinant dextran sucrase Escherichia coli suitable for diglycoside transfer function
A technology for dextran and transfer functions, which is applied in the field of construction of recombinant dextran sucrase Escherichia coli, can solve the problems of cumbersome separation and purification process, low yield and the like, and achieves the effects of reducing production cost, high conversion rate, and simple separation and purification
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] A method for constructing a recombinant dextran sucrase Escherichia coli suitable for diglycoside transfer function, using the P473S / P856S double mutant thermostable dextran sucrase gene as a template, mutating asparagine at position 555 to glutamine and then recombining transformation The recombinant dextran sucrase Escherichia coli BL21(DE3) / dex-YG-TrMU202001 genetically engineered bacteria obtained from BL21(DE3) Escherichia coli with suitable diglycoside transfer function. The BL21(DE3) / dex-YG-TrMU202001 genetically engineered bacterium is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and is classified as suitable for diglycoside transfer. Functional recombinant dextran sucrase Escherichia coli BL21 (DE3), the preservation date is August 10, 2020, and the preservation number is CGMCC No.20509.
[0035] The construction method specificall...
Embodiment 2
[0051] Expression of Recombinant Dextran Sucrase Escherichia coli BL21(DE3) / dex-YG-TrMU202001 Genetic Engineering Bacteria Suitable for Diglycoside Transfer Function
[0052] Inoculate the genetically engineered bacteria BL21(DE3) / dex-YG-TrMU202001 into LB medium containing 40-60 μg / ml kanamycin at an inoculum size of 0.5%, rotate at 250r / min, and cultivate at 37°C for 14 hours; Take 2 mL of the above culture solution and add it to 200 mL of medium A, culture on a shaker at 37°C, when the enriched culture solution is diluted 10 times with distilled water, the OD 600 At 0.20-0.24 hours, 500 μL IPTG can be added to induce enzyme production. After induction and fermentation at 25°C for 3.5-4 hours, it is broken and centrifuged, and the samples taken are analyzed by SDS-PAGE electrophoresis. The expressed protein of recombinant dextran sucrase The molecular weight is about 170KDa, consistent with the predicted value. Wherein, each liter of the A medium contains 5g glycerol, 5g gl...
Embodiment 3
[0054] The fermentative production of recombinant dextran sucrase Escherichia coli BL21(DE3) / dex-YG-TrMU202001 genetically engineered bacteria suitable for diglycoside transfer function specifically includes the following steps:
[0055] Inoculate the genetically engineered bacteria BL21(DE3) / dex-YG-TrMU202001 into LB medium containing 40-60 μg / ml kanamycin at a volume fraction of 0.5%, at a speed of 250 r / min, and cultivate at 37°C 14 hours; draw 2 mL from the above culture solution and add it to 200 mL of A medium, place it on a shaker at 37°C for culture, when the enriched culture solution is diluted 10 times with distilled water, the OD 600 At 0.20~0.24, add 500μL IPTG to induce enzyme production, keep at 25°C to induce fermentation for 3.5~4 hours, centrifuge the bacterial suspension after induction fermentation at 0°C, 6000r / min for 12~15min, and centrifuge The tube corresponds to a bottle of bacterial suspension, then add distilled water to shake and wash, and centrifug...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More