A kind of dextran sucrase and its application
A technology of dextran sucrase and dextran, which is applied in the fields of genetic engineering and enzyme engineering, can solve problems such as difficult product separation, many impurities in cell nuclei proteins, and difficulty in controlling the size of dextran molecules, and achieves a broad application prospect. Effect
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Embodiment 1
[0032] Example 1 Screening and identification of strains producing dextran sucrase
[0033] Step 1: Take 1 mL of kimchi fermentation liquid in 9 mL of sterilized saline, and dilute to 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 , take 0.1mL of each gradient dilution solution into the selective plate, culture at 28°C for 2~3d, after the colony grows, pick a typical colony, and streak on the MRS plate containing vancomycin (30μl / mL) , placed in a 28°C incubator for anaerobic culture for 2-3 days, and observed the growth of the colony. If the colony grows, then further select a single colony and culture it on the MRS solid medium containing vancomycin (30 μl / mL), and repeat After 2 to 3 times of the above steps, the strains confirmed as pure cultures by microscopic examination and with typical Leuconostoc cell morphology were transferred to MRS solid medium, and stored at low temperature for later use.
[0034] Step 2: Inoculate the strains isolated in step 1 on the surface of the sol...
Embodiment 2
[0035] Example 2 Construction of Recombinant Escherichia coli Producing Glucan Sucrase
[0036] Step 1: Leuconostoma enterica Leuconostoc mesenteroides M8 was activated by test tube culture. The liquid volume in the test tube was 5 mL, the inoculum volume was 1%, the culture temperature was 30°C, and the culture time was 10-12 h. The vigorous growth of the strain was obtained, and the bacteria sludge was obtained by centrifugation at 10,000 rpm. Gram-positive bacteria extraction genome kit to extract Leuconostoc enterica Leuconostoc mesenteroides The genome of M8 was used as a PCR template.
[0037] Step 2: Dextran Sucrase Dsr Acquisition of U gene and preparation of linearized cloning vector
[0038]Upstream primers:
[0039] 5'-CGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTC-3'
[0040] Downstream primers:
[0041] 5'-ACGGAGCTCGAATTCTTATATAGTATTTAAACGCTGTCTCTCTC-3'
[0042] by Leuconostoc mesenteroides The genomic DNA of M8 was used as a template, and PCR amplification ...
Embodiment 3
[0048] Example 3 Constructing strains to ferment and prepare dextran sucrase Dsr u
[0049] Seed medium (g / L): yeast powder 5, peptone 10, NaCl 10, pH 7.0, sterilized at 121°C for 15 min.
[0050] Fermentation medium (g / L): yeast powder 24, peptone 12, KH 2 PO 4 2.31,K 2 HPO 4 16.43, glucose 10, sterilized at 121°C for 15 min.
[0051] Production method: Take a single colony on the plate and inoculate it into a test tube with a liquid volume of 5 mL, and incubate at 30°C and 200 rpm for 12 hours. Prepare the primary seed solution, inoculate the primary seed solution into a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, culture at 200 rpm, 30°C for 0–4 h, and then add 0.1–1.0 mM IPTG to induce Dsr For the expression of the target protein, continue to culture at 25~30°C for 24 hours. After stopping the fermentation, centrifuge the culture solution at 10,000 r / min for 10 minutes, discard the supernatant, resuspend the pellet with pH 6.2 phosphate buffer...
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