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

Active Publication Date: 2019-07-02
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When producing dextran by direct fermentation, the composition of the fermentation liquid is complex, resulting in difficulties in product separation, difficulty in controlling the molecular size of dextran, and many impurities such as nucleoproteins. However, the use of pure enzymes to catalyze the preparation of dextran can overcome these shortcomings and produce Dextran of high product quality

Method used

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  • A kind of dextran sucrase and its application
  • A kind of dextran sucrase and its application
  • A kind of dextran sucrase and its application

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses dextransucrase. An amino acid sequence of the dextransucrase is shown as SEQ ID NO.2, and a nucleotide sequence encoding the enzyme (the dextransucrase) is shown as SEQ ID NO.1. A genetically engineered bacterium containing a dextransucrase gene is obtained by constructing recombinant plasmid by virtue of the dextransucrase gene and then transforming the recombinant plasmid into a host bacterium. The invention also provides an application of the dextransucrase provided by the invention in preparing insoluble glucan by catalyzing sucrose, wherein the dextransucrase, under a special catalysis condition, can effectively catalyze the sucrose so as to prepare the insoluble glucan, and meanwhile, the yield of the glucan can reach 90% or above.

Description

technical field [0001] The invention relates to the fields of genetic engineering and enzyme engineering, in particular to a novel dextran sucrase and a method for catalyzing and preparing water-insoluble glucan. Background technique [0002] Dextran (Glucan) is a polymer with glucose as a monomer, which is widely used in the fields of medicine, fine chemical industry, petroleum exploration, food, cosmetics, etc. According to the type of glycosidic bond, it can be divided into α, glucan sugars and beta-glucans. Among α-glucans, dextran (dextran) is widely used in research. Dextran has a relatively high molecular weight and is mainly connected by D-glucopyranose with α, 1-6 bonds. The branch point has α, 1- 2. Formed by 1-3, 1-4 glycosidic linkages. With the different types of microorganisms and growth conditions, their structure is also different. Due to the difference in structure, dextran can be divided into water-insoluble dextran and water-soluble dextran. Water-inso...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/53C12N15/70C12N1/21C12P19/18C12P19/08C12R1/19
CPCC12N9/1051C12N15/70C12N2800/101C12P19/08C12P19/18C12Y204/01005
Inventor 姜岷张敏古蕾孔祥平马江锋董维亮吴昊
Owner NANJING TECH UNIV
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