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Method for constructing recombinant bacillus subtilis with high-yield hyaluronic acid oligosaccharide

A technology of hyaluronic acid oligosaccharide and Bacillus subtilis is applied in the field of bioengineering, which can solve the problem of low production intensity and achieve the effects of improving production intensity, increasing yield and reducing the phenomenon of bacterial autolysis.

Active Publication Date: 2019-07-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing microorganisms used for the synthesis of hyaluronic acid oligosaccharides have low production intensity (19.5g / L of fermentation 100h yield)

Method used

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  • Method for constructing recombinant bacillus subtilis with high-yield hyaluronic acid oligosaccharide
  • Method for constructing recombinant bacillus subtilis with high-yield hyaluronic acid oligosaccharide
  • Method for constructing recombinant bacillus subtilis with high-yield hyaluronic acid oligosaccharide

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 recombinant plasmid and H6Ha sz / pm Construction of / DUMS / sr5 recombinant bacteria

[0044] 1.1 Integration of expression genes glmM, glmS, glmU, tuaD and leech-derived hyaluronan hydrolase gene H6LHyal

[0045] Select yoyJ-yoyA, lytH, lytD, lytG, and lytC in the Bacillus subtilis genome as integration sites, and integrate the pathway genes glmM, glmS, glmU, tuaD and leech-derived hyaluronan hydrolase gene H6LHyal into these sites, respectively, P 43 A promoter is a promoter.

[0046] Taking the integration of the H6LHyal gene into the lytC site of the genome as an example, the integration method is as follows: first, construct a DNA recombinant fragment lytC(shang)-lox71-zeocin-lox66-P containing 5 modules through homologous recombination 43 -H6LHyal[SP(yweA)]--lytC(xia), which respectively include: ①A DNA fragment larger than 500bp upstream of the lytC site on the genome of B. subtilis168 (using primers lytC(shang)-F / lytC(shang)-R from B. subtilis168 g...

Embodiment 2

[0066] Example 2 H6Ha sz / pm Fermentation results of / DUMS / sr5 recombinant bacteria

[0067] 2.1 Culture method

[0068] Seed culture: inoculate the seeds (using LB medium) in the 50ml shaking tube into a 500mL Erlenmeyer flask containing 50mL seed medium, and add kanamycin at a final concentration of 50μg / ml at the beginning of the cultivation, 5μg / ml Erythromycin, 20μg / ml bleomycin, 25μg / ml chloramphenicol and 1.5mmol / L IPTG inducer, cultured at 37°C, 200r / min for 12-16 hours.

[0069] Shake flask fermentation: Transfer the seed culture solution to a 500mL Erlenmeyer flask containing 50mL fermentation medium according to the inoculation amount of 10%, and add the final concentration of 50μg / ml kanamycin, 5μg / ml erythromycin, 20μg / ml ml bleomycin, 25 μg / ml chloramphenicol and 1.5 mmol / L IPTG inducer, cultured at 37°C and 200 r / min for 24 hours.

[0070] Fermentation in 3L fermenter: put the seed solution into a 3L fermenter containing 1.35L fermentation medium according to ...

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Abstract

The invention discloses a method for constructing recombinant bacillus subtilis with high-yield hyaluronic acid oligosaccharide, and belongs to the technical field of bioengineering. The method includes selecting yoyJ-yoyA, lytH, lytD, lytG and lytC as integration loci, and integrating pathway genes glmM, glmS, glum and tuaD and a leech-derived hyaluronan hydrolase gene H6LHyal into a bacillus subtilis genome, which not only improves the yield of hyaluronic acid oligosaccharide but also increases the biomass and facilitates separation and purification of the hyaluronic acid oligosaccharide inthe later stage. The yield of the hyaluronic acid oligosaccharide reaches 36.04g / L when fermented in a 3L fermentation tank for 40h. The hyaluronic acid at the 3L fermentation tank level has the average molecular weight HA Mw of about 3.3*104 Da. The method lays a foundation for high-efficiency fermentation of a microbial system to prepare the hyaluronic acid oligosaccharide, and is suitable for industrial production application.

Description

technical field [0001] The invention relates to a method for constructing a recombinant Bacillus subtilis with high yield of hyaluronic acid oligosaccharides, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (HA for short), also known as hyaluronic acid, is a mucopolysaccharide widely used in medicine, cosmetics, food and other fields. The biological activity and use effect of HA are directly determined by its relative molecular weight (Mw). Compared with high-molecular-weight HA (molecular weight above 1 million Da), small-molecule hyaluronic acid, such as hyaluronic acid oligosaccharides (molecular weight less than 10,000 Da), has more important physiological activities and special physiological functions. Hyaluronic acid Acid oligosaccharides can penetrate into the dermis, have a slight expansion of capillaries, increase blood circulation, improve intermediary metabolism, and promote skin nutrient absorption. It has a stron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/125
CPCC07K14/32C12N9/1051C12N9/2474C12P19/26C12Y204/01212
Inventor 康振韦朝宝陈坚堵国成
Owner JIANGNAN UNIV
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