A kind of recombinant Bacillus subtilis with high chondroitin production and its application

A technology of Bacillus subtilis and chondroitin, applied in the field of bioengineering, can solve problems such as high cost, low yield, and inability to meet market demand, and achieve the effect of increasing production and sufficient supply

Active Publication Date: 2019-08-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The method has low yield and high cost, and a large amount of industrial wastewater produced by alkaline hydrolysis has adverse effects on the environment
At the same time, the shortage of raw materials and the complexity of the downstream purification process limit the acquisition of chondroitin sulfate, which cannot meet the growing market demand
Moreover, in the long run, the safety management of animal-derived drugs is becoming increasingly stringent, and the extraction of chondroitin sulfate from animals may be restricted from the crude drug market

Method used

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  • A kind of recombinant Bacillus subtilis with high chondroitin production and its application
  • A kind of recombinant Bacillus subtilis with high chondroitin production and its application
  • A kind of recombinant Bacillus subtilis with high chondroitin production and its application

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Experimental program
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Effect test

Embodiment 1

[0028] Construction of the recombinant Bacillus subtilis of embodiment 1 co-expression pathway gene

[0029] First prepare B.subtilis E168C competent cells, the required reagent is inorganic salt mother solution (g / L): K 2 HPO 4 140, KH 2 PO 4 60, (NH 4 ) 2 SO 4 20, (Na 3 C 6 h 5 o 7 2H 2 O)10, MgSO4 7H 2 O2; GMI solution (every 100mL): inorganic salt mother solution 9.6mL, 20% glucose 2.5mL, 5% hydrolyzed casein 0.4mL, 10% yeast juice 1mL; GMII solution (every 100mL): inorganic salt mother solution 9.7mL, 20 % glucose 2.5mL, 5% hydrolyzed casein 0.08mL, 10% yeast juice 0.04mL, 1M MgCl 2 0.25mL, 1M CaCl 2 0.05mL. Inoculate a single colony of B.subtilis E168C in 5mL of GMI solution, culture at 30°C and 125rpm for 16h, transfer 2mL of culture solution to 18mL of GMI solution, culture at 37°C and 200rpm for 3.5h, then transfer 10mL of culture solution to 90mL of GMII solution , cultured at 37°C and 200rpm for 90min, centrifuged at 4°C and 5000g for 10min to collect...

Embodiment 2

[0040] Shake flask fermentation of embodiment 2 recombinant bacillus subtilis

[0041] Single colonies of the 4 strains of recombinant Bacillus subtilis constructed above and the control bacterium B. subtilis E168C were picked respectively, inoculated in the seed medium, and cultured overnight at 200 rpm at 37°C for 16 hours. Be 10% inoculum by volume ratio and transfer in 25mL fermentation medium (250mL shaking flask), fermentation medium is: 20g / L yeast powder, 50g / L sucrose, potassium sulfate 3.9g / L, magnesium sulfate 1.5g / L, 50mM phosphate buffer, pH 7.0, in which 50μg / mL kanamycin should be added to the culture medium of recombinant bacteria. Culture at 200rpm at 37°C for 54h, and add xylose mother liquor 2h after inoculation to make the final concentration 20g / L for induction.

[0042] After the fermentation liquid sampled in shake flask culture was centrifuged at 10000g for 5min, add 3 times the volume of absolute ethanol to the supernatant, mix well and place it at 4...

Embodiment 3

[0047] 3L tank fed-batch culture of the recombinant Bacillus subtilis B.subtilis E168C / pP43-DU-PBMS of embodiment 3

[0048] Inoculate a single colony of recombinant bacteria into 150mL seed medium, cultivate at 37°C and 200r / min for 16h, and transfer the seed culture solution to a 3L fermenter containing 1.35L fermentation medium with an inoculum volume of 10% by volume. And add 50 μg / mL kanamycin. Add xylose solution 2h after inoculation to make the final concentration 20g / L for induction. Use 5M NaOH solution to control pH to 7.0, temperature to 37°C, stirring speed to 600r / min within 8h after inoculation, 800r / min after 8h, and ventilation to 2.0vvm. The feeding material liquid is 800g / L sucrose mother liquor, and when the sucrose concentration in the fermentation liquid is lower than 5g / L, start feeding, and maintain the residual sugar concentration at 0-5g / L. During 8-12h, the flow rate was 7.5, 7.5, 15, 10g / L / h, respectively, and thereafter the flow rate of 5g / L / h was...

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Abstract

The invention discloses a recombinant bacillus subtilis for highly producing chondroitin and application of the recombinant bacillus subtilis, belonging to the technical field of bioengineering. By optimizing synthetic routes of the recombinant bacillus subtilis for producing the chondroitin, namely carrying out coexpression on key enzyme genes in a UDP-GlcUA route and a UDP-GlcNAc route, the yield of the chondroitin is increased. The recombinant bacillus subtilis provided by the invention can meet the requirements of medical and health services and food safety, and the endotoxin and pathogenic infection risks are avoided; the accumulation of the chondroitin can be maximum, namely 6.06g / L, and the molecular weight is 78.64KD. According to the research of the invention, a new strategy is provided for the high production of the chondroitin, on one hand, the recombinant bacillus subtilis can be applied to other glycosaminoglycans, and on the other hand, a foundation is laid for the industrial large-scale production of the chondroitin.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis with high chondroitin production and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (chondroitin sulfate) is a member of the glycosaminoglycan (GAG) family, which is formed by sulfuration at carbon 4 or carbon 6 of GalNAc after the sugar chain of precursor chondroitin is generated. . Chondroitin is a kind of 4-GlcA-β-1,3-GalNAc-β-1 (GlcUA: glucuronic acid, GalNAc: acetylgalactosamine) disaccharide monomers connected alternately by β-1,3 bonds. into polysaccharides. According to its chemical composition and structural differences, it can be divided into A, B, C, D, E, F, H, etc. CS-A and CS-C are the most common. Chondroitin sulfate generally contains 50-70 disaccharide units, and its molecular weight is between 10,000-50,000 Daltons. Chondroitin sulfate has a variety of pharmacological and physiological functions, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/26C12R1/125
CPCC12N9/0006C12N9/104C12N9/1241C12N9/90C12P19/04C12P19/26C12Y207/07009C12Y504/02002C12Y504/0201
Inventor 康振陈坚堵国成张琳培王浩周正雄
Owner JIANGNAN UNIV
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