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Method for producing and preparing ultra-high molecular weight hyaluronic acid by microbial fermentation method

A technology for ultra-high molecular weight and hyaluronic acid, which is applied in the field of production and preparation of ultra-high molecular weight hyaluronic acid, which can solve the problems of high cost and difficult methods for purifying hyaluronic acid, so as to increase the molecular weight, easy to control the fermentation process, and widely used foreground effect

Active Publication Date: 2022-01-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ultra-high molecular weight hyaluronic acid obtained by artificial extraction is far from meeting the needs of the society, and the method of purifying hyaluronic acid is more difficult and costly
Streptococcus zooepidemicus can cause various inflammations and sepsis, and has certain risks in terms of industrial production safety.

Method used

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  • Method for producing and preparing ultra-high molecular weight hyaluronic acid by microbial fermentation method
  • Method for producing and preparing ultra-high molecular weight hyaluronic acid by microbial fermentation method
  • Method for producing and preparing ultra-high molecular weight hyaluronic acid by microbial fermentation method

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Embodiment 1

[0031] Embodiment 1: low-temperature fermentation of recombinant Corynebacterium glutamicum produces ultra-high molecular weight hyaluronic acid

[0032] Step 1: Construct recombinant plasmid to express Pasteurella multocida derived hyaluronan synthase pmHasA

[0033] According to the genome sequence of Pasteurella multocida, the hyaluronic acid synthase gene pmHasA was synthesized by GENEWIZ (Suzhou). The sequence of pmHasA is shown in Gene ID: 11805012. Using the synthesized pmHasA gene sequence as a template, primers pmhasA-F and pmhasA-R were designed for PCR amplification, and then the pmHasA gene sequence was connected to the plasmid pXMJ19 by the T5 exonuclease-dependent assembly (TEDA) method to construct the recombinant expression plasmid pXMJ19- pm has A.

[0034] Table 1 constructs the primers used for the recombinant plasmid expressing hyaluronan synthase

[0035]

[0036] Step 2: construct the recombinant plasmid that RBS weakens and expresses hyaluronan synt...

Embodiment 2

[0070] Embodiment 2: Recombinant Corynebacterium glutamicum fermentation tank horizontal low-temperature fermentation produces ultra-high molecular weight hyaluronic acid

[0071] 1. Medium

[0072] Fermentation medium: corn steep liquor dry powder 20g / L, (NH4) 2 SO 4 20g / L, KH 2 PO 4 1g / L, K 2 HPO 4 1g / L, MgSO 4 0.25g / L, MOPS (3-morpholine propanesulfonic acid) 4.2g / L, glucose concentration 40g / L.

[0073] 2. Culture method

[0074] Pick the monoclonal cells from the plate, place them in a shaking tube filled with 5mL of BHIS medium, and culture them overnight at 28°C and 220rpm.

[0075] with initial OD 600 Take the seed liquid cultivated overnight for an inoculum size of 0.1, and transfer it to a baffle shake flask containing 25 mL of fermentation medium. OD 600 After rising to 10-15, transfer to a 3L fermenter with 10% inoculum, so that the initial OD of the bacteria in the fermenter 600 1-1.5. After culturing at 28℃, 220rpm for 3h 600 After rising to abo...

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Abstract

The invention discloses a method for producing and preparing ultra-high molecular weight hyaluronic acid through a microbial fermentation method, and belongs to the technical field of bioengineering. According to the invention, a hyaluronic acid synthase encoding gene pmhasA is expressed in corynebacterium glutamicum in a free manner, and a synthetic route of hyaluronic acid is constructed; the expression of synthase pmHAS is weakened by adopting a method for weakening the strength of RBS; the hyaluronic acid synthetase encoding gene pmhasA is modified by adopting a strategy of site-specific mutagenesis; in the corynebacterium glutamicum, a synthetic pathway enzyme encoding gene of precursor UDP-glucuronic acid and UDP-N-acetylglucosamine is expressed in an enhanced manner through a combined regulation strategy; a fermentation process is optimized, and a low-temperature fermentation strategy is adopted; and the molecular weight of the hyaluronic acid produced by the recombinant corynebacterium glutamicum reaches 5-9 MDa. The recombinant corynebacterium glutamicum constructed by the invention is suitable for industrial production and application.

Description

technical field [0001] The invention relates to a method for producing ultra-high molecular weight hyaluronic acid by microbial fermentation, and belongs to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (Hyaluronan, HA) is a macromolecular acidic mucopolysaccharide widely present in the extracellular space of microorganisms, composed of (1-β-4)D-glucuronic acid (1-β-3)N-acetyl- A linear polyanionic electrolyte composed of disaccharide repeating units of D-glucosamine. The viscous physical properties of HA make it an important role in ophthalmic surgery, arthritis treatment, and cosmetic manufacturing. Hyaluronic acid is needed for the structure of cell tissue, water regulation of extracellular fluid, lubrication and wound healing. [0003] Compared with ordinary molecular weight hyaluronic acid, ultra-high molecular weight hyaluronic acid (molecular weight higher than 6MDa) has more important physiological functions. It has a more ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/15
CPCC12P19/26C12N9/1051C12N9/1096C12N9/0006C12N9/90C12N9/1241C12N9/1029C12Y204/01212C12Y101/01022C12Y206/01016C12Y504/02002C12Y207/07009C12Y203/01008Y02A50/30
Inventor 康振王阳堵国成汪昊陈坚
Owner JIANGNAN UNIV
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