Method for synthesizing hyaluronic acid from monosaccharides as initial substrates by multienzyme coupling

A hyaluronic acid and monosaccharide technology, applied in the field of enzymatic synthesis of hyaluronic acid and multi-enzyme coupling synthesis of hyaluronic acid, can solve the problems of in-depth enzymatic synthesis of hyaluronic acid, cumbersome product purification steps, Acid pollution and other problems, to achieve the effect of mild conditions, easy operation, and reduce production costs

Inactive Publication Date: 2015-02-18
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Potential problems with hyaluronic acid contamination in animal tissue extraction, such as residual animal viruses
The second is the production of hyaluronic acid by microbial fermentation. Microbial fermentation is the main method for hyaluronic acid production at present, but microbial fermentation has problems such as long fermentation period and cumbersome product purification steps.
[0005] The reported enzymatic synthesis of hyaluronic acid is mainly catalyzed by hyaluronan synthase, which requires the use of expensive sugar nucleotides: uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate -Glucuronic acid (UDP-GlcA) as a substrate severely restricts the in-depth development of enzymatic synthesis of hyaluronic acid
[0006] After searching, there are related enzymatic synthesis of sugar nucleotides starting from monosaccharides, coupled with monosaccharides as substrates and hyaluronidase synthesis with sugar nucleotides as substrates, multi-enzyme coupling synthesis of hyaluronic acid The method of acidic acid has not been reported yet

Method used

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  • Method for synthesizing hyaluronic acid from monosaccharides as initial substrates by multienzyme coupling
  • Method for synthesizing hyaluronic acid from monosaccharides as initial substrates by multienzyme coupling
  • Method for synthesizing hyaluronic acid from monosaccharides as initial substrates by multienzyme coupling

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 A method for synthesizing hyaluronic acid by multi-enzyme coupling initiated by monosaccharide

[0040] Proceed as follows:

[0041] (1) Preparation of catalytic reaction enzyme solution

[0042] N-acetylglucosamine kinase, derived from Bifidobacterium longum (Bifidobacterium longum), recombinant vector construction and protein expression methods refer to (Nishimoto, M., and Kitaoka, M. (2007) Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I / galacto-N-biose metabolic pathway in Bifidobacterium longum. Appl Environ Microbiol 73, 6444-6449);

[0043] Glucuronokinase, derived from Arabidopsis thaliana, recombinant expression vector construction and protein expression method see (Pieslinger, A.M., Hoepflinger, M.C., and Tenhaken, R. (2010) Cloning of glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars. J Biol Chem 285,2902-2910);

[0044] N-acetylglucosamine t...

Embodiment 2

[0071] Example 2 A method for synthesizing hyaluronic acid by multi-enzyme coupling initiated by monosaccharide

[0072] The method for the multi-enzyme coupling synthesis of hyaluronic acid initiated by monosaccharide as described in Example 1, the difference is:

[0073] The volume ratio of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) reaction solution and uridine diphosphate-glucuronic acid (UDP-GlcA) reaction solution described in step (6) is 1:0.8 mixing, 28 Incubate in a water bath at ±1°C for 10±2 minutes, and add a hyaluronidase solution of 40% of the volume of the mixture during incubation.

Embodiment 3

[0074] Example 3 A method for synthesizing hyaluronic acid by multi-enzyme coupling initiated by monosaccharide

[0075] The method for the multi-enzyme coupling synthesis of hyaluronic acid initiated by monosaccharide as described in Example 1, the difference is:

[0076] The volume ratio of the uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) reaction solution and the uridine diphosphate-glucuronic acid (UDP-GlcA) reaction solution described in step (6) is 1:1.2 mixing, Incubate in a water bath at 28±1°C for 10±2 minutes, add a hyaluronidase solution with a volume of 60% of the mixture during incubation, and the reaction time is 48 hours.

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Abstract

The invention discloses a method for synthesizing hyaluronic acid from monosaccharides as initial substrates by multienzyme coupling. Two monosaccharides are used as initial substrates to synthesize the hyaluronic acid by coupling of the multienzyme system. The method couples the enzyme-process synthesis of sugar nucleotide by using monosaccharides as the substrates and the hyaluronidase-process synthesis by using the sugar nucleotide as the substrate; the monosaccharides used as the initial substrates are low in price and easy to obtain, thereby avoiding the supply of the expensive sugar nucleotide and greatly lowering the production cost of the enzyme process for synthesizing hyaluronic acid; and meanwhile, the synthesis of hyaluronic acid by multienzyme coupling can effectively avoid the defect of endotoxin residues in the hyaluronic acid microbial fermentation production technique. The method has the advantages of simple technique, mild conditions and no pollution, is easy to operate and suitable for large-scale synthesis of hyaluronic acid, and has wide application prospects.

Description

technical field [0001] The invention relates to a method for enzymatically synthesizing hyaluronic acid, in particular to a method for synthesizing hyaluronic acid by multi-enzyme coupling starting with monosaccharide, and belongs to the technical field of oligosaccharide synthesis and preparation. Background technique [0002] Hyaluronic acid (hyaluronan, hyaluronic acid, HA) is a long-chain acidic mucopolysaccharide formed by repeating disaccharide units. In the disaccharide unit, there is a molecule of N-acetylglucosamine and a molecule of glucuronic acid [-β3- GlcNAc-β1-4-GlcA-1-] link composition. Hyaluronic acid is the simplest glycosaminoglycan in nature. It is not covalently linked to proteins, has no sulfuric acid or isomerization modification, and exists in free form. With its unique molecular structure and physical and chemical properties, hyaluronic acid shows a variety of important physiological functions in the body. In addition, hyaluronic acid can also affe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26
Inventor 房俊强王鹏王帅帅付玄
Owner SHANDONG UNIV
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