A Novel Hyaluronic Acid Fusion Enzyme Anti-feedback Inhibition and Its Preparation Method and Enzyme Cutting Process

A hyaluronic acid, feedback inhibition technology, applied in biochemical equipment and methods, enzymes, introduction of foreign genetic material using vectors, etc., can solve the limitation of hyaluronic acid thorough enzyme digestion preparation and production, animal immune source infection, substrate Low affinity and other problems, to achieve the effect of solving the inhibition of enzyme cleavage substrate, improving the efficiency of enzyme cleavage and high expression efficiency

Active Publication Date: 2022-05-17
NANJING TZONE BIOLOGICAL SCI & TECH
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commercialized hyaluronidase is mainly bovine testicular HAase prepared from bovine testicular tissue, but this product is of poor quality, expensive and has the risk of animal immune source infection
And existing hyaluronan hydrolase also all has a serious defect, and its substrate affinity is low, along with the deepening of enzymatic cleavage, the reduction of hyaluronic acid molecular weight, the binding constant of hyaluronan hydrolase and substrate reduces, The reduction of enzyme cleavage rate, this phenomenon is also called feedback inhibition of enzyme cleavage substrates, which severely limits the complete enzyme digestion of hyaluronic acid and the preparation and production of high-purity hyaluronic acid oligosaccharides

Method used

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  • A Novel Hyaluronic Acid Fusion Enzyme Anti-feedback Inhibition and Its Preparation Method and Enzyme Cutting Process
  • A Novel Hyaluronic Acid Fusion Enzyme Anti-feedback Inhibition and Its Preparation Method and Enzyme Cutting Process
  • A Novel Hyaluronic Acid Fusion Enzyme Anti-feedback Inhibition and Its Preparation Method and Enzyme Cutting Process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This embodiment is a preparation method of a novel hyaluronic acid fusion enzyme resistant to feedback inhibition. The novel hyaluronic acid fusion enzyme is obtained by recombining the gene encoding hyaluronan-binding protein and the gene encoding hyaluronan hydrolase into yeast for fusion expression, and the hyaluronic acid-binding protein part can be combined with the substrate hyaluronic acid The specific binding is beneficial to the function of the hyaluronan hydrolase part, solves the problem of the inhibition of the enzyme cleavage substrate existing in the hyaluronidase catalyzed reaction, and significantly improves the enzyme cleavage efficiency.

[0043] In this example, Saccharomyces cerevisiae is used as the host, and the genes of hyaluronan-binding protein and leech hyalurolase are introduced into the host through genetic engineering technology, and the repeated amino acid sequences of 9 glycines and serines (9 GS sequences) are used as Linker Linking bindi...

Embodiment 2

[0055] This example is to investigate the effect of the addition amount of the inducer galactose on the induced expression of the novel hyaluronic acid fusion enzyme. The fusion enzyme gene sequence construction and plasmid recombination were carried out according to the preparation method in Example 1, and the obtained recombinant plasmid was introduced into Saccharomyces cerevisiae, and then inoculated into a 500 mL conical flask containing 50 mL of YPD medium. The speed of the shaker was 200 r / min, and the culture was carried out for 24 hours to obtain the seed solution of the expression bacteria; then the seed solution of the expression bacteria was transferred to a 10L shake flask containing 3L YPD medium according to the volume concentration of 10%, and continued at 32 °C, 200 r / Fermentation was carried out for about 10 hours under the condition of 7 When cells / ml, different concentrations of galactose were added overnight at 30°C to induce the expression of the novel ...

Embodiment 3

[0060] In this example, the enzymatic activity of the novel hyaluronic acid fusion enzyme prepared in Example 1 was detected by the plate diffusion method. The principle of plate diffusion detection of enzyme activity is: using the permeability of agarose plate, the permeated enzyme solution undergoes enzymatic hydrolysis reaction with macromolecular hyaluronic acid in the plate, and using cetylpyridine chloride and macromolecular hyaluronic acid. The non-colored part (hydrolyzed transparent circle) can be visually seen in the color development, so as to judge the enzymatic activity of hyaluronic acid.

[0061] In this example, the novel hyaluronic acid fusion enzyme was introduced into Saccharomyces cerevisiae after the recombinant plasmid constructed by linking the hyaluronic acid-binding protein and the leech hyaluronic acid hydrolase gene according to the preparation method in Example 1, containing 50 mL of YPD In a 500 mL Erlenmeyer flask of culture medium, at a temperatu...

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Abstract

The invention discloses a novel hyaluronic acid fusion enzyme that is anti-feedback inhibition and its preparation method and enzymatic cleavage process. In the present invention, for the first time, the hyaluronic acid binding protein coding gene and the leech hyaluronan hydrolase coding gene are fused and expressed in yeast A novel hyaluronic acid fusion enzyme was obtained. The hyaluronic acid binding protein is connected with the leech hyaluronan hydrolase through a Linker formed by repeating amino acid sequences of 9 glycines and serines, and fused with 6 histidine sequences as tags. The novel hyaluronic acid fusion enzyme of the present invention, its hyaluronic acid binding protein part can specifically bind to the substrate hyaluronic acid, which is beneficial to the function of the hyaluronic acid hydrolase part, and solves the feedback inhibition of hyaluronidase catalytic reaction Problems, significantly improved enzyme cutting efficiency, has good industrial production value and economic value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to hyaluronan hydrolase, in particular to a novel hyaluronan fusion enzyme with anti-feedback inhibition, a preparation method and an enzyme cutting process. Background technique [0002] Hyaluronic acid (HA), also known as hyaluronic acid, is an acidic mucopolysaccharide polymerized with D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAC) as disaccharide units. D-glucuronic acid and N-acetylglucosamine are linked by β-1,3-glycosidic bonds, and the disaccharide units are linked by β-1,4-glycosidic bonds. Commercial HA often exists in the form of sodium salt, which is white powder or fibrous solid. Hyaluronan oligosaccharides (o-Has) refers to oligosaccharides with relative molecular mass less than 10KDa formed by the degradation of HA, including hyaluronic acid tetrasaccharide (o-Ha4), hyaluronic acid hexasaccharide (o-Has) -Ha6), hyaluronic acid octasaccharide (o-Ha8), hyaluronic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C07K14/47C12N15/81C12N15/62C12N15/66C12P19/26C12P19/14
Inventor 徐林金玉徐念沁徐松泉鲁锦标常开金曹晶晶戴超
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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