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Method for enzymatic degradation of hyaluronic acid or salt thereof

A technology of hyaluronidase and hyaluronic acid, which is applied in the field of hyaluronic acid or its salt enzyme degradation, can solve the problems of hyaluronidase activity reduction, bovine serum albumin is expensive, and cannot be applied on a large scale, so as to improve The effect of enzymatic efficiency

Active Publication Date: 2020-10-16
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experiments have shown that adding non-catalytic proteins, such as bovine serum albumin (BSA), to the hyaluronic acid hydrolysis solution, these proteins can compete with hyaluronidase, thereby liberating the hyaluronidase in the enzymatic hydrolysis solution, Restore the activity of hyaluronidase, but the high concentration of BSA will also lead to the reduction of hyaluronidase activity
In addition, bovine serum albumin is expensive and cannot be widely used in the production process of hyaluronic acid enzymatic hydrolysis

Method used

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  • Method for enzymatic degradation of hyaluronic acid or salt thereof
  • Method for enzymatic degradation of hyaluronic acid or salt thereof
  • Method for enzymatic degradation of hyaluronic acid or salt thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. To 1.5m 3 Add 1m to the stainless steel dissolution tank 3 Purify water, heat up to 40°C, adjust the pH to 6.0, set the stirring speed to 20rpm, and add 400g of glucose and 50g of glucose oxidase to the dissolution tank sequentially while stirring.

[0049] 2. After stirring for 3 minutes, slowly add 300 kg of sodium hyaluronate with a molecular weight of 1000 kDa, and stir for 0.5 to 1 hour after the hyaluronic acid is completely added.

[0050] 3. Add 8.0×10 to the sodium hyaluronate solution 10 IU of hyaluronidase until the enzymatic hydrolysis reaches the desired molecular weight, and the enzymatic hydrolysis ends.

[0051] 4. Add ethanol to the enzymolysis solution until the alcohol content of the enzymolysis solution reaches 25%, stir for 0.5-1 hour, filter, and collect the filtrate.

[0052] 5. Add 20 times the volume of ethanol to the filtrate. At this time, the alcohol content of the filtrate is about 92%, and sodium hyaluronate is precipitated. The prec...

Embodiment 2

[0055] 1. To 1.5m 3 Add 1m to the stainless steel dissolution tank 3 Purify water, heat up to 40°C, adjust the pH to 6.0, set the stirring speed to 20rpm, and add 200g of glucose and 30g of glucose oxidase to the dissolution tank sequentially while stirring.

[0056] 2. After stirring for 3 minutes, slowly add 100 kg of calcium hyaluronate with a molecular weight of 2500 kDa, and stir for 0.5 to 1 hour after the hyaluronic acid is completely added.

[0057] 3. Add 5.0×10 to the sodium hyaluronate solution 11 IU of hyaluronidase until the enzymatic hydrolysis reaches the desired molecular weight, and the enzymatic hydrolysis ends.

[0058] 4. Add ethanol to the enzymolysis solution until the alcohol content reaches 20%, stir for 0.5-1 hour, filter, and collect the filtrate.

[0059] 5. Add 3 times the volume of ethanol to the filtrate, the alcohol content of the filtrate is about 76% at this time, and sodium hyaluronate is precipitated. The precipitate is dehydrated with et...

Embodiment 3

[0062] 1. To 1.5m 3 Add 1m to the stainless steel dissolution tank 3 Purify water, heat up to 40°C, adjust the pH to 6.0, set the stirring speed to 20rpm, and add 100g of glucose and 10g of glucose oxidase to the dissolution tank sequentially while stirring.

[0063] 2. After stirring for 3 minutes, slowly add 50 kg of potassium hyaluronate with a molecular weight of 3000 kDa, and stir for 0.5 to 1 hour after the hyaluronic acid is completely added.

[0064] 3. Add 5.0×10 to the sodium hyaluronate solution 8 IU of hyaluronidase until the enzymatic hydrolysis reaches the desired molecular weight, and the enzymatic hydrolysis ends.

[0065] 4. Add ethanol to the enzymolysis solution until the alcohol content reaches 35%, stir for 0.5-1 hour, filter, and collect the filtrate.

[0066] 5. Add 5 times the volume of ethanol to the filtrate, and the alcohol content of the filtrate is about 80% at this time, to obtain sodium hyaluronate precipitation. The precipitate is dehydrated...

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Abstract

The invention relates to a method for enzymatic degradation of hyaluronic acid or a salt thereof. The method comprises the following steps: preparing a substrate solution: mixing glucose oxidase, glucose, hyaluronic acid or a salt thereof and water to generate the substrate solution; performing enzymolysis: adding hyaluronidase or chondroitinase into the substrate solution for enzymolysis to obtain enzymatic hydrolysate; and performing purification: inactivating the enzymatic hydrolysate to obtain low-molecular hyaluronic acid or oligomeric hyaluronic acid or a salt of the low-molecular hyaluronic acid or oligomeric hyaluronic acid. According to the method, glucose oxidase is added, so that the activity of hyaluronidase is improved, oxygen in the hyaluronic acid / salt enzymatic hydrolysateis removed, oxidative browning is reduced or prevented, meanwhile, growth of bacteria in the enzymatic hydrolysate is remarkably inhibited, no preservative needs to be additionally added in the enzymolysis process, and the quality of the low-molecular hyaluronic acid or the oligomeric hyaluronic acid or the salt thereof is improved.

Description

technical field [0001] The present application generally relates to the field of preparation and purification of hyaluronic acid, and specifically relates to a method for enzymatic degradation of hyaluronic acid or its salt. Background technique [0002] Hyaluronic acid (HA for short), also known as hyaluronic acid or hyaluronic acid, is composed of (1-3)-2-acetylamino-2-deoxy-D-glucose (1-4)-D-glucuronic acid disaccharide High-molecular-weight linear mucopolysaccharides composed of repeating units widely exist in the vitreous body, skin, and umbilical cord of humans and animals, and are indispensable key functional substances in the human body. [0003] Hyaluronic acid has many different functions according to its molecular weight. Usually, we define hyaluronic acid with molecular weight ≥1000KDa as high molecular hyaluronic acid, hyaluronic acid with molecular weight 10KDa~1000KDa as low molecular hyaluronic acid, and hyaluronic acid with molecular weight ≤10KDa as oligom...

Claims

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Application Information

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IPC IPC(8): C12P19/26C12N9/99
CPCC12P19/26C12N9/99
Inventor 乔莉苹王珊珊石艳丽郭学平李德杰于鲁孟周宁
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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