阅读说明：本技术 一种透明质酸或其盐酶降解的方法 (Method for enzymatic degradation of hyaluronic acid or salt thereof ) 是由 乔莉苹 王珊珊 石艳丽 郭学平 李德杰 于鲁孟 周宁 于 2020-06-15 设计创作，主要内容包括：本申请涉及一种透明质酸或其盐酶降解的方法。该方法包括：底物溶液配制：混合葡萄糖氧化酶、葡萄糖、透明质酸或其盐和水以生成底物溶液；酶解：向底物溶液加入透明质酸酶或硫酸软骨素酶进行酶解,得到酶解液；纯化：对酶解液进行灭活得到低分子透明质酸或寡聚透明质酸或其盐。本申请的方法中添加了葡萄糖氧化酶,提高了透明质酸酶的活性,并去除透明质酸/盐酶解液中的氧气,减少或防止氧化褐变的发生,同时显著抑制酶解液中细菌的生长,酶解过程中不用额外添加防腐剂,提高低分子透明质酸或寡聚透明质酸或其盐的品质。(The present application 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 produce a substrate solution; enzymolysis: adding hyaluronidase or chondroitinase sulfate into the substrate solution for enzymolysis to obtain an enzymolysis solution; and (3) purification: inactivating the enzymolysis solution to obtain low molecular hyaluronic acid or oligomeric hyaluronic acid or salt thereof. The method has the advantages that the glucose oxidase is added, so that the activity of the hyaluronidase is improved, oxygen in the hyaluronic acid/salt enzymolysis liquid is removed, the occurrence of oxidative browning is reduced or prevented, the growth of bacteria in the enzymolysis liquid is obviously inhibited, no preservative is 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.)

1. A method for enzymatic degradation of hyaluronic acid or a salt thereof, comprising:

preparing a substrate solution: mixing glucose oxidase, glucose, hyaluronic acid or a salt thereof, and water to produce a substrate solution;

enzymolysis: adding hyaluronidase or chondroitinase into the substrate solution for enzymolysis to obtain an enzymolysis solution;

and (3) purification: inactivating the enzymolysis solution to obtain the low molecular hyaluronic acid or oligomeric hyaluronic acid or salt thereof.

2. The method according to claim 1, wherein the purification step comprises adding ethanol to the enzymatic hydrolysate to obtain a mixed solution, stirring, and filtering to obtain a filtrate containing the low molecular weight hyaluronic acid or the oligomeric hyaluronic acid or the salt thereof;

preferably, the volume percentage concentration of the ethanol in the mixed solution is 20-50%;

more preferably, the volume percentage concentration of the ethanol in the mixed solution is 20-35%.

3. The method of claim 1, further comprising precipitation and dehydration drying after said purification;

preferably, the precipitation is to add an organic solvent into the filtrate, and mix the mixture evenly to obtain the precipitate of the low molecular weight hyaluronic acid or the oligomeric hyaluronic acid or the salt thereof;

preferably, the dehydration drying is to separate the precipitate, and the low molecular weight hyaluronic acid or oligomeric hyaluronic acid or salt thereof is obtained by dehydration drying.

4. The method according to claim 3, wherein the organic solvent is added in an amount of 3 to 20 times the volume of the filtrate in the precipitation.

5. The method according to claim 1, wherein the glucose oxidase is glucose oxidase from any source, preferably glucose oxidase which is commercialized; more preferably food grade or premium grade pure glucose oxidase; glucose oxidase produced by Penicillium is also preferred.

6. The method according to claim 1, wherein the enzyme activity unit of the glucose oxidase is not less than 10 ten thousand U/g;

the addition amount of the glucose oxidase is 10-100 g of the glucose oxidase added into every 1000L of the substrate solution; preferably, 10g to 50g of the glucose oxidase is added into every 1000L of the substrate solution.

7. The method of claim 1, wherein the hyaluronic acid or salt thereof is any high molecular hyaluronic acid or high molecular hyaluronate, any low molecular hyaluronic acid or low molecular weight hyaluronate;

the mass volume concentration of the hyaluronic acid or the salt thereof in the substrate solution is 5-30%.

8. The method according to claim 1, characterized in that the mass volume concentration of the glucose in the substrate solution is between 0.1 and 1%, preferably between 0.1 and 0.4%.

9. The method of claim 1, wherein the hyaluronidase or chondroitinase is a microbial hyaluronidase, a snake venom-derived hyaluronidase, a leech-derived hyaluronidase, a mammalian-derived hyaluronidase, or a chondroitinase; more preferably a hyaluronidase produced by Bacillus (Bacillus sp.); most preferred is hyaluronidase produced by fermentation of Bacillus (Bacillus sp.) A50, CGMCC NO. 5744.

10. The method according to claim 1, wherein the hyaluronidase or chondroitinase is added in an amount of 1 × 10 per 1kg of the hyaluronic acid or salt thereof⁷～5×10⁹IU of said hyaluronidase or chondroitinase.

Technical Field

The application relates to the field of hyaluronic acid preparation and purification, in particular to a method for degrading hyaluronic acid or hyaluronic acid salt by enzyme.

Background

Hyaluronic Acid (HA), also known as Hyaluronic acid or Hyaluronic acid, is a high molecular weight linear mucopolysaccharide composed of (1-3) -2-acetamido-2-deoxy-D-glucose (1-4) -D-glucuronic acid disaccharide repeating units, widely exists in vitreous bodies, skins and umbilical cords of human beings and animals, and is an indispensable key functional substance in human bodies.

Hyaluronic acid has many different functions depending on its molecular weight. Generally, hyaluronic acid with the molecular weight more than or equal to 1000KDa is defined as high molecular hyaluronic acid, hyaluronic acid with the molecular weight between 10KDa and 1000KDa is defined as low molecular hyaluronic acid, and hyaluronic acid with the molecular weight less than or equal to 10KDa is defined as oligomeric hyaluronic acid.

With the reduction of the molecular weight of hyaluronic acid, particularly when the molecular weight is reduced to be below 10KDa, hyaluronic acid is more and more easily oxidized to generate oxidation browning, and finally the color of the hyaluronic acid solution is darkened, and the whiteness of the powder precipitated by alcohol is reduced and yellowed. When the preparation of low molecular and oligomeric hyaluronic acid and the salt thereof by using hyaluronidase or chondroitinase is finished, the hyaluronidase needs to be inactivated. The traditional method is high-pH or high-temperature inactivation, but low-molecular and oligomeric hyaluronic acid and salt thereof are more easily oxidized and browned under the condition of high pH or high temperature. The use of low pH to inactivate hyaluronidase can cause severe corrosion to the equipment used for enzymolysis. How to perform antioxidant protection on hyaluronic acid in the enzymolysis and enzyme inactivation processes of hyaluronic acid and salt thereof becomes a difficult problem.

In addition, since Lenorm Lenonid reported in 2009 (The hyaluronic-protein complexes at lewis strength strain: How The hyaluronic activity is controlled by The bovine bone repair alum, Matrix Biology Volume 28, Issue6, July 2009, Pages 365-. As the molecular weight of the hyaluronic acid decreases, the complex gradually dissociates, releasing the hyaluronidase. Experiments show that non-catalytic proteins, such as Bovine Serum Albumin (BSA), are added into the hyaluronic acid enzymatic hydrolysate and can generate a competitive effect with hyaluronidase, so that the hyaluronidase in the enzymatic hydrolysate is liberated, and the activity of the hyaluronidase is recovered, but the reduction of the activity of the hyaluronidase can also be caused by the overhigh concentration of the BSA. In addition, bovine serum albumin is expensive and cannot be applied to the hyaluronic acid enzymolysis production process on a large scale.

Disclosure of Invention

The purpose of the present application is to provide a method for enzymatic degradation of hyaluronic acid or a salt thereof.

The application provides a method for enzymatic degradation of hyaluronic acid or a salt thereof, comprising:

preparing a substrate solution: mixing glucose oxidase, glucose, hyaluronic acid or a salt thereof, and water to produce a substrate solution;

enzymolysis: adding hyaluronidase or chondroitinase into the substrate solution for enzymolysis to obtain an enzymolysis solution;

and (3) purification: inactivating the enzymolysis solution to obtain the low molecular hyaluronic acid or oligomeric hyaluronic acid or salt thereof.

Alternatively, according to the above method, the purifying step comprises adding ethanol to the enzymatic hydrolysate to obtain a mixed solution, stirring, and filtering to obtain a filtrate containing the low molecular weight hyaluronic acid or the oligomeric hyaluronic acid or the salt thereof. Preferably, the volume percentage concentration of the ethanol in the mixed solution is 20-50%. More preferably, the volume percentage concentration of the ethanol in the mixed solution is 20-35%.

Optionally, the method further comprises precipitation and dehydration drying after the purification. Preferably, the precipitation is to add an organic solvent to the filtrate and mix the mixture evenly to obtain the precipitate of the low molecular weight hyaluronic acid or the oligomeric hyaluronic acid or the salt thereof. Preferably, the dehydration drying is to separate the precipitate, and the low molecular weight hyaluronic acid or oligomeric hyaluronic acid or salt thereof is obtained by dehydration drying.

Optionally, according to the above method, in the precipitating, the ethanol is added in an amount of 3 to 20 times the volume of the filtrate.

Alternatively, the glucose oxidase is any source of glucose oxidase, according to the method described above. The glucose oxidase is preferably a commercially available glucose oxidase. More preferably food grade or premium grade pure glucose oxidase. Glucose oxidase produced by Penicillium is also preferred.

Optionally, according to the method, the enzyme activity unit of the glucose oxidase is more than or equal to 10 ten thousand U/g.

Optionally, according to the method, the addition amount of the glucose oxidase is 10-100 g of the glucose oxidase per 1000L of the substrate solution. Preferably, 10g to 50g of the glucose oxidase is added into every 1000L of the substrate solution.

Optionally, the hyaluronic acid or salt thereof is any high molecular hyaluronic acid or high molecular hyaluronate, any low molecular hyaluronic acid or low molecular weight hyaluronate according to the above method.

Optionally, according to the above method, the mass volume concentration of the hyaluronic acid or salt thereof in the substrate solution is between 5% and 30%.

Optionally, according to the above method, the mass volume concentration of the glucose in the substrate solution is 0.1 to 1%. Preferably 0.1 to 0.4 per mill.

Optionally, the hyaluronidase or chondroitinase is a microbial hyaluronidase, a snake venom derived hyaluronidase, a leech derived hyaluronidase, a mammalian source hyaluronidase, or a chondroitinase, according to the above-described method. More preferably, the hyaluronidase is produced by Bacillus (Bacillus sp.). Most preferred is hyaluronidase produced by fermentation of Bacillus (Bacillus sp.) A50, CGMCC NO. 5744.

Optionally, according to the above method, the hyaluronidase or chondroitinase is added in an amount of 1 × 10/1 kg of the hyaluronic acid or salt thereof⁷～5×10⁹IU of said hyaluronidase or chondroitinase.

According to the method, the glucose oxidase is innovatively added in the process of preparing the low-molecular and oligomeric hyaluronic acid/salt through hyaluronic acid/salt enzymolysis, so that the hyaluronidase and hyaluronic acid/salt complex is prevented from being generated when the ratio of the hyaluronidase/hyaluronic acid/salt is too low, and the enzymolysis efficiency is improved.

According to the method, the glucose oxidase and the glucose are innovatively added in the process of preparing the low-molecular and oligomeric hyaluronic acid/salt through hyaluronic acid/salt enzymolysis, so that oxygen in enzymolysis liquid is removed, and the oxidation browning of the low-molecular and oligomeric hyaluronic acid/salt is reduced.

After enzymolysis is finished, hyaluronidase and glucose oxidase are inactivated and filtered by low-alcohol content ethanol, and then final low-molecular and oligomeric hyaluronate is obtained through alcohol precipitation and drying, so that the traditional alkaline inactivation or high-temperature inactivation mode is abandoned, and oxidation of low-molecular and oligomeric hyaluronic acid/salt during alkaline inactivation or high-temperature enzyme inactivation is avoided.

Drawings

Fig. 1 is a schematic flow chart of a method for enzymatic degradation of hyaluronic acid or a salt thereof according to an embodiment of the present application.

Detailed Description

The following detailed description of the present application, taken in conjunction with the accompanying drawings and examples, is provided to enable the aspects of the present application and its advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the present application.