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Extraction method for hyaluronidase

A technology of hyaluronidase and acid solution, applied in the biological field, can solve the problem that the potency can only reach 320iu/mg, and achieve obvious economic and social benefits, high potency, and low yield

Active Publication Date: 2013-04-24
QINGDAO JIULONG BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wang Yan, Zhou Shumin, etc. once published a paper called "Research on Hyaluronidase Extraction Technology" in "Heilongjiang Medicine" in Volume 15 (1) in 2002. The traditional process of extracting hyaluronidase from sheep testis and the new process improved by them on this basis, but its potency can only reach 320iu / mg. On the basis of the traditional process, the company changed the formula of acid extraction, etc. technology, the potency of hyaluronidase reached 340iu / mg

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1 extraction:

[0023] Weigh 5 kg of minced pulp, add 6.5 L of acid solution, stir and leach for 4 hours, filter the leachate, and adjust the pH of the filtrate to 3.20.

[0024] Add 1.2 L of acid solution to the filter residue, stir for 1 hour, then filter again, and adjust the pH of the filtrate to 3.18.

[0025] 2 crude:

[0026] Combine 7.8 L of the two filtrates, add 1638 g of solid ammonium sulfate under continuous stirring, let it stand for 12 hours after completely dissolving, filter, and leave the solid. Measure 6.6 L of the filtrate, add 1914 g of ammonium sulfate under continuous stirring, let it stand for 12 hours after completely dissolving, filter, and combine the two solids to obtain 646.35 g of crude product.

[0027] 3 refined:

[0028] 3.1 Dialysis

[0029] The crude product was dissolved in a small amount of water, adjusted to pH 4.5, wrapped in a dialysis bag, and dialyzed in phosphate-citric acid buffer for 24 hours.

[0030] 3.2 Pyrogen treatm...

Embodiment 2

[0035] 1 extraction:

[0036] Weigh 400 g of minced pulp, add 0.5 L of acid solution, stir and leach for 4 hours, filter the leachate, and adjust the pH of the filtrate to 3.96.

[0037] Add 0.1 L of acid solution to the filter residue, stir for 1 hour, then filter again, and adjust the pH of the filtrate to 4.05.

[0038] 2 crude:

[0039] Combine 618mL of the two filtrates, add 130g of solid ammonium sulfate under constant stirring, let it stand for 12 hours after completely dissolving, filter, and leave the solid. Measure 523 mL of the filtrate, add 152 g of ammonium sulfate under continuous stirring, let it stand for 12 hours after completely dissolving, filter, and combine the two solids to obtain 52.13 g of crude product.

[0040] 3 refined:

[0041] 3.1 Dialysis

[0042] The crude product was dissolved in a small amount of water, adjusted to pH 4.5, wrapped in a dialysis bag, and dialyzed in phosphate-citric acid buffer for 24 hours.

[0043] 3.2 Pyrogen treatment:...

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PUM

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Abstract

The invention provides an extraction method for hyaluronidase. Acid liquid is adopted to extract and ammonium sulfate is adopted to salt out in order to obtain a hyaluronidase crude product; and a hyaluronidase competitive product is obtained by implementing dialysis, pyrogen treatment and freeze-drying for the crude product. The method chooses sheep testis as a raw material, and adopts the high-speed centrifugal extraction method to separate and purify the hyaluronidase, so that the titer of the hyaluronidase can achieve 340 iu / mg.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting hyaluronidase from fresh animal testes. Background technique [0002] Hyaluronidase, also known as hyaluronidase, is abundant in the testes of mammals. It can hydrolyze hyaluronic acid and significantly reduce its viscosity, which is beneficial for sperm to enter the egg during fertilization. It also exists in sperm, submandibular gland, bee venom, snake venom, skin, spleen, leech and lysosome of cells. [0003] The stability of hyaluronidase is good, and the activity will not be lost when heated at 42°C for 60 minutes; 80% of the activity can be retained when heated at 100°C for 5 minutes. Cooling after heat inactivation can restore some vitality. The enzyme is still relatively stable at pH below 5 or above pH 8. It is easy to inactivate in low-concentration aqueous solution, but it can be protected by adding 0.2% or 0.5% gum arabic or 0.2% gelatin. Fe 2...

Claims

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Application Information

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IPC IPC(8): C12N9/42
Inventor 刘乃山宋超龙刘翠珍朱琳
Owner QINGDAO JIULONG BIO PHARMA
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