A method for preparing hyaluronidase using porcine testis

A hyaluronidase and testis technology, applied in the biological field, can solve the problems of cumbersome extraction steps, long time and low yield, and achieve the effects of short extraction time, improved product activity and yield, and high extraction efficiency.

Active Publication Date: 2013-01-02
西藏天虹科技股份有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing hyaluronidase from bovine testis through process optimization, so as to overcome the problems of cumbersome extraction steps, long time and low yield in the existing process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Use fresh bovine testicles as raw material, remove the inner and outer epididymis, wash with distilled water, twist into minced pulp; add NaCl extract with a concentration of 6%, the ratio of bovine testicles to extract is 1:10, adjust the pH to 4.5, and the temperature to 35 ℃, 40min under 40W ultrasonic intensity, the specific activity of hyaluronidase reaches 283U / mg; the extract is centrifuged at 3000r / min for 15min to remove impurities; The temperature is 30°C, the elution flow rate is 25mL / h, and the elution is 5h; the chromatographic solution is freeze-dried to obtain the finished product. Hyaluronidase product activity 874.3U / mg, yield 9.28%.

Embodiment 2

[0027] Use fresh bovine testicles as raw materials, remove the inner and outer epididymis, wash with distilled water, and twist into a slurry; add NaCl extract with a concentration of 7%, the ratio of bovine testicles to extract is 1:11, adjust the pH to 4.5, and the temperature to 35 ℃, extracted under 40W ultrasonic intensity for 40min, the specific activity of hyaluronidase reached 279.3U / mg; the extract was centrifuged at 3000r / min for 15min to remove impurities; the dextran gel was swollen and packed into a column, eluted with deionized water, and washed The elution temperature is 30°C, the elution flow rate is 25mL / h, and the elution is 5h; the chromatographic solution is freeze-dried to obtain the finished product. Hyaluronidase product activity 869U / mg, yield 9.25%.

Embodiment 3

[0029] Use fresh bovine testicles as raw material, remove the inner and outer epididymis, wash with distilled water, twist into minced pulp; add NaCl extract with a concentration of 6%, the ratio of bovine testicles to extract is 1:10, adjust the pH to 4.5, and the temperature to 35 ℃, 45W ultrasonic intensity for 40min extraction, the specific activity of hyaluronidase reached 286.8U / mg; the extract was centrifuged at 3000r / min for 15min to remove impurities; the dextran gel was swollen and packed into a column, eluted with deionized water, and washed with The elution temperature is 30°C, the elution flow rate is 25mL / h, and the elution is 5h; the chromatographic solution is freeze-dried to obtain the finished product. Hyaluronidase product activity 892.1U / mg, yield 9.31%.

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Abstract

The invention discloses a method for preparing hyaluronidase using porcine testis, which includes the steps of using fresh porcine testis as raw material, removing internal and external epididymis, mashing into slurry, performing ultrasonic extraction, centrifugating, subjecting the extract to a Sephadex G-75 chromatography, and freeze-drying to obtain pure product of hyaluronidase. The product has an activity above 700U / mg and a yield up to 9.2%, thereby greatly improving the product quality. The process has the advantages of high purity, high yield, high safety, no pollution, and low time consumption, is suitable for industrialized production, and can meet the need of international research and development.

Description

technical field [0001] The invention belongs to the field of biotechnology and provides a method for preparing hyaluronidase from bovine testes. Background technique [0002] In 1928, Duran Reynals discovered hyaluronidase and called it a diffusion factor. In 1940, Chain and Duthie named it hyaluronidase (HAase). Hyaluronidase is a decomposing enzyme of mucopolysaccharides, which can promote the skin to absorb nutrients such as water, trace elements, vitamins, etc., and can increase the permeability of tissues, thereby promoting the subcutaneous infusion and local accumulation of exudate or blood. Absorption; in terms of biochemistry, this enzyme can be used for the identification of hyaluronic acid, sodium hyaluronate and chondroitin sulfate, etc. [0003] At present, hyaluronidase used in the market is mostly extracted from the testes of mammals. The general process for extracting hyaluronidase from the testes of cattle (sheep) is as follows: after the testes of cattle ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26
Inventor 高聚霞
Owner 西藏天虹科技股份有限责任公司
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