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Method for removing hybrid protein from lumbrokinase crude product

A technology for removing impurity proteins and lumbrokinase, applied in the field of separation, can solve the problems of not taking into account the extraction and separation of active components, complex operation, large loss of enzymes, etc., and achieve precise control of operating parameters and less loss of enzyme activity. , reduce the effect of protein denaturation

Inactive Publication Date: 2006-04-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent 00132716.X discloses a separation process of lumbrokinase, including salt treatment, washing, adding buffer, pulping, centrifugation, affinity chromatography, ion exchange chromatography and other steps. The process is complicated to operate and the loss of enzyme larger
And this separation process does not take into account the extraction and separation of other active components in earthworms

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Take an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 30% (volume percentage) and a crude enzyme concentration of 4% (mass percentage), and repeatedly stir to dissolve and filter out insolubles. Then 20 mL of the solution was added to an autoclave preheated to 25 °C. Then, pressurized CO with a preheating temperature of 25°C was introduced. 2 Stir at the same time to increase the pressure to 7.0MPa, and adjust the operating temperature to 25°C. At this time, the pH of the solution is 4.4, and the precipitation phenomenon in the kettle is obvious. Keep it under high pressure for 40 minutes. Precipitation is complete, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 98.6%, and the purification factor of this process is 2.53. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, forming pressurized CO 2 - Ethanol-wa...

Embodiment 2

[0012] Example 2: Take an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 40% (volume percentage) and a crude enzyme concentration of 6% (mass percentage), stir repeatedly to dissolve, and filter out insolubles. Then 20 mL of the solution was added to an autoclave preheated to 25 °C. Then, pressurized CO with a preheating temperature of 25°C was introduced. 2 Stir at the same time to increase the pressure to 4.5MPa, and adjust the operating temperature to 25°C. The precipitation phenomenon in the kettle is obvious. Keep it under high pressure for 55 minutes. The impurity protein in the crude product of lumbrokinase is completely precipitated under this condition, while the active ingredient remains. remain in solution. Then the precipitate is separated, the enzyme activity yield is 94.1%, and the purification factor of this process is 2.41. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, fo...

Embodiment 3

[0013] Example 3: Take an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 35% (volume percentage) and a crude enzyme concentration of 2% (mass percentage), stir repeatedly to dissolve, and filter out insolubles. Then 20 mL of the solution was added to an autoclave preheated to 20°C. Then, pressurized CO with a preheating temperature of 20°C was introduced. 2 Stir at the same time to increase the pressure to 3.5MPa, and adjust the operating temperature to 20°C. The precipitation phenomenon in the kettle is obvious. Keep it under high pressure for 30 minutes. The foreign protein in the crude product of lumbrokinase is completely precipitated under this condition, while the active ingredient is still remain in solution. Then the precipitate is separated, the enzyme activity yield is 97.9%, and the purification factor of this process is 1.11. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, form...

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PUM

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Abstract

The invention discloses a method for removing impurity proteins from crude lumbrokinase. The steps of the method are as follows: 1) First, dissolve the crude lumbrokinase in an ethanol-water solution, prepare a solution with a concentration of 2-7% by mass of the crude enzyme and 0-40% by volume of ethanol, and filter out after repeated stirring and dissolving Insoluble matter; 2) Then, take 20mL of the above mixed solution and add it into an autoclave with a preheating temperature of 20-40°C, and then pass it into a pressurized CO with a preheating temperature of 20-40°C. 2 Stir at the same time to increase the pressure to 3.5-8.5MPa, adjust the pH of the solution to 4.4, and keep it under high pressure for 20-60 minutes. The foreign protein in the crude product of lumbrokinase is completely precipitated under this condition, while the active ingredient remains in the solution. , and then separate the precipitate. The invention has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and the loss of enzyme activity is small. It is a method for separating and purifying lumbrokinase with precisely controlled operating parameters and no fluctuations in the operating process.

Description

technical field [0001] The present invention relates to a separation method, in particular to a separation method using pressurized CO 2 A method for separating and purifying lumbrokinase by isoelectric precipitation of impurity proteins as a volatile acid and ethanol as an auxiliary precipitating agent. Background technique [0002] Lumbrokinase (lumbrokinase) is a general term for multi-component proteins extracted from earthworms with fibrinolytic enzyme activity, and it is one of the most promising fibrinolytic drugs at present. Existing literature shows that lumbrokinase is a group of serine proteases with a molecular weight of 20-40 kD and activity of kinase and plasmin, and the isoelectric points of most of the active components are concentrated below 4.0. In addition, earthworms also contain some other biologically active ingredients, so it is of great significance to separate and purify lumbrokinase in a mild and environmentally friendly way for the separation of v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64
Inventor 姚善泾关怡新齐祥明
Owner ZHEJIANG UNIV