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: 2005-03-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 othe

Method used

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Examples

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Embodiment 1

[0011] The technical solutions of the present invention will be further specifically described below through specific examples. 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. Th...

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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Abstract

The process of removing hybrid protein from crude lumbrokinase product includes the following steps: dissolving crude lumbrokinase product in alcohol-water solution to compound solution with coarse lumbrokinase concentration of 2-7 wt% and alcohol concentration of 0-40 vol% via stirring and filtering out insoluble matter; and adding 20 ml of the said mixed solution into high pressure reactor preheated to 20-40 deg.c, introducing CO2 of 20-40 deg.c temperature and 3.5-8.0 MPa pressure, regulating pH to 4.4, maintaining for 20-60 min to deposit hybrid protein while maintaining the active component in the solution, and separating the precipitate. The present invention has the features of mild operation condition, capacity of obtaining other active components, less loss of enzyme activity, etc. and the process has precisely controlled operation parameters and no fluctuation in the operation 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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IPC IPC(8): C12N9/64
Inventor 姚善泾关怡新齐祥明
Owner ZHEJIANG UNIV
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