CN1587398A - Method for removing hybrid protein from lumbrokinase crude product - Google Patents

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

Method for removing impurity protein from lumbrokinase crude product

technical field

The invention relates to a separation method, in particular to a method for separating and purifying lumbrokinase by isoelectric precipitation of miscellaneous proteins using pressurized _CO2 as a volatile acid and ethanol as an auxiliary precipitating agent.

Background technique

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 various nutritional and pharmaceutical active ingredients. At present, lumbrokinase is a rough processed product obtained by crushing fresh wormwood, centrifuging to collect the supernatant, then salting out the supernatant to obtain a precipitate, and then desalting and freeze-drying [1]. 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.

references

[1] Wang Dongpeng, Sun Guibao, Research on Extraction Technology of Highly Active Lumbrokinase, Ningxia Science and Technology, 2001, (6), 44-45

Contents of the invention

The purpose of the present invention is to provide a method for removing impurity proteins from crude lumbrokinase.

The steps of the method are as follows:

1) First, dissolve the crude product of lumbrokinase in an ethanol-water solution, prepare a solution with a crude enzyme concentration of 2 to 7% (mass percentage), and an ethanol concentration of 0 to 40% (volume percentage), and repeatedly stir to dissolve and filter out the insoluble thing;

2) Then, take 20mL of the above mixed solution and put it into an autoclave with a preheating temperature of 20-40°C, and then feed in pressurized _CO2 with a preheating temperature of 20-40°C and a pressure of 3.5-8.5MPa while stirring and adjusting The pH of the solution is 4.4, and it is kept under high pressure for 20 to 60 minutes. The impurity proteins in the crude lumbrokinase are completely precipitated under this condition, while the active components still remain in the solution, and then the precipitate can be separated.

Compared with the traditional isoelectric precipitation method, the present invention has less loss of enzyme activity, and has more advantages than the traditional isoelectric precipitation system, such as relatively mild operating conditions, and can simultaneously obtain multiple active ingredients in crude lumbrokinase . In addition, the addition of ethanol can facilitate the precipitation of highly water-soluble proteins, and the amount of ethanol added as an auxiliary agent is much less than that of the organic solvent precipitation method, so the loss of enzyme activity is very small. The use of pressurized _CO2 to adjust the pH can avoid the situation of excessive acid or low local pH when adjusting the pH of the solution with inorganic acids, etc., and reduce the protein denaturation caused by it. Since CO ₂ mostly exists in the form of molecules in the solution, the ionic strength of the solution will not increase too much, so it is more suitable for pH adjustment in isoelectric precipitation than inorganic acids. The invention solves the technical problems existing in the prior art such as harsh operating conditions, inability to simultaneously obtain other active components in earthworm enzymes, and large enzyme loss, and provides a relatively mild operating condition that can simultaneously obtain other active components. The invention discloses a method for separating and purifying lumbrokinase with substantially no loss of enzyme activity. The invention also solves the technical problems in the prior art that the operating conditions are difficult to be accurately controlled and the operating process fluctuates greatly, and provides a method for separating and purifying lumbrokinase with precisely controlled operating parameters and no fluctuations in the operating process.

Detailed ways

The present invention adopts pressurized CO ₂ as volatile acid and ethanol as precipitant aid to form electroprecipitation technology such as pressurized CO ₂ -ethanol-water system protein; the separation and purification steps include the preparation of lumbrokinase ethanol-water solution, using pressurized CO _2. Adjust the pH of the solution to the required range, filter the precipitated miscellaneous protein, and lumbrokinase still remains in the filtrate. In order to realize this process, first prepare ethanol-water solution of appropriate concentration of lumbrokinase, then take an appropriate amount of solution into the autoclave, and then pass in pressurized CO ₂ to adjust the pH of the solution to the required range, because lumbrokinase The precipitation pH of some kinase-inactive proteins in the crude product is between 3.0 and 5.0, but lumbrokinases do not precipitate in this range, so when the pH of the solution is adjusted to this range with CO ₂ , the kinase-inactive proteins in lumbrokinase It will precipitate out, so as to achieve the effect of separation and purification. The pressurized CO ₂ -ethanol-water system used in the present invention is used for isoelectric precipitation of proteins, and the autoclave preheating temperature and CO ₂ preheating temperature of the separation and purification method of lumbrokinase are 20-40°C, and the CO ₂ pressure is 3.5 ～8.5MPa, the mass percent concentration of cellulase is 2～7%, the volume percent concentration of ethanol solution is 0～40%, and the _CO2 pressure maintenance time is 20～60 minutes.

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 while stirring to increase the pressure to 7.0MPa, and the operating temperature was adjusted to 25°C. At this time, the pH of the solution was 4.4, and precipitation in the kettle was obvious. Minutes, the impurity proteins in the crude lumbrokinase were completely precipitated under this condition, while the active ingredient remained in the solution. Then the precipitate is separated, the enzyme activity yield is 98.6%, and the purification factor of this process is 2.53. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

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 feed _pressurized CO with a preheating temperature of 25°C while stirring to increase the pressure to 4.5 MPa, and adjust the operating temperature to 25°C. The precipitation phenomenon in the kettle is obvious, and the impurity in the crude product of lumbrokinase is kept at high pressure for 55 minutes. The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 94.1%, and the purification factor of this process is 2.41. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

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 feed _pressurized CO with a preheating temperature of 20°C while stirring to increase the pressure to 3.5 MPa, and adjust the operating temperature to 20°C. Precipitation in the kettle is obvious. Keep it under high pressure for 30 minutes. Impurities in the crude product of lumbrokinase The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 97.9%, and the purification factor of this process is 1.11. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

Embodiment 4: Weigh an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 0% (volume percentage) and a crude enzyme concentration of 5% (mass percentage), and stir repeatedly to dissolve and then filter out the insoluble matter. Then 20 mL of the solution was added to an autoclave preheated to 40°C. Then feed _pressurized CO with a preheating temperature of 40°C while stirring to increase the pressure to 8.5 MPa, and adjust the operating temperature to 40°C. The precipitation phenomenon in the kettle is obvious. Keep it under high pressure for 20 minutes, and the impurities in the crude product of lumbrokinase The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 98.1%, and the purification factor of this process is 1.29. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

Example 5: 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 3% (mass percentage), stir repeatedly to dissolve, and filter out insolubles. Then 20 mL of the solution was added to an autoclave preheated to 35 °C. Then feed _pressurized CO with a preheating temperature of 35°C while stirring to increase the pressure to 6.0 MPa, and adjust the operating temperature to 35°C. The precipitation phenomenon in the kettle is obvious, and the impurity in the crude product of lumbrokinase is kept at high pressure for 60 minutes. The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 97.6%, and the purification factor of this process is 1.63. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

Example 6: Weighing an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 15% and a crude enzyme concentration of 7%, stirring repeatedly to dissolve and then filtering out insoluble matter. Then 20 mL of the solution was added to an autoclave preheated to 30°C. Then feed _pressurized CO with a preheating temperature of 30°C while stirring to increase the pressure to 5.5 MPa, and adjust the operating temperature to 30°C. The precipitation phenomenon in the kettle is obvious, and the impurity in the crude product of lumbrokinase is kept at high pressure for 50 minutes. The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 97.3%, and the purification factor of this process is 2.99. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

Example 7: Weigh an appropriate amount of crude lumbrokinase to prepare a solution with an ethanol concentration of 25% (volume percentage) and a crude enzyme concentration of 4% (mass percentage), stir repeatedly to dissolve, and filter out insoluble matter. Then 20 mL of the solution was added to an autoclave preheated to 30°C. Then, _pressurized CO with a preheating temperature of 30°C was introduced while stirring to increase the pressure to 8.0 MPa, and the operating temperature was adjusted to 30°C. The precipitation phenomenon in the kettle was obvious, and the impurity in the crude product of lumbrokinase was kept under high pressure for 35 minutes. The protein is completely precipitated under these conditions, while the active ingredient remains in solution. Then the precipitate is separated, the enzyme activity yield is 98.9%, and the purification factor of this process is 2.61. The method adopts pressurized CO ₂ as volatile acid and ethanol as precipitating agent to form a pressurized CO ₂ -ethanol-water system protein isoelectroprecipitation technology to precipitate foreign proteins in crude lumbrokinase. The method has the characteristics of relatively mild operating conditions, other active components can be obtained at the same time, and there is basically no loss of enzymes.

Claims (6)

1. remove the method for foreign protein in the Lumbrukinase crude product, it is characterized in that the step of method is as follows:

1) at first, the Lumbrukinase crude product is dissolved in the ethanol-water solution, preparing thick enzyme mass percent concentration is 2～7%, and the ethanol concentration of volume percent is 0～40% solution, repeatedly elimination insolubles after the stirring and dissolving;

2) then, getting the above-mentioned mixing solutions of 20mL, to join preheating temperature be in 20～40 ℃ of autoclaves, and feeding preheating temperature then is 20～40 ℃, and pressure is the pressurization CO of 3.5～8.5MPa ₂Stir simultaneously, the pH of regulator solution is 4.4, under high pressure keeps 20～60 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution, precipitate and separate is gone out to get final product again.

2. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said autoclave preheating temperature and CO ₂Preheating temperature is 25～35 ℃.

3. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said CO ₂Pressure is 6.0～8.0MPa.

4. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said enzyme mass percent concentration is 3～6%.

5. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said volumes of aqueous ethanol percentage concentration is 15～35%.

6. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said CO ₂It is 30～50 minutes that pressure is held time.