CN108642031B - Elastase and extraction method thereof - Google Patents
Elastase and extraction method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6448—Elastases, e.g. pancreatic elastase (3.4.21.36); leukocyte elastase (3.4.31.37)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21011—Elastase (3.4.21.11)
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Abstract
The invention relates to the technical field of biological pharmacy, and provides an extraction method of elastase, which comprises the following steps: fully reacting the pig pancreas meat pulp with 0.1-0.5M, pH of 4.0-7.0 first phosphate buffer solution, and performing solid-liquid separation to obtain a primary extracting solution; passing the primary extract through an ultrafiltration membrane to obtain a secondary extract; equilibrating a chromatographic column filled with CM Bestarose FF by using a second phosphate buffer solution with the concentration of 0.02-0.1M, pH being 4.5-6.5; adjusting the pH value of the secondary extracting solution and filtering to obtain a sample loading solution; enabling the sample liquid to pass through the column at the speed of 2-4 ml/min; washing with a second phosphate buffer solution after the sample loading is finished; eluting with a third phosphate buffer solution of which the concentration is 4.5-6.5 and the concentration is 0.5-1.2M, pH, and collecting an eluent; and (4) obtaining the elastase solution by using an ultrafiltration membrane. The elastase prepared by the method has high quality. The invention also provides the elastase prepared by the method.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, and particularly relates to elastase and an extraction method thereof.
Background
The elastase has high medicinal and commercial values, and is mainly used for treating hyperlipidemia, and preventing atherosclerosis and fatty liver in medical clinic. Can also be used for tenderizing meat and aquatic product. However, the activity of elastase obtained by the existing extraction method of elastase is not high enough, so the extraction method still needs to be improved.
In view of this, the present application is specifically made.
Disclosure of Invention
The invention provides an extraction method of elastase, aiming at solving the problem of the activity of elastase prepared by the existing extraction method of elastase.
The invention provides an elastase which has a higher activity value.
The invention is realized by the following steps:
an extraction method of elastase, comprising the following steps:
the extraction step comprises: mixing the pig pancreas meat pulp with a first phosphate buffer solution with the concentration of 0.1-0.5M, pH being 4.0-7.0, which is 1-10 times of the mass of the pig pancreas meat pulp, fully reacting the pig pancreas meat pulp with the first phosphate buffer solution, and performing solid-liquid separation to obtain a primary extracting solution;
a desalting step: concentrating and desalting the primary extracting solution by a molecular weight ultrafiltration membrane of 3000-20000 Da to obtain a secondary extracting solution with the electric conductivity of less than 5 ms;
resin balancing step: equilibrating a chromatographic column filled with CM Bestarose FF by using a second phosphate buffer solution with the concentration of 0.02-0.1M, pH of 4.5-6.5;
a sample liquid preparation step: adjusting the pH value of the secondary extracting solution to be the same as that of the second phosphate buffer solution, and filtering to obtain a sample solution;
a sample loading step: enabling the sample liquid to pass through the column at the speed of 2-4 ml/min;
a washing step: washing 1-5 column volumes with a second phosphate buffer solution after the sample loading is finished;
an elution step: eluting 2-8 column volumes by using a third phosphate buffer solution with the concentration of 0.5-1.2M, pH being 4.5-6.5, and collecting eluent;
a desalting step: concentrating and desalting the mixture by using a 3000-20000 Da molecular weight ultrafiltration membrane until the electric conductivity is less than 500us to obtain the elastase solution.
Further, in a preferred embodiment of the present invention, the method further comprises a drying step: and drying the elastase solution to obtain the elastase powder.
Further, in a preferred embodiment of the present invention, the drying is freeze drying.
Further, in a preferred embodiment of the present invention, a sterilization step is further included before drying.
Further, in a preferred embodiment of the present invention, the sterilization step is to filter the elastase solution through a 0.1-0.22 μm filter.
Further, in the preferred embodiment of the present invention, solid-liquid separation is performed by filtration.
Further, in a preferred embodiment of the present invention, the step of fully reacting the porcine pancreas meat pulp with the first phosphate buffer is to stir the mixture for 4 to 16 hours.
Further, in the preferred embodiment of the present invention, the temperature of the mixture is controlled to be 10-15 ℃ and the pH is controlled to be 5.5-6.0 during the stirring process.
Further, in a preferred embodiment of the present invention, the step of grinding the porcine pancreas to obtain the porcine pancreas meat pulp is further included before mixing the porcine pancreas meat pulp with the first phosphate buffer.
An elastase is extracted by the above extraction method.
The invention has the beneficial effects that: according to the extraction method of the elastase, which is designed by the design, the extraction steps are reasonably arranged, the molar concentrations and the pH values of the first phosphate buffer solution, the second phosphate buffer solution and the third phosphate buffer solution are set within a preferred range, the sample solution passes through a column at a proper speed by adopting a chromatographic column filled with CM Bestarose FF, and the elastase with high activity and high quality is obtained by reasonably washing, eluting and separating after passing through the column.
The elastase obtained by the design is extracted by the extraction method of the elastase provided by the invention, so that the elastase has high activity value and good quality.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following provides a specific description of the method for extracting elastase.
An extraction method of elastase, comprising the following steps:
s1, extraction step: mixing the pig pancreas meat pulp with a first phosphate buffer solution with the concentration of 0.1-0.5M, pH being 4.0-7.0, wherein the concentration of the first phosphate buffer solution is 1-10 times of the mass of the pig pancreas meat pulp, fully reacting the pig pancreas meat pulp with the first phosphate buffer solution, and carrying out solid-liquid separation to obtain a primary extracting solution.
Specifically, the pig pancreas frozen and stored in a freezer is unfrozen and then placed in a pulping machine to be stirred and crushed to obtain the pig pancreas meat pulp. And fully mixing the obtained porcine pancreas meat pulp with a first phosphate buffer solution with the mass of 1-10 times of that of the porcine pancreas meat pulp so as to fully react the porcine pancreas meat pulp with the first phosphate buffer solution and fully dissolve out the elastase. Specifically, the concentration of the first phosphate buffer solution is 0.1-0.5M, pH and is 4.0-7.0, and the first phosphate buffer solution can be NaH2PO4And Na2HPO4The mixed solution may be KH2PO4And K2HPO4And (3) stirring the mixed solution of the porcine pancreas meat pulp and the first phosphate buffer solution for 4-16 hours to ensure that the elastase is completely dissolved, preferably, keeping the temperature of the mixed solution at 10-15 ℃ and the pH value at 5.5-6.0 in a thinner process to ensure that the elastase contained in the mixed solution keeps higher activity. After stirring, carrying out solid-liquid separation, wherein in the invention, the mode which is simple and convenient to operate is filtration to obtain primary extracting solution, and the mode for carrying out solid-liquid separation in the invention can also be centrifugation for a period of time and then taking supernatant.
S2, desalting step: and (3) concentrating and desalting the primary extracting solution by passing through a 3000-20000 Da molecular weight ultrafiltration membrane to obtain a secondary extracting solution with the electric conductivity less than 5 ms.
S3, resin balancing: and (3) balancing the chromatographic column filled with CM Bestarose FF by using a second phosphate buffer solution with the concentration of 0.02-0.1M, pH of 4.5-6.5.
Specifically, a chromatographic column filled with CM Bestarose FF is balanced by using a second phosphate buffer solution until the pH value and the conductivity of an effluent are the same as those of the second phosphate buffer solution, or the pH value is within the range of 4.5-6.0, and the conductivity is close to that of the second phosphate buffer solution. The second phosphate buffer solution may be NaH2PO4And Na2HPO4The mixed solution may be KH2PO4And K2HPO4The solution was mixed. CM Bestarose FF is available directly.
S4, a sample loading solution preparation step: and adjusting the pH value of the secondary extracting solution to be the same as that of the second phosphate buffer solution, and filtering to obtain a sample solution.
Specifically, the pH of the secondary extract obtained by the desalting step is different from that of the second phosphate buffer, and the secondary extract needs to be in accordance with the pH in the chromatography column or within a predetermined range (i.e., the pH range of the second phosphate buffer) during column passing, so that the extraction effect is good, and if the pH of the secondary extract is not within the pH range of the second phosphate buffer, the pH of the secondary extract needs to be adjusted to be the same as the pH of the second phosphate buffer, and then the secondary extract is filtered to obtain the sample solution, otherwise, the step is unnecessary. Naturally, when the pH of the prepared sample solution is the same as the pH of the second phosphate, the elastase extraction effect is the best.
S5, a sample loading step: and enabling the sample solution to pass through the column at the speed of 2-4 ml/min.
S6, washing step: and washing 1-5 column volumes by using a second phosphate buffer solution after the sample loading is finished.
S7, elution step: eluting 2-8 column volumes by using a third phosphate buffer solution with the concentration of 0.5-1.2M, pH being 4.5-6.5, and collecting the eluent.
S8, desalting step: concentrating and desalting the mixture by using a 3000-20000 Da molecular weight ultrafiltration membrane until the electric conductivity is less than 500us to obtain the elastase solution.
Specifically, after the elastase liquid is obtained by concentrating and desalting through a 3000-20000 Da molecular weight ultrafiltration membrane, the method further comprises the step of sterilizing the elastase liquid, wherein the sterilization method comprises the steps of passing the elastase liquid through a 0.1-0.22 mu m filter membrane and intercepting bacteria.
Sterilizing, and freeze drying to obtain elastase powder
The following will specifically describe the method for extracting elastase provided by the present invention with reference to specific examples.
Example 1
This example provides an elastase and a method for its extraction.
A method for extracting elastase comprises: the method comprises the following steps:
and (3) putting the thawed porcine pancreas into a pulping machine, and crushing into porcine pancreas meat pulp. Mixing and stirring the pig pancreas meat pulp and a first phosphate buffer solution with the mass concentration of 0.5M, pH being 4.0 being 1 time, keeping the temperature within 10-15 ℃ and the pH within 5.5-6.0 in the stirring process, stirring for 4 hours, and filtering to obtain a primary extracting solution.
Concentrating and desalting the primary extract with ultrafiltration membrane with molecular weight of 3000Da to obtain secondary extract with conductivity less than 5 ms.
The column containing CM Bestarose FF was equilibrated with a second phosphate buffer at a concentration of 0.02M, pH of 4.5 until the effluent pH was also 4.5.
Adjusting the pH of the secondary extract to be the same as the pH of the second phosphate buffer solution, namely 4.5, and filtering to obtain a sample solution.
The sample was passed through the column at a rate of 4 ml/min. After loading, 5 column volumes were washed with the second phosphate buffer. The eluate was collected by eluting 8 column volumes with a third phosphate buffer solution with a concentration of 1.2M, pH of 4.5.
Concentrating with ultrafiltration membrane of 3000Da molecular weight, desalting to electric conductance less than 500us to obtain elastase solution. Then, the elastase solution is filtered by a filter membrane with the diameter of 0.1 mu m for sterilization to obtain the sterile elastase solution. Finally, freeze-drying the sterile elastase solution to obtain the elastase powder.
The obtained elastase powder is the elastase provided in this example.
Example 2
This example provides an elastase and a method for its extraction.
A method for extracting elastase comprises: the method comprises the following steps:
and (3) putting the thawed porcine pancreas into a pulping machine, and crushing into porcine pancreas meat pulp. Mixing and stirring the pig pancreas meat pulp and a first phosphate buffer solution with the concentration of 0.1M, pH being 7.0 being 10 times of the mass of the pig pancreas meat pulp, keeping the temperature within 10-15 ℃ and the pH within 5.5-6.0 in the stirring process, stirring for 16 hours, and filtering to obtain a primary extracting solution.
Concentrating and desalting the primary extract with a molecular weight of 20000Da with ultrafiltration membrane to obtain secondary extract with conductivity of less than 5 ms.
The column containing CM Bestarose FF was equilibrated with a second phosphate buffer solution at a concentration of 0.1M, pH of 6.5 until the effluent pH was also 6.5.
Adjusting the pH of the secondary extracting solution to be the same as that of the second phosphate buffer solution, namely 6.5, and filtering to obtain a sample solution.
The sample was passed through the column at a rate of 2 ml/min. After loading, wash 1 column volume with the second phosphate buffer. The eluate was collected by eluting 2 column volumes with a third phosphate buffer solution with a concentration of 0.5M, pH of 6.5.
Concentrating with 20000Da ultrafiltration membrane to desalt until electric conductivity is less than 500us to obtain elastase solution. Then, the elastase solution is filtered by a filter membrane with the diameter of 0.22 μm for sterilization to obtain the sterile elastase solution. Finally, freeze-drying the sterile elastase solution to obtain the elastase powder.
The obtained elastase powder is the elastase provided in this example.
Example 3
This example provides an elastase and a method for its extraction.
A method for extracting elastase comprises: the method comprises the following steps:
and (3) putting the thawed porcine pancreas into a pulping machine, and crushing into porcine pancreas meat pulp. Mixing and stirring the pig pancreas meat pulp and a first phosphate buffer solution with the mass concentration of 0.2M, pH being 5.5 times of that of the pig pancreas meat pulp, keeping the temperature within 10-15 ℃ and the pH within 5.5-6.0 in the stirring process, stirring for 8 hours, and filtering to obtain a primary extracting solution.
Concentrating and desalting the primary extract with a molecular weight of 10000Da by using an ultrafiltration membrane to obtain a secondary extract with the conductivity of less than 5 ms.
The column containing CM Bestarose FF was equilibrated with a second phosphate buffer solution at a concentration of 6.0 at 0.05M, pH until the effluent pH was also 6.0.
Adjusting the pH of the secondary extracting solution to be the same as that of the second phosphate buffer solution, namely 6.0, and filtering to obtain a sample solution.
The sample was passed through the column at a rate of 3 ml/min. After loading, wash 2 column volumes with the second phosphate buffer. The eluate was collected by eluting 5 column volumes with a third phosphate buffer solution with a concentration of 1M, pH of 6.0.
Concentrating with 10000Da ultrafiltration membrane until the electric conductivity is less than 500us to obtain elastase solution. Then, the elastase solution is filtered by a filter membrane with the diameter of 0.20 mu m for sterilization to obtain the sterile elastase solution. Finally, freeze-drying the sterile elastase solution to obtain the elastase powder.
The obtained elastase powder is the elastase provided in this example.
Example 4
This example provides an elastase and a method for its extraction.
A method for extracting elastase comprises: the method comprises the following steps:
and (3) putting the thawed porcine pancreas into a pulping machine, and crushing into porcine pancreas meat pulp. Mixing and stirring the pig pancreas meat pulp and a first phosphate buffer solution with the concentration of 0.3M, pH being 5 times of the mass of the pig pancreas meat pulp, keeping the temperature within 10-15 ℃ and the pH within 5.5-6.0 in the stirring process, stirring for 10 hours, and filtering to obtain a primary extracting solution.
Concentrating and desalting the primary extract with a molecular weight of 5000Da with ultrafiltration membrane to obtain secondary extract with conductivity of less than 5 ms.
The column containing CM Bestarose FF was equilibrated with a second phosphate buffer at a concentration of 0.07M, pH of 5.5 until the effluent pH was also 5.5.
Adjusting the pH of the secondary extract to be the same as that of the second phosphate buffer solution, namely 5.5, and filtering to obtain a sample solution.
The sample was passed through the column at a rate of 2 ml/min. After loading, wash 2 column volumes with the second phosphate buffer. The eluate was collected by eluting 3 column volumes with a third phosphate buffer solution with a concentration of 0.7M, pH of 5.5.
Concentrating with ultrafiltration membrane of 5000Da molecular weight, desalting to electric conductance less than 500us to obtain elastase solution. Then, the elastase solution is filtered by a filter membrane with the diameter of 0.15 μm for sterilization to obtain the sterile elastase solution. Finally, freeze-drying the sterile elastase solution to obtain the elastase powder.
The obtained elastase powder is the elastase provided in this example.
Example 5
This example provides an elastase and a method for its extraction.
A method for extracting elastase comprises: the method comprises the following steps:
and (3) putting the thawed porcine pancreas into a pulping machine, and crushing into porcine pancreas meat pulp. Mixing and stirring the pig pancreas meat pulp and a first phosphate buffer solution with the concentration of 0.4M, pH being 6 which is 8 times of the mass of the pig pancreas meat pulp, keeping the temperature within 10-15 ℃ and the pH within 5.5-6.0 in the stirring process, stirring for 12 hours, and filtering to obtain a primary extracting solution.
Concentrating and desalting the primary extract with a molecular weight of 20000Da with ultrafiltration membrane to obtain secondary extract with conductivity of less than 5 ms.
The column containing CM Bestarose FF was equilibrated with a second phosphate buffer at a concentration of 0.09M, pH of 5.0 until the effluent pH was also 5.0.
Adjusting the pH of the secondary extracting solution to be the same as that of the second phosphate buffer solution, namely 5.0, and filtering to obtain a sample solution.
The sample was passed through the column at a rate of 3 ml/min. After loading, wash 4 column volumes with the second phosphate buffer. The eluate was collected by eluting 6 column volumes with a third phosphate buffer solution with a concentration of 0.6M, pH of 5.
Concentrating with 20000Da ultrafiltration membrane to desalt until electric conductivity is less than 500us to obtain elastase solution. Then, the elastase solution is filtered by a filter membrane with the diameter of 0.1 mu m for sterilization to obtain the sterile elastase solution. Finally, freeze-drying the sterile elastase solution to obtain the elastase powder.
The obtained elastase powder is the elastase provided in this example.
Comparative example 1
This comparative example is substantially the same as example 1 except that the column passing speed of the sample liquid was 5 ml/min.
Comparative example 2
This comparative example is substantially the same as example 2 except that the column passing speed of the sample liquid was 1 ml/min.
Comparative example 3
This comparative example is essentially the same as example 3, except that a DEAE anion exchange chromatography column is used in place of the column packed with CM Bestarose FF.
Comparative example 4
This comparative example is essentially the same as example 3, except that the column packed with CM Bestarose FF was replaced with an Amberlite CG-50 macroporous cationic resin column.
Examples of the experiments
The activities of examples 1 to 5 and comparative examples 1 to 4 were measured by reference to the drug standard method of the ministry of health of the people's republic of China and recorded in Table 1. And the indices of elastase obtained in example 3 were measured and recorded in table 2.
The activity detection method comprises the following steps:
preparation of control solutions: accurately weighing Congo red-elastin 100mg, placing in 100ml measuring flask, adding control elastase solution 20ml, placing in 37 deg.C water bath, shaking continuously until completely dissolving, adding phosphate buffer (pH6.0)50ml, and diluting with borate buffer (pH8.8) to scale.
Preparation of a test solution: taking a proper amount of the product, precisely weighing, placing in a mortar, adding a small amount of borate buffer solution (pH8.8) and about 4 drops of sodium hydroxide solution (0.1mol/L), grinding to dissolve, transferring to a 100ml measuring flask, and diluting to scale with borate buffer solution (pH 8.8). The elastase content was about 8 units per 1 ml.
Drawing a standard curve: the control solutions 0, 2.0, 4.0, 6.0, 8.0 and 10.0ml were precisely measured, mixed solutions (1:1) to 10ml of borate buffer (pH8.8) and phosphate buffer (pH6.0) were added, and absorbance was measured at a wavelength of 495nm by a spectrophotometric method (appendix 20 page of second part of the 1985 version of Chinese pharmacopoeia) with a zero tube as a blank. The substrate amount was plotted as a horizontal coordinate, and the absorbance was plotted as a vertical coordinate.
The determination method comprises the following steps: taking 3 test tubes with the diameter of about 20mm, respectively adding 20mg of Congo red-elastin and 3.0ml of borate buffer (pH8.8), placing the test tubes in a 37 ℃ water bath for preheating for 10 minutes, sequentially adding 2.0ml of borate buffer (pH8.8) into the first tube, sequentially adding 2.0ml of test solution preheated to 37 ℃ into the second tube and the third tube, timing, placing the test solution in the 37 ℃ water bath for continuous shaking for 20 minutes (accurate timing), adding 5.0ml of phosphate buffer (pH6.0), shaking uniformly, centrifuging at 2,500 rpm/min for 20 minutes, accurately sucking 2.0ml of supernatant, adding a mixed solution (1:1) to 4.0 ml) of borate buffer (pH8.8) and phosphate buffer (pH6.0), blank the first tube, and measuring the absorbance at the wavelength of 495nm by spectrophotometry (appendix 20 pages of the 1985 edition two parts). And (4) searching corresponding substrate amount from the standard curve, and calculating according to unit definition and conversion into unit number of activity.
Under the above conditions, the amount of enzyme required to hydrolyze 1mg of congo red-elastin in 20 minutes is one unit of elastase activity.
TABLE 1 Activity (U/mg) of Elastase obtained in each example and comparative example
As can be seen from Table 1, the ear activity values of the elastase prepared in examples 1-5 of the present invention are all higher than 90U/mg. The fact that the column-passing speed of comparative example 1 is higher than that of example 1, the activity value of the elastase obtained by final extraction is lower than that of example 1, the column-passing speed of comparative example 2 is lower than that of example 2, and the activity value of the elastase obtained by final extraction is lower than that of example 2 indicates that the activity of the elastase obtained by extraction is not the best when the speed of the sample solution passing through the column is out of the range required by the present invention. Comparing comparative example 3 and comparative example 4 with example 3, it is evident that the activity of the obtained elastase is lower, indicating that extraction of elastase using a chromatography column equipped with CM Bestarose FF leads to higher activity values of the extracted elastase.
TABLE 2 indices of elastase obtained in example 3
| Index (I) | The result of the detection |
| Traits | Off-white color |
| Residue on ignition | 0.8％ |
| Potency (U/mg) | 95.8 |
| Bacterium (per g) | ＜1000 |
| Mold, yeast (per/g) | ＜10 |
As can be seen from Table 1, the elastase ignition residue obtained in example 3 of the present invention was low in content, high in activity, low in bacteria content and almost free from mold yeasts. From this, it is inferred that the elastase produced by the present invention has good quality.
In summary, the elastase extracted by the method for extracting elastase provided by the invention has the advantages that the extraction steps are reasonably arranged, the molar concentrations and the pH values of the first phosphate buffer solution, the second phosphate buffer solution and the third phosphate buffer solution are set within a preferred range, the chromatographic column filled with CM Bestarose FF is adopted to pass the sample solution through the column at a proper speed, and the reasonable detergent is used for eluting and separating after passing through the column to obtain the elastase with high activity and high quality.
The elastase provided by the invention is extracted by the extraction method of the elastase, so that the elastase has high activity value and good quality.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The extraction method of elastase is characterized by comprising the following steps:
the extraction step comprises: mixing the pig pancreas meat pulp with a first phosphate buffer solution with the concentration of 0.1-0.5M, pH being 4.0-7.0, wherein the concentration of the first phosphate buffer solution is 1-10 times of the mass of the pig pancreas meat pulp, fully reacting the pig pancreas meat pulp with the first phosphate buffer solution, and performing solid-liquid separation to obtain a primary extracting solution;
a desalting step: concentrating and desalting the primary extracting solution by passing through a molecular weight ultrafiltration membrane of 3000-20000 Da to obtain a secondary extracting solution with the electric conductivity of less than 5 ms;
resin balancing step: equilibrating a chromatographic column filled with CM Bestarose FF by using a second phosphate buffer solution with the concentration of 0.02-0.1M, pH of 4.5-6.5;
a sample liquid preparation step: adjusting the pH value of the secondary extracting solution to be the same as that of the second phosphate buffer solution, and filtering to obtain a sample loading solution;
a sample loading step: enabling the sample loading liquid to pass through the column at the speed of 2-4 ml/min;
a washing step: washing 1-5 column volumes with a second phosphate buffer solution after the sample loading is finished;
an elution step: eluting 2-8 column volumes by using a third phosphate buffer solution with the concentration of 0.5-1.2M, pH being 4.5-6.5, and collecting eluent;
a desalting step: concentrating and desalting the mixture by using a 3000-20000 Da molecular weight ultrafiltration membrane until the electric conductivity is less than 500us to obtain the elastase solution.
2. The method for extracting elastase as claimed in claim 1, further comprising a drying step of: and drying the elastase solution to obtain the elastase powder.
3. The method for extracting elastase according to claim 2, wherein the drying method is freeze drying.
4. The method for extracting elastase as claimed in claim 2, which further comprises a sterilization step before drying.
5. The method for extracting elastase according to claim 4, wherein the step of sterilizing comprises passing the elastase solution through a 0.1 to 0.22 μm filter.
6. The method for extracting elastase according to claim 1, wherein the solid-liquid separation is performed by filtration.
7. The method for extracting elastase according to claim 1, wherein the step of sufficiently reacting the porcine pancreas meat slurry with the first phosphate buffer solution comprises stirring the mixture for 4 to 16 hours.
8. The method for extracting elastase as claimed in claim 7, wherein the temperature of the mixture is controlled to 10-15 ℃ and the pH is controlled to 5.5-6.0 during the stirring process.
9. The method for extracting elastase as claimed in claim 1, which further comprises crushing porcine pancreas to obtain said porcine pancreas meat pulp before mixing said porcine pancreas meat pulp with said first phosphate buffer.
10. An elastase extracted by the method for extracting elastase according to any one of claims 1 to 9.
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