Novel earthworm protease separation and purification process

Abstract

The invention relates to the technical field of protease separation, in particular to a novel earthworm protease separation and purification process. According to the present invention, the ultra-high speed separation, the molecular weight spectrum comparison extraction and the affinity chromatography purification scheme are adopted, such that the different biological macromolecules in the earthworm can be effectively separated, the earthworm protease-containing cell lysis solution can be concentrated, and most of the impurities can be removed so as to obtain the high-purity protease solution, the method is simple in operation and does not depend on complex chemical reactions, contact between protein and chemical reagents can be reduced, meanwhile, damage of high temperature to earthworm protease is avoided through a low-temperature centrifugation scheme, the enzymatic activity of earthworm protease is kept, high-purity and high-activity earthworm protease is obtained through affinity chromatography purification, operation is easy, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of protease separation, and in particular to a novel process for separating and purifying earthworm protease. Background Art

[0002] Earthworm protease is a proteolytic enzyme that can hydrolyze peptide bonds in protein molecules to release amino acids or small peptide chains. It can decompose various types of proteins and has extremely strong proteolytic ability. In the food industry, it is used to hydrolyze proteins, improve food taste, and increase nutritional value. In the medical field, it is used to promote digestion and improve gastrointestinal function. In the fields of environmental protection and agriculture, it is often used to deal with the decomposition of organic waste and soil improvement. Therefore, how to efficiently and cost-effectively separate and purify earthworm protease has become a research focus in various fields.

[0003] Existing methods for isolating and purifying earthworm protease include freeze-centrifugation, solvent extraction, and salting-out. Freeze-centrifugation involves freezing earthworm tissue and then centrifuging it to extract the protein. This method is simple to operate, but it can easily lead to a decrease in protease activity and cannot produce highly pure protein. Solvent extraction uses solvents such as alcohols and ethers to extract earthworm protease, but this method often loses some valuable protease and is difficult to remove solvent residues. Salting-out uses salt solutions of varying concentrations to precipitate the protein, but this method has low purification efficiency and is not suitable for the extraction of heat-sensitive proteins. Therefore, a more efficient and sophisticated separation and purification process is needed.

[0004] In response to the above problems, the present invention provides a novel process for separating and purifying earthworm protease. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel process for separating and purifying earthworm protease, which can significantly improve the activity and purity of earthworm protease under large-scale production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a novel process for separating and purifying earthworm protease, comprising the following steps:

[0007] S1: Fresh earthworm protease raw materials collected from the organic environment are transported to the unloading area in the factory, damaged earthworm protease raw materials are removed, and the raw materials are transferred to the ultrasonic cleaning instrument for ultrasonic cleaning to remove dirt and impurities. The raw materials are then sent to the foreign matter detector to detect the presence of foreign matter.

[0008] S2: Turn on the ice maker. After ice making is complete, cut the earthworm protease raw material, which has been checked for foreign matter, into small pieces and place them on ice. Use a mortar and pestle to freeze-grind for 20-30 minutes, then place the pieces in a homogenizer to break them up, thereby obtaining the earthworm protease raw material tissue.

[0009] S3: Place the earthworm protease raw material tissue in 0.1 M phosphate buffer solution with a pH of 7.4 on ice, and add 0.1 M PMSF. Then transfer the phosphate buffer solution containing the earthworm protease raw material tissue to a centrifuge tube and place it in an ultrasonic disruptor for 20 minutes;

[0010] S4: Precool the centrifuge to 4°C, centrifuge at 5000-6000 rpm, and centrifuge for 10-15 minutes at 4°C. After centrifugation, transfer the supernatant to a new centrifuge tube and filter using a 0.45 μm filter. Collect the sieved supernatant into a centrifuge tube precooled to 4°C to obtain the protein extract.

[0011] S5: Pre-cool the ultracentrifuge to 4°C, place the protein extract in the ultracentrifuge, and centrifuge at 400,000 x g for 1.5-2 hours. After centrifugation, transfer the supernatant to different new centrifuge tubes to obtain different layers of earthworm protease, and store them in a -80°C refrigerator.

[0012] S6: Take 20ul of earthworm protease from different layers and determine the protein concentration of earthworm protease in different layers using the BCA method. Then prepare polyacrylamide gel, take 20ul of earthworm protease from different layers and dilute them into 1mg / ml different determination protein solutions using 0.1M phosphate buffer solution;

[0013] S7: Add 2 mg SDS and 5 μl β-mercaptoethanol to 100 μl of the different assay protein solutions, then incubate in a 55°C metal bath for 15 min to ensure complete protein denaturation. Load 20 μl of the denatured different assay protein solutions into the polyacrylamide gel sample wells and simultaneously load 20 μl of the Protein Ladder gel for electrophoresis for 28 min.

[0014] S8: After the electrophoresis is completed, the gel is stained with Coomassie Brilliant Blue to obtain a protein map, and different layers of earthworm proteases of 20-40 kDa are labeled to obtain earthworm proteases of different purposes. The earthworm proteases of different purposes are combined to obtain recombinant earthworm proteases;

[0015] S9: NI poured on ice 2+ -NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles on the column bed, pre-equilibrated with 15 ml of eluent, loaded with sample, and injected recombinant earthworm protease into the NI 2+ -NTA affinity chromatography column, repeated 3 times, then eluted with 7.5 ml of eluent, repeated 3 times, and the eluent was collected to obtain eluted earthworm protease;

[0016] S10: The eluted earthworm protease is transferred to a nanofiltration membrane, and both sides of the nanofiltration membrane are clamped with a nanofiltration clamp. The nanofiltration membrane containing the eluted earthworm protease is then placed in the nanofiltrate and nanofiltration is performed at 4°C for 8 hours. After the nanofiltration is completed, the purified earthworm protease is obtained.

[0017] S11: Transfer the purified earthworm protease to the freeze dryer, and verify the integrity of the door seal to ensure the normal operation of the freeze dryer. After verifying that the door seal is intact, turn on the vacuum pump and pump the freeze dryer to a vacuum degree of 15 Pa. Freeze-dry for 8 hours to obtain the earthworm protease.

[0018] Furthermore, the mass ratio of 0.1M FMSF to 0.1M phosphate in step S3 is 1:100; the ultrasonic crushing program of the ultrasonic crusher in step S3 is ultrasonication for 2 seconds, pause for 10 seconds, and the power is 400W;

[0019] Furthermore, the wavelength of the spectrum measurement of the BCA method in step S6 is 562 nm; the concentration of the polyacrylamide gel in step S6 is 12%; the molecular weight of the protein ladder in step S7 is 10-80 kDa;

[0020] Furthermore, the eluent in step S9 is a 0.5 mmol / L ammonium sulfide solution; the pore size of the nanofiltration membrane in step S10 is a 20 kDa MWCO nanofiltration membrane; and the nanofiltrate in step S10 is a saturated PEG-8000 solution.

[0021] In summary, due to the adoption of the above-mentioned technical scheme, the beneficial effects of the present invention are as follows: the present invention uses ultra-high-speed separation, molecular weight profile comparison extraction and affinity chromatography purification scheme, which can effectively separate different biological macromolecules in earthworms, and can concentrate the cell lysate containing earthworm protease and remove most impurities, thereby obtaining a high-purity protease solution, and does not rely on complex chemical reactions, and can reduce the contact between protein and chemical reagents. At the same time, the low-temperature centrifugation scheme avoids the destruction of earthworm protease by high temperature, thereby maintaining the enzymatic activity of earthworm protease, and obtaining high-purity and strong-activity earthworm protease through affinity chromatography purification, which is simple to operate and suitable for industrial production. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0024] Example 1

[0025] 1: Fresh earthworm protease raw materials collected from the organic environment are transported to the unloading area in the factory, damaged earthworm protease raw materials are removed, and transferred to the ultrasonic cleaning instrument for ultrasonic cleaning to remove dirt and impurities, and then sent to the foreign matter detector to detect the presence of foreign matter;

[0026] 2. Turn on the ice maker. After ice making is complete, cut the earthworm protease raw material into small pieces and place them on ice. Use a grinding mortar and pestle to freeze-grind for 30 minutes, then place it in a homogenizer to break it up to obtain the earthworm protease raw material tissue.

[0027] 3. On ice, place 1 L of lumiprotease tissue in 0.1 M phosphate buffer solution (pH 7.4) and 10 ml of 0.1 M PMSF. Transfer the phosphate buffer solution containing the lumiprotease tissue to a centrifuge tube and place it in an ultrasonic disruptor. Ultrasonicate for 2 seconds, pause for 10 seconds, and ultrasonicate for 20 minutes at 400 W.

[0028] 4: Pre-cool the centrifuge to 4°C, centrifuge at 6000 rpm, and centrifuge for 10 minutes at 4°C. After centrifugation, transfer the supernatant to a new centrifuge tube and filter it using a 0.45 μm filter. Collect the sieved supernatant into a centrifuge tube pre-cooled to 4°C to obtain the protein extract.

[0029] 5. Pre-cool the ultracentrifuge to 4°C, place the protein extract in the ultracentrifuge, and centrifuge at 400,000 x g for 1.5 hours. After centrifugation, transfer the supernatant to different new centrifuge tubes to obtain different layers of earthworm protease and store them in a -80°C refrigerator.

[0030] 6: Take 20ul of earthworm protease from different layers and measure the protein concentration of earthworm protease in different layers using the BCA method at a wavelength of 562nm. Then prepare a 12% polyacrylamide gel and take 20ul of earthworm protease from different layers. Use 0.1M phosphate buffer solution to dilute the earthworm protease from different layers to 1mg / ml different measurement protein solutions;

[0031] 7: Add 2 mg SDS and 5 μl β-mercaptoethanol to 100 μl of different protein solutions, then place in a 55°C metal bath for 15 minutes to ensure complete protein denaturation. Load 20 μl of the denatured different protein solutions into the polyacrylamide gel sample wells and simultaneously load 20 μl of the Protein Ladder gel for electrophoresis for 28 minutes.

[0032] 8: After the electrophoresis is completed, the gel is stained with Coomassie Brilliant Blue to obtain a protein map. Different layers of earthworm proteases of 20-40 kDa are labeled to obtain earthworm proteases of different purposes. The earthworm proteases of different purposes are combined to obtain recombinant earthworm proteases.

[0033] 9: Pour NI on ice 2+ -NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles on the column bed, pre-equilibrated with 15ml of 0.5mmol / L ammonium sulfide solution, loaded with sample, and injected recombinant earthworm protease into the NI 2+ -NTA affinity chromatography column, repeated 3 times, then eluted with 7.5 ml of 0.5 mmol / L ammonium sulfide solution, repeated 3 times, and collected 0.5 mmol / L ammonium sulfide solution to obtain eluted earthworm protease;

[0034] 10: Transfer the eluted earthworm protease to a 20kDa MWCO nanofiltration membrane, clamp the two sides of the nanofiltration membrane with a nanofiltration clamp, then place the 20kDa MWCO nanofiltration membrane containing the eluted earthworm protease into a saturated PEG-8000 solution and nanofilter at 4°C for 8 hours to obtain purified earthworm protease.

[0035] 11: Transfer the purified earthworm protease to the freeze dryer, and verify the integrity of the door seal to ensure the normal operation of the freeze drying chamber. After verifying that the door seal is intact, turn on the vacuum pump and evacuate the freeze dryer to a vacuum degree of 15 Pa. Freeze dry for 8 hours to obtain the earthworm protease prepared in Example 1.

[0036] Table 1, parameter table of reagents used in Example 1

[0037] Drug name source model parameter PMSF Aladdin P408676 1ml SDS Aladdin S774281 500g β-Mercaptoethanol Aladdin M301574 100ml Ammonium sulfide solution Aladdin A305309 500ml

[0038] Example 2

[0039] 1: Fresh earthworm protease raw materials collected from the organic environment are transported to the unloading area in the factory, damaged earthworm protease raw materials are removed, and transferred to the ultrasonic cleaning instrument for ultrasonic cleaning to remove dirt and impurities, and then sent to the foreign matter detector to detect the presence of foreign matter;

[0040] 2. Turn on the ice maker. After ice making is complete, cut the earthworm protease raw material into small pieces and place them on ice. Use a grinding mortar and pestle to freeze-grind for 20-30 minutes, then place it in a homogenizer to break it up to obtain the earthworm protease raw material tissue.

[0041] 3. On ice, place 1 L of lumiprotease tissue in 0.1 M phosphate buffer solution (pH 7.4) and 10 ml of 0.1 M PMSF. Transfer the phosphate buffer solution containing the lumiprotease tissue to a centrifuge tube and place it in an ultrasonic disruptor. Ultrasonicate for 2 seconds, pause for 10 seconds, and ultrasonicate for 20 minutes at 400 W.

[0042] 4: Pre-cool the centrifuge to 4°C, 5000 rpm, centrifuge for 15 minutes at 4°C. After centrifugation, transfer the supernatant to a new centrifuge tube and filter using a 0.45 μm filter. Collect the sieved supernatant into a centrifuge tube pre-cooled to 4°C to obtain the protein extract.

[0043] 5: Pre-cool the ultracentrifuge to 4°C, place the protein extract in the ultracentrifuge, and centrifuge at 400,000 x g for 2 hours. After centrifugation, transfer the supernatant to different new centrifuge tubes to obtain different layers of earthworm protease and store them in a -80°C refrigerator.

[0044] 6: Take 20ul of earthworm protease from different layers and measure the protein concentration of earthworm protease in different layers using the BCA method at a wavelength of 562nm. Then prepare a 12% polyacrylamide gel and take 20ul of earthworm protease from different layers. Use 0.1M phosphate buffer solution to dilute the earthworm protease from different layers to 1mg / ml different measurement protein solutions;

[0045] 7: Add 2 mg SDS and 5 μl β-mercaptoethanol to 100 μl of different protein solutions, then place in a 55°C metal bath for 15 minutes to ensure complete protein denaturation. Load 20 μl of the denatured different protein solutions into the polyacrylamide gel sample wells and simultaneously load 20 μl of the Protein Ladder gel for electrophoresis for 28 minutes.

[0046] 8: After the electrophoresis is completed, the gel is stained with Coomassie Brilliant Blue to obtain a protein map. Different layers of earthworm proteases of 20-40 kDa are labeled to obtain earthworm proteases of different purposes. The earthworm proteases of different purposes are combined to obtain recombinant earthworm proteases.

[0047] 9: Pour NI on ice 2+ -NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles on the column bed, pre-equilibrated with 15ml of 0.5mmol / L ammonium sulfide solution, loaded with sample, and injected recombinant earthworm protease into the NI 2+ -NTA affinity chromatography column, repeated 3 times, then eluted with 7.5 ml of 0.5 mmol / L ammonium sulfide solution, repeated 3 times, and collected 0.5 mmol / L ammonium sulfide solution to obtain eluted earthworm protease;

[0048] 10: Transfer the eluted earthworm protease to a 20kDa MWCO nanofiltration membrane, clamp the two sides of the nanofiltration membrane with a nanofiltration clamp, then place the 20kDa MWCO nanofiltration membrane containing the eluted earthworm protease into a saturated PEG-8000 solution and nanofilter at 4°C for 8 hours to obtain purified earthworm protease.

[0049] 11: Transfer the purified earthworm protease to the freeze dryer and verify the integrity of the door seal to ensure the normal operation of the freeze dryer. After verifying that the door seal is intact, turn on the vacuum pump and pump the freeze dryer to a vacuum degree of 15Pa. Freeze dry for 8 hours to obtain earthworm protease.

[0050] Table 2, parameters of reagents used in Example 2

[0051] Drug name source model parameter PMSF Aladdin P408676 1ml SDS Aladdin S774281 500g β-Mercaptoethanol Aladdin M301574 100ml Ammonium sulfide solution Aladdin A305309 500ml

[0052] Comparative Example 1

[0053] Example 2 of the Chinese patent publication number CN108048434A was selected as comparative example 1.

[0054] Comparative Example 2

[0055] Example 1 of the Chinese patent publication number CN1454994A was selected as comparative example 2.

[0056] Enzyme activity test

[0057] The enzymatic activity of the earthworm proteases prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 was tested according to GB / T 21793-2008 "Determination of Enzyme Activity in Bioenzyme Preparations";

[0058] Prepare 1 mg / mL casein solution as a substrate for the enzyme reaction, and add 0.5 ml of casein solution to each test tube; then add 50 ul of earthworm protease prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 to each test tube (before addition, dilute the earthworm protease prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 to 1 mg / 50 ul using PBS buffer); set the reaction temperature to 37°C and react at this temperature for 30 minutes; after the reaction is completed, add 1 ml of TCA solution to each test tube to terminate the enzymatic reaction, then use a centrifuge at 4°C, 6000 rpm for 15 minutes, take the supernatant and transfer it to a clean centrifuge tube, add 0.5 ml of Ninhydrin reagent, use a spectrophotometer to measure the absorbance at a wavelength of 570 nm, and calculate the earthworm protease activity prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 using the standard curve.

[0059] Table 3, Enzyme activity test results

[0060] Grouping Enzyme activity Example 1 586 U / mg Example 2 612 U / mg Comparative Example 1 454 U / mg Comparative Example 2 424 U / mg

[0061] Enzyme purity test

[0062] BCA working solution was prepared by mixing BCA reagent and copper sulfate in a mass ratio of 50:1. Then, bovine protein serum was used to prepare standard solutions of 0ug / ml, 10ug / ml, 20ug / ml, 40ug / ml, 80ug / ml, and 100ug / ml. 200ul of BCA working solution was added to each standard solution and then transferred to a 37°C incubator for incubation for 30min. After incubation, the absorbance of the standard solution was measured at 562nm using a spectrophotometer. A standard curve was drawn with the concentration of the standard solution as the horizontal axis and the absorbance as the vertical axis.

[0063] Use PBS buffer to dilute the earthworm protease prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 by 10 times, take 200ul of the diluted earthworm protease prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 and mix them with 200ul of BCA working solution, then transfer them to a 37°C incubator and incubate for 30 minutes. After the incubation is completed, use a spectrophotometer to measure the absorbance of the standard solution at 562nm, and use the standard curve to calculate the protein concentration according to the absorbance value of the sample.

[0064] Table 4, Enzyme purity test results

[0065] Grouping Enzyme purity Example 1 11.32mg / ml Example 2 11.56mg / ml Comparative Example 1 5.47mg / ml Comparative Example 2 4.82mg / ml

[0066] By analyzing Table 3 and Table 4, it can be seen that the earthworm proteases prepared in Example 1 and Example 2 not only have better enzyme activity, but also have significantly higher enzyme purity than that of Comparative Example 1 and Comparative Example 2. Among them, the earthworm protease prepared in Comparative Example 2 has the best enzyme activity and enzyme purity, and its enzyme activity can reach more than 600 U / mg.

[0067] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A novel process for separating and purifying earthworm protease, characterized in that: The following steps are involved: S1: Fresh earthworm protease raw materials collected from the organic environment are transported to the unloading area in the factory, damaged earthworm protease raw materials are removed, and the raw materials are transferred to the ultrasonic cleaning instrument for ultrasonic cleaning to remove dirt and impurities. The raw materials are then sent to the foreign matter detector to detect the presence of foreign matter. S2: Turn on the ice maker. After ice making is complete, cut the earthworm protease raw material, which has been checked for foreign matter, into small pieces and place them on ice. Use a mortar and pestle to freeze-grind for 20-30 minutes, then place the pieces in a homogenizer to break them up, thereby obtaining the earthworm protease raw material tissue. S3: Place the earthworm protease raw material tissue in 0.1 M phosphate buffer solution with a pH of 7.4 on ice, and add 0.1 M PMSF. Then transfer the phosphate buffer solution containing the earthworm protease raw material tissue to a centrifuge tube and place it in an ultrasonic disruptor for 20 minutes; S4: Precool the centrifuge to 4°C, centrifuge at 5000-6000 rpm, and centrifuge for 10-15 minutes at 4°C. After centrifugation, transfer the supernatant to a new centrifuge tube and filter using a 0.45 μm filter. Collect the sieved supernatant into a centrifuge tube precooled to 4°C to obtain the protein extract. S5: Pre-cool the ultracentrifuge to 4°C, place the protein extract in the ultracentrifuge, and centrifuge at 400,000 x g for 1.5-2 hours. After centrifugation, transfer the supernatant to different new centrifuge tubes to obtain different layers of earthworm protease, and store them in a -80°C refrigerator. S6: Take 20ul of earthworm protease from different layers and determine the protein concentration of earthworm protease in different layers using the BCA method. Then prepare a 12% polyacrylamide gel and take 20ul of earthworm protease from different layers. Use 0.1M phosphate buffer solution to dilute the earthworm protease from different layers to 1mg / ml different measurement protein solutions. S7: Add 2 mg SDS and 5 μl β-mercaptoethanol to 100 μl of the different assay protein solutions, then incubate in a 55°C metal bath for 15 min to ensure complete protein denaturation. Load 20 μl of the denatured different assay protein solutions into the polyacrylamide gel sample wells and simultaneously load 20 μl of the Protein Ladder gel for electrophoresis for 28 min. S8: After the electrophoresis is completed, the gel is stained with Coomassie Brilliant Blue to obtain a protein map, and different layers of earthworm proteases of 20-40 kDa are labeled to obtain earthworm proteases of different purposes. The earthworm proteases of different purposes are combined to obtain recombinant earthworm proteases; S9: NI poured on ice 2+ -NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles on the column bed, pre-equilibrated with 15 ml of eluent, loaded with sample, and injected recombinant earthworm protease into the NI 2+ -NTA affinity chromatography column, repeated 3 times, then eluted with 7.5 ml of eluent, repeated 3 times, and the eluent was collected to obtain eluted earthworm protease; S10: The eluted earthworm protease is transferred to a nanofiltration membrane, and both sides of the nanofiltration membrane are clamped with a nanofiltration clamp. The nanofiltration membrane containing the eluted earthworm protease is then placed in the nanofiltrate and nanofiltration is performed at 4°C for 8 hours. After the nanofiltration is completed, the purified earthworm protease is obtained. S11: Transfer the purified earthworm protease to the freeze dryer, and verify the integrity of the door seal to ensure the normal operation of the freeze dryer. After verifying that the door seal is intact, turn on the vacuum pump and pump the freeze dryer to a vacuum degree of 15 Pa. Freeze-dry for 8 hours to obtain the earthworm protease.

2. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The mass ratio of 0.1 M FMSF and 0.1 M phosphate described in step S3 is 1:

100.

3. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The ultrasonic crushing program of the ultrasonic crusher described in step S3 is ultrasonication for 2 seconds, pause for 10 seconds, and the power is 400W.

4. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The spectrum measurement wavelength of the BCA method described in step S6 is 562 nm.

5. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The concentration of the polyacrylamide gel in step S6 is 12%.

6. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The molecular weight of the Protein Ladder described in step S7 is 10-80 kDa.

7. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The eluent in step S9 is a 0.5 mmol / L ammonium sulfide solution.

8. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The pore size of the nanofiltration membrane in step S10 is a 20 kDa MWCO nanofiltration membrane.

9. A novel process for separating and purifying earthworm protease according to claim 1, characterized in that: The nanofiltrate described in step S10 is a saturated PEG-8000 solution.