Preparation method and application of lumbrukinase freeze-dried powder

Abstract

The invention discloses a preparation method of high-activity lumbrukinase freeze-dried powder, which mainly comprises the following steps: cleaning, homogenizing and centrifuging frozen fresh earthworms to remove precipitates, and then carrying out step-by-step alcohol precipitation; carrying out cooling control on the combined lumbrukinase extraction supernatant, and carrying out step-by-step alcohol precipitation to obtain a lumbrukinase crude product; and the concentrations of ethanol used in each step of alcohol precipitation are controlled to be different. And redissolving the precipitate, and adsorbing and eluting with macroporous anion resin to obtain the high-activity lumbrukinase freeze-dried powder. According to the preparation method provided by the invention, the difficulties of using a large amount of ammonium sulfate and post-treatment desalination in the production process are avoided, meanwhile, macroporous anion resin adsorption purification is used for replacing chromatography system purification, the production cost is reduced, the production process is simple, the product activity is high, and large-scale industrial production is easy.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the technical field of preparation of high-activity lumbrokinase. Background Art

[0002] Lumbrokinase, also known as earthworm fibrinolytic enzyme, was first extracted and isolated from earthworms in 1983. The 2010 edition of the Pharmacopoeia standard defines it as a group of proteolytic enzymes extracted and prepared from artificially cultivated Eisenia fetida. It has a molecular weight of 20~40 KD, an isoelectric point of 3~5, is very stable below 60°C, and has the most optimal activity at pH 7~9. It has both kinase and fibrinolytic effects. Lumbrokinase has been proven to have good clinical application value. It is a multi-molecule recombinant oral preparation that has a special affinity for thrombus (fibrin), can track thrombus dissolution, effectively dissolve microthrombus, improve microcirculation, strengthen cardiovascular and cerebrovascular collateral circulation, openly repair damaged endothelial cells in blood vessels, increase vascular elasticity, improve vascular oxygen supply function, reduce blood viscosity, reduce platelet aggregation rate, and inhibit the re-formation of thrombus. It repairs necrotic brain cells around thrombus formation and saves the penumbra. It has been widely used in clinical practice and is increasingly used in the prevention and treatment of cardiovascular, cerebrovascular, endocrine, respiratory and other diseases.

[0003] At present, the method for preparing lumbrokinase is mainly to obtain lumbrokinase by crude separation from fresh earthworms by ammonium sulfate precipitation method and purification by dextran gel-ion exchange chromatography. The disadvantage is that a large amount of ammonium sulfate is used in production, and post-treatment desalination is very difficult. The main method of industrial desalination is dialysis chromatography, etc., which leads to complex operation and production process and high cost; and the production cycle is long and the activity is reduced. Patent CN102660523A discloses a method for preparing low-immunogenic lumbrokinase, which first obtains crude lumbrokinase by homogenate extraction of fresh earthworms and step-by-step precipitation of ammonium sulfate, then redissolves and desalts the obtained crude lumbrokinase, and then elutes and purifies the desalted crude product by ion exchange column, collects the components with fibrinolytic activity, freeze-dries and concentrates, separates the concentrated lumbrokinase solution by gel filtration chromatography, detects fibrinolytic activity by agarose-fibrin plate method, collects the second elution peak with fibrinolytic activity, and has a molecular weight of 23500-27500 Da, and the elution peak is dialyzed, desalted, and freeze-dried at low temperature, and then molecularly modified with activated monomethoxy polyethylene glycol and dextran, and a lumbrokinase with an activity of 31400U / mg can be obtained. This preparation process uses step-by-step precipitation of ammonium sulfate, and the post-treatment requires long-term desalting and purification by ion chromatography, and the production cost is relatively high. Summary of the invention

[0004] The purpose of the present invention is to disclose a method for preparing lumbrokinase with high activity and simple process, which solves the problems of complicated process steps, high production cost and low enzyme activity in the existing lumbrokinase production process. The specific scheme is as follows: A method for preparing lumbrokinase freeze-dried powder, which can remove impurities and obtain crude lumbrokinase by step-by-step alcohol precipitation of earthworm homogenate with ethanol, and then purify the obtained crude lumbrokinase by macroporous anion resin adsorption to obtain high-activity lumbrokinase freeze-dried powder.

[0005] A further improvement is that the activity of the prepared lyophilized powder of lumbrokinase is not less than 45,000 U / mg.

[0006] A further improvement is that the protein content of the prepared lyophilized powder of lumbrokinase is mainly distributed in the range of 20 kDa to 50 kDa.

[0007] A further improvement is that the preparation of the lyophilized lumbrokinase powder includes the following steps: Step 1: Thawing and washing earthworms, placing frozen fresh earthworms at room temperature to thaw naturally, and washing the thawed fresh earthworms with clean water to wash away sludge and sticky substances; Then, homogenize and wash, add 0.14M NaCl homogenization buffer to the washed earthworms at a ratio of m:v=1:1 to crush and homogenize, stir for 30 minutes after crushing, and centrifuge the obtained earthworm homogenate to collect the supernatant solution; then wash with the homogenization buffer in the centrifugal precipitate at a ratio of m:v=1:1, stir at room temperature for 30 minutes, centrifuge to collect the supernatant solution, and combine the extracted supernatants; wash twice, and combine to obtain the final lumbrokinase extraction supernatant; step three: stepwise alcohol precipitation, cool the combined lumbrokinase extraction supernatant, control the temperature at 4-10℃, perform stepwise alcohol precipitation to obtain the crude lumbrokinase, and control the ethanol concentration used in each step of alcohol precipitation to be different.

[0008] The further improvement is that it also includes the following steps: Step 4: Redissolution, using purified water to redissolve the crude lumbrokinase, adjusting the pH with 1M NaOH, adding purified water and controlling the conductivity of the solution; Step 5: Macroporous anion resin purification, adding activated macroporous strong anion resin or macroporous weak anion resin to the re-dissolved sample, stirring and exchanging for 2-4 hours, and draining; adding 0.5-0.8 M NaCl solution to the adsorbed resin for elution, stirring and eluting for 1-2 hours, draining, and eluting with elution buffer, and draining; Step 6: Desalting and concentrating, ultrafiltration of the obtained eluate through an ultrafiltration system, the size of the ultrafiltration membrane package is 10000Da, desalting and dehydrating, and controlling the conductivity of the solution to ≤200 us / cm; Step 7: Filtration and freeze-drying, filtering the ultrafiltered sample with a filter element, freezing the filtered solution at -30~-40℃ for 6 hours, heating and crushing to obtain lumbrokinase freeze-dried powder.

[0009] A further improvement is that in the step of cooling and performing stepwise alcohol precipitation in step three, 95% by volume ethanol is added to the supernatant of the lumbrokinase extraction, the ethanol concentration is controlled at 30-45%, the mixture is allowed to stand for 4-6 hours, and then centrifuged to collect the supernatant; 95% by volume ethanol is continued to be added to the supernatant solution after centrifugation, the ethanol concentration is controlled at 70-80%, the mixture is allowed to stand for 4-6 hours, and then centrifuged to collect the precipitate, which is the crude lumbrokinase.

[0010] A further improvement is that in step 4, 1M NaOH is used to adjust the pH to 7.5-8.5, and purified water is added to control the solution conductivity to be no higher than 5 ms / cm.

[0011] A further improvement is that the elution buffer in step 5 is specifically 0.8-1.0M NaCl buffer.

[0012] A further improvement is that in step seven, the temperature is controlled at 30-40° C. for drying and crushing to obtain lyophilized powder of lumbrokinase.

[0013] The invention also discloses lyophilized powder of lumbrokinase prepared according to the method, which can be used for skin care products and preventive applications in the fields of cardiovascular and cerebrovascular, endocrine, respiratory systems, etc.

[0014] The purpose of the present invention is to disclose a method for preparing lumbrokinase with high activity and simple process, which solves the problems of complicated process steps, high production cost and low enzyme activity in the existing lumbrokinase production process. The present invention provides a process that can realize the industrial production of lumbrokinase, which is to perform step-by-step alcohol precipitation of earthworm homogenate with ethanol to remove impurities and retain highly active lumbrokinase to obtain crude lumbrokinase. The crude lumbrokinase is then purified by macroporous anion resin adsorption to obtain lyophilized powder of lumbrokinase with an activity of not less than 45,000 U / mg, avoiding the investment in high-cost equipment and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a protein electrophoresis gel image; Among them, the protein gel sample information: from left to right are protein marker, supernatant of initial extraction of lumbrokinase, 45% alcohol precipitation, crude lumbrokinase after 75% alcohol precipitation, and lyophilized powder of lumbrokinase after resin purification. DETAILED DESCRIPTION

[0016] The following examples are included to illustrate preferred embodiments of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in practicing the present invention. Based on this disclosure, those skilled in the art will recognize that many changes may be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, that is, any equivalent results obtained by utilizing the contents of the present specification, equivalent process changes, or direct or indirect application in other related technical fields, shall be included within the scope of protection of this patent. Example 1

[0017] Take the frozen fresh earthworms, thaw them and wash them: place the frozen fresh earthworms at room temperature to thaw naturally, use clean water to wash the thawed fresh earthworms, and wash away the sludge and sticky substances. Secondly, homogenize and wash: take 1kg of washed earthworms, add 0.14M NaCl homogenization buffer to the washed earthworms at a ratio of m:v=1:1 to crush and homogenize, stir for 30 minutes after crushing, and centrifuge the homogenate to collect the supernatant solution. Then wash the centrifuged precipitate with homogenization buffer at a ratio of m:v=1:1, stir at room temperature for 30 minutes, centrifuge to collect the supernatant solution, and combine the extracted supernatants. Wash twice, and after merging, obtain the final lumbrokinase extraction supernatant, that is, obtain the final earthworm homogenate.

[0018] Perform alcohol precipitation step by step, cool the combined lumbrokinase extraction supernatant, control the temperature at 10℃, then add 95% ethanol to the lumbrokinase extraction supernatant, control the ethanol concentration at 40%, let it stand for 4 hours, centrifuge and collect the supernatant by centrifugation. Continue to add 95% ethanol to the supernatant solution after centrifugation, control the ethanol concentration at 75%, let it stand for 4 hours, centrifuge and collect the precipitate by centrifugation, which is the crude lumbrokinase. Redissolve: Use purified water to re-dissolve the crude lumbrokinase, adjust the pH to 7.5 with 1M NaOH, and add an appropriate amount of purified water to control the solution conductivity to be no higher than 5 ms / cm.

[0019] The obtained crude lumbrokinase was purified by macroporous anion resin: the activated macroporous strong anion resin was added to the reconstituted sample, stirred and exchanged for 4 hours, and then dried. 0.8 M NaCl solution was added to the adsorbed resin for elution, stirred and eluted for 2 hours, dried, and rinsed with elution buffer, and dried. Desalting and concentration: the eluate was ultrafiltered by an ultrafiltration system, the size of the ultrafiltration membrane package was 10000Da, desalting and dehydrating, and the solution conductivity was controlled to be ≤200 us / cm. Filtration and freeze-drying: the ultrafiltration sample was filtered by a filter element, the filtered solution was frozen at -30℃ for 6 hours, and then continued to heat to 35℃ for drying, and crushed to obtain lumbrokinase freeze-dried powder.

[0020] Through the above steps, 7.5 g of white lumbrokinase powder was obtained after freeze-drying and crushing. The lumbrokinase enzyme activity was detected by the fiber plate method, and the activity of the freeze-dried powder was 45850 U / mg. Example 2

[0021] First, take the frozen fresh earthworms and thaw and wash them: place the frozen fresh earthworms at room temperature to thaw naturally, use clean water to wash the thawed fresh earthworms, and wash away the sludge and sticky substances; secondly, homogenize and wash: take 1kg of washed earthworms, add 0.14M NaCl homogenization buffer to the washed earthworms at a ratio of m:v=1:1 to crush and homogenize, stir for 30 minutes after crushing, and centrifuge the homogenate to collect the supernatant solution. Then wash the centrifuged precipitate with homogenization buffer at a ratio of m:v=1:1, stir at room temperature for 30 minutes, centrifuge to collect the supernatant solution, and combine the extracted supernatants. Wash twice, and after merging, obtain the final lumbrokinase extraction supernatant, that is, obtain the final earthworm homogenate.

[0022] Perform step-by-step alcohol precipitation, cool the combined lumbrokinase extraction supernatant, control the temperature at 10°C, then add 95% ethanol to the lumbrokinase extraction supernatant, control the ethanol concentration at 45%, let it stand for 4 hours, centrifuge, and collect the supernatant by centrifugation. Continue to add 95% ethanol to the supernatant solution after centrifugation, control the ethanol concentration at 75%, let it stand for 4 hours, centrifuge, and collect the precipitate by centrifugation, which is the crude lumbrokinase. Redissolve: Use purified water to re-dissolve the crude lumbrokinase, adjust the pH to 7.5 with 1M NaOH, and add an appropriate amount of purified water to control the solution conductivity to be no higher than 5 ms / cm.

[0023] The obtained crude lumbrokinase was purified by macroporous anion resin, and the activated macroporous strong anion resin was added to the reconstituted sample, stirred and exchanged for 4 hours, and then dried. 1.0 M NaCl solution was added to the adsorbed resin for elution, stirred and eluted for 2 hours, and then dried, and rinsed with elution buffer and dried. Desalting and concentration: The eluate was ultrafiltered by an ultrafiltration system, and the size of the ultrafiltration membrane package was 10000Da. Desalting and dehydration were performed, and the conductivity of the solution was controlled to be ≤200 us / cm. Filtration and freeze-drying: The ultrafiltration sample was filtered by a filter element, and the filtered solution was frozen at -30℃ for 6 hours, and then continued to heat to 35℃ for drying, and crushed to obtain lyophilized powder of lumbrokinase.

[0024] Through the above steps, 6.5 g of white lumbrokinase powder was obtained after freeze-drying and crushing. The lumbrokinase enzyme activity was detected by the fiber plate method, and the activity of the freeze-dried powder was 50647 U / mg. Example 3

[0025] Take the frozen fresh earthworms, thaw them and wash them: put the frozen fresh earthworms to thaw naturally at room temperature, use clean water to wash the thawed fresh earthworms, wash away the sludge and sticky substances; then homogenize and wash: take 1kg of washed earthworms, add 0.14M NaCl homogenization buffer to the washed earthworms at a ratio of m:v=1:1 to crush and homogenize, stir for 30 minutes after crushing, centrifuge the homogenate to collect the supernatant solution. Then wash the centrifuged precipitate with homogenization buffer at a ratio of m:v=1:1, stir at room temperature for 30 minutes, centrifuge to collect the supernatant solution, and combine the supernatants. Wash twice, and after merging, obtain the final lumbrokinase extraction supernatant, that is, obtain the final earthworm homogenate.

[0026] Perform step-by-step alcohol precipitation, cool the combined lumbrokinase extraction supernatant, control the temperature at 10°C, then add 95% ethanol to the lumbrokinase extraction supernatant, control the ethanol concentration at 40%, let it stand for 4 hours, centrifuge, and collect the supernatant by centrifugation. Continue to add 95% ethanol to the supernatant solution after centrifugation, control the ethanol concentration at 75%, let it stand for 4 hours, centrifuge, and collect the precipitate by centrifugation, which is the crude lumbrokinase. Redissolve: Use purified water to re-dissolve the crude lumbrokinase, adjust the pH to 8.5 with 1 M NaOH, and add an appropriate amount of purified water to control the solution conductivity to be no higher than 5 ms / cm.

[0027] The obtained crude lumbrokinase was purified by macroporous anion resin, and the activated macroporous weak anion resin was added to the reconstituted sample, stirred and exchanged for 4 hours, and then dried. 1.0 M NaCl solution was added to the adsorbed resin for elution, stirred and eluted for 2 hours, and then dried, and rinsed with elution buffer and dried. Desalting and concentration: The eluate was ultrafiltered by an ultrafiltration system, and the size of the ultrafiltration membrane package was 10000Da. Desalting and dehydration were performed, and the conductivity of the solution was controlled to be ≤200 us / cm. Finally, filtration and freeze-drying were performed: the ultrafiltration sample was filtered by a filter element, and the filtered solution was frozen at -30℃ for 6 hours, and then continued to heat to 35℃ for drying, and crushed to obtain lumbrokinase freeze-dried powder.

[0028] After the steps of this example are performed, 7.2 g of white lumbrokinase powder can be obtained after freeze-drying and crushing. The lumbrokinase enzyme activity is detected by the fiber plate method, and the activity of the freeze-dried powder is 48432 U / mg.

[0029] The above descriptions are only embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent results obtained by utilizing the contents of the present invention specification through equivalent process transformation, or direct or indirect application in other related technical fields, should be included in the protection scope of this patent.

Claims

1. A method for preparing lyophilized powder of lumbrokinase, characterized in that: By subjecting earthworm homogenate to step-by-step ethanol precipitation, impurity proteins can be removed to obtain crude lumbrokinase, which is then purified by macroporous anion resin adsorption to obtain highly active lumbrokinase freeze-dried powder.

2. The method for preparing a lyophilized powder of lumbrokinase according to claim 1, characterized in that: The activity of the lyophilized powder of lumbrokinase prepared is not less than 45,000 U / mg.

3. The method for preparing a lyophilized powder of lumbrokinase according to claim 1, characterized in that: The protein distribution of the prepared lyophilized powder of lumbrokinase is between 20 kDa and 50 kDa.

4. The method for preparing a lyophilized powder of lumbrokinase according to claim 1, characterized in that: The preparation of the lumbrokinase freeze-dried powder comprises the following steps: Step 1: Thawing and cleaning earthworms: Place frozen fresh earthworms at room temperature to thaw naturally, and use clean water to clean the thawed fresh earthworms to remove mud and sticky substances; Step 2: homogenization and washing, according to the ratio of m:v=1:1, add 0.14M NaCl homogenization buffer to the washed earthworms for crushing and homogenization, stir for 30 minutes after crushing, and centrifuge the obtained homogenate to collect the supernatant solution; then wash the centrifuged precipitate with homogenization buffer according to the ratio of m:v=1:1, stir at room temperature for 30 minutes, centrifuge to collect the supernatant solution, and combine the extracted supernatants; wash twice, and after combining, obtain the final lumbrokinase extraction supernatant, that is, obtain the final earthworm homogenate; Step 3: Step by step alcohol precipitation, cool down the combined lumbrokinase extraction supernatant, and control the temperature at 4-10℃. The crude lumbrokinase was obtained by stepwise alcohol precipitation, and the ethanol concentration used in each step of alcohol precipitation was controlled to be different.

5. The method for preparing a lyophilized powder of lumbrokinase according to claim 4, characterized in that: The following steps are also included: Step 4: Redissolve the crude lumbrokinase with purified water, adjust the pH with 1M NaOH, add purified water and control the solution conductivity; Step 5: Purification with macroporous anion resin: add activated macroporous strong anion resin or macroporous weak anion resin to the reconstituted sample, stir and exchange for 2-4 h, and drain; add 0.5-0.8 M NaCl solution to the adsorbed resin for elution, stir and elute for 1-2 h, drain, and rinse with elution buffer, and drain; Step 6: Desalting and concentrating, the obtained eluate is ultrafiltered through an ultrafiltration system, the size of the ultrafiltration membrane package is 10000Da, desalting and dehydrating, and the solution conductivity is controlled to be ≤200 us / cm; Step 7: Filtration and freeze-drying. Filter the sample after ultrafiltration with a filter element, freeze the filtered solution at -30~-40℃ for 6h, warm it up, and crush it to obtain lyophilized powder of lumbrokinase.

6. The method for preparing a lyophilized powder of lumbrokinase according to claim 4, characterized in that: The step of stepwise alcohol precipitation after cooling in step three is to add 95% ethanol by volume to the supernatant of lumbrokinase extraction, control the ethanol concentration at 30-45%, let it stand for 4-6 hours and then centrifuge to collect the supernatant; continue to add 95% ethanol by volume to the supernatant solution after centrifugation, control the ethanol concentration at 70-80%, let it stand for 4-6 hours and then centrifuge to collect the precipitate, which is the crude lumbrokinase.

7. The method for preparing a lyophilized powder of lumbrokinase according to claim 5, characterized in that: In the step 4, 1 M NaOH is used to adjust the pH to 7.5-8.5, and purified water is added to control the solution conductivity to be no higher than 5 ms / cm.

8. The method for preparing a lyophilized powder of lumbrokinase according to claim 5, characterized in that: The elution buffer in step 5 is specifically 0.8-1.0 M NaCl buffer.

9. The method for preparing a lyophilized powder of lumbrokinase according to claim 5, characterized in that: In the step 7, the temperature is controlled at 30-40° C. for drying, and the lumbrokinase freeze-dried powder is obtained by crushing.

10. The lyophilized powder of lumbrokinase obtained by the preparation method according to any one of claims 1 to 9 can be used for preventive applications in the fields of skin care products and cardiovascular, endocrine, and respiratory systems.

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