CN111154734A - Preparation method of antarctic krill thrombolytic enzyme - Google Patents
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Abstract
The invention discloses a antarctic krill thrombolytic enzyme and a preparation method thereof. Which comprises the following steps: 1) crushing Antarctic krill at low temperature; 2) mixing with pre-cooled 0.1-0.3mol/L, pH 6-8 phosphate buffer solution, and centrifuging; 3) mixing the supernatant with 20-75% (w/v) ammonium sulfate, precipitating at 4 deg.C overnight, centrifuging, dissolving the precipitate in 2-10mM CaCl2Dialyzing and desalting at 4 ℃ in Tris-HCl buffer solution with the pH value of 7.0-7.5 for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum; 4) then Sepharose F ion exchange column chromatography is adopted, 40-85% (w/v) ammonium sulfate is precipitated at 4 ℃ overnight, dialyzed and desalted, and dialyzate is frozen and concentrated and is frozen and dried in vacuum; 5) and performing Sephadex G-75 gel filtration chromatography, performing overnight precipitation with 60-95% (w/v) ammonium sulfate, dialyzing, desalting, freeze-concentrating the dialyzate, and performing vacuum freeze-drying to obtain the antarctic krill thrombolytic enzyme. The method has the advantages of obviously improving the separation efficiency of the target product, realizing the high-efficiency separation of the antarctic krill enzyme and obviously increasing the additional value of the antarctic krill product, along with high yield.
Description
Technical Field
The invention belongs to the field of marine biological resource utilization, and particularly relates to a preparation method of antarctic krill thrombolytic enzyme.
Background
The thrombotic diseases mainly comprise two types: arterial thromboembolic disorders and venous thromboembolic disorders.
Arterial thrombosis is primarily due to rupture of the atheromatous plaque. Atherosclerotic plaques are formed by the deposition of lipids and the accumulation of foam cells on the walls of arterial blood vessels. The atherosclerotic plaque and subendothelial collagen fibers come into contact with blood components, and the coagulation process is initiated, eventually leading to thrombus formation. Venous thrombosis is primarily caused by changes in the composition of blood or slowing or stopping blood flow or changes in the walls of the veins.
At present, three main methods for treating thrombotic diseases include surgery, taking anticoagulant, injecting thrombolytic agent and the like. Thrombolytic therapy includes intra-arterial thrombolysis, intra-venous thrombolysis, arteriovenous combined thrombolysis, device-assisted thrombolysis, ultrasonic thrombolysis and other methods.
The development of thrombolytic agents is divided into three generations: the first generation of thrombolytic agents include Streptokinase (SK) and Urokinase (UK), which have good thrombolytic effect but lack fibrin selectivity, and can activate plasminogen on the surface of thrombus, as well as free plasminogen in plasma, so that antiplasmin is largely consumed and fibrinogen is degraded, and systemic bleeding side effects are generated, and SK repeated use is likely to cause anaphylaxis. UK is a proteolytic enzyme extracted from human urine, and can activate plasminogen and prevent thrombosis. Because of being derived from human bodies, having no specific antigenicity and high thrombolysis efficiency, the domestic cerebral thrombosis thrombolysis treatment still takes UK as the main treatment. However, UK has a short half-life (about 15min), and is prone to systemic bleeding when administered in large amounts for a long period of time, and is generally not effective when administered orally. The defects are mainly that the bleeding side effect is large, and the like, and the traditional Chinese medicine is eliminated.
Second generation thrombolytic agents include: tissue plasminogen activator (t-PA), prourokinase (pro-UK) and p-methoxybenzoyl (anisoyl) plasminogen streptokinase activator complex (APSAC). t-PA and pro-UK have obvious fibrin affinity in vitro and in animal experiments, and have small bleeding side effect. The second generation thrombolytic agents were used in the first clinical setting with the most experience and are more common. The method is simple and convenient to operate, timely in administration and free of special equipment, so that the method is still widely applied up to now. The main disadvantages are large dosage, low specificity, poor curative effect and high possibility of bleeding complications.
Third generation thrombolytics are t-PA variants, such as TNKase (teneplase, TNK-t-PA), Monteplase, Lanoteplase (Lanateplase, n-PA) and the like. The third generation thrombolytic agent has the characteristics of rapid thrombolysis, opening blocked coronary artery, recovering blood circulation and high cure rate, does not need intravenous injection in hospitals, does not need to adjust dosage due to weight, has long half-life period and the like. The disadvantage is the high price.
With the aging population, the incidence of thrombotic diseases tends to increase year by year. WHO predicts that by 2020, stroke will be one of the leading diseases worldwide causing death and disability, while ischemic stroke will account for 85%. The patients above 1/3 in stroke survivors become disabled and place a heavy burden on families and society, so the prevention and treatment of stroke, especially for ischemic stroke, is of great importance. In view of the limited sources, complex production process, high price and other factors of the current commercial thrombolytic drug, the yield and the use of the product are limited, and the treatment of patients is directly influenced. Therefore, there is a need to develop thrombolytic enzymes with wide sources and good effect.
Antarctic krill (Euphausia superba) is a small planktonic crustacean living in Antarctic sea area, and the amount of biological resources is huge; due to the special habitat, the biological activity-enhancing agent has the potential of generating special bioactive substances, and therefore, has good development and application potential. Research shows that the antarctic krill enzyme has the characteristics of high activity and low temperature resistance. Although antarctic krill has a highly efficient enzyme system, its activity is affected by in vivo enzyme inhibitors; the enzyme system of the live antarctic krill is in a dynamic equilibrium state; after the Antarctic krill is dead, the dynamic equilibrium is broken and autolysis of the Antarctic krill occurs. Based on the characteristics of high activity and low temperature resistance of the antarctic krill enzyme, the antarctic krill enzyme has wide application potential in the fields of food, medicine, washing and the like.
The antarctic krill enzyme has good effect on treating cardiovascular and cerebrovascular diseases. Therefore, research on a preparation method and application of antarctic krill thrombolytic enzyme is needed to promote deep development of the value of antarctic krill.
Disclosure of Invention
The invention provides the antarctic krill thrombolytic enzyme and the preparation method thereof aiming at the problem of lack of the antarctic krill thrombolytic enzyme.
The technical scheme of the invention is as follows: a preparation method of antarctic krill thrombolytic enzyme comprises the following steps:
1) crushing the euphausia superba at low temperature, and controlling the average particle size to be 0.5-1.5cm to obtain a crushed euphausia superba;
2) mixing the crushed Antarctic krill with 0.1-0.3mol/L, pH 6-8 phosphate buffer pre-cooled at 4 ℃ at a solid-to-liquid ratio of 1:2-1:8(w/v), stirring for 1-5h, centrifuging for 20-40min at 4 ℃ at 8000-;
3) mixing the first extract of antarctic krill thrombolytic enzyme with 20-75% (w/v) ammonium sulfate at equal volume, and precipitating at 4 deg.C overnight; centrifuging at 8000-; dissolving the precipitate in a solvent containing 2-10mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with pH of 7.0-7.5 at 4 ℃ for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a second extract of the streptokinase of the Antarctic krill;
4) subjecting the second extract of the antarctic krill thrombolytic enzyme to Sepharose F ion exchange column chromatography, performing linear gradient elution by using 0-0.1M NaCl solution, evaluating the thrombolytic activity of different components, and collecting the eluate with strong thrombolytic activity; adding 40-85% (w/v) ammonium sulfate in the same volume at 4 ℃ for overnight precipitation; centrifuging at 8000-; dissolving the precipitate in CaCl containing 2-10mM2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0-7.5 at the temperature of 4 ℃ for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a third extract of the antarctic krill thrombolytic enzyme;
5) performing Sephadex G-75 gel filtration chromatography on the third extract of the antarctic krill thrombolytic enzyme, eluting with 0.1M phosphate buffer solution with pH7.5 and containing 0-0.05M NaCl, evaluating the thrombolytic activity of different components, and collecting the eluate with strong thrombolytic activity; adding equal volume60-95% (w/v) ammonium sulfate at 4 ℃ overnight; centrifuging at 8000-; dissolving the precipitate in CaCl containing 2-10mM2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0-7.5 at 4 ℃ for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain the antarctic krill thrombolytic enzyme.
In step 1), the antarctic krill is fresh or frozen antarctic krill, preferably frozen antarctic krill. The low temperature is 0-8 ℃, preferably 4 ℃. The low-temperature crushing is preferably performed by a wet low-temperature shearing machine. The average particle size is preferably controlled to be between 0.8 and 1.0 cm.
In the invention, the centrifugation is carried out at 4 ℃ and 8000-.
The invention has the beneficial effects that: the method provided by the invention can realize the efficient preparation of the thrombolytic enzyme of the antarctic krill, optimize the processing technology of the antarctic krill and improve the industrial value of the antarctic krill. According to the invention, the raw materials are treated by adopting a low-temperature shearing technology, the separation of the enzyme is realized by adopting a chromatography and ammonium sulfate precipitation technology, the separation efficiency of the target product is obviously improved, and the high-efficiency separation of the antarctic krill enzyme is realized. The method is expected to promote the improvement of the additional value of the euphausia superba. The invention has simple technical route and easy realization; safe operation and little environmental pollution. The method is applied to the antarctic krill, has high enzyme yield, and can obviously increase the additional value of the antarctic krill product.
Detailed Description
The present invention will be more clearly and completely described below in connection with preferred embodiments.
Example 1
Preparation method of antarctic krill thrombolytic enzyme
Step 1)
The frozen Antarctic krill is crushed by a wet low-temperature (4 ℃) shearing machine, and the average particle size is controlled to be 0.5 cm.
Step 2)
Adding 0.1mol/L, pH 6 phosphate buffer pre-cooled at 4 deg.C with solid-to-liquid ratio of 1:2(w/v) into the pulverized material of frozen Euphausia superba, and mixing well. After stirring for 1h, centrifuging (4 ℃, 10000r/min, centrifugation for 20min) to obtain a supernatant which is a first extract of antarctic krill thrombolytic enzyme.
Step 3)
The first extract of the antarctic krill thrombolytic enzyme is added with equal volume of 20% (w/v) ammonium sulfate and mixed evenly, and the mixture is precipitated overnight at 4 ℃. Centrifuging at 4 deg.C and 10000r/min for 20min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 2mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution (pH 7.0) (4 ℃, dialyzing for 12h), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a second extract of the antarctic krill thrombolytic enzyme.
Step 4)
And (3) performing Sepharose F ion exchange column chromatography on the second extract of the antarctic krill thrombolytic enzyme, performing linear gradient elution by adopting a 0-0.1MNaCl solution, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 40% (w/v) ammonium sulfate was added for precipitation (overnight at 4 ℃). Centrifuging at 4 deg.C and 10000r/min for 20min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 2mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0 (4 ℃, dialyzing for 12 hours), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a third extract of the antarctic krill thrombolytic enzyme.
Step 5)
And (3) performing Sephadex G-75 gel filtration chromatography on the third extract of the antarctic krill thrombolytic enzyme, eluting by 0.1M phosphate buffer solution with pH7.5 and containing 0-0.05MNaCl, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 60% (w/v) ammonium sulfate was added for precipitation (4 ℃, overnight). Centrifuging at 4 deg.C and 10000r/min for 20min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 2mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0-7.5 (4 ℃, dialyzing for 12h), freezing and concentrating the dialyzate, and carrying out vacuum freeze drying to obtain the antarctic krill thrombolytic enzyme.
Example 2
Preparation method of antarctic krill thrombolytic enzyme
Step 1)
The frozen Antarctic krill is crushed by a wet low-temperature (4 ℃) shearing machine, and the average particle size is controlled to be 1.5 cm.
Step 2)
Adding 0.3mol/L, pH 8 phosphate buffer pre-cooled at 4 deg.C with solid-to-liquid ratio of 1:8(w/v) into the pulverized material of frozen Euphausia superba, and mixing well. After stirring for 5h, centrifuging (4 ℃, 10000r/min, centrifugation 40min), and obtaining supernatant as the first extract of the antarctic krill thrombolytic enzyme.
Step 3)
The first extract of the antarctic krill thrombolytic enzyme is added with 75% (w/v) ammonium sulfate with the same volume and mixed evenly, and the mixture is precipitated overnight at 4 ℃. Centrifuging at 4 deg.C and 10000r/min for 40min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in 10mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution (pH 7.5) (4 ℃, dialyzing for 36h), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a second extract of the antarctic krill thrombolytic enzyme.
Step 4)
And (3) performing Sepharose F ion exchange column chromatography on the second extract of the antarctic krill thrombolytic enzyme, performing linear gradient elution by adopting a 0-1.0MNaCl solution, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 85% (w/v) ammonium sulfate was added for precipitation (overnight at 4 ℃). Centrifuging at 4 deg.C and 10000r/min for 40min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in 10mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with pH of 7.5 (4 ℃, dialyzing for 36h), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a third extract of the antarctic krill thrombolytic enzyme.
Step 5)
And (3) performing Sephadex G-75 gel filtration chromatography on the third extract of the antarctic krill thrombolytic enzyme, eluting by 0.1M phosphate buffer solution with pH7.5 and containing 0-0.05MNaCl, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 95% (w/v) ammonium sulfate was added for precipitation (4 ℃, overnight). Centrifuging at 4 deg.C and 10000r/min for 40min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in 10mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.5 (4 ℃, dialyzing for 36 hours), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain the antarctic krill thrombolytic enzyme.
Example 3
Preparation method of antarctic krill thrombolytic enzyme
Step 1)
The frozen antarctic krill is crushed by a wet low-temperature (4 ℃) shearing machine, and the average particle size is controlled to be 1.0 cm.
Step 2)
Adding 0.2mol/L, pH7 phosphate buffer pre-cooled at 4 deg.C with solid-to-liquid ratio of 1:5(w/v) into the pulverized material of frozen Euphausia superba, and mixing well. After stirring for 3h, centrifuging (4 ℃, 10000r/min, centrifuging for 30min) to obtain a supernatant which is the first extract of the antarctic krill thrombolytic enzyme.
Step 3)
The first extract of the antarctic krill thrombolytic enzyme is added with equal volume of 45% (w/v) ammonium sulfate and mixed evenly, and the mixture is precipitated overnight at 4 ℃. Centrifuging at 4 deg.C and 10000r/min for 30min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 7mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0 (4 ℃, dialyzing for 24 hours), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a second extract of the antarctic krill thrombolytic enzyme.
Step 4)
And (3) performing Sepharose F ion exchange column chromatography on the second extract of the antarctic krill thrombolytic enzyme, performing linear gradient elution by adopting a 0-0.1MNaCl solution, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 65% (w/v) ammonium sulfate was added for precipitation (overnight at 4 ℃). Centrifuging at 4 deg.C and 10000r/min for 30min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 7mM CaCl2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0 (4 ℃, dialyzing for 24 hours), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a third extract of the antarctic krill thrombolytic enzyme.
Step 5)
And (3) performing Sephadex G-75 gel filtration chromatography on the third extract of the antarctic krill thrombolytic enzyme, eluting by 0.1M phosphate buffer solution with pH7.5 and containing 0-0.05MNacl, evaluating the thrombolytic activity of different components, and collecting the eluent part with strong thrombolytic activity. An equal volume of 85% (w/v) ammonium sulfate was added for precipitation (4 ℃, overnight). Centrifuging at 4 deg.C and 10000r/min for 30min, discarding supernatant, and retaining precipitate. The precipitate was dissolved in a solution containing 7mM CaCl2pH of 7.0TriDialyzing and desalting in s-HCl buffer solution (4 ℃, dialyzing for 24h), freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain the antarctic krill thrombolytic enzyme.
Comparative example 1
The same procedure as in example 1 was repeated except that the ammonium sulfate precipitation step was not used in the conventional production method.
Even if the same technological process is adopted, the final separation is not complete due to lack of ammonium sulfate precipitation link. Therefore, the separation of the antarctic krill thrombolytic enzyme cannot be realized.
Comparative example 2
The original preparation method is the same as example 1 except that the dialysis step is not performed.
Even if the same process flow is adopted, the final separation is not thorough due to the lack of a dialysis link. Therefore, the separation and purification of the antarctic krill thrombolytic enzyme cannot be realized.
Effect example 1
The yield and the purification fold of the thrombolytic enzyme prepared from antarctic krill in examples 1-3 and comparative examples 1-2 are shown in table 1.
TABLE 1 Effect of preparation conditions on thrombolytic enzyme of Antarctic krill
Effect example 2
The total enzyme activity, total protein, specific activity, yield and purification fold of the antarctic krill thrombolytic enzyme extract obtained in each step of example 3 are shown in table 2. Wherein, the measuring and calculating method comprises the following steps:
preparation of fibrin plate: adding 2.5g of agarose into 50mL of physiological saline, dissolving, placing in a 45 ℃ water bath for heat preservation for 30min, adding 1mL of 200U/mL thrombin, uniformly mixing, dissolving 0.2 g of fibrinogen in 50mL of barbital buffer solution (pH 7), placing in a 45 ℃ water bath for heat preservation for 5min, respectively taking 5mL of thrombin solution and 5mL of fibrinogen solution, uniformly mixing, quickly pouring into a plate with the diameter of 9cm, horizontally placing for 30min, and placing in a 4 ℃ refrigerator for later use.
And (3) enzyme activity determination: three holes are punched on the prepared flat plate by adopting a rubber head dropper, then 10 mu L of enzyme solution is loaded by a microsyringe, the flat plate is placed in a constant temperature box at 37 ℃ for heat preservation for 18h, the length of two vertical diameters of the lysis ring is photographed and measured, the average value is taken, the area of the lysis ring is calculated, and then the enzyme activity unit is calculated. Definition of enzyme activity units: one unit of enzyme activity was taken as the amount of enzyme per 1 mm square of increase in the area of the lysostane under the above conditions. See table 2.
And (3) specific activity determination: 10 mul each of urokinase samples (100, 200, 300, 400, 500U/mL) are spotted on a newly prepared fibrin plate, the fibrin plate is placed for 10min, the fibrin plate is moved to a 37 ℃ incubator, the incubation is carried out for 18h, then the fibrin plate is taken out, the diameter of a lysostan is measured, and the area of each lysostan is calculated. And calculating the activity of the sample according to the standard curve by taking the area of the lysis ring as an abscissa and the enzyme activity as an ordinate. See table 2.
Protein content determination: the protein content was determined by the Bradford (1976) method (i.e., Coomassie Brilliant blue method). See table 2.
Table 2 example 3 effect of preparation procedure on antarctic krill thrombolytic enzyme
Effect example 3
The activity of the antarctic krill thrombolytic enzyme is evaluated by adopting a coagulation experiment. The thrombolytic effect of the thrombolytic enzyme of antarctic krill obtained in example 3 was examined by the following method:
fresh chicken is taken for coagulation, cut into 0.3cm multiplied by 0.3cm distilled water and washed, 0.5g of each five equal parts are accurately weighed and respectively added into five test tubes, 4mL of enzyme solution is added into each tube, one test tube is added with sodium phosphate buffer solution as a control, and sampling detection is carried out on time. See table 3.
TABLE 3 thrombolytic effect of antarctic krill thrombolytic enzyme obtained in example 3
The above description merely illustrates some embodiments of the present invention, and does not intend to limit the scope of the present invention. The invention extends to any novel feature or any novel combination of features disclosed herein, and any novel method or process steps or novel combination disclosed. It is intended that all technical solutions and inventive concepts according to the present invention and all technical equivalents thereof which are equivalent or modified by those skilled in the art are included in the scope of the present invention.
Claims (7)
1. A preparation method of antarctic krill thrombolytic enzyme is characterized by comprising the following steps:
1) crushing the euphausia superba at low temperature, and controlling the average particle size to be 0.5-1.5cm to obtain a crushed euphausia superba;
2) mixing the crushed Antarctic krill with 0.1-0.3mol/L, pH 6-8 phosphate buffer pre-cooled at 4 ℃ at a solid-to-liquid ratio of 1:2-1:8(w/v), stirring for 1-5h, centrifuging for 20-40min at 4 ℃ at 8000-;
3) mixing the first extract of antarctic krill thrombolytic enzyme with 20-75% (w/v) ammonium sulfate at equal volume, and precipitating at 4 deg.C overnight; centrifuging at 8000-; dissolving the precipitate in CaCl containing 2-10mM2Dialyzing and desalting in Tris-HCl buffer solution with pH of 7.0-7.5 at 4 ℃ for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain a second extract of the streptokinase of the Antarctic krill;
4) subjecting the second extract of the antarctic krill thrombolytic enzyme to Sepharose F ion exchange column chromatography, performing linear gradient elution by using 0-0.1MNaCl solution, evaluating the thrombolytic activity of different components, and collecting the eluate with strong thrombolytic activity; adding 40-85% (w/v) ammonium sulfate in the same volume at 4 ℃ for overnight precipitation; centrifuging at 8000-; dissolving the precipitate in CaCl containing 2-10mM2Dialyzing and desalting in Tris-HCl buffer solution with pH of 7.0-7.5 at 4 deg.C for 12-36h, freeze-concentrating the dialyzed solution, and vacuum freeze-drying to obtain Euphausia superba solutionA third extract of the enzyme;
5) performing Sephadex G-75 gel filtration chromatography on the third extract of the antarctic krill thrombolytic enzyme, eluting with 0.1M phosphate buffer solution with pH7.5 and containing 0-0.05MNaCl, evaluating the thrombolytic activity of different components, and collecting the eluate with strong thrombolytic activity; adding an equal volume of 60-95% (w/v) ammonium sulfate for overnight precipitation at 4 ℃; centrifuging at 8000-; dissolving the precipitate in CaCl containing 2-10mM2Dialyzing and desalting in Tris-HCl buffer solution with the pH value of 7.0-7.5 at 4 ℃ for 12-36h, freezing and concentrating the dialyzate, and freeze-drying in vacuum to obtain the antarctic krill thrombolytic enzyme.
2. The method according to claim 1, wherein in step 1), the antarctic krill is fresh or frozen antarctic krill.
3. The method according to claim 1, wherein the low temperature in step 1) is 4 ℃.
4. The method of claim 1, wherein the cryo-disruption is disrupted using a wet cryo-shearer.
5. The method according to claim 1, wherein the average particle size is controlled to be 0.8 to 1.0 cm.
6. The method according to claim 1, wherein the centrifugation is performed at 4 ℃ and 8000-.
7. The antarctic krill thrombolytic enzyme prepared by the method of any one of claims 1 to 6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0170115A1 (en) * | 1984-07-20 | 1986-02-05 | Kao Corporation | Thrombus dissolvent |
| CN1181018A (en) * | 1995-02-08 | 1998-05-06 | 费尔森医疗有限公司 | Multifunctional enzyme |
-
2019
- 2019-09-23 CN CN201910899601.3A patent/CN111154734A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0170115A1 (en) * | 1984-07-20 | 1986-02-05 | Kao Corporation | Thrombus dissolvent |
| CN1181018A (en) * | 1995-02-08 | 1998-05-06 | 费尔森医疗有限公司 | Multifunctional enzyme |
Non-Patent Citations (2)
| Title |
|---|
| ZHIQIANG WU等: "Purification and Characterization of Cold Adapted Trypsins from Antarctic krill (Euphausia superba)", 《INT J PEPT RES THER》 * |
| 燕梦雅等: "南极磷虾来源酶的研究进展", 《食品工业科技》 * |
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