CN1253564C - Method for removing hybrid protein from lumbrokinase crude product - Google Patents
Method for removing hybrid protein from lumbrokinase crude product Download PDFInfo
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- CN1253564C CN1253564C CN 200410054043 CN200410054043A CN1253564C CN 1253564 C CN1253564 C CN 1253564C CN 200410054043 CN200410054043 CN 200410054043 CN 200410054043 A CN200410054043 A CN 200410054043A CN 1253564 C CN1253564 C CN 1253564C
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- Prior art keywords
- lumbrukinase
- crude product
- solution
- foreign protein
- preheating temperature
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- 238000000034 method Methods 0.000 title claims abstract description 44
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 39
- 239000012043 crude product Substances 0.000 title claims abstract description 38
- 108010070324 lumbrokinase Proteins 0.000 title abstract description 3
- 108090000790 Enzymes Proteins 0.000 claims abstract description 32
- 102000004190 Enzymes Human genes 0.000 claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000003756 stirring Methods 0.000 claims abstract description 20
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 13
- 239000002244 precipitate Substances 0.000 claims description 10
- 230000008030 elimination Effects 0.000 claims description 9
- 238000003379 elimination reaction Methods 0.000 claims description 9
- 239000012467 final product Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 239000000243 solution Substances 0.000 abstract 5
- 239000000470 constituent Substances 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000011259 mixed solution Substances 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 230000000717 retained effect Effects 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 28
- 238000001556 precipitation Methods 0.000 description 25
- 238000005516 engineering process Methods 0.000 description 14
- 239000002253 acid Substances 0.000 description 13
- 239000004480 active ingredient Substances 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 238000005303 weighing Methods 0.000 description 7
- 241000361919 Metaphire sieboldi Species 0.000 description 5
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- 102000020233 phosphotransferase Human genes 0.000 description 3
- 108010088842 Fibrinolysin Proteins 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 229940012957 plasmin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000243686 Eisenia fetida Species 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 102000010404 kinase activity proteins Human genes 0.000 description 1
- 108040005184 kinase activity proteins Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a method for removing hybrid protein from a lumbrokinase crude product. The method comprises the following steps: 1) firstly, dissolving the lumbrukinase crude product in alcohol-water solution to prepare solution of which the concentration of crude enzyme of the mass percent of 2 to 7% and the concentration of the alcohol volume percent of 0 to 40%, and filtering insoluble substances after repeatedly stirring and dissolving the solution; 2) then, adding 20mL of the mixed solution to an autoclave of which the preheating temperature is from 20 DEG C to 40 DEG C, supplying pressurization CO2 of which the preheating temperature is from 20 DEG C to 40 DEG C and the pressure is from 3.5 to 8.5MPa and stirring, regulating the pH value of the solution to 4.4, and keeping the pH value in high pressure for 20 to 60 minutes; at the time, the hybrid protein in the lumbrukinase crude product is completely settled under the condition, and active components are still retained in the solution; separating settlement materials. The present invention has the characteristics of relatively mild operating condition, possibility of simultaneously obtaining other active constituents, small enzyme activity loss, etc.; the present invention is a lumbrukinase separation and purification method having accurate controllable operation parameters and no fluctuation in operation process.
Description
Technical field
The present invention relates to a kind of separation method, especially relate to a kind of employing pressurization CO
2As volatile acid, ethanol separates the method for purification Lumbrukinase as the isoelectric precipitation foreign protein that helps precipitation agent.
Background technology
Lumbrukinase (lumbrokinase) is the proteinic general name of the polycomponent with plasmin activity that extracts in earthworm, and being has one of fibrinolytic medicine of DEVELOPMENT PROSPECT at present.Existing document shows, Lumbrukinase is that one group of molecular weight is 20～40kD, has the serine protease of kinases and plasmin activity, wherein concentrates on below 4.0 than the multiple active components iso-electric point.In addition, also contain some other bioactive ingredients in the earthworm, thereby adopt way separation purification Lumbrukinase gentle, environmental protection to have more important meaning separating of multiple nutrients and effective ingredient for wherein.Lumbrukinase is that fresh Eisenia foetida is smashed to pieces at present, centrifugal collection supernatant, and saltouing from supernatant liquor then obtains precipitation, the roughly processed product [1] after the desalination freeze-drying then.Patent 00132716.X discloses a kind of separating technology of Lumbrukinase, comprise salt handle, clean, with damping fluid, slurrying, centrifugal, affinity chromatography, steps such as ion exchange chromatography, this process operation complexity, the enzyme loss amount is bigger.And this separating technology is not considered the extraction and the separation problem of other active ingredients in the earthworm.
Reference
[1] Wang Dongpeng, Sun Guibao, high actirity earthworm kinase Study on extraction, Ningxia science and technology, 2001, (6), 44-45
Summary of the invention
The purpose of this invention is to provide the method for removing foreign protein in a kind of Lumbrukinase crude product.The step of method is as follows:
1) at first, the Lumbrukinase crude product is dissolved in the ethanol-water solution, preparing thick enzyme concn is 2～7% (mass percents), and alcohol concn is the solution of 0～40% (volume percent), repeatedly elimination insolubles after the stirring and dissolving;
2) then, getting the above-mentioned mixing solutions of 20mL, to join preheating temperature be in 20～40 ℃ of autoclaves, feeds preheating temperature then and be 20～40 ℃ pressurization CO
2Stir simultaneously, make pressure increase to 3.5～8.5MPa, the pH of regulator solution is 4.4, under high pressure kept 20～60 minutes, foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution, precipitate and separate is gone out to get final product again.
The present invention compares with traditional isoelectric precipitation method, and enzyme is lived in losing and lacked, and has more advantage than traditional isoelectric precipitation system again simultaneously, and is relatively gentleer as operational condition, and can obtain the various active composition in the Lumbrukinase crude product simultaneously.The alcoholic acid adding can make water-soluble stronger protein be easy to be precipitated out in addition, and institute's adding assistant amount of alcohol will be lacked much than organic solvent precipitation method, thereby enzyme is lived loss seldom.Adopt pressurization CO
2Excessive acid or the low excessively situation of local pH when the adjusting of carrying out pH can be avoided with regulator solution pH such as mineral acids reduce the protein denaturation of therefore bringing.Because CO
2Therefore exist with molecular form mostly in solution, this makes solution ion strength can too much not raise, thereby is more suitable for pH regulator in the isoelectric precipitation than mineral acid.The invention solves existing in prior technology operational condition harshness, can't obtain other activeconstituents in the earthworm enzyme simultaneously, technical problems such as the enzyme loss amount is bigger, provide a kind of operational condition gentle relatively, can obtain other active ingredient simultaneously, the separating and purifying method of enzyme break-even substantially Lumbrukinase alive.Technical problems such as the present invention has also solved the existing in prior technology operational condition and has been difficult to accurate control, and the operating process fluctuation is bigger provide a kind of operating parameters accurately to control, the separating and purifying method of the pulsation-free a kind of Lumbrukinase of operating process.
Embodiment
The present invention adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology; Separate the preparation that purification step comprises the Lumbrukinase ethanol-water solution, with pressurization CO
2PH value of solution is transferred to required scope, the foreign protein that filtering-depositing comes out, and Lumbrukinase still remains in the filtrate.For realizing this process, at first prepare the ethanol-water solution of the Lumbrukinase of suitable concn, get an amount of solution then and join in the autoclave, feed pressurization CO then
2, be used for the pH of regulator solution to required scope, because the proteinic precipitation of some the no kinase activities pH in the Lumbrukinase crude product is between 3.0～5.0, and the Lumbrukinase class does not precipitate in this scope, thereby uses CO
2When pH value of solution was transferred to this scope, the no kinase activity protein in the Lumbrukinase will be precipitated out, and separated the effect of purifying thereby reach.Pressurization CO of the present invention
2-ethanol-water system is used for isoelectric precipitation protein, the autoclave preheating temperature and the CO of the separating and purifying method of Lumbrukinase
2Preheating temperature is 20～40 ℃, CO
2Pressure is 3.5～8.5MPa, and the cellulase mass percent concentration is 2～7%, and the ethanol concentration of volume percent is 0～40%, CO
2It is 20～60 minutes that pressure is held time.
Below by specific embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 30% (volume percent), and thick enzyme concn is the solution of 4% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 25 ℃.Then feed the pressurization CO of 25 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 7.0MPa, and regulate service temperature to 25 ℃, this moment, the pH of solution was 4.4, and deposited phenomenon is obvious in the still, under high pressure keeps 40 minutes, foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 98.6%, the purifying multiple 2.53 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 2: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 40% (volume percent), and thick enzyme concn is the solution of 6% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 25 ℃.Then feed the pressurization CO of 25 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 4.5MPa, and regulate service temperature to 25 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 55 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 94.1%, the purifying multiple 2.41 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 3: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 35% (volume percent), and thick enzyme concn is the solution of 2% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 20 ℃.Then feed the pressurization CO of 20 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 3.5MPa, and regulate service temperature to 20 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 30 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 97.9%, the purifying multiple 1.11 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 4: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 0% (volume percent), and thick enzyme concn is the solution of 5% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 40 ℃.Then feed the pressurization CO of 40 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 8.5MPa, and regulate service temperature to 40 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 20 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 98.1%, the purifying multiple 1.29 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 5: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 30% (volume percent), and thick enzyme concn is the solution of 3% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 35 ℃.Then feed the pressurization CO of 35 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 6.0MPa, and regulate service temperature to 35 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 60 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 97.6%, the purifying multiple 1.63 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 6: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 15%, and thick enzyme concn is 7% solution, elimination insolubles after the stirring and dissolving repeatedly.The solution of getting 20mL then joins in the autoclave that is preheated to 30 ℃.Then feed the pressurization CO of 30 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 5.5MPa, and regulate service temperature to 30 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 50 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 97.3%, the purifying multiple 2.99 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Embodiment 7: taking by weighing an amount of Lumbrukinase crude product, to be mixed with alcohol concn be 25% (volume percent), and thick enzyme concn is the solution of 4% (mass percent), repeatedly elimination insolubles after the stirring and dissolving.The solution of getting 20mL then joins in the autoclave that is preheated to 30 ℃.Then feed the pressurization CO of 30 ℃ of preheating temperatures
2Stir simultaneously, make pressure increase to 8.0MPa, and regulate service temperature to 30 ℃, deposited phenomenon is obvious in the still, under high pressure keeps 35 minutes, and the foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution.Then precipitate and separate is come out, enzyme yield alive is 98.9%, the purifying multiple 2.61 of this process.Present method adopts pressurization CO
2As volatile acid, ethanol forms pressurization CO as helping precipitation agent
2-ethanol-water system protein isoelectric precipitation technology is precipitated out the foreign protein in the Lumbrukinase crude product.It is gentle relatively that this method has operational condition, can obtain other active ingredient simultaneously, the characteristics of the basic free of losses of enzyme etc.
Claims (5)
1. remove the method for foreign protein in the Lumbrukinase crude product, it is characterized in that the step of method is as follows:
1) at first, the Lumbrukinase crude product is dissolved in the ethanol-water solution, preparing thick enzyme mass percent concentration is 2～7%, and the ethanol concentration of volume percent is 0～40% solution, repeatedly elimination insolubles after the stirring and dissolving;
2) then, getting the above-mentioned mixing solutions of 20mL, to join preheating temperature be in 20～40 ℃ of autoclaves, feeds preheating temperature then and be 20～40 ℃ pressurization CO
2Stir simultaneously, make pressure increase to 3.5～8.5MPa, the pH of regulator solution is 4.4, under high pressure kept 20～60 minutes, foreign protein in the Lumbrukinase crude product is precipitated out under this condition fully, and activeconstituents still remains in the solution, precipitate and separate is gone out to get final product again.
2. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said autoclave preheating temperature and CO
2Preheating temperature is 25～35 ℃.
3. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said thick enzyme mass percent concentration is 3～6%.
4. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said ethanol concentration of volume percent is 15～35%.
5. remove the method for foreign protein in a kind of Lumbrukinase crude product according to claim 1, it is characterized in that said CO
2It is 30～50 minutes that pressure is held time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054043 CN1253564C (en) | 2004-08-24 | 2004-08-24 | Method for removing hybrid protein from lumbrokinase crude product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054043 CN1253564C (en) | 2004-08-24 | 2004-08-24 | Method for removing hybrid protein from lumbrokinase crude product |
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| Publication Number | Publication Date |
|---|---|
| CN1587398A CN1587398A (en) | 2005-03-02 |
| CN1253564C true CN1253564C (en) | 2006-04-26 |
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| CN 200410054043 Expired - Fee Related CN1253564C (en) | 2004-08-24 | 2004-08-24 | Method for removing hybrid protein from lumbrokinase crude product |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP5548931B1 (en) * | 2013-09-03 | 2014-07-16 | ワキ製薬株式会社 | Earthworm dry powder manufacturing method |
| CN110698529A (en) * | 2019-11-19 | 2020-01-17 | 湖南新合新生物医药有限公司 | Preparation method of eplerenone intermediate △ 9,11 alkenyl ester |
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