CN100537757C - A kind of purification process of Nattokinase - Google Patents
A kind of purification process of Nattokinase Download PDFInfo
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- CN100537757C CN100537757C CNB2007100274868A CN200710027486A CN100537757C CN 100537757 C CN100537757 C CN 100537757C CN B2007100274868 A CNB2007100274868 A CN B2007100274868A CN 200710027486 A CN200710027486 A CN 200710027486A CN 100537757 C CN100537757 C CN 100537757C
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- nattokinase
- elutriant
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- lyophilize
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Abstract
The invention discloses a kind of purification process of Nattokinase.Purification process of the present invention is that the Bacillus natto fermented liquid is centrifugal, gets supernatant liquor, filters, and filtrate is collected the elutriant that contains Nattokinase through ion exchange column, promptly obtains the Nattokinase lyophilisate through lyophilize.This purification process technology is simple, easy to operate, can large-scale production, and the product purity height has a extensive future.
Description
Technical field
The present invention relates to Nattokinase, particularly relate to a kind of purification process of Nattokinase.
Background technology
The intravital thrombus of people often causes serious diseases such as vascular embolization, cerebral thrombosis apoplexy, myocardial infarction, lung infraction, polarity pulmonary heart disease.Thromboembolic disease has characteristics such as sickness rate height, disability rate height, mortality ratio height and recurrence rate height, is greatly endangering human life and health, and people expect to research and develop and to produce the thrombolytic drug of more high-efficiency high-qualities in clinical application.(Nattokinase NK) finds so far that from 1987 progressively obtained research extensively and profoundly, its amidohydrolase activity, fibrinolytic and thrombus dissolving effect are proved to Nattokinase.And Nattokinase is compared with thrombolytic drugs such as used streptokinase, urokinase, rt-PAs, have safe, the effect directly rapidly, easily be absorbed by the body, the specificity height, in vivo longer duration, oral and the injection all can wait advantage, can be used for the thrombolytics or the protective foods of Development of New Generation, have very tempting bright prospects.
Problems such as the purification process of existing Nattokinase exists technology loaded down with trivial details mostly, and is not easy to operate, and purity is low.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists on the existing natto kinase purifying method, provide a kind of technology simple, easy to operate, can large-scale production, the natto kinase purifying method that product purity is high.
A kind of purification process of Nattokinase comprises the steps:
(1) fermentation liquor pretreatment: the Bacillus natto fermented liquid is centrifugal, collect supernatant liquor, supernatant liquor was collected cleaner liquid with 0.2～0.45 μ m membrane filtration;
(2) ion exchange column purifying: the used gel of ion exchange chromatography is CM SepharoseFast Flow, gel earlier with pH6.8, contain 6～70mM the phosphate buffered saline buffer balance good; Regulated the ionic concn of cleaner liquid, making its specific conductivity is 1.6～2.2mS/cm, with the flow velocity of 6～15mL/min it is added in post; With the phosphate buffered saline buffer washing column bed of pH6.8,6mM, contain the SODIUM PHOSPHATE, MONOBASIC of 6mM and the elutriant line style gradient elution of 0～1M sodium-chlor with pH6.8 more earlier, flow velocity is 5～10mL/min; (or packing into earlier before adding post in the plastic tank through the cleaner liquid of crossing of regulating ionic concn, add the 2L good gel of balance, transferring pH is 6.0, and with stirrer whip attachment 30 minutes at a slow speed, absorption is finished, and filters clear liquid, detergent gel, gel upper prop; Use the phosphate buffered saline buffer balance pillar of pH6.8,6mM again, pH6.8 use in the back, contains the eluant solution of 6mM SODIUM PHOSPHATE, MONOBASIC and 100mM sodium-chlor, and flow velocity is 30～100mL/min) to be 280nm place detection with ultraviolet spectrophotometer at wavelength, collection OD
280It is the elutriant more than 0.01;
(3) lyophilize: the Nattokinase elutriant through the membrane filtration degerming, is added additive and excipient, packing, lyophilize obtains the Nattokinase lyophilisate.
In above-mentioned purification process, step (1) described with the Bacillus natto fermented liquid centrifugal be to adopt high speed freezing centrifuge at 4～10 ℃, centrifugal 5～10min under the speed of 8000～16000rpm; Or adopt fermentor tank interlayer method of cooling to be cooled to 4～15 ℃, centrifugal with the high speed tubular-bowl centrifuge with the speed of 10000～16000rpm.
In above-mentioned purification process, the used post specification of the described ion exchange chromatography of step (2) is 3.5～5.5 centimetres of internal diameters, and is high 20～30 centimetres; Or be 16～20 centimetres of internal diameters, high 38～60 centimetres.
In above-mentioned purification process, the described filter membrane of step (3) is 0.2 μ m filter membrane.
In above-mentioned purification process, the described additive of step (3) is a tween-80, and consumption is 0.05%, and described excipient is a N.F,USP MANNITOL, and consumption is 2%.
In above-mentioned purification process, it is packing again behind the 1000IU/mL that described adding additive of step (3) and excipient are regulated concentration.
In above-mentioned purification process, the described lyophilize of step (3) is meant: be-40 ℃ in freezing temp, the vacuum-drying temperature is lyophilize 30～36 hours under 5～10 ℃ of conditions.
Compared with prior art, the present invention has following beneficial effect: purification process of the present invention is that the Bacillus natto fermented liquid is centrifugal, gets supernatant liquor, filter, filtrate is collected the elutriant that contains Nattokinase through ion exchange column, promptly obtains the Nattokinase lyophilisate through lyophilize.This purification process technology is simple, easy to operate, can large-scale production, and the product purity height has a extensive future.
Embodiment
Embodiment 1
1. fermentation liquor pretreatment
Use high speed freezing centrifuge under 4 ℃ the Bacillus natto fermented liquid,, collect supernatant liquor with the centrifugal 10min of the speed of 8000rpm.Supernatant liquor was collected cleaner liquid with 0.45 μ m membrane filtration, and it is standby to make upper prop liquid.
2. ion exchange chromatography purifying
The used gel of ion exchange chromatography is CM Sepharose Fast Flow, and the post specification is 3.5 centimetres of internal diameters, and is high 20 centimetres; Gel successively with pH6.8, contain 70mM and 6mM the phosphate buffered saline buffer balance good; Regulated the ionic concn (specific conductivity is adjusted to 1.8mS/cm) of cleaner liquid; The cleaner liquid of crossing that mixes up ionic concn is added in the post with the speed of 10mL/min; With the phosphate buffered saline buffer washing column bed of pH6.8,6mM, with the elutriant linear gradient elution of pH6.8, the SODIUM PHOSPHATE, MONOBASIC that contains 6mM and 1M sodium-chlor, flow velocity is 6mL/min more earlier; Is that the 280nm place is detected with ultraviolet spectrophotometer at wavelength, collects OD
280It is the elutriant more than 0.01.
3. lyophilize
With the Nattokinase elutriant with 0.2 μ m membrane filtration degerming, add 0.05% tween-80 and 2% N.F,USP MANNITOL, adjusting concentration is 1000IU/mL, with the packing of 10mL borosilicate injection bottle, loading amount is the 1mL/ bottle, in freezing temp is-40 ℃, and the vacuum-drying temperature is lyophilize 30 hours under 10 ℃ of conditions, obtains the Nattokinase lyophilized powder.
Embodiment 2
1. fermentation liquor pretreatment
Use high speed freezing centrifuge under 10 ℃ Nattokinase,, collect supernatant liquor with the centrifugal 5min of the speed of 12000rpm.Supernatant liquor was collected cleaner liquid with 0.2 μ m membrane filtration, and was standby as upper prop sample liquid.
2. ion exchange chromatography purifying
The used gel of ion exchange chromatography is CM Sepharose Fast Flow, and the post specification is 5.5 centimetres of internal diameters, and is high 30 centimetres; Gel earlier, the back with pH6.8, contain 70mM and 6mM the phosphate buffered saline buffer balance good; Regulated the ionic concn (specific conductivity is adjusted to 2.0mS/cm) of cleaner liquid; The cleaner liquid of crossing that mixes up ionic concn is added in the post with the speed of 15mL/min; Earlier with the phosphate buffered saline buffer washing column bed of pH6.8,6mM, again with pH6.8, contain the elutriant linear gradient elution of 6mM SODIUM PHOSPHATE, MONOBASIC and 0.5M sodium-chlor, flow velocity is 10mL/min; Detect at wavelength 280nm place with ultraviolet spectrophotometer, collect OD
280It is the elutriant more than 0.01.
3. lyophilize
With the Nattokinase elutriant collected through 0.2 μ m membrane filtration degerming, add 0.05% tween-80 and 2% N.F,USP MANNITOL, adjusting concentration is 1000IU/mL, with the packing of 10mL borosilicate injection bottle, loading amount is the 1mL/ bottle, in freezing temp is-40 ℃, and the vacuum-drying temperature is lyophilize 36 hours under 5 ℃ of conditions, promptly obtains the Nattokinase lyophilized powder.
Embodiment 3
1. fermentation liquor pretreatment
The Bacillus natto fermented liquid is cooled to 15 ℃ by fermentor tank interlayer method of cooling, centrifugal with the high speed tubular-bowl centrifuge with the speed of 16000rpm, collect supernatant liquor.Supernatant liquor was collected cleaner liquid with 0.2 μ m membrane filtration, and was standby as upper prop sample liquid.
2. ion exchange chromatography purifying
The used gel of ion exchange chromatography is CM Sepharose Fast Flow, and the post specification is 16 centimetres of internal diameters, and is high 38 centimetres; Gel earlier, the back with pH6.0, contain 70mM and 6mM the phosphate buffered saline buffer balance good; Regulate the ionic concn of having prepared (specific conductivity is adjusted to 2.2mS/cm) of crossing cleaner liquid, pack in the plastic tank, add the 2L CM Sepharose FastFlow gel that balance is good, transferring pH is 6.0, with stirrer whip attachment 30 minutes at a slow speed, absorption is finished, and filters clear liquid, detergent gel, the gel upper prop; Again with pH6.8, contain the phosphate buffered saline buffer balance pillar of 6mM, at last with pH6.8, contain the elutriant wash-out of 6mM SODIUM PHOSPHATE, MONOBASIC and 100mM sodium-chlor, flow velocity is 50mL/min; Detect at wavelength 280nm place with ultraviolet spectrophotometer, collect OD
280It is the elutriant more than 0.01.
3. lyophilize
With the Nattokinase elutriant collected through 0.2 μ m membrane filtration degerming, add additive 0.05% tween-80, excipient 2% N.F,USP MANNITOL, adjusting concentration is 1000IU/mL, with the packing of 10mL borosilicate injection bottle, loading amount is the 1mL/ bottle, in freezing temp is-40 ℃, and the vacuum-drying temperature is lyophilize 32 hours under 8 ℃ of conditions, promptly obtains the Nattokinase lyophilisate.
Claims (7)
1. the purification process of a Nattokinase comprises the steps:
(1) fermentation liquor pretreatment: the Bacillus natto fermented liquid is centrifugal, collect supernatant liquor, supernatant liquor was collected cleaner liquid with 0.2～0.45 μ m membrane filtration;
(2) ion exchange column purifying: the used gel of ion exchange chromatography is CM SepharoseFast Flow, gel earlier with pH6.8, contain 6～70mM the phosphate buffered saline buffer balance good; Regulated the ionic concn of cleaner liquid, making its specific conductivity is 1.6～2.2mS/cm, flow velocity with 6～15mL/min adds it in post, use the phosphate buffered saline buffer washing column bed of pH6.8,6mM earlier, contain the SODIUM PHOSPHATE, MONOBASIC of 6mM and the elutriant line style gradient elution of 0～1M sodium-chlor with pH6.8 again, flow velocity is 5～10mL/min, is that the 280nm place is detected with ultraviolet spectrophotometer at wavelength, collects OD
280It is the elutriant more than 0.01; Or, add the 2L good gel of balance packing into earlier before adding post in the plastic tank through the cleaner liquid of crossing of regulating ionic concn, transferring pH is 6.0, with stirrer whip attachment 30 minutes at a slow speed, absorption is finished, and filters clear liquid, detergent gel, the gel upper prop is used the phosphate buffered saline buffer balance pillar of pH6.8,6mM again, back pH6.8, the eluant solution that contains 6mM SODIUM PHOSPHATE, MONOBASIC and 100mM sodium-chlor, flow velocity is 30～100mL/min, is that the 280nm place is detected with ultraviolet spectrophotometer at wavelength, collects OD
280It is the elutriant more than 0.01;
(3) lyophilize: the Nattokinase elutriant through the membrane filtration degerming, is added additive and excipient, packing, lyophilize obtains the Nattokinase lyophilisate.
2. purification process as claimed in claim 1, it is characterized in that step (1) described with the Bacillus natto fermented liquid centrifugal be to adopt high speed freezing centrifuge at 4～10 ℃, centrifugal 5～10min under the speed of 8000～16000rpm; Or adopt fermentor tank interlayer method of cooling to be cooled to 4～15 ℃, centrifugal with the high speed tubular-bowl centrifuge with the speed of 10000～16000rpm.
3. purification process as claimed in claim 1 is characterized in that the used post specification of the described ion exchange chromatography of step (2) is 3.5～5.5 centimetres of internal diameters, and is high 20～30 centimetres; Or be 16～20 centimetres of internal diameters, high 38～60 centimetres.
4. purification process as claimed in claim 1 is characterized in that the described filter membrane of step (3) is 0.2 μ m filter membrane.
5. purification process as claimed in claim 1 is characterized in that the described additive of step (3) is a tween-80, and consumption is 0.05%; Described excipient is a N.F,USP MANNITOL, and consumption is 2%.
6. purification process as claimed in claim 1 is characterized in that it is packing again behind the 1000IU/mL that described adding additive of step (3) and excipient are regulated concentration.
7. purification process as claimed in claim 1 is characterized in that the described lyophilize of step (3) is meant: be-40 ℃ in freezing temp, the vacuum-drying temperature is lyophilize 30～36 hours under 5～10 ℃ of conditions.
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| CNB2007100274868A CN100537757C (en) | 2007-04-10 | 2007-04-10 | A kind of purification process of Nattokinase |
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| CNB2007100274868A CN100537757C (en) | 2007-04-10 | 2007-04-10 | A kind of purification process of Nattokinase |
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| CN100537757C true CN100537757C (en) | 2009-09-09 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101508983B (en) * | 2009-03-26 | 2011-11-02 | 河北省生物研究所 | Method for separation and purification of natto kinase by using soybean particle as affinity matrix |
| CN108865978A (en) * | 2018-07-25 | 2018-11-23 | 辽宁润基生物科技有限公司 | A method of separation and purifying excretion body |
| CN114250216B (en) * | 2021-12-23 | 2024-03-26 | 浙江工业大学 | Nattokinase separation and purification method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453356A (en) * | 2002-04-26 | 2003-11-05 | 同联集团沈阳抗生素厂 | Natto kinase purifying process and its freeze dried powder for injection |
| CN1594560A (en) * | 2004-06-29 | 2005-03-16 | 浙江大学 | Method for separating and purifying natto kinase by ion exchange |
| WO2006025276A1 (en) * | 2004-08-31 | 2006-03-09 | Kumamoto University | Remedy/preventive for ophthalmic diseases containing nattokinase |
| CN1766096A (en) * | 2004-10-27 | 2006-05-03 | 北京北大维信生物科技有限公司 | Nattokinase purification process and microcapsule formulation process |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453356A (en) * | 2002-04-26 | 2003-11-05 | 同联集团沈阳抗生素厂 | Natto kinase purifying process and its freeze dried powder for injection |
| CN1594560A (en) * | 2004-06-29 | 2005-03-16 | 浙江大学 | Method for separating and purifying natto kinase by ion exchange |
| WO2006025276A1 (en) * | 2004-08-31 | 2006-03-09 | Kumamoto University | Remedy/preventive for ophthalmic diseases containing nattokinase |
| CN1766096A (en) * | 2004-10-27 | 2006-05-03 | 北京北大维信生物科技有限公司 | Nattokinase purification process and microcapsule formulation process |
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Granted publication date: 20090909 Termination date: 20140410 |