CN1453356A - Natto kinase purifying process and its freeze dried powder for injection - Google Patents
Natto kinase purifying process and its freeze dried powder for injection Download PDFInfo
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- CN1453356A CN1453356A CN 02109538 CN02109538A CN1453356A CN 1453356 A CN1453356 A CN 1453356A CN 02109538 CN02109538 CN 02109538 CN 02109538 A CN02109538 A CN 02109538A CN 1453356 A CN1453356 A CN 1453356A
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Abstract
The present invention relates to biochemical medicine. The natto kinase purifying process includes the steps of: dissolving coarse natto kinase in water in concentration of 0.1-10 wt%; setting at 0-55 deg.c for 0.5 hr to 6 months and/or salt separation, centrifugation or filtering to eliminate insoluble matter, slat separating supernatant to collect the precipitate; and chromatography for further purification to obtain purified natto kinase product with single band or quasi-single band. The freeze dried powder of natto kinase is prepared with the purified natto kinase product and through adding additives for injection, mixing and freeze drying.
Description
Technical field:
The present invention belongs to biochemical drug, mainly relates to the improvement of natto kinase purifying process and producing of freeze-dried powder.
Background technology:
Natto (Natto) is a kind of traditional tempeh in Japan, is similar to the water Salt black bean in the Chinese tasteless preserved soybean, imports Japan into from the Tang Dynasty.The unique smell of natto is by a kind of Bacillus subtilus---and natto bacillus (Bacillus subtilis/natto) fermentative action produces.[1] such as Sami was reported in first and has a kind of enzyme with fibrinolytic effect in the natto in 1987---and Nattokinase (Nattokinase, NK).Subsequently, find that Nattokinase is derived from the natto bacillus in the natto, be a kind of subtilyne (Subtilisin) class serine protease, initial synthetic product is the preceding Nattokinase former (pre-pro-NK) of 381 amino-acid residues, 106 amino-acid residues of excision N end form the single chain polypeptide [2] with 275 amino-acid residues in secretion process.Iso-electric point (PI value) is 8.7, and molecular weight is 28,000Dalton[3].Pharmacological evaluation proof Nattokinase has high fibrinolytic ability [1,4], belongs to s-generation thrombolytics [5].Not only have stronger direct fibrin degradation effect, also have the effect of plasminogen activation.Can not only intravenously administrable, oral also effectively [4], and nontoxic [6].
But the purifying process of Nattokinase adopts following key step at present: hydrophobic chromatography (filler is Butyl-Toyopearl), ion-exchange (filler is CM-Toyopearl), gel-filtration (SephadexG-50) and dialysis, centrifugal, concentrated etc. just can obtain the elaboration (SDS-PAGE is the wall scroll band) [3] of Nattokinase, it is various that shortcoming is that it produces step, is unfavorable for amplifying, producing.
In recent years, China is also very active to the research of Nattokinase, Nattokinase has been carried out gene clone [7].The research work [8] of carrying out Nattokinase protective foods and oral capsule medicine for treating thrombus thing that also has, but the present elaboration of Nattokinase of still not finding is made the report of the research of injection.
Summary of the invention:
The present invention solves the complicated and various problem of step of present natto kinase purifying process, and the problem of not having the Nattokinase freeze-dried powder, a kind of simple natto kinase purifying process of natto kinase purifying process step and injection lyophilized powder pin goods produced are provided.
Natto kinase purifying process of the present invention adopts following technical scheme:
1, the Nattokinase crude product is soluble in water, ratio is 1～100 milligram: 1 milliliter, placing temperature is 0 ℃～55 ℃, and the time is 30 minutes～6 months.The SDS-PAGE of this product (sodium laurylsulfonate---polyacrylamide gel electrophoresis) is shown as almost one or a master tape.
2, the Nattokinase crude product is soluble in water, ratio is 1～100 milligram: 1 milliliter, adding saltout with salt (as, ammonium sulfate concentrations is 1.3～2.1 mol), centrifugal or filter to remove insoluble substance.Supernatant or filtrate add again saltout with salt (as, ammonium sulfate concentrations is 3.5～5.5 mol), collecting precipitation can obtain the Nattokinase product of a master tape.
3, go up method 1 or 2, or 1 and 2 products that obtain, available column chromatography method a kind of (as, hydrophobic chromatography, gel-filtration, ion-exchange, reversed phase chromatography etc.) be further purified, can obtain SDS-PAGE and be the wall scroll band or the elaboration of the Nattokinase of wall scroll band almost.Column chromatography can not only make protein be further purified, and can also remove other impurity.The fibrin plate Faxian shows that elaboration improves more than four times than the supernatant liquor specific activity of crude product.
Wherein, the preparation of Nattokinase crude product (number of patent application is 02116667.6), roughly process is as follows, natto bacillus (Bacillus Subtilis/natto HW9302, CGMCC No.0718), through suitable condition fermentation, fermented liquid carries out centrifugation, removes sediment, on reset and add processing such as ethanol sedimentation, collecting precipitation, drying gets the Nattokinase crude product.Show 15 bands of having an appointment through SDS-PAGE (Coomassie brilliant blue R250 dyeing and argentation), molecular weight ranges is about 8,000～12,000 Dalton, comprising the Nattokinase band that is slightly smaller than 30,000 Dalton.It is 150～500 urokinase units/milligram that the fibrin plate method is measured activity.
The beneficial effect of the purifying process of Nattokinase crude product:
Existing natto kinase purifying process, adopt hydrophobic chromatography simultaneously, ion-exchange, three step of gel-filtration column chromatography and a plurality of separating step, and the present invention adopts primary column chromatography, as being the filled column chromatography with Octyl Separose 4 FastFlow, the productive rate height, speed is fast, but ambient operation, condition is easy to control, and it is convenient to handle.The present invention finds that also Nattokinase is relatively stable, and other protein is degraded comparatively fast relatively, thereby the purifying of Nattokinase crude product is greatly simplified.In fact, from the supernatant of the centrifugal back of fermented liquid gained, by alcohol precipitation (getting crude product), thermal destruction (or saltouing), these main three steps of chromatography, basically finished purifying, technology is greatly simplified, the product purity height, cost is low, and yield is higher, can be mass-produced.The fibrin plate Faxian shows that elaboration improves more than four times than the supernatant liquor specific activity of crude product.
Injection freeze-dried product of the present invention adopts following technical scheme: with the additives (comprising excipient) of Nattokinase elaboration of the present invention and suitable injection as Dextran 40, mixing, lyophilize, the injection freeze-dried product.
The beneficial effect of freeze-dried powder goods:
This injection freeze-dried product, if can be used for clinical, administration fast, rapid-action, enzyme acts on thrombus directly into blood.These goods are activity stabilized.Clarity and particulate matter are up to specification.Bacterial endotoxin inspection and sterility test are qualified.
Embodiment:
Embodiment 1:
Get 191 milligrams of Nattokinase crude products, be dissolved in the phosphate buffered saline buffer (PH7.0,0.05 mol) of 20 milliliter of 1.5 mol ammonium sulfate, room temperature is placed and is spent the night, and 2000 rev/mins, centrifugal 15 minutes, draw supernatant, add an amount of ammonium sulfate, final concentration is 4.5 mol, after room temperature is placed and is spent the night, again through 2000 rev/mins, centrifugal 15 minutes, remove supernatant, get precipitation, make it be dissolved in 20 milliliters of 1.4-1.8 mol ammonium sulfate phosphate buffered saline buffers (PH7.0,0.05 mol) fully.Last hydrophobic chromatography post [filler: OctylSepharose 4 Fast Flow, 2.5 * 60 centimetres, give earlier with 1.4-1.8 mol ammonium sulfate phosphate buffer (PH6.0～12.0,0.01～0.5 mol) balance], gradient elution, be that ammonium sulfate concentrations is zero by reducing to by 1.4～1.8 mol, the time is about 6 hours.Beginning that big peak, a non-activity of passing through is arranged.Near zero the time, occur an active climax in ammonium sulfate concentrations, collect this peak, the ultra-filtration desalination gets 15 milliliters of Nattokinase elaboration, and yield is 1.0% (elaboration protein milligram ÷ crude product milligram * 100%).
The Nattokinase elaboration is the wall scroll band through SDS-PAGE (Coomassie brilliant blue R250 dyeing and argentation), and molecular weight is 28,000～30,000 Dalton; It is 1000 urokinase units per ml that the fibrin plate method is measured activity; It is 127.3 mcg/ml that lowry method (micromethod) is surveyed protein content; Specific activity is 7855 urokinase units/milligram protein.
Embodiment 2:
Get 150 milligrams of Nattokinase crude products, be dissolved in 20 ml phosphate buffers (PH7.4,0.02 mol), 43 ℃ of water-baths 20 hours.2000 rev/mins, centrifugal 15 minutes, get and reset and add an amount of ammonium sulfate, making final concentration is 1.4～1.8 mol.With [the filler: Octyl Sepharose 4Fast Flow of hydrophobic chromatography post on whole supernatants, give earlier with 1.4-1.8 mol ammonium sulfate phosphate buffered saline buffer (PH6.0～12.0,0.01～0.5 mol) balance], gradient elution, be that ammonium sulfate concentrations is zero by reducing to by 1.4～1.8 mol, begin to have big peak, a non-activity of passing through.When ammonium sulfate concentrations near zero the time, an active climax appears, collect this peak, the ultra-filtration desalination gets 40 milliliters of Nattokinase elaboration, yield is 2.0% (elaboration protein milligram ÷ crude product milligram * 100%).
The Nattokinase elaboration is the wall scroll band through SDS-PAGE (Coomassie brilliant blue R250 dyeing and argentation), and molecular weight is 28,000～30,000 Dalton; It is 623.7 urokinase units per ml that the fibrin plate method is measured activity; It is 77.5 mcg/ml that the lowry method is measured protein content; Specific activity is 8048 urokinase units/milligram protein.
Embodiment 3:
Get 240 milligrams of Nattokinase crude products and be dissolved in 10 ml phosphate buffers (PH7.4,0.02 mol), 43 ℃ of water-baths 20 hours, 2000 rev/mins, centrifugal 15 minutes, go precipitation, get supernatant, add an amount of ammonium sulfate, make its final concentration reach 4.5 mol, 3000 rev/mins, centrifugal 20 minutes, remove supernatant, precipitation adds 3 ml phosphate buffer (PH7.4,0.02 mol), make dissolving.Last gel-filtration column [filler: Sephadex G-75, Sweden's import packing, China Drug Co. sells, 2.6 * 100 centimetres, give earlier with phosphate buffered saline buffer [PH7.4,0.02 mol) balance], with phosphate buffered saline buffer (PH7.4,0.02 mol) wash-out, high reactivity peak occurs earlier, Yi Dafeng is arranged at last, non-activity, elution time is about 16 hours.Collect active climax, the ultra-filtration desalination concentrate 50 milliliters of Nattokinase elaboration, yield is 1.8% (elaboration protein milligram ÷ crude product milligram * 100%).
The Nattokinase elaboration is the wall scroll band through SDS-PAGE (Coomassie brilliant blue R250 dyeing and argentation) demonstration, and molecular weight is 28,000～30,000 Dalton; It is 661 urokinase units per ml that the fibrin plate method is measured activity; It is 87.8 mcg/ml that the lowry method is measured protein content; Specific activity is 7528 urokinase units/milligram protein.
Embodiment 4:
It is 1000 urokinase units per ml that the Nattokinase elaboration is adjusted to concentration, each cillin bottle adds 3%～10% Dextran 20-80), mixing, be packed as 1.25 milliliters/cillin bottle, lyophilize (30 handkerchiefs, minimum temperature is-40 ℃) 24 hours, tamponade promptly gets the injection freeze-dried product of Nattokinase.
Nattokinase elaboration SDS-PAGE demonstration is the wall scroll band, and molecular weight is 28,000～30,000 Dalton; It is 1050 urokinase units per ml that the fibrin plate method is measured activity; It is 130.5 mcg/ml that the lowry method is measured protein content; Clarity and particulate matter are up to specification; Bacterial endotoxin inspection and sterility test are qualified.Preserve after 6 months for 4 ℃-10 ℃, repetition measurement SDS-PAGE still is the wall scroll band, and molecular weight is 28,000～30,000 Dalton; It is 1118 urokinase units per ml that the fibrin plate method is measured activity; It is 128.6 mcg/ml that the lowry method is measured protein content; Clarity and particulate matter are up to specification; Bacterial endotoxin inspection and sterility test are qualified.
Reference:
1.Sumi?H，Hamada?H，Tsushima?H，etal.A?novel?fibrinolyticenzyme(Nattokinase)in?the?vegetable?cheese?natto；a?typical?and?popularsoybea?food?inthe?Japanese?diat.Experientia，1987，43：1110～1111
2.Nakamura?T，Yamagata?Y，Ichishima?E.Nucleotide?sequence?of?thesubtilisin?NAT?gene，aprN?of?Bacillus?subtilis(natto).Biosci?Biotech?Biochem，1992，56(1)：1869-1871
3.Fujita?M，Nomura?K，Hong?K，etal.Purification?and?characterization?of?astrong?fibrinolytic?enzyme(Nattokinase)in?the?vegetable?cheese?natto，a?popularsoybean?Fermented?food?in?Japan.Biochem?Biophys?Research?Communication，1993，197(3)：1340～1347.
4.Sumi?H，Hamada?H，Nakanishi?H，etal.Enhancement?of?the?fibrinolyticactivity?in?plasma?by?oral?administration?of?nattokinase.Acta?Haematol，1990，84：139-143.
5. Zou and prosperous. the development of thrombolytics and research. Chinese Pharmaceutical Journal, 1997,32 (5): 263-267
6. Li Jing, Zhang Yun lake. the acute toxicity test of Nattokinase capsule. the Applied Microbiology Inst., Heilongjiang Academy of Sciences
7. Liu Bei territory, official's filial piety group, Song Houyan. gene clone that Nattokinase is former and the expression in intestinal bacteria. Shanghai Medical Univ's journal, 1999,26 (6): 401～404
8. Liu Yu peak, Wang Jinying, grandson's bank sheath or bow case etc., Nattokinase preparation-grace is opened capsular production technique pilot scale research. biotechnology, 2000,10 (2): 1～3
Claims (6)
1, Nattokinase purifying crude process using following steps:
(1) the Nattokinase crude product is soluble in water, ratio is 1～100 milligram: 1 milliliter, placing temperature is 0 ℃～55 ℃, and the time is 30 minutes～6 months; The SDS-PAGE of this product is shown as almost a band or a master tape.
(2) the Nattokinase crude product is soluble in water, ratio is 1～100 milligram: 1 milliliter, adding saltouts uses salt, and insoluble substance is removed in centrifugal or filtration; Supernatant or filtrate add to saltout again uses salt, collecting precipitation;
(3) go up method (1) or (2), or (1) and (2) product of obtaining, a kind of of available column chromatography method is further purified, and can obtain the wall scroll band or the elaboration of the Nattokinase of wall scroll band almost.
2, natto kinase purifying process according to claim 1, it is characterized in that getting 191 milligrams of Nattokinase crude products, be dissolved in the phosphate buffered saline buffer (PH7.0 of 20 milliliter of 1.5 mol ammonium sulfate, 0.05 mol), room temperature is placed and is spent the night, and 2000 rev/mins, centrifugal 15 minutes, draw supernatant, add an amount of ammonium sulfate, final concentration is 4.5 mol, after room temperature is placed and spent the night, again through 2000 rev/mins, centrifugal 15 minutes, remove supernatant, get precipitation, make it be dissolved in 20 milliliters of 1.4-1.8 mol ammonium sulfate phosphate buffered saline buffers (PH6.0-12.0,0.01-0.5 mol) fully; Last hydrophobic chromatography post, filler are Octyl Sepharose4 Fast Flow, with 1.4-1.8 mol ammonium sulfate phosphate buffered saline buffer (PH6.0-12.0,0.01-0.5 mol) gradient elution, time is about 6 hours, collects active climax, and the ultra-filtration desalination gets 15 milliliters of Nattokinase elaboration.
3, natto kinase purifying process according to claim 1 is characterized in that getting 150 milligrams of Nattokinase crude products, is dissolved in 20 ml phosphate buffers (PH7.4,0.02 mol), 43 ℃ of water-baths 20 hours; 2000 rev/mins, centrifugal 15 minutes, get and reset and add an amount of ammonium sulfate, making final concentration is the 1.4-1.8 mol; With hydrophobic chromatography post on whole supernatants, filler is Octyl Separose 4 Fast Flow, with 1.4-1.8 mol ammonium sulfate phosphate buffered saline buffer (PH6.0-12.0,0.01-0.5 mol) gradient elution, collect active climax, the ultra-filtration desalination gets 40 milliliters of Nattokinase elaboration.
4, natto kinase purifying process according to claim 1 is characterized in that getting 240 milligrams of Nattokinase crude products, is dissolved in 10 ml phosphate buffer (PH7.4,0.02 mol), 43 ℃ of water-baths 20 hours, 2000 rev/mins, centrifugal 15 minutes, go precipitation, get supernatant, add an amount of ammonium sulfate, make its final concentration reach 4.5 mol, 3000 rev/mins, centrifugal 20 minutes, remove supernatant, precipitation adds 3 ml phosphate buffer (PH7.4,0.02 mol), make dissolving; Last gel-filtration column, filler be Sephadex G-75 with phosphate buffered saline buffer (PH7.4,0.02 mol) wash-out, elution time is about 16 hours; Collect active climax, get 50 milliliters of Nattokinase elaboration.
5, producing of injection freeze-dried product is the injection additives that the Nattokinase elaboration added appropriate amount, mixing, lyophilize, promptly.
6, injection freeze-dried product according to claim 5 produces, it is characterized in that it is 1000 urokinase units per ml that the Nattokinase elaboration is adjusted to concentration, each cillin bottle adds 3%～10% Dextran 20-80, mixing, be packed as 1.25 milliliters/cillin bottle, lyophilize 24 hours, promptly.
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| CN 02109538 CN1453356A (en) | 2002-04-26 | 2002-04-26 | Natto kinase purifying process and its freeze dried powder for injection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295326C (en) * | 2004-04-30 | 2007-01-17 | 中国科学院过程工程研究所 | Process for separating and purifying natto kinase by reverse micelle method |
| CN100537757C (en) * | 2007-04-10 | 2009-09-09 | 广州市微生物研究所 | A kind of purification process of Nattokinase |
-
2002
- 2002-04-26 CN CN 02109538 patent/CN1453356A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295326C (en) * | 2004-04-30 | 2007-01-17 | 中国科学院过程工程研究所 | Process for separating and purifying natto kinase by reverse micelle method |
| CN100537757C (en) * | 2007-04-10 | 2009-09-09 | 广州市微生物研究所 | A kind of purification process of Nattokinase |
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