CN1594560A - Method for separating and purifying natto kinase by ion exchange - Google Patents
Method for separating and purifying natto kinase by ion exchange Download PDFInfo
- Publication number
- CN1594560A CN1594560A CN 200410025769 CN200410025769A CN1594560A CN 1594560 A CN1594560 A CN 1594560A CN 200410025769 CN200410025769 CN 200410025769 CN 200410025769 A CN200410025769 A CN 200410025769A CN 1594560 A CN1594560 A CN 1594560A
- Authority
- CN
- China
- Prior art keywords
- supernatant liquor
- centrifugal
- 10000rpm
- elutriant
- collecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940086319 nattokinase Drugs 0.000 title claims abstract description 40
- 108010073682 nattokinase Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000005342 ion exchange Methods 0.000 title description 5
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 238000001556 precipitation Methods 0.000 claims abstract description 24
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 20
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 17
- 238000010828 elution Methods 0.000 claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims description 51
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 48
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 24
- 239000000872 buffer Substances 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 13
- 230000004151 fermentation Effects 0.000 claims description 13
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 238000011033 desalting Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 8
- 230000008014 freezing Effects 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 6
- 229920002684 Sepharose Polymers 0.000 claims description 2
- 239000012505 Superdex™ Substances 0.000 claims description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims 1
- 238000005341 cation exchange Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 244000063299 Bacillus subtilis Species 0.000 abstract description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 abstract 3
- 102000001253 Protein Kinase Human genes 0.000 abstract 2
- 238000002523 gelfiltration Methods 0.000 abstract 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract 1
- 230000003213 activating effect Effects 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 229910052698 phosphorus Inorganic materials 0.000 abstract 1
- 239000011574 phosphorus Substances 0.000 abstract 1
- 208000007536 Thrombosis Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 239000003527 fibrinolytic agent Substances 0.000 description 2
- 235000013557 nattō Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 108010046845 tryptones Proteins 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 102000005686 Serum Globulins Human genes 0.000 description 1
- 108010045362 Serum Globulins Proteins 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007813 chromatographic assay Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 229960003487 xylose Drugs 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for separating and purifying Natto kinase mainly using ion exchange chromatography. The steps are composed of crude enzyme liquid preparation, gel chromatography and ion exchange chromatography, activating bacillus subtilis through slant medium, medium culture and fermenting in ferment medium, natto kinase being secreted to fermented solution by bacillus subtilis, centrifuging to collecting upper clear solution, then proceeding (NH#-[4])#-[2]SO#-[4] graded precipitation, redissolving using phosphorus acid buffer, adding redissolved solution to gel chromatography column to proceeding gel filtration, collecting elution peak containg natto kinase, adding fraction of having natto kinase activity from gel filtration to inon exchange chromatography column, proceeding adsorbing, collecting elution peak containg natto kinase, freeze drying to get purified natto kinase. The invention has merits of condition, low cost, simple process , easy operation ,high purity and short production period, etc.
Description
Technical field
The present invention relates to a kind of is the method for main means separating and purifying nattokinase with ion-exchange.
Background technology
The thrombotic diseases serious threat human life with healthy, and its M ﹠ M occupies first of the various diseases.Thrombus is the prefered method of this class disease of treatment.But as urokinase, streptokinase, rt-PA etc., there are short, various shortcomings such as side effect big, cost an arm and a leg of transformation period in current thrombolytics, is difficult to become suitable popular medicine.Palpus in 1987 sees that foreign firm has found a kind of enzyme with strong fibrinolytic in the traditional fermented food natto of Japan, name into Nattokinase (nattokinase, NK).Studies show that, Nattokinase is a kind of serine protease, and energy is the inside and outside thrombus of dissolve body significantly, obviously shortens the dissolution time (ELT) of euglobulin, and can stimulate venous endothelial cell to produce plasminogen activator (t-PA), thereby more effectively bring into play the thrombolysis effect.Therefore Nattokinase is a kind of very potential new oral thrombolytic drug.
Fujita etc. are separating and purifying nattokinase from the solid fermentation natto, technology is: physiological saline extracting Nattokinase, adding ammonium sulfate spends the night under 25% saturation ratio, again in 4 ℃, the centrifugal 40min of 7000r/min, collect Butyl-Toyopearl drainage column on the supernatant, collect the ammonium sulfate precipitation that active peak carries out saturation ratio 25% once more, get the ammonium sulfate precipitation that supernatant carries out saturation ratio 60%, go up the CM-Toyopearl ion exchange column then, collect active peak and go up Sephadex-G50 again, collect active peak and dialysed overnight.This technological operation complexity, yield is low, and the final product that obtains is seldom.The fermentation of Yu Li Hua Yisan times of ethanol sedimentation is centrifugal to go out supernatant liquor behind the bacterium, goes up Hitrap CM-Sepharose-FF 1ml column chromatography purification then, and ult rec is 85.4%, and the purifying multiple is 8.1 times.This method rate of recovery height, but operation capacity is limited, and the production cost height does not fit into the preparation of extensive Nattokinase.
Summary of the invention
The purpose of this invention is to provide that a kind of cost is low, the cycle is short, method is simple, be easy to grasp, product purity is high is the method for main means separating and purifying nattokinase with ion-exchange.
Its method steps is as follows:
1) engineering bacterium fermentation
Single bacterium colony of picking expression Nattokinase inserts the seed culture medium of 10～200ml from solid medium, shaking table was cultivated 8～25 hours under 150～300rpm, insert in the fermention medium by 1%～5% inoculum size,, cultivated 100～150 hours at 28～37 ℃, 150～300rpm bottom fermentation jar again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 0～20 ℃ with the centrifugal 8～15min of the speed of 2000～10000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 10～40% saturation ratios
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 2000～10000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 60～85%, 2000～10000rpm centrifugal collecting precipitation is resuspended in precipitation in the phosphoric acid buffer of 10～100mM, pH6～8, and the centrifugal 10～30min of 2000～10000rpm collects supernatant liquor, and is standby in 0～20 ℃ of preservation;
3) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 10～100cm/h added in the gel chromatography column, phosphoric acid buffer with 10～200mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 9～15ml place, and collecting liquid long-pending is 3～6ml;
4) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 15～75cm/h added in the chromatography column, NaCl with the phosphoric acid buffer that contains 0.2～2M, pH6～8 and 0.1～1M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 0～10 ℃ of condition;
5) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 16-32h in deionized water after, place that freezing 12-24h carries out lyophilize with it under-20 ℃ of refrigerators.Be 24～48h time of drying.
Advantage of the present invention:
1) separating technology is simple, with short production cycle, product purity is high
This separation purifying process is made up of ammonium sulfate precipitation, gel chromatography and ion chromatography operating unit.Ammonium sulfate precipitation is about 5 hours, and gel chromatography is about 3 hours, and ion exchange chromatography is about 2 hours, and the final Nattokinase that obtains is single band after the sds gel electrophoresis analysis, illustrates that the product purity that obtains is very high;
2) action condition gentleness, instant effect
The iso-electric point of Nattokinase is 8.7, utilizes cationite, relies on electrostatic interaction to separate with other protein under non-denatured state with medium under the pH that is lower than electricity such as Nattokinase electricity, the action condition gentleness;
3) production automation level height, easy to operate
This production technique adopts KTA explorer 100 chromatographic assay systems, and whether the existence of the online detection Nattokinase of absorption value that can be by wavelength 280nm, 260nm;
4) occupation of land is little, cost is low
This separation production operation only needs more than 20 square metre laboratory to carry out.
This bacterial classification is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on June 7th, 2004.Numbering CGMCC No.1177 registers on the books at the center of containing.
Embodiment
Below in conjunction with embodiment the present invention is elaborated:
In order to ion exchange chromatography is that the separation purification method of main means prepares Nattokinase, and its concrete steps are as follows:
1) liquid fermenting of Nattokinase
Single bacterium colony of picking expression Nattokinase inserts the seed culture medium of 10～200ml from the consubstantiality substratum, shaking table was cultivated 8～25 hours under 150～300rpm, insert in the fermention medium by 1%～5% inoculum size, cultivated 100～150 hours at 28～37 ℃, 150～300rpm bottom fermentation jar again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 0～20 ℃ with the centrifugal 8～15min of the speed of 2000～10000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 10～40% saturation ratios
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 2000～10000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 60～85%, 2000～10000rpm centrifugal collecting precipitation is resuspended in precipitation in the phosphoric acid buffer of 10～100mM, pH6～8, and the centrifugal 10～30min of 2000～10000rpm collects supernatant liquor, and is standby in 0～20 ℃ of preservation;
3) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 10～100cm/h added in the gel chromatography column, phosphoric acid buffer with 10～200mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 9～15ml place, and collecting liquid long-pending is 3～6ml;
4) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 15～75cm/h added in the chromatography column, NaCl with the phosphoric acid buffer that contains 0.2～20mM, pH6～8 and 0.1～1M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 0～10 ℃ of condition;
5) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 16-32h in deionized water after, place freezing 12-24h under-20 ℃ of refrigerators, again it is carried out lyophilize, be 24～48h time of drying;
Equipment used and material are as follows in the present invention:
1) bacterial classification
Bacterial classification is Bacillus subtilis MLH-02, and bacterium numbering is CGMCC No.1177;
2) substratum
Seed culture medium (g/l): LB liquid nutrient medium;
Fermention medium (g/l): yeast extract 5, Tryptones 22.7, wood sugar 25, Na
2HPO
45.37, NaH
2PO
41, CaCl
20.2, MgSO
40.5, pH7.0;
Solid medium (g/l): yeast extract 5, Tryptones 10, NaCl 5.0, and pH 7.0, agar 8;
3) equipment
Electrophoresis apparatus (Mini-Proten
3cell) available from Bio-RAD company; Chromatographic system ( KTA explorer100), chromatography column (XK16/20), gel (Superdex 75), Ion Exchange Medium (SP Sepharose FastFlow) are available from Amersham Pharmacia Biotech AB.Sweden; Other reagent are commercially available analytical reagent.
Embodiment 1
1) engineering bacterium fermentation
Single bacterium colony that picking is expressed Nattokinase from solid medium inserts the seed culture medium of 10ml, and shaking table was cultivated 8 hours under the 150rpm, insert in the fermention medium by 1% inoculum size, and, cultivated 100 hours at 28 ℃, 150rpm bottom fermentation jar again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 0 ℃ with the centrifugal 8min of the speed of 2000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 10% saturation ratio
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 2000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 60%, the 2000rpm centrifugal collecting precipitation is resuspended in precipitation in the phosphoric acid buffer of 10mM, pH6, and the centrifugal 10min of 2000rpm collects supernatant liquor, and is standby in 0 ℃ of preservation;
3) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 10cm/h added in the gel chromatography column, phosphoric acid buffer with 10mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 9 places, and collecting liquid long-pending is 3;
4) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 15cm/h added in the chromatography column, carrying out linear gradient elution with 0.1 NaCl with identical linear velocity with the phosphoric acid buffer that contains 0.2M, pH6, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 0 ℃ of condition;
5) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 16h in deionized water after, place that freezing 12h carries out lyophilize with it under-20 ℃ of refrigerators.Be 24h time of drying.
Embodiment 2
1) engineering bacterium fermentation
Single bacterium colony that picking is expressed Nattokinase from solid medium inserts the seed culture medium of 200ml, and shaking table was cultivated 25 hours under the 300rpm, insert in the fermention medium by 5% inoculum size, and, cultivated 150 hours at 37 ℃, 300rpm bottom fermentation jar again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 20 ℃ with the centrifugal 15min of the speed of 10000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 40% saturation ratio
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 10000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 85%, the 10000rpm centrifugal collecting precipitation is resuspended in precipitation in the phosphoric acid buffer of 100mM, pH8, and the centrifugal 30min of 10000rpm collects supernatant liquor, and is standby in 20 ℃ of preservations;
3) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 100cm/h added in the gel chromatography column, phosphoric acid buffer with 200mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 15ml place, collects the long-pending 6ml of being of liquid;
4) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 75cm/h added in the chromatography column, usefulness contains the phosphoric acid buffer of 2M, pH8 and the NaCl of 1M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 10 ℃ of conditions;
5) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 32h in deionized water after, place that freezing 24h carries out lyophilize with it under-20 ℃ of refrigerators.Be 48h time of drying;
Embodiment 3
1) engineering bacterium fermentation
Single bacterium colony that picking is expressed Nattokinase from solid medium inserts the seed culture medium of 50ml, and shaking table was cultivated 10 hours under the 240rpm, inserts in the fermention medium by 4% inoculum size, cultivates 120 hours at 30 ℃, 220rpm bottom fermentation jar again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 4 ℃ with the centrifugal 20min of the speed of 8000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 20% saturation ratio
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 8000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 60%, the 8000rpm centrifugal collecting precipitation is resuspended in precipitation in the phosphoric acid buffer of 10mM pH6.4, and the centrifugal 20min of 8000rpm collects supernatant liquor, and is standby in 4 ℃ of preservations;
3) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 30cm/h added in the gel chromatography column, phosphoric acid buffer with 10mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 9～15ml place, collects the long-pending 6ml of being of liquid;
4) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 90cm/h added in the chromatography column, NaCl with phosphoric acid buffer that contains 20mM, pH6.4 and 1M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 4 ℃ of conditions;
5) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 16h in deionized water after, place freezing 12h under-20 ℃ of refrigerators, again it is carried out lyophilize, be 48h time of drying.
Claims (3)
1. one kind is the method for main means separating and purifying nattokinase with ion exchange chromatography, it is characterized in that: its method steps is as follows:
1) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 0～20 ℃ with the centrifugal 8～15min of the speed of 2000～10000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 10～40% saturation ratios
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 2000～10000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 60～85%, 2000～10000rpm centrifugal collecting precipitation is dissolved in precipitation in the phosphoric acid buffer of 5～100mM, pH5～8 again, and the centrifugal 10～30min of 2000～10000rpm collects supernatant liquor, and is standby in 0～20 ℃ of preservation;
2) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 10～100cm/h added in the gel chromatography column, phosphoric acid buffer with 10～200mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 9～15ml place, and collecting liquid long-pending is 3～6ml;
3) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 15～75cm/h added in the chromatography column, NaCl with the phosphoric acid buffer that contains 0.2～2M, pH6～8 and 0.1～1M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 0～10 ℃ of condition;
4) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 16-32h in deionized water after, place freezing 12～24h under-20 ℃ of refrigerators, again it is carried out lyophilize, be 24～48h time of drying.
2. according to claim 1 a kind of be the method for main means separating and purifying nattokinase with ion exchange chromatography, it is characterized in that: its method steps is as follows:
1) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 0～4 ℃ with the centrifugal 8～10min of the speed of 8000～10000rpm, collect supernatant liquor, in supernatant liquor, add (the NH of 10～30% saturation ratios
4)
2SO
4Precipitation, the centrifugal collection supernatant liquor of 8000～10000rpm continues to add (NH in supernatant liquor
4)
2SO
4Saturation ratio to 75～85%, 8000～10000rpm centrifugal collecting precipitation is dissolved in precipitation in the phosphoric acid buffer of 10～30mM, pH6～7 again, and the centrifugal 15～25min of 8000～10000rpm collects supernatant liquor, and is standby in 0～4 ℃ of preservation;
2) gel chromatography separates
With chromatographic system the linear velocity of supernatant liquor with 10～30cm/h added in the gel chromatography column, phosphoric acid buffer with 10～20mM finishes to going out the peak with identical linear velocity washing, is that 280nm and 260nm detect with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant at 10～13ml place, and collecting liquid long-pending is 3～6ml;
3) ion exchange chromatography separates
With chromatographic system the linear velocity of elutriant liquid with 30～45cm/h added in the chromatography column, NaCl with the phosphoric acid buffer that contains 0.2～1M, pH6～7 and 0.1～5M carries out linear gradient elution with identical linear velocity, is that 280nm, 260nm detect with Ultraviolet Detector at wavelength.Collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 0～4 ℃ of condition;
4) activated protein is collected in lyophilize
The active eluant of collecting carried out dialysis desalting 18～22h in deionized water after, place freezing 14～18h under-20 ℃ of refrigerators, again it is carried out lyophilize, be 36～40h time of drying.
3. according to claim 2 a kind of be the method for main means separating and purifying nattokinase with ion exchange chromatography, it is characterized in that: its method steps is as follows:
1) engineering bacterium fermentation
Single bacterium colony of picking expression Nattokinase inserts the seed culture medium of 10～200ml from solid medium, shaking table was cultivated 8～25 hours under 150～300rpm, insert in the fermention medium by 1%～5% inoculum size, cultivated 100～150 hours at 28～37 ℃, 150～300rpm bottom fermentation jar, above-mentioned required bacterium numbering is CGMCC No.1177 again;
2) ammonium sulfate precipitation
With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 20min of the speed of 10000rpm, the thalline of leaving away is collected supernatant liquor, adds (the NH of 20% saturation ratio in supernatant liquor
4)
2SO
4, left standstill 4 hours, collect supernatant liquor with the centrifugal 25min of 10000rpm again, continue in supernatant liquor, to add (NH then
4)
2SO
4Saturation ratio to 60% with the centrifugal 25min collecting precipitation of the speed of 10000rpm, is resuspended in precipitation in the phosphoric acid buffer of 10mM pH7.0, and centrifugal again 25min collects supernatant liquor under the 10000rpm, and is standby in 4 ℃ of preservations;
3) gel chromatography separates
With automatic chromatographic system the 1mL supernatant liquor being added the post bed height with the linear velocity of 30.0cm/h is in Superdex 75 gel chromatography columns of 12cm, phosphoric acid buffer with 10mM finishes to going out the peak with the linear velocity washing identical with sample introduction, is to detect under 280nm, the 260nm with Ultraviolet Detector at wavelength, collecting retention volume is the elutriant that is about the 50.0ml place, collects the long-pending 25mL of being of liquid;
4) ion exchange chromatography separates
With chromatographic system active elution peak being added cation exchange medium with 2.5mL with the linear velocity of 75cm/h is SP Sepharose Fast Flow, the post height is in the XK16/20 chromatography column of 10cm, carry out linear gradient elution with phosphoric acid buffer that contains 10mM, pH6.4 and 1M NaCl, is to detect under 280nm, the 260nm with Ultraviolet Detector at wavelength, collection contains the active peak elutriant of Nattokinase, and elutriant is preserved standby under 4 ℃ of conditions;
5) lyophilize
The active eluant of collecting carried out dialysis desalting 16h in deionized water after, place that freezing 12h carries out lyophilize with it under-20 ℃ of refrigerators.Be 32h time of drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025769 CN1594560A (en) | 2004-06-29 | 2004-06-29 | Method for separating and purifying natto kinase by ion exchange |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025769 CN1594560A (en) | 2004-06-29 | 2004-06-29 | Method for separating and purifying natto kinase by ion exchange |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1594560A true CN1594560A (en) | 2005-03-16 |
Family
ID=34663833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410025769 Pending CN1594560A (en) | 2004-06-29 | 2004-06-29 | Method for separating and purifying natto kinase by ion exchange |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1594560A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100537757C (en) * | 2007-04-10 | 2009-09-09 | 广州市微生物研究所 | A kind of purification process of Nattokinase |
| CN103589706A (en) * | 2013-04-25 | 2014-02-19 | 珠海市御品堂生物科技有限公司 | Purification method and preparation method for Nattokinase |
| CN105566476A (en) * | 2015-12-28 | 2016-05-11 | 中国农业大学 | Preparation method of antioxidant protein in rana japonica oil |
| CN107897718A (en) * | 2017-11-27 | 2018-04-13 | 荣成市飞创科技有限公司 | A kind of preparation method of Nattokinase tablet |
| CN113736684A (en) * | 2021-06-25 | 2021-12-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermentation of American ginseng endophyte |
| CN114250216A (en) * | 2021-12-23 | 2022-03-29 | 浙江工业大学 | Method for separating and purifying nattokinase |
-
2004
- 2004-06-29 CN CN 200410025769 patent/CN1594560A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100537757C (en) * | 2007-04-10 | 2009-09-09 | 广州市微生物研究所 | A kind of purification process of Nattokinase |
| CN103589706A (en) * | 2013-04-25 | 2014-02-19 | 珠海市御品堂生物科技有限公司 | Purification method and preparation method for Nattokinase |
| CN103589706B (en) * | 2013-04-25 | 2016-03-30 | 珠海市御品堂生物科技有限公司 | The method of purification of Nattokinase and preparation method |
| CN105566476A (en) * | 2015-12-28 | 2016-05-11 | 中国农业大学 | Preparation method of antioxidant protein in rana japonica oil |
| CN107897718A (en) * | 2017-11-27 | 2018-04-13 | 荣成市飞创科技有限公司 | A kind of preparation method of Nattokinase tablet |
| CN113736684A (en) * | 2021-06-25 | 2021-12-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermentation of American ginseng endophyte |
| CN113736684B (en) * | 2021-06-25 | 2023-11-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermenting American ginseng endophyte |
| CN114250216A (en) * | 2021-12-23 | 2022-03-29 | 浙江工业大学 | Method for separating and purifying nattokinase |
| CN114250216B (en) * | 2021-12-23 | 2024-03-26 | 浙江工业大学 | Nattokinase separation and purification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2012532932A5 (en) | ||
| CN106701719B (en) | Fermentation and co-production of vitamin K by utilizing bacillus natto2Method for producing nattokinase | |
| RU2012104778A (en) | METHOD AND SYSTEM FOR PRODUCING BOTULINIC NEUROTOXIN | |
| CN101235394B (en) | Method for separating and extracting fumaric acid | |
| CN101316608A (en) | Bacillus subtilis novel bacterial strain and its usage in preparation of medicament for treating thrombus disease | |
| CN107354188A (en) | The technique of ETEC JL GlcN fermenting and producing N acetylglucosamines | |
| CN101555503A (en) | Method for separating and extracting L-arabinose from waste wood sugar mother liquid from wood sugar production | |
| CN1594560A (en) | Method for separating and purifying natto kinase by ion exchange | |
| CN101412993A (en) | Solid fermentation method of natto kinase | |
| CN101591628A (en) | Acinetobacter junii X8 and the application in the preparation algin catenase thereof | |
| CN100999746A (en) | Method for synthesizing gamma-D-glutamyl-L-tryptophan by enzyme method | |
| CN105331562A (en) | Bacillus amyloliquefaciens Q-426 and lipopeptid separation method thereof | |
| CN104402976B (en) | A kind of method preparing enramycin fine powder | |
| CN101962637A (en) | Cordyceps militaris plasmin and culturing method thereof | |
| CN105713069A (en) | Bacilysin purification method | |
| CN1300311C (en) | Preparation method of chitin incision enzyme | |
| CN114250216B (en) | Nattokinase separation and purification method | |
| CN102604917A (en) | Preparation method of high-purity nattokinase | |
| CN101058804A (en) | Method for purifying nattokinase | |
| CN1680550A (en) | Purification of recommbined human urokinase zymogen | |
| CN104774794A (en) | Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same | |
| CN105219754A (en) | A kind of preparation method of Actinomycete fibrinolytic enzyme | |
| CN1101406C (en) | Polyose with fungal cell wall structure and its preparing process and application | |
| CN103865851B (en) | One strain Pseudomonas aeruginosa is used for the preparation of kappa-carrageenan enzyme | |
| CN1295326C (en) | Process for separating and purifying natto kinase by reverse micelle method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |