NO142226B - PROCEDURE FOR THE EXTRACTION OF STRIPTKINASE - Google Patents
PROCEDURE FOR THE EXTRACTION OF STRIPTKINASE Download PDFInfo
- Publication number
- NO142226B NO142226B NO740336A NO740336A NO142226B NO 142226 B NO142226 B NO 142226B NO 740336 A NO740336 A NO 740336A NO 740336 A NO740336 A NO 740336A NO 142226 B NO142226 B NO 142226B
- Authority
- NO
- Norway
- Prior art keywords
- streptokinase
- extraction
- molar
- cellulose
- value
- Prior art date
Links
- 238000000605 extraction Methods 0.000 title claims description 4
- 108010023197 Streptokinase Proteins 0.000 claims description 24
- 229960005202 streptokinase Drugs 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 239000000706 filtrate Substances 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 4
- 239000003463 adsorbent Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000000356 contaminant Substances 0.000 claims description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 3
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 3
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 2
- 239000001913 cellulose Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108010011834 Streptolysins Proteins 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- -1 carboxymethyl polysaccharide Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960004533 streptodornase Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
Oppfinnelsen vedrører en fremgangsmåte til utvinning The invention relates to a method for extraction
av streptokinase som etter ytterligere behandling kan finne an- of streptokinase which after further treatment can find
vendelse som terapeutikum. reversal as a therapeutic.
Den fibrinolytiske virkning av kulturfiltrater av The fibrinolytic action of culture filtrates of
mange streptokokkstammer er kjent. Som stoff som er ansvarlig for dette ble det oppdaget streptokinase. Det dannes ved siden av mange andre stoffer som f.eks. streptodornase, streptolysin eller hyaluronidase under bestemte kulturbetingelser. For en terapeutisk anvendelse er det derfor ubetinget nødvendig en ad- many streptococcal strains are known. Streptokinase was discovered as the substance responsible for this. It is formed next to many other substances such as e.g. streptodornase, streptolysin or hyaluronidase under specific culture conditions. For a therapeutic application it is therefore absolutely necessary an ad-
skillelse fra næringsbestanddelene og de andre bakteriestoffveks-lingsprodukter. separation from the nutritional components and the other bacterial metabolic products.
En av de herved opptredende hovedvanskeligheter ligger One of the main difficulties arising in this way lies
i håndteringen av de derved dannede relativt store væskemengder, in the handling of the resulting relatively large amounts of liquid,
forbundet med relativt små streptokinasemengder. De tidligere metoder anvender til konsentrering utfellingsreaksjoner med etanol, metanol, ammoniumsulfat eller andre oppløsningsmidler. Ulempen ved disse metoder består på den ene side i ofte ikke meget høye utbytter, i forbruk av store mengder tilsetningsstoffer, forbundet med en primær volumøkning og i relativt liten rensefaktor. associated with relatively small amounts of streptokinase. The previous methods use precipitation reactions with ethanol, methanol, ammonium sulphate or other solvents for concentration. The disadvantage of these methods consists, on the one hand, in often not very high yields, in the consumption of large amounts of additives, associated with a primary volume increase and in a relatively small purification factor.
Videre er det kjent en fremgangsmåte til rensing av streptokinase, idet denne fremgangsmåte er karakterisert ved at man adsorberer en fra streptokokkfiltratet utvunnet streptokinase- Furthermore, a method for purifying streptokinase is known, as this method is characterized by adsorbing a streptokinase extracted from the streptococcal filtrate
holdig vandig pufferoppløsning med molaritet på 0,01 med en pH- containing aqueous buffer solution with a molarity of 0.01 with a pH
verdi på 5,0 på karboksymetylpolysakkarid, utvasker med puffer-oppløsning av samme molaritet og eluerer hovedfraksjonen av streptokinase med 0,2 molar pufferoppløsning av pH-verdi 5,5. value of 5.0 on carboxymethyl polysaccharide, wash out with buffer solution of the same molarity and elute the main fraction of streptokinase with 0.2 molar buffer solution of pH value 5.5.
Fra norsk patent 113.095 er det på den annen side From Norwegian patent 113,095 it is on the other side
kjent en fremgangsmåte til rensing av rå streptokinase, idet denne known a method for the purification of crude streptokinase, as this
fremgangsmåte er karakterisert ved at streptokinase adsorberes ved molare fosfatpufferkonsentrasjoner fra 0,005 til 0,015 på celluloseioneutvekslerharpiks og deretter eluerer forurensninger ved gradienteluering ved hjelp av fosfatpuffere i første rekke ved en molarkonsentrasjon fra 0,02 til 0,06 og deretter elueres streptokinase ved molare konsentrasjoner fra 0,06 til 0,15. method is characterized in that streptokinase is adsorbed at molar phosphate buffer concentrations from 0.005 to 0.015 on cellulose ion exchange resin and then contaminants are eluted by gradient elution using phosphate buffers in the first place at a molar concentration from 0.02 to 0.06 and then streptokinase is eluted at molar concentrations from 0, 06 to 0.15.
Denne fremgangsmåte gjennomføres hele tiden ved pH 7,5. This procedure is carried out all the time at pH 7.5.
En tilsvarende fremgangsmåte er også kjent fra US-patent 344404-5, idet det likeledes er omtalt en finrensing av rå streptokinase ved behandling med DEAE-cellulose. Streptokinase elueres deretter fra cellulosen med 0,1 til 0,15 molar fosfat-puf f er . A similar method is also known from US patent 344404-5, as a fine purification of crude streptokinase by treatment with DEAE cellulose is also described. Streptokinase is then eluted from the cellulose with 0.1 to 0.15 molar phosphate buffer.
Den foreliggende oppfinnelse har til formål å tilveie-bringe en fremgangsmåte til utvinning av streptokinase fra kulturfiltrater av streptokokker, som forbinder høye utbytter, lite materialforbruk og en høy rensningseffekt. The purpose of the present invention is to provide a method for extracting streptokinase from culture filtrates of streptococci, which combines high yields, low material consumption and a high purification effect.
Oppfinnelsen vedrører en fremgangsmåte til utvinning av streptokinase fra kulturfiltrater av streptokokker, hvor streptokinasen utfelles på et adsorberende materiale ved pH-verdier under 3,5 og etter fjerning av forurensninger ved vask elueres med en pufferoppløsning med alkalisk pH-verdi, hvoretter streptokinasen utvinnes fra eluatet, idet fremgangsmåten er karakterisert ved at det som adsorberende materiale anvendes karboksymetylcellulose og forurensninger fjernes ved vask med fortynnet iseddik. The invention relates to a method for the recovery of streptokinase from culture filtrates of streptococci, where the streptokinase is precipitated on an adsorbent material at pH values below 3.5 and after removal of impurities by washing is eluted with a buffer solution with an alkaline pH value, after which the streptokinase is recovered from the eluate , the method being characterized in that carboxymethyl cellulose is used as adsorbent material and contaminants are removed by washing with diluted glacial acetic acid.
Ved dekantering, filtrering eller sentrifugering By decanting, filtering or centrifuging
lar CM-cellulosen seg adskille lett med den bundne streptokinase. Ved utvasking av CM-cellulosen med 0,01 N eddiksyre lar de fleste medrevne næringsbestanddeler seg fjerne. Streptokinasen gjen-vinnes ved eluering med 0,05 molar fosfatpuffer, som er 0,1 molar med hensyn på natriumklorid, ved pH=8,0. I eluatet kan streptolysinet etter kjente fremgangsmåter ved tilsetning av CaC^ fjernes på det kalsiumfosfat som danner seg, idet samtidig den største del av forurensninger fjernes. Det således dannede produkt utfelles enten med etanol eller ammoniumsulfat på kjent måte, dialyseres mot vann og lyofiliseres. Det inneholder ca. 30.000 _Christensen-enh.eter pr. mg protein og er fritt for streptolysin. allows the CM-cellulose to dissociate easily with the bound streptokinase. By washing out the CM cellulose with 0.01 N acetic acid, most entrained nutrients can be removed. The streptokinase is recovered by elution with 0.05 molar phosphate buffer, which is 0.1 molar with respect to sodium chloride, at pH=8.0. In the eluate, the streptolysin can be removed by known methods by adding CaC^ on the calcium phosphate that forms, while at the same time the largest part of impurities is removed. The product thus formed is precipitated either with ethanol or ammonium sulfate in a known manner, dialyzed against water and lyophilized. It contains approx. 30,000 _Christensen units per mg protein and is free of streptolysin.
Som næringssubstrat for dyrking av streptokokkene tjener tryptisk kaseinpepton med glukose, salt- og vitamintil-setninger. Andre næringsbunner gir dårligere utbytter. Tryptic casein peptone with glucose, salt and vitamin additives serves as a nutrient substrate for growing the streptococci. Other nutrients give poorer yields.
Det kreves fortrinnsvis ingen dyre tilsetningsstoffer, Preferably no expensive additives are required,
den praktiske utførelse er enkel, den anvendte CM-cellulose er regenererbar, utbyttene svinger mellom 60 og 90% og det ekstra- the practical implementation is simple, the CM cellulose used is renewable, the yields fluctuate between 60 and 90% and the extra-
herte kulturfiltrat er praktisk talt sterilt og kan kasseres uten ytterligere behandling. Ved felling av streptokinase på en ione-utveksler kan det videre ved bestemte ekstraheringsbetingelser fjernes hovedmengden av forurensninger uten ekstra arbeids<p>rosess. hardened culture filtrate is practically sterile and can be discarded without further treatment. By precipitating streptokinase on an ion exchanger, the main amount of contaminants can be removed without additional work under specific extraction conditions.
Oppfinnelsen skal forklares nærmere ved hjelp av et The invention shall be explained in more detail by means of a
eksempel: Example:
10 g CM-cellulose ekvilibreres med 500 ml 0,2 N 10 g of CM cellulose is equilibrated with 500 ml of 0.2 N
eddiksyre i 30 minutter og vaskes deretter med 500 ml 0,01 N acetic acid for 30 minutes and then washed with 500 ml of 0.01 N
eddiksyre. Den således forberedte karboksymetylcellulose setter man til 5 liter streptokokk-kulturfiltrat, hvis pH-verdi på for- acetic acid. The carboxymethylcellulose prepared in this way is added to 5 liters of streptococcus culture filtrate, whose pH value of
hånd ble innstilt til 3,0. Etter to timers omrøring filtrerer man over en grov glassfritte (G2) og kasserer filtratet. CM-cellulo- hand was set to 3.0. After stirring for two hours, filter over a coarse glass frit (G2) and discard the filtrate. CM Cellulose
sen vaskes med 0,5 1 0,01 N eddiksyre. Deretter eluerer man streptokinasen med 1 liter fosfatpuffer (0,05 molar fosfat, 0,1 then wash with 0.5 1 0.01 N acetic acid. The streptokinase is then eluted with 1 liter of phosphate buffer (0.05 molar phosphate, 0.1
molar NaCl, pH=8,0). pH-verdien i eluatet ble innstilt til 7,0 molar NaCl, pH=8.0). The pH value in the eluate was adjusted to 7.0
og streptolysinet utfelt med 10 ml 1 molar CaCl2 ved konstant holdt pH-verdi. Utfellingen ble bortsentrifugert etter 30 minutter og kassert. Streptokinasen felles ved pH 5,5 med 350 g ammonium- and the streptolysin precipitated with 10 ml of 1 molar CaCl2 at a constant pH value. The precipitate was centrifuged off after 30 minutes and discarded. The streptokinase is combined at pH 5.5 with 350 g of ammonium
sulfat (pr. liter). Den dannede utfelling oppløser man i ca. sulphate (per litre). The formed precipitate is dissolved in approx.
60 ml vann, innstiller suspensjonens pH-verdi til 9,0, dialyserer sulfatfri og lyofiliserer. 60 ml of water, adjust the pH value of the suspension to 9.0, dialyze sulfate-free and lyophilize.
Man får 450 mg streptokinase med en titer på 2,5x10<4 >Christensen-enheter pr. mg. Det tilsvarer 80% av utgangsaktivi-teten. For terapeutiske formål kan det dannede produkt videre- You get 450 mg of streptokinase with a titer of 2.5x10<4 >Christensen units per mg. This corresponds to 80% of the output activity. For therapeutic purposes, the product formed can further
renses etter en av de kjente fremgangsmåter. is cleaned according to one of the known methods.
Claims (1)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD16861573A DD110885A1 (en) | 1973-02-02 | 1973-02-02 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| NO740336A NO740336A (en) | 1974-08-05 |
| NO142226B true NO142226B (en) | 1980-04-08 |
| NO142226C NO142226C (en) | 1980-07-16 |
Family
ID=5489948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO740336A NO142226C (en) | 1973-02-02 | 1974-02-01 | PROCEDURE FOR THE EXTRACTION OF STRIPTKINASE |
Country Status (7)
| Country | Link |
|---|---|
| AT (1) | AT331749B (en) |
| CS (1) | CS177967B1 (en) |
| DD (1) | DD110885A1 (en) |
| DK (1) | DK136531C (en) |
| HU (1) | HU169641B (en) |
| NO (1) | NO142226C (en) |
| RO (1) | RO62678A (en) |
-
1973
- 1973-02-02 DD DD16861573A patent/DD110885A1/xx unknown
-
1974
- 1974-01-15 AT AT31574A patent/AT331749B/en not_active IP Right Cessation
- 1974-01-30 HU HUAE000403 patent/HU169641B/hu unknown
- 1974-01-31 DK DK50474A patent/DK136531C/en active
- 1974-02-01 RO RO7749474A patent/RO62678A/en unknown
- 1974-02-01 CS CS69774A patent/CS177967B1/en unknown
- 1974-02-01 NO NO740336A patent/NO142226C/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO142226C (en) | 1980-07-16 |
| ATA31574A (en) | 1975-12-15 |
| CS177967B1 (en) | 1977-08-31 |
| RO62678A (en) | 1978-02-15 |
| DK136531B (en) | 1977-10-24 |
| NO740336A (en) | 1974-08-05 |
| DK136531C (en) | 1978-03-20 |
| HU169641B (en) | 1977-02-28 |
| AT331749B (en) | 1976-08-25 |
| DD110885A1 (en) | 1975-01-12 |
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