CN103667223B - A kind of method of sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone - Google Patents
A kind of method of sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone Download PDFInfo
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- CN103667223B CN103667223B CN201310647981.4A CN201310647981A CN103667223B CN 103667223 B CN103667223 B CN 103667223B CN 201310647981 A CN201310647981 A CN 201310647981A CN 103667223 B CN103667223 B CN 103667223B
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- acetone
- kallikrein
- sodium chloride
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21008—Kallikrein (3.4.21.8)
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Abstract
The invention discloses a kind of method of sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone. Technical scheme of the present invention is to extract, precipitate pig pancreas to make after acetone powder taking pig pancreas as raw material, the Extraction of acetic acid of applying in a flexible way, acetone precipitation, and ether defatting dehydration, traditional protein purification technology such as sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetone, prepare kallikrein. The present invention has with short production cycle, simple to operate, to chemistry and Heat stability is good, feature with low cost, is highly suitable for the large production of scale.
Description
Technical field
The present invention relates to biological technical field, specifically extract, precipitate pig taking pig pancreas as raw materialPancreas is made after acetone powder, the Extraction of acetic acid of applying in a flexible way, acetone precipitation, and ether defatting dehydration, sodium chloride-ammoniacal liquor,Traditional protein purification technology such as the collaborative removal of impurities of acetone, prepare kallikrein.
Background technology
Kallikrein (kallikein) is the natural Enzymes medicine that separation and purification makes from pig pancreas, facesOn bed, claim again callicrein, there is the capillary of unfolding and arteriolar effect, be commonly used to clinically controlTreat high fat of blood, prevent and treat the various diseases such as fatty liver, atherosclerotic, hypertension, angina pectoris, it openSend out and there is important medical science and social value. In China, kallikrein is produced and is also had very large development skyBetween.
In modern Bio-pharmaceutical Industry research, expert adopts ion-exchange chromatography, resin adsorption chromatography etc. each morePlant high-end chromatographic separation technology purification and obtain various enzymes and albumen. And select mainly the complying with of ion exchange resinAccording to be separated object character with separate object, comprise liberation characteristic, the molecule of separated object and major impurityThe character of amount, concentration, stability, medium of living in and the actual conditions of separation and requirement. Wherein most importantOne be to ensure to separate object and the absorption affinity of major impurity to resin according to separation requirement and isolating environmentThere is enough difference. In separated object, impurity is more, and this otherness obviously can be more not obvious. Large in realityIn production, in raw material, all contain impurity, if before adopting these high-end chromatographic techniques, noRaw material or semifinished product are carried out to pretreatment, will certainly be because the impurity content in raw material be too muchShorten the life-span of Ion Exchange Medium, reduce resin adsorption efficiency, and can have hangover in wash-outPhenomenon, extends manufacture cycle, and affects product yield and specific activity. As utilizations such as Li LihuanAmberlite-CG50 is separation and purification kallikrein from pancreatin, obtains by chromatographic separation technologyThe kallikrein specific activity, the purity that arrive are higher.
Kallikrein be taking pig pancreas as raw material, therefore, must to raw material carry out pretreatment,Purification obtains rough kallikrein, and then adopts ion-exchange chromatography to refineProduce.
The present invention is apply in a flexible way Extraction of acetic acid, acetone precipitation, ether defatting, dehydration, sodium chlorideTraditional protein purification technology such as-ammoniacal liquor, the collaborative removal of impurities of acetone are prepared kallikrein from pig pancreas,This technology is simple to operate, low production cost not only, and has avoided kallikrein degraded to loseLive, improve product specific activity.
Summary of the invention
The object of the invention is purifying and extract kallikrein from qualified pig pancreas.
Technical scheme of the present invention is: extract, precipitate pig pancreas taking pig pancreas as raw material and make after acetone powder, flexiblyUse Extraction of acetic acid, acetone precipitation, ether defatting dehydration, the tradition such as sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetoneProtein purification technology, prepares kallikrein. The present invention has with short production cycle, simple to operate, one-tenthThe feature that this is cheap, is highly suitable for the large production of scale. Comprise the steps:
(1) Extraction of acetic acid: pig pancreas is made after acetone powder, added acetum and stir extraction,Centrifugal, obtain supernatant;
(2) acetone precipitation: collect supernatant, add acetone precipitation, acetone, second for precipitationEther degreasing, dehydration, dry, obtain kallikrein crude product;
(3) sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetone: the 2-10 DEG C that crude product is doubly measured with 45-55Sodium chloride solution dissolves, and adjusts PH to 7.5-8.5 with ammoniacal liquor, is uniformly mixed, and filters, and obtains filtrate;Filtrate is chilled to 0-5 DEG C,, add 2-10 DEG C of acetone to make solution concentration reach 40%, stir evenly, leave standstill10-14 hour, centrifugal, obtain supernatant; By adding in supernatant, 2-10 DEG C of acetone is dense to solutionDegree reaches 60%, leaves standstill 4-8 hour, centrifugal, collecting precipitation. Acetone, ether washing for precipitation,Dry, obtain kallikrein;
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
The described pig pancreas that extracts taking pig pancreas as raw material, precipitates is made after acetone powder, the vinegar of applying in a flexible wayAcid extractants, acetone precipitation, ether defatting dehydration, sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetone etc.Modern protein purification technology, prepares kallikrein, comprises the steps:
1, Extraction of acetic acid: pig pancreas 10kg is made after acetone powder, add the vinegar of 30 times of amountsAcid solution, stirs and extracts, centrifugal, obtains supernatant 30L.
2, acetone precipitation: collect supernatant, add acetone precipitation. Acetone, ether for precipitationDegreasing, dehydration, dry, obtain kallikrein crude product 96g.
3, sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetone: 8 DEG C of sodium chloride by crude product by 50 times of amountsSolution dissolves, and adjusts PH to 8.0 with ammoniacal liquor, is uniformly mixed, and filters, and obtains filtrate; By filtrateBe chilled to 2 DEG C, add 5 DEG C of acetone to make solution concentration reach 40%, stir evenly, leave standstill 12 hours, fromThe heart, obtains supernatant; Reach add 5 DEG C of acetone to solution concentrations in supernatant at 60%, leave standstill 4Hour, centrifugal, collecting precipitation. For precipitation, acetone, ether washing, dry, obtains kassinin kinin and dischargeEnzyme 64g.
The above, be only preferred embodiment of the present invention, is not that the present invention is done to itThe restriction of its form, any those skilled in the art may utilize the technology of above-mentioned announcementContent is changed or is modified as the equivalent embodiment of equivalent variations. But everyly do not depart from thisBright technical scheme content, any letter of above embodiment being done according to technical spirit of the present inventionSingle amendment, equivalent variations and remodeling, still belong to the protection domain of technical solution of the present invention.
Claims (1)
1. a method for sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone, is characterized in that,The method comprises the steps:
1, Extraction of acetic acid: pig pancreas 10kg is made after acetone powder, add the vinegar of 30 times of amountsAcid solution, stirs and extracts, centrifugal, obtains supernatant 30L;
2, acetone precipitation: collect supernatant, add acetone precipitation; Acetone, ether for precipitationDegreasing, dehydration, dry, obtain kallikrein crude product 96g;
3, sodium chloride-ammoniacal liquor, the collaborative removal of impurities of acetone: 8 DEG C of sodium chloride by crude product by 50 times of amountsSolution dissolves, and adjusts PH to 8.0 with ammoniacal liquor, is uniformly mixed, and filters, and obtains filtrate; Filtrate is coldTo 2 DEG C, add 5 DEG C of acetone to make solution concentration reach 40%, stir evenly, leave standstill 12 hours, centrifugal,Obtain supernatant; Reach add 5 DEG C of acetone to solution concentrations in supernatant at 60%, leave standstill 4 hours,Centrifugal, collecting precipitation; For precipitation, acetone, ether washing, dry, obtains kallikrein 64g.
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| CN201310647981.4A CN103667223B (en) | 2013-11-27 | 2013-11-27 | A kind of method of sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone |
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| CN201310647981.4A CN103667223B (en) | 2013-11-27 | 2013-11-27 | A kind of method of sodium chloride-ammoniacal liquor, the collaborative purifying kallikrein of acetone |
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| CN103667223A CN103667223A (en) | 2014-03-26 |
| CN103667223B true CN103667223B (en) | 2016-05-11 |
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| CN104513816A (en) * | 2014-12-23 | 2015-04-15 | 青岛康原药业有限公司 | Method for extracting kallikein through acetic acid and medicine composition containing kallikein |
| CN104531647A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for purifying pancreatic kininogenase by using synergism of sodium chloride-ammonia water and acetone and pharmaceutical composition containing pancreatic kininogenase |
| CN105385670A (en) * | 2015-11-24 | 2016-03-09 | 青岛康原药业有限公司 | Method for highly purifying kallidin zymogen and drug composition improving stability of kallidin zymogen |
| CN105400758A (en) * | 2015-11-26 | 2016-03-16 | 青岛康原药业有限公司 | Method for purifying pancreatic kininogenase through synergism of sodium chloride-ammonia water and acetone and medicine composition for improving stability of pancreatic kininogenase |
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| CN102864134A (en) * | 2012-06-28 | 2013-01-09 | 江苏天福莱集团有限公司 | Extraction technology for squid viscera enzyme |
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Denomination of invention: A Method for Synergistic Purification of Kallikrein with Sodium Chloride Ammonia and Acetone Effective date of registration: 20230428 Granted publication date: 20160511 Pledgee: Weihai commercial bank Limited by Share Ltd. Qingdao branch Pledgor: QINGDAO KANGYUAN PHARMACEUTICAL Co.,Ltd. Registration number: Y2023980039608 |