CN103740686B - A kind of method with Extraction of acetic acid kallikrein - Google Patents
A kind of method with Extraction of acetic acid kallikrein Download PDFInfo
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- CN103740686B CN103740686B CN201310638710.2A CN201310638710A CN103740686B CN 103740686 B CN103740686 B CN 103740686B CN 201310638710 A CN201310638710 A CN 201310638710A CN 103740686 B CN103740686 B CN 103740686B
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- acetone
- kallikrein
- extraction
- acetic acid
- pig pancreas
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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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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of method with Extraction of acetic acid kallikrein. 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 traditional protein purification technology such as ether defatting dehydration, prepare kallikrein. The present invention is simple to operate, with short production cycle, 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 pancreas taking pig pancreas as raw materialMake after acetone powder the Extraction of acetic acid of applying in a flexible way, acetone precipitation, traditional protein purifications such as ether defatting dehydrationTechnology, prepares 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.
The technique of traditional purifying kallikrein is because complex process, cost are higher; Ion-exchange chromatography isThe common method that is applicable to kallikrein suitability for industrialized production at present; And selection ion exchange resin is mainAccording to be separated object character and separate object, comprise separated object and major impurity liberation characteristic, divideThe character of sub-amount, concentration, stability, medium of living in and the actual conditions of separation and requirement. Wherein the heaviestOne that wants is to ensure to separate object and the absorption of major impurity to resin according to separation requirement with isolating environmentPower has enough difference. In separated object, impurity is more, and this otherness obviously can be more not obvious. In realityIn large production, in raw material, all contain impurity, if before adopting these high-end chromatographic techniques,Raw material or semifinished product are not carried out to pretreatment, will certainly be too much because of the impurity content in raw materialAnd shorten life-span of Ion Exchange Medium, reduce resin adsorption efficiency, and can exist and drag in wash-outTail phenomenon, extends manufacture cycle, and affects product yield and specific activity.
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, traditional eggs such as ether defatting, dehydrationWhite purification technique is prepared kallikrein from pig pancreas, and this technology is simple to operate, production cost not onlyCheap, and avoided kallikrein degraded inactivation, improve product specific activity.
Summary of the invention
The object of the invention is from qualified pig pancreas, to extract kallikrein with acetum.
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, traditional protein purification technology such as ether defatting dehydration, prepare kassinin kinin and dischargeEnzyme. The present invention has with short production cycle, and feature simple to operate, with low cost is very applicableIn the large production of scale. Comprise the steps:
1, Extraction of acetic acid: pig pancreas is made after acetone powder, added 18～22 times of amounts0.01～0.03mol/L acetum, stirs and extracts 10～14 hours in 10 DEG C, centrifugal, obtainsClear liquid; Residue is added to 8～12 times of amount 0.01～0.03mol/L acetums again to be stirred and carriesGet 4～8 hours, centrifugal, merge twice supernatant;
2, acetone precipitation: collect supernatant, add 2～10 DEG C of acetone that the concentration of solution is reached33%, stir evenly, filter, obtain filtrate; The concentration of 2～10 DEG C of acetone to solution will be added in filtrateReach 70%, stir evenly, leave standstill 3～6 hours, centrifugal, collecting precipitation. Acetone, second for precipitationEther degreasing, dehydration, 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, traditional protein purification technology such as ether defatting dehydration, prepare kassinin kinin and releasePut enzyme, comprise the steps:
1, Extraction of acetic acid: pig pancreas 10Kg is made after acetone powder, add 20 times of amounts0.02mol/L acetum, stirs and extracts 12 hours in 10 DEG C, centrifugal, obtains supernatant; WillResidue adds 10 times of amount 0.02mol/L acetums and again stirs extraction 6 hours, centrifugal,Merge supernatant 29.8L twice.
2, acetone precipitation: collect supernatant, add 5 DEG C of acetone to make the concentration of solution reach 33%,Stir evenly, filter, obtain filtrate; Reach 70% by adding 5 DEG C of acetone to the concentration of solution in filtrate,Stir evenly, leave standstill 4 hours, centrifugal, collecting precipitation. Acetone, ether defatting, dehydration for precipitation,Dry, obtain kallikrein 97g.
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. by a method for Extraction of acetic acid kallikrein, it is characterized in that, the method comprises as followsStep:
1, Extraction of acetic acid: pig pancreas 10Kg is made after acetone powder, add 20 times of amounts0.02mol/L acetum, stirs and extracts 12 hours in 10 DEG C, centrifugal, obtains supernatant; WillResidue adds 10 times of amount 0.02mol/L acetums and again stirs extraction 6 hours, centrifugal,Merge supernatant 29.8L twice;
2, acetone precipitation: collect supernatant, add 5 DEG C of acetone to make the concentration of solution reach 33%,Stir evenly, filter, obtain filtrate; Reach 70% by adding 5 DEG C of acetone to the concentration of solution in filtrate,Stir evenly, leave standstill 4 hours, centrifugal, collecting precipitation; Acetone, ether defatting, dehydration for precipitation,Dry, obtain kallikrein 97g.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310638710.2A CN103740686B (en) | 2013-11-29 | 2013-11-29 | A kind of method with Extraction of acetic acid kallikrein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310638710.2A CN103740686B (en) | 2013-11-29 | 2013-11-29 | A kind of method with Extraction of acetic acid kallikrein |
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| Publication Number | Publication Date |
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| CN103740686A CN103740686A (en) | 2014-04-23 |
| CN103740686B true CN103740686B (en) | 2016-05-11 |
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| CN201310638710.2A Active CN103740686B (en) | 2013-11-29 | 2013-11-29 | A kind of method with Extraction of acetic acid kallikrein |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105400762A (en) * | 2015-11-26 | 2016-03-16 | 青岛康原药业有限公司 | Method for extracting pancreatic kininogenase with acetic acid and pharmaceutical composition capable of improving stability of pancreatic kininogenase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1336434A (en) * | 2000-08-01 | 2002-02-20 | 上海惠海生化制品厂 | Prepn. and affinity chromatographic purification process of kallidinogen enzyme |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1336434A (en) * | 2000-08-01 | 2002-02-20 | 上海惠海生化制品厂 | Prepn. and affinity chromatographic purification process of kallidinogen enzyme |
Non-Patent Citations (2)
| Title |
|---|
| 离子交换和亲和层析技术纯化制备猪胰激肽释放酶;姜春华;《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》;20061015;第23页表2-1,第24页第3-14行 * |
| 胰激肤释放酶生产新工艺研究;赵世燊等;《中国生化药物杂志》;19901231;第28页右栏第5-11行 * |
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| CN103740686A (en) | 2014-04-23 |
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