CN105368810A - Method for preparing chymotrypsin - Google Patents
Method for preparing chymotrypsin Download PDFInfo
- Publication number
- CN105368810A CN105368810A CN201510826531.0A CN201510826531A CN105368810A CN 105368810 A CN105368810 A CN 105368810A CN 201510826531 A CN201510826531 A CN 201510826531A CN 105368810 A CN105368810 A CN 105368810A
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- China
- Prior art keywords
- acetone
- solution
- add
- quimotrase
- acid buffer
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 108090000317 Chymotrypsin Proteins 0.000 title claims abstract description 6
- 229960002376 chymotrypsin Drugs 0.000 title abstract description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 44
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 210000000496 pancreas Anatomy 0.000 claims abstract description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 8
- 241001465754 Metazoa Species 0.000 claims abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 108010019160 Pancreatin Proteins 0.000 claims description 7
- 239000012043 crude product Substances 0.000 claims description 7
- 229940055695 pancreatin Drugs 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 3
- 230000018044 dehydration Effects 0.000 claims description 3
- 238000006297 dehydration reaction Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 5
- 238000001556 precipitation Methods 0.000 abstract description 5
- 238000001742 protein purification Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108010067035 Pancrelipase Proteins 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 238000009938 salting Methods 0.000 abstract 1
- 238000005185 salting out Methods 0.000 abstract 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 3
- 206010030113 Oedema Diseases 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 208000002177 Cataract Diseases 0.000 description 1
- 108010038061 Chymotrypsinogen Proteins 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 206010018833 Haematocoele Diseases 0.000 description 1
- 208000005873 Hematocele Diseases 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000001886 ciliary effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 201000004920 hematocele of tunica vaginalis testis Diseases 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
-
- 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)
- C12Y304/21001—Chymotrypsin (3.4.21.1)
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for preparing chymotrypsin. The technical scheme includes that animal pancreases are ground for preparing the chymotrypsin by flexible application of modern protein purification technologies including pancreatic enzyme hydrolysis, acetone precipitation, phosphate buffer salting in, ammonium sulfate salting out and the like. The method for preparing the chymotrypsin is simple in operation, low in cost and quite suitable for large-scale production.
Description
Technical field
The present invention relates to biological technical field, pancreatin hydrolysis of specifically applying in a flexible way, acetone precipitation, phosphoric acid buffer salt be molten, the modern Protein purification techniques such as ammonium sulfate precipitation, from qualified animal pancreas, prepare the method for Quimotrase.
Background technology
Quimotrase chymotrypsin is a kind of typical serine protease, vertebrate digestive ferment.EC3.421.1。With the form biosynthesizing of enzyme precursor material chymotrypsinogen in pancreas, go out with pancreatic secretion.Be subject to certain decomposition of trypsinase and Quimotrase at small intestine, be transformed into active Quimotrase.
Quimotrase economic worth is high, mainly as medicine in medical: (1). for wound or wound healing after operation (2) anti-inflammatory and prevent local edema, hematocele, after (3) operation on breast of spraining hemotoncus (4) this product such as edema, otitis media, rhinitis to the selective dissolution of eyeball ciliary ligament, therefore can be used for cataract extraction, lens is removed with comparalive ease.
Medicinal Quimotrase mainly extracts from Pancreas Bovis seu Bubali, and the present invention is the modern Protein purification techniques such as pancreatin hydrolysis, acetone precipitation, phosphoric acid buffer salt are molten, ammonium sulfate precipitation of applying in a flexible way, use the low-cost auxiliary materials such as phosphoric acid salt, acetone, ammonium sulfate, prepare Quimotrase, not only reduce cost to a certain extent, and the yield of product also can reach 200,000 units/kg pancreas.
Summary of the invention
The object of the invention is to extract Quimotrase from qualified animal pancreas.
Technical scheme of the present invention is: by qualified animal pancreas after rubbing, and the modern Protein purification techniques such as the purification of pancreatin hydrolysis of applying in a flexible way, acetone precipitation, phosphoric acid buffer and ammonium sulfate precipitation and crystallization, prepare the method for Quimotrase.The present invention is simple to operate, cost is low, is very suitable for the large production of mass-producing.Comprise the steps:
(1) be hydrolyzed: fresh or freezing Pancreas Sus domestica is rubbed and is placed in container, add the acetone of 2 ~ 5 times amount less than 0 DEG C, dehydration 0.5 ~ 1.5 hour is stirred at about 0 DEG C, centrifugal throw out, add water and 0.01 ~ 0.02 times amount pancreatin stirring and dissolving of 0.5 ~ 1.5 times amount, be adjusted to pH value to 6.0 ~ 9.0 with 6% ~ 10% sodium hydroxide solution, in 15 DEG C ~ 25 DEG C insulated and stirred 1 ~ 3 hour, add diatomite more fully to stir, filter;
(2) precipitate: collection filtrate and 1.5 ~ 3 times amount acetone stir evenly, leave standstill 10 ~ 14 hours, siphon discards upper strata acetone clear liquid, divides 3 ~ 4 washing precipitates with 1.5 ~ 2 times amount acetone, filters and dries, dry, obtains Quimotrase crude product;
(3) purify: crude product and 45 ~ 60 times amount phosphoric acid buffers are stirred 0.5 ~ 1.5 hour, leave standstill 15 ~ 25 hours in 15 DEG C ~ 25 DEG C, the diatomite adding its amount about 3% ~ 7% according to the volume of solution is mixed thoroughly, filters.In filtrate, add ammonium sulfate, reach 35% ~ 45% to ammonium sulfate concentrations.Filter, thing of must saltouing; By saltouing, thing is dissolved in carbonic acid buffer in the ratio of 1g:8 ~ 10ml again, then under agitation dropwise adds saturated ammonium sulphate solution, until micro-muddy, leave standstill 48 ~ 72 hours, slowly form needle crystal at 0 DEG C ~ 8 DEG C, filter;
(4) dry: leaching crystal also rinses 3 ~ 5 times with water, lyophilize, Quimotrase.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
Described utilizes the modern Protein purification techniques such as the purification of pancreatin hydrolysis, acetone precipitation, phosphoric acid buffer and ammonium sulfate crystallization, prepares the method for Quimotrase, comprises the steps:
1. be hydrolyzed: fresh pig pancreas is rubbed and is placed in container, add the acetone of 3 times amount 0 DEG C, dehydration 1 hour is stirred at about 0 DEG C, centrifugal throw out, add 1 times of water gaging and 0.015 times amount pancreatin stirring and dissolving, be adjusted to pH value to 7.5 with 8% sodium hydroxide solution, in 20 DEG C of insulated and stirred 2 hours, add 0.01 times amount diatomite more fully to stir, filter.
2. precipitate: collection filtrate and 2 times amount acetone stir evenly, leave standstill 12 hours, siphon discards upper strata acetone clear liquid, divides 4 washing precipitates with 2 times amount acetone, filters and dries, dry, about obtains 120g Quimotrase crude product.
3. purify: crude product and 52 times amount phosphoric acid buffers are uniformly mixed 1 hour, leave standstill 20 hours in 20 DEG C, the diatomite adding its amount about 5% is mixed thoroughly, filters.In filtrate, add ammonium sulfate reach 40% to concentration, filter, about 35g saltouts thing; By saltouing, thing is dissolved in carbonic acid buffer in the ratio of 1g:10ml again, then under agitation dropwise adds saturated ammonium sulphate solution, until micro-muddy, leave standstill 72 hours, slowly form needle crystal at 4 DEG C, filter.
4. dry: leaching crystal also rinses 3 times with water, lyophilize, about can obtain 4.5g Quimotrase.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (5)
1. prepare a method for Quimotrase, it is characterized in that, the method comprises the steps:
(1) be hydrolyzed: fresh or freezing animal pancreas rubbed and be placed in container, add acetone and stir dehydration, centrifugal throw out, adds water and pancreatin stirring and dissolving, is adjusted to pH value, insulated and stirred with sodium hydroxide solution, then adds diatomite and fully stir, and filters;
(2) precipitate: collect filtrate, add acetone and stir evenly, leave standstill, siphon discards upper strata acetone clear liquid, then uses washing with acetone throw out, filters and dries, dry; Obtain Quimotrase crude product;
(3) purify: crude product phosphoric acid buffer is dissolved, is uniformly mixed, leave standstill, then add diatomite and mix thoroughly, filter; Ammonium sulfate is added, thing of can saltouing after filtration in filtrate; Thing of saltouing is dissolved in carbonic acid buffer again, then under agitation dropwise adds saturated ammonium sulphate solution, leaves standstill, and filters;
(4) leaching crystal and with water rinse, lyophilize, Quimotrase.
2. method according to claim 1, is characterized in that: in step (1), and the ratio of acetone and pancreas is 2 ~ 5L:1kg, acetone temperature less than 0 DEG C; Concentration of sodium hydroxide solution is 6% ~ 10%; The pH value of solution is adjusted to 6.0 ~ 9.0; In 15 DEG C ~ 25 DEG C insulated and stirred about 1 ~ 3 hour; Before filtration, add diatomite and fully stir.
3. method according to claim 1, is characterized in that: in step (2), and the volume ratio of acetone and filtrate is 1.5 ~ 3:1; Time of repose is: 10 ~ 14 hours; After abandoning supernatant, use washing with acetone throw out.
4. method according to claim 1, is characterized in that: in step (3), the ratio of crude product and phosphoric acid buffer is: 1g:45 ~ 60ml; Solution leaves standstill 15 ~ 25 hours in 15 DEG C ~ 25 DEG C; The diatomite of its amount 3% ~ 7% weight is added according to the volume of solution; The amount adding ammonium sulfate in filtrate reaches 35% ~ 45% for making the concentration of solution; The ratio of thing and carbonic acid buffer of saltouing is: 1g:8 ~ 10ml; Add the amount of saturated ammonium sulphate solution for micro-muddy to solution; Dwell temperature is: 0 DEG C ~ 8 DEG C, and the time is: 48 ~ 72 hours.
5. method according to claim 1, is characterized in that: the chymotrypsin protein enzyme crystal obtained will carry out lyophilize.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510826531.0A CN105368810A (en) | 2015-11-25 | 2015-11-25 | Method for preparing chymotrypsin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510826531.0A CN105368810A (en) | 2015-11-25 | 2015-11-25 | Method for preparing chymotrypsin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105368810A true CN105368810A (en) | 2016-03-02 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510826531.0A Withdrawn CN105368810A (en) | 2015-11-25 | 2015-11-25 | Method for preparing chymotrypsin |
Country Status (1)
| Country | Link |
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| CN (1) | CN105368810A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018138292A1 (en) * | 2017-01-26 | 2018-08-02 | Enzymatica Ab | A polypeptide having protease activity for use in treating otitis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944641A (en) * | 2006-10-26 | 2007-04-11 | 上海林叶生物科技有限公司 | Method for preparing high purity chymotrypsin |
| WO2011126957A1 (en) * | 2010-04-05 | 2011-10-13 | Avantor Performance Materials, Inc. | Process for trypsin and chymotrypsin purification utilizing hydrophobic interaction chromatography |
| CN103667225A (en) * | 2013-11-23 | 2014-03-26 | 青岛康原药业有限公司 | Method for preparing elastase |
-
2015
- 2015-11-25 CN CN201510826531.0A patent/CN105368810A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944641A (en) * | 2006-10-26 | 2007-04-11 | 上海林叶生物科技有限公司 | Method for preparing high purity chymotrypsin |
| WO2011126957A1 (en) * | 2010-04-05 | 2011-10-13 | Avantor Performance Materials, Inc. | Process for trypsin and chymotrypsin purification utilizing hydrophobic interaction chromatography |
| CN103667225A (en) * | 2013-11-23 | 2014-03-26 | 青岛康原药业有限公司 | Method for preparing elastase |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018138292A1 (en) * | 2017-01-26 | 2018-08-02 | Enzymatica Ab | A polypeptide having protease activity for use in treating otitis |
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| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
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| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20160302 |