CN105238771A - Method for extracting urokinase and ulinastatin from urine - Google Patents
Method for extracting urokinase and ulinastatin from urine Download PDFInfo
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- CN105238771A CN105238771A CN201510691424.1A CN201510691424A CN105238771A CN 105238771 A CN105238771 A CN 105238771A CN 201510691424 A CN201510691424 A CN 201510691424A CN 105238771 A CN105238771 A CN 105238771A
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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)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
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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)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
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Abstract
The invention provides a method for extracting urokinase and ulinastatin from urine. The method comprises the following steps: collecting the urine, and adjusting the pH value of the urine; adding silica gel powder into the urine, and stirring so that urokinase protein in the urine is adsorbed by the silica gel; acquiring the silica gel through a filtering method, and adjusting the pH value of the filtered urine; adding chitin into the filtered urine so that ulinastatin protein in the urine is adsorbed by the chitin; acquiring urokinase protein fluid and ulinastatin protein fluid from the silica gel and the chitin, respectively; and adding the urokinase protein fluid into saturated water, and extracting to obtain urokinase, and adding ammonium sulfate into the ulinastatin protein fluid, and extracting to obtain ulinastatin. According to the method provided by the invention, the urokinase crude product and the ulinastatin crude product can be extracted from the urine, so that the economic value of the urine is greatly improved.
Description
Technical field
The present invention relates to biological extraction technology, particularly relate to a kind of method extracting urokinase and ulinastatin from urine.
Background technology
In recent years, in man's urine, extract the proteolytic enzyme such as urokinase, ulinastatin crude product company and individual are a lot, but ubiquity extract urokinase, ulinastatin crude product technology not good enough, cause the extracted amount of urokinase, ulinastatin little, active low, cause a large amount of wastes, affect the problem of the economic worth of urine.Therefore, how improving urokinase, the extraction yield of ulinastatin crude product and quality, is a research very with application prospect.The research can make the urine of collection, turns waste into wealth, and the economic worth of urine is promoted further.
Summary of the invention
Technical problem solved by the invention is to provide a kind of method extracting urokinase and ulinastatin from urine, and it can improve the seed output and quality that urine is extracted, and greatly promotes the economic worth of urine.
For solving the problems of the technologies described above, the present invention adopts a kind of method extracting urokinase and ulinastatin from urine, comprising:
Collect urine and regulate between its pH value to 6.0-6.5;
In urine, add Powdered silica gel and stir, making urokinase albumen in urine by silica gel adsorption;
Obtain the silica gel in urine by filter type and regulate between the pH value of the urine after filtering to 5.3-5.7;
Add chitin in urine after foregoing filtration and stir, the ulinastatin albumen in urine is adsorbed by chitin;
Urokinase protein liquid and ulinastatin protein liquid is obtained respectively from aforementioned silica gel and chitin;
Urokinase protein liquid is joined in saturation water to extract and obtain urokinase, in ulinastatin protein liquid, add ammonium sulfate extraction obtain ulinastatin.
As the further improvement of the technical program, collect urine and regulate between its pH value to 6.0-6.5, specifically comprising:
Flush urinal is exempted from utilization and urine collected by pipeline;
The pH value to 6.3 of urine is regulated with hydrochloric acid.
As the further improvement of the technical program, in urine, add Powdered silica gel and stir, specifically comprising: to add the ratio of 20-25 kilogram of silica gel in urine per ton, silica gel and urine mixed and stirs.
As the further improvement of the technical program, obtain the silica gel in urine by filter type and regulate between the pH value of the urine after filtering to 5.3-5.7, specifically comprising:
Filter described urine with gauze pocket, and collect the silica gel being adsorbed with urokinase albumen, the pH value of the urine after filtration is adjusted to 5.5 by the mode adopting gradation to inject hydrochloric acid.
As the further improvement of the technical program, add chitin in the urine after foregoing filtration and stir, specifically comprising:
To add the ratio of 5 kilograms of chitins in urine per ton, chitin and urine are mixed and stirs, and gradation adds hydrochloric acid and defoamer, make urine pH value remain on 5.5.
As the further improvement of the technical program, from aforementioned silica gel and chitin, obtain urokinase protein liquid and ulinastatin protein liquid respectively, specifically comprise:
Collect with gauze pocket and be adsorbed with the silica gel of urokinase albumen, and the chromatographic solution that Image processing obtains containing urokinase albumen is carried out to silica gel, then wash-out process is carried out to chromatographic solution obtain urokinase protein liquid;
Collect the chitin being adsorbed with ulinastatin albumen with gauze pocket, chitin clear water is embathed, and wash-out process is carried out to chitin, obtain ulinastatin protein liquid.
As the further improvement of the technical program, Image processing is carried out to silica gel, specifically comprises:
Silica gel is injected in chromatography column, silica gel is washed with clear water, the proportioning scavenging solution being not less than 1:8 in the ratio of silica gel and scavenging solution again cleans, obtain the chromatographic solution containing urokinase albumen, described scavenging solution adds 82 grams of SODIUM PHOSPHATE, MONOBASIC by every 50 kg of water to be allocated and adds ammoniacal liquor modulation pH value to 8.9 ± 0.1.
As the further improvement of the technical program, wash-out process is carried out to chromatographic solution, specifically comprises:
The ratio of 3 liters of elutriants is joined in 1 liter of silica gel, with elutriant, wash-out collection is carried out to the urokinase albumen in chromatographic solution, obtain urokinase protein liquid, described elutriant adds 3.5 kilograms of ammonium chlorides by every 50 kg of water to be allocated and adds ammoniacal liquor modulation pH value to 10.0-11.0.
As the further improvement of the technical program, wash-out process is carried out to chitin, specifically comprises:
The chitin being adsorbed with ulinastatin albumen is put into elutriant stir, discharge by the ulinastatin albumen adsorbed, described elutriant is mixed in the ratio of 1:2 by secondary saturation water and water, and adds sheet alkali modulation pH value to 9.0-10.0.
As the further improvement of the technical program, urokinase protein liquid is joined in saturation water to extract and obtain urokinase, specifically comprise: the ratio of joining 2 liters of saturation waters in 1 liter of urokinase protein liquid, described urokinase protein liquid is joined in saturation water, after leaving standstill through at least 10 hours, adopt and filter extraction urokinase containing soaking diatomaceous vacuum funnel;
In ulinastatin protein liquid, add ammonium sulfate extraction obtain ulinastatin, specifically comprise: will commercial sulphuric acid ammonium be added in the liquid discharging ulinastatin albumen and stir, and proportion remains between 26-28, after leaving standstill through at least 5 hours, adopt and filter extraction ulinastatin containing soaking diatomaceous vacuum funnel.
The present invention extracts crude urokinase and ulinastatin crude product in urine, adds the seed output and quality of extraction, substantially increases the economic worth of urine.
Accompanying drawing explanation
Fig. 1 is the schema extracting the method for urokinase and ulinastatin from urine of the present invention.
Embodiment
Refer to shown in Fig. 1, the invention provides a kind of method extracting urokinase and ulinastatin from urine, comprise following S101 to S106 step, specifically:
s101: collect urine and regulate between its pH value to 6.0-6.5
In present pre-ferred embodiments, described " S101: collect urine and regulate between its pH value to 6.0-6.5 " specifically comprises: utilize and exempt from flush urinal and pipeline collection urine; The pH value to 6.3 of urine is regulated with hydrochloric acid.In the present embodiment, urine is injected generation stirred pot, stir while slowly inject the pH value that dilute hydrochloric acid (hydrochloric acid and water ratio are 1:5) debugs urine, be stabilized in till 6.3 until pH value.
s102: add Powdered silica gel and stir in urine, makes urokinase albumen in urine by silica gel adsorption
In present pre-ferred embodiments, described " S102: add Powdered silica gel and stir in urine " specifically comprises: to add the ratio of 20-25 kilogram of silica gel in urine per ton, silica gel and urine mixed and stir.
s103: obtain the silica gel in urine by filter type and regulate between the pH value of the urine after filtering to 5.3-5.7
In present pre-ferred embodiments, described " S103: obtain the silica gel in urine by filter type; and regulate between the pH value of the urine after filtering to 5.3-5.7 " specifically comprise: filter described urine with gauze pocket, and collect the silica gel being adsorbed with urokinase albumen, the pH value of the urine after filtration is adjusted to 5.5, in the present embodiment, the mode that hydrochloric acid gradation can be adopted to inject when pH value regulates is carried out, and adds defoamer to reduce foam volume simultaneously.
s104: add chitin in the urine after foregoing filtration and stir, makes the ulinastatin albumen in urine be adsorbed by chitin
In present pre-ferred embodiments, described " S104: add chitin in the urine after foregoing filtration and stir " specifically comprises: to add the ratio (i.e. 200:1) of 5 kilograms of chitins in urine per ton, chitin and urine mixed and stir.In the present embodiment, can chitin and urine are blended in stirred pot, carry out the stirring of 1.5 hours, and gradation adds hydrochloric acid and defoamer, urine pH value is remained unchanged, namely still between 5.3-5.7, preferably 5.5.
s105: obtain urokinase protein liquid and ulinastatin protein liquid respectively from aforementioned silica gel and chitin
In present pre-ferred embodiments, described " S105: obtain urokinase protein liquid and ulinastatin protein liquid respectively from aforementioned silica gel and chitin " specifically comprises:
Collect with gauze pocket and be adsorbed with the silica gel of urokinase albumen, and the chromatographic solution that Image processing obtains containing urokinase albumen is carried out to silica gel, then wash-out process is carried out to chromatographic solution obtain urokinase protein liquid.Wherein, described " Image processing " comprising: injected by silica gel in chromatography column, with clear water by silica gel washes clean, scavenging solution (ratio of silica gel and scavenging solution is not less than 1:8) is used to clean to remove foreign protein again, obtain the chromatographic solution containing urokinase albumen, and the composition proportion of described scavenging solution is: every 50 kg of water add 82 grams of SODIUM PHOSPHATE, MONOBASIC, and add pH value to 8.9 ± 0.1 that ammoniacal liquor modulation makes scavenging solution; Described " wash-out process " comprising: carry out wash-out collection with elutriant (joining the ratio of 3 liters of elutriants in 1 liter of silica gel) to the urokinase albumen in chromatographic solution, obtain urokinase protein liquid, and the composition proportion of described elutriant is: every 50 kg of water add 3.5 kilograms of ammonium chlorides, and add ammoniacal liquor modulation make the pH value of elutriant to 10.0-11.0.
Collect the chitin being adsorbed with ulinastatin albumen with gauze pocket, chitin clear water is embathed totally, and carries out wash-out process, obtain ulinastatin protein liquid.Wherein, described " wash-out process " comprising: the chitin being adsorbed with ulinastatin albumen is put into elutriant and stirs, discharge by the ulinastatin albumen adsorbed, and the composition proportion of described elutriant is: secondary saturation water and water mix with the ratio of 1:2, and add sheet alkali modulation make the pH value of elutriant to 9.0-10.0.
s106: urokinase protein liquid is joined extraction in saturation water and obtain urokinase, add ammonium sulfate extraction and obtain ulinastatin in ulinastatin protein liquid
In present pre-ferred embodiments, described " joined by urokinase protein liquid in saturation water to extract and obtain urokinase " specifically comprises: joined by described urokinase protein liquid in saturation water (joining the ratio of 2 liters of saturation waters in 1 liter of urokinase protein liquid), after leaving standstill, (preferably more than 10 hours) are filtered and are extracted urokinase (being now crude product), wherein, described saturation water is the mixing solutions of commercial sulphuric acid ammonium and water, and density is 27.5 ± 0.5g/cm
3, filter when extracting and select vacuum funnel, and diatomite that soak, that hit tight is set in vacuum funnel, form the filter sieve that 5-6cm is thick.
In present pre-ferred embodiments, described " in ulinastatin protein liquid, add ammonium sulfate extraction obtain ulinastatin " specifically comprises: will add commercial sulphuric acid ammonium in the liquid discharging ulinastatin albumen and stir, proportion remains between 26-28, leave standstill (more than 5 hours) and filter extraction ulinastatin (being now crude product) afterwards, wherein, filter when extracting and also select vacuum funnel, and diatomite that soak, that hit tight is set in vacuum funnel, form the filter sieve that 5-6cm is thick.
In preferred embodiment, the described method extracting urokinase and ulinastatin from urine also comprises:
s107: described urokinase and ulinastatin are sieved process, and freezen protective.in the present embodiment, the thermal creep stress-10 of described freezen protective is to-20 degree.
s108: recycle described silica gel and chitin.
In the present embodiment, in order to improve the utilization ratio of resource, the present invention also recycles with the silica gel crossed and chitin, that is: the dilute hydrochloric acid (1:1) putting into triplication with the silica gel crossed is stirred 1 hour, keep pH value be less than or equal to 1, then by clear water stirring and washing until pH value is more than or equal to 6.0; By the chitin after use, first soak more than 2 hours with clear water, then dewater, then use 1% soaking with sodium hydroxide more than 6 hours, finally rinse to identical with the pH value of water with clear water.
The above; it is only most preferred embodiment of the present invention; not any pro forma restriction is done to the present invention; any those of ordinary skill in the art; do not departing under technical solution of the present invention ambit; utilize the method content of above-mentioned announcement to make many possible variations and modification to technical solution of the present invention, all belong to the scope of claims protection.
Claims (10)
1. from urine, extract a method for urokinase and ulinastatin, it is characterized in that, comprising:
Collect urine and regulate between its pH value to 6.0-6.5;
In urine, add Powdered silica gel and stir, making urokinase albumen in urine by silica gel adsorption;
Obtain the silica gel in urine by filter type and regulate between the pH value of the urine after filtering to 5.3-5.7;
Add chitin in urine after foregoing filtration and stir, the ulinastatin albumen in urine is adsorbed by chitin;
Urokinase protein liquid and ulinastatin protein liquid is obtained respectively from aforementioned silica gel and chitin;
Urokinase protein liquid is joined in saturation water to extract and obtain urokinase, in ulinastatin protein liquid, add ammonium sulfate extraction obtain ulinastatin.
2. method according to claim 1, is characterized in that: collect urine and regulate between its pH value to 6.0-6.5, specifically comprising:
Flush urinal is exempted from utilization and urine collected by pipeline;
The pH value to 6.3 of urine is regulated with hydrochloric acid.
3. method according to claim 2, is characterized in that: in urine, add Powdered silica gel and stir, specifically comprising: to add the ratio of 20-25 kilogram of silica gel in urine per ton, silica gel and urine mixed and stir.
4. method according to claim 3, is characterized in that: obtain the silica gel in urine by filter type and regulate between the pH value of the urine after filtering to 5.3-5.7, specifically comprising:
Filter described urine with gauze pocket, and collect the silica gel being adsorbed with urokinase albumen, the pH value of the urine after filtration is adjusted to 5.5 by the mode adopting gradation to inject hydrochloric acid.
5. method according to claim 4, is characterized in that: add chitin in the urine after foregoing filtration and stir, specifically comprising:
To add the ratio of 5 kilograms of chitins in urine per ton, chitin and urine are mixed and stirs, and gradation adds hydrochloric acid and defoamer, make urine pH value remain on 5.5.
6. method according to claim 5, is characterized in that: from aforementioned silica gel and chitin, obtain urokinase protein liquid and ulinastatin protein liquid respectively, specifically comprise:
Collect with gauze pocket and be adsorbed with the silica gel of urokinase albumen, and the chromatographic solution that Image processing obtains containing urokinase albumen is carried out to silica gel, then wash-out process is carried out to chromatographic solution obtain urokinase protein liquid;
Collect the chitin being adsorbed with ulinastatin albumen with gauze pocket, chitin clear water is embathed, and wash-out process is carried out to chitin, obtain ulinastatin protein liquid.
7. method according to claim 6, is characterized in that: carry out Image processing to silica gel, specifically comprise:
Silica gel is injected in chromatography column, silica gel is washed with clear water, the proportioning scavenging solution being not less than 1:8 in the ratio of silica gel and scavenging solution again cleans, obtain the chromatographic solution containing urokinase albumen, described scavenging solution adds 82 grams of SODIUM PHOSPHATE, MONOBASIC by every 50 kg of water to be allocated and adds ammoniacal liquor modulation pH value to 8.9 ± 0.1.
8. method according to claim 7, is characterized in that: carry out wash-out process to chromatographic solution, specifically comprise:
The ratio of 3 liters of elutriants is joined in 1 liter of silica gel, with elutriant, wash-out collection is carried out to the urokinase albumen in chromatographic solution, obtain urokinase protein liquid, described elutriant adds 3.5 kilograms of ammonium chlorides by every 50 kg of water to be allocated and adds ammoniacal liquor modulation pH value to 10.0-11.0.
9. method according to claim 6, is characterized in that: carry out wash-out process to chitin, specifically comprise:
The chitin being adsorbed with ulinastatin albumen is put into elutriant stir, discharge by the ulinastatin albumen adsorbed, described elutriant is mixed in the ratio of 1:2 by secondary saturation water and water, and adds sheet alkali modulation pH value to 9.0-10.0.
10. method according to claim 6, is characterized in that:
Urokinase protein liquid is joined in saturation water to extract and obtain urokinase, specifically comprise: the ratio of joining 2 liters of saturation waters in 1 liter of urokinase protein liquid, described urokinase protein liquid is joined in saturation water, after leaving standstill through at least 10 hours, adopt and filter extraction urokinase containing soaking diatomaceous vacuum funnel;
In ulinastatin protein liquid, add ammonium sulfate extraction obtain ulinastatin, specifically comprise: will commercial sulphuric acid ammonium be added in the liquid discharging ulinastatin albumen and stir, and proportion remains between 26-28, after leaving standstill through at least 5 hours, adopt and filter extraction ulinastatin containing soaking diatomaceous vacuum funnel.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105753976A (en) * | 2016-05-13 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for purifying ulinastatin based on cation exchange resin |
| CN105753977A (en) * | 2016-05-13 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for large-scale production of high-purity ulinastatin (namely human urinary trypsin inhibitor) |
| CN105837685A (en) * | 2016-05-13 | 2016-08-10 | 广东天普生化医药股份有限公司 | Method for purifying ulinastatin based on anion exchange resin |
| CN106866812A (en) * | 2017-02-27 | 2017-06-20 | 日照岚山生化制品有限公司 | A kind of method that various Urine proteins are extracted in the urine from women |
| CN107936114A (en) * | 2017-11-08 | 2018-04-20 | 广东天普生化医药股份有限公司 | A kind of method based on cation exchange resin purifying ulinastatin |
| CN111185141A (en) * | 2019-12-31 | 2020-05-22 | 江西浩然生物制药有限公司 | Preparation method and application of modified silica gel for extracting urine protein |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105753976A (en) * | 2016-05-13 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for purifying ulinastatin based on cation exchange resin |
| CN105753977A (en) * | 2016-05-13 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for large-scale production of high-purity ulinastatin (namely human urinary trypsin inhibitor) |
| CN105837685A (en) * | 2016-05-13 | 2016-08-10 | 广东天普生化医药股份有限公司 | Method for purifying ulinastatin based on anion exchange resin |
| CN106866812A (en) * | 2017-02-27 | 2017-06-20 | 日照岚山生化制品有限公司 | A kind of method that various Urine proteins are extracted in the urine from women |
| CN107936114A (en) * | 2017-11-08 | 2018-04-20 | 广东天普生化医药股份有限公司 | A kind of method based on cation exchange resin purifying ulinastatin |
| CN107936114B (en) * | 2017-11-08 | 2019-01-22 | 广东天普生化医药股份有限公司 | A method of ulinastatin is purified based on cation exchange resin |
| CN111185141A (en) * | 2019-12-31 | 2020-05-22 | 江西浩然生物制药有限公司 | Preparation method and application of modified silica gel for extracting urine protein |
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Application publication date: 20160113 |