CN1067403C - Direct high-purity peptide-inhibiting enzyme extraction method from ox lungs - Google Patents
Direct high-purity peptide-inhibiting enzyme extraction method from ox lungs Download PDFInfo
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- CN1067403C CN1067403C CN96120494A CN96120494A CN1067403C CN 1067403 C CN1067403 C CN 1067403C CN 96120494 A CN96120494 A CN 96120494A CN 96120494 A CN96120494 A CN 96120494A CN 1067403 C CN1067403 C CN 1067403C
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- lung
- trasylol
- rubs
- rise
- trypsin inhibitor
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Abstract
The present invention relates to a method for preparing trasylol with high purity from the lung of an ox, which comprises: a chemically modified chitin active carrier in a Chinese patent with the application number of 96120646.2 is coupled with trypsinase to be made into chitin immobilized trypsinase affinity adsorbent; the lung of an ox is then statically absorbed and dynamically eluted. The specific activity of the obtained trasylol reaches more than 5000 kiu/mg and is far higher than the specific activity of 3600 kiu/mg specified in Chinese pharmacopoeia.
Description
The present invention relates to a kind of method of directly producing the high purity Trypsin inhibitor,Trasylol, particularly adopt the chitosan-immobilized trypsinase of chemical modification and modification to make affinity adsorbent is directly produced the high purity Trypsin inhibitor,Trasylol from the ox lung method from the ox lung.
Aprotinin bovine records first on the Chinese Pharmacopoeia of 90 years versions and is the medicinal zymin of therapeutic, it is a kind of non-specific broad-spectrum protease inhibitor, multiple enzymes such as trypsinase to the people have very strong restraining effect, it is the specific medicament for the treatment of diseases such as acute and chronic pancreatitis, along with the widespread use at pharmaceutical sector, its demand rises year by year in recent years.Though it is many to prepare the method for Trypsin inhibitor,Trasylol at present both at home and abroad, but it is all similar at all to follow it, for example: C.A.Vol:78 (1973), the Trypsin inhibitor,Trasylol crude extract that from the ox lung, extracts that 144649b reported, become raw product through twice ammonium sulfate precipitation, make the Trypsin inhibitor,Trasylol finished product through affinity chromatography again.This kind method is commonly referred to as two step method, and its shortcoming is that sulphur ammonium consumption is big, and the production cycle is long, and is big for environment pollution, and cost is also high.
Journal of biological chemistry in 1992, Vol.8, N0.4, Aug, 1992, P418-423 though also report adopts the affinity adsorbent that the pearl Sepharose is made as carrier, directly produces the method for high purity Trypsin inhibitor,Trasylol from the ox lung, but its shortcoming is that the physical strength of this sorbent material is little, contamination resistance is poor, and working conditions is strict harsh, as P
HLess than 2 or temperature just can not use when surpassing 40 ℃, so up to now can't industrialization.
The object of the present invention is to provide the affinity adsorbent of a kind of chitin activating carriers (Chinese patent application number be 96120646.2) preparation with chemical modification and modification, directly from the ox lung, produce the method for high purity Trypsin inhibitor,Trasylol, its advantage is that the ratio vigor of resulting product Trypsin inhibitor,Trasylol is up to more than the 5000kiu/mg, activity recovery is up to 83%, at P
HLess than 2 or temperature be higher than the preparation method of also spendable high purity Trypsin inhibitor,Trasylol under 40 ℃ the environment.
Embodiment of the present invention are as follows:
(1) affinity adsorbent is synthetic: be about to Chinese patent application and number be the chitin activating carriers of 96120646.2 chemical modification and modification and at P
HBe 8 borate buffer down and bovine trypsin carry out coupling and form affine absorption agent;
(2) from the ox lung, prepare Trypsin inhibitor,Trasylol.With the ox lung with sulphuric acid extraction, remove slag, transfer P with sodium hydroxide again
HValue leaves standstill centrifugal removing slag, and the affine absorption agent that adds (1) in supernatant liquor carries out the Static Adsorption dynamic desorption and makes the high purity aprotinin bovine.(generally being) to represent the height of purity than the size of vigor
Below preference to the detailed description of the invention, but and do not mean that limitation of the scope of the invention.
Example 1:
(a) with 100 milliliters the above-mentioned chemical modification and the chitin activating carriers deionized water wash of modification, putting into volume then is 1000 milliliters of general reactors that have stirring, adds 200 milliliters of P
HBe 8 borate buffer, stir that adding trypsin is 1200kiu/mg than vigor) 500 milligrams, stirred 4 hours, under 5 ° ± 1 ℃, left standstill 20 hours, the immigration sand core funnel filters, solids with 0.5 rub/liter the Nacl solution washing, use P again
HBe 1.5 salt acid elution, filter with deionized water wash at last, all solids thing is put into 100 milliliters of borate buffer solution (P
HBe 7.8) in, get 100 milliliters of chitosan kind immobilizing trypsinase affinity adsorbents, record than vigor 〉=13000kiu/g;
(b) get 500 grams the degas pipe and the ox lung of impurity in addition, be cut into small pieces, rub with mincer, be broken into rotten slurry with general high-speed tissue mashing machine again, move in 4000 milliliters of general reactors of band stirring, add 3000 milliliters in water, stir, transfer to the P of solution with 2N sulfuric acid
HBe 1.5, filter that filtrate is heated to 80 ℃, adds activated carbon decolorizing, is incubated 2 hours, filter that filtrate is transferred P with 10% sodium hydroxide
HBe 8, standing over night, centrifugal, it is standby to get 2500 milliliters of supernatant liquors (recording total activity is 600,000 kiu).In the general reactor that the band of again these 2500 milliliters of supernatant liquors being packed into stirs, add 100 milliliters of the affinity adsorbents of (a) preparation, at room temperature whip attachment is 24 hours, and the affinity adsorbent after static state is fully adsorbed is packed in 150 milliliters the chromatography column, uses P
HBe the washing of 8 borax calcium chloride balance liquid balance, with elutriant 1 KCl that rubs/rise, 0.5 NaAC that rubs/rise, 0.5 solution that rubs/rise HAC, P
HBe 1.5 to carry out wash-out, collect P
HBe 2 elutriant, get the former medicine of Trypsin inhibitor,Trasylol that total activity is 49.8 ten thousand kiu-20 ℃ of following freeze-drying after the ultrafiltration, recording than vigor is 5000kiu/mg, and more much higher than the 3600kiu/mg of Chinese Pharmacopoeia regulation, activity recovery is 83%.
Example 2: the P that removes (a) borate buffer solution in the stage
HBe 8, add-on is 120 milliliters, adds trypsinase and be 300 milligrams and (b) to get supernatant liquor in the stage be outside 1500 milliliters, and all the other conditions are identical that total activity be 300,000 kiu are than the former medicine of the Trypsin inhibitor,Trasylol of vigor 5200kiu/mg with example 1.
Advantage of the present invention be the ratio vigor of the former medicine of Aprotinin prepared up to more than the 5000kiu/mg, live The property rate of recovery is up to 83%.
Claims (1)
1. method of directly producing the high purity Trypsin inhibitor,Trasylol from the ox lung is characterized in that:
(1) affinity adsorbent is synthetic:
With Chinese patent application number is that 96120646.2 the chemical modification and the chitin active carrier of modification are put into P
H8.0 borate buffer in, add trypsinase, under agitation filtered in 20 hours in 4-6 ℃ of reaction, solids washs and uses P with 0.5 sodium chloride solution that rubs/rise
H1.5 the salt acid elution washes filtration again with water, solids is put into P
H7.8-8.0 borate buffer solution make chitosan kind immobilizing trypsinase affinity adsorbent;
(2) prepare Trypsin inhibitor,Trasylol from the ox lung:
The ox lung is rubbed, add water and transfer to P with 2N sulfuric acid
H=1.5, to filter, filtrate adds gac in 80 ℃ of heating down, decolorization filtering, filtrate transfers to P with 10% sodium hydroxide
H=8.0, leave standstill, centrifugal, get supernatant liquor, add the affinity adsorbent of (1) making and carry out Static Adsorption, then, use P in the chromatography column of packing into
H8.0 borax calcium chloride balance liquid balance, with 1 Repone K that rubs/rise, 0.5 sodium-acetate and the 0.5 acetic acid (P that rubs/rise that rubs/rise
H1.5) wash-out, collect P
H=2 elutriant forms in-20 ℃ of following freeze-drying after ultrafiltration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96120494A CN1067403C (en) | 1996-11-18 | 1996-11-18 | Direct high-purity peptide-inhibiting enzyme extraction method from ox lungs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96120494A CN1067403C (en) | 1996-11-18 | 1996-11-18 | Direct high-purity peptide-inhibiting enzyme extraction method from ox lungs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1160720A CN1160720A (en) | 1997-10-01 |
| CN1067403C true CN1067403C (en) | 2001-06-20 |
Family
ID=5126376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96120494A Expired - Fee Related CN1067403C (en) | 1996-11-18 | 1996-11-18 | Direct high-purity peptide-inhibiting enzyme extraction method from ox lungs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1067403C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103897054B (en) * | 2014-04-15 | 2017-03-15 | 南昌市万华生化制品有限公司 | A kind of synthesis of ox lung Aprotinin opposite sex preparative chromatography medium and application |
| CN111714469B (en) * | 2019-03-22 | 2023-10-03 | 苏州特瑞药业股份有限公司 | Thymalfasin preparation and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE232431C (en) * | ||||
| CN1155581A (en) * | 1996-11-15 | 1997-07-30 | 安徽省生物研究所 | Chemically modified chitin activating carriers |
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1996
- 1996-11-18 CN CN96120494A patent/CN1067403C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE232431C (en) * | ||||
| CN1155581A (en) * | 1996-11-15 | 1997-07-30 | 安徽省生物研究所 | Chemically modified chitin activating carriers |
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| Publication number | Publication date |
|---|---|
| CN1160720A (en) | 1997-10-01 |
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| C14 | Grant of patent or utility model | ||
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