CN101768582B - Production process for modifying SOD - Google Patents
Production process for modifying SOD Download PDFInfo
- Publication number
- CN101768582B CN101768582B CN2010101371852A CN201010137185A CN101768582B CN 101768582 B CN101768582 B CN 101768582B CN 2010101371852 A CN2010101371852 A CN 2010101371852A CN 201010137185 A CN201010137185 A CN 201010137185A CN 101768582 B CN101768582 B CN 101768582B
- Authority
- CN
- China
- Prior art keywords
- sod
- enzyme liquid
- sod enzyme
- production
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a production process for modifying SOD. The production process comprises the following three steps: preparing SOD enzyme solution by using animal blood; performing modification reaction of the SOD enzyme solution; and preparing SOD freeze-dry powder by using the SOD enzyme solution. In the production process, the SOD modification process and the SOD extraction-purification process are realized on the same production line, so that the production ring is simplified, the production cost is saved by about 20 percent, and the SOD activity is better protected by the process than by the method for preparing raw solution by dissolving the SOD dry powder; the addition of lauroyl chloride and alkali solution changes the environment and the process of the modification chemical reaction, but hardly causes the fluctuation of the pH value of the SOD enzyme solution, the reaction process is easily controlled, the production process is suitable for mass production, and the activity loss of the SOD is less; in the whole production process, the acetone addition process in the conventional process is eliminated, so that the product safety is guaranteed, and the activity loss of the SOD is less.
Description
Technical field
The invention belongs to technological field of biochemistry, relate in particular to the technical field of producing of a kind of modification superoxide-dismutase (SOD).
Background technology
Free radical theory is in life science, and the theory of more and more being accepted extensively by people about the old and feeble theory of life entity, this theory think that radical is to cause the aging major reason that reaches many diseases of life entity.80～90% aging, degenerative disease are all relevant with radical according to estimates; Comprising cancer, senile dementia, Parkinson's disease, skin pigment deposition, cataract, heart trouble etc., keep healthy physique and keep young figure most important so remove radical pair.
And SOD is a superoxide-dismutase is single-minded free-radical scavengers.SOD is a kind of extensively being present in animal, plant and the mikrobe, has the enzyme of catalysis ultra-oxygen anion free radical generation disproportionation reaction, and its catalytic disproportionation reaction formula is following:
·O
2 -+·O
2 -+2H
+→H
2O
2+O
2
SOD can remove the ultra-oxygen anion free radical (biologic garbage) in the human body cell; Thereby be described as the biologic garbage street cleaner of human body; And radical is crossed and is caused more than 80 kind of disease at most, so the human body timely and appropriate discovery replenishes SOD, just can prevent even treat the multiple disease that too much causes because of radical; Have functions such as anti-oxidant, radioprotective, anticancer change, anti-inflammatory; Be used for treatment or assisting therapy cardiovascular and cerebrovascular diseases, autoimmune disorder, blood reperfusion damage, chronic inflammatory diseases and cancer etc., and can be used for daily anti-ageing healthcare, control geriatric disease and chronic disease etc.
Up to now, the modification technique of SOD has multiple, like Yuan Qinsheng with cycloheptaamylose modification SOD (medicine biotechnology [J], 1996; 3 (2): 87-90) with Yin Liang with PEG modification SOD (biotechnology [J]; 2003:8), macromolecular cpds such as cycloheptaamylose and PEG are made modifier, and the molecular weight of modification SOD is increased considerably; Reach 300～400kd; Be prone to cause blood vessel blockage during intravenous injection, and the loss of living of SOD enzyme is big when modifying, the enzyme that lose about 40% is lived.
Also there is the scholar to come modification SOD, like " preparation of LAURIC ACID 99 MIN modification SOD and stability study thereof " (Wuhan University's journal (natural science edition) [J], 1998 of Zou Guolin with small numerator modified dose of LAURIC ACID 99 MIN etc.; 49 (81)) and Yan Jiaqi " preparation of LAURIC ACID 99 MIN modification SOD and property research " (biological chemistry and biophysics progress [J], 1,994 44 (4):; 21 (2): 154-157), also have " preparation of Triple Pressed Stearic Acid modification SOD and character thereof " (journal of biological chemistry [J], 1996 of Zou state woods; 12 (1): 64-67) etc.Yan Jiaqi professor's modifying method need be the LAURIC ACID 99 MIN activation, and activation is the chemical reaction process of a complicacy, and reaction not exclusively causes the residual of chemical reagent such as sulfur oxychloride, pyridine easily, influences security of products.Zou Guolin professor's modifying method is to do raw material with SOD dry powder, has increased production cost and lost enzyme alive; And in modification process; Be added to lauroyl chloride once in the SOD enzyme liquid earlier, can cause the rapid decline of pH value and the lauroyl chloride partial concn of SOD enzyme liquid too high, cause the loss of activity of SOD big; About 20%, reaction process is wayward and be not suitable for large-scale production.
In addition, modify the back in the present modifying method and precipitate collection SOD, influenced security of products, also reduced the activity of SOD with virulent acetone.
Summary of the invention
The present invention discloses a kind of SOD according to deficiency of the prior art and has modified working method, and the present invention realizes through following technical scheme.
SOD modifies working method, may further comprise the steps:
(1) prepares SOD enzyme liquid by animal blood;
(2) step (1) SOD enzyme liquid modification reaction;
(3) step (2) SOD enzyme liquid prepares the SOD lyophilized powder.
The preparation of said step (1) SOD enzyme liquid is: animal clot through emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatography production process, is collected and obtained blue-greenish colour SOD enzyme liquid, according to volume, activity and A
280Value is calculated the addition of lauroyl chloride.Above-mentioned emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatography production process are conventional production process, see " extraction of CuZn-SOD research in the animal clot " (renewable resource research [J], 2001 (2): 34-36) of thorough method etc.The addition of said reckoning lauroyl chloride; Be the protein concentration that detects SOD enzyme liquid according to the Lowry method, obtain regression equation, different detection equipment, testing conditions; The regression equation that obtains has certain difference; Try to achieve protein content by regression equation, multiply by experience factor 1.4 again, just obtain the addition of lauroyl chloride.
Said step (2) is in the SOD enzyme liquid of step (1) preparation, to carry out;
The concrete grammar of step (2) is: SOD enzyme liquid is poured in the reaction kettle of jacketed, stirs, and drips the 2M alkaline solution and regulates pH value to 9.0; Be heated to 36～45 ℃, and keep constant temperature; Slowly drip a certain amount of lauroyl chloride then continuously, drip the 2M alkaline solution simultaneously, make the pH value of SOD enzyme liquid maintain about 9.0 all the time; Reacted 1 hour, wherein lauroyl chloride added at preceding about 20 minutes and finishes; It is stable to keep pH that the slow continuously dropping of alkaline solution runs through entire reaction course; After modification reaction finishes, be cooled to 4 ℃ rapidly, centrifugal, the SOD enzyme liquid that obtains modifying.
Said alkaline solution can be potassium hydroxide solution or sodium hydroxide solution and other basic soln one of them, also can be two or more mixed solution in them.The sodium hydroxide solution of preferred 2M.
Said step (3) SOD enzyme liquid prepare the SOD lyophilized powder be SOD enzyme liquid behind the modification reaction that step (2) is obtained through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, obtain the modification SOD lyophilized powder.Above-mentioned is conventional production process through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, " purifying of superoxide dismutase from pig blood and property research thereof " (Acta Biochimica et Biophysica Sinica [J], 1987 of seeing that Yuan Qin gives birth to etc.; 19 (1): 22-25).
Principle of the present invention: lauroyl chloride acts on the Methionin at the nonactive position of SOD molecular surface under weak basic condition, and with the ε-NH of Methionin
2Covalent attachment forms a kind of acidylate SOD, has strengthened the rigid conformation of SOD; Spatial obstacle that acidylate produced and electrostatic repulsion; Stopped proteolytic ferment and the attack of other suppressor factor to SOD, the secondary key of stablizing the SOD molecular conformation is protected, the stability of SOD improves.
The present invention is that the SOD enzyme liquid of chromatographic column serves as to modify object under extracting in the purification production with SOD, measures the volume and the A of SOD enzyme liquid
280Be worth, record the activity of SOD enzyme liquid, just can extrapolate the amount of the lauroyl chloride that will add, like this, the modification process of SOD and extraction and purification process are realized on same production line, reduced production link, practiced thrift about 20% production cost.Secondly, in modification process, directly make modifier with the high purity lauroyl chloride; The more important thing is that lauroyl chloride is slowly to add reaction gradually, adding needs about 20 minutes time approximately; When adding lauroyl chloride, add the alkaline solution liquid of 2M and regulate the pH value, the pH value all-the-time stable that makes enzyme liquid is about 9.0; Like this, the pH value of SOD enzyme liquid fluctuation is little, and reaction process is controlled easily and is suitable for large-scale production; And the SOD loss of activity is few, whole modification process, the loss of activity of SOD in 5%, can not cause the disposable adding of lauroyl chloride after, the pH value of SOD enzyme liquid sharply descends and causes the big phenomenon of SOD loss of activity; After above-mentioned modification reaction finished, SOD enzyme liquid was produced the modification SOD lyophilized powder through cooling, centrifugal, sterilization, ultrafiltration and concentration, lyophilize, does not add objectionable impuritiess such as acetone, guaranteed security of products, and the SOD loss of activity is few.
In sum; The present invention and prior art relatively have modification process and the extraction and purification process of following helpfulness: SOD and on same production line, realize; Reduced production link; Practiced thrift about 20% production cost, compared, protected the activity of SOD preferably with SOD dry powder dissolving preparation stoste method; The adding method of lauroyl chloride and alkaline solution has changed the environment and the process of modifying chemical reaction, makes the pH value fluctuation of SOD enzyme liquid little, and reaction process is controlled easily and is suitable for large-scale production, and the SOD loss of activity is few; In the full scale production process, do not have the acetone of traditional technology to add technology, guaranteed security of products, and the SOD loss of activity is few.
Embodiment
Below in conjunction with embodiment the present invention is further specified.
Embodiment 1
1,0.5 ton of pig clot is through production processes such as emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatographys, and collecting and obtaining activity is 10.33 ten thousand U/ml, A
280Value is 14.6, volume is the blue-greenish colour SOD enzyme liquid of 2.65L, and the addition of extrapolating lauroyl chloride is 46ml.
2, SOD enzyme liquid is poured in the 10L glass reaction still of jacketed, starts whipping appts, and the NaOH liquid that drips 2M is regulated pH value to 9.0.The chuck hot water circulation is heated to 40 ℃ then, and keeps constant temperature; Drip continuously slowly the 46ml lauroyl chloride then, drip 2M NaOH liquid simultaneously, the pH value of SOD enzyme liquid is maintained about 9.0 all the time, reacted 1 hour, wherein lauroyl chloride added at preceding about 20 minutes and finishes; And the interpolation of 2M NaOH liquid runs through entire reaction course, but addition reduces gradually.After modification reaction finished, SOD enzyme liquid is cooled to 4 ℃ rapidly with heat exchanger, and was centrifugal, the SOD enzyme liquid that obtains modifying.
3, the SOD enzyme liquid of modifying is produced 20.4 gram modification SOD lyophilized powders through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, and its specific activity is 1.21 * 10
4U/mg albumen, protein content are 98.7%.
Embodiment 2
1,2.0 tons of pig clots are through production processes such as emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatographys, and collecting and obtaining activity is 15.27 ten thousand U/ml, A
280Value is 30.8, volume is the blue-greenish colour SOD enzyme liquid of 6.1L, and the addition of extrapolating lauroyl chloride is 112ml.
2, SOD enzyme liquid is poured in the 10L glass reaction still of jacketed, starts whipping appts, and the KOH liquid that drips 2M is regulated pH value to 9.0.The chuck hot water circulation is heated to 42 ℃ then, and keeps constant temperature; Drip continuously slowly the 112ml lauroyl chloride then, drip 2M KOH liquid simultaneously, the pH value of SOD enzyme liquid is maintained about 9.0 all the time, reacted 1 hour, wherein lauroyl chloride added at preceding about 20 minutes and finishes; And the interpolation of 2M KOH liquid runs through entire reaction course, but addition reduces gradually.After modification reaction finishes, be cooled to 4 ℃ rapidly with heat exchanger, centrifugal, the SOD enzyme liquid that obtains modifying.
3, the SOD enzyme liquid of modifying is produced 90.4 gram modification SOD lyophilized powders through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, and its specific activity is 9.72 * 10
3U/mg albumen, protein content are 96.6%.
Embodiment 3
1,2.5 tons of ox blood pieces are through production processes such as emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatographys, and collecting and obtaining activity is 16.4 ten thousand U/ml, A
280Value is 42.3, volume is the blue-greenish colour SOD enzyme liquid of 7.8L, and the addition of extrapolating lauroyl chloride is 147ml.
2, SOD enzyme liquid is poured in the 10L glass reaction still of jacketed, starts whipping appts, and the NaOH liquid that drips 2M is regulated pH value to 9.0.The chuck hot water circulation is heated to 45 ℃ then, and keeps constant temperature; Drip continuously slowly the 147ml lauroyl chloride then, drip 2M NaOH liquid simultaneously, the pH value of SOD enzyme liquid is maintained about 9.0 all the time, reacted 1 hour, wherein lauroyl chloride added at preceding about 20 minutes and finishes; And the interpolation of 2M NaOH liquid runs through entire reaction course, but addition reduces gradually.After modification reaction finishes, be cooled to 4 ℃ rapidly with heat exchanger, centrifugal, the SOD enzyme liquid that obtains modifying.
3, the SOD enzyme liquid of modifying is produced 110.8 gram modification SOD lyophilized powders through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, and its specific activity is 1.08 * 10
4U/mg albumen, protein content are 97.8%.
The foregoing description is to specify of the present invention, is not construed as limiting the invention, and any change in suitable implication with claims of the present invention and the scope all should be in protection scope of the present invention.
Claims (3)
1. a SOD modifies working method, and may further comprise the steps: (1) animal blood prepares SOD enzyme liquid; (2) SOD enzyme liquid modification reaction; (3) step (2) SOD enzyme liquid prepares the SOD lyophilized powder; It is characterized in that:
Said step (1) animal blood prepare SOD enzyme liquid be with animal clot through emulsification, haemolysis, continuously centrifuged, ultrafiltration and concentration, thermally denature, column chromatography production process, collect and to obtain blue-greenish colour SOD enzyme liquid, according to volume, activity and A
280Value is calculated the addition of lauroyl chloride;
Said step (2) is in the SOD enzyme liquid of step (1) preparation, to carry out;
The concrete grammar of step (2) is: SOD enzyme liquid is poured in the reaction kettle of jacketed, stirs, and the alkaline solution that drips 2M is regulated pH value to 9.0; Be heated to 36～45 ℃, and keep constant temperature; Slowly drip a certain amount of lauroyl chloride then continuously, drip the alkaline solution of 2M simultaneously, make the pH value of SOD enzyme liquid maintain about 9.0 all the time; Reacted 1 hour, wherein lauroyl chloride added at preceding about 20 minutes and finishes, and it is stable to keep pH that slowly the dripping continuously of alkaline solution run through entire reaction course; After reaction finishes, be cooled to 4 ℃ rapidly, centrifugal, the SOD enzyme liquid that obtains modifying;
Said step (3) SOD enzyme liquid prepare the SOD lyophilized powder be SOD enzyme liquid behind the modification reaction that step (2) is obtained through the sterilization of bacteriological filtration film, ultrafiltration and concentration, lyophilize, obtain the modification SOD lyophilized powder.
2. SOD according to claim 1 modifies working method, and it is characterized in that: said alkaline solution can be potassium hydroxide solution or one of sodium hydroxide solution and other basic soln, also can be two or more mixed solution in them.
3. SOD according to claim 2 modifies working method, it is characterized in that: the sodium hydroxide solution of preferred 2M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101371852A CN101768582B (en) | 2010-04-01 | 2010-04-01 | Production process for modifying SOD |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101371852A CN101768582B (en) | 2010-04-01 | 2010-04-01 | Production process for modifying SOD |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101768582A CN101768582A (en) | 2010-07-07 |
| CN101768582B true CN101768582B (en) | 2012-04-25 |
Family
ID=42501683
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101371852A Expired - Fee Related CN101768582B (en) | 2010-04-01 | 2010-04-01 | Production process for modifying SOD |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101768582B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215247A (en) * | 2013-04-11 | 2013-07-24 | 莫宏春 | Method of modifying natural superoxide dismutase(SOD) enzyme solution, posttreatment modified SOD enzyme solution and myristic acid-SOD lyophilized powder |
| CN105543185B (en) * | 2016-03-10 | 2019-01-11 | 甘肃养泰和生物科技有限公司 | A kind of production method extracted from animal clot, purify SOD freeze-dried powder |
| CN109112119B (en) * | 2018-08-28 | 2022-04-26 | 佛山科学技术学院 | Preparation method of chemically modified duck blood SOD preparation |
| CN109438590B (en) * | 2018-11-13 | 2021-04-27 | 广西科学院 | Agarose modification method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260355C (en) * | 2004-05-09 | 2006-06-21 | 沈阳五爱康田生物制品有限责任公司 | Superoxide dismutase complex enzyme industrialized production method using corn as material and its special device for germination |
| CN1721519A (en) * | 2005-06-30 | 2006-01-18 | 河南省百泉春酒业有限公司 | Process for making modified SOD wine |
-
2010
- 2010-04-01 CN CN2010101371852A patent/CN101768582B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101768582A (en) | 2010-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103484513B (en) | Method for preparing small-molecule oligomeric hyaluronic acid through enzyme method | |
| BR112014010922B1 (en) | PROCESS OF EXTRACTION OF POLISACARIDES FROM BROWN ALGAE | |
| CN105018555A (en) | Preparation method of giant salamander skin collagen peptide | |
| CN101768582B (en) | Production process for modifying SOD | |
| CN104277134B (en) | A kind of preparation method and applications of the dictyophora fungus polysaccharide-chelates of zinc with anti-tumor activity | |
| CN102224879A (en) | Preparation method and application of champignon polypeptide | |
| CN107188990A (en) | The method that chondroitin sulfate is extracted in sturgeon bone | |
| CN106866834B (en) | A kind of method and its application for preparing the efficiently fucoidin of customization molecular weight | |
| CN104498564A (en) | Low molecular weight chondroitin sulfate preparation method | |
| CN105111277A (en) | Preparation method of ginseng peptide | |
| CN103289967A (en) | Extraction method for extracting superoxide dismutase from shenzhou grass | |
| CN100467061C (en) | Scorpionfish-ink polysaccharide and its preparation | |
| CN103804522B (en) | A kind of method improving heparin sodium purity | |
| CN102898540A (en) | Extraction technology of hyaluronic acid | |
| CN101307338A (en) | Method for preparing reducing coenzyme Q10 | |
| CN105886569A (en) | Preparation method of theaflavin | |
| CN104666518A (en) | Composition containing extracts of cichorium intybus and poncirus trifoliata for strengthening skin barrier | |
| CN103215247A (en) | Method of modifying natural superoxide dismutase(SOD) enzyme solution, posttreatment modified SOD enzyme solution and myristic acid-SOD lyophilized powder | |
| CN110856744A (en) | New application of dendrobe polypeptide | |
| CN111909248A (en) | Method for industrially extracting amylase inhibitor | |
| CN105543185B (en) | A kind of production method extracted from animal clot, purify SOD freeze-dried powder | |
| CN104725532B (en) | A kind of method of chondroitin sulfate and dermatan sulfate content in accurate quantification control heparin/heparan | |
| CN101967499A (en) | Method for producing reduced coenzyme Q10 | |
| CN107163163A (en) | A kind of processing method of chondroitin sulfate product | |
| CN104163877B (en) | The preparation method of chondroitin sulfate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: JIANG ZHONGLI Free format text: FORMER OWNER: CHENGDU XINYA BIOENGINEERING CO., LTD. Effective date: 20120625 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 610031 CHENGDU, SICHUAN PROVINCE TO: 610036 CHENGDU, SICHUAN PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20120625 Address after: 610036 3 sets of Jinniu native bridge village, Chengdu, Sichuan, Jinniu District Patentee after: Jiang Zhongli Address before: 610031, Sichuan, Chengdu province Qingyang dragon industrial port, East Sea 3 Patentee before: Chengdu Xinya Bioengineering Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120425 Termination date: 20180401 |