CN109879931A - A method of utilizing crystallisation separating-purifying superoxide dismutase - Google Patents
A method of utilizing crystallisation separating-purifying superoxide dismutase Download PDFInfo
- Publication number
- CN109879931A CN109879931A CN201910203596.8A CN201910203596A CN109879931A CN 109879931 A CN109879931 A CN 109879931A CN 201910203596 A CN201910203596 A CN 201910203596A CN 109879931 A CN109879931 A CN 109879931A
- Authority
- CN
- China
- Prior art keywords
- superoxide dismutase
- crystal
- solution
- separating
- crystallisation
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a kind of methods using crystallisation separating-purifying superoxide dismutase, broken bacterial fermentation medium pH value is adjusted to acidity with hydrochloric acid, after standing, centrifuging and taking supernatant, after supernatant is concentrated by ultrafiltration, it is separately added into precipitating reagent and seed crystal, it is put into temperature-controlled box after mixing until crystal is precipitated, after cleaning crystal three times, after continuing crystallization more than twice, freeze-drying, obtains finished product.Not only process is simple for this method, at low cost, and does not use any chromatographic technique, avoids the defect of chromatographic technique.This method has also obtained superoxide dismutase crystal while obtaining Superoxide Dismutase of Higher Purity.Obtained superoxide dismutase purity reaches 96%, Rate activity 4523U/mg.Therefore, this method is both a kind of novel separating and purifying technology of alternative chromatography, additionally provides a kind of method for preparing superoxide dismutase crystal, has broad application prospects.
Description
Technical field
The invention belongs to crystallization of protein and purified technology of protein field, are related to a kind of super using crystallisation separating-purifying
The method of superoxide dismutase is super using crystallisation separating-purifying especially in the protein solution without purification by chromatography
The method of superoxide dismutase.
Background technique
Pharmaceutical grade protein research and development and production are very important component parts in Bio-pharmaceutical Industry.In its research and development and production
In the process, it is related to the integrated application of multiple technologies.With the investment of huge development resources, upstream technology have been achieved with it is important into
Exhibition.In contrast, the downstream technique part progress of pharmaceutical grade protein research and development and production is relatively slow, constantly obtains in upstream technology
Under the background of progress, downstream technique is increasingly becoming the bottleneck of whole process.Wherein, scale separating and purifying technology is albumen
The key of matter drug downstream production process, currently, widely used chromatography is there are problems, column effect is low, filler is expensive,
Service life is short, multistage combination, complete equipment is needed to need the defect of huge capital investment, can not cope with upstream technology and change
Kind bring high titre and high yield.Solve the problems, such as that this is the urgent need of bio-pharmaceutical industry downstream technique development.Therefore, it sends out
Exhibition novel protein scale separating and purifying technology not only conforms with China's innovation driving development strategy, and as the great war of country
Slightly demand, has been put into " National Program for Medium-to Long-term Scientific and Technological Development (2006-2020) ".
In novel separating and purifying technology development process, crystallization is had broad application prospects with its exclusive advantage.Knot
Brilliant process in one step, both shortens separation, purifying, upgrade set production procedure, meets current intensive manufacture
Trend, does not need the tomography devices using complex and expensive yet, and fund and equipment investment significantly reduce.Pharmaceutical grade protein crystal form preparation
The unique advantages such as lower viscosity when with high activity, high-purity, high-biocompatibility, longer half-life period, high concentration administration,
This makes crystal form pharmaceutical grade protein have broad application prospects.
Superoxidase (Superoxide dismutase, SOD, EC 1.15.1.1) is that one kind can be catalyzed super oxygen yin
Ion radical (O2ˉ) enzyme of molecular oxygen and hydrogen peroxide is converted by disproportionated reaction, be widely present in microorganism, plant with
And in animal.Clinically it is used for the treatment of cardiovascular and cerebrovascular disease, rheumatoid arthritis, burn, chemotherapy etc..Currently, existing
Some superoxide dismutase separating and purifying technologies are all made of traditional chromatography, purify superoxide dismutase using crystallisation
At home and abroad it is not reported.
There are problems for chromatography used at present, and column effect is low, filler is expensive, service life is short, needs multi-cascade
The defect that huge capital investment is needed with, complete equipment, can not cope with upstream technology improves bring high titre and high yield
Amount.
Summary of the invention
Technical problems to be solved
In order to avoid the shortcomings of the prior art, the present invention proposes a kind of utilization crystallisation separating-purifying superoxides discrimination
The method for changing enzyme adjusts broken bacterial fermentation medium pH value to acidity with hydrochloric acid, and after standing, centrifuging and taking supernatant will be upper
After clear ultrafiltration concentration, it is separately added into precipitating reagent and seed crystal, is put into temperature-controlled box after mixing until crystal precipitation, cleans crystal
After three times, after continuing crystallization more than twice, freeze-drying obtains finished product.
Technical solution
A method of utilizing crystallisation separating-purifying superoxide dismutase, it is characterised in that steps are as follows:
Step 1: after bacterial fermentation culture solution is crushed using Ultrasonic Cell Disruptor, centrifuging and taking supernatant, with hydrochloric acid regulatory protein
Matter solution ph is to acidity, and after 0 DEG C of -4 DEG C of standing 1h -3h, centrifuging and taking supernatant obtains protein solution after ultrafiltration concentration；
Step 2: precipitating reagent being dissolved in 0.1-5mM, in the citric acid solution that pH value is 3.0-5.5, with protein solution
After the mixing of 1:1 volume ratio, 10-200 μ L seed crystals are added, are placed in temperature-controlled box, the stirred crystallization in 4 DEG C-20 DEG C；The precipitating
Agent is PEG8000, and concentration is 20-80mg/mL；
Step 3: precipitating will be taken after the solution centrifugation after the completion of crystallization, gained precipitating is that superoxide dismutase crystal is mixed
Close object；
Step 4: superoxide dismutase crystal cleaning solution is added, after being slowly stirred mixing, supernatant is abandoned in centrifugation, and repeating should
Process obtains superoxide dismutase crystal repeatedly thoroughly to clean crystal；The crystal cleaning solution is 20-100mg/mL's
PEG8000 solution；
Step 5: gained crystal being lyophilized, obtains white powder superoxide dismutase finished product, or be not lyophilized, is directly made
For crystal formulations application.
The protein solution of solution step of replacing 2 after the superoxide dismutase dissolution of crystals obtained for step 4, is adopted
It repeats to crystallize repeatedly with crystallization condition identical with step 2, keeps obtained superoxide dismutase crystal purity higher.
The bacterial fermentation culture solution is the fermentation liquid of the engineering bacteria with source of people Expression of Superoxide Dismutase ability.
The pH value of the step 1 is 4.0-6.0.
Ultrafiltration retaining molecular weight used in the ultrafiltration of the step 1 is 3-10KDa, and pressure is 0.2-0.45MPa.
The step 4 is repeatedly three times.
The step 5 is repeatedly more than two times.
Beneficial effect
A kind of method using crystallisation separating-purifying superoxide dismutase proposed by the present invention, is purified by crystallisation
Superoxide dismutase proteins are a kind of novel methods for isolating and purifying superoxide dismutase, and not only process is simple for this method,
It is at low cost, and any chromatographic technique is not used, avoid the defect of chromatographic technique.This method is obtaining high-purity superoxides
While mutase, superoxide dismutase crystal is had also obtained.Obtained superoxide dismutase purity reaches 96%, than work
Power is 4523U/mg.Therefore, this method is both a kind of novel separating and purifying technology of alternative chromatography, additionally provides a kind of system
The method of standby superoxide dismutase crystal, has broad application prospects.
Specific embodiment
Now in conjunction with embodiment, the invention will be further described:
Technical solution: broken bacterial fermentation medium pH value is adjusted to acidity, after standing, in centrifuging and taking with hydrochloric acid
Clearly, after supernatant being concentrated by ultrafiltration, it is separately added into precipitating reagent and seed crystal, is put into temperature-controlled box after mixing up to crystal precipitation,
After cleaning crystal three times, after continuing crystallization more than twice, freeze-drying obtains finished product.
Specific step is as follows:
(1) it is crushed bacterial fermentation culture solution (power 30%, opens 5s, stops 3s, is crushed 1h) using Ultrasonic Cell Disruptor, 4 DEG C,
12000rpm is centrifuged 40min, takes supernatant, with hydrochloric acid regulatory protein matter solution ph to acidity, after 4 DEG C of standing 1h, and 4 DEG C,
12000rpm is centrifuged 40min centrifuging and taking supernatant, and supernatant is concentrated by ultrafiltration to 10-50mg/mL；
(2) precipitating reagent is dissolved in 0.1mM, in the citric acid solution of pH 3.0, the PEG 8000 for being configured to 40mg/mL is heavy
Shallow lake agent solution is added in the protein solution in step (1) by the volume ratio of 1:1, and 10-200 μ L seed crystals are added after mixing,
It is placed in temperature-controlled box, the stirred crystallization at 1-30 DEG C, mixing speed is set as 100-800rpm；
(3) 4 DEG C, supernatant is abandoned in 12000rpm centrifugation, and gained precipitating is superoxide dismutase mixed crystal；
(4) superoxide dismutase crystal cleaning solution is added in mixed crystal, after being slowly stirred mixing, centrifugation is abandoned
Supernatant repeats the process three times thoroughly to clean crystal.
(5) with identical crystallization condition, method for crystallising repeats crystallization more than twice, to obtain the super oxygen of higher purity
Object is disproportionated enzyme crystal.
(6) gained crystal is lyophilized, obtains white powder superoxide dismutase finished product.
Embodiment 1:
(1) it is crushed bacterial fermentation culture solution (power 30%, opens 5s, stops 3s, is crushed 1h) using Ultrasonic Cell Disruptor,
12000rpm is centrifuged 40min, takes supernatant, with hydrochloric acid conditioning solution pH=5.0, after 4 DEG C of standings 1h, and 12000rpm centrifugation 40min
After take supernatant, pressure be 0.5Mpa under, supernatant is concentrated into 30mg/mL by the ultrafiltration membrane for being 3KDa with molecular cut off；
(2) precipitating reagent is dissolved in 0.1mM, in the citric acid solution of pH 3.0, the PEG 8000 for being configured to 40mg/mL is heavy
Shallow lake agent solution is added in the protein solution in step (1) by the volume ratio of 1:1, and the seed crystal of 200uL is added after mixing,
It is placed in stirred crystallization in 4 DEG C of temperature-controlled boxs, mixing speed 200rpm；
(3) 4 DEG C of solution, the 12000rpm centrifugation 30min after the completion of step (2) crystallization are taken into precipitating, gained precipitating is
Superoxide dismutase mixed crystal；
(4) 8000 solution of PEG of 40mg/mL is added in the solution obtained in step (3), after being slowly stirred mixing, from
The heart abandons supernatant, repeats the process three times thoroughly to clean crystal.
(5) with identical crystallization condition, method for crystallising repeats crystallization more than twice, to obtain the super oxygen of higher purity
Object is disproportionated enzyme crystal.
(6) crystalloid solution obtained by step (5) is freeze-dried, obtaining superoxide dismutase purity is 95%, Rate activity
For 4335U/mg.
Embodiment 2:
(1) it is crushed bacterial fermentation culture solution (power 30%, opens 5s, stops 3s, is crushed 1h) using Ultrasonic Cell Disruptor,
12000rpm is centrifuged 40min, takes supernatant, with hydrochloric acid conditioning solution pH=4.5, after 4 DEG C of standings 1h, and 12000rpm centrifugation 40min
After take supernatant, pressure be 0.5Mpa under, supernatant is concentrated into 40mg/mL by the ultrafiltration membrane for being 3KDa with molecular cut off；
(2) precipitating reagent is dissolved in 0.2mM, in the citric acid solution of pH 3.5, the PEG 8000 for being configured to 40mg/mL is heavy
Shallow lake agent solution is added in the protein solution in step (1) by the volume ratio of 1:1, and the seed crystal of 200uL is added after mixing,
It is placed in stirred crystallization in 4 DEG C of temperature-controlled boxs, mixing speed 250rpm；
(3) 4 DEG C of solution, the 12000rpm centrifugation 30min after the completion of step (2) crystallization are taken into precipitating, gained precipitating is
Superoxide dismutase mixed crystal；
(4) 8000 solution of PEG of 60mg/mL is added in the solution obtained in step (3), after being slowly stirred mixing, from
The heart abandons supernatant, repeats the process three times thoroughly to clean crystal.
(5) with identical crystallization condition, method for crystallising repeats crystallization more than twice, to obtain the super oxygen of higher purity
Object is disproportionated enzyme crystal.
(6) crystalloid solution obtained by step (5) is freeze-dried, obtaining superoxide dismutase purity is 96%, Rate activity
For 4523U/mg.
Embodiment 3:
(1) it is crushed bacterial fermentation culture solution (power 30%, opens 5s, stops 3s, is crushed 1h) using Ultrasonic Cell Disruptor,
12000rpm is centrifuged 40min, takes supernatant, with hydrochloric acid conditioning solution pH=4.0, after 4 DEG C of standings 1h, and 12000rpm centrifugation 40min
After take supernatant, pressure be 0.5Mpa under, supernatant is concentrated into 35mg/mL by the ultrafiltration membrane for being 3KDa with molecular cut off；
(2) precipitating reagent is dissolved in 0.2mM, in the citric acid solution of pH 3.5, the PEG 8000 for being configured to 60mg/mL is heavy
Shallow lake agent solution is added in the protein solution in step (1) by the volume ratio of 1:1, and the seed crystal of 200uL is added after mixing,
It is placed in stirred crystallization in 4 DEG C of temperature-controlled boxs, mixing speed 250rpm；
(3) 4 DEG C of solution, the 12000rpm centrifugation 30min after the completion of step (2) crystallization are taken into precipitating, gained precipitating is
Superoxide dismutase mixed crystal；
(4) 8000 solution of PEG of 60mg/mL is added in the solution obtained in step (3), after being slowly stirred mixing, from
The heart abandons supernatant, repeats the process three times thoroughly to clean crystal.
(5) with identical crystallization condition, method for crystallising repeats crystallization more than twice, to obtain the super oxygen of higher purity
Object is disproportionated enzyme crystal.
(6) crystalloid solution obtained by step (5) is freeze-dried, obtaining superoxide dismutase purity is 93%, Rate activity
For 3869U/mg.
Claims (7)
1. a kind of method using crystallisation separating-purifying superoxide dismutase, it is characterised in that steps are as follows:
Step 1: after being crushed bacterial fermentation culture solution using Ultrasonic Cell Disruptor, centrifuging and taking supernatant is molten with hydrochloric acid regulatory protein matter
Liquid pH value is to acidity, and after 0 DEG C of -4 DEG C of standing 1h -3h, centrifuging and taking supernatant obtains protein solution after ultrafiltration concentration；
Step 2: precipitating reagent being dissolved in 0.1-5mM, in the citric acid solution that pH value is 3.0-5.5, presses 1:1 with protein solution
After volume ratio mixing, 10-200 μ L seed crystals are added, are placed in temperature-controlled box, the stirred crystallization in 4 DEG C-20 DEG C；The precipitating reagent is
PEG8000, concentration are 20-80mg/mL；
Step 3: precipitating will be taken after the solution centrifugation after the completion of crystallization, gained precipitating is the mixing of superoxide dismutase crystal
Object；
Step 4: superoxide dismutase crystal cleaning solution is added, after being slowly stirred mixing, supernatant is abandoned in centrifugation, repeats the process
Repeatedly thoroughly to clean crystal, superoxide dismutase crystal is obtained；The crystal cleaning solution is 20-100mg/mL's
PEG8000 solution；
Step 5: gained crystal being lyophilized, white powder superoxide dismutase finished product is obtained, or be not lyophilized, directly as crystalline substance
Body preparation application.
2. utilizing the method for crystallisation separating-purifying superoxide dismutase according to claim 1, it is characterised in that: for
The protein solution of solution step of replacing 2 after the superoxide dismutase dissolution of crystals that step 4 obtains, using with step 2 phase
Same crystallization condition repeats to crystallize repeatedly, keeps obtained superoxide dismutase crystal purity higher.
3. the method according to claim 1 or claim 2 using crystallisation separating-purifying superoxide dismutase, it is characterised in that:
The bacterial fermentation culture solution is the fermentation liquid of the engineering bacteria with source of people Expression of Superoxide Dismutase ability.
4. utilizing the method for crystallisation separating-purifying superoxide dismutase according to claim 1, it is characterised in that: described
The pH value of step 1 is 4.0-6.0.
5. utilizing the method for crystallisation separating-purifying superoxide dismutase according to claim 1, it is characterised in that: described
Ultrafiltration retaining molecular weight used in the ultrafiltration of step 1 is 3-10KDa, and pressure is 0.2-0.45MPa.
6. utilizing the method for crystallisation separating-purifying superoxide dismutase according to claim 1, it is characterised in that: described
Step 4 is repeatedly three times.
7. utilizing the method for crystallisation separating-purifying superoxide dismutase according to claim 1, it is characterised in that: described
Step 5 is repeatedly more than two times.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810497873.6A CN108659096A (en) | 2018-05-23 | 2018-05-23 | A method of improving crystallization of protein success rate in crystallization plates surface grafting charge |
| CN2018104978736 | 2018-05-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109879931A true CN109879931A (en) | 2019-06-14 |
Family
ID=63777368
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810497873.6A Withdrawn CN108659096A (en) | 2018-05-23 | 2018-05-23 | A method of improving crystallization of protein success rate in crystallization plates surface grafting charge |
| CN201910203596.8A Pending CN109879931A (en) | 2018-05-23 | 2019-03-18 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810497873.6A Withdrawn CN108659096A (en) | 2018-05-23 | 2018-05-23 | A method of improving crystallization of protein success rate in crystallization plates surface grafting charge |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN108659096A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1760172A (en) * | 2004-10-26 | 2006-04-19 | 邓希贤 | Tetra-(alpha-amino-acid or dipeptide) complexes of di-copper and synthetic method, and SOD loke activity |
| EP1745802A1 (en) * | 2005-07-20 | 2007-01-24 | Kreatech Biotechnology B.V. | Method of conjugating therapeutic compounds to cell targeting moieties via metal complexes |
| CN104396707A (en) * | 2014-06-19 | 2015-03-11 | 东北林业大学 | Method for improving stress resistance of pinus sylvestris seedling |
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
-
2018
- 2018-05-23 CN CN201810497873.6A patent/CN108659096A/en not_active Withdrawn
-
2019
- 2019-03-18 CN CN201910203596.8A patent/CN109879931A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1760172A (en) * | 2004-10-26 | 2006-04-19 | 邓希贤 | Tetra-(alpha-amino-acid or dipeptide) complexes of di-copper and synthetic method, and SOD loke activity |
| EP1745802A1 (en) * | 2005-07-20 | 2007-01-24 | Kreatech Biotechnology B.V. | Method of conjugating therapeutic compounds to cell targeting moieties via metal complexes |
| CN104396707A (en) * | 2014-06-19 | 2015-03-11 | 东北林业大学 | Method for improving stress resistance of pinus sylvestris seedling |
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108659096A (en) | 2018-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110423241B (en) | Process for preparing cephalosporin C sodium salt and 7-aminocephalosporanic acid | |
| JP2011517553A (en) | Method for producing galactooligosaccharides by free cells | |
| CN108285912A (en) | A method of fermentation prepares extraction pharmaceutical grade valine | |
| CN108486074A (en) | A method of utilizing crystallisation separating-purifying superoxide dismutase | |
| CN109337883A (en) | A kind of α -1,2- fucosyltransferase and its application in milk powder | |
| CN109486894A (en) | A kind of production method of Valine | |
| EP4442694A1 (en) | New crystal form of trisaccharide | |
| CN117089503B (en) | Escherichia coli K-12 MG1655 BLBYZT6 and application thereof | |
| CN105348122A (en) | Method for purifying L-alanine final mother liquor | |
| CN102676604A (en) | Method for preparing high-purity galacto-oligosaccharide by continuous simulated moving bed chromatography separation | |
| CN112778149A (en) | Method for extracting and separating beta-alanine from fermentation liquor | |
| CN108285913B (en) | Process for preparing and extracting L-glutamine | |
| CN114181268A (en) | Method for co-producing erythritol and arabinose by xylose mother liquor | |
| CN104974032A (en) | Method of separation and extraction of D-lactic acid from sodium D-lactate fermentation liquid | |
| CN109879931A (en) | A method of utilizing crystallisation separating-purifying superoxide dismutase | |
| CN109136299B (en) | Method for preparing, extracting and purifying threonine | |
| JP2022514350A (en) | A method for separating biomass from a solution containing biomass and at least one oligosaccharide. | |
| CN102827884B (en) | Method for extracting L-lactic acid produced in corn soaking process | |
| CN113005161B (en) | Preparation method of polysialic acid and polysialic acid product | |
| CN104789607B (en) | A kind of method that Integrated process prepares lactic acid and/or lactate | |
| CN115678925A (en) | Preparation method of calcium propionate | |
| CN109021066A (en) | A kind of method of broth extraction glutathione | |
| CN108315363B (en) | Citric acid fermentation method for continuously supplementing nitrogen | |
| CN102690333B (en) | Preparation method of high-purity teicoplanin | |
| CN111235192B (en) | Process for producing, separating and purifying L-phenylalanine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190614 |