CN102746396A - Method for extracting and transferring bovine serum albumin by preparing Cm-s-Cm type surfactant into reverse micelle - Google Patents
Method for extracting and transferring bovine serum albumin by preparing Cm-s-Cm type surfactant into reverse micelle Download PDFInfo
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- CN102746396A CN102746396A CN2011104027300A CN201110402730A CN102746396A CN 102746396 A CN102746396 A CN 102746396A CN 2011104027300 A CN2011104027300 A CN 2011104027300A CN 201110402730 A CN201110402730 A CN 201110402730A CN 102746396 A CN102746396 A CN 102746396A
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- bovine serum
- serum albumin
- active agent
- reverse micelle
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Abstract
The invention relates to a method for extracting and transferring bovine serum albumin by preparing a Cm-s-Cm type surfactant into a reverse micelle, and belongs to the technical field of reverse micelle extraction in biological engineering. The method comprises the following steps: preparing a Cm-s-Cm-type surfactant into a reverse micelle; preparing a bovine serum albumin solution; carrying out primary extraction; carrying out secondary extraction; and carrying out vacuum freeze-drying to obtain the bovine serum albumin product. According to the present invention, the reverse micelle formed from the Cm-s-Cm-type surfactant is adopted to carry out extraction (the primary extraction) on the bovine serum albumin without addition of an inorganic salt so as to completely transfer the water phase bovine serum albumin to the reverse micelle; and during transferring of the bovine serum albumin to the water phase again from the reverse micelle (the secondary extraction stage), adjustment of the pH value of the water is only required without addition of an inorganic salt so as to efficiently enrich the BSA in the water phase. The method of the present invention has the following advantages that: the operation is simple, addition of an inorganic salt is not required, the use amount of the surfactant is less, and the natural activity of the bovine serum albumin is not affected.
Description
Technical field
The invention belongs to inverse micelle abstraction technique field in the biotechnology, the inverse micelle abstraction that particularly a kind of novel surfactant forms shifts the method for bovine serum albumin.
Background technology
Reverse micelle is a kind of molecular assembly molectron that tensio-active agent forms in non-polar solvent.It has special nanometer space---" pond ", should " pond " can enzyme and protein etc. be solubilized into wherein, and for they provide a special microenvironment, make it be water molecules on every side, thereby can well keep its natural radioactivity.
Reverse micelle is a kind of method of protein separation, transfer, enrichment and purifying to the liquid-liquid extraction of bovine serum albumin, comprises preceding extraction (being about to bovine serum albumin is extracted in the reverse micelle) and back extraction (be about to bovine serum albumin and be transferred to water once more from reverse micelle) two steps.The general tensio-active agent of in inverse micelle abstraction bovine serum albumin technology, using is 1,4-two (2-ethylhexyl) succinate sodium sulfonate (AOT) and cetyl trimethylammonium bromide (CTAB).Though AOT very easily forms reverse micelle, when it extracted bovine serum albumin, not only consumption big (0.16 M, i.e. 70 g/L), and needs 2 M inorganic salt (KCl) exist could guarantee extraction efficiency.When CTAB is used for the bovine serum albumin extraction,, consumption could guarantee extraction efficiency though well below AOT (the CTAB consumption can not be lower than 0.04 M, i.e. 14.5 g/L), still requiring the inorganic salt (NaBr) of at least 1.7 M to exist.Under the situation of salt-free existence, these two kinds of tensio-active agents approach 0 to the extraction efficiency of bovine serum albumin.The existence of high salt concentration is not only made troubles to the subsequent purification of bovine serum albumin, simultaneously to the requirement of processing unit also than higher, cause corrosion to prevent the relevant processing unit of salt pair.In addition; Remain in the bovine serum albumin extraction process in following unavoidable unfavorable factor: (1) AOT concentration is easy to make water emulsification when high; (2) CTAB in extraction process easily and bovine serum albumin form white insoluble mixture; (3) bovine serum albumin content to be extracted must not be higher than 6 g/L, otherwise percentage extraction can sharply reduce.The present invention shifts the extraction that a kind of novel surfactant is used for bovine serum albumin, and not only dosage of surfactant few (getting final product greater than 3 g/L) need not to add inorganic salt, and waits that the content that extracts bovine serum albumin may be up to 28 g/L.In addition, the used tensio-active agent of the present invention reaches in concentration under the situation of 90 g/L can not cause water emulsification yet, also can not form insoluble mixture with bovine serum albumin.
Summary of the invention
The purpose of this invention is to provide a kind of simple to operate, dosage of surfactant is few, need not inorganic salt can extract the transfer bovine serum albumin, and makes bovine serum albumin keep the C of natural radioactivity
m-s-C
mThe method of bovine serum albumin is shifted in the anti-glue extraction of type tensio-active agent.
The inventive method is: carry out preceding extraction with reverse micelle after bovine serum albumin is mixed with bovine serum albumen solution to be extracted; Carry out the back extraction with damping fluid again after obtaining organic phase; Centrifuging and taking gets the bovine serum albumin aqueous solution, obtains bovine serum albumin dry powder through vacuum lyophilization again; Characteristics of the present invention are: adopt C
m-s-C
mType tensio-active agent preparation reverse micelle.
The present invention uses C
m-s-C
mThe reverse micelle that the type tensio-active agent forms extracts (extraction promptly) to bovine serum albumin; In extraction process, only need a spot of tensio-active agent; And under salt-free condition, just can make the bovine serum albumin of water be transferred to reverse micelle fully, this process operation is simple, in the extraction process; Bovine serum albumin is surrounded by the water surrounding in the reverse micelle, can make BSA keep natural radioactivity.The extraction stages in the back is about to BSA and is transferred to once more the process of water from reverse micelle, only needs the pH value of regulator solution just can make bovine serum albumin shift and be enriched in aqueous phase.The inventive method is simple to operate, need not inorganic salt, under the few situation of dosage of surfactant, can extract the transfer bovine serum albumin efficiently, and can keep the natural radioactivity of bovine serum albumin.
In addition, reverse micelle according to the invention is by C
m-s-C
mType tensio-active agent, normal paraffin, short chain alcohol and water are formed.
C according to the invention
m-s-C
mType surfactant structure formula is:
Wherein, 8≤m≤16, s=8.
Suitably increase surfactant concentration and help improving preceding extraction efficiency, but the diffusion that concentration too greatly may impede protein matter, and extraction efficiency reduces on the contrary before making.So C in the said reverse micelle
m-s-C
mThe type surfactant concentrations is 0.003 g/ml ~ 0.09 g/ml.
A spot of solubility promoter (being short chain alcohol) helps the formation of reverse micelle, but solubility promoter too much can influence the polarity of solution and the size of reverse micelle, and extraction efficiency reduces before causing.So the volume ratio of normal paraffin and short chain alcohol is 12 ~ 5: 1 in the reverse micelle according to the invention.
Water-content directly influences the size of reverse micelle, and water-content is too small or cross the formation that mostly is unfavorable for reverse micelle.So water and C in the reverse micelle according to the invention
m-s-C
mThe mol ratio of type tensio-active agent is 5 ~ 20: 1.
Normal paraffin according to the invention is any one in Skellysolve A, normal hexane or the octane.
Short chain alcohol according to the invention is any one in Pentyl alcohol, n-hexyl alcohol or the n-Octanol.
Because the iso-electric point of bovine serum albumin is 4.7, control pH value is greater than its iso-electric point, and bovine serum albumin is electronegative, help strengthening it with tensio-active agent of the present invention between electrostatic interaction, the preceding extraction efficiency of raising.So the present invention is that 5 ~ 10 damping fluid dissolving bovine serum albumin is processed bovine serum albumen solution to be extracted with pH.
The pH of the used damping fluid of extraction is 3.0 ~ 4.5 behind the present invention.The present invention is controlled at the pH of damping fluid in the iso-electric point scope less than BSA, the BSA positively charged, thus cause between the tensio-active agent head base in it and the reverse micelle kernel Coulomb repulsion taking place and be discharged from reverse micelle water nuclear, help the back extraction process.
Embodiment
1, preparation reverse micelle:
Earlier with normal paraffin and short chain alcohol with 12 ~ 5: 1 volume ratio is mixed, the formation non-polar solvent.
Again with 3 ~ 90 g C
m-s-C
mThe type tensio-active agent is dissolved in the 1 L non-polar solvent, adds 1 ~ 35 mL water again, fully vibrates to the solution clear, is reverse micelle.
The preparation of table 1 reverse micelle
| ? | Tensio-active agent | m (tensio-active agent) | V Non-polar solvent | V Water | C (tensio-active agent) |
| Scheme one | C 12C 8C 12 | 10 g | 1 L | 3.9 mL | 0.014 M |
| Scheme two | C 12C 8C 12 | 20 g | 1 L | 7.7 mL | 0.028 M |
| Scheme three | C 16C 8C 16 | 10 g | 1 L | 3.3 mL | 0.012 M |
| Scheme four | C 16C 8C 16 | 15 g | 1 L | 5.0 mL | 0.018 M |
| Scheme five | C 16C 8C 16 | 20 g | 1 L | 6.7 mL | 0.024 M |
2, preparation bovine serum albumen solution to be extracted:
The preparation of damping fluid:
With Sodium phosphate, dibasic and Hydrocerol A or acetic acid and sodium-acetate, join the pH scope and be Sodium phosphate, dibasic-citrate buffer solution (0.01 M) of 4.7 ~ 8.
With glycocoll and sodium hydroxide, join the pH scope and be glycocoll-sodium hydrate buffer solution (0.01 M) of 9 ~ 10.
Get above two kinds of damping fluids and dissolve bovine serum albumin respectively, form bovine serum albumen solution to be extracted.
3, preceding extraction
The reverse micelle of above preparation and bovine serum albumen solution to be extracted are shaken under 10 min and 14000 rpm behind centrifugal 15 min through vortex, get upper strata reverse micelle clear liquid.
Calculate the efficient of preceding extraction respectively through following formula:
Preceding extraction information slip
| ? | pH ） | Preceding extraction efficiency * | Wait to extract bovine serum albumin concentration ** |
| Scheme one | 7 | 100% | ≤ 22 mg/ml |
| Scheme two | 7 | 100% | ≤ 28 mg/ml |
| Scheme three | 7 | 100% | ≤ 8.6 mg/ml |
| Scheme four | 7 | 100% | ≤ 14 mg/ml |
| Scheme five | 7 | 100% | ≤ 18 mg/ml |
Present method need not that extra adding inorganic salt can make bovine serum albumin be extracted to organic phase fully in system, and this is that other tensio-active agent can't be accomplished.
(1) as with other tensio-active agent such as CTAB or AOT, reach 100 % as if extraction efficiency before making, the concentration of waiting to extract bovine serum albumin in the bovine serum albumen solution must not be higher than 6 mg/ml; (2) among the present invention, when wait to extract in the bovine serum albumen solution bovine serum albumin concentration be higher than give numerical value in the form after, the used reverse micelle of present method can not guarantee that bovine serum albumin 100 %'s is extracted into reverse micelle from water
4, back extraction
The preparation of back collection liquid: with Sodium phosphate, dibasic and Hydrocerol A or acetic acid and sodium-acetate, join the pH scope and be acetic acid-sodium-acetate buffer (0.01 M) of 3 ~ 4.5, this solution is the liquid that afterwards comes together.
Be 1:1 by volume, back collection liquid mixed with the inverse micellar solution of step 2 gained mutually that vortex shakes 10 min and behind centrifugal 15 min under 14000 rpm, takes off a layer bovine serum albumen solution.
Calculate the efficient of back extraction through following formula:
Back extraction information slip
| ? | pH | Back extraction efficiency |
| Scheme one | 4.3 | 76% |
| Scheme two | 4.3 | 89% |
| Scheme three | 4.3 | 91% |
| Scheme four | 4.3 | 92% |
| Scheme five | 4.3 | 96% |
Present method need not that extra adding inorganic salt can make bovine serum albumin from reverse micelle, be extracted into water efficiently in system.As not adding inorganic salt, other tensio-active agent is 0 to the back percentage extraction of BSA.
5, lyophilize
With the bovine serum albumen solution of step 4 gained, be that 50 Pa, temperature are-60 ℃ of following lyophilize 36 h at pressure, obtain pure bovine serum albumin dry powder.
Claims (9)
1.C
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin; Comprise bovine serum albumin is mixed with and carry out preceding extraction with reverse micelle after waiting to extract bovine serum albumen solution; Carry out the back extraction with damping fluid again after obtaining organic phase; Centrifuging and taking gets the bovine serum albumin aqueous solution, obtains bovine serum albumin dry powder through vacuum lyophilization again; It is characterized in that adopting C
m-s-C
mType tensio-active agent preparation reverse micelle.
2. according to the said C of claim 1
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that said C
m-s-C
mType surfactant structure formula is:
Wherein, 8≤m≤16, s=8.
3. according to claim 1 or 2 said C
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that said reverse micelle is by C
m-s-C
mType tensio-active agent, normal paraffin, short chain alcohol and water are formed C in the said reverse micelle
m-s-C
mThe type surfactant concentrations is 0.003 g/ml ~ 0.09 g/ml.
4. according to the said C of claim 3
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that the volume ratio of normal paraffin and short chain alcohol is 12 ~ 5: 1 in the said reverse micelle.
5. according to the said C of claim 3
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that water and C in the said reverse micelle
m-s-C
mThe mol ratio of type tensio-active agent is 5 ~ 20: 1.
6. according to the said C of claim 3
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that said normal paraffin is any one in Skellysolve A, normal hexane or the octane.
7. according to the said C of claim 3
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that said short chain alcohol is any one in Pentyl alcohol, n-hexyl alcohol or the n-Octanol.
8. according to the said C of claim 1
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that with pH being 5 ~ 10 damping fluid dissolving bovine serum albumin, processes bovine serum albumen solution.
9. according to the said C of claim 1
m-s-C
mType tensio-active agent inverse micelle abstraction shifts the method for bovine serum albumin, it is characterized in that the pH of the damping fluid that the back extraction is used is 3.0 ~ 4.5.
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Cited By (3)
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| CN105399650A (en) * | 2015-12-16 | 2016-03-16 | 齐鲁工业大学 | Method for extracting alliin through reverse micelles system |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105399650A (en) * | 2015-12-16 | 2016-03-16 | 齐鲁工业大学 | Method for extracting alliin through reverse micelles system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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