CN1153604C - Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon - Google Patents
Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon Download PDFInfo
- Publication number
- CN1153604C CN1153604C CNB001327062A CN00132706A CN1153604C CN 1153604 C CN1153604 C CN 1153604C CN B001327062 A CNB001327062 A CN B001327062A CN 00132706 A CN00132706 A CN 00132706A CN 1153604 C CN1153604 C CN 1153604C
- Authority
- CN
- China
- Prior art keywords
- halogenated hydrocarbons
- ctab
- back extraction
- mixed reversed
- micelle
- 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
Images
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The present invention belongs to the field of separating and purifying biological substances, particularly to a method for enhancing the extraction rate and the back extraction rate of reverse micelle relative biological substances by adding halogenated hydrocarbon. The method adds the halogenated hydrocarbon in cation surfactant, and the extraction rate and the back extraction rate of formed cation-halogenated-hydrocarbon mixing reverse micelle relative biological substances are enhanced. The method can remarkably enhance the back extraction rate of the reverse micelle relative biological substances under wide pH ranges and different ionic strength, and can remarkably reduce time that a reverse micelle phase achieves back extraction balance; the method has the outstanding advantage that the reverse micelle phase can be cyclically used.
Description
The invention belongs to biological substance separation and purification field, be particularly related to and add halogenated hydrocarbons to the middle mutually formation cation-halogenated hydrocarbons mixed reversed micelle phase of cationic reverse micelle, thus biological substance extraction yield and the interpolation halogenated hydrocarbons raising relative biological substance extraction yield of reverse micelle of back extraction ratio and the methods of back extraction ratio such as the relative protein of raising reverse micelle.
Reverse micelle is the molecule aggregate that surfactant forms in organic solvent mutually, bioactivators such as little pond energy solubilising protein wherein.By regulating conditions such as aqueous pH values, ionic strength, can optionally extract target protein.In theory, reverse micelle phase method extracting protein matter only needs by extraction and back extraction process, can realize the concentrated and purified of protein, its operating procedure is simple, compare with traditional protein stripping technique such as salt precipitation, gel permeation chromatography, ion-exchange chromatography etc., but have that treating capacity is big, selectivity good and advantage such as continued operation, be specially adapted to the extensive separation and purification of biological product, because of it might reduce the production cost of biological product significantly.But reverse micelle phase method extracting protein matter fails to realize industrialization so far, and reason is a lot, and one of them is that the back extraction process of this method is very difficult.
It is a lot of in the factor of water and the alternate distribution of reverse micelle to influence protein, kind as aqueous pH values, ionic strength and salt, the use of the type of organic facies surfactant, concentration, cosurfactant whether, consumption what and solvent types, the temperature and pressure of extraction process etc.Wherein the most important thing is the pH value and the ionic strength of water.When using cationic surface active agent to form the reverse micelle phase time, aqueous pH values is higher than the isoelectric point pI of protein, helps the extraction of protein; When using anionic surfactant to form the reverse micelle phase time, aqueous pH values is lower than the isoelectric point pI of protein, helps the extraction of protein.When the water ionic strength is low, help the extraction of protein; And the water ionic strength is unfavorable for the extraction of protein when higher.
As everyone knows, because there is very high mass transfer interfacial resistance, the back extraction of protein is that the transfer of protein from reverse micelle to strip aqueous is very difficult.It is generally acknowledged that carry out back extraction under the condition that is unfavorable for extracting, protein should be realized complete back extraction.As if but the back extraction of protein is not an equilibrium process, even in very high ionic strength with under the pH condition that is unfavorable for fully extracting, the back extraction of protein can not realize fully, even back extraction ratio is very low or back extraction does not take place fully.
In recent years, many researchers have carried out new trial, propose the new method of a series of raising reverse micelle method protein back extraction ratios.These new methods are: l) Ermin (Prikl Biokhim Kikrobiol, 1988,24,42-49) and Woll (Biotechnol.Prog., 1989,5,57-62) wait the people to attempt adding second kind of solvent and destroy the reverse micelle phase, thereby discharge the protein of reverse micelle in mutually; 2) Dekker (Chem.Eng.J., 1991,46, B69-74) wait the people by increase earlier the back extraction temperature then centrifugation method isolate unnecessary water, condensing protein is in isolated water; 3) Carison (Biotechnol.Prog.1992,8,85-90) wait the people to add 10% to 50% isopropyl alcohol to strip aqueous, realize the back extraction of protein; 4) Leser (Chimia, 1990,44,270-282) add silica gel and absorb moisture, thereby protein is separated with organic facies; 5) Gupta (Biotechnol.Bioeng., 1994,44,830-836) make the reverse micelle phase dehydration by molecular sieve, realize separating of protein and organic facies; 6) Hayes (Biotechnol.Bioeng., 1998,59 (5), 557-566) add a spot of cosurfactant, the back extraction of realization protein to the W/O microemulsion; 7) Jarudilokkul (Biotechnol.Bioeng., 1999,62 (5), 593-601) have the surfactant of opposite charges by interpolation, under less salt and gentle pH condition, realized the back extraction of protein.
Though above several method can be from the reverse micelle protein that is separated out, the method for only adding isopropyl alcohol and adding the opposite charges surfactant is easy to realize industrialized production, and other are several only research value.And the shortcoming of adding the method for isopropyl alcohol to be the ability of reverse micelle phase reextraction protein sharply descend, cause organic facies not recycle; The shortcoming of adding the oppositely charged surfactant is to destroy the reverse micelle phase fully, and organic facies loses the ability of extracting protein matter.Therefore, the back extraction problem of reverse micelle phase extracting protein matter does not solve at all.
The objective of the invention is to extract mutually the problem of biological substance process back extraction difficulty at reverse micelle, a kind of method that halogenated hydrocarbons improves reverse micelle phase extraction yield and back extraction ratio of adding is provided, this method can improve relative biological substance extraction yield of cationic reverse micelle and back extraction ratio simultaneously, and reverse micelle can recycle mutually.
Embodiment of the present invention are as follows:
Interpolation halogenated hydrocarbons provided by the invention improves the method for relative biological substance extraction yield of reverse micelle and back extraction ratio, and its processing step is as follows:
The preparation by cationic surface active agent, cosurfactant, halogenated hydrocarbons, water and organic solvent form cation-the halogenated hydrocarbons mixed reversed micelle mutually;
Being formulated as follows of described cation-halogenated hydrocarbons mixed reversed micelle phase: at first take by weighing or pipette a certain amount of cationic surface active agent, and to wherein adding cosurfactant, halogenated hydrocarbons, water and organic solvent successively, mix, leave standstill after the vibration, dissolve fully to cationic surface active agent and halogenated hydrocarbons, add the organic solvent constant volume again, promptly make cation-halogenated hydrocarbons mixed reversed micelle phase;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the cosurfactant that is added: 0.50-20;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the halogenated hydrocarbons that is added: 0.5-10;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the water that is added: 0.5-3;
Cationic surface active agent the concentration of the cation of being prepared-halogenated hydrocarbons mixed reversed micelle in mutually be 10 mMs-200 mM/liter;
With cation-halogenated hydrocarbons mixed reversed micelle and aqueous phase extracted in 1: 1-1: 50 ratio is mixed, and extracts;
With cation-halogenated hydrocarbons mixed reversed micelle and strip aqueous in 1: 1-50: 1 ratio is mixed, and carries out back extraction;
Described halogenated hydrocarbons is chloralkane RCl, bromo alkane RBr, alkane iodide RI;
Alkyl R among described chloralkane RCl, bromo alkane RBr, the alkane iodide RI is a straight or branched;
Described alkyl R can be the alkyl of 4 carbon to 20 carbon atoms;
Described cationic surface active agent is trialkyl methyl quaternary ammonium, dialkyl dimethyl quaternary ammonium salt, alkyl trimethyl quaternary ammonium salt;
Described cosurfactant is the alkyl alcohols;
Described organic solvent is single liquid alkane or mixes liquid alkane.
The present invention adds halogenated hydrocarbons and forms cation-halogenated hydrocarbons mixed reversed micelle phase in single cationic reverse micelle phase system, to produce the effect of two aspects: the one, the size of increase reverse micelle phase, the positive charge that the cationic surfactant band is certain, it is a kind of lewis acid, and the polar head of halogenated hydrocarbons has lone pair electrons, is a kind of lewis base.The halogenated hydrocarbons polar head has reduced phase repulsive interaction between the cationic surfactant polar head in the appearance of reverse micelle phase inwall, thereby the reverse micelle phase transformation is big, can hold bigger biological tissue's molecule, has the extracting power bigger mutually than single reverse micelle; The 2nd, reduce the interfacial resistance of reverse micelle phase, help biological tissue's turnover reverse micelle phase, thereby in higher ionic strength be unfavorable for improving under the pH value condition of biological tissue's extraction the back extraction ratio of biological tissue.
In addition, the halogenated hydrocarbons with hydrophobic tail is less in the solubility of water, and the overwhelming majority is dissolved in organic solvent, and the loss of extraction process seldom very helps recycling of reverse micelle phase.If reverse micelle because halogenated hydrocarbons is dissolved in the back extraction ratio of water reduction to biological tissue, can add small amounts of halocarbon in organic facies mutually.
Be applicable to that the biological tissue that the inventive method is separated comprises protein, enzyme, amino acid and nucleic acid etc.
Interpolation halogenated hydrocarbons provided by the invention improves the method for relative biological substance extraction yield of reverse micelle and back extraction ratio, can significantly increase the extraction yield of the relative biological tissue of reverse micelle, under very wide pH scope and different ionic strength, can significantly improve simultaneously the back extraction ratio of the relative biological tissue of reverse micelle, significantly reduce reverse micelle and reach the back extraction balance time mutually, its outstanding reverse micelle that is also advantageous in that can recycle mutually.
Further describe the present invention below in conjunction with drawings and Examples:
The schematic diagram that the extraction yield that the single reverse micelle of accompanying drawing 1 extracts BSA mutually with cation-halogenated hydrocarbons mixed reversed micelle mutually changes with the variation of aqueous phase extracted pH value;
Accompanying drawing 2 cations-halogenated hydrocarbons mixing mixed reversed micelle extracts the schematic diagram that the extraction yield of BSA changes with the halogenated hydrocarbons addition mutually;
The back extraction ratio of accompanying drawing 3 cations-halogenated hydrocarbons mixing mixed reversed micelle phase back extraction BSA changes the schematic diagram of (KBr that strip aqueous contains 1.0mol/L) with strip aqueous pH value;
The back extraction ratio of accompanying drawing 4 cations-halogenated hydrocarbons mixing mixed reversed micelle phase back extraction BSA changes the schematic diagram of (KBr that strip aqueous contains 1.0mol/L) with the back extraction time;
(strip aqueous contains KBr to the back extraction ratio of accompanying drawing 5 cations-halogenated hydrocarbons mixing mixed reversed micelle phase back extraction BSA, pH=4.3) and the schematic diagram that changes with the variation of strip aqueous ionic strength;
(strip aqueous contains KCl to the back extraction ratio of accompanying drawing 6 cations-halogenated hydrocarbons mixing mixed reversed micelle phase back extraction BSA, pH=4.3) and the schematic diagram that changes with the variation of strip aqueous ionic strength;
Wherein :-■-be softex kw (being abbreviated as CTAB)
-●-be the CTAB+ chlorobutane
-▲-is the CTAB+ bromooctane
--be the CTAB+ iodobutane
Embodiment 1: extract BSA with method of the present invention:
In the present embodiment, cationic surfactant is selected CTAB (softex kw is analyzed pure) for use; Cosurfactant is selected n-hexyl alcohol for use; Halogenated hydrocarbons is selected neoprene hydrocarbon, bromooctane, butyl iodide respectively for use; Organic solvent is selected benzinum for use; Hydromining distilled water;
Aqueous phase extracted is to contain the buffer solution of different pH of bovine serum albumin(BSA) (BSA) of the potassium chloride of 0.10 mol and 1.0 mg/ml (in the pH value is the 3-6 scope, uses the citric acid-trisodium citrate buffer solution of 0.05 mol; In the pH value is the 6-8 scope, uses the Na of 0.05 mol
2HPO
4-NaH
2PO
4Buffer solution; The pH value is used the borax-hydrochloride buffer of 0.05 mol for the 8-11 scope);
Its concrete processing step is as follows:
1. prepare CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle phase and single CTAB reverse micelle respectively mutually:
To prepare 100 milliliters of CTAB-chlorobutane mixed reversed micelles is example mutually, its process for preparation is as follows: take by weighing 1.822 gram CTAB, and to wherein adding 20 milliliters of n-hexyl alcohols (cosurfactant), 5 milliliters chlorobutane, 50 milliliters benzinum and 1.2 milliliters distilled water successively, vibration is left standstill, dissolve fully to CTAB and chlorobutane, add benzinum and be settled to 100 milliliters, mix, thereby make 100 milliliters CTAB-chlorobutane mixed reversed micelle phase;
Adopt the process for preparation mutually identical to prepare 100 milliliters of CTAB-bromooctane mixed reversed micelles phases and 100 milliliters of CTAB-iodobutane mixed reversed micelle phases respectively with preparation CTAB-chlorobutane mixed reversed micelle;
The process for preparation of single CTAB reverse micelle phase is except that not adding the halogenated hydrocarbons, and is identical with the process for preparation of above-mentioned mixed reversed micelle phase;
CTAB-chlorobutane mixed reversed micelle mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of CTAB-chlorobutane mixed reversed micelle phase volume, the volume of chlorobutane accounts for 5% of CTAB-chlorobutane mixed reversed micelle phase volume, the volume of distilled water accounts for 1.2% of CTAB-chlorobutane mixed reversed micelle phase volume, and all the other are benzinum;
CTAB-bromooctane mixed reversed micelle mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of CTAB-bromooctane mixed reversed micelle phase volume, the volume of bromooctane accounts for 5% of CTAB-bromooctane mixed reversed micelle phase volume, the volume of distilled water accounts for 1.2% of CTAB-bromooctane mixed reversed micelle phase volume, and all the other are benzinum;
CTAB-iodobutane mixed reversed micelle mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of CTAB-iodobutane mixed reversed micelle phase volume, the volume of iodobutane accounts for 5% of CTAB-iodobutane mixed reversed micelle phase volume, the volume of distilled water accounts for 1.2% of CTAB-iodobutane mixed reversed micelle phase volume, and all the other are benzinum;
Single CTAB reverse micelle mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for single CTAB reverse micelle phase volume 1.2%, all the other are benzinum;
2. extraction process is as follows; In 5 triangular flasks that fill the equal-volume aqueous phase extracted, add isopyknic CTAB-chlorobutane mixed reversed micelle phase respectively, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle phase, single CTAB reverse micelle phase, under room temperature (30 degrees centigrade), 150 times/minute vibrations 20 minutes, separation is promptly finished in the centrifugal phase-splitting of 3500rpm;
Measure the organic facies Protein content with ultraviolet method, the Brandford method is measured the protein content of water, its result as shown in Figure 1, among Fig. 1, the curve that the extraction yield of-■-extract mutually for single CTAB reverse micelle BSA changes with the variation of aqueous phase extracted pH value;-● the curve that-extraction yield that extracts BSA for CTAB-chlorobutane mixed reversed micelle mutually changes with the variation of aqueous phase extracted pH value; The curve that-▲-extracts BSA mutually for CTAB-bromooctane mixed reversed micelle extraction yield changes with the variation of aqueous phase extracted pH value; The curve that the extraction yield of--extract mutually for CTAB-iodobutane mixed reversed micelle BSA changes with the variation of aqueous phase extracted pH value; As shown in Figure 1, under very wide pH value, the extraction yield of the relative BSA of CTAB-halogenated hydrocarbons mixed reversed micelle is than the extraction yield height of the relative BSA of single CTAB reverse micelle.
Embodiment 2: extract BSA with method of the present invention:
Selected cationic surfactant, cosurfactant, halogenated hydrocarbons and the distilled water of present embodiment is all identical with embodiment 1, and selecting n-hexane for use is organic solvent; The pH value of extraction water is 7.0.
Respectively preparation have different halogenated hydrocarbons percents by volume CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle mutually and single CTAB reverse micelle mutually; Compound method is with embodiment 1, the CTAB-halogenated hydrocarbons mixed reversed micelle that obtains mutually in, the concentration of CTAB be 200 mMs/liter, the volume of n-hexyl alcohol all accounts for 20% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, the volume of distilled water all accounts for 3.0% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, in CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle mutually in, the volume of its halogenated hydrocarbons accounts for 1% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume respectively, 2%, 3%, 4% and 5%, all the other are n-hexane;
2. extraction process is with embodiment 1, and its extraction results is seen accompanying drawing 2, among Fig. 2, and-■-be the extraction yield that single CTAB reverse micelle extracts BSA mutually;-● the curve that-extraction yield that extracts BSA for CTAB-chlorobutane mixed reversed micelle mutually changes with the variation of halogenated hydrocarbons addition; The curve that-▲-extracts BSA mutually for CTAB-bromooctane mixed reversed micelle extraction yield changes with the variation of halogenated hydrocarbons addition;--extract the curve that the variation of the extraction yield halogenated hydrocarbons addition of BSA changes mutually for CTAB-iodobutane mixed reversed micelle; As can be seen from Figure 2, be 7.0 o'clock in the pH value, the extraction yield of CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase and the relative BSA of CTAB-iodobutane mixed reversed micelle is than the extraction yield height of the relative BSA of single CTAB reverse micelle.Wherein, the extraction yield of the relative BSA of CTAB-bromooctane mixed reversed micelle improves with the increase of halogenated hydrocarbons addition.
Embodiment 3: extract and back extraction BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1, and the pH value is 9.10; Strip aqueous is the buffer solution of different pH values that contains the KBr of 1.0 mol;
1. prepare CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle phase and single CTAB reverse micelle respectively mutually; Compound method is with embodiment 1, three kinds of CTAB-halogenated hydrocarbons mixed reversed micelle phases that obtain, wherein the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol all accounts for 20% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, the volume of halogenated hydrocarbons accounts for 4.0% of CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle phase volume respectively, what the volume of distilled water all accounted for CTAB-halogenated hydrocarbons mixed reversed micelle phase volume is 1.2%, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for single CTAB reverse micelle phase volume 20%, the volume of distilled water accounts for 1.2% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
3. back extraction process is as follows: add isopyknic strip aqueous and the organic facies that contains the BSA of 1.0 mg/ml in 50 milliliters of triangular flasks, 250 times/minute vibrations one hour, the centrifugal phase-splitting of 4000rpm.The Brandford method is measured the protein content of strip aqueous, and it the results are shown in accompanying drawing 3, among Fig. 3, and the curve of-■-change with the variation of strip aqueous pH value for the back extraction ratio of single CTAB reverse micelle phase back extraction BSA;-●-curve that changes with the variation of strip aqueous pH value for the back extraction ratio of CTAB-chlorobutane mixed reversed micelle phase back extraction BSA; The curve that-▲-changes with the variation of strip aqueous pH value for the back extraction ratio of CTAB-bromooctane mixed reversed micelle phase back extraction BSA; The curve of--change with the variation of strip aqueous pH value for the back extraction ratio of CTAB-iodobutane mixed reversed micelle phase back extraction BSA; As can be seen from Figure 3, when strip aqueous contains the KBr of 1.0 mol, CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle in the pH value is the scope of 3.5-5.0 to the back extraction ratio of BSA back extraction ratio height than the relative BSA of single CTAB reverse micelle, especially the back extraction ratio of the relative BSA of CTAB-iodobutane mixed reversed micelle (comprises the isoelectric point pI of pH value greater than BSA) in very wide pH value scope has very high back extraction ratio to BSA, and under the same case, the extraction yield of single CTAB reverse micelle phase is almost nil.
Embodiment 4: extract and back extraction BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1, and the pH value is 9.10; Strip aqueous be contain 1.0 mol KBr, the pH value is 4.30 buffer solution;
1. prepare CTAB-chlorobutane mixed reversed micelle phase respectively, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, three kinds of CTAB-halogenated hydrocarbons mixed reversed micelle phases that obtain, the concentration of its CTAB be 50 mMs/liter, the volume of n-hexyl alcohol all accounts for 20% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, chlorobutane, bromooctane, the volume of iodobutane accounts for 4.0% of CTAB-halogenated hydrocarbons reverse micelle phase volume respectively, the volume of distilled water all accounts for 1.2% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for 1.2% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
3. back extraction process adopts the different back extraction time with embodiment 3, and it the results are shown in accompanying drawing 4, among Fig. 4, and the curve of-■-change with the variation of back extraction time for the back extraction ratio of single CTAB reverse micelle phase back extraction BSA;-●-curve that changes with the variation of back extraction time for the back extraction ratio of CTAB-chlorobutane mixed reversed micelle phase back extraction BSA; The curve that-▲-changes with the variation of back extraction back extraction time for the back extraction ratio of CTAB-bromooctane mixed reversed micelle phase back extraction BSA; The curve of--change with the variation of back extraction back extraction time for the back extraction ratio of CTAB-iodobutane mixed reversed micelle phase back extraction BSA; As can be seen from Figure 4, when strip aqueous pH value is 4.30, when containing the KBr of 1.0 mol, CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle are on good terms and are significantly reduced the CTAB system and reach the back extraction balance time, and single CTAB reverse micelle needs the long time just can reach the back extraction balance mutually; Through the same mixture time, the protein transfer rate of mixed reversed micelle phase is higher than the protein transfer rate of single reverse micelle phase.
Embodiment 5: extract and back extraction BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1, and the pH value is 9.10; KBr, the pH value that strip aqueous contains variable concentrations is 4.3 buffer solution;
1. prepare CTAB-chlorobutane mixed reversed micelle phase respectively, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, three kinds of CTAB-halogenated hydrocarbons mixed reversed micelles that obtain mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol all accounts for 20% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, chlorobutane, bromooctane, the volume of iodobutane accounts for 2.0% of CTAB-halogenated hydrocarbons reverse micelle phase volume respectively, the volume of distilled water all accounts for 1.2% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for 1.2% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
3. back extraction process is with embodiment 3, and it the results are shown in accompanying drawing 5, among Fig. 5, and the curve of-■-change with the variation of strip aqueous ionic strength for the back extraction ratio of single CTAB reverse micelle phase back extraction BSA;-●-curve that changes with the variation of strip aqueous ionic strength for the back extraction ratio of CTAB-chlorobutane mixed reversed micelle phase back extraction BSA; The curve that-▲-changes with the variation of strip aqueous ionic strength for the back extraction ratio of CTAB-bromooctane mixed reversed micelle phase back extraction BSA; The curve of--change with the variation of back extraction strip aqueous ionic strength for the back extraction ratio of CTAB-iodobutane mixed reversed micelle phase back extraction BSA; As can be seen from Figure 5, when strip aqueous contains different KBr concentration, when the pH value was 4.30 buffer solution, the back extraction ratio of CTAB-chlorobutane mixed reversed micelle phase, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle relative BSA of more single CTAB reverse micelle under lower KBr concentration was high a lot.
Embodiment 6: extract and back extraction BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1, and the pH value is 9.10; Strip aqueous be contain variable concentrations potassium chloride, the pH value is 4.30 buffer solution;
1. prepare CTAB-chlorobutane mixed reversed micelle phase respectively, CTAB-bromooctane mixed reversed micelle phase, CTAB-iodobutane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, three kinds of CTAB-halogenated hydrocarbons mixed reversed micelles that obtain mutually in, the concentration of C-B be 50 mMs/liter, the volume of n-hexyl alcohol all accounts for 20% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, chlorobutane, bromooctane, the volume of iodobutane accounts for 4.0% of CTAB-halogenated hydrocarbons reverse micelle phase volume respectively, the volume of distilled water accounts for 1.2% of CTAB-halogenated hydrocarbons mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for 1.2% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
4. back extraction process is with embodiment 3, and it the results are shown in accompanying drawing 6, among Fig. 6, and the curve of-■-change with the variation of strip aqueous ionic strength for the back extraction ratio of single CTAB reverse micelle phase back extraction BSA;-●-curve that changes with the variation of strip aqueous ionic strength for the back extraction ratio of CTAB-chlorobutane mixed reversed micelle phase back extraction BSA; The curve that-▲-changes with the variation of strip aqueous ionic strength for the back extraction ratio of CTAB-bromooctane mixed reversed micelle phase back extraction BSA; The curve of--change with the variation of back extraction strip aqueous ionic strength for the back extraction ratio of CTAB-iodobutane mixed reversed micelle phase back extraction BSA; As can be seen from Figure 6, when strip aqueous pH value is 4.30, when containing the potassium chloride of variable concentrations, the back extraction ratio of CTAB-bromooctane mixed reversed micelle phase, the CTAB-iodobutane mixed reversed micelle relative BSA of more single CTAB reverse micelle under lower potassium chloride concentration is high a lot.
Embodiment 7: extract BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1;
1. prepare respectively CTAB-bromooctane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, the CTAB-bromooctane mixed reversed micelle that obtains mutually in, the concentration of CTAB be 10 mMs/liter, the volume of n-hexyl alcohol accounts for 5% of CTAB-bromooctane mixed reversed micelle phase volume, the volume of bromooctane accounts for 2.0% of CTAB-bromooctane reverse micelle phase volume, and the volume of distilled water accounts for 0.5% of CTAB-bromooctane mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 10 mMs/liter, the volume of n-hexyl alcohol accounts for 5% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for 0.5% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
Its experimental result shows that the extraction yield of the relative BSA of CTAB-bromooctane mixed reversed micelle is than single CTAB
The extraction yield height of the relative BSA of reverse micelle.Present embodiment also can adopt trimethyl alkyl (C
10-C
16) ammonium chloride or trimethyl alkyl (C
17-C
20) ammonium chloride, preparation trimethyl alkyl (C
10-C
16) ammonium chloride-bromooctane mixed reversed micelle phase or trimethyl alkyl (C
17-C
20) ammonium chloride-bromooctane mixed reversed micelle phase, its extraction yield to BSA also is higher than single trimethyl alkyl (C
10-C
16) ammonium chloride reverse micelle phase or trimethyl alkyl (C
17-C
20) extraction yield of the relative BSA of ammonium chloride reverse micelle.
Embodiment 8: extract and back extraction BSA with method of the present invention:
In the present embodiment, aqueous phase extracted is with embodiment 1, and strip aqueous is with embodiment 3;
1. prepare dialkyl group (C respectively
10-C
16) dimethyl ammonium bromide-bromooctane mixed reversed micelle phase and single dialkyl group (C
10-C
16) dimethyl ammonium bromide reverse micelle phase; Compound method is with embodiment 1, the dialkyl group (C that obtains
10-C
16) dimethyl ammonium bromide-bromooctane mixed reversed micelle mutually in, dialkyl group (C
10-C
16) concentration of dimethyl ammonium bromide be 100 mMs/liter, the volume of n-hexyl alcohol accounts for dialkyl group (C
10-C
16) dimethyl ammonium bromide-bromooctane mixed reversed micelle phase volume 10%, the volume of bromooctane accounts for dialkyl group (C
10-C
16) dimethyl ammonium bromide-bromooctane mixed reversed micelle phase volume 10%, the volume of distilled water accounts for dialkyl group (C
10-C
16) dimethyl ammonium bromide-bromooctane mixed reversed micelle phase volume 1.0%, all the other are benzinum; Single dialkyl group (C
10-C
16) dimethyl ammonium bromide reverse micelle mutually in, dialkyl group (C
10-C
16) concentration of dimethyl ammonium bromide be 100 mMs/liter, the volume of n-hexyl alcohol accounts for single dialkyl group (C
10-C
16) dimethyl ammonium bromide reverse micelle phase volume 10%, the volume of distilled water accounts for single dialkyl group (C
10-C
16) dimethyl ammonium bromide reverse micelle phase volume 1.0%, all the other are benzinum;
2. extraction process is with embodiment 1;
3. back extraction process is with embodiment 3, and reverse micelle is 50: 1 with the volume ratio of strip aqueous;
Its experimental result shows that the interpolation of bromooctane can significantly improve dialkyl group (C
10-C
16) extraction yield of the relative BSA of dimethyl ammonium bromide reverse micelle.Present embodiment also can adopt chlorination dimethyl two positive hot ammoniums or chlorination dimethyl dialkyl (C
16-C
20) ammonium, preparation chlorination dimethyl two positive hot ammoniums-bromooctane mixed reversed micelle phase or chlorination dimethyl dialkyl (C
16-C
20) ammonium-bromooctane mixed reversed micelle phase, its extraction yield to BSA also is higher than single chlorination dimethyl two positive hot ammonium reverse micelle or chlorination dimethyl dialkyl (C
16-C
20) the ammonium reverse micelle is to the extraction yield of BSA.
Embodiment 9: extract AMS with method of the present invention:
In the present embodiment, aqueous phase extracted is the buffer solution of different pH that contains the AMS (AM) of the potassium chloride of 0.10 mol and 1.0 mg/ml;
Preparation Aliquat 336 (trialkyl methyl ammonium chloride)-bromooctane mixed reversed micelle mutually with single Aliquat336 reverse micelle mutually, compound method is with embodiment 1, the mixed reversed micelle that obtains mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 0.5% of mixed reversed micelle phase volume, the volume of bromooctane accounts for 0.5% of mixed reversed micelle phase volume, and the volume of distilled water accounts for 0.5% of mixed reversed micelle phase volume, and all the other are benzinum; Single Aliquat 336 reverse micelles mutually in, the concentration of Aliquat 336 be 50 mMs/liter, the volume of n-hexyl alcohol accounts for 0.5% of single Aliquat 336 reverse micelle phase volumes, and the volume of distilled water accounts for 0.5% of single Aliquat 336 reverse micelle phase volumes, and all the other are benzinum;
2. extraction process is with embodiment 1, and aqueous phase extracted is 50: 1 with reverse micelle volume ratio mutually;
Its experimental result shows, the extraction yield height of the relative AMS of more single Aliquat 336 reverse micelles of the extraction yield of the relative AMS of Aliquat 336-bromooctane mixed reversed micelle.Present embodiment, also can adopt methyl chloride three hot ammoniums or methyl chloride three positive 12 ammoniums, preparation methyl chloride three hot ammoniums-bromooctane mixed reversed micelle mutually or methyl chloride three positive 12 ammoniums-bromooctane mixed reversed micelle phase, its extraction yield to AMS also is higher than single methyl chloride three hot ammonium reverse micelles or the methyl chloride three positive 12 ammonium reverse micelles extraction yield to AMS.
Embodiment 10:
In the present embodiment, aqueous phase extracted is with embodiment 1, and strip aqueous is with embodiment 3;
1. prepare respectively CTAB-bromo-octadecane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, the CTAB-bromo-octadecane mixed reversed micelle phase that obtains, the concentration of its CTAB be 50 mMs/liter, the volume of n-octyl alcohol accounts for 20% of CTAB-bromo-octadecane mixed reversed micelle phase volume, the volume of bromo-octadecane accounts for 2.0% of CTAB-bromo-octadecane reverse micelle phase volume, the volume of distilled water accounts for 1.2% of CTAB-bromo-octadecane mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-octyl alcohol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water account for single CTAB reverse micelle phase volume 1.2% all the other for benzinum;
2. extraction process is with embodiment 1;
3. back extraction process is with embodiment 3;
Its experimental result shows that the extraction yield of the relative BSA of CTAB-bromo-octadecane mixed reversed micelle is than the extraction yield height of the relative BSA of single CTAB reverse micelle.Present embodiment, also can adopt chloro-octane, chloroheptane, iodo-octane or iodohexane, preparation CTAB-chloro-octane mixed reversed micelle phase, CTAB-chloroheptane mixed reversed micelle phase, CTAB-iodo-octane mixed reversed micelle phase or CTAB-iodohexane mixed reversed micelle phase, its extraction yield and back extraction ratio to BSA all is higher than extraction yield and the back extraction ratio of single CTAB reverse micelle to BSA.
Embodiment 11:
In the present embodiment, aqueous phase extracted is with embodiment 1;
1. prepare respectively CTAB-(1-bromo-3-methyl) pentane mixed reversed micelle mutually with single CTAB reverse micelle mutually, compound method is with embodiment 1, the CTAB-that obtains (1-bromo-3-methyl) pentane mixed reversed micelle mutually in, the concentration of CTAB be 50 mMs/liter, the volume of n-butanol accounts for 20% of CTAB-(1-bromo-3-methyl) pentane mixed reversed micelle phase volume, the volume of (1-bromo-3-methyl) pentane all accounts for 1.0% of CTAB-(1-bromo-3-methyl) pentane reverse micelle phase volume, the volume of distilled water accounts for 1.2% of CTAB-(1-bromo-3-methyl) pentane mixed reversed micelle phase volume, and all the other are benzinum; The concentration of single CTAB reverse micelle phase CTAB be 50 mMs/liter, the volume of n-butanol accounts for 20% of single CTAB reverse micelle phase volume, the volume of distilled water accounts for 1.2% of single CTAB reverse micelle phase volume, all the other are benzinum;
2. extraction process is with embodiment 1;
Its experimental result shows that the extraction yield of CTAB-(1-bromo-3-methyl) the relative BSA of pentane mixed reversed micelle is than the extraction yield height of the relative BSA of single CTAB reverse micelle.
Claims (8)
1. one kind is added the method that halogenated hydrocarbons improves relative biological substance extraction yield of reverse micelle and back extraction ratio, and its processing step is as follows:
1) preparation by cationic surface active agent, cosurfactant, halogenated hydrocarbons, water and organic solvent form cation-the halogenated hydrocarbons mixed reversed micelle mutually;
Being formulated as follows of described cation-halogenated hydrocarbons mixed reversed micelle phase: at first take by weighing or pipette a certain amount of cationic surface active agent, and to wherein adding cosurfactant, halogenated hydrocarbons, water and organic solvent successively, evenly mix, leave standstill after the vibration, dissolve fully to cationic surface active agent and halogenated hydrocarbons, add the organic solvent constant volume again, promptly make cation-halogenated hydrocarbons mixed reversed micelle phase;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the cosurfactant that is added: 0.50-20;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the halogenated hydrocarbons that is added: 0.5-10;
The cation of being prepared-halogenated hydrocarbons mixed reversed micelle is 100 with the volume ratio of the water that is added: 0.5-3;
Cationic surface active agent the concentration of the cation of being prepared-halogenated hydrocarbons mixed reversed micelle in mutually be 10 mMs-200 mM/liter;
2) with cation-halogenated hydrocarbons mixed reversed micelle and aqueous phase extracted in 1: 1-1: 50 ratio is mixed, and extracts;
3) with cation-halogenated hydrocarbons mixed reversed micelle and strip aqueous in 1: 1-50: 1 ratio is mixed, and carries out back extraction.
2. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 1, it is characterized in that: described halogenated hydrocarbons is chloralkane RCl, bromo alkane RBr, alkane iodide RI.
3. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 2, it is characterized in that: the alkyl among described chloralkane RCl, bromo alkane RBr, the alkane iodide RI is a straight or branched.
4. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 3, it is characterized in that: the alkyl R among described halogenated hydrocarbons chloralkane RCl, bromo alkane RBr, the alkane iodide RI is the alkyl of 4 carbon to 20 carbon atoms.
5. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 2, it is characterized in that: described halogenated hydrocarbons is that the alkyl R among chloralkane RCl, bromo alkane RBr, the alkane iodide RI is the alkyl of 4 carbon to 20 carbon atoms.
6. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 1, it is characterized in that: described cationic surface active agent is trialkyl methyl quaternary ammonium, dialkyl dimethyl quaternary ammonium salt, alkyl trimethyl quaternary ammonium salt.
7. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 1, it is characterized in that: described cosurfactant is the alkyl alcohols.
8. improve the method for relative biological substance extraction yield of reverse micelle and back extraction ratio by the described interpolation halogenated hydrocarbons of claim 1, it is characterized in that: described organic solvent is single liquid alkane or mixes liquid alkane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001327062A CN1153604C (en) | 2000-11-13 | 2000-11-13 | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001327062A CN1153604C (en) | 2000-11-13 | 2000-11-13 | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1353001A CN1353001A (en) | 2002-06-12 |
| CN1153604C true CN1153604C (en) | 2004-06-16 |
Family
ID=4595341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001327062A Expired - Fee Related CN1153604C (en) | 2000-11-13 | 2000-11-13 | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1153604C (en) |
-
2000
- 2000-11-13 CN CNB001327062A patent/CN1153604C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1353001A (en) | 2002-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR940010283B1 (en) | Process for the extraction and purification of proteins from culture media | |
| CN1396176A (en) | Method for purifying drug active protein by cation exchange chromatogrphy | |
| CN105017412A (en) | Method for separating high-purity bovine serum albumin from bovine serum | |
| CN106008705A (en) | Method for separating and purifying phycocyanin by means of combination of two aqueous phase extraction and ultrasonic waves | |
| CN1548420A (en) | Extraction process of astaxanthin in phaffiarhodozyma | |
| CN1153604C (en) | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biological substance by adding halohydrocarbon | |
| CN101619098B (en) | Backward extraction method for extracting rapeseed protein by reverse micelle | |
| CN1153603C (en) | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biologic substance by adding aldone ester | |
| CN103833840A (en) | Method for extracting high-density lipoprotein and separating and purifying apolipoprotein apoA-I from human plasma | |
| CN111676261A (en) | Preparation process of high-purity recombinant interleukin-2 | |
| CN100455592C (en) | Method for extracting glutathione from glutathione fermented broth | |
| CN1132649C (en) | Process for increasing extraction rate and back-extraction rate of anti-coagulation relative biologic substance by adding amine extractants | |
| Zhu et al. | In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems | |
| US5462863A (en) | Isolation of Hepatitis B surface antigen from transformed yeast cells | |
| EP0295859A2 (en) | Production of proteins in active forms | |
| Wu et al. | Improving glutathione extraction from crude yeast extracts by optimizing aqueous two-phase system composition and operation conditions | |
| Dong et al. | Effect of the spacer of gemini surfactants on reverse micellar extraction of bovine serum albumin | |
| CN102464713A (en) | Preparation method of follicle-stimulating hormone | |
| CN1958603A (en) | Method for purifying human chorionic gonadotropin | |
| CN87104013A (en) | The combination medicament of froth flotation zircon | |
| CN102286069B (en) | Method for primarily purifying glutathione fermentation liquid by precipitation and separation with organic solvent | |
| CN1473843A (en) | Industrially extracting, renaturation and purifying method for recombined TRAILinclusion body protein | |
| CN100345609C (en) | Phase formation agent for liquid-liquid-liquid tri-phase system | |
| CN1749267A (en) | Protein separating and purifying method | |
| CN108727487B (en) | Liquid membrane extraction method of hirudin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |