CN101564656A - Preparation of nanometer composite affinity membrane used for rapidly and efficiently separating and purifying thiol protease - Google Patents
Preparation of nanometer composite affinity membrane used for rapidly and efficiently separating and purifying thiol protease Download PDFInfo
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- CN101564656A CN101564656A CNA2009100524330A CN200910052433A CN101564656A CN 101564656 A CN101564656 A CN 101564656A CN A2009100524330 A CNA2009100524330 A CN A2009100524330A CN 200910052433 A CN200910052433 A CN 200910052433A CN 101564656 A CN101564656 A CN 101564656A
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Abstract
The invention relates to a preparation of a nanometer composite affinity membrane used for rapidly and efficiently separating and purifying thiol protease, which comprises the steps of: (1) using a mixed solution of hexafluoroisopropanol (HFIP) and formic acid as the solvent system and doping the mixed solution in a reactor; (2) adding polyamide 6 and chitosan powder in the reactor; (3) putting the reactor in a water bath oscillator to obtain polyamide 6/chitosan spinning solution; (4) using the spinning solution to perform the electrospinning to prepare a nanometer fiber membrane and then drying the nanometer fiber membrane; (5) cutting the nanometer fiber membrane into circular membranes and then activating the circular membranes; (6) putting the circular membranes into dye solution for reaction after a plurality of times of washing; (7) adding NaCl solution for treatment; (8) adding Na2CO3 for fixation reaction; and (9) cooling and washing. The preparation of the nanometer composite affinity membrane used for rapidly and efficiently separating and purifying thiol protease has the advantages of simple operation, short time and large adsorbing capacity; the prepared nanometer affinity membrane is rapid, simple, cheap and efficient, can efficiently purify the thiol protease and is applicable to scale production.
Description
Technical field
The invention belongs to the preparation field of nanometer composite affinity membrane, particularly relate to the preparation of the nanometer composite affinity membrane that is used for rapidly and efficiently separating and purifying thiol protease.
Background technology
In recent years, growing along with life science, biotechnology, pharmacy and medical skill, people are also more and more higher to the separation of large biological molecule, extraction and refining requirement.Affinity chromatography is a kind of separation and purification of biological macromolecule technology commonly used in laboratory and the industry.It is that promptly specific reversible combination makes large biological molecule be separated with dissociating according to the affinity between large biological molecule and the specific immobilized aglucon.It has characteristics such as operating condition gentleness, pollution-free, no phase transformation, but film separation process equipment simple, be easy to amplify, low, the fast continued operation of separating rate of cost, all obtained application in many aspects.
Shitosan (CS) is a natural polymer, contains extremely strong reactive group amino and hydroxyl in the molecule, has advantages such as excellent solvent-resistance and alkali resistance.It is compound on the perforated membrane of various materials, can makes the composite hyperfiltration membrane in different apertures.At present, chitosan polymer is used to prepare infiltration evaporation (or being pervaporation) composite membrane mostly, and carries out blending with shitosan and synthetic high polymer nylon, makes ultrafiltration composite membrane and does not appear in the newspapers as yet.
Summary of the invention
Technical problem to be solved by this invention provides the preparation of the nanometer composite affinity membrane that is used for rapidly and efficiently separating and purifying thiol protease, and this method is simple to operate, and consuming time less, adsorbance is big; The nanometer affinity membrane of preparing is quick, easy, cheap, efficient, but the efficiently purifying thiol protease is applicable to large-scale production.
The preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease of the present invention comprises:
(1) with volume ratio be 9～12: 1 hexafluoroisopropanol (1,1,1,3,3,3-hexafluoroisopropanol) and the formic acid mixed liquor as dicyandiamide solution, blending is gone in the reaction vessel, temperature is 40～60 ℃, water-bath oscillating reactions 20～45min;
(2) nylon 6 and shitosan powder are slowly added in the above-mentioned reaction vessel under stirring condition, continue to stir 2～3h to complete swelling, wherein the mass concentration of nylon 6 particles is 5～8wt%, and chitosan mass concentration is 0～2wt%;
(3) above-mentioned reaction vessel is placed in the water bath chader, under reflux condensation mode, slowly be heated to 40～60 ℃, vibration 15h～30h gets nylon 6/ shitosan spinning solution to dissolving fully;
(4) extract nylon 6/ shitosan spinning solution with syringe, be fixed on the electrostatic spinning apparatus, regulate spinning parameter and carry out electrospinning, electrostatic pressure is 10～18kv; Receiving screen adopts the reception of aluminium foil ground connection, and the distance of syringe needle and receiving screen is 10～20cm, gets nylon 6/ shitosan superfine nano tunica fibrosa;
(5) film of collecting is utilized baking oven, vacuum desiccator drying successively;
(6) dried nylon 6/ chitosan nano fiber membrane is cut into the circular film that diameter is 47mm, puts into 50～60 ℃, concentration is to activate 20-30min in the NaOH solution of 10～15wt%;
(7) nylon 6/ chitosan nano fiber membrane after the activation behind the hot water wash several, is put in the dye solution and is reacted;
(8) add the NaCl solution-treated, dyestuff is adsorbed onto on nylon 6/ chitosan nano fiber membrane;
(9) regulate bath temperature, add Na
2CO
3Carry out the fixation reaction;
(10) cooling after fully washing with hot water, methyl alcohol, NaCl, urea, distilled water successively, or is stored in the Tris-HCl buffer solution, and obtaining with the dyestuff is the nylon 6/ chitosan nano fiber affinity membrane of aglucon.
The mass concentration of described step (1) hexafluoroisopropanol (HIFP) is 99%～100%, and water content is less than 0.1%.
Syringe specification in the described step (4) is 5ml, and the syringe needle internal diameter is about 0.4～0.7mm; The ejection flow velocity is 0.5～2ml/h.
Serial dried drying method in the described step (5) is earlier with dry 12h under the spun film room temperature, puts into 90 ℃ of drying 4～6h of baking oven then, puts into 60～70 ℃ of dry 24h of vacuum desiccator at last.
Described step (6) dyestuff is dyestuff Cibacron Blue F3GA, and concentration is 5～15mg/ml.
Described step (7) NaCl solution-treated is that NaCl solution with 20～30wt% is at 40～80 ℃ of water-bath oscillation treatment 20～45min.
Described step (8) Na
2CO
3The fixation reaction is with 25～30wt%Na
2CO
3At 60～90 ℃ of water-bath oscillating reactions 3～6h.
Washing in the described step (9) is to be 99.5～100% methyl alcohol, 2M NaCl, the washing of 6M urea with 40 ℃ of-50 ℃ of hot water, content, the washing degree be the cleaning solution after washing be colourless.
Prepared affinity membrane is applied to the separation and purification of thiol protease and uses.
This method is main spinning material with nylon 6, and mix be rich in hydrophily functional group-OH ,-NH
2Shitosan, by adjusting solvent and relevant spinning condition parameter, successful realization blending; Utilizing the nano composite material that is obtained to be raw material, is aglucon with the dyestuff, and the nanometer affinity membrane of preparing is quick, easy, cheap, efficient, but the efficiently purifying thiol protease is applicable to large-scale production.
Method of electrostatic spinning is a kind of important method for preparing superfine fibre, it has significantly different with traditional method, as tractive force, with several thousand to several ten thousand volt high-pressure electrostatics on polymer solution or the melt band, charged polymer liquid drops under the effect of electric field force and is stretched by electrostatic force.When electric field force was enough big, the polymer drop can overcome surface tension and form the injection thread, and thread is solvent evaporation or curing in course of injection, finally drops on the receiving system, has formed the fibrofelt of similar nonwoven shape.The fiber that makes with method of electrostatic spinning is much thinner than conventional spinning method, and diameter generally arrives hundreds of nanometers at tens nanometer, and minimum diameter can be to 1nm.
Electrospinning process can be the different polymer of kind more than 50, for example polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, polyaniline, polyacrylonitrile etc. be spun into diameter range from less than several nanometers to the superfine fibre that surpasses 1 μ m, resulting electrospun fibers is collected in and is deposited as non-weaving cloth on the negative plate.The nylon 6/nanometer fiber of electrostatic spinning preparation can prepare high performance polyamide fibre nanofiber on the one hand, improves the wearability of nylon fiber; The fiber that blending in the natural polymers blending of some biologically active such as the nylon polymer is become can have both advantages concurrently on the other hand, the toughness of existing nylon, wearability, given composite membrane new biological nature again, extensive application aspect a lot.
The membrane matrix material has important effect in the preparation of affinity membrane, because it is not only determining the spacerarm and the aglucon kind of institute's bonding, but also directly determining the effect of separating.Cellulose is the maximum natural polymer of occurring in nature content, it also is one of medium that is used for the earliest affine separation, because cellulosic molecule contains a large amount of-OH, thereby give cellulose good hydrophilicity, biocompatibility and reactable, and cellulose membrane is cheap, cost is low when being used for affine separation, is fit to industrial applications.In recent years, cellulose is applied to more and more cause in biochemical the separation people's extensive attention.
Beneficial effect
(1) method of the present invention is simple to operate, and consuming time less, adsorbance is big;
(2) raw material used in the present invention are cheap and easy to get, convenient sources, but itself contain abundant response function group, need not modification and handle, time saving and energy saving, reduced preparation cost; Be convenient to extract on a large scale purifying;
(3) it is quick, easy, cheap, efficient that we invent the nanometer affinity membrane of preparing, but the efficiently purifying thiol protease is applicable to large-scale production.
Description of drawings
Fig. 1 is that nylon 6/ chitosan nano fiber of electrostatic spinning preparation and the affinity membrane stereoscan photograph that obtains through processing: A are nylon 6/ chitosan nano fiber without any processing; B is an affinity membrane;
Fig. 2 is the influence curve of the variation of reaction density to papain absorption;
Fig. 3 is the dynamic absorption test curve to papain.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Be used for the preparation of the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease, comprise:
(1) with volume ratio is 9～12: 1 hexafluoroisopropanol (1,1,1,3,3,3-hexafluoroisopropanol) and the formic acid mixed liquor as dicyandiamide solution, blending is gone in the reaction vessel, temperature is 40 ℃, water-bath oscillating reactions 20～45min, the wherein mass concentration of hexafluoroisopropanol＞99%;
(2) nylon 6 and shitosan powder are slowly added in the above-mentioned reaction vessel under stirring condition, continue to stir 2～3h to complete swelling, wherein the mass concentration of nylon 6 is 5wt%, and chitosan mass concentration is 2wt%;
(3) above-mentioned reaction vessel is placed in the water bath chader, under reflux condensation mode, slowly be heated to 40 ℃, vibration 15h～30h gets nylon 6/ shitosan spinning solution to dissolving fully;
(4) extract nylon 6/ shitosan spinning solution with syringe, be fixed on the electrostatic spinning apparatus, regulate spinning parameter and carry out electrospinning, electrostatic pressure is 10～18kv; Receiving screen adopts the reception of aluminium foil ground connection, and the distance of syringe needle and receiving screen is 10～20cm, gets nylon 6/ shitosan superfine nano tunica fibrosa;
(5) earlier with dry 12h under the spun film room temperature, put into 90 ℃ of drying 4～6h of baking oven then, put into 60～70 ℃ of dry 24h of vacuum desiccator at last;
(6) dried nylon 6/ chitosan nano fiber membrane is cut into the circular film that diameter is 47mm, puts into 50～60 ℃, concentration is to activate 20-30min in 10～15% the NaOH solution;
(7) nylon 6/ chitosan nano fiber membrane after the activation, with hot water wash for several times after, put into concentration and be in the dyestuff Cibacron Blue F3GA solution of 5～15mg/ml and react;
(8) with the NaCl solution of 20～30wt% at 40 ℃ of water-bath oscillation treatment 20～45min, dyestuff is adsorbed onto on nylon 6/ chitosan nano fiber membrane;
(9) regulate bath temperature, use 25wt%Na
2CO
3At 90 ℃ of water-bath oscillating reactions 3～6h;
(10) cooling, successively with the methyl alcohol, 2M NaCl, the washing of 6M urea that with 50 ℃ of hot water, content are 99.5～100%, after distilled water fully washs, the washing degree be the cleaning solution after washing be colourless or be stored in the Tris-HCl buffer solution that obtaining with dyestuff Cibacron Blue F3GA is the nylon 6/ chitosan nano fiber affinity membrane of aglucon.
Embodiment 2
Take by weighing 6 groups of each 0.1g cellulose affinity membranes, the buffer solution of adding and concentration are respectively 0,0.5,1.0,1.5, in the enzyme liquid of 2.0mg/ml, and 37 ℃ of water-baths vibration 3h measure before and after the absorption absorbance at the 280nm place; Calculate the concentration of that papain of adsorbance maximum, as following experiment.Use under the same terms without the cellulose membrane of any processing and do parallel contrast experiment, measure the non-specific adsorption amount, contrast with the affinity membrane adsorbance.Find that the suitableeest is 2.0mg/ml to the enzyme amount.Fig. 2 is the adsorption curve of nylon 6/ chitosan nano fiber of affinity membrane and unprocessed mistake to the variable concentrations papain.
Embodiment 3
20 affinity membranes are built up membrane stack, the film bridge of packing into.At room temperature, at first with buffer solution balance membrane stack, the papaya powder solution that will be made into buffer solution is with sample on the suitable flow velocity, then with buffer solution flushing membrane stack, to remove the albumen that is not adsorbed on the striping, until the absorbance A that flows out liquid then
280Value is near 0, and is last, with the papain that suitable eluent wash-out is adsorbed, is in charge of the collection component, and every pipe component volume is 4ml, measures the absorbance A of each component
280Value is with A
280Be ordinate, volume is an abscissa, obtains the absorb-elute curve of papain.Fig. 3 is the dynamic absorption test curve to papain.
Claims (8)
1. be used for the preparation of the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease, comprise:
(1) with volume ratio be 9～12: 1 hexafluoroisopropanol HFIP and formic acid mixed liquor as dicyandiamide solution, blending is gone in the reaction vessel, temperature is 40～60 ℃, water-bath oscillating reactions 20～45min;
(2) nylon 6 and shitosan powder are slowly added in the above-mentioned reaction vessel under stirring condition, continue to stir 2～3h to complete swelling, wherein nylon 6 granular mass concentration are 5～8wt%, and the mass concentration of shitosan is 0～2wt%;
(3) above-mentioned reaction vessel is placed in the water bath chader, under reflux condensation mode, slowly be heated to 40～60 ℃, vibration 15h～30h gets nylon 6/ shitosan spinning solution to dissolving fully;
(4) extract nylon 6/ shitosan spinning solution with syringe, be fixed on the electrostatic spinning apparatus, regulate spinning parameter and carry out electrospinning, electrostatic pressure is 10～18kv; Receiving screen adopts the reception of aluminium foil ground connection, and the distance of syringe needle and receiving screen is 10～20cm, gets nylon 6/ shitosan superfine nano tunica fibrosa, and is dry then;
(5) dried nylon 6/ chitosan nano fiber membrane is cut into circular film, puts into 50～60 ℃, concentration is to activate 20-30min in the NaOH solution of 10～15wt%;
(6), behind the hot water wash several, put in the dye solution and react with above-mentioned tunica fibrosa;
(7) with the NaCl solution of 20～30wt% at 40 ℃～80 ℃ water-bath oscillation treatment 20～45min, dyestuff is adsorbed onto on nylon 6/ chitosan nano fiber membrane;
(8) regulate bath temperature, with 25～30wt%Na
2CO
3At 60 ℃～90 ℃ water-bath oscillating reactions 3～6h;
(9) cooling, washing, or be stored in the Tris-HCl buffer solution, obtaining with the dyestuff is the nylon 6/ chitosan nano fiber affinity membrane of aglucon.
2. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the mass concentration of described step (1) hexafluoroisopropanol HIFP is 99%～100%, and water content is less than 0.1%.
3. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the syringe specification in the described step (4) is 5ml, and the syringe needle internal diameter is about 0.4～0.7mm; The ejection flow velocity is 0.5～2ml/h.
4. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1, it is characterized in that: the drying means in the described step (4) is earlier with dry 12h under the spun film room temperature, put into 90 ℃ of drying 4～6h of baking oven then, put into 60～70 ℃ of dry 24h of vacuum desiccator at last.
5. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the diameter of described step (5) circular film is 47mm.
6. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: described step (6) dyestuff is dyestuff Cibacron Blue F3GA, and concentration is 5～15mg/ml.
7. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1, it is characterized in that: the washing in the described step (9) is to be 99.5～100% methyl alcohol, 2～3M NaCl, the washing of 3～6M urea with 40～50 ℃ of hot water, content, the washing degree be the cleaning solution after washing be colourless.
8. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: described nanometer composite affinity membrane is applied to the separation and purification of thiol protease.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107530639A (en) * | 2015-04-17 | 2018-01-02 | Emd密理博公司 | The method for using target biomaterial in the nanofibre hyperfiltration membrane purification of samples operated with tangential flow filtration mode |
| CN112962216A (en) * | 2021-02-07 | 2021-06-15 | 宁波工程学院 | Preparation method of nylon 6/chitosan/precious metal nano-fiber |
| US11154821B2 (en) | 2011-04-01 | 2021-10-26 | Emd Millipore Corporation | Nanofiber containing composite membrane structures |
| US12059644B2 (en) | 2014-06-26 | 2024-08-13 | Emd Millipore Corporation | Filter structure with enhanced dirt holding capacity |
-
2009
- 2009-06-03 CN CNA2009100524330A patent/CN101564656A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11154821B2 (en) | 2011-04-01 | 2021-10-26 | Emd Millipore Corporation | Nanofiber containing composite membrane structures |
| US12059644B2 (en) | 2014-06-26 | 2024-08-13 | Emd Millipore Corporation | Filter structure with enhanced dirt holding capacity |
| CN107530639A (en) * | 2015-04-17 | 2018-01-02 | Emd密理博公司 | The method for using target biomaterial in the nanofibre hyperfiltration membrane purification of samples operated with tangential flow filtration mode |
| US10675588B2 (en) | 2015-04-17 | 2020-06-09 | Emd Millipore Corporation | Method of purifying a biological material of interest in a sample using nanofiber ultrafiltration membranes operated in tangential flow filtration mode |
| CN107530639B (en) * | 2015-04-17 | 2021-02-09 | Emd密理博公司 | Method for purifying target biological material in sample using nanofiber ultrafiltration membrane operating in tangential flow filtration mode |
| CN112962216A (en) * | 2021-02-07 | 2021-06-15 | 宁波工程学院 | Preparation method of nylon 6/chitosan/precious metal nano-fiber |
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Open date: 20091028 |