CN105131329B - A kind of preparation method and application of the polyvinyl alcohol crosslinked affinity membrane of macropore chitosan of chelated metal ions - Google Patents
A kind of preparation method and application of the polyvinyl alcohol crosslinked affinity membrane of macropore chitosan of chelated metal ions Download PDFInfo
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- CN105131329B CN105131329B CN201510671237.7A CN201510671237A CN105131329B CN 105131329 B CN105131329 B CN 105131329B CN 201510671237 A CN201510671237 A CN 201510671237A CN 105131329 B CN105131329 B CN 105131329B
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Abstract
The present invention relates to a kind of preparation method and applications of the polyvinyl alcohol crosslinked affinity membrane of macropore chitosan of chelated metal ions.The preparation of film includes：Chitosan, polyvinyl alcohol, silica gel carry out being mixed to prepare chitosan polyvinyl alcohol film；Drilling is handled using sodium hydroxide solution；It is added into epoxychloropropane and sodium hydroxide, obtains the polyvinyl alcohol crosslinked film of macropore chitosan of network structure；It is dipped in into the sodium carbonate liquor containing iminodiacetic acid；The film after drying is immersed into the solution containing bivalent metal ion again, it can obtain the polyvinyl alcohol crosslinked affinity membrane of macropore chitosan of chelated metal ions, it has stronger mechanical strength, and has efficient affine adsorption capacity to the protein containing histidine.It is applied to the separation rich in histidine protein matter or enzyme, it is main prepared including crude enzyme liquid, film to the specific affine absorption rich in histidine protein matter or enzyme, the elution of adhesion protein, film regeneration Four processes.
Description
Technical field
The present invention relates to a kind of preparation method of the macropore chitosan of chelated metal ions-polyvinyl alcohol crosslinked affinity membrane and
Using belonging to the preparation of new material, belong to biochemistry interdisciplinary field.
Background technology
Obtained along with the fast development of the related discipline such as life science and bioengineering, particularly genetic engineering huge
Achievement, people can produce required target product in cell, therefore the requirement that isolates and purifies to biological product is also got over
Come higher.Affinity chromatography is a kind of protein purification important method, and it has very high selectivity and separating property with larger load
Amount.Affinity chromatography group will be made up of affiliation carrier and affinity ligand.Traditional carrier is expensive with aglucon, is not suitable for advising greatly
The production of mould.Chitosan is former to be had substantial amounts of amino and hydroxyl activity functional group and has preferable film forming extensively, is a kind of
Good affine membrane material.
There is the shortcomings that physics, chemical constitution are unstable, and mechanical strength is poor for chitosan affinity membrane at present.It will directly inhale
When the affinity membrane of attached metal ion is used for absorption rich in histidine protein, because metal ion particle is smaller, intensive is attached
On affinity membrane.Protein belongs to macromolecular, with film during Action of Metal Ions there is larger steric hindrance, hinder
Application of the chitosan in affinity chromatography.
This method is prepared for a kind of macropore chitosan-polyvinyl alcohol crosslinked affinity membrane, by adding polyvinyl alcohol so that shell
Glycan occurs physical-chemical reaction with polyvinyl alcohol and forms a kind of high polymer, increases the mechanical strength of affinity membrane, utilizes epoxy chlorine
Propane is as crosslinking agent so that the chitosan polyvinyl alcohol film of chain turns into network structure, adds film physical and chemical stability
Can, the use range of affinity membrane under various conditions is expanded, is not also dissolved in acid condition such as.By connecting on affinity membrane
The different chelating agent and spacerarm such as iminodiacetic acid, ciba blue dyestuff or trihydroxy methyl ethylenediamine are connect, effectively prevent gold
Belong to ion leakage, and reduce the space resistance of metal ion and target protein, increase its adsorption capacity to albumen, and have
Target protein is eluted from affinity membrane beneficial to eluent.
The content of the invention
It is an object of the invention to overcome the defect of prior art, there is provided a kind of macropore shell of chelated metal ions gathers
The preparation method of sugar-polyvinyl alcohol crosslinked affinity membrane, its affinity membrane prepared have stronger mechanical strength, and with showing
The film for having preparation is compared to the protein adsorption ability of histidine mark, be the advantage is that the loss it is avoided that metal ion, is subtracted
Small steric hindrance between metal ion and target protein, increases its adsorbance to target protein and can be effectively mesh
Mark albumen elutes from film, while has played putamina glycan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions to egg
White adsorption capacity, the synergy with the rejection effect of film in itself.The inventive method has a step efficient absorption albumen, operation
Simplicity, repeat and utilize, energy consumption is low, there is potential industrial applications value.
What the present invention was realized in：
The present invention provides a kind of preparation method of the macropore chitosan of chelated metal ions-polyvinyl alcohol crosslinked affinity membrane,
Comprise the following steps：Step 1：The polyvinyl alcohol that chitosan that concentration is 1%-3% is 3%-9% with concentration by volume 1：
(2-3) is mixed, and adds silica gel, and 24-30 hours are reacted in 50-60 DEG C of water-bath, and reaction mixture pours into film mould
Plate, obtain chitosan-polyvinyl alcohol film；Step 2：Chitosan-polyvinyl alcohol film is soaked using sodium hydroxide solution, obtains hole
Footpath is 5-50nm macropore chitosan-polyvinyl alcohol film；Step 3：Macropore chitosan-polyvinyl alcohol film is added to epoxy chlorine
In the mixed solution of propane and sodium hydroxide, at a temperature of 50-60 DEG C, cross-linking reaction is carried out, obtains the macropore shell of network structure
Glycan-polyvinyl alcohol crosslinked film；Step 4：Macropore chitosan-polyvinyl alcohol film of network structure is immersed to containing imino group
In the sodium carbonate liquor of oxalic acid, ciba blue dyestuff or trihydroxy methyl ethylenediamine, 8-12 hours are reacted in 50-60 DEG C of water-bath
Afterwards, rinsed with water, and dried at room temperature；The film after drying is immersed into the solution containing bivalent metal ion again, carried out
Chelated metal ions, you can obtain macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions.
Further, the chitosan is dissolved in aqueous acid, and the polyvinyl alcohol is soluble in water.
Further, the bivalent metal ion is copper ion, nickel ion or cobalt ions.
Further, the concentration of the bivalent metal ion is 0.05-0.1mol/L.
Further, the solution containing bivalent metal ion is CuCl2Solution or NiCl2Solution or CoCl2Solution.
The present invention also provides a kind of application of macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions,
The macropore chitosan of the chelated metal ions-polyvinyl alcohol crosslinked affinity membrane is used for the separation of albumen or enzyme rich in histidine
And purifying.
Further, the macropore chitosan of the chelated metal ions-polyvinyl alcohol crosslinked affinity membrane is to rich in histidine
Albumen or enzyme the step of being separated and being purified it is as follows：Step 1：The preparation of mixed enzyme solution, it is 0.2-1mg/ml's to take concentration
Crude protein mixed liquor or crude enzyme liquid rich in histidine protein matter；Step 2：Protein adsorption, above-mentioned mixed enzyme solution 5mL is taken, respectively
Macropore chitosan-polyvinyl alcohol crosslinked affinity membrane 0.1g of chelated metal ions is added, adjusts pH 4-8 and ion concentration 0.1-
0.5mol/L, in 4-25 DEG C of temperature to adsorbing 1-5 hours rich in histidine protein or enzyme spcificity；Step 3：Target protein or
Enzyme elutes, and takes the affinity membrane of adsorbed target albumen in step 2, is that 2-10ml concentration is 0.05-0.1mol/L imidazoles using volume
Affinity membrane is eluted, is separated protein macromolecule with imidazoles small-molecule substance using dialysis process, obtains the mesh rich in histidine
Mark albumen or enzyme；Step book four：The renaturation of film, by the affinity membrane in step 3 in the EDTA that 5ml concentration is 0.1-0.2mol/L
Solution, temperature are 25-30 DEG C, soak 2-3h, with deionized water rinsing 2-10 time, drying at room temperature, and chelated metal ions again, suction
Attached target protein, operation is repeated a number of times, realizes recycling for film.
The invention has the advantages that：
The preparation method of the macropore chitosan of chelated metal ions provided by the invention-polyvinyl alcohol crosslinked affinity membrane, lead to
Crossing the affinity membrane that above-mentioned steps prepare has stronger mechanical strength, and has efficient parent to the protein containing histidine
And adsorption capacity.Compared with the film of existing preparation is to the protein adsorption ability of histidine mark, it the advantage is that it is avoided that gold
Belong to the loss of ion, reduce the steric hindrance between metal ion and target protein, increase its absorption to target protein
Measure and effectively target protein can be eluted from film, while played putamina glycan-polyethylene of chelated metal ions
Alcohol and cross linking affinity membrane is to the adsorption capacity of albumen, the synergy with the rejection effect of film in itself.The inventive method has a step
Efficient absorption albumen, it is easy to operate, repeat and utilize, energy consumption is low, there is potential industrial applications value.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only part of the embodiment of the present invention, rather than whole embodiments.It is common based on the embodiment in the present invention, this area
All other embodiment that technical staff is obtained on the premise of creative work is not made, belong to what the present invention protected
Scope.
The embodiment of the present invention provides a kind of preparation of the macropore chitosan of chelated metal ions-polyvinyl alcohol crosslinked affinity membrane
Method, comprise the following steps：
Step 1：The polyvinyl alcohol that chitosan that concentration is 1%-3% is 3%-9% with concentration by volume 1：(2-3)
Mixed, and add 200-300 mesh silica gel 2.5%-7.5% simultaneously, be well mixed, 24-30 is reacted in 50-60 DEG C of water-bath
Hour, reaction mixture pours into film template, obtains chitosan-polyvinyl alcohol film.Wherein, described concentration is 1%-3%'s
Chitosan is dissolved in 2-5% acetic acid aqueous solution, and described concentration is that 3%-9% polyvinyl alcohol is soluble in water.At this preferably
In embodiment, chitosan and polyvinyl alcohol by volume 1：2 to 3：8 any proportion is mixed.
Step 2：Chitosan-polyvinyl alcohol film is soaked using 1-3mol/L sodium hydroxide solution, soak time 1-
3 hours, macropore chitosan-polyvinyl alcohol film that aperture is 5-50nm is obtained, the permeability rate of film is 1-50ml (cm2·min)。
Step 3：Macropore chitosan-polyvinyl alcohol film is added into the mixed solution of epoxychloropropane and sodium hydroxide,
At a temperature of 50-60 DEG C, cross-linking reaction 2-3 hours are carried out, obtain macropore chitosan-polyvinyl alcohol crosslinked film of network structure；
Wherein, the content of epoxychloropropane is 10%-20%.By infrared spectrum understand chitosan-polyvinyl alcohol film in may contain-
O-H associations ,-C-H ,-C-O-, R-NO2With the functional group such as-C-N- ,-O-H- freely forms to-O-H after being crosslinked by epoxychloropropane
Transformation is closed ,-c h bond diminishes R-NO2,-C-N keys die down.So that single chitosan-polyvinyl alcohol turns into network structure, stability
It can increase so that its mechanical strength is higher.
Step 4：Macropore chitosan-polyvinyl alcohol film of network structure is immersed to containing iminodiacetic acid, ciba blue
In the sodium carbonate liquor of dyestuff or trihydroxy methyl ethylenediamine, after reacting 8-12 hours in 50-60 DEG C of water-bath, rinsed with water, and
Dry at room temperature；The film after drying is immersed into the solution containing bivalent metal ion again, continues 2-3 hours, carries out chela
Metal ion, you can obtain macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions.The divalent metal from
Son is copper ion, nickel ion or cobalt ions, and the concentration of the bivalent metal ion is 0.05-0.1mol/L, is preferably implemented at this
In example, the solution containing bivalent metal ion is CuCl2Solution or NiCl2Solution or CoCl2Solution.
Further, in step 4, the concentration of sodium carbonate is 1-3mol/L, iminodiacetic acid and ciba blue dyestuff
Concentration is 0.1-1mol/L；Or the concentration of sodium carbonate is 1-3mol/L, the content of trihydroxy methyl ethylenediamine is 5%-10%.
Macropore chitosan-polyvinyl alcohol crosslinked affinity membrane connection iminodiacetic acid, chelates copper ion, purifies histidine mark
The schematic construction of the protein of note is as follows：
The specific experimental group of the present invention is as follows：
The present invention also provides a kind of application of macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions,
The present invention preparation method prepare chelated metal ions macropore chitosan-polyvinyl alcohol crosslinked affinity membrane can be used for separation and
Protein or enzyme of the purifying rich in histidine, its main process are：
(1) preparation of protein solution or crude enzyme liquid
Take the crude protein mixed liquor or crude enzyme liquid (such as concentration 0.2- rich in histidine protein matter that concentration is 0.2-1mg/ml
The Recombinant SHMT (SHMT) that 1mg/ml bovine serum albumin(BSA) (BSA) or concentration is 0.5-1.3mg/ml is thick
Ball is immunized in enzyme liquid or restructuring pfu crude enzyme liquids or 0.2-1.5mol/L hydantoin enzymes or 0.2-1mol/L that concentration is 0.4-0.8mg/ml
Albumen or 0.2-1mol/L recombinant penicillin acylases, are not limited to listed crude enzyme liquid).
BSA contains on surface 17 histidine residues, and copper ion can be with the histidine residues phase interaction exposed to BSA surfaces
With copper ion has 6 coordination sites, wherein 3 points are combined with IDA, 3 points can be combined with BSA in addition.Recombinate SHMT, restructuring
The thick enzymes of pfu, recombinant penicillin acylase contain histidine mark.Lysozyme is made up of 18 kinds of 129 amino acid residues
Single peptide chain.It is rich in basic amino acid, has 4 pairs of disulfide bond to maintain enzyme configuration, is a kind of alkaline protein, and its N-terminal is to rely ammonia
Acid, C-terminal are leucine.Hydantoin enzyme is a kind of metal-dependent enzymes, and activated centre is mainly in combination with bivalent cation ion, for activity
Institute is necessary；Immunoglobulin is immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immune ball
Protein D (IgD) and immunoglobulin E (IgE).
(2) protein adsorption
Each 5ml of mixed enzyme solution in (1) is taken, is separately added into the macropore chitosan of chelated metal ions-polyvinyl alcohol crosslinked
Affinity membrane 0.1-1g, pH=4-8 is adjusted, temperature is 4-25 DEG C, ion concentration 0.1-0.5mol/L, adsorbs 1-5 hours.
(3) albumen wash-out
The affinity membrane of adsorbed target albumen in (2) is taken, is 2-10ml using volume, concentration is 0.05-0.1mol/L imidazoles
Affinity membrane is eluted, is separated protein macromolecule with imidazoles small-molecule substance using dialysis process, obtains the mesh rich in histidine
Mark albumen or enzyme；
(4) renaturation of film
By the affinity membrane in (3) in the EDTA solution that 5ml concentration is 0.1-0.2mol/L, temperature is 25-30 DEG C, soaks 2-
3h.With deionized water rinsing 2-10 times, drying at room temperature, chelated metal ions, adsorbed target albumen, are repeated a number of times behaviour again
Make, realize recycling for film.
Specific embodiment is as follows：
The result of above-mentioned experiment shows that macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of preparation connects iminodiacetic acid (salt)
Sour (IDA), ciba blue dyestuff (Cibacron blue 3GA), trihydroxy methyl ethylenediamine (TED) can effectively chelated mineral from
Son, avoid the loss of metal ion.There is larger adsorbance to the albumen of histidine mark, have in the separation of protein
Larger application prospect.
In summary, the system of the macropore chitosan of chelated metal ions provided by the invention-polyvinyl alcohol crosslinked affinity membrane
Preparation Method, the affinity membrane prepared by above-mentioned steps have a stronger mechanical strength, and with the film of existing preparation to group
The protein adsorption ability of His tag is compared, and the advantage is that the loss it is avoided that metal ion, reduce metal ion with
Steric hindrance between target protein, increase its adsorbance to target protein and effectively target protein can be washed from film
Take off, while played affinity membrane to the adsorption capacity of albumen and the synergy of the rejection effect of film in itself.Present invention side
Method has a step efficient absorption albumen, easy to operate, repeats and utilizes, and energy consumption is low, has potential industrial applications value etc. excellent
Point.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (7)
- A kind of 1. preparation method of the macropore chitosan of chelated metal ions-polyvinyl alcohol crosslinked affinity membrane, it is characterised in that bag Include following steps：Step 1：The polyvinyl alcohol that chitosan that concentration is 1%-3% is 3%-9% with concentration by volume 1：（2-3）Mixed, And silica gel is added, 24-30 hours are reacted in 50-60 DEG C of water-bath, reaction mixture pours into film template, obtains chitosan-poly- Vinyl alcohol film；Step 2：Chitosan-polyvinyl alcohol film is soaked using sodium hydroxide solution, the macropore shell that aperture is 5-50nm is obtained and gathers Sugar-polyvinyl alcohol film；Step 3：Macropore chitosan-polyvinyl alcohol film is added into the mixed solution of epoxychloropropane and sodium hydroxide, in At a temperature of 50-60 DEG C, cross-linking reaction is carried out, obtains macropore chitosan-polyvinyl alcohol crosslinked film of network structure；Step 4：Macropore chitosan-polyvinyl alcohol film of network structure is immersed to containing iminodiacetic acid, ciba blue dyestuff Or in the sodium carbonate liquor of trihydroxy methyl ethylenediamine, after reacting 8-12 hours in 50-60 DEG C of water-bath, rinsed with water, and in room Dried under temperature；The film after drying is immersed into the solution containing bivalent metal ion again, carries out chelated metal ions, you can To macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions.
- 2. the preparation method of the macropore chitosan of chelated metal ions as claimed in claim 1-polyvinyl alcohol crosslinked affinity membrane, It is characterized in that：The chitosan is dissolved in acetic acid aqueous solution, and the polyvinyl alcohol is soluble in water.
- 3. the preparation method of the macropore chitosan of chelated metal ions as claimed in claim 1-polyvinyl alcohol crosslinked affinity membrane, It is characterized in that：The bivalent metal ion is copper ion, nickel ion or cobalt ions.
- 4. the preparation side of the macropore chitosan of the chelated metal ions as described in claim 1 or 3-polyvinyl alcohol crosslinked affinity membrane Method, it is characterised in that：The concentration of the bivalent metal ion is 0.05-0.1 mol/L.
- 5. the preparation side of the macropore chitosan of the chelated metal ions as described in claim 1 or 3-polyvinyl alcohol crosslinked affinity membrane Method, it is characterised in that：The solution containing bivalent metal ion is CuCl2Solution or NiCl2Solution or CoCl2Solution.
- 6. a kind of application of macropore chitosan-polyvinyl alcohol crosslinked affinity membrane of chelated metal ions as claimed in claim 1, It is characterized in that：The macropore chitosan of the chelated metal ions-polyvinyl alcohol crosslinked affinity membrane is used for the egg rich in histidine White or enzyme separation and purifying.
- 7. the application of the macropore chitosan of chelated metal ions as claimed in claim 6-polyvinyl alcohol crosslinked affinity membrane, it is special Sign is：The macropore chitosan of the chelated metal ions-polyvinyl alcohol crosslinked affinity membrane is to albumen or enzyme rich in histidine The step of being separated and being purified is as follows：Step 1：The preparation of mixed enzyme solution, take the crude protein mixed liquor rich in histidine protein matter that concentration is 0.2-1mg/ml Or crude enzyme liquid；Step 2：Protein adsorption, above-mentioned mixed enzyme solution 5mL is taken, be separately added into macropore chitosan-polyethylene of chelated metal ions Alcohol and cross linking affinity membrane 0.1g, pH 4-8 and ion concentration 0.1-0.5mol/L are adjusted, in 4-25 DEG C of temperature to rich in histidine egg White or enzyme spcificity absorption 1-5 hours；Step 3：Target protein or enzyme elution, the affinity membrane of adsorbed target albumen in step 2 is taken, be that 2-10ml is dense using volume Spend and elute affinity membrane for 0.05-0.1mol/L imidazoles, divided protein macromolecule and imidazoles small-molecule substance using dialysis process From obtaining the target protein or enzyme rich in histidine；Step 4：The renaturation of film, by the affinity membrane in step 3 in the EDTA solution that 5ml concentration is 0.1-0.2mol/L, temperature For 25-30 DEG C, 2-3h is soaked, with deionized water rinsing 2-10 times, drying at room temperature, chelated metal ions again, adsorbed target egg In vain, operation is repeated a number of times, realizes recycling for film.
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| CN107376872B (en) * | 2017-08-06 | 2020-01-21 | 桂林理工大学 | Preparation method of chitosan-soybean protein composite porous microspheres for lead adsorption |
| CN108855005A (en) * | 2018-06-29 | 2018-11-23 | 成都纺织高等专科学校 | A kind of ternary blends and preparation method thereof for adsorbing coloring agent |
| CN108728269B (en) * | 2018-08-03 | 2020-12-18 | 冠道（广州）科技有限公司 | Ultra-strong cleaning nano coral calcium powder cleaning agent and preparation method thereof |
| CN109400816B (en) * | 2018-08-24 | 2020-10-30 | 哈尔滨工业大学 | Ultraviolet-curable color-changing gel resin and synthesis method thereof, and color changing and fading method of gel |
| CN111408354B (en) * | 2020-03-30 | 2022-11-25 | 杭州师范大学钱江学院 | Solid phase extracting agent based on bio-fiber mask leftover material and preparation method thereof |
| CN112604506B (en) * | 2020-10-22 | 2022-11-25 | 山东东岳高分子材料有限公司 | Preparation method of polytetrafluoroethylene membrane for catalytic dye decolorization and application method of polytetrafluoroethylene membrane |
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