CN1296032A - Polyacrylonitrile magnetic pearl and preparation process and application thereof - Google Patents
Polyacrylonitrile magnetic pearl and preparation process and application thereof Download PDFInfo
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- CN1296032A CN1296032A CN 00127460 CN00127460A CN1296032A CN 1296032 A CN1296032 A CN 1296032A CN 00127460 CN00127460 CN 00127460 CN 00127460 A CN00127460 A CN 00127460A CN 1296032 A CN1296032 A CN 1296032A
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- polyacrylonitrile
- magnetic
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- pearl
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Abstract
The persent invention adots polyacrylonitrile and water-soluble magnetic fluid a smain raw materials, and provides its preparation method which includes the following steps: firstly, dispersing the water-soluble magnetic fluid in N.N-dimethyl formamide (DMF) solution of polyacrylonitrile, then spraying and pelleting to obtain polyacrylonitrile magnetic beads, and then cross-linking the enzyme on the surface of hydrogenated polyacrylonitrile magnetic bead, and after the catalytic reaction is completed, utilizing magnetic field for recovery so as to bring the immobilization predominance into play. Said invention magnetic bead possesses excellent performance, its immobilized quantity is high, immobilization process is simple, and it is convenient for separation and recovery.
Description
The present invention relates to a kind of magnetic bio material and preparation method thereof and, relate in particular to a kind of polyacrylonitrile magnetic pearl and its production and application as the application of fixed enzyme vector.
Immobilized enzyme is that water miscible enzyme is handled with physics or chemical process, makes it to become the water-fast enzyme derivative that still has enzymic activity.Immobilized enzyme not only should have the catalytic characteristics of high-level efficiency under the height specificity of enzyme and the mild conditions, also should have certain mechanical strength, makes production serialization, automatization, does not bring impurity into, and product is refining easily, and yield is higher.Simultaneously, immobilized enzyme also should be able to use repeatedly, and has advantages of higher stability.Along with developing rapidly of biotechnology, the application of immobilized enzyme in industrial production is increasingly extensive, plays crucial effect in food, pharmaceutical manufacturing.What special use chemical technology was difficult to carry out is grasping, and just is easier to solve with immobilized enzyme.
Can be a lot of as the material of fixed enzyme vector, as macroporous resin, chitosan, chitin polysaccharide etc.But find that in production reality their unit mass carrier fixed enzyme amounts are very limited.(CN), can be hydrogenated is amino (NH to polyacrylonitrile because acrylonitrile monemer all contains an itrile group
3), realize the immobilization of enzyme easily.Yet, document (Japanese published unexamined patent applications No.121592/1976 and No.7485/1979, Auslegeschrift No.122633) report, even polyacrylonitrile is after its hydrogenation, the ability of its adsorptive enzyme is still lower, and absorption is very unstable, and enzyme is easy to discharge from carrier surface.
In order to overcome above-mentioned difficulties, United States Patent (USP) U.S.P.4486549 adopts the polyacrylonitrile of the poroid fibrous texture of hydrogenant as enzyme carrier, and experiment is found, had amino (NH
3) poroid polyacrylonitrile fibre the adsorptive capacity of enzyme and binding ability are all improved greatly, can reach 2 to 30 times.Yet, this method, recovery that an outstanding problem that faces is an immobilization material and recycling be difficulty relatively.
The magnetic bead that polymer embedded nano magnetic particle forms can the various functional moleculars of grafting to its surface modification, as enzyme, antibody, DNA etc.Utilize externally-applied magnetic field to reclaim and to reuse.But used polymer magnetic bead mostly is the vinyl aromatic polymers microballon at present, the report of the typical visible USP4358388 of preparation method.Yet adopt this class methods, be difficult to make polyacrylonitrile magnetic pearl.Because magnetic nanoparticle is difficult to be dispersed in the vinyl cyanide, and can not the polymerization balling-up.
One of purpose of the present invention is to solve the above-mentioned defective that exists in the prior art, discloses a kind of enzyme adsorptive capacity height, the polyacrylonitrile magnetic pearl that separation cycle is short, carrier recovery efficient is high;
Two of purpose of the present invention is to provide above-mentioned polyacrylonitrile magnetic pearl preparation method;
Three of purpose of the present invention is the application of above-mentioned polyacrylonitrile magnetic pearl as fixed enzyme vector.
Design of the present invention is such:
(1) adopting polyacrylonitrile and water-soluble magnetic fluid is main raw material, earlier water-soluble magnetic fluid is dispersed in the N of polyacrylonitrile, and in dinethylformamide (DMF) solution, mist projection granulating obtains polyacrylonitrile magnetic pearl then.
(2) enzyme is linked to polyacrylonitrile magnetic pearl surface after the hydrogenation, after catalyzed reaction finishes, utilizes magnetic field to reclaim, thus the performance immobilized dominant.
The said magnetic bead of the present invention is made of polyacrylonitrile and magnetic nanoparticle, and its component and weight percent content are:
Polyacrylonitrile 40～90%
Magnetic nanoparticle 10～60%
Said magnetic nanoparticle is Fe
3O
4
Preferred weight percent content is:
Polyacrylonitrile 60～90%
Magnetic nanoparticle 10～40%
Most preferredly be:
Polyacrylonitrile 70～80%
Magnetic nanoparticle 20～30%
The diameter of magnetic bead is between 0.2～7 μ m, and the best is 1～5 μ m.
The preparation of above-mentioned polyacrylonitrile magnetic pearl comprises the steps:
1. the nanometer Fe of superparamagnetism
3O
4The preparation of particle suspension:
Said superparamagnetism is meant that this magnetic particle has under the action of a magnetic field and shows magnetic, removes the character of promptly not having remanent magnetism behind the magnetic field.
Alkali is added ferrous iron and ferric vitriol or muriate mixed aqueous solution, and trivalent iron salt and ferrous mol ratio are 1.0～3.0, preferably 1.8～2.2, can obtain the size and the magnetic property of satisfactory superparamagnetic material like this.
Divalent transition metal salt such as chromium, cobalt, copper, magnesium, manganese, nickel, zinc salt and their mixture can partly or entirely replace divalent iron salt;
Said alkali is one or more in the mineral alkalis such as ammoniacal liquor, sodium hydroxide, potassium hydroxide, yellow soda ash, sodium bicarbonate or salt of wormwood, preferably sodium hydroxide or ammoniacal liquor;
The add-on of alkali is preferably 10～11 so that the pH value of solution reaches 8～12 for well;
Preparation condition is at 55～90 ℃, reacts, and obtains the nanometer Fe of black superparamagnetism
3O
4Particle suspension.
2. the nanometer Fe of superparamagnetism
3O
4The preparation of particle:
In above-mentioned nanometer Fe
3O
4The anion surfactant that adds 5～15% weight ratios in the particle suspension again, as Sodium dodecylbenzene sulfonate, sodium oleate, sodium lauryl sulphate etc., behind slaking 30～60min, be cooled to room temperature, transfer pH to 4～6 with acid as HCl, filter then, washing, lyophilize promptly obtains the nanometer Fe of superparamagnetism
3O
4Particle;
3. the preparation of polyacrylonitrile magnetic pearl:
In above-mentioned nanometer Fe
3O
4The anion surfactant that adds 5～15% weight ratios in the particle as Sodium dodecylbenzene sulfonate, sodium oleate, sodium lauryl sulphate etc., is made weight percent concentration and be 10～70% water miscible magnetic fluid, and is standby;
Method according to routine prepares polyacrylonitrile, and this method is a prior art, is summarized as follows:
Take by weighing 0.1～0.5g Diisopropyl azodicarboxylate and pour in the small beaker of 500mL, add 20～40mL vinyl cyanide, 1.0～2.0mL methymethacrylate is stirred to Diisopropyl azodicarboxylate and dissolves fully.Polyvinyl alcohol water solution 20～the 80mL of adding 0.5～2.5%, deionized water 60～100mL, behind the homodisperse, be poured in the there-necked flask reactor, under 60～80 ℃ of agitation conditions, react discharging in 3～5 hours again, with deionized water 250mL water dialysis 3～5 times, vacuum-drying obtains polyacrylonitrile;
N with polyacrylonitrile, in dinethylformamide (DMF) solution, be mixed with the solution that concentration is 0.5～3g/1000mL, again above-mentioned water-soluble magnetic fluid is dispersed in the solution of above-mentioned polyacrylonitrile, be mixed with magnetic fluid content and be 10～50% mixing solutions, carry out mist projection granulating with spray-drier then, the drying temperature of spray-drier is 160～200 ℃, feeding rate is 1.0～4.0mL/s, the rotating disk speed of rotation is 20000～40000r/min, obtains the said polyacrylonitrile magnetic pearl of the present invention.
Above-mentioned polyacrylonitrile magnetic pearl can be used as a kind of fixed enzyme vector, is used for fixing the biological enzyme of iso-electric point slant acidity, as penicillin acylase, E.C. 1.1.99.1, glucose oxidase etc.
This polyacrylonitrile magnetic pearl has very excellent performance, and the comparable polyacrylonitrile fibre of its fixed amount or other fixation supports are high more than 30%, and immobilization process is simple, the Separation and Recovery facility.
Concrete application method is as follows:
It is the LiAlH of 0.01～0.05g/mL that said polyacrylonitrile magnetic pearl is placed concentration
4Anhydrous ether solution in, back flow reaction is 10 minutes～30 hours in 40～80 ℃ water-bath, uses anhydrous diethyl ether, dilute hydrochloric acid, deionized water, alkaline solution and deionized water wash polyacrylonitrile magnetic pearl then successively, obtains a kind of hydrogenant polyacrylonitrile magnetic pearl;
Polyacrylonitrile magnetic pearl and LiAlH
4Additional proportion be 1: 1～1: 4;
With above-mentionedization hydrogenant polyacrylonitrile magnetic pearl place that concentration is 10～20%, pH is 8～9 glutaraldehyde and borate buffer solution, fully stir down at 0 ℃, in magnetic field, be that 8～9 borate buffer solution and pH are the phosphoric acid acid salt aqueous solution washing of 7.5 0.1M then with pH, hydrogenation polyacrylonitrile magnetic pearl after the washing is put into the test tube of the phosphate buffer soln (pH is 7.5) that is equipped with enzyme rapidly, under 0～40 ℃ condition, reacted 0.5～5 hour, then magnetic bead is isolated solution, promptly obtain to contain the polyacrylonitrile magnetic pearl of enzyme.
The ratio of polyacrylonitrile magnetic pearl and glutaraldehyde and borate buffer solution is 1: 40～1: 100;
The enzyme adsorptive capacity height of said polyacrylonitrile magnetic pearl, and the reaction recyclable repeated use in back.Recovery method is as follows:
After containing the polyacrylonitrile magnetic pearl process catalyzed reaction of enzyme, with magnetic field reaction soln is separated, detailed process is as follows: the mother liquor that will contain polyacrylonitrile magnetic pearl places the magnetic field of 1 tesla, because the magnetic that comprises in the magnetic bead is enrichment rapidly under the effect in magnetic field, can separate, through washing, just reusable then.
Further illustrate content of the present invention below in conjunction with embodiment, but these embodiment do not limit protection scope of the present invention.
Embodiment 1
With concentration is that the NaOH aqueous solution of 1.0mol/L slowly adds the FeCl that concentration is 1.0mol/L
2With concentration be the FeCl of 2.0mol/L
3Mixed aqueous solution (1: 2, mol ratio) is to react under 10 the condition at 85 ℃, pH, obtains nanometer Fe
3O
4Particle suspension;
Add the sodium oleate of 10% (weight), slaking 30 seconds is cooled to room temperature,, filters washing, lyophilize, the nanometer Fe of acquisition superparamagnetism then with the pH to 5 of HCl regulator solution
3O
4Particle;
Then in above-mentioned nanometer Fe
3O
4Middle again sodium oleate and the water that adds 10% (weight) of particle is made water miscible magnetic fluid, uses electron microscope observation, and its particle diameter is 8～12nm.
Embodiment 2
Taking by weighing 0.2 gram Diisopropyl azodicarboxylate pours in the small beaker of 500mL, add the 30mL vinyl cyanide, 1.5mL methymethacrylate is stirred to Diisopropyl azodicarboxylate and dissolves fully, adds 2% polyvinyl alcohol water solution 50mL, deionized water 80mL, be poured into behind the homodisperse in the reaction flask, again 70 ℃ of following stirring reactions 4 hours, with deionized water dialysis 5 times, vacuum-drying obtains polyacrylonitrile.
Embodiment 3
The polyacrylonitrile that embodiment 2 is obtained is dissolved in N ' the N-dimethylformamide, be mixed with the solution that concentration is the 1.5g/mL milliliter, water miscible magnetic fluid with embodiment 1 is dispersed in the above-mentioned polyacrylonitrile solution again, be mixed with magnetic fluid content and be 30% mixing solutions, carry out mist projection granulating with spray-drier then, the drying temperature of spray-drier is 180 ℃, and feeding rate is 2.0mL/s, the rotating disk rotating speed is 40000r/min, obtains polyacrylonitrile magnetic pearl.Recording median size with the laser light scattering particle size analyzer is 1.6 μ m.
Embodiment 4
The anhydrous diethyl ether and the 1 gram LiAlH that in 250 ml flasks, add 120 milliliters
4Add the polyacrylonitrile magnetic pearl of 2.5 gram embodiment 3 then, back flow reaction is 30 minutes under 40 ℃ water-bath, after reaction finishes, use anhydrous diethyl ether, dilute hydrochloric acid, deionized water, alkaline solution and deionized water wash polyacrylonitrile magnetic pearl successively, obtain a kind of hydrogenant polyacrylonitrile magnetic pearl.Record Fe with the thermogravimetric instrument
3O
4Content is 23.2%.
Embodiment 5
With the hydrogenation polyacrylonitrile magnetic pearl adding 15mL concentration of 180mg embodiment 4 is 12.5%, pH is in 8.5 the glutaraldehyde and borate buffer solution, fully stir down at 0 ℃, activation 30min, in magnetic field, be the washing of 8.5 borate buffer solution then with pH, hydrogenation polyacrylonitrile magnetic pearl after the washing is put into the enzyme work that is equipped with 10 milliliters rapidly be the E.C. 1.1.99.1 solution of 23u/mL, the concussion reaction is 2 hours under 30 ℃ condition, use pH=7.5 successively, 7.0 phosphate buffer soln under the action of a magnetic field, magnetic bead is washed, promptly obtain to contain the polyacrylonitrile magnetic pearl of E.C. 1.1.99.1.Live by detecting enzyme, the content that records the immobilized E.C. 1.1.99.1 of polyacrylonitrile magnetic pearl is 45u/mL.After the immobilized enzyme of magnetic bead carried out catalyzed reaction, reclaim with magnetic field separation, magnetic bead is reusable again.
Claims (10)
1. a polyacrylonitrile magnetic pearl is characterized in that, component and weight percent content are:
Polyacrylonitrile 40～90%
Magnetic nanoparticle 10～60%.
Said magnetic nanoparticle is Fe
3O
4
2. magnetic bead as claimed in claim 1 is characterized in that, weight percent content is:
Polyacrylonitrile 60～90%
Magnetic nanoparticle 10～40%.
3. magnetic bead as claimed in claim 2 is characterized in that, weight percent content is:
Polyacrylonitrile 70～80%
Magnetic nanoparticle 20～30%.
4. as the arbitrary described magnetic bead of claim 1～3, it is characterized in that the diameter of magnetic bead is 0.2～7 μ m.
5. magnetic bead as claimed in claim 4 is characterized in that, the diameter of magnetic bead is 1～5 μ m.
6. as the preparation method of the arbitrary described magnetic bead of claim 1～3, it is characterized in that, comprise the steps:
1. the nanometer Fe of superparamagnetism
3O
4The preparation of particle suspension:
Alkali is added ferrous iron and ferric vitriol or muriate mixed aqueous solution, and trivalent iron salt and ferrous mol ratio are 1.0～3.0; Said alkali is mineral alkali, and the add-on of alkali makes the pH value of solution reach 8～12;
2. the nanometer Fe of superparamagnetism
3O
4The preparation of particle:
In above-mentioned nanometer Fe
3O
4Add anion surfactant in the particle suspension, transfer pH to 4～6, adopt conventional method to collect the nanometer Fe of superparamagnetism then with acid
3O
4Particle;
3. the preparation of polyacrylonitrile magnetic pearl:
In above-mentioned nanometer Fe
3O
4Add anion surfactant in the particle again, make water miscible magnetic fluid, standby;
Polyacrylonitrile is dissolved in the solvent, more above-mentioned water-soluble magnetic fluid is dispersed in the solution of above-mentioned polyacrylonitrile, carry out mist projection granulating then, promptly obtain polyacrylonitrile magnetic pearl.
7. the preparation method of magnetic bead as claimed in claim 6 is characterized in that, said mineral alkali is sodium hydroxide or ammoniacal liquor, and the add-on of alkali makes the pH value of solution reach 10～11, and trivalent iron salt and ferrous mol ratio are 1.8～2.2.
8. the preparation method of magnetic bead as claimed in claim 6 is characterized in that, partly or entirely replaces divalent iron salt with chromium, cobalt, copper, magnesium, manganese, nickel or zinc salt and their mixture.
9. the preparation method of magnetic bead as claimed in claim 7 is characterized in that, partly or entirely replaces divalent iron salt with chromium, cobalt, copper, magnesium, manganese, nickel or zinc salt and their mixture.
10. as the application of the arbitrary described magnetic bead of claim 1～3, it is characterized in that, can be used as a kind of fixed enzyme vector, be used for fixing the biological enzyme of iso-electric point slant acidity.
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| CN 00127460 CN1296032A (en) | 2000-11-21 | 2000-11-21 | Polyacrylonitrile magnetic pearl and preparation process and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1975429B (en) * | 2006-12-14 | 2011-11-02 | 复旦大学 | Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof |
| CN107418949A (en) * | 2017-07-31 | 2017-12-01 | 苏州凯邦生物技术有限公司 | A kind of carrier material fixed for cellulase and preparation method thereof |
-
2000
- 2000-11-21 CN CN 00127460 patent/CN1296032A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1975429B (en) * | 2006-12-14 | 2011-11-02 | 复旦大学 | Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof |
| CN107418949A (en) * | 2017-07-31 | 2017-12-01 | 苏州凯邦生物技术有限公司 | A kind of carrier material fixed for cellulase and preparation method thereof |
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