CN110330607A - It is a kind of for separating the preparation method and applications of the magnetic molecularly imprinted polymer of antalzyme protein - Google Patents
It is a kind of for separating the preparation method and applications of the magnetic molecularly imprinted polymer of antalzyme protein Download PDFInfo
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- CN110330607A CN110330607A CN201910718479.5A CN201910718479A CN110330607A CN 110330607 A CN110330607 A CN 110330607A CN 201910718479 A CN201910718479 A CN 201910718479A CN 110330607 A CN110330607 A CN 110330607A
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/305—Addition of material, later completely removed, e.g. as result of heat treatment, leaching or washing, e.g. for forming pores
- B01J20/3057—Use of a templating or imprinting material ; filling pores of a substrate or matrix followed by the removal of the substrate or matrix
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
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Abstract
The invention discloses a kind of for separating the preparation method and applications of the magnetic molecularly imprinted polymer of antalzyme protein, passes through 3- (methacryloxypropyl) propyl trimethoxy silicane and Fe first3O4It reacts and the double bond containing magnetic nano-particle in surface is made, by the double bond containing magnetic nano-particle in surface, N- (3- dimethylamino-propyl) Methacrylamide, N, N '-methylene-bisacrylamide, methacrylic acid and lysozyme are placed in reaction vessel, magnetic molecularly imprinted polymer is prepared in the method for magnetic nano-particle surface free radical copolymerzation with cross-linking by initiator ammonium persulfate APS and tetramethylethylenediamine TEMED, then the method by dialysing in water completely removes template molecule lysozyme and magnetic molecularly imprinted polymer for separating antalzyme protein is finally made.The present invention has higher surface area and more accurate recognition site by the magnetic molecularly imprinted polymer of the method preparation in magnetic particle surface grafting nanogel, has preferably identification separating effect to template molecule.
Description
Technical field
The invention belongs to the synthesis technical fields of molecularly imprinted polymer, and in particular to one kind is for separating antalzyme protein
Magnetic molecularly imprinted polymer preparation method and applications.
Background technique
Lysozyme is also known as muramidase or N-acetylmuramide lycanohydrlase, is that a kind of can hydrolyze sticks polysaccharide in pathogenic bacteria
Alkaline enzyme, have the effects that antibacterial, anti-inflammatory, antiviral.Lysozyme is clinically effective antiphlogistic, in food antiseptic and
Genetic engineering etc. is also widely used.Bacteriolyze enzyme source is very wide, is found in people, animal, plant and microorganism,
Wherein content highest in egg white, therefore, industrial production lysozyme is often using egg as raw material.The side of lysozyme is extracted from egg
There are many method report, there is direct crystallization method, affinity chromatography, ion-exchange, the precipitation method, aqueous two-phase extraction, ultrafiltration, the precipitation method
Deng.Various method advantage and disadvantage are different, and the quality and production cost of product are also different.Currently, the precipitation method are industrially to apply
The method of comparative maturity, although obtained product purity height, high income, activity are also relatively good, production cost is higher, and
It operates sufficiently complex.
Molecularly imprinted polymer (MIP) is the artificial receptors material that a kind of pair of specific target molecules have selectivity and high parent's property
Material is widely used in chemical sensor, medicine because it has many advantages, such as that stability is high, synthetic method is simple and low in cost
The fields such as object transmission, separation and artificial antibody.Lysozyme molecular imprinting polymer combines magnetic feature, on the one hand to lysozyme
There are single-minded identification and separating capacity, on the other hand can achieve the purpose that be repeatedly circulated by simple magnetism.Herein
The present invention has synthesized the magnetic molecularly imprinted polymer for capableing of specific recognition template molecule lysozyme, and applied to right in egg white
The separation and Extraction of lysozyme provides a kind of simple, effective method for the extraction separation of lysozyme from egg white.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of simple processes and low-cost for separating lysozyme egg
The magnetic molecularly imprinted polymer of the preparation method of white magnetic molecularly imprinted polymer, this method preparation has higher surface
Long-pending and more accurate recognition site, can be applied to the separation and Extraction of lysozyme from egg white, therefore the magnetic molecularly imprinted polymer
Can more effectively from egg white specificity extraction isolate lysozyme.
The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of for separating the magnetism of antalzyme protein
The preparation method of molecularly imprinted polymer, it is characterised in that detailed process are as follows: pass through 3- (methacryloxypropyl) propyl three first
Methoxy silane and Fe3O4React be made the double bond containing magnetic nano-particle in surface, by the double bond containing magnetic nano-particle in surface,
N- (3- dimethylamino-propyl) Methacrylamide, N, N '-methylene-bisacrylamide, methacrylic acid and lysozyme are placed in instead
It answers in container, is crosslinked by initiator ammonium persulfate APS and tetramethylethylenediamine TEMED in magnetic nano-particle surface free radical
The method of copolymerization prepares magnetic molecularly imprinted polymer, then the method by dialysing in water completely removes template molecule lyase bacterium most
Being made eventually being capable of magnetism point effective, that exclusively extraction separates lysozyme and recycled and recycled by Magneto separate from egg white
Sub- imprinted polymer；Specific synthetic route in preparation process are as follows:
。
It further limits, the specific preparation process of the double bond containing magnetic nano-particle in surface are as follows: in round-bottomed flask
Fe is added3O4Nanoparticle 0.1g, 3- (methacryloxypropyl) propyl trimethoxy silicane 0.1-1g and toluene 3-20mL, in N2
In 60-80 DEG C of reaction 24-48h under atmosphere, the double bond containing magnetism in surface is dried to obtain by sufficiently being washed after Magneto separate with methanol
Nanoparticle；Specific synthetic route in preparation process are as follows:
。
It further limits, the specific preparation process of the magnetic molecularly imprinted polymer are as follows: successively add in round-bottomed flask
Enter the double bond containing magnetic nano-particle 10-50mg in surface, function monomer N- (3- dimethylamino-propyl) Methacrylamide 0.1-
5mmol and methacrylic acid 0.2-10mmol, template molecule lysozyme 1-4mmol, crosslinking agent N, N '-methylene-bisacrylamide
0.4-0.8mmol and lauryl sodium sulfate 30mg adds the phosphate buffer solution 20-100mL of pH=7.2, is then added
Initiator tetramethylethylenediamine 30 μ L and ammonium persulfate 30mg is sealed after leading to nitrogen 1h and in 60-80 DEG C of reaction 24-48h, reaction
Pass through Magneto separate after the completion and dialyse in water and completely remove template molecule lyase bacterium, is then used in 40 DEG C of vacuum drying
Separate the magnetic molecularly imprinted polymer of antalzyme protein.
Magnetic molecularly imprinted polymer of the present invention extracts the application of separation antalzyme protein, the magnetism in egg white
Molecularly imprinted polymer can effectively, exclusively extract separation lysozyme, the magnetic molecule after adsorbing lysozyme from egg white
Imprinted polymer is recycled and is recycled after passing through Magnetic Isolation.
Magnetic molecularly imprinted polymer of the present invention extracts the application of separation antalzyme protein, feature in egg white
It is detailed process are as follows: the phosphate buffer solution of 50mL, pH=7.2 are added first in 1mL egg white, ultrasound is sufficiently mixed
Centrifuge separation after even takes the above egg white solution 10mL that 10-50mg magnetic molecularly imprinted polymer is added and is uniformly dispersed, shakes in 25 DEG C
Bed concussion 16-24h realizes that the specificity of lyase bacterium in egg white extracts separation.
Compared with the prior art, the present invention has the following advantages: 1, the present invention pass through in magnetic particle surface grafting nanometer solidifying
The magnetic molecularly imprinted polymer of the method preparation of glue has higher surface area and more accurate recognition site, to template molecule
With better identification separating effect；2, Fe in magnetic molecularly imprinted polymer produced by the present invention3O4Presence have be conducive to magnetism
The recycling of molecularly imprinted polymer is recycled.
Detailed description of the invention
A is the scanning electron microscope (SEM) photograph that magnetic molecularly imprinted polymer is made in embodiment 2 in Fig. 1, and b is that magnetism is made in embodiment 2
The scanning electron microscope (SEM) photograph of the non-imprinted polymer of molecule；
A is the electrophoresis result of the solution after extracting separation lysozyme in egg white of embodiment 3 in Fig. 2, and b is embodiment 4 from egg white
The electrophoresis result of solution after separating lysozyme is extracted, c is the electrophoresis knot of the solution after extracting separation lysozyme in egg white of embodiment 5
Fruit, d are the electrophoresis result of the solution after extracting separation lysozyme in egg white of embodiment 6.
Specific embodiment
Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this
The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair
Bright range.
Embodiment 1
The preparation of surface magnetic nano-particle containing double bond
Fe is added in round-bottomed flask3O4Nanoparticle 0.1g, 3- (methacryloxypropyl) propyl trimethoxy silicane 0.1g and
Toluene 3mL, in N2In 80 DEG C of reaction 48h under atmosphere, surface is dried to obtain containing double bond by sufficiently being washed after Magneto separate with methanol
Magnetic nano-particle.
Embodiment 2
The preparation of magnetic molecularly imprinted polymer and the non-imprinted polymer of magnetic molecule
The double bond containing magnetic nano-particle 50mg in surface, function monomer N- (3- dimethylamino third are sequentially added in round-bottomed flask
Base) Methacrylamide 0.1mmol and methacrylic acid 0.2mmol, template molecule lysozyme 1mmol, crosslinking agent N, N '-methylene
Base bisacrylamide 0.4mmol and lauryl sodium sulfate 30mg adds the phosphate buffer solution 20mL of pH=7.2, then
Be added initiator tetramethylethylenediamine 30 μ L and ammonium persulfate 30mg, lead to nitrogen 1h after seal and in 60 DEG C of reaction 48h, reacted
Completely remove template molecule lyase bacterium at rear by Magneto separate and in water dialysis, then in 40 DEG C of vacuum drying obtain for point
Magnetic molecularly imprinted polymer from antalzyme protein.In order to compare the effect of the non-trace of trace, simultaneously synthesizing magnetic molecule
Non- imprinted polymer, specific synthetic method are same as above, and template molecule lysozyme is not added in synthesis process only.
Embodiment 3
The phosphate buffer solution of 50mL, pH=7.2, centrifugation point after ultrasound is sufficiently mixed uniformly are added first in 1mL egg white
From taking above egg white solution 10mL that magnetic molecularly imprinted polymer made from 50mg embodiment 2 is added and be uniformly dispersed, shaken in 25 DEG C
Bed concussion takes supernatant afterwards for 24 hours, and high performance liquid chromatography (HPLC) detects imprinting efficiency, and examines separating effect by SDS method.
Embodiment 4
The phosphate buffer solution of 50mL, pH=7.2, centrifugation point after ultrasound is sufficiently mixed uniformly are added first in 1mL egg white
From taking above egg white solution 10mL that the magnetic molecularly imprinted polymer of 3 magnetic recovery of 50mg embodiment is added and be uniformly dispersed, in 25
The concussion of DEG C shaking table takes supernatant afterwards for 24 hours, and high performance liquid chromatography (HPLC) detects imprinting efficiency, and examines separation by SDS method
Effect.
Embodiment 5
The phosphate buffer solution of 50mL, pH=7.2, centrifugation point after ultrasound is sufficiently mixed uniformly are added first in 1mL egg white
From taking above egg white solution 10mL that the magnetic molecularly imprinted polymer of 4 magnetic recovery of 50mg embodiment is added and be uniformly dispersed, in 25
The concussion of DEG C shaking table takes supernatant afterwards for 24 hours, and high performance liquid chromatography (HPLC) detects imprinting efficiency, and examines separation by SDS method
Effect.
Embodiment 6
The phosphate buffer solution of 50mL, pH=7.2, centrifugation point after ultrasound is sufficiently mixed uniformly are added first in 1mL egg white
From taking above egg white solution 10mL that the magnetic molecularly imprinted polymer of 5 magnetic recovery of 50mg embodiment is added and be uniformly dispersed, in 25
The concussion of DEG C shaking table takes supernatant afterwards for 24 hours, and high performance liquid chromatography (HPLC) detects imprinting efficiency, and examines separation by SDS method
Effect.
The present invention extracts separation to lysozyme from egg white specificity by research magnetic molecularly imprinted polymer and passes through
The method of Magnetic Isolation extracts separation repeatedly, has shown that the magnetic molecularly imprinted polymer of preparation has very high molecule by research
Imprinting efficiency and good specificity.
Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (5)
1. a kind of for separating the preparation method of the magnetic molecularly imprinted polymer of antalzyme protein, it is characterised in that detailed process
Are as follows: pass through 3- (methacryloxypropyl) propyl trimethoxy silicane and Fe first3O4It reacts and the double bond containing magnetic Nano in surface is made
Particle, by the double bond containing magnetic nano-particle in surface, N- (3- dimethylamino-propyl) Methacrylamide, N, N '-di-2-ethylhexylphosphine oxide
Acrylamide, methacrylic acid and lysozyme are placed in reaction vessel, pass through initiator ammonium persulfate APS and tetramethylethylenediamine
TEMED prepares magnetic molecularly imprinted polymer in the method for magnetic nano-particle surface free radical copolymerzation with cross-linking, then by water
The method of dialysis completely remove template molecule lyase bacterium finally be made can effectively, exclusively from egg white extract separation lysozyme
And the magnetic molecularly imprinted polymer for being recycled and being recycled by Magneto separate；Specific synthetic route in preparation process are as follows:
。
2. it is according to claim 1 for separating the preparation method of the magnetic molecularly imprinted polymer of antalzyme protein,
It is characterized in that the specific preparation process of the double bond containing magnetic nano-particle in the surface are as follows: Fe is added in round-bottomed flask3O4Nanometer
Particle 0.1g, 3- (methacryloxypropyl) propyl trimethoxy silicane 0.1-1g and toluene 3-20mL, in N2In 60-80 under atmosphere
DEG C reaction 24-48h, be dried to obtain the double bond containing magnetic nano-particle in surface by sufficiently being washed after Magneto separate with methanol；Preparation
Specific synthetic route in the process are as follows:
。
3. it is according to claim 1 for separating the preparation method of the magnetic molecularly imprinted polymer of antalzyme protein,
It is characterized in that the specific preparation process of the magnetic molecularly imprinted polymer are as follows: surface is sequentially added in round-bottomed flask containing double bond
Magnetic nano-particle 10-50mg, function monomer N- (3- dimethylamino-propyl) Methacrylamide 0.1-5mmol and methyl-prop
Olefin(e) acid 0.2-10mmol, template molecule lysozyme 1-4mmol, crosslinking agent N, N '-methylene-bisacrylamide 0.4-0.8mmol and
Lauryl sodium sulfate 30mg adds the phosphate buffer solution 20-100mL of pH=7.2, and initiator tetramethyl is then added
Ethylenediamine 30 μ L and ammonium persulfate 30mg, lead to nitrogen 1h after seal and in 60-80 DEG C of reaction 24-48h, pass through magnetic after the reaction was completed
It separates and dialysis completely removes template molecule lyase bacterium in water, then obtain in 40 DEG C of vacuum drying for separating lysozyme egg
White magnetic molecularly imprinted polymer.
4. magnetic molecularly imprinted polymer made from method described in any one of -3 extracts in egg white according to claim 1
The application of antalzyme protein is separated, it is molten which can effectively, exclusively extract separation from egg white
Bacterium enzyme, the magnetic molecularly imprinted polymer after adsorbing lysozyme is by recycling and recycling after Magnetic Isolation.
5. magnetic molecularly imprinted polymer made from method described in any one of -3 extracts in egg white according to claim 1
Separate the application of antalzyme protein, it is characterised in that detailed process are as follows: the phosphorus of 50mL, pH=7.2 are added first in 1mL egg white
Hydrochlorate buffer solution, centrifuge separation after ultrasound is sufficiently mixed uniformly take the above egg white solution 10mL that 10-50mg magnetic molecule print is added
Mark polymer is simultaneously uniformly dispersed, and realizes that the specificity of lyase bacterium in egg white extracts separation in 25 DEG C of shaking table concussion 16-24h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113368837A (en) * | 2021-05-26 | 2021-09-10 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102145279A (en) * | 2010-02-05 | 2011-08-10 | 华中科技大学 | Method for preparing lysozyme molecular imprinting nano particles with magnetic responsiveness and extremely high adsorption capacity |
| CN103100377A (en) * | 2013-02-27 | 2013-05-15 | 江苏大学 | Green preparation method of magnetic molecular imprinting sorbent |
| CN104356323A (en) * | 2014-10-13 | 2015-02-18 | 北京大学 | Magnetic molecularly imprinted nano-particle as well as preparation method and application thereof |
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- 2019-08-05 CN CN201910718479.5A patent/CN110330607A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102145279A (en) * | 2010-02-05 | 2011-08-10 | 华中科技大学 | Method for preparing lysozyme molecular imprinting nano particles with magnetic responsiveness and extremely high adsorption capacity |
| CN103100377A (en) * | 2013-02-27 | 2013-05-15 | 江苏大学 | Green preparation method of magnetic molecular imprinting sorbent |
| CN104356323A (en) * | 2014-10-13 | 2015-02-18 | 北京大学 | Magnetic molecularly imprinted nano-particle as well as preparation method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113368837A (en) * | 2021-05-26 | 2021-09-10 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
| CN113368837B (en) * | 2021-05-26 | 2022-05-17 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
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Application publication date: 20191015 |