CN105200029B - A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization - Google Patents
A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization Download PDFInfo
- Publication number
- CN105200029B CN105200029B CN201510701434.9A CN201510701434A CN105200029B CN 105200029 B CN105200029 B CN 105200029B CN 201510701434 A CN201510701434 A CN 201510701434A CN 105200029 B CN105200029 B CN 105200029B
- Authority
- CN
- China
- Prior art keywords
- sphere
- immobilization
- chitosan
- earthworm fibrinolysin
- magnetic micro
- 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.)
- Active
Links
Abstract
The invention discloses a kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization, enzymatic activity is 40 60U/mg.Preparation method is that first arrowhead trypsin inhibitor is dissolved in the buffer solution of pH7~9 to obtain albumen enzyme buffer liquid, it adds it to again in the chitosan magnetic micro-sphere through overactivation, the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor is obtained after 4~7h of water-bath oscillation coupling；It is added into the thick enzyme solutions of earthworm fibrinolysin again and vibrates 0.5 3h of curing reaction in 30 60 DEG C of water bath with thermostatic control shaking tables；After reaction, washed repeatedly into cleaning solution inspection do not measure albumen until, magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.The present invention is coupled with magnetic microsphere using arrowhead trypsin inhibitor as aglucon, with isolating and purifying for earthworm fibrinolysin, improves the selectivity that isolates and purifies of earthworm fibrinolysin, and magnetic microsphere is easy to detach with earthworm fibrinolysin.
Description
Technical field
The invention belongs to magnetic microsphere technical field, it is specifically related to the magnetic microsphere for immobilization earthworm fibrinolysin
Preparation method.
Background technology
Magnetic macromolecular microsphere is a kind of novel magnetic materials that developed recently gets up, and is by proper method by magnetic nothing
Machine particle is combined what is formed to have certain magnetic and special construction complex microsphere with organic polymer.Magnetic composite microsphere is not only
Numerous characteristics with common polymer microsphere are also with magnetic responsiveness, so can not only pass through the side such as copolymerization and surface modification
Method assigns its surface functional group (such as-OH ,-COOH ,-CHO ,-NH2, etc.), moreover it is possible to there is guiding work(outside plus under magnetic fields
Energy.Magnetic microsphere immobilised enzymes, cell separation, Separation of Proteins and purifying, immunoassays, targeted drug and DNA separation with
The fields such as nucleic acid hybridization have a wide range of applications.The method for being usually used in preparing magnetic microsphere at present has investment, suspends and gather
Magnetic microsphere grain size wider distribution legal, that these methods are prepared, it is in irregular shape.
Arrowhead protease inhibitors are the multifunctional dual-head protease inhibitors isolated and purified from arrowhead bulb, can be divided
For inhibitor A and B (API-A, API-B), the two can inhibit a variety of serine proteases, their molecular weight and soybean Kunizt
Inhibitor is close, but has larger difference on rejection characteristic, and also without homology in structure.Its peptide chain is by 150 amino
Sour residue is formed, Arg and Lys residues have respectively constituted two activated centres of inhibitor, in the Arg activity of inhibitor A, B
The heart is Arg-Tys-Lys (76~78), and the sequence in the activated centres Lys is Lys-Ser (44~45), the work with Kazal inhibitor
Property center is similar.In addition, there is a single-minded inhibition in the activated centres Lys to trypsase, and the activated centres Arg then show it is multi-functional,
There is inhibiting effect to multiple protein enzyme.
Earthworm fibrinolysin is also known as Lumbrokinase, is the both fibres with direct solution fibrin extracted out of earthworm body
Antiplasmin activity, and the active class protein of Plasminogen activation with class urokinase can dissolve outmoded thrombus but also suppression
New thrombosis is made, experiment shows that the enzyme has anti-freezing, molten fine, improvement hemorheology properties, is prevention and treatment thrombus disease
The active drug of disease, the enzyme belong to serine protease, can be by serpin such as soybean trypsin inhibitor
Inhibit, clinical application in recent years shows that it has in terms of the thrombus diseases such as treatment cerebral thrombus, hemiplegia, myocardial infarction
Have the advantages that quick, curative effect is good, oral effectively and safer.Numerous studies show no matter earthworm fibrinolysin is injected intravenously
Or oral medication, thrombolytic effect is all had, is a kind of thrombotic disease medicine ideal at present, before there is wide exploitation
Scape.The extraction of usual fibrinolysin be homogenized, saltoutd by earthworm, gel chromatography, ion-exchange chromatography and high performance liquid chromatography
(HPLC) etc. series of steps are isolated and purified to obtain, and the fibrinolysin purity that this method is extracted is high, but due to purifying work
Sequence complexity is of high cost, and time-consuming, and economic benefit is low, is only used for laboratory research, is difficult to industrial production, how to obtain steady
The earthworm fibrinolysin of fixed high activity can be that a road is opened up in the utilization of earthworm resource.
There is presently no be used for immobilization earthworm fibrinolytic about arrowhead trypsin inhibitor coupled chitosan magnetic microsphere
The report of enzyme.
Invention content
The purpose of the present invention is to provide a kind of magnetic microspheres and preparation method thereof of immobilization earthworm fibrinolysin.It is with kind
Mushroom trypsin inhibitor is aglucon, is coupled with magnetic microsphere, with isolating and purifying for earthworm fibrinolysin, improves earthworm
Fibrinolysin isolates and purifies selectivity, and magnetic microsphere is easy to detach with earthworm fibrinolysin.
Technical scheme is as follows：
A kind of magnetic microsphere for earthworm fibrinolysin immobilization, enzymatic activity 40-60U/mg, in pH 6.5~8.0
Between stability be maintained at 78% or more, storage time is in 30 days for the magnetic microsphere of earthworm fibrinolysin immobilization
Enzymatic activity is 82% or more.
The preparation method of the magnetic microsphere for earthworm fibrinolysin immobilization of the present invention comprising following steps：
(1) coupling of chitosan magnetic micro-sphere：Arrowhead trypsin inhibitor is dissolved in the buffer solution of pH 7~9 and is obtained
Albumen enzyme buffer liquid, then add it in the chitosan magnetic micro-sphere through overactivation, water-bath oscillation 4~7h of coupling, oscillation temperature
Degree 40~60 DEG C, after obtain coupling arrowhead trypsin inhibitor chitosan magnetic micro-sphere；
(2) immobilization of earthworm fibrinolysin：The chitosan magnetic micro-sphere for being coupled arrowhead trypsin inhibitor is added again
The thick enzyme solutions of earthworm fibrinolysin vibrate curing reaction 0.5-3h in 30-60 DEG C of water bath with thermostatic control shaking table；After reaction, spend from
Sub- water and the phosphate buffer that pH is 6-8 wash the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until washing
Until inspection does not measure albumen in liquid, magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
As the preferred of technical solution, the buffer solution in the step (1) is Tris-HCl buffer solutions.The protease is slow
Fliud flushing and the liquid-solid ratio of the chitosan magnetic micro-sphere of activation are 10~25: 1,4~6h of water-bath oscillation coupling.This process conditions obtains
The chitosan magnetic micro-sphere performance of the coupling white enzyme inhibitor of arrowhead arrived is especially good.
As the preferred of technical solution, in the step (2), the magnetic crust of every gram of coupling arrowhead trypsin inhibitor is poly-
The thick enzyme solutions of earthworm fibrinolysin that 1~5ml enzyme activity is ten thousand U/mL of 1-2 are then added in sugared microballoon.
The preparation of the chitosan magnetic micro-sphere activated in the present invention be by chitosan magnetic micro-sphere, be added NaOH solution and
NaBH4Solution vibrates 30~40min in 30~80 DEG C of water-baths, and it is activator to add dimethyl sulfoxide (DMSO), adds volume fraction
For 10%~60% epoxychloropropane, after oscillation activates 2~10h, filters, be washed with distilled water the magnetism that can be obtained activation
Chitosan microball.
As the further preferred of technical solution, the weight ratio of the chitosan magnetic micro-sphere and NaOH solution is 1: 5~
10, chitosan magnetic micro-sphere and NaBH4The weight ratio of solution is 1: 4~12；Every gram of chitosan magnetic micro-sphere adds the volume fraction to be
40%~60% 1.5~9mL of dimethyl sulfoxide (DMSO), epoxychloropropane are 1~12mL.
As the further preferred of technical solution, a concentration of 1.2~2.0mol/L, NaBH of NaOH solution4Solution it is dense
Degree is 5~10g/L, and the volume fraction of epoxychloropropane is 30%~50%, magnetic when 2~6h of oscillation activation at 30~50 DEG C
The activation effect of chitosan microball is best.
The preparation method of chitosan magnetic micro-sphere in the present invention：
(1) it is 20~40: 1 mixing according to weight ratio by atoleine and Span-80,1~2h systems is stirred at 30~50 DEG C
For at oil phase；
(2) a certain amount of chitosan is weighed, is dissolved with 3~5% acetum, shell is obtained after chitosan is completely dissolved
Fe is added in glycan solution3O4Powder, 10~20min of ultrasonic disperse, is made water phase；
(3) it is 1: 5~6 according to volume ratio by water phase and oil phase, water phase is slowly added in oil phase, 0.5~1h of stirring makes
Solution forms emulsion system；
(4) glutaraldehyde that mass fraction is 25% is added dropwise into emulsion system again, continues 1~2h of stirring, uses NaOH solution
PH to 9~10 is adjusted, stops reaction after the reaction was continued 1~2h, magnet separation, with petroleum ether, ethyl alcohol and deionized water are washed into
Property, vacuum drying, you can obtain chitosan magnetic micro-sphere.
As the preferred of technical solution, ferroso-ferric oxide is Nano grade used in the present invention, and the chitosan of preparation is molten
A concentration of 5~10mg/mL of liquid, speed of agitator are 600~700rpm/min, the grain for the chitosan magnetic micro-sphere being prepared
Diameter is minimum, is 215nm.
Compared with prior art, the present invention having the following advantages that：
1. the preparation method of the present invention is simple, operation is easy, processing procedure is mild, not high to equipment requirement.
2. the arrowhead trypsin inhibitor of the conjugated magnetic chitosan microball used in the present invention is aglucon, Lys activity
There is single-minded inhibition at center to trypsase, and is reversible, and there is inhibitor effect in the activated centres Arg to multiple protein enzyme, and two
The purity and specificity of earthworm fibrinolysin can be improved in person's Reverse transcriptase.
3. the earthworm fibrinolysin of the present invention is fixed on magnetic microsphere, because microballoon has magnetism, in the effect of externally-applied magnetic field
Under, fibrinolysin can soon be detached with reactant.And the pH stability of immobilization fibrinolysin is preferable, it can be in pH 6.5~8.0
In the range of, stability is maintained at 78% or more, and thermal stability heats, relative activity remains to compared with resolvase height at 60 DEG C
70% or more, and the storage of long period, enzymatic activity is kept to keep 82% or more.
Description of the drawings
Fig. 1 is 7000 times of electron microscope of the chitosan magnetic micro-sphere of the present invention；
Fig. 2 is 100,000 times of electron microscope of the chitosan magnetic micro-sphere of the present invention；
Fig. 3 is the infrared spectrum analysis figure of the chitosan magnetic micro-sphere of the present invention, 3415cm in figure-1It is stretching for N-H and O-H
Contracting vibration peak, 2923cm-1And 2855cm-1It is the characteristic absorption peak of C-H in aliphatic, 1617cm-1It is the characteristic absorption of amide
Peak, 1431cm-1It is-CH3Eigen vibration peak, 1119cm-1It is the stretching vibration absworption peak of C-O, 620cm-1There is Fe3O4's
Characteristic absorption peak.
Fig. 4 is the pH stability diagrams of resolvase and immobilization earthworm fibrinolysin；
Fig. 5 is the storage stability figure of resolvase and immobilization earthworm fibrinolysin；
Fig. 6 is the thermal stability figure of resolvase and immobilization earthworm fibrinolysin.
Specific embodiment
It is further illustrated the present invention below by specific embodiment.It should be understood that the embodiment of the present invention is only
For the present invention, rather than limiting the invention, to the letter of the preparation method of the present invention under the concept thereof of the present invention
Single improve belongs to the scope of protection of present invention.
In embodiment, (a) particle diameter distribution of chitosan magnetic micro-sphere, (b) activation grade measure the side by recording below
Method is measured.
(a) particle diameter distribution
Chitosan magnetic micro-sphere is scattered in ethyl alcohol, a few drop dispersant Tween-20s are added, ultrasonic disperse 10min is used
Nanometer laser Particle Size Analyzer carries out Lido measurement.
(b) activation grade
Using sodium thiosulfate titration, the microballoon after 0.1g washings are drained is weighed, the 1.3moL/L of 1.5mL is added
Hypo solution, the oscillating reactions 30min in 40 DEG C of water-bath constant temperature oscillators, after completion of the reaction, be added dropwise 1 drop phenolphthalein refer to
Show agent, with standard titration with hydrochloric acid to the colourless rear stopping titration of solution, records the volume change of the front and back standard hydrochloric acid of titration.Epoxy
Base modification density is calculated with following formula：
Embodiment 1
The span-80 of the atoleine and 2mL that measure 100mL is added in the three-necked flask of 250mL, and 45 DEG C of water-bath heat preservations are stirred
It mixes 1h and oil phase is made；The chitosan for weighing 0.2g is dissolved with the acetum of 20mL3%, after chitosan is completely dissolved, is added
The Fe of 0.6g3O4, ultrasonic disperse 10min；It is prepared into the chitosan solution of a concentration of 5mg/ml, is then added in oil phase, 300rpm
After stirring half an hour makes solution form emulsion system, the glutaraldehyde that 3mL mass is 25% is slowly added dropwise, continues to stir 1h, use
Stop reaction after the NaOH solution tune pH to 10 or so of 1.0mol/L, the reaction was continued 1h, magnet separation, with petroleum ether, ethyl alcohol and
Deionized water is washed, vacuum drying, you can obtain chitosan magnetic micro-sphere.
The particle size of the chitosan magnetic micro-sphere is shown in table 1.
Embodiment 2
The span-80 of the atoleine and 3mL that measure 100mL is added in the three-necked flask of 250mL, and 30 DEG C of water-bath heat preservations are stirred
It mixes 2h and oil phase is made；The chitosan for weighing 0.8g is dissolved with the acetum of 20mL5%, after chitosan is completely dissolved, is added
The Fe of 0.6g3O4, ultrasonic disperse 20min；It is prepared into the chitosan solution of a concentration of 6.5mg/ml, is then added in oil phase,
After 400rpm stirring half an hours make solution form emulsion system, the glutaraldehyde that 5mL mass is 25% is slowly added dropwise, continues to stir
2h stops reaction, magnet separation, with petroleum ether, second with the NaOH solution tune pH to 9 or so of 1.0mol/L after the reaction was continued 2h
Alcohol and deionized water washing, vacuum drying, you can obtain chitosan magnetic micro-sphere.
The particle size of the chitosan magnetic micro-sphere is shown in table 1.
Embodiment 3
The span-80 of the atoleine and 2.5mL that measure 100mL is added in the three-necked flask of 250mL, 40 DEG C of water-bath heat preservations
Oil phase is made in stirring 2h；The chitosan for weighing 1g is dissolved with the acetum of 20mL3%, after chitosan is completely dissolved, is added
The Fe of 0.6g3O4, ultrasonic disperse 15min；It is prepared into the chitosan solution of a concentration of 8.1mg/ml, is then added in oil phase,
After 500rpm stirring half an hours make solution form emulsion system, the glutaraldehyde that 10mL mass is 25% is slowly added dropwise, continues to stir
1.5h is mixed, with the NaOH solution tune pH to 9.5 or so of 1.0mol/L, stops reaction after the reaction was continued 1.5h, stone is used in magnet separation
Oily ether, ethyl alcohol and deionized water washing, vacuum drying, you can obtain chitosan magnetic micro-sphere.
The particle size of the chitosan magnetic micro-sphere is shown in table 1.
Embodiment 4
The span-80 of the atoleine and 2mL that measure 100mL is added in the three-necked flask of 250mL, and 45 DEG C of water-bath heat preservations are stirred
It mixes 1.5h and oil phase is made；The chitosan for weighing 1.2g is dissolved with the acetum of 20mL3%, after chitosan is completely dissolved, is added
Enter the Fe of 0.6g3O4, ultrasonic disperse 10min；It is prepared into the chitosan solution of a concentration of 10mg/ml, is then added in oil phase,
After 600rpm stirring half an hours make solution form emulsion system, the glutaraldehyde that 3mL mass is 25% is slowly added dropwise, continues to stir
1h stops reaction, magnet separation, with petroleum ether, second with the NaOH solution tune pH to 10 or so of 1.0mol/L after the reaction was continued 1h
Alcohol and deionized water washing, vacuum drying, you can obtain chitosan magnetic micro-sphere.
The particle size of the chitosan magnetic micro-sphere is shown in table 1.
Embodiment 5
The span-80 of the atoleine and 2mL that measure 100mL is added in the three-necked flask of 250mL, 45 DEG C of water-bath heat preservations；
The chitosan for weighing 0.2g is dissolved with the acetum of 20mL3%, after chitosan is completely dissolved, the Fe of 0.6g is added3O4, surpass
Sound disperses 10min；Chitosan solution is added in oil phase, after 700rpm stirring half an hours make solution form emulsion system, is delayed
The slow glutaraldehyde that 3mL mass is added dropwise and is 25%, continues to stir 1h, with the NaOH solution tune pH to 10 or so of 1.0mol/L, continues
Stop reaction after reacting 1h, magnet separation, with petroleum ether, ethyl alcohol and deionized water are washed, vacuum drying, you can obtain magnetic crust
Glycan microballoon.
The particle size of the chitosan magnetic micro-sphere is shown in table 1.
Chitosan magnetic micro-sphere particle diameter distribution is shown in Fig. 1, Fig. 2, from fig. 1, it can be seen that chitosan and Fe3O4Nano-particle
The composite particles grain size of formation is between 100-300nm, which disperses in the reaction system conducive to microballoon and Magneto separate, and
The microballoon of small particle has the specific surface area of bigger, can be with the more enzymes of covalent bond, while can also reduce substrate and product
Spread the influence of limitation.Its infrared spectrum analysis figure is referring to Fig. 3.
The grain size of 1 chitosan magnetic micro-sphere of table
Embodiment 6
Chitosan magnetic micro-sphere that 0.25g embodiments 5 are prepared is weighed in conical flask, 25mL 1.5mol/L are added
NaOH solution and 150mL 10g/L NaBH4, 30min is vibrated in thermostatic control oscillator vibration, reaction temperature is 30 DEG C, then
Using the dimethyl sulfoxide (DMSO) of volume fraction 50% as activating solvent, the epoxychloropropane of volume fraction 40% is added, it is anti-after persistent oscillation
After answering 5h, filter, in distilled water flushing to filtrate without epoxy root and hydroxyl, you can the chitosan magnetic activated is micro-
Ball.
The detection of epoxy root：The filtrate of 2mL is taken, 1 drop phenolphthalein is instilled, the Na of the 1.3moL/L of same volume is added2S2O3,
Oscillation makes its reaction, if solution does not redden, epoxy group wash clean.Detection hydroxy：The filtrate of 2mL is taken, 1 drop phenol is added
Phthalein, if solution does not redden, hydroxyl wash clean.
The activation grade result of the chitosan magnetic micro-sphere is shown in table 2.
Embodiment 7
It weighs in the chitosan magnetic micro-sphere conical flask that 0.25g embodiments 5 are prepared, is added 25mL2.0mol/L's
The NaBH of NaOH solution and 100mL20g/L4, 60min is vibrated in thermostatic control oscillator vibration, reaction temperature is 35 DEG C, then with body
The dimethyl sulfoxide (DMSO) of fraction 45% is activating solvent, and the epoxychloropropane of volume fraction 30% is added, and continues oscillating reactions 3h
Afterwards, filter, in distilled water flushing to filtrate without epoxy root and hydroxyl, you can the chitosan magnetic micro-sphere activated.
The detection of epoxy root：The filtrate of 2mL is taken, 1 drop phenolphthalein is instilled, the Na of the 1.3moL/L of same volume is added2S2O3,
Oscillation makes its reaction, if solution does not redden, epoxy group wash clean.Detection hydroxy：The filtrate of 2mL is taken, 1 drop phenol is added
Phthalein, if solution does not redden, hydroxyl wash clean.
The activation grade result of the chitosan magnetic micro-sphere is shown in table 2.
Embodiment 8
It weighs in the chitosan magnetic micro-sphere conical flask that 0.25g embodiments 5 are prepared, is added 20mL2.2mol/L's
The NaBH of NaOH solution and 100mL10g/L4, 45min is vibrated in thermostatic control oscillator vibration, reaction temperature is 50 DEG C, then with body
The dimethyl sulfoxide (DMSO) of fraction 40% is activating solvent, and the epoxychloropropane of volume fraction 35% is added, and continues oscillating reactions
After 3.5h, filter, in distilled water flushing to filtrate without epoxy root and hydroxyl, you can the chitosan magnetic activated is micro-
Ball.
The detection of epoxy root：The filtrate of 2mL is taken, 1 drop phenolphthalein is instilled, the Na of the 1.3moL/L of same volume is added2S2O3,
Oscillation makes its reaction, if solution does not redden, epoxy group wash clean.Detection hydroxy：The filtrate of 2mL is taken, 1 drop phenol is added
Phthalein, if solution does not redden, hydroxyl wash clean.
The activation grade result of the chitosan magnetic micro-sphere is shown in table 2.
Embodiment 9
It weighs in the chitosan magnetic micro-sphere conical flask that 0.25g embodiments 5 are prepared, is added 31mL1.8mol/L's
The NaBH of NaOH solution and 50mL25g/L4, 30min is vibrated in thermostatic control oscillator vibration, reaction temperature is 60 DEG C, then with body
The dimethyl sulfoxide (DMSO) of fraction 35% is activating solvent, and the epoxychloropropane of volume fraction 45% is added, and continues oscillating reactions
After 4.5h, filter, in distilled water flushing to filtrate without epoxy root and hydroxyl, you can the chitosan magnetic activated is micro-
Ball.
The detection of epoxy root：The filtrate of 2mL is taken, 1 drop phenolphthalein is instilled, the Na of the 1.3moL/L of same volume is added2S2O3,
Oscillation makes its reaction, if solution does not redden, epoxy group wash clean.Detection hydroxy：The filtrate of 2mL is taken, 1 drop phenol is added
Phthalein, if solution does not redden, hydroxyl wash clean.
The activation grade result of the chitosan magnetic micro-sphere is shown in table 2.
Embodiment 10
It weighs in the chitosan magnetic micro-sphere conical flask that 0.25g embodiments 5 are prepared, is added 25mL 2.5mol/L's
The NaBH4 of NaOH solution and 125mL 20g/L vibrates 45min in thermostatic control oscillator vibration, and reaction temperature is 70 DEG C, then with
The dimethyl sulfoxide (DMSO) of volume fraction 30% is activating solvent, and the epoxychloropropane of volume fraction 60% is added, and continues oscillating reactions
After 4h, filter, in distilled water flushing to filtrate without epoxy root and hydroxyl, you can the chitosan magnetic micro-sphere activated.
The detection of epoxy root：The filtrate of 2mL is taken, 1 drop phenolphthalein is instilled, the Na of the 1.3moL/L of same volume is added2S2O3,
Oscillation makes its reaction, if solution does not redden, epoxy group wash clean.Detection hydroxy：The filtrate of 2mL is taken, 1 drop phenol is added
Phthalein, if solution does not redden, hydroxyl wash clean.
The activation grade result of the chitosan magnetic micro-sphere is shown in table 2.
The activation grade measurement result of 2 chitosan magnetic micro-sphere of table
Embodiment 11
(1) coupling of chitosan magnetic micro-sphere：The arrowhead trypsin inhibitor of 40mg is taken to be dissolved in 3mL pH=7.0's
Albumen enzyme buffer liquid is obtained in Tris-HCl buffer solutions, and it is micro- to add it to the chitosan magnetic that 0.25g is activated by embodiment 10
In ball, 45 DEG C of water bath with thermostatic control oscillation coupling 5h take out, magnet separation, washing obtains the magnetic of coupling arrowhead trypsin inhibitor
Property chitosan microball.
Raffinate is collected, the volume of raffinate is measured, the amount of arrowhead trypsin inhibitor in raffinate is measured according to BAPNA methods,
And Conjugate ratio is calculated.The Conjugate ratio result of the magnetic microsphere is shown in table 3.
(2) immobilization of earthworm fibrinolysin：Earthworm fibrinolysin extract is dissolved and determined with the phosphate buffer of pH=8.0
Hold to 50mL, a concentration of 10,000 U/mL of earthworm fibrinolysin crude enzyme liquid, 1.25mL is taken to be added to 0.25g coupling arrowhead trypsase
In the chitosan magnetic micro-sphere of inhibitor, 1.5h are vibrated in 40 DEG C of waters bath with thermostatic control, take out, repeatedly with deionized water and phosphate buffer
The chitosan magnetic micro-sphere crude product for washing immobilization earthworm fibrinolysin, until inspection does not measure albumen in cleaning solution, magnet separation
Collect the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
Cleaning solution is collected, the activity of earthworm fibrinolysin in stoste and cleaning solution is measured with fibrin plate method, using admittedly
Enzyme activity difference calculates immobilized enzyme before and after fixedization.
The enzyme activity of immobilised enzymes is shown in table 4.
Embodiment 12
(1) coupling of chitosan magnetic micro-sphere：The arrowhead trypsin inhibitor of 40mg is taken to be dissolved in 5mL pH=8.0's
Albumen enzyme buffer liquid is obtained in Tris-HCl, is added it in the chitosan magnetic micro-sphere that 0.3g is activated by embodiment 10,50
DEG C water bath with thermostatic control oscillation coupling 4.5h, takes out, magnet separation, washing obtains the magnetic crust of coupling arrowhead trypsin inhibitor
Glycan microballoon.
Raffinate is collected, the volume of raffinate is measured, the amount of arrowhead trypsin inhibitor in raffinate is measured according to BAPNA methods,
And Conjugate ratio is calculated.The Conjugate ratio result of the magnetic microsphere is shown in table 3.
(2) immobilization of earthworm fibrinolysin：Earthworm fibrinolysin extract is dissolved and determined with the phosphate buffer of pH=7.0
Hold to 25ml, a concentration of 20,000 U/mL of earthworm fibrinolysin crude enzyme liquid, 2.25mL is taken to be added to 0.75g coupling arrowhead trypsase suppressions
In the chitosan magnetic micro-sphere of preparation, 2.0h is vibrated in 35 DEG C of waters bath with thermostatic control, takes out, and the phosphoric acid for being 6-8 with deionized water and pH is slow
Fliud flushing washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until inspection does not measure albumen in cleaning solution,
Magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
Cleaning solution is collected, the activity of earthworm fibrinolysin in stoste and cleaning solution is measured with fibrin plate method, using admittedly
Enzyme activity difference calculates immobilized enzyme before and after fixedization.
The enzyme activity of immobilised enzymes is shown in table 4.
Embodiment 13
(1) coupling of chitosan magnetic micro-sphere：The arrowhead trypsin inhibitor of 50mg is taken to be dissolved in 4mLpH=7.5's
Albumen enzyme buffer liquid is obtained in Tris-HCl, is added it in the chitosan magnetic micro-sphere that 0.2g is activated by embodiment 10,60
DEG C water bath with thermostatic control oscillation coupling 5.5h, takes out, magnet separation, washing obtains the magnetic crust of coupling arrowhead trypsin inhibitor
Glycan microballoon.
Raffinate is collected, the volume of raffinate is measured, the amount of arrowhead trypsin inhibitor in raffinate is measured according to BAPNA methods,
And Conjugate ratio is calculated.
The Conjugate ratio result of the magnetic microsphere is shown in table 3.
(2) immobilization of earthworm fibrinolysin：Earthworm fibrinolysin extract is dissolved and determined with the phosphate buffer of pH=7.5
Hold to 50mL, a concentration of 1.5 ten thousand U/mL of earthworm fibrinolysin crude enzyme liquid, 3mL is taken to be added to 0.5g coupling arrowhead trypsase suppressions
In the chitosan magnetic micro-sphere of preparation, 2.5h is vibrated in 30 DEG C of waters bath with thermostatic control, takes out, and the phosphoric acid for being 6-8 with deionized water and pH is slow
Fliud flushing washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until inspection does not measure albumen in cleaning solution,
Magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
Cleaning solution is collected, the activity of earthworm fibrinolysin in stoste and cleaning solution is measured with fibrin plate method, using admittedly
Enzyme activity difference calculates immobilized enzyme before and after fixedization.
The enzyme activity of immobilised enzymes is shown in table 4.
Embodiment 14
(1) coupling of chitosan magnetic micro-sphere：The arrowhead trypsin inhibitor of 45mg is taken to be dissolved in 5mL pH=8.5's
Albumen enzyme buffer liquid is obtained in Tris-HCl buffer solutions, adds it to the chitosan magnetic micro-sphere that 0.2g is activated by embodiment 10
In, 50 DEG C of water bath with thermostatic control oscillation coupling 4.0h take out, magnet separation, washing obtains the magnetic of coupling arrowhead trypsin inhibitor
Property chitosan microball.
Raffinate is collected, the volume of raffinate is measured, the amount of arrowhead trypsin inhibitor in raffinate is measured according to BAPNA methods,
And Conjugate ratio is calculated.
The Conjugate ratio result of the magnetic microsphere is shown in table 3.
(2) immobilization of earthworm fibrinolysin：Earthworm fibrinolysin extract is dissolved and determined with the phosphate buffer of pH=6.5
Hold to 50ml, a concentration of 20,000 U/mL of earthworm fibrinolysin crude enzyme liquid, 1.0mL is taken to be added to 0.5g coupling arrowhead trypsase suppressions
In the chitosan magnetic micro-sphere of preparation, 0.5h is vibrated in 60 DEG C of waters bath with thermostatic control, takes out, and the phosphoric acid for being 6-8 with deionized water and pH is slow
Fliud flushing washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until inspection does not measure albumen in cleaning solution,
Magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
Cleaning solution is collected, the activity of earthworm fibrinolysin in stoste and cleaning solution is measured with fibrin plate method, using admittedly
Enzyme activity difference calculates immobilized enzyme before and after fixedization.
The enzyme activity of immobilised enzymes is shown in table 4.
Embodiment 15
(1) coupling of chitosan magnetic micro-sphere：The arrowhead trypsin inhibitor of 40mg is taken to be dissolved in 5mL pH=9.0's
Albumen enzyme buffer liquid is obtained in Tris-HCl buffer solutions, adds it to the chitosan magnetic micro-sphere that 0.2g is activated by embodiment 10
In, 30 DEG C of water bath with thermostatic control oscillation coupling 7h take out, and magnet separation, washing collects raffinate, measures the volume of raffinate, according to
BAPNA methods measure the amount of arrowhead trypsin inhibitor in raffinate, and Conjugate ratio is calculated.
The Conjugate ratio result of the magnetic microsphere is shown in table 3.
(2) immobilization of earthworm fibrinolysin：Earthworm fibrinolysin extract is dissolved and determined with the phosphate buffer of pH=6.0
Hold to 50ml, a concentration of 1.5 ten thousand U/mL of earthworm fibrinolysin crude enzyme liquid, 5.0mL is taken to be added to 1g coupling arrowhead trypsase suppressions
In the chitosan magnetic micro-sphere of preparation, 3.0h is vibrated in 45 DEG C of waters bath with thermostatic control, takes out, and the phosphoric acid for being 6-8 with deionized water and pH is slow
Fliud flushing washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until inspection does not measure albumen in cleaning solution,
Magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization.
Cleaning solution is collected, the activity of earthworm fibrinolysin in stoste and cleaning solution is measured with fibrin plate method, using admittedly
Enzyme activity difference calculates immobilized enzyme before and after fixedization.
The enzyme activity of immobilised enzymes is shown in table 4.
Table 3 is coupled the chitosan magnetic micro-sphere Conjugate ratio of arrowhead trypsin inhibitor
Table 4 is used for the magnetic microsphere enzyme activity determination result of earthworm fibrinolysin immobilization
Embodiment 16
Measure immobilization earthworm fibrinolysin pH stability
The magnetic microsphere and earthworm fibrinolysin for earthworm fibrinolysin immobilization of the present invention of identical enzyme concentration is dissociated
Liquid is respectively 5.5,6.5,7.5,8.5,9.5 lower 37 DEG C of water bath processing 30min in pH, measures enzyme activity, untreated enzyme activity is
100%.The pH stability diagrams of resolvase and immobilization earthworm fibrinolysin are obtained, as shown in Figure 4.Immobilization earthworm is fine as seen from the figure
The resistance to acid and alkali of lyase is good compared with resolvase, and for stability range also compared with pH wide, resolvase is to have most highly active at 7.5 in pH,
Immobilised enzymes is to have most highly active at 7.0 in pH, this is that its conformation is changed after enzyme is fixed on carrier so that pH is steady
It is qualitative to change.
Measure immobilization earthworm fibrinolysin storage stability
The magnetic microsphere and earthworm fibrinolysin for earthworm fibrinolysin immobilization of the present invention of identical enzyme concentration is dissociated
Liquid is placed in 4 DEG C of refrigerators and preserves, and is sampled every 5d and surveys enzyme activity, until 30d.Enzyme activity is measured, untreated enzyme activity is 100%.
The storage stability figure of resolvase and immobilization earthworm fibrinolysin is obtained, as shown in Figure 5.
As shown in Figure 5, with the variation of storage time, the activity of resolvase and immobilization earthworm fibrinolysin all gradually under
Drop, at first 5 days of storage, the activity of resolvase and immobilization earthworm fibrinolysin was without significant change, when storage 10 days, dissociated
The opposite enzyme activity of enzyme is 80%, and the enzyme activity of immobilization earthworm fibrinolysin still has 98%, and the activity of resolvase drastically declines later,
When by 30 days, only retain 32%, and the activity of immobilization earthworm fibrinolysin still has 82%, the results showed that, earthworm fibrinolysin is through solid
Surely change rear stability to be greatly improved.
Measure immobilization earthworm fibrinolysin thermal stability
The magnetic microsphere and earthworm fibrinolysin for earthworm fibrinolysin immobilization of the present invention of identical enzyme concentration is dissociated
Liquid water-bath 30min at 30,40,50,55,60 DEG C, measures enzyme activity, and not thermally treated enzyme activity is 100%., calculate residual enzyme
It lives than q,
In formula：Enzyme activity, U when 0min under c0- different temperatures；
Enzyme activity when ct-tmin, U.
The thermal stability figure of resolvase and immobilization earthworm fibrinolysin is obtained, as shown in Figure 6.It will be appreciated from fig. 6 that resolvase and
It is similar that immobilization earthworm fibrinolysin with respect to enzyme activity varies with temperature trend, and as temperature increases, opposite enzyme activity declines, but in phase
Under the conditions of synthermal, the relative activity of the relative activity specific ionization earthworm fibrinolysin of immobilization earthworm fibrinolysin is high, illustrates through magnetic
The earthworm fibrinolysin thermal stability of earthworm fibrinolysin specific ionization state after property is chitin microspheric immobilized is good.
Claims (8)
1. a kind of preparation method of magnetic microsphere for earthworm fibrinolysin immobilization, it is characterised in that：It includes the following steps：
The coupling of chitosan magnetic micro-sphere：Arrowhead trypsin inhibitor is dissolved in the buffer solution of pH 7 ~ 9 to obtain protease slow
Fliud flushing, then add it in the chitosan magnetic micro-sphere through overactivation, water-bath oscillation coupling 4 ~ 7 h, vibration temperature 30-
60 DEG C, obtain the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor；
The immobilization of earthworm fibrinolysin：Earthworm fibrinolytic is added in the chitosan magnetic micro-sphere for being coupled arrowhead trypsin inhibitor again
The thick enzyme solutions of enzyme vibrate curing reaction 0.5-3 h in 30-60 DEG C of water bath with thermostatic control shaking table；After reaction, with deionized water and
PH is that the phosphate buffer of 6-8 washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until being examined in cleaning solution
Until not measuring albumen, magnet separates and collects the magnetic microsphere that can be obtained for earthworm fibrinolysin immobilization, and enzymatic activity is
40-60 U/mg, the stability between pH 6.5 ~ 8.0 are maintained at 78% or more, and storage time is fine for earthworm in 30 days
The enzymatic activity of the magnetic microsphere of lyase immobilization is 82% or more.
2. the preparation method of the magnetic microsphere according to claim 1 for earthworm fibrinolysin immobilization, it is characterised in that：
The step（1）Middle albumen enzyme buffer liquid and the liquid-solid ratio of the chitosan magnetic micro-sphere of activation are 10 ~ 25:1, water-bath oscillation coupling
4~6 h。
3. the preparation method of the magnetic microsphere according to claim 1 for earthworm fibrinolysin immobilization, it is characterised in that：
The step（1）In buffer solution be Tris-HCl buffer solutions.
4. the preparation method of the magnetic microsphere according to claim 1 for earthworm fibrinolysin immobilization, it is characterised in that：
The step（2）In, it is 1-2 that 1 ~ 5 mL enzyme activity, which is then added, in the chitosan magnetic micro-sphere of every gram of coupling arrowhead trypsin inhibitor
The thick enzyme solutions of earthworm fibrinolysin of ten thousand U/mL.
5. the preparation method of the magnetic microsphere according to claim 1 for earthworm fibrinolysin immobilization, it is characterised in that：
The preparation of the chitosan magnetic micro-sphere of the activation is that NaOH solution and NaBH is added in chitosan magnetic micro-sphere4Solution, 30
30 ~ 40 min are vibrated in ~ 60 DEG C of water-baths, and it is activator to add dimethyl sulfoxide (DMSO), add the ring that volume fraction is 10% ~ 60%
Oxygen chloropropane after oscillation activates 2 ~ 10 h, filters, is washed with distilled water the chitosan magnetic micro-sphere activated.
6. the preparation method of the magnetic microsphere according to claim 5 for earthworm fibrinolysin immobilization, it is characterised in that：
The weight ratio of the chitosan magnetic micro-sphere and NaOH solution is 1:5 ~ 10, chitosan magnetic micro-sphere and NaBH4The weight of solution
Than being 1:4~12；Every gram of chitosan magnetic micro-sphere adds volume fraction to be 40% ~ 60% 1.5 ~ 9 mL of dimethyl sulfoxide (DMSO), epoxy chloropropionate
Alkane is 1 ~ 12 mL.
7. the preparation method of the magnetic microsphere according to claim 5 for earthworm fibrinolysin immobilization, it is characterised in that：
A concentration of 1.2 ~ 2.0 mol/L, NaBH of the NaOH solution4A concentration of 5 ~ 10 g/L of solution, the volume of epoxychloropropane
Score is 30% ~ 50%, 2 ~ 6 h of oscillation activation at 30 ~ 50 DEG C.
8. special according to the preparation method of any magnetic microsphere for earthworm fibrinolysin immobilization of claim 5 or 7
Sign is：The preparation method of the chitosan magnetic micro-sphere：
（1）According to weight ratio it is 30 ~ 50 by atoleine and Span-80:1 mixing stirs 1 ~ 2 h at 30 ~ 50 DEG C and is prepared into oil
Phase；
（2）A certain amount of chitosan is weighed, is dissolved with 3 ~ 5% acetum, obtain a concentration of 5 after chitosan is completely dissolved ~
Fe is added in the chitosan solution of 10 mg/mL3O4Powder, 10 ~ 20 min of ultrasonic disperse, is made water phase；
（3）According to volume ratio it is 1 by water phase and oil phase:5 ~ 6, water phase is slowly added in oil phase, 0.5 ~ 1.0 h of stirring makes molten
Liquid forms emulsion system；
（4）The glutaraldehyde that mass fraction is 25% is added dropwise into emulsion system again, continues 1 ~ 2 h of stirring, extremely with NaOH solution tune pH
Stop reaction after 9 ~ 10, the reaction was continued 1 ~ 2 h, magnet separation, with petroleum ether, ethyl alcohol and deionized water are washed to neutrality, vacuum
It is dry to get to chitosan magnetic micro-sphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510701434.9A CN105200029B (en) | 2015-10-27 | 2015-10-27 | A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510701434.9A CN105200029B (en) | 2015-10-27 | 2015-10-27 | A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105200029A CN105200029A (en) | 2015-12-30 |
| CN105200029B true CN105200029B (en) | 2018-10-16 |
Family
ID=54947991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510701434.9A Active CN105200029B (en) | 2015-10-27 | 2015-10-27 | A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105200029B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389916A (en) * | 2017-06-26 | 2017-11-24 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method of CD133 immunomagnetic beadses |
| CN114377626A (en) * | 2021-12-29 | 2022-04-22 | 苏州百奥吉生物科技有限公司 | Novel composite polysaccharide protein A magnetic bead and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673367A (en) * | 2005-03-23 | 2005-09-28 | 河北大学 | A method for separating earthworm plasminogen single component |
| CN103289981A (en) * | 2013-06-24 | 2013-09-11 | 广州润虹医药科技有限公司 | A preparation method for lyophilized powder of earthworm fibrinolytic enzymes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006023530A2 (en) * | 2004-08-16 | 2006-03-02 | Massachusetts Institute Of Technology | Compositions and methods for enhancing structural and functional nervous system reorganization and recovery |
-
2015
- 2015-10-27 CN CN201510701434.9A patent/CN105200029B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673367A (en) * | 2005-03-23 | 2005-09-28 | 河北大学 | A method for separating earthworm plasminogen single component |
| CN103289981A (en) * | 2013-06-24 | 2013-09-11 | 广州润虹医药科技有限公司 | A preparation method for lyophilized powder of earthworm fibrinolytic enzymes |
Non-Patent Citations (6)
| Title |
|---|
| Enzyme encapsulation in magnetic chitosan-Fe3O4 microparticles;Tales Alexandre Costa-Silva 等;《J Microencapsul》;20140901;第1-6页，尤其是第2页左栏Enzyme immobilisation onto magnetic chitosan部分 * |
| Preparation of Magnetic Chitosan Nanoparticles As Support for Cellulase Immobilization;Limin Zang 等;《Ind. Eng. Chem. Res.》;20141231;第3448-3454页 * |
| 京尼平交联壳聚糖微球的制备及理化性质;熊华 等;《南昌大学学报（工科版）》;20131231;第35卷(第4期);第307-312页，尤其是第308页1.2.1壳聚糖微球的制备方法 * |
| 壳聚糖固定化蚯蚓纤溶酶及其性质;武金霞 等;《河北大学学报（自然科学版）》;20080331;第28卷(第2期);第193-197页，尤其是材料与方法部分 * |
| 磁性蚯蚓纤溶酶脂质体的制备;静天玉 等;《河北大学学报（自然科学版）》;19991231;第19卷(第4期);第360-363页，尤其是摘要、讨论部分 * |
| 蚯蚓纤溶酶的分离纯化及抗栓活性研究;何执中 等;《中国生化药物杂志》;20011231;第22卷(第6期);第284-286页，尤其是摘要、第286页右栏 讨论部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105200029A (en) | 2015-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102964539B (en) | The preparation method of porous magnetic polystyrene microsphere | |
| Penefsky et al. | [19] Extraction of water-soluble enzymes and proteins from membranes | |
| Shu et al. | Conjugation of a water-soluble gadolinium endohedral fulleride with an antibody as a magnetic resonance imaging contrast agent | |
| CN102219800B (en) | Organic substituted boric acid ester, boron affinity functional material using organic substituted boric acid ester as functional monomer as well as preparation and application of organic substituted boric acid ester | |
| CN102603896B (en) | Nano graphene oxide protein complex, preparation method and applications thereof | |
| CN105200029B (en) | A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization | |
| CN103131728B (en) | The Graphene genophore of a kind of multifunction and gene transfection agent based on this genophore and preparation method thereof | |
| CN102659979A (en) | Preparation method of double-hydrophilic temperature response polymer | |
| CN105164152A (en) | Gla domains as targeting agents | |
| CN102703411B (en) | Aramagnetic epoxy group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof | |
| CN105200030B (en) | A kind of preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin | |
| CN103361267B (en) | Preparation method and application of micro-enzyme reactor based on magnetic functionalized graphene oxide | |
| CN105203752A (en) | Magnetic platelet adsorption particle and preparation method thereof | |
| Tarragó et al. | Identification by 19F NMR of traditional Chinese medicinal plants possessing prolyl oligopeptidase inhibitory activity | |
| CN104945510A (en) | Sodium alginate immunomagnetic beads as well as preparation method and application thereof | |
| CN106093377A (en) | A kind of test kit for the quick coupling of albumen, antibody, enzyme and nucleic acid and preparation method thereof | |
| Li et al. | Metal–Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis | |
| Qiao et al. | Fabricating bimetal organic material capsules with a commodious microenvironment and synergistic effect for glycosyltransferase | |
| Shi et al. | Hierarchical magnetic nanoparticles for highly effective capture of small extracellular vesicles | |
| CN105200031B (en) | A kind of magnetic microsphere of immobilization earthworm fibrinolysin and preparation method thereof | |
| CN101716495A (en) | Sulfonylurea weedicide molecularly imprinted polymer microballoon and preparation method thereof | |
| CN104498464B (en) | A kind of method extracting white-browed snake venom blood coagulation enzyme from agkistrodon halys ussuriensis snake venom | |
| CN101284660A (en) | Fullerene water solution and method for making same | |
| CN105823764A (en) | Calix[4]arene adenine derivative-oxidized graphene compound, preparation method and applications thereof | |
| CN103320420A (en) | Magnetic crosslinking lipase aggregation as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |