CN105200030B - A kind of preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin - Google Patents
A kind of preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin Download PDFInfo
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- CN105200030B CN105200030B CN201510701431.5A CN201510701431A CN105200030B CN 105200030 B CN105200030 B CN 105200030B CN 201510701431 A CN201510701431 A CN 201510701431A CN 105200030 B CN105200030 B CN 105200030B
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Abstract
The invention discloses a kind of preparation methods of the magnetic microsphere of immobilization earthworm fibrinolysin, first pig lung Aprotinin is dissolved in the buffer of pH7~9 and obtains albumen enzyme buffer liquid, it is added it in the chitosan magnetic micro-sphere through overactivation again, obtains the chitosan magnetic micro-sphere of coupling pig lung Aprotinin after 4~8h of water-bath oscillation coupling；It is added into the thick enzyme solutions of earthworm fibrinolysin again and vibrates curing reaction 0.5-3h in 30-60 DEG C of water bath with thermostatic control shaking table；After reaction, washed repeatedly into cleaning solution inspection do not measure albumen until, magnet separates and collects the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained, enzymatic activity 15-30U/mg.The present invention is coupled with magnetic microsphere using pig lung Aprotinin 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 separate 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 technique
Magnetic macromolecular microsphere is a kind of novel magnetic materials that developed recently gets up, and is by proper method by magnetic nothing
What machine particle was formed in conjunction with organic polymer has certain magnetic and special construction complex microsphere.Magnetic composite microsphere is not only
Numerous characteristics with common polymer microsphere also have 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 function under the action of an external magnetic field
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 partial size wider distribution legal, that these methods are prepared, it is in irregular shape.
Aprotinin is a kind of serpin, is the trypsase extracted from ox pancreas by Kunitz earliest
Inhibitor, and the kallikrein inhibitor that extracts from the tissue such as ox lung, pancreas, salivary gland, are present in that animal device is dirty, male
In urine, its chemical composition and structure of the Aprotinin of separate sources is different.The Aprotinin extracted from ox lung or ox pancreas,
Relative molecular weight is 6200, the basic polypeptide being made of 58 amino acid residues, its primary structure and secondary structure is
It illustrates, intramolecular has 3 pairs of disulfide bond, and entire molecule is in pears type, and activated centre is lysine, positioned at the top of molecule, due to two
Sulfide linkage content is high, and specific space structure can not only keep in the solution stable, moreover it is possible to be subjected to quite violent processing without
It is destroyed.It is all very stable to high temperature, acid, alkali and enzyme, and 100 DEG C are heated in diluted acid, is heated in 2.5% trichloroacetic acid
80 DEG C of activity are not lost yet.
Earthworm fibrinolysin is also known as Lumbrokinase, is extracted out of earthworm body both with the fibre of direct solution fibrin
Antiplasmin activity, and the active class protein of Plasminogen activation with class urokinase can dissolve outmoded thrombus but also suppression
New thrombosis is made, it is to prevent and treat thrombus disease that experiment, which shows that the enzyme has anticoagulant, molten fine, improvement hemorheology properties,
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 thrombosis, hemiplegia, myocardial infarction
Have the advantages that quick, curative effect is good, oral effectively and safer.A large number of studies show that no matter earthworm fibrinolysin is injected intravenously
Or oral administration, thrombolytic effect is all had, is a kind of thrombotic disease therapeutic agent 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, the fibrinolysin purity is high that this method is extracted, but due to purifying work
Sequence complexity is at 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 open up a road for the utilization of earthworm resource.
There is presently no the reports that immobilization earthworm fibrinolysin is used for about pig lung Aprotinin coupled chitosan magnetic microsphere.
Summary of the invention
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 pig
Lung Aprotinin is aglucon, is coupled with magnetic microsphere, with isolating and purifying for earthworm fibrinolysin, improves earthworm fibrinolysin
Selectivity is isolated and purified, and magnetic microsphere is easy to separate with earthworm fibrinolysin.
Technical scheme is as follows:
A kind of magnetic microsphere of immobilization earthworm fibrinolysin, enzymatic activity 15-30U/mg, between pH 6.5~8.0
Stability be maintained at 70% or more, storage time enzymatic activity of the magnetic microsphere of immobilization earthworm fibrinolysin in 30 days is
78% or more.
The preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin of the invention comprising following steps:
(1) coupling of chitosan magnetic micro-sphere: pig lung Aprotinin is dissolved in and obtains protease in the buffer of pH 7~9 and delays
Fliud flushing, then add it in the chitosan magnetic micro-sphere through overactivation, water-bath oscillation 4~8h of coupling, vibration temperature 45~60
DEG C, after obtain coupling pig lung Aprotinin chitosan magnetic micro-sphere；
(2) earthworm fibrinolytic the immobilization of earthworm fibrinolysin: is added in the chitosan magnetic micro-sphere for being coupled pig lung Aprotinin again
The thick enzyme solutions of enzyme vibrate curing reaction 1-4h in 30-60 DEG C of water bath with thermostatic control shaking table；After reaction, it is with deionized water and pH
7~9 phosphate buffer washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until detecting not in cleaning solution
Out until albumen, magnet separates and collects the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained.
As a preferred option of the technical scheme, the buffer in the step (1) is boric acid-borate buffer solution.The protease
The liquid-solid ratio of buffer and the chitosan magnetic micro-sphere of activation is 100~150: 1,4~6h of water-bath oscillation coupling.This technique item
The chitosan magnetic micro-sphere performance for the coupling pig lung Aprotinin that part obtains is especially good.
As a preferred option of the technical scheme, in the step (2), the chitosan magnetic micro-sphere of every gram of coupling pig lung Aprotinin is then
The thick enzyme solutions of earthworm fibrinolysin that 1~5ml enzyme activity is ten thousand U/mL of 0.8-2 are added.
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 30min in 40~70 DEG C of water-baths, and adding dimethyl sulfoxide is activator, and adding volume fraction is
20%~60% epoxychloropropane after oscillation activates 4~8h, filters, is washed with distilled water the magnetic crust that activation can be obtained
Glycan microballoon.
As the further preferred of technical solution, the weight ratio of the chitosan magnetic micro-sphere and NaOH solution is 1: 4~
9, chitosan magnetic micro-sphere and NaBH4The weight ratio of solution is 1: 4~11；Every gram of chitosan magnetic micro-sphere adds the volume fraction to be
40%~60% 3~8mL of dimethyl sulfoxide, epoxychloropropane are 3~10mL.
As the further preferred of technical solution, the concentration of NaOH solution is 1.0~2.4mol/L, NaBH4Solution it is dense
Degree is 5~10g/L, and the volume fraction of epoxychloropropane is 20%~60%, magnetic when 4~10h of oscillation activation at 30~70 DEG C
The activation effect of property chitosan microball is best.
The preparation method of chitosan magnetic micro-sphere in the present invention:
It (1) is 20~40: 1 mixing according to weight ratio by atoleine and Span-80, in 30~50 DEG C of stirring 1~2h systems
For at oily phase；
(2) a certain amount of chitosan is weighed, is dissolved with 3~5% acetum, obtains shell 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 with oily phase by water phase, water phase is slowly added in oily 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, chitosan magnetic micro-sphere can be obtained in vacuum drying.
As a preferred option of the technical scheme, ferroso-ferric oxide used in the present invention is Nano grade, and the chitosan of preparation is molten
Liquid concentration is 5~10mg/mL, and speed of agitator is 600~700rpm/min, the grain for the chitosan magnetic micro-sphere being prepared
Diameter is minimum, is 215nm.
Compared with the prior art, the invention has the following advantages:
1. preparation method of the invention is simple, operation is easy, treatment process is mild, not high to equipment requirement.
2. the present invention using pig lung Aprotinin as aglucon, is coupled, with for earthworm fibrinolysin with chitosan magnetic micro-sphere
It isolates and purifies, improves the selectivity that isolates and purifies of earthworm fibrinolysin, and chitosan magnetic micro-sphere is easy to and earthworm fibrinolysin
Separation.
3. earthworm fibrinolysin of the invention is fixed on magnetic microsphere, because microballoon has magnetism, in the effect of externally-applied magnetic field
Under, fibrinolysin can soon be separated 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 70% or more, and thermal stability is high compared with resolvase,
Detailed description of the invention
Fig. 1 is 7000 times of electron microscope of chitosan magnetic micro-sphere of the invention；
Fig. 2 is 100,000 times of electron microscope of chitosan magnetic micro-sphere of the invention；
Fig. 3 is the infrared spectrum analysis figure of chitosan magnetic micro-sphere of the 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 diagram 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
The present invention is further illustrated 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 preparation method of the invention under concept thereof of the 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, measurement pass through the side recorded below
Method is measured.
(a) particle diameter distribution
It disperses chitosan magnetic micro-sphere in ethyl alcohol, a few drop dispersing agent Tween-20s is 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 washing is 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 titration front and back standard hydrochloric acid.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 oily 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 that concentration is 5mg/ml, is then added in oily 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
Chitosan magnetic micro-sphere can be obtained in deionized water washing, vacuum drying.
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 oily 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 that concentration is 6.5mg/ml, is then added in oily phase,
After 400rpm stirring half an hour makes 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 after the reaction was continued 2h with the NaOH solution tune pH to 9 or so of 1.0mol/L
Chitosan magnetic micro-sphere can be obtained in pure and mild deionized water washing, vacuum drying.
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
Oily 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 that concentration is 8.1mg/ml, is then added in oily phase,
After 500rpm stirring half an hour makes 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
Chitosan magnetic micro-sphere can be obtained in oily ether, ethyl alcohol and deionized water washing, vacuum drying.
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 oily 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 that concentration is 10mg/ml, is then added in oily phase,
After 600rpm stirring half an hour makes 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 after the reaction was continued 1h with the NaOH solution tune pH to 10 or so of 1.0mol/L
Chitosan magnetic micro-sphere can be obtained in pure and mild deionized water washing, vacuum drying.
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 oily phase, after 700rpm stirring half an hour makes 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, magnetic crust is can be obtained in ethyl alcohol and deionized water washing, vacuum drying
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 Fe3O4Nanoparticle
The composite particles partial 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 bigger specific surface area, 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 partial size of 1 chitosan magnetic micro-sphere of table
Embodiment 6
Chitosan magnetic micro-sphere that 0.25g embodiment 5 is prepared is weighed in conical flask, 25mL 1.5mol/L is 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 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, with distilled water flushing into filtrate without epoxy root and hydroxyl, the chitosan magnetic that activation can be obtained is micro-
Ball.
The detection of epoxy root: taking the filtrate of 2mL, instills 1 drop phenolphthalein, 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: taking the filtrate of 2mL, and 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 embodiment 5 is 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 of fraction 45% is activating solvent, and the epoxychloropropane of volume fraction 30% is added, and continues oscillating reactions 3h
Afterwards, it filters, the chitosan magnetic micro-sphere of activation can be obtained without epoxy root and hydroxyl into filtrate with distilled water flushing.
The detection of epoxy root: taking the filtrate of 2mL, instills 1 drop phenolphthalein, 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: taking the filtrate of 2mL, and 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 embodiment 5 is 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 of fraction 40% is activating solvent, and the epoxychloropropane of volume fraction 35% is added, and continues oscillating reactions
After 3.5h, filter, with distilled water flushing into filtrate without epoxy root and hydroxyl, the chitosan magnetic that activation can be obtained is micro-
Ball.
The detection of epoxy root: taking the filtrate of 2mL, instills 1 drop phenolphthalein, 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: taking the filtrate of 2mL, and 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 embodiment 5 is 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 of fraction 35% is activating solvent, and the epoxychloropropane of volume fraction 45% is added, and continues oscillating reactions
After 4.5h, filter, with distilled water flushing into filtrate without epoxy root and hydroxyl, the chitosan magnetic that activation can be obtained is micro-
Ball.
The detection of epoxy root: taking the filtrate of 2mL, instills 1 drop phenolphthalein, 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: taking the filtrate of 2mL, and 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 embodiment 5 is prepared, is added 25mL 2.5mol/L's
The NaBH of NaOH solution and 125mL 20g/L4, 45min is vibrated in thermostatic control oscillator vibration, reaction temperature is 70 DEG C, then with
The dimethyl sulfoxide of volume fraction 30% is activating solvent, and the epoxychloropropane of volume fraction 60% is added, and continues oscillating reactions
After 4h, filters, the chitosan magnetic micro-sphere of activation can be obtained without epoxy root and hydroxyl into filtrate with distilled water flushing.
The detection of epoxy root: taking the filtrate of 2mL, instills 1 drop phenolphthalein, 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: taking the filtrate of 2mL, and 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 pig lung Aprotinin of 100mg is taken to be dissolved in boric acid-boron of 20mL pH=7.0
Albumen enzyme buffer liquid is obtained in sand buffer, is added it in the chitosan magnetic micro-sphere that 0.2g is activated by embodiment 10,50
DEG C water bath with thermostatic control oscillation coupling 5h, takes out, magnet separation, washing obtains the chitosan magnetic micro-sphere of coupling pig lung Aprotinin.
Raffinate is collected, the volume of raffinate is measured, the amount of pig lung Aprotinin in raffinate is measured according to BAPNA method, and is calculated
To Conjugate ratio.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, the concentration of earthworm fibrinolysin crude enzyme liquid is 10,000 U/mL, and 1.25mL is taken to be added to 0.25g coupling pig lung Aprotinin
In chitosan magnetic micro-sphere, 1.5h is vibrated in 40 DEG C of waters bath with thermostatic control, takes out, washs fixation repeatedly with deionized water and phosphate buffer
The chitosan magnetic micro-sphere crude product for changing earthworm fibrinolysin, until into cleaning solution, inspection does not measure albumen, magnet is separated and collected
Obtain the magnetic microsphere of immobilization earthworm fibrinolysin.
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 pig lung Aprotinin of 100mg is taken to be dissolved in boric acid-boron of 30mL pH=7.5
Albumen enzyme buffer liquid is obtained in sand buffer, is added it in the chitosan magnetic micro-sphere that 0.25g is activated by embodiment 10,45
DEG C water bath with thermostatic control oscillation coupling 4h, takes out, magnet separation, washing obtains the chitosan magnetic micro-sphere of coupling pig lung Aprotinin.
Raffinate is collected, the volume of raffinate is measured, the amount of pig lung Aprotinin in raffinate is measured according to BAPNA method, and is calculated
To Conjugate ratio.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, earthworm fibrinolysin crude enzyme liquid concentration is 20,000 U/mL, and 2.25mL is taken to be added to the magnetic of 0.75g coupling pig lung Aprotinin
Property chitosan microball in, 2.0h are vibrated in 35 DEG C of waters bath with thermostatic control, take out, with deionized water and pH be 7-8 phosphate buffer repeatedly
The chitosan magnetic micro-sphere crude product for washing immobilization earthworm fibrinolysin, until into cleaning solution, inspection does not measure albumen, magnet separation
Collect the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained.
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 pig lung Aprotinin of 120mg is taken to be dissolved in boric acid-borax of 35mL pH=9
Albumen enzyme buffer liquid is obtained in buffer, is added it in the chitosan magnetic micro-sphere that 0.35g is activated by embodiment 10,55 DEG C
Water bath with thermostatic control oscillation coupling 5.5h, takes out, magnet separation, washing obtains the chitosan magnetic micro-sphere of coupling pig lung Aprotinin.
Raffinate is collected, the volume of raffinate is measured, the amount of pig lung Aprotinin in raffinate is measured according to BAPNA method, and is calculated
To Conjugate ratio.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, the concentration of earthworm fibrinolysin crude enzyme liquid is 10,000 U/mL, and 2mL is taken to be added to the magnetism of 0.5g coupling pig lung Aprotinin
In chitosan microball, 2.5h is vibrated in 30 DEG C of waters bath with thermostatic control, takes out, and the phosphate buffer for being 8-9 with deionized water and pH is washed repeatedly
The chitosan magnetic micro-sphere crude product for washing immobilization earthworm fibrinolysin, until into cleaning solution, inspection does not measure albumen, magnet separation is received
The magnetic microsphere of immobilization earthworm fibrinolysin can be obtained in collection.
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 pig lung Aprotinin of 140mg is taken to be dissolved in boric acid-boron of 50mL pH=7.0
Albumen enzyme buffer liquid is obtained in sand, is added it in the chitosan magnetic micro-sphere that 0.4g is activated by embodiment 10,60 DEG C of constant temperature
Water-bath oscillation coupling 8.0h, takes out, magnet separation, washing obtains the chitosan magnetic micro-sphere of coupling pig lung Aprotinin.
Raffinate is collected, the volume of raffinate is measured, the amount of trypsin inhibitor in raffinate is measured according to BAPNA method, and is counted
Calculation obtains Conjugate ratio.
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, the concentration of earthworm fibrinolysin crude enzyme liquid is 0.8 ten thousand U/mL, and 1.5mL is taken to be added to 0.5g coupling pig lung Aprotinin
In chitosan magnetic micro-sphere, 4.0h is vibrated in 40 DEG C of waters bath with thermostatic control, takes out, and the phosphate buffer for being 7-8 with deionized water and pH is anti-
The chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin is washed in after backwashing, until into cleaning solution, inspection does not measure albumen, magnet point
From the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained in collection.
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 pig lung Aprotinin of 120mg is taken to be dissolved in boric acid-boron of 30mL pH=8.5
Albumen enzyme buffer liquid is obtained in sand buffer, is added it in the chitosan magnetic micro-sphere that 0.25g is activated by embodiment 10,70
DEG C water bath with thermostatic control oscillation coupling 5h, takes out, and magnet separation, washing collects raffinate, measures the volume of raffinate, is surveyed according to BAPNA method
Determine the amount of pig lung Aprotinin 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, the concentration of earthworm fibrinolysin crude enzyme liquid is 1.5 ten thousand U/mL, and 2.5mL is taken to be added to the magnetic of 1g coupling pig lung Aprotinin
Property chitosan microball in, 3.0h are vibrated in 45 DEG C of waters bath with thermostatic control, take out, with deionized water and pH be 8-9 phosphate buffer repeatedly
The chitosan magnetic micro-sphere crude product for washing immobilization earthworm fibrinolysin, until into cleaning solution, inspection does not measure albumen, magnet separation
Collect the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained.
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.
The chitosan magnetic micro-sphere Conjugate ratio of the coupling pig lung Aprotinin of table 3
The magnetic microsphere enzyme activity determination result of 4 immobilization earthworm fibrinolysin of table
Embodiment 16
(1) immobilization earthworm fibrinolysin pH stability is measured
By the magnetic microsphere and earthworm fibrinolysin free liquid of the immobilization earthworm fibrinolysin of the invention of identical enzyme concentration, divide
37 DEG C of water bath processing 30min under being not 5.5,6.5,7.5,8.5,9.5 in pH, measure enzyme activity, untreated enzyme activity is
100%.The pH stability diagram of resolvase and immobilization earthworm fibrinolysin is 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 7.5 in pH, this is that its conformation is changed after enzyme to be fixed on to carrier, so that pH is steady
It is qualitative to change.
(2) immobilization earthworm fibrinolysin storage stability is measured
The magnetic microsphere of the immobilization earthworm fibrinolysin of the invention of identical enzyme concentration and earthworm fibrinolysin free liquid are set
It is saved in 4 DEG C of refrigerators, is sampled every 5d and survey enzyme activity, until 30d.Enzyme activity is measured, untreated enzyme activity is 100%.It obtains
The storage stability figure of resolvase and immobilization earthworm fibrinolysin, 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 storing 10 days, dissociated
The opposite enzyme activity of enzyme is 75%, and the enzyme activity of immobilization earthworm fibrinolysin still has 93%, and the activity of resolvase sharply declines later,
When by 30 days, only retain 32%, and the activity of immobilization earthworm fibrinolysin still has 78%, the results showed that, earthworm fibrinolysin is through solid
Surely change rear stability to be greatly improved.
(3) immobilization earthworm fibrinolysin thermal stability is measured
The magnetic microsphere of the immobilization earthworm fibrinolysin of the invention of identical enzyme concentration and earthworm fibrinolysin free liquid are existed
30, water-bath 30min at 40,50,55,60 DEG C, measures enzyme activity, and not thermally treated enzyme activity is 100%., calculate remnant enzyme activity ratio
Q,
In formula: c0Enzyme activity when 0min, U under different temperatures；
ctEnzyme activity when-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 the magnetic microsphere of immobilization earthworm fibrinolysin, it is characterised in that: it includes the following steps:
(1) coupling of chitosan magnetic micro-sphere: pig lung Aprotinin being dissolved in the buffer of pH 7 ~ 9 and obtains albumen enzyme buffer liquid,
It being added it in the chitosan magnetic micro-sphere through overactivation again, water-bath oscillation 4 ~ 8 h of coupling, vibration temperature is 40-70 DEG C,
Obtain the chitosan magnetic micro-sphere of coupling pig lung Aprotinin；
(2) it is thick that earthworm fibrinolysin the immobilization of earthworm fibrinolysin: is added in the chitosan magnetic micro-sphere for being coupled pig lung Aprotinin again
Enzyme solutions vibrate curing reaction 1-4 h in 30-60 DEG C of water bath with thermostatic control shaking table；After reaction, with deionized water and pH be 7 ~
9 phosphate buffer washs the chitosan magnetic micro-sphere crude product of immobilization earthworm fibrinolysin repeatedly, until inspection does not measure in cleaning solution
Until albumen, magnet separates and collects the magnetic microsphere that immobilization earthworm fibrinolysin can be obtained, and enzymatic activity is 15-30 U/mg,
Stability between pH 6.5 ~ 8.0 is maintained at 70% or more, the magnetic of storage time immobilization earthworm fibrinolysin in 30 days
Property microballoon enzymatic activity be 78% or more.
2. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 1, it is characterised in that: described
Albumen enzyme buffer liquid and the liquid-solid ratio of the chitosan magnetic micro-sphere of activation are 100 ~ 150:1 in step (1), water-bath oscillation coupling 4 ~
6 h。
3. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 1, it is characterised in that: described
Buffer in step (1) is boric acid-borate buffer solution.
4. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 1, it is characterised in that: described
In step (2), it is fine that the earthworm that 1 ~ 5 mL enzyme activity is ten thousand U/mL of 0.8-2 is then added in the chitosan magnetic micro-sphere of every gram of pig lung Aprotinin
The thick enzyme solutions of lyase.
5. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 1, it is characterised in that: described
The preparation of the chitosan magnetic micro-sphere of activation is that NaOH solution and NaBH4 solution is added, 30 ~ 80 in chitosan magnetic micro-sphere
30 min are vibrated in DEG C water-bath, and adding dimethyl sulfoxide is activator, add the epoxy chloropropionate that volume fraction is 20% ~ 60%
Alkane after oscillation activates 4 ~ 10 h, filters, is washed with distilled water the chitosan magnetic micro-sphere activated.
6. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 5, it is characterised in that: described
The weight ratio of chitosan magnetic micro-sphere and NaOH solution is 1:4 ~ 9, and the weight ratio of chitosan magnetic micro-sphere and NaBH4 solution is 1:
4~11；Every gram of chitosan magnetic micro-sphere adds volume fraction to be 40% ~ 65% 1.5 ~ 10 mL of dimethyl sulfoxide, and epoxychloropropane is
1.5~12.5 mL。
7. the preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin according to claim 5, it is characterised in that: described
The concentration of NaOH solution is 1.0 ~ 2.4 mol/L, and the concentration of NaBH4 solution is 5 ~ 10 g/L, the volume fraction of epoxychloropropane
It is 20% ~ 60%, 4 ~ 10 h of oscillation activation at 30 ~ 70 DEG C.
8. according to the preparation method of the magnetic microsphere of any immobilization earthworm fibrinolysin of claim 5 or 7, feature exists
In: the preparation method of the chitosan magnetic micro-sphere:
(1) atoleine and Span-80 are mixed according to weight ratio for 30 ~ 50:1, is prepared into oil in 30 ~ 50 DEG C of 1 ~ 2 h of stirring
Phase；
(2) weigh a certain amount of chitosan, with 3 ~ 5% acetum dissolve, obtained after chitosan is completely dissolved concentration be 5 ~
The chitosan solution of 10 mg/mL, is added Fe3O4 powder, and water phase is made in 10 ~ 20 min of ultrasonic disperse；
(3) it is 1:5 ~ 6 according to volume ratio with oily phase by water phase, water phase is slowly added in oily phase, 0.5 ~ 1 h of stirring makes solution
Form emulsion system；
(4) 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 dries to get chitosan magnetic micro-sphere is arrived.
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