CN105200029A - Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof - Google Patents
Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof Download PDFInfo
- Publication number
- CN105200029A CN105200029A CN201510701434.9A CN201510701434A CN105200029A CN 105200029 A CN105200029 A CN 105200029A CN 201510701434 A CN201510701434 A CN 201510701434A CN 105200029 A CN105200029 A CN 105200029A
- Authority
- CN
- China
- Prior art keywords
- sphere
- chitosan
- earthworm fibrinolysin
- magnetic micro
- earthworm
- 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.)
- Granted
Links
Abstract
The invention discloses a magnetic microsphere for earthworm fibrinolytic enzyme immobilization and a preparation method thereof. Enzymatic activity of the magnetic microsphere is 40-60 U/mg. The preparation method comprises the following steps: firstly dissolving a sagittaria sagittifolia trypsin inhibitor in a buffer solution of pH 7-9 so as to obtain a protease buffer solution, adding the protease buffer solution into activated magnetic chitosan microspheres; carrying out water-bath shaking and coupling for 4-7 h so as to obtain sagittaria sagittifolia trypsin inhibitor-coupled magnetic chitosan microspheres; adding the sagittaria sagittifolia trypsin inhibitor-coupled magnetic chitosan microspheres into a crude enzyme solution of earthworm fibrinolytic enzyme and shaking in a thermostatic shaking bath of 30-60 DEG C and carrying out a curing reaction for 0.5-3 h; repeatedly washing after the reaction until no protein is detected from the washing liquid, and carrying out magnetic separation and collection so as to obtain the magnetic microsphere for earthworm fibrinolytic enzyme immobilization. According to the invention, the sagittaria sagittifolia trypsin inhibitor used as ligand is coupled with the magnetic microspheres, and the magnetic microspheres are used in separation and purification of earthworm fibrinolytic enzyme to enhance separation and purification selectivity of earthworm fibrinolytic enzyme. In addition, the magnetic microspheres are easy to separate from earthworm fibrinolytic enzyme.
Description
Technical field
The invention belongs to magnetic microsphere technical field, specifically relate to the preparation method of the magnetic microsphere for immobilization earthworm fibrinolysin.
Background technology
Magnetic macromolecular microsphere is a kind of novel magnetic materials that developed recently gets up, and is magnetic inorganic particle to be combined by proper method the complex microsphere with certain magnetic and special construction formed with organic polymer.Numerous characteristics that magnetic composite microsphere not only has common polymer microsphere also have magnetic responsiveness, so can not only give its surface functional group (as-OH ,-COOH ,-CHO ,-NH by the method such as copolymerization and surface modification
2, etc.), guide function can also be had under additional the action of a magnetic field.Magnetic microsphere is separated at immobilized enzyme, cellular segregation, protein separation and purifying, immunoassay, targeted drug and DNA and has a wide range of applications with the field such as nucleic acid hybridization.The method being usually used in preparing magnetic microsphere at present has entrapping method, suspension polymerization, the magnetic microsphere particle diameter wider distribution that these methods prepare, out-of-shape.
Arrowhead protease inhibitors is the multifunctional dual-head proteinase inhibitor of separation and purification from arrowhead bulb, it can be divided into inhibitor A and B (API-A, API-B), both can suppress multiple serine protease, their molecular weight is close with soybean Kunizt inhibitor, but there is larger difference on rejection characteristic, and also without homology in structure.Its peptide chain is made up of 150 amino-acid residues, Arg and Lys residue constitutes two active centre of inhibitor respectively, the Arg active centre of inhibitor A, B is Arg-Tys-Lys (76 ~ 78), the sequence in Lys active centre is Lys-Ser (44 ~ 45), similar with the active centre of Kazal inhibitor.In addition, there is single-minded suppression in Lys active centre to trypsinase, and Arg active centre then shows multi-functional, has restraining effect to multiple protein enzyme.
Earthworm fibrinolysin is also known as Lumbrukinase, be extract in earthworm body both there is direct fibrinolytic plasmin activity, there is again the class protein of the Plasminogen activation activity of class urokinase, outmoded thrombus can be dissolved and can suppress new thrombosis again, experiment shows that this enzyme has anti-freezing, molten fibre, improve hemorheology properties, it is the active drug of prevention and therapy thrombus disease, this enzyme belongs to serine protease, can by serpin as Trypsin inhibitor SBTI suppresses, clinical application in recent years shows, it is in treatment cerebral thrombosis, hemiplegia, the thrombus disease aspects such as myocardial infarction have instant effect, curative effect is good, effectively oral, and the advantage such as safer.Large quantity research shows earthworm fibrinolysin no matter intravenous injection or oral administration, all has thrombolytic effect, is a kind of thrombus disease medicine ideal at present, has wide DEVELOPMENT PROSPECT.The extraction of usual plasmin is by earthworm homogenate, saltout, the series of steps such as gel chromatography, ion exchange chromatography and high performance liquid chromatography (HPLC) is carried out separation and purification and obtained, it is high that the method extracts the plasmin purity obtained, but because the complicated cost of purification procedures is high, long economic benefit consuming time is low, laboratory study can only be used for, be difficult to industrial production, how obtain stable highly active earthworm fibrinolysin, a road can be opened up for the utilization of earthworm resource.
The report of immobilization earthworm fibrinolysin is not also used at present about arrowhead trypsin inhibitor coupled chitosan magnetic microsphere.
Summary of the invention
The object of the present invention is to provide magnetic microsphere of a kind of immobilization earthworm fibrinolysin and preparation method thereof.It for aglucon with arrowhead trypsin inhibitor, with magnetic microsphere coupling, uses the separation and purification being used for earthworm fibrinolysin, improve the separation and purification selectivity of earthworm fibrinolysin, and magnetic microsphere is easy to be separated with earthworm fibrinolysin.
Technical scheme of the present invention is as follows:
A kind of for the immobilized magnetic microsphere of earthworm fibrinolysin, its enzymic activity is 40-60U/mg, stability between pH6.5 ~ 8.0 remains on more than 78%, and storage time was more than 82% for the enzymic activity of the immobilized magnetic microsphere of earthworm fibrinolysin in 30 days.
Preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin of the present invention, it comprises the steps:
(1) coupling of chitosan magnetic micro-sphere: arrowhead trypsin inhibitor is dissolved in the damping fluid of pH7 ~ 9 and obtains proteolytic enzyme damping fluid, joined again in the chitosan magnetic micro-sphere of overactivation, water-bath vibration coupling 4 ~ 7h, vibration temperature 40 ~ 60 DEG C, after obtain the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor;
(2) immobilization of earthworm fibrinolysin: again the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor is added the thick enzyme solution of earthworm fibrinolysin and to vibrate in 30-60 DEG C of water bath with thermostatic control shaking table curing reaction 0.5-3h; After reaction terminates, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Preferred as technical scheme, the damping fluid in described step (1) is Tris-HCl damping fluid.The liquid-solid ratio of the chitosan magnetic micro-sphere of described proteolytic enzyme damping fluid and activation is 10 ~ 25: 1, water-bath vibration coupling 4 ~ 6h.The chitosan magnetic micro-sphere performance of the white enzyme inhibitors of coupling arrowhead that these processing condition obtain is good especially.
Preferred as technical scheme, in described step (2), the chitosan magnetic micro-sphere of every gram of coupling arrowhead trypsin inhibitor then adds 1 ~ 5ml enzyme and lives as the thick enzyme solution of earthworm fibrinolysin of 1-2 ten thousand U/mL.
The preparation of the chitosan magnetic micro-sphere activated in the present invention is by chitosan magnetic micro-sphere, adds NaOH solution and NaBH
4solution, at 30 ~ 80 DEG C of water-baths vibration, 30 ~ 40min, then to add dimethyl sulfoxide (DMSO) be activator, add the epoxy chloropropane that volume fraction is 10% ~ 60% again, after vibration activation 2 ~ 10h, suction filtration, can obtain the chitosan magnetic micro-sphere activated with distilled water wash.
As technical scheme further preferably, the weight ratio of described chitosan magnetic micro-sphere and NaOH solution is 1: 5 ~ 10, chitosan magnetic micro-sphere and NaBH
4the weight ratio of solution is 1: 4 ~ 12; Every gram of chitosan magnetic micro-sphere adds dimethyl sulfoxide (DMSO) 1.5 ~ 9mL that volume fraction is 40% ~ 60%, and epoxy chloropropane is 1 ~ 12mL.
As technical scheme further preferably, the concentration of NaOH solution is 1.2 ~ 2.0mol/L, NaBH
4the concentration of solution is 5 ~ 10g/L, and the volume fraction of epoxy chloropropane is 30% ~ 50%, vibrate at 30 ~ 50 DEG C activation 2 ~ 6h time chitosan magnetic micro-sphere activation effect best.
The preparation method of chitosan magnetic micro-sphere in the present invention:
(1) be 20 ~ 40: 1 mixing by whiteruss and Span-80 according to weight ratio, stir 1 ~ 2h at 30 ~ 50 DEG C and be prepared into oil phase;
(2) take a certain amount of chitosan, the acetum with 3 ~ 5% dissolves, and obtains chitosan solution, add Fe after chitosan dissolves completely
3o
4powder, ultrasonic disperse 10 ~ 20min, obtains aqueous phase;
(3) be 1: 5 ~ 6 by aqueous phase and oil phase according to volume ratio, aqueous phase slowly added in oil phase, stirs 0.5 ~ 1h and make solution form emulsion system;
(4) in emulsion system, the glutaraldehyde that massfraction is 25% is dripped again, continue stirring 1 ~ 2h, pH to 9 ~ 10 are adjusted by NaOH solution, stopped reaction after continuation reaction 1 ~ 2h, magnet is separated, and with sherwood oil, ethanol and deionized water wash are to neutral, vacuum-drying, can obtain chitosan magnetic micro-sphere.
Preferred as technical scheme, Z 250 used in the present invention is Nano grade, and the chitosan solution concentration of preparation is 5 ~ 10mg/mL, and mixing speed is 600 ~ 700rpm/min, the particle diameter of the chitosan magnetic micro-sphere prepared like this is minimum, is 215nm.
Compared with prior art, tool has the following advantages in the present invention:
1. preparation method of the present invention simple, operate easy, treating processes is gentle, not high to equipment requirements.
2. the arrowhead trypsin inhibitor of the conjugated magnetic chitosan microball that the present invention is used is aglucon, there is single-minded suppression in its Lys active centre to trypsinase, and be reversible, and there is inhibitor effect in Arg active centre to multiple protein enzyme, both competitive inhibition can improve purity and the specificity of earthworm fibrinolysin.
3. earthworm fibrinolysin of the present invention is fixed on magnetic microsphere, and because microballoon has magnetic, under the effect of externally-applied magnetic field, plasmin can be separated soon with reactant.And the pH stability of immobilization plasmin is better, can in the scope of pH6.5 ~ 8.0, stability remains on more than 78%, thermostability comparatively resolvase is high, and heat at 60 DEG C, relative reactivity still can keep more than 70%, and the storage of long period, enzymic activity keeps more than 82%.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum figure of 7000 times of chitosan magnetic micro-sphere of the present invention;
Fig. 2 is the Electronic Speculum figure of 100,000 times of chitosan magnetic micro-sphere of the present invention;
Fig. 3 is the Infrared spectroscopy figure of chitosan magnetic micro-sphere of the present invention, 3415cm in figure
-1the stretching vibration peak of N-H and O-H, 2923cm
-1and 2855cm
-1the charateristic avsorption band of C-H in aliphatics, 1617cm
-1the charateristic avsorption band of acid amides, 1431cm
-1-CH
3eigen vibration peak, 1119cm
-1the stretching vibration absorption peak of C-O, 620cm
-1there is Fe
3o
4charateristic avsorption band.
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 thermostability figure of resolvase and immobilization earthworm fibrinolysin.
Specific embodiment
The present invention is further illustrated below by specific embodiment.It should be understood that embodiments of the invention are only used for the present invention, instead of limitation of the present invention, under concept thereof of the present invention, all the scope of protection of present invention is belonged to the simple modifications of preparation method of the present invention.
In an embodiment, (a) size distribution of chitosan magnetic micro-sphere, (b) degree of activation, the method measured by recording below measures.
(a) size distribution
Chitosan magnetic micro-sphere is scattered in ethanol, adds several dispersion agent tween 20s, ultrasonic disperse 10min, use nanometer laser particle-size analyzer to carry out Lido mensuration.
(b) degree of activation
Adopt sodium thiosulfate titration, take 0.1g and wash the microballoon after draining, add the hypo solution of the 1.3moL/L of 1.5mL, oscillatory reaction 30min in 40 DEG C of water-bath constant temperature oscillators, after completion of the reaction, drip 1 phenolphthalein indicator, with standard salt acidometric titration to the colourless rear stopping titration of solution, record the volume change of standard hydrochloric acid before and after titration.The following formula of epoxy group modified density calculates:
Embodiment 1
The span-80 of the whiteruss and 2mL that measure 100mL adds in the there-necked flask of 250mL, and 45 DEG C of water bath heat preservations stir 1h and make oil phase; Take the chitosan of 0.2g, dissolve with the acetum of 20mL3%, after chitosan dissolves completely, add the Fe of 0.6g
3o
4, ultrasonic disperse 10min; Be prepared into the chitosan solution that concentration is 5mg/ml, then add in oil phase, 300rpm stirs half hour makes solution be formed after emulsion system, and slowly dripping 3mL quality is the glutaraldehyde of 25%, continue to stir 1h, adjust about pH to 10 by the NaOH solution of 1.0mol/L, stopped reaction after continuation reaction 1h, magnet is separated, with sherwood oil, ethanol and deionized water wash, vacuum-drying, can obtain chitosan magnetic micro-sphere.
The size of this chitosan magnetic micro-sphere illustrates in Table 1.
Embodiment 2
The span-80 of the whiteruss and 3mL that measure 100mL adds in the there-necked flask of 250mL, and 30 DEG C of water bath heat preservations stir 2h and make oil phase; Take the chitosan of 0.8g, dissolve with the acetum of 20mL5%, after chitosan dissolves completely, add the Fe of 0.6g
3o
4, ultrasonic disperse 20min; Be prepared into the chitosan solution that concentration is 6.5mg/ml, then add in oil phase, 400rpm stirs half hour makes solution be formed after emulsion system, and slowly dripping 5mL quality is the glutaraldehyde of 25%, continue to stir 2h, adjust about pH to 9 by the NaOH solution of 1.0mol/L, stopped reaction after continuation reaction 2h, magnet is separated, with sherwood oil, ethanol and deionized water wash, vacuum-drying, can obtain chitosan magnetic micro-sphere.
The size of this chitosan magnetic micro-sphere illustrates in Table 1.
Embodiment 3
The span-80 of the whiteruss and 2.5mL that measure 100mL adds in the there-necked flask of 250mL, and 40 DEG C of water bath heat preservations stir 2h and make oil phase; Take the chitosan of 1g, dissolve with the acetum of 20mL3%, after chitosan dissolves completely, add the Fe of 0.6g
3o
4, ultrasonic disperse 15min; Be prepared into the chitosan solution that concentration is 8.1mg/ml, then add in oil phase, 500rpm stirs half hour makes solution be formed after emulsion system, and slowly dripping 10mL quality is the glutaraldehyde of 25%, continue to stir 1.5h, adjust about pH to 9.5 by the NaOH solution of 1.0mol/L, stopped reaction after continuation reaction 1.5h, magnet is separated, with sherwood oil, ethanol and deionized water wash, vacuum-drying, can obtain chitosan magnetic micro-sphere.
The size of this chitosan magnetic micro-sphere illustrates in Table 1.
Embodiment 4
The span-80 of the whiteruss and 2mL that measure 100mL adds in the there-necked flask of 250mL, and 45 DEG C of water bath heat preservations stir 1.5h and make oil phase; Take the chitosan of 1.2g, dissolve with the acetum of 20mL3%, after chitosan dissolves completely, add the Fe of 0.6g
3o
4, ultrasonic disperse 10min; Be prepared into the chitosan solution that concentration is 10mg/ml, then add in oil phase, 600rpm stirs half hour makes solution be formed after emulsion system, and slowly dripping 3mL quality is the glutaraldehyde of 25%, continue to stir 1h, adjust about pH to 10 by the NaOH solution of 1.0mol/L, stopped reaction after continuation reaction 1h, magnet is separated, with sherwood oil, ethanol and deionized water wash, vacuum-drying, can obtain chitosan magnetic micro-sphere.
The size of this chitosan magnetic micro-sphere illustrates in Table 1.
Embodiment 5
The span-80 of the whiteruss and 2mL that measure 100mL adds in the there-necked flask of 250mL, 45 DEG C of water bath heat preservations; Take the chitosan of 0.2g, dissolve with the acetum of 20mL3%, after chitosan dissolves completely, add the Fe of 0.6g
3o
4, ultrasonic disperse 10min; Chitosan solution is added in oil phase, 700rpm stirs half hour makes solution be formed after emulsion system, slow dropping 3mL quality is the glutaraldehyde of 25%, continues to stir 1h, adjusts about pH to 10 by the NaOH solution of 1.0mol/L, stopped reaction after continuation reaction 1h, magnet is separated, with sherwood oil, and ethanol and deionized water wash, vacuum-drying, can obtain chitosan magnetic micro-sphere.
The size of this chitosan magnetic micro-sphere illustrates in Table 1.
Chitosan magnetic micro-sphere size distribution shown in Fig. 1, Fig. 2, as can be seen from Figure 1, chitosan and Fe
3o
4the composite particles particle diameter that nanoparticle is formed is between 100-300nm, this particle diameter is beneficial to microballoon to be disperseed and Magneto separate in reaction system, and the microballoon of small particle size has larger specific surface area, can the more enzyme of covalent attachment, also can reduce the impact of substrate and product diffusional limitation simultaneously.Its Infrared spectroscopy figure is see Fig. 3.
The particle diameter of table 1 chitosan magnetic micro-sphere
Embodiment 6
Take chitosan magnetic micro-sphere that 0.25g embodiment 5 prepares in Erlenmeyer flask, add the NaOH solution of 25mL1.5mol/L and the NaBH of 150mL10g/L
4vibrate 30min in water bath with thermostatic control vibrator, temperature of reaction is 30 DEG C, again with the dimethyl sulfoxide (DMSO) of volume fraction 50% for activating solvent, add the epoxy chloropropane of volume fraction 40%, after continuing oscillatory reaction 5h, suction filtration, with in distilled water flushing to filtrate without epoxy root and hydroxide radical, can obtain activate chitosan magnetic micro-sphere.
The detection of epoxy root: the filtrate of getting 2mL, instills 1 phenolphthalein, adds the Na of the 1.3moL/L of same volume
2s
2o
3, vibration makes it react, if solution does not redden, then and epoxy group(ing) wash clean.Detection hydroxy: the filtrate of getting 2mL, adds 1 phenolphthalein, if solution does not redden, then and hydroxide radical wash clean.
The degree of activation result of this chitosan magnetic micro-sphere illustrates in table 2.
Embodiment 7
Take in the chitosan magnetic micro-sphere Erlenmeyer flask that 0.25g embodiment 5 prepares, add the NaOH solution of 25mL2.0mol/L and the NaBH of 100mL20g/L
4vibrate 60min in water bath with thermostatic control vibrator, temperature of reaction is 35 DEG C, again with the dimethyl sulfoxide (DMSO) of volume fraction 45% for activating solvent, add the epoxy chloropropane of volume fraction 30%, after continuing oscillatory reaction 3h, suction filtration, with in distilled water flushing to filtrate without epoxy root and hydroxide radical, can obtain activate chitosan magnetic micro-sphere.
The detection of epoxy root: the filtrate of getting 2mL, instills 1 phenolphthalein, adds the Na of the 1.3moL/L of same volume
2s
2o
3, vibration makes it react, if solution does not redden, then and epoxy group(ing) wash clean.Detection hydroxy: the filtrate of getting 2mL, adds 1 phenolphthalein, if solution does not redden, then and hydroxide radical wash clean.
The degree of activation result of this chitosan magnetic micro-sphere illustrates in table 2.
Embodiment 8
Take in the chitosan magnetic micro-sphere Erlenmeyer flask that 0.25g embodiment 5 prepares, add the NaOH solution of 20mL2.2mol/L and the NaBH of 100mL10g/L
4vibrate 45min in water bath with thermostatic control vibrator, temperature of reaction is 50 DEG C, again with the dimethyl sulfoxide (DMSO) of volume fraction 40% for activating solvent, add the epoxy chloropropane of volume fraction 35%, after continuing oscillatory reaction 3.5h, suction filtration, with in distilled water flushing to filtrate without epoxy root and hydroxide radical, can obtain activate chitosan magnetic micro-sphere.
The detection of epoxy root: the filtrate of getting 2mL, instills 1 phenolphthalein, adds the Na of the 1.3moL/L of same volume
2s
2o
3, vibration makes it react, if solution does not redden, then and epoxy group(ing) wash clean.Detection hydroxy: the filtrate of getting 2mL, adds 1 phenolphthalein, if solution does not redden, then and hydroxide radical wash clean.
The degree of activation result of this chitosan magnetic micro-sphere illustrates in table 2.
Embodiment 9
Take in the chitosan magnetic micro-sphere Erlenmeyer flask that 0.25g embodiment 5 prepares, add the NaOH solution of 31mL1.8mol/L and the NaBH of 50mL25g/L
4vibrate 30min in water bath with thermostatic control vibrator, temperature of reaction is 60 DEG C, again with the dimethyl sulfoxide (DMSO) of volume fraction 35% for activating solvent, add the epoxy chloropropane of volume fraction 45%, after continuing oscillatory reaction 4.5h, suction filtration, with in distilled water flushing to filtrate without epoxy root and hydroxide radical, can obtain activate chitosan magnetic micro-sphere.
The detection of epoxy root: the filtrate of getting 2mL, instills 1 phenolphthalein, adds the Na of the 1.3moL/L of same volume
2s
2o
3, vibration makes it react, if solution does not redden, then and epoxy group(ing) wash clean.Detection hydroxy: the filtrate of getting 2mL, adds 1 phenolphthalein, if solution does not redden, then and hydroxide radical wash clean.
The degree of activation result of this chitosan magnetic micro-sphere illustrates in table 2.
Embodiment 10
Take in the chitosan magnetic micro-sphere Erlenmeyer flask that 0.25g embodiment 5 prepares, add the NaOH solution of 25mL2.5mol/L and the NaBH4 of 125mL20g/L, vibrate 45min in water bath with thermostatic control vibrator, temperature of reaction is 70 DEG C, then with the dimethyl sulfoxide (DMSO) of volume fraction 30% for activating solvent, adds the epoxy chloropropane of volume fraction 60%, after continuing oscillatory reaction 4h, suction filtration, with in distilled water flushing to filtrate without epoxy root and hydroxide radical, can obtain activate chitosan magnetic micro-sphere.
The detection of epoxy root: the filtrate of getting 2mL, instills 1 phenolphthalein, adds the Na of the 1.3moL/L of same volume
2s
2o
3, vibration makes it react, if solution does not redden, then and epoxy group(ing) wash clean.Detection hydroxy: the filtrate of getting 2mL, adds 1 phenolphthalein, if solution does not redden, then and hydroxide radical wash clean.
The degree of activation result of this chitosan magnetic micro-sphere illustrates in table 2.
The degree of activation measurement result of table 2 chitosan magnetic micro-sphere
Embodiment 11
(1) coupling of chitosan magnetic micro-sphere: the arrowhead trypsin inhibitor getting 40mg is dissolved in the Tris-HCl damping fluid of 3mLpH=7.0 and obtains proteolytic enzyme damping fluid, be added in the chitosan magnetic micro-sphere that 0.25g activates by embodiment 10,45 DEG C of water bath with thermostatic control vibration coupling 5h, take out, magnet is separated, washing, obtains the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor.
Collect raffinate, measure the volume of raffinate, measure the amount of arrowhead trypsin inhibitor in raffinate according to BAPNA method, and calculate Conjugate ratio.The Conjugate ratio result of this magnetic microsphere illustrates in table 3.
(2) immobilization of earthworm fibrinolysin: the phosphoric acid buffer of earthworm fibrinolysin extract pH=8.0 is dissolved and is settled to 50mL, the concentration of earthworm fibrinolysin crude enzyme liquid is 10,000 U/mL, getting 1.25mL is added in the chitosan magnetic micro-sphere of 0.25g coupling arrowhead trypsin inhibitor, 40 DEG C of water bath with thermostatic control vibration 1.5h, take out, with the chitosan magnetic micro-sphere crude product of deionized water and phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Collect washings, measure the activity of earthworm fibrinolysin in stoste and washings with fibrin plate method, utilize the difference alive of enzyme before and after immobilization to calculate immobilized enzyme.
The enzyme of immobilized enzyme is lived and is illustrated in table 4.
Embodiment 12
(1) coupling of chitosan magnetic micro-sphere: the arrowhead trypsin inhibitor getting 40mg is dissolved in the Tris-HCl of 5mLpH=8.0 and obtains proteolytic enzyme damping fluid, be added in the chitosan magnetic micro-sphere that 0.3g activates by embodiment 10,50 DEG C of water bath with thermostatic control vibration coupling 4.5h, take out, magnet is separated, washing, obtains the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor.
Collect raffinate, measure the volume of raffinate, measure the amount of arrowhead trypsin inhibitor in raffinate according to BAPNA method, and calculate Conjugate ratio.The Conjugate ratio result of this magnetic microsphere illustrates in table 3.
(2) immobilization of earthworm fibrinolysin: the phosphoric acid buffer of earthworm fibrinolysin extract pH=7.0 is dissolved and is settled to 25ml, earthworm fibrinolysin crude enzyme liquid concentration is 20,000 U/mL, getting 2.25mL is added in the chitosan magnetic micro-sphere of 0.75g coupling arrowhead trypsin inhibitor, 35 DEG C of water bath with thermostatic control vibration 2.0h, take out, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Collect washings, measure the activity of earthworm fibrinolysin in stoste and washings with fibrin plate method, utilize the difference alive of enzyme before and after immobilization to calculate immobilized enzyme.
The enzyme of immobilized enzyme is lived and is illustrated in table 4.
Embodiment 13
(1) coupling of chitosan magnetic micro-sphere: the arrowhead trypsin inhibitor getting 50mg is dissolved in the Tris-HCl of 4mLpH=7.5 and obtains proteolytic enzyme damping fluid, be added in the chitosan magnetic micro-sphere that 0.2g activates by embodiment 10,60 DEG C of water bath with thermostatic control vibration coupling 5.5h, take out, magnet is separated, washing, obtains the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor.
Collect raffinate, measure the volume of raffinate, measure the amount of arrowhead trypsin inhibitor in raffinate according to BAPNA method, and calculate Conjugate ratio.
The Conjugate ratio result of this magnetic microsphere illustrates in table 3.
(2) immobilization of earthworm fibrinolysin: the phosphoric acid buffer of earthworm fibrinolysin extract pH=7.5 is dissolved and is settled to 50mL, the concentration of earthworm fibrinolysin crude enzyme liquid is 1.5 ten thousand U/mL, getting 3mL is added in the chitosan magnetic micro-sphere of 0.5g coupling arrowhead trypsin inhibitor, 30 DEG C of water bath with thermostatic control vibration 2.5h, take out, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Collect washings, measure the activity of earthworm fibrinolysin in stoste and washings with fibrin plate method, utilize the difference alive of enzyme before and after immobilization to calculate immobilized enzyme.
The enzyme of immobilized enzyme is lived and is illustrated in table 4.
Embodiment 14
(1) coupling of chitosan magnetic micro-sphere: the arrowhead trypsin inhibitor getting 45mg is dissolved in the Tris-HCl damping fluid of 5mLpH=8.5 and obtains proteolytic enzyme damping fluid, be added in the chitosan magnetic micro-sphere that 0.2g activates by embodiment 10,50 DEG C of water bath with thermostatic control vibration coupling 4.0h, take out, magnet is separated, washing, obtains the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor.
Collect raffinate, measure the volume of raffinate, measure the amount of arrowhead trypsin inhibitor in raffinate according to BAPNA method, and calculate Conjugate ratio.
The Conjugate ratio result of this magnetic microsphere illustrates in table 3.
(2) immobilization of earthworm fibrinolysin: the phosphoric acid buffer of earthworm fibrinolysin extract pH=6.5 is dissolved and is settled to 50ml, the concentration of earthworm fibrinolysin crude enzyme liquid is 20,000 U/mL, getting 1.0mL is added in the chitosan magnetic micro-sphere of 0.5g coupling arrowhead trypsin inhibitor, 60 DEG C of water bath with thermostatic control vibration 0.5h, take out, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Collect washings, measure the activity of earthworm fibrinolysin in stoste and washings with fibrin plate method, utilize the difference alive of enzyme before and after immobilization to calculate immobilized enzyme.
The enzyme of immobilized enzyme is lived and is illustrated in table 4.
Embodiment 15
(1) coupling of chitosan magnetic micro-sphere: the arrowhead trypsin inhibitor getting 40mg is dissolved in the Tris-HCl damping fluid of 5mLpH=9.0 and obtains proteolytic enzyme damping fluid, be added in the chitosan magnetic micro-sphere that 0.2g activates by embodiment 10,30 DEG C of water bath with thermostatic control vibration coupling 7h, take out, magnet is separated, washing, collect raffinate, measure the volume of raffinate, measure the amount of arrowhead trypsin inhibitor in raffinate according to BAPNA method, and calculate Conjugate ratio.
The Conjugate ratio result of this magnetic microsphere illustrates in table 3.
(2) immobilization of earthworm fibrinolysin: the phosphoric acid buffer of earthworm fibrinolysin extract pH=6.0 is dissolved and is settled to 50ml, the concentration of earthworm fibrinolysin crude enzyme liquid is 1.5 ten thousand U/mL, getting 5.0mL is added in the chitosan magnetic micro-sphere of 1g coupling arrowhead trypsin inhibitor, 45 DEG C of water bath with thermostatic control vibration 3.0h, take out, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
Collect washings, measure the activity of earthworm fibrinolysin in stoste and washings with fibrin plate method, utilize the difference alive of enzyme before and after immobilization to calculate immobilized enzyme.
The enzyme of immobilized enzyme is lived and is illustrated in table 4.
The chitosan magnetic micro-sphere Conjugate ratio of table 3 coupling arrowhead trypsin inhibitor
Table 4 is for earthworm fibrinolysin immobilized magnetic microsphere enzyme activity determination result
Embodiment 16
Measure immobilization earthworm fibrinolysin pH stability
Of the present invention for the immobilized magnetic microsphere of earthworm fibrinolysin and earthworm fibrinolysin free liquid by same enzyme concentration, be 5.5,6.5,7.5,8.5,9.5 times 37 DEG C of water bath processing 30min at pH respectively, measure enzyme and live, undressed enzyme work is 100%.Obtain the pH stability diagram of resolvase and immobilization earthworm fibrinolysin, as shown in Figure 4.Good compared with resolvase of the resistance to acids and bases of immobilization earthworm fibrinolysin as seen from the figure, its stable range is also wide compared with pH, resolvase is that 7.5 places have most high reactivity at pH, immobilized enzyme is that 7.0 places have most high reactivity at pH, this is that after enzyme is fixed on carrier, its conformation there occurs change, and pH stability is changed.
Measure immobilization earthworm fibrinolysin storage stability
Same enzyme concentration of the present invention is placed in 4 DEG C of refrigerators for the immobilized magnetic microsphere of earthworm fibrinolysin and earthworm fibrinolysin free liquid preserve, surveys enzyme every 5d sampling and live, until 30d.Mensuration enzyme is lived, and undressed enzyme work is 100%.Obtain the storage stability figure of resolvase and immobilization earthworm fibrinolysin, as shown in Figure 5.
As shown in Figure 5, along with the change of storage time, the activity of resolvase and immobilization earthworm fibrinolysin all little by little declines, at first 5 days of storage, the activity of resolvase and immobilization earthworm fibrinolysin does not all have considerable change, when storing 10 days, the relative enzyme work of resolvase is 80%, and the enzyme work of immobilization earthworm fibrinolysin still has 98%, the activity of resolvase sharply declines afterwards, during by 30 days, only retain 32%, and the activity of immobilization earthworm fibrinolysin still has 82%, result shows, earthworm fibrinolysin is greatly improved through immobilization rear stability.
Measure immobilization earthworm fibrinolysin thermostability
By same enzyme concentration of the present invention for the immobilized magnetic microsphere of earthworm fibrinolysin and earthworm fibrinolysin free liquid water-bath 30min at 30,40,50,55,60 DEG C, measuring enzyme and live, is 100% without heat treated enzyme work., calculate remnant enzyme activity than q,
In formula: under c0-differing temps, during 0min, enzyme is lived, U;
During ct-tmin, enzyme is lived, U.
Obtain the thermostability figure of resolvase and immobilization earthworm fibrinolysin, as shown in Figure 6.As shown in Figure 6, resolvase enzyme relative to immobilization earthworm fibrinolysin is lived and is varied with temperature similar trend, along with temperature raises, relative enzyme is lived and is declined, but under uniform temp condition, the relative reactivity of the relative reactivity specific ionization earthworm fibrinolysin of immobilization earthworm fibrinolysin is high, and the earthworm fibrinolysin Heat stability is good of the earthworm fibrinolysin specific ionization state after chitosan magnetic micro-sphere immobilization is described.
Claims (10)
1. for the immobilized magnetic microsphere of earthworm fibrinolysin, it is characterized in that: its enzymic activity is 40-60U/mg.
2. according to claim 1 for the immobilized magnetic microsphere of earthworm fibrinolysin, it is characterized in that: its stability between pH6.5 ~ 8.0 remains on more than 78%, storage time was more than 82% for the enzymic activity of the immobilized magnetic microsphere of earthworm fibrinolysin in 30 days.
3., as claimed in claim 1 or 2 for a preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin, it is characterized in that: it comprises the steps:
(1) coupling of chitosan magnetic micro-sphere: arrowhead trypsin inhibitor is dissolved in the damping fluid of pH7 ~ 9 and obtains proteolytic enzyme damping fluid, joined again in the chitosan magnetic micro-sphere of overactivation, water-bath vibration coupling 4 ~ 7h, vibration temperature is 30-60 DEG C, obtains the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor;
(2) immobilization of earthworm fibrinolysin: again the chitosan magnetic micro-sphere of coupling arrowhead trypsin inhibitor is added the thick enzyme solution of earthworm fibrinolysin and to vibrate in 30-60 DEG C of water bath with thermostatic control shaking table curing reaction 0.5-3h; After reaction terminates, it is the chitosan magnetic micro-sphere crude product of the phosphoric acid buffer repetitive scrubbing immobilization earthworm fibrinolysin of 6-8 with deionized water and pH, to inspection in washings does not measure albumen, magnet separated and collected can obtain for the immobilized magnetic microsphere of earthworm fibrinolysin.
4. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 3, it is characterized in that: in described step (1), the liquid-solid ratio of the chitosan magnetic micro-sphere of proteolytic enzyme damping fluid and activation is 10 ~ 25: 1, water-bath vibration coupling 4 ~ 6h.
5. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 3, is characterized in that: the damping fluid in described step (1) is for being Tris-HCl damping fluid.
6. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 3, it is characterized in that: in described step (2), the chitosan magnetic micro-sphere of every gram of coupling arrowhead trypsin inhibitor then adds 1 ~ 5ml enzyme and lives as the thick enzyme solution of earthworm fibrinolysin of 1-2 ten thousand U/mL.
7. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 3, is characterized in that: the preparation of the chitosan magnetic micro-sphere of described activation is by chitosan magnetic micro-sphere, adds NaOH solution and NaBH
4solution, at 30 ~ 60 DEG C of water-baths vibration, 30 ~ 40min, then to add dimethyl sulfoxide (DMSO) be activator, add the epoxy chloropropane that volume fraction is 10% ~ 60% again, after vibration activation 2 ~ 10h, suction filtration, can obtain the chitosan magnetic micro-sphere activated with distilled water wash.
8. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 7, is characterized in that: the weight ratio of described chitosan magnetic micro-sphere and NaOH solution is 1: 5 ~ 10, chitosan magnetic micro-sphere and NaBH
4the weight ratio of solution is 1: 4 ~ 12; Every gram of chitosan magnetic micro-sphere adds dimethyl sulfoxide (DMSO) 1.5 ~ 9mL that volume fraction is 40% ~ 60%, and epoxy chloropropane is 1 ~ 12mL.
9. the preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin according to claim 7, it is characterized in that: the concentration of described NaOH solution is 1.2 ~ 2.0mol/L, the concentration of NaBH4 solution is 5 ~ 10g/L, the volume fraction of epoxy chloropropane is 30% ~ 50%, and vibrate activation 2 ~ 6h at 30 ~ 50 DEG C.
10., according to the arbitrary described preparation method for the immobilized magnetic microsphere of earthworm fibrinolysin of claim 7 or 9, it is characterized in that: the preparation method of described chitosan magnetic micro-sphere:
(1) be 30 ~ 50: 1 mixing by whiteruss and Span-80 according to weight ratio, stir 1 ~ 2h at 30 ~ 50 DEG C and be prepared into oil phase;
(2) take a certain amount of chitosan, the acetum with 3 ~ 5% dissolves, and obtains the chitosan solution that concentration is 5 ~ 10mg/mL, add Fe after chitosan dissolves completely
3o
4powder, ultrasonic disperse 10 ~ 20min, obtains aqueous phase;
(3) be 1: 5 ~ 6 by aqueous phase and oil phase according to volume ratio, aqueous phase slowly added in oil phase, stirs 0.5 ~ 1h and make solution form emulsion system;
(4) in emulsion system, the glutaraldehyde that massfraction is 25% is dripped again, continue stirring 1 ~ 2h, pH to 9 ~ 10 are adjusted by NaOH solution, stopped reaction after continuation reaction 1 ~ 2h, magnet is separated, and with sherwood oil, ethanol and deionized water wash are to neutral, vacuum-drying, can obtain 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 true CN105200029A (en) | 2015-12-30 |
| CN105200029B 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) |
Cited By (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 (3)
| 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 |
| US20060104969A1 (en) * | 2004-08-16 | 2006-05-18 | Massachusetts Institute Of Technology | Compositions and methods for enhancing structural and functional nervous system reorganization and recovery |
| 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 |
-
2015
- 2015-10-27 CN CN201510701434.9A patent/CN105200029B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060104969A1 (en) * | 2004-08-16 | 2006-05-18 | Massachusetts Institute Of Technology | Compositions and methods for enhancing structural and functional nervous system reorganization and recovery |
| 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 |
|---|
| LIMIN ZANG 等: "Preparation of Magnetic Chitosan Nanoparticles As Support for Cellulase Immobilization", 《IND. ENG. CHEM. RES.》 * |
| TALES ALEXANDRE COSTA-SILVA 等: "Enzyme encapsulation in magnetic chitosan-Fe3O4 microparticles", 《J MICROENCAPSUL》 * |
| 何执中 等: "蚯蚓纤溶酶的分离纯化及抗栓活性研究", 《中国生化药物杂志》 * |
| 武金霞 等: "壳聚糖固定化蚯蚓纤溶酶及其性质", 《河北大学学报（自然科学版）》 * |
| 熊华 等: "京尼平交联壳聚糖微球的制备及理化性质", 《南昌大学学报（工科版）》 * |
| 静天玉 等: "磁性蚯蚓纤溶酶脂质体的制备", 《河北大学学报（自然科学版）》 * |
Cited By (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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105200029B (en) | 2018-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tarannum et al. | Perspective and application of molecular imprinting approach for antibiotic detection in food and environmental samples: A critical review | |
| CN102344151B (en) | Prussian blue nano-scale hollow olivary microballoons | |
| CN102964539B (en) | The preparation method of porous magnetic polystyrene microsphere | |
| Zhang et al. | Efficient synthesis of molecularly imprinted polymers with enzyme inhibition potency by the controlled surface imprinting approach | |
| CN105200029A (en) | Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof | |
| Berka et al. | Relation between aggregation kinetics and the structure of kaolinite aggregates | |
| Krishnamurthy et al. | Inactivation of lysozyme by sonication under conditions relevant to microencapsulation | |
| CN105200030A (en) | Preparation method of magnetic microspheres for immobilizing earthworm fibrinolytic enzyme | |
| Gupta et al. | Differential effect of surfactants tetra-n-butyl ammonium bromide and N-Cetyl-N, N, N-trimethyl ammonium bromide bound to nano-cellulose on binding and sustained release of some non-steroidal anti-inflammatory drugs | |
| CN104844768A (en) | Preparation method and application of core-shell structure temperature sensitive magnetic protein imprinted microspheres | |
| CN103289981A (en) | A preparation method for lyophilized powder of earthworm fibrinolytic enzymes | |
| Fan et al. | Effects of ionic liquids on the hydrolysis of casein by lumbrokinase | |
| CN105200031A (en) | Magnetic microsphere for immobilizing earthworm fibrinolytic enzyme and preparation method thereof | |
| CN107312767B (en) | Combined immobilized beta-glucosidase particle and preparation method thereof | |
| Sandefur et al. | Pyrimethamine-resistant dihydrofolate reductase enzymes of Plasmodium falciparum are not enzymatically compromised in vitro | |
| CN100584944C (en) | Trimeresurus albolabris defibrase preparation method | |
| Landaburu et al. | Proteolytic and polymerase activity of thrombin | |
| Yurko et al. | Design of biomedical nanodevices for dissolution of blood clots | |
| CN103169957A (en) | Nano thrombin and preparation method thereof | |
| Upadhyay et al. | Evaluation of acetylcholinesterase and butyrylcholinesterase inhibitory activity of Huperzine-A; in silico and in vitro studies | |
| CN106932506A (en) | A kind of slow controlled-release solid formulations detection pre-treating method of hydrogel matrix type | |
| Liang et al. | Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system | |
| CN102660523B (en) | Low-immunogenicity lumbrukinase, preparation method and application thereof | |
| CN104419695A (en) | Purification method of chymotrypsinogen bionic affinity material and purification method of chymotrypsin | |
| ITO et al. | Encapsulation of human urokinase in rabbit erythrocytes and its disposition in the circulation system in rabbits |
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 |