CN103882002B - The preparation of a kind of immobilization proteinase reagent and application thereof - Google Patents
The preparation of a kind of immobilization proteinase reagent and application thereof Download PDFInfo
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Abstract
The invention discloses preparation and the application thereof of a kind of immobilization proteinase reagent.A kind of immobilized enzyme disclosed by the invention is made up of hydrophobic carrier material and the protease being fixed on described hydrophobic carrier material.The magnetic nano particle immobilized trypsin that the two kinds of polymer chains of different nature hydrophilic, hydrophobic utilizing surface Atom Transfer Radical Polymerization method (SI ATRP) to prepare respectively disclosed by the invention are modified, achieve the enzymolysis quick, efficient, comprehensive of albumen, and immobilization proteinases different for two kinds of hydrophilic, hydrophobic character is used in combination, achieve complementary enzymolysis, can effectively reduce the enzymolysis bias that carrier selectivity causes, improve the comprehensive of protein digestion, thus dramatically increase the qualification quantity of protein and peptide fragment.
Description
Technical field
The present invention relates to preparation and the application thereof of a kind of immobilization proteinase reagent.
Background technology
As the priority research areas of genome times afterwards comprehensively, proteomics research can not only explaining for life mechanics
Bright provide fundamental basis, also can the diagnosis of disease, treat, prevent, the aspect such as infective pathogen pathogenesis and new drug development
Play a role.After calendar year 2001 is chosen as the six big hot research fields of 21 century by " science " magazine, proteomics research is subject to
Arrive the extensive concern of scientist.Being most widely used proteomics research strategy at present is " shot gun method " (shot-gun
Method) strategy.In this strategy, the qualitative and quantitative information of protein is by carrying out corresponding enzymatic hydrolysate peptide fragment point
Analysis is obtained, the most quickly, efficiently, completely protein digestion just become protein group accurately, high throughput identification and quantitative
Extremely important link in research.But, in order to avoid protease self enzymolysis in traditional in-solution digestion, often must use relatively
Low protease and substrate protein white matter ratio (protease: protein substrate=1:50), therefore enzyme digestion reaction typically requires longer
Incubation time (12-20 hour), and the sample for high complexity is difficult to complete enzymolysis, thus limit dividing of sample
Analysis flux, the sensitivity of identification of proteins and the accuracy of quantitative study, it is impossible to meet that protein science is extensive, high throughput analysis
Requirement.
Based on above reason, develop protein digestion technology fast and efficiently, for the qualification of complex proteins group sample
It is the most necessary.Enzyme immobilization technology avoids the problem of protease self enzymolysis, therefore can use higher enzyme/substrate
Ratio, thus significantly accelerate the process of enzyme digestion reaction, has recyclable simultaneously and the plurality of advantages such as reuses. numerous
Carrier Materials of Immobilized Enzyme, such as silicon dioxide microsphere, polymeric film, Monolithic Columns, (Xu, F.et in mesoporous material substrate
al.Anal.Chem.2010,82,10045-10051.Spross,J.et al.Anal.Chem.2010,82,1434-
1443.Qian, K.et al.Anal.Chem.2009,81,5749-5756.), magnetic nanoparticle is because of its bigger ratio table
Area and be easily achieved the characteristics such as Magnetic Isolation highlight advantage (Lin, S.et al.J.Proteome Res.2008,7,
1297-1307.).But the most commonly used carries out, at substrate material surface, the method that monolayer enzyme is fixing, makes unit mass fix
The enzyme supported quantity of change enzyme material is amassed by substrate material surface and is limited, thus limits the further raising of enzymolysis efficiency.With
Time existing immobilized enzyme reagent all use single carrier material, the inevitable carrier affinity difference institute to protein itself
The enzymolysis bias caused, causes protein digestion comprehensive not, have impact on protein and the coverage rate of peptide fragment qualification.
Transfer Radical Polymerization (Atom Transfer Radical Polymerization, ATRP) is
MatyjaszewkiK. a kind of controllable free radical polymerization process that professor group proposed first in nineteen ninety-five.Obtain since more than ten years
The extensive concern of international academic community and industrial quarters.ATRP method has that resulting polymer structural controllability is good, molecular weight distribution
Narrow, suitable monomers scope is wide, reaction condition requires the features such as moderate.Surface Atom Transfer Radical Polymerization method (SI-
ATRP) it is a kind of method ATRP initiator being fixed on and causing polymer growth after material surface in situ, now has been widely used
Modify and functionalization in the surface of material.
Summary of the invention
It is an object of the invention to provide preparation and the application thereof of a kind of immobilization proteinase reagent.
A kind of immobilized enzyme that the present invention provides, this immobilized enzyme is by hydrophobic carrier material and is fixed on described hydrophobicity
Protease composition on carrier material；
Described hydrophobic carrier material is that hydrophobic monomer is with surface Atom Transfer Radical Polymerization initiator
The surface of granule there is atom transfer radical polymerization and the product that obtains；Wherein, described hydrophobic monomer aggregates into polymerization
Chain；The polymeric chain that described hydrophobic monomer is polymerized and described granule are bonded by C-Si-O；
Described protease is linked by carbonnitrogen bond with the polymeric chain on described hydrophobic carrier material.
In above-mentioned immobilized enzyme, described granule is magnetic nanoparticle；
Described magnetic nanoparticle is specially the magnetic nanoparticle of Silica-coated；
The described granule with surface Atom Transfer Radical Polymerization initiator for causing atom transfer certainly by surface
The surface being fixedly connected on described granule by base polymerization initiator is made；
One end of described surface Atom Transfer Radical Polymerization initiator is the coupling agent being combined with silicone hydroxyl, separately
One end is atom transfer radical polymerization initiator；
Described hydrophobic monomer is the (+)-2,3-Epoxy-1-propanol esters monomer containing epoxy radicals, specially methyl propenoic acid glycidyl
Ester；
Described protease is specially trypsin, protein incision enzyme, intracellular protein enzyme or chymase.
In any of the above-described described immobilized enzyme, the method for described fixing connection is that described surface causes atom transfer freely
It is covalently bound that base polymerization initiator and the silicone hydroxyl generation dehydration of described particle surface generate siliconoxygen bond；
Described coupling agent is silane coupler；
Described silane coupler be specially APTES, 3-TSL 8330 or
Gamma-mercaptopropyltriethoxysilane；
The initiator of described atom transfer radical polymerization is 2-bromine isobutyl acylbromide, alpha-brominated isoamyl acylbromide or α-bromine propionyl
Bromine；
Described silane coupler is connected by amido link with described atom transfer radical polymerization initiator.
In any of the above-described described immobilized enzyme, the preparation side of described surface Atom Transfer Radical Polymerization initiator
Method is as follows: silane coupler and Bronsted acid agent for capturing is reacted in ice bath, adds atom transfer radical polymerization and draw
Send out agent, react, to obtain final product；
The molar ratio of described silane coupler, atom transfer radical polymerization initiator and Bronsted acid agent for capturing is
0.5-1:1:1, specially 0.8:1:1.
In any of the above-described described immobilized enzyme, described with surface Atom Transfer Radical Polymerization initiator
Grain preparation method is as follows: the magnetic nanoparticle of described Silica-coated is carried out acid-alkali treatment and exposes silicone hydroxyl, by it
In solvent, mix generation dehydration with surface Atom Transfer Radical Polymerization initiator and generate siliconoxygen bond, to obtain final product；
Described solvent be in ethanol, methanol and Hexalin any one；
The temperature of described dehydration is 20-30 DEG C；
The time of described dehydration is 1-24 hour, specially 10 hours；
Described magnetic nanoparticle is 1 with the mass ratio of the initiator of described surface Atom Transfer Radical Polymerization:
0.37-7.4, specially 1:0.74.
In any of the above-described described immobilized enzyme, the preparation method of described hydrophobic carrier material is as follows: by hydrophobicity list
Body, catalyst, part and solvent mixing, obtain mixed liquor 1；Cause atom transfer freely with described with surface mixed liquor 1
The granule mixing of base polymerization initiator, obtains mixed liquor 2；Removing the oxygen in mixed liquor 2, Atom Transfer Radical Polymerization is anti-
Should, to obtain final product；
Described catalyst is the halogenide of any one metal following: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and Pb；
Described part be following any one: N, N, N', N'', N''-PMDETA, 2,2'-bipyridyl,
Tetramethylethylenediamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl triens and three (2-dimethylaminoethyl) amine；
Solvent for use be following any one: methanol, ethanol, Hexalin, dimethyl sulfoxide, oxolane；
Described hydrophobic monomer, catalyst, the mol ratio of part are 200:0.5-5:0.75-7.5, specially 200:1:
1.5；
The method that described protease is fixed on described hydrophobic carrier material is as follows:
(1) by hydrophobic carrier material functional, the carrier material of functionalization is obtained；
(2) carry out the fixing of protease on the carrier material of the functionalization obtained in step (1), obtain securing protease
Functional supports；
(3) the residue functional group on the functional supports securing protease is closed, obtain purpose immobilization albumen
Enzyme；
Method fixing described in described step (2) is that described protease is dissolved in the ammonium bicarbonate soln of pH8.0, adds
Sodium cyanoborohydride obtains mixed liquor；Mixed liquor is joined in the carrier material of the functionalization described in step (1), reaction, then will
Granule takes out and get final product；
The method closed described in described step (3) be with ethylaminoethanol phosphate buffer and step (2) obtain
Secure the functional supports mixing of protease, react and get final product；
Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be 5%-20%, be specially
10%；
The method of described functionalization is aldehyde radical and/or chemical modification；
Described aldehyde radicalization is specially and is mixed with the dilute sulfuric acid of 0.2M by described hydrophobic carrier material, lucifuge reaction at 25 DEG C
Obtain；
Described protease is connected by carbonnitrogen bond with the carrier material of described functionalization；
Described protease is 1:10-10:1, specially 2:1 with the mass ratio of the carrier material of described functionalization；
Described functional group is the group being combined with described protease；
Described functional group is positioned on the polymeric chain of described carrier material.
A kind of immobilized enzyme mixture falls within protection scope of the present invention, by any of the above-described described immobilized enzyme and with
Hydrophilic carrier material is the immobilized enzyme composition of substrate；
Any of the above-described described immobilized enzyme and the mass ratio of the described immobilized enzyme with hydrophilic carrier material as substrate
It is specially 1:1；
The described immobilized enzyme with hydrophilic carrier material as substrate is by hydrophilic carrier material and is fixed on described hydrophilic
Property carrier material on protease composition；
Described protease is specially trypsin, protein incision enzyme, intracellular protein enzyme or chymase；
Described protease is linked by carbonnitrogen bond with described hydrophilic carrier material；
Described hydrophilic carrier material is that hydrophilic monomer draws with surface Atom Transfer Radical Polymerization described
There is atom transfer radical polymerization and the product that obtains in the surface of the granule sending out agent；Wherein, described hydrophilic monomer aggregates into
Polymeric chain；The polymeric chain that described hydrophilic monomer is polymerized and described granule are bonded by C-Si-O；
One end of the surface Atom Transfer Radical Polymerization initiator of described granule is the idol being combined with silicone hydroxyl
Connection agent, the other end is atom transfer radical polymerization initiator；
Described coupling agent is silane coupler；
Described silane coupler is APTES, 3-TSL 8330 or γ-mercapto
Propyl-triethoxysilicane；
The initiator of described atom transfer radical polymerization is 2-bromine isobutyl acylbromide, alpha-brominated isoamyl acylbromide or α-bromine propionyl
Bromine；
Described silane coupler is connected by amido link with the initiator of described atom transfer radical polymerization；
Described hydrophilic monomer is specially containing the hydrophilic monomer of monosaccharide group, acrylic monomer, butylene acids list
Body or amylene acrylic monomer；
Described monosaccharide is specially glucosamine or epichitosamine；
Described hydrophilic monomer is specially 2-methacrylic acid 3-Glucoamino propyl ester monomer；
Described granule is magnetic nanoparticle；
Described magnetic nanoparticle is specially the magnetic nanoparticle of Silica-coated；
Described 2-methacrylic acid 3-Glucoamino propyl ester monomer is prepared as follows: by Glycidyl methacrylate
Glyceride aoxidizes, and obtains the glycidyl methacrylate of oxidation；By glucosamine with sodium cyanoborohydride in first
Alcohol mixes, obtains the methanol solution of glucosamine；By the glycidyl methacrylate of oxidation and glucosamine
Methanol solution carries out schiff base reaction and obtains reactant liquor；Reactant liquor noble gas is dried up to paste, to obtain final product；
Described noble gas is specially nitrogen.
In above-mentioned immobilized enzyme mixture, the preparation method of described hydrophilic carrier material is as follows: by hydrophilic monomer, urge
Agent, part and solvent mixing, obtain mixed liquor 1；By mixed liquor 1 with described with surface Atom Transfer Radical Polymerization
The granule mixing of initiator, obtains mixed liquor 2；Removing the oxygen in mixed liquor 2, Atom Transfer Radical Polymerization reacts,
To reactant liquor；Residual reactant in reactant liquor is removed, obtains the hydrophilic carrier material of immobilization proteinase；
Described catalyst is the halogenide of any one metal following: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and Pb；
Described part be following any one: N, N, N', N'', N''-PMDETA, 2,2'-bipyridyl,
Tetramethylethylenediamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl triens and three (2-dimethylaminoethyl) amine；
Solvent for use be following any one: methanol, ethanol, Hexalin, dimethyl sulfoxide, oxolane；
Described hydrophilic monomer, catalyst, the mol ratio of part are 200:0.5-5:0.75-7.5, specially 200:1:
1.5；
The synthetic method of described immobilization proteinase is as follows:
(1) by carrier material functionalization, the carrier material of functionalization is obtained；
(2) carry out the fixing of protease on the carrier material of the functionalization obtained in step (1), obtain securing protease
Functional supports；
(3) the residue functional group on the functional supports securing protease is closed, obtain purpose immobilization albumen
Enzyme；
Method fixing described in described step (2) is that described protease is dissolved in the ammonium bicarbonate soln of pH8.0, adds
Sodium cyanoborohydride obtains mixed liquor；Mixed liquor is joined in the carrier material of the functionalization described in step (1), reaction, then will
Granule takes out and get final product；
The method closed described in described step (3) be with ethylaminoethanol phosphate buffer and step (2) obtain
Secure the functional supports mixing of protease, react and get final product；
Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be 5%-20%, be specially
10%；
Described carrier material is to be polymerized with the polymeric chain being polymerized by hydrophilic monomer or hydrophobic monomer by surface
The granule of polymeric chain；
The method of described functionalization is aldehyde radical and/or chemical modification；
The vicinal diamines of the monosaccharide group on polymeric chain that described hydrophilic monomer is specifically polymerized by described aldehyde radicalization
Thaumatropy is that aldehyde radical carries out aldehyde radical, obtains aldehyde radical carrier；
Or,
The cycloalkyl groups on polymeric chain that hydrophobic monomer is specifically polymerized by described aldehyde radicalization is converted into aldehyde radical,
Obtain aldehyde radical carrier；
Described protease is 1:10-10:1, specially 2:1 with the mass ratio of the carrier material of described functionalization；
Described protease is connected by carbonnitrogen bond with the carrier material of described functionalization；
Described functional group is the group being combined with described protease；
Described functional group is positioned on the polymeric chain of described carrier material.
A kind of method of enzymolysis protein falls within protection scope of the present invention, comprises the steps: to treat the albumen of enzymolysis
Carrying out degeneration, obtain degeneration treats enzymolysis protein；Any of the above-described described immobilized enzyme mixture and degeneration treated enzymolysis egg
White mix homogeneously in the phosphate buffer of pH=8.025-100mM ammonium bicarbonate soln or pH=7.825-100mM, hatches,
Obtain enzymolysis solution；
The method that the described albumen treating enzymolysis carries out degeneration is specific as follows: addition DTT in the described albumen treating enzymolysis, and 37
DEG C water-bath is reduced, adds IAA and place in dark place and be alkylated.
The application in proteolysis of any of the above-described described immobilized enzyme falls within protection scope of the present invention；
Or,
The application in proteolysis of any of the above-described described immobilized enzyme mixture falls within protection scope of the present invention；
Described albumen is specially yeast whole protein.
The present invention utilizes surface Atom Transfer Radical Polymerization method (SI-ATRP) to be prepared for hydrophilic, hydrophobic two respectively
The magnetic nano particle immobilized trypsin that kind of polymer chain of different nature is modified, it is achieved that albumen quick, efficient, complete
Face enzymolysis, and immobilization proteinases different for two kinds of hydrophilic, hydrophobic character is used in combination by the present invention, it is achieved complementary enzymolysis,
Can effectively reduce the enzymolysis bias that carrier selectivity causes, improve the comprehensive of protein digestion, thus dramatically increase protein
Qualification quantity with peptide fragment.The method that the present invention provides can be applicable to utilize " shot gun method " protein group to identify, and strategy is to protein
Before being analyzed identifying.
The technical scheme that the present invention provides has the advantage that
One, the polymer chain that the magnetic nanoparticle surface of Silica-coated is prepared by SI-ATRP method is modified, polymerization
Thing side chain with a large amount of aldehyde functions, and noncrosslinking polymer chain be alternatively arranged as timbering material support 3 D stereo,
Multilamellar protease is fixed.This special immobilization way ensure that the protease being positioned at internal layer can also be sent out with protein substrate
Raw effectively contact.Therefore, compared with the immobilized enzyme using conventional monolayers covering pattern to prepare, this polymer-modified magnetic Nano
Granule not only can substantially increase the supported quantity of the protease of unit mass immobilized enzyme reagent, and is favorably improved immobilization egg
White enzyme and the collision probability of protein substrate, improve the availability of protease；
Two, with by compared with trypsin is fixed on immobilized enzyme prepared by hard substrate material surface, it is polymerized at this
Trypsin in thing modification immobilizing enzyme be fixed on softness polymer chain on, have bigger degree of freedom, beneficially enzyme with
Being fully contacted of protein substrate, thus promote the carrying out of enzymolysis；
Three, need within 12-20 hour, just can complete enzymolysis with conventional solution enzymolysis compared with, polymer-modified magnetic nanoparticle
Immobilized enzyme completes protein digestion required time extremely short (1 minute), is greatly accelerated sample treatment speed；
Four, this immobilization proteinase convenience easy to use.After enzymolysis completes, enzymolysis can be produced by a step Magnetic Isolation
Thing separates with magnetic-particle immobilization proteinase；
Five, compared with immobilization proteinase prepared by carrier single with existing employing, this double carrier immobilization proteinase is held concurrently
Have hydrophilic and hydrophobic two kinds of character, hydrophilic and hydrophobic protein are all had preferable affinity, such that it is able to be effectively improved albumen
Matter enzymolysis comprehensive, dramatically increases the qualification quantity of protein and peptide fragment, is therefore particularly suitable for complex proteins group sample
Analyzing and processing；
Six, due to this immobilization proteinase reagent, there is the highest enzymolysis efficiency and comprehensive, hence help to improve multiple
Heteroproteins group sample carries out qualification overburden depth during qualitative analysis and the reliability of result with " shot gun method " qualification strategy；
Seven, these two kinds of immobilized enzyme can repeatedly use.After using, can be reclaimed by Magnetic Isolation every time.Warp
Repeatedly using, enzymolysis efficiency has no decline.
Accompanying drawing explanation
Fig. 1 is technical scheme and the double carrier that SI-ATRP method prepares double carrier immobilizing trypsinase hydrophilic, hydrophobic
The flow chart of immobilizing trypsinase enzymolysis protein matter.
Fig. 2 is the MALDI-TOF-MS spectrogram of GMA-G.
Fig. 3 is the thermogravimetric analysis of the magnetic nanoparticle that different surfaces is modified.
Fig. 4 is the static contact angle analysis of the magnetic nanoparticle that different surfaces is modified.
Fig. 5 is aqueous phase and the organic facies dispersion photo of the magnetic nanoparticle that different surfaces is modified.
Fig. 6 is the non-specific adsorption research to protein of the magnetic nanoparticle of different surfaces modification.
Fig. 7 is hydrophobic GMA-Trypsin and two kinds of immobilization proteinase enzymatic hydrolysate of hydrophilic GMA-G-Trypsin hydrophobic
Property GRAVY Distribution value figure.
Fig. 8 is hydrophobic GMA-Trypsin, the enzymolysis qualification result of hydrophilic GMA-G-Trypsin and CITD.
Fig. 9 is the enzymolysis qualification result of CITD and solution.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Material therefor is not as provided preparation method, and being can be by being either commercially available.
Ferroferric oxide magnetic nanoparticle (particle diameter 20nm) is purchased from Beijing Deco Dao Jin Science and Technology Ltd..
The preparation method of the ferroferric oxide magnetic nanoparticle of the Silica-coated in following embodiment is as follows: will
2.31g ferroferric oxide magnetic nanoparticle joins ultrasonic 60min in 40ml dehydrated alcohol, obtains suspension, to this suspension
Middle addition 1.5ml volumn concentration be 25% ammonia, 6ml water, 1.5ml tetraethyl orthosilicate, be stirred vigorously in 40 DEG C of water-baths
After 2h, after ultrasonic 60min, with 40ml ethanol rinse granule 3 times, then it is resuspended in 40mL dehydrated alcohol, 60 DEG C of backflows
Heating 12h, the granule finally obtained is the ferroferric oxide magnetic nanoparticle of Silica-coated, it is again suspended from
In 60mL dehydrated alcohol, room temperature preservation is standby.
Embodiment 1, the synthesis of carrier material of immobilization proteinase
SI-ATRP method prepares the technical scheme of double carrier immobilizing trypsinase hydrophilic, hydrophobic and double carrier is fixed
Change the flow chart of enzyme enzymolysis protein matter as shown in Figure 1.
One, the synthesis of SI-ATRP initiator
APTES is used to synthesize SI-ATRP initiator, this initiation with 2-bromine isobutyryl bromine reaction
One end of agent is the silane coupler being combined with Silica-coated magnetic nanoparticle surface, and the other end is ATRP initiator.
Specifically comprise the following steps that and 8mmol3-aminopropyltriethoxywerene werene and 10mmol triethylamine are joined 12.5ml oxolane
In, it is passed through nitrogen deoxygenation ice bath 30min simultaneously after mixing, obtains silane coupler with this, afterwards by 10mmol2-bromine isobutyryl
Bromine (ATRP initiator) is slowly dropped in mixed liquor, and 25 DEG C are stirred vigorously 4h(maintaining nitrogen purge), after finally solution being filtered
Be dried under vacuum to the 1/3 of initial volume to remove oxolane and triethylamine, centrifugal after remove precipitation, nitrogen is available after drying up
Yellow, viscous SI-ATRP initiator, airtight 4 DEG C of preservations after inflated with nitrogen.
Two, SI-ATRP initiator is fixing
Utilize the ferroferric oxide magnetic nanoparticle of the silane coupler on SI-ATRP initiator and Silica-coated
It is covalently bound that the silicone hydroxyl generation dehydration on surface generates siliconoxygen bond, thus SI-ATRP initiator is fixed on magnetic-particle
On surface.Specifically comprise the following steps that first passing through acid-alkali treatment exposes the ferroferric oxide magnetic nanoparticle of Silica-coated
The silicone hydroxyl on (following abbreviation magnetic nanoparticle) surface, i.e. use 0.1M HCl solution immersion magnetic nanoparticle, 30 minutes
After HCl solution is removed, cleaning magnetic nanoparticle with water is about 7 to pH value of solution, re-uses 0.1M NaOH solution and soaks magnetic
Property nano-particle, after 30 minutes, NaOH solution is removed, cleaning magnetic nanoparticle with water is about 7 to pH.Afterwards by step
The ethanol solution containing 500 μMs of SI-ATRP initiators of one preparation mixes (SI-with the magnetic nanoparticle after acid-alkali treatment
The mass ratio of the magnetic nanoparticle after ATRP initiator and acid-alkali treatment is 0.74:1), go after reacting 10 hours at 20-30 DEG C
Except residue SI-ATRP initiator, and cleaning magnetic nanoparticle with methanol solution, nitrogen dries up.
Three, the synthesis of 2-methacrylic acid 3-Glucoamino propyl ester (GMA-G) monomer
In 674.53 μ l glycidyl methacrylate (GMA), add 0.2M sulphuric acid, be positioned in 50 DEG C of water-baths and add
After heat carries out oxidation reaction 4 hours, obtain the GMA of oxidation.Separately measure 20 ml methanol, be gradually added 1.1 grams of glucosamine,
Stirring is to dissolving, and adds the sodium cyanoborohydride of 5mg/ml.The GMA that gained aoxidizes is added dropwise to continuously stirred amino
In the methanol solution of glucose, 20-30 DEG C of stirring carries out schiff base reaction 4 hours in yellow transparent solution, and will prepare
Solution nitrogen dries up and evaporates in paste, obtains GMA-G monomer, places 4 DEG C of preservations, the MALDI-of GMA-G after airtight inflated with nitrogen
TOF-MS spectrogram as in figure 2 it is shown, Fig. 2 shows that the molecular weight of GMA-G hydrophilic monomer is 303, table on MALDI-TOF-MS spectrogram
Now for hydrogenation peak, m/z=304, it coincide with theoretical molecular.
Four, SI-ATRP reaction is caused to generate hydrophobic or hydrophilic polymer chains at magnetic nanoparticle surface in situ
Monomer used be hydrophobicity GMA or one end of step 3 synthesis with the double bond other end with the hydrophilic of glucose
GMA-G。
By GMA or GMA-G monomer, catalyst cuprous bromide, part N, N, N', N'', N''-PMDETA
Add in solvent (GMA uses Hexalin, and GMA-G uses methanol) according to the ratio of mol ratio 200/200:1:1.5, supersound process
It is made to dissolve and mix homogeneously.The surface above-mentioned mixed liquor and step 2 prepared has secured the magnetic of SI-ATRP initiator
Nano-particle mixes, and uses the oxygen in the method removing system of the freezing-evacuation-nitrogen that thaws-rush, so circulation 3 times, it
Seal with sealed membrane afterwards, cause ST-ATRP reaction 6 hours (GMA) or 24 hours in the 20-30 DEG C of surface in situ at silica filler
(GMA-G).Repeatedly clean with methanol, remove residual reactant after having reacted, obtain hydrophobicity GMA or hydrophilic GMA-G and gather
The carrier material of the immobilization proteinase that the magnetic nanoparticle that compound is modified, the i.e. present invention provide.
Five, the property analysis of three kinds of magnetic-particles
(1) the polymer-modified magnetic nanoparticle of 500mgGMA, the magnetic that 500mgGMA-G is polymer-modified are weighed respectively
Property nano-particle and 500mg parcel silicon dioxide magnetic nanoparticle carry out thermogravimetric analysis, result is as shown in Figure 3.
In Fig. 3, (a) is the thermal gravimetric analysis curve of the magnetic nanoparticle of Silica-coated；B () is GMA-G polymer
The thermal gravimetric analysis curve of the magnetic nanoparticle modified；C () is that the thermogravimetric analysis of magnetic nanoparticle polymer-modified for GMA is bent
Line.
The polymer-modified magnetic nanoparticle of GMA, GMA-G by thermal decomposition thus cause mass loss polymer
Layer and the most heat decomposable magnetic-particle core two parts form.As can be seen from Figure 3, the magnetic Nano of Silica-coated
Grain is with the raising of heat treatment temperature without obvious mass loss, and the magnetic nanoparticle that GMA-G is polymer-modified, with heat treatment temperature
The raising of degree, shows obvious mass loss (about 25%), and the magnetic nanoparticle that GMA is polymer-modified, with heat treatment temperature
Raising, show obvious mass loss (about 25%) equally.Prove former on magnetic nanoparticle surface by SI-ATRP method
It is highly effective that position polymerization generates GMA or GMA-G polymer.Simultaneously as the high controllability of SI-ATRP method, magnetic
Grain surface aggregate generates the amount of GMA or GMA-G polymer and can be effectively controlled by changing polymerization time, thus realizes egg
Effective control of white enzyme supported quantity.
(2) weighing the magnetic nanoparticle that 500mg GMA polymer chain is modified respectively, 500mg GMA-G polymer chain is repaiied
The magnetic nanoparticle of decorations and the magnetic nanoparticle of 500mg parcel silicon dioxide, carry out static contact angle experiment.Result such as figure
Shown in 4.
In Fig. 4, (a) is the static contact angle photo of the magnetic nanoparticle of Silica-coated；B () is GMA polymer
The static contact angle photo of the magnetic nanoparticle that chain is modified；C () is the quiet of the magnetic nanoparticle of GMA-G polymer chain modification
State contact angle photo.
Fig. 4 shows, the static contact angle after the magnetic nanoparticle press mold of Silica-coated is 78.6 °, GMA polymer
Static contact angle after the magnetic nanoparticle press mold modified is 50.5 °, magnetic nanoparticle press mold polymer-modified for GMA-G
After static contact angle be 0 °.Data above illustrates, silica surface has extremely strong hydrophobicity, and GMA polymer has suitable
In hydrophobicity, and GMA-G polymer has extremely strong hydrophilic.
(3) the hydrophilic and hydrophobic difference of different polymer-modified magnetic nanoparticles can be by it in different solutions system
The difference of middle degree of scatter proves further.Weigh the magnetic nanoparticle of 100mg Silica-coated respectively, 100mgGMA gathers
Magnetic nanoparticle and the magnetic nanoparticle of 100mg GMA-G polymer chain modification that compound chain is modified are scattered in acetonitrile respectively
Or in water, result is as shown in Figure 5.
In Fig. 5, the aqueous phase of the magnetic nanoparticle that (a) is respectively Silica-coated with (b) disperses photo mutually with acetonitrile；
C () is respectively the aqueous phase of the magnetic nanoparticle that GMA polymer chain is modified with (d) and disperses photo with acetonitrile mutually；(e) and (f) point
The aqueous phase of the magnetic nanoparticle do not modified for GMA-G polymer chain disperses photo mutually with acetonitrile.
Fig. 5 shows, the magnetic nanoparticle of Silica-coated is reunited in aqueous phase completely, is deposited in bottom EP pipe, and
Acetonitrile mutually in dispersed；GMA polymer chain modify magnetic nanoparticle can disperse in aqueous phase, but acetonitrile mutually in
Dispersibility more preferable, degree of scatter is higher；The magnetic nanoparticle that GMA-G polymer chain is modified disperses at aqueous phase camber, and
Acetonitrile mutually in substantially reunite, be deposited in bottom EP pipe.Data above demonstrates again that the magnetic nanoparticle of Silica-coated
Having a stronger hydrophobicity, the magnetic nanoparticle that GMA polymer chain is modified has a moderate hydrophobicity, and GMA-G polymer
The magnetic nanoparticle that chain is modified has extremely strong hydrophilic.
(4) use dynamic light scattering that three kinds of granules are carried out diameter characterization.Result shows, the magnetic of Silica-coated
Property nano particle diameter be 57.4nm, GMA polymer chain modify magnetic nanoparticle particle diameter be 73.7nm, GMA-G polymer
The particle diameter of the magnetic nanoparticle that chain is modified is 85.9nm.
(5) weighing the magnetic nanoparticle that 500mg GMA polymer chain is modified respectively, 500mg GMA-G polymer chain is repaiied
The magnetic nanoparticle of decorations and the magnetic nanoparticle of 500mg Silica-coated, carry out the non-specific suction of fluoroscopic examination albumen
Attached experiment, result is as shown in Figure 6.
In Fig. 6, (a) is that the fluorescence after fluorescent protein B SA-FITC is hatched with the magnetic nanoparticle of Silica-coated is aobvious
Micro mirror photo, (d) is with PBS(pH=7.4) clean after fluorescent microscopy images；B () is fluorescent protein B SA-FITC and GMA
The magnetic nanoparticle that polymer chain is modified hatch after fluorescent microscopy images, (e) is for PBS(pH=7.4) clean after glimmering
Light microscope photo；The magnetic nanoparticle that c () is fluorescent protein B SA-FITC modifies with GMA-G polymer chain hatch after glimmering
Light microscope photo, (f) is with PBS(pH=7.4) clean after fluorescent microscopy images.
Fig. 6 shows, the magnetic nanoparticle of parcel silicon dioxide has strong non-specific adsorption, warp to fluorescin
Still there is a large amount of fluorescin to remain in particle surface after PBS, illustrate that silica surface hydrophobic is too strong, easily cause egg
The non-specific adsorption of white matter/peptide fragment, causes sample loss, and the magnetic nanoparticle of parcel silicon dioxide should not be as immobilization
The carrier material of protease.Although the magnetic nanoparticle that hydrophobic GMA polymer chain is modified also can adsorb fluorescin, but warp
After PBS, granule does not has substantially fluorescin remain, illustrate that it has the hydrophobicity of appropriateness, can carry as hydrophobicity
Body materials application is in immobilization proteinase.The magnetic nanoparticle that hydrophilic GMA-G polymer chain is modified, does not has fluorescin
Any non-specific adsorption, illustrates that it has good hydrophilic, can be used for preparing immobilization as hydrophilic carrier material
Enzyme reagent.
Embodiment 2, tryptic fixing
One, the aldehyde radical of magnetic nanoparticle
The aldehyde radical functionalization of the polymer lateral chain of the magnetic nanoparticle that GMA-G polymer chain is modified, concrete grammar is as follows:
The magnetic nanoparticle that 10mM sodium metaperiodate aqueous solution is modified with GMA-G polymer chain is mixed homogeneously, lucifuge at 20-30 DEG C
React 2 hours.The methanol aqueous solution using volumn concentration to be 50% after having reacted repeatedly cleans, removes residual reactant.
The aldehyde radical functionalization of the polymer lateral chain of the magnetic nanoparticle that GMA polymer chain is modified, concrete grammar is as follows: use
The magnetic nanoparticle mix homogeneously that GMA polymer chain is modified by the dilute sulfuric acid of 0.2M, at room temperature lucifuge is reacted 2 hours.Instead
The methanol aqueous solution that volumn concentration should be used to be 50% after completing repeatedly cleans, removes residual reactant.
Two, on the magnetic-particle that GMA, GMA-G polymer chain of aldehyde radical is modified, carry out trypsin to fix
Concrete grammar is as follows, trypsin is dissolved in 50mM ammonium bicarbonate soln (pH=8.0), and adds 5mg/ml cyano group
Sodium borohydride, obtains mixed liquor, the most tryptic final concentration of 2mg/ml.Solution after 1ml mix homogeneously is joined and contains
Have in the magnetic particle solution that GMA or the GMA-G polymer chain of 1mg aldehyde radical is modified, 4 DEG C reaction 12 hours after use Magnet will
Magnetic nanoparticle is adsorbed in tube wall, removes supernatant.By trypsin solution ultraviolet at 280nm before and after immobilization
The Trypsin enzyme amount of residual in the change calculations supernatant of absorption value, thus show that trypsin is at GMA or GMA-G polymer chain
The supported quantity on magnetic nanoparticle modified is respectively 183.1 μ g/mg, 170.4 μ g/mg.Use 50mM ammonium hydrogen carbonate afterwards
Solution cleans magnetic nanoparticle, removes residual reactant.
Three, the closing of the polymer lateral chain residue aldehyde radical of the magnetic-particle that GMA, GMA-G polymer chain is modified
Dose volume percentage composition be the ethylaminoethanol of 10% phosphate buffer, by its magnetic Nano with step 2
Granule mixes, and reacts 4 hours at 4 DEG C.Repeatedly clean granule with 50mM ammonium bicarbonate soln afterwards, remove residual reactant and get final product
Magnetic-particle immobilized enzyme (GMA-Trypsin) and the magnetic-particle of GMA-G polymer chain modification that GMA polymer chain is modified are solid
Surely enzyme (GMA-G-Trypsin) is changed.
The magnetic-particle immobilized enzyme (GMA-Trypsin) of embodiment 3, GMA polymer chain modification and GMA-G polymer chain
The functional analysis of the magnetic-particle immobilized enzyme (GMA-G-Trypsin) modified
One, the extraction of yeast whole protein
(1) whole protein extracting solution: 50mM Tris-HCl (pH=8.0), 8M carbamide, 2mM EDTA.
(2) in 500 μ l whole protein extracting solution, 10 μ l " cocktail " formula protease inhibitor is added (purchased from Roche moral
State), join equipped with in saccharomycetic test tube after mix homogeneously, sonicated cells.At 4 DEG C, 20000g is centrifuged 20 points afterwards
Clock, removes unbroken cell and fragment, and solution is yeast whole protein.
(3) in yeast whole protein solution, add the cold acetone solution (-20 DEG C of pre-coolings) of 4-5 times of volume, put for-20 DEG C
After putting more than 2 hours, 4 DEG C of 12000g are centrifuged 10 minutes, carefully draw supernatant, retain precipitation.
(4) precipitation is positioned in fume hood, makes residue acetone fully volatilize.The albumen obtained by acetone precipitation is again
It is dissolved in 8M urea buffer solution and use Bradford method to measure protein concentration.Yeast whole protein immobilized enzyme enzyme action
Two, preparation 2mg/ml yeast whole protein solution, adds DTT, reduces 4 hours, add IAA afterwards in 37 DEG C of water-baths
Place in dark place and be alkylated for 1 hour.50mM ammonium bicarbonate soln, mix homogeneously is added in the protein solution handled well
After be divided into 4 parts, first part add 10 μ l GMA polymer chains modify magnetic nano particle immobilized enzyme (GMA-Trypsin)
(the magnetic nano particle immobilized enzyme that in first part, GMA polymer chain is modified is 10:1 with the mass ratio of albumen to be degraded),
Second part adds the magnetic nano particle immobilized enzyme (GMA-G-Trypsin) (second part that 10 μ l GMA-G polymer chains are modified
The magnetic nano particle immobilized enzyme that middle GMA-G polymer chain is modified is 10:1 with the mass ratio of albumen to be degraded), the 3rd part
Add the mixture (each 5 μ l, mixed enzymolysis (CITD)) the (the 3rd of two kinds of immobilized enzyme of GMA-Trypsin and GMA-G-Trypsin
In Fen, two kinds of immobilized enzyme, mass ratioes of albumen to be degraded are 5:5:1), after hatching 1 minute in 37 DEG C of water-baths respectively, make
With Magnet, magnetic nano particle immobilized enzyme is adsorbed in tube wall, draws supernatant.4th part, according to conventional enzymatic hydrolysis condition, adds matter
Amount ratio for 1:50(trypsin: treat protein degradation matter) trypsin, be placed in after 37 DEG C of water-baths are hatched 12 hours add body
Long-pending percentage composition is that the TFA of 0.1% is by trypsin inactivation.Take three kinds of immobilized enzyme enzymolysis and conventional soln enzymatic hydrolysate respectively
Use chromatography-electrospray-ionization/mass spectrometry combination to identify, and data qualification obtained use MASCOT software to carry out searching storehouse,
Result is as shown in Table 1 and Table 2.
The repeatability analysis of table 1 hydrolysis result
The mutual coverage rate analysis of table 2 hydrolysis result
Table 1 shows, uses the hydrophobic GMA-Trypsin enzymatic yeast whole protein of two batches, at same experiment condition
Under, 527 and 510 protein, 1860 and 1785 peptide fragments can be identified respectively, repeatability is respectively up to 89.1% and 83.9；
Use the hydrophilic GMA-G-Trypsin enzymatic yeast whole protein of two batches, under same experiment condition, can identify respectively
To 560 and 533 protein, 1847 and 1744 peptide fragments, repeatability is respectively up to 86.0% and 76.3%.Data above illustrates,
GMA-Trypsin, GMA-G-Trypsin of two batches is respectively provided with good enzymolysis repeatability, and identification of proteins repeatability can
Reaching more than 85%, peptide fragment identifies that repeatability, up to more than 75%, can meet the demand of complex proteins sample enzymolysis.
Due to the high controllability of SI-ATRP method, the polymer wrapped amount on polymer hybrid magnetic nanoparticle surface can
Accurately controlled by polymerization time, nucleocapsid structure, surface polymer content and the supported quantity of protease between different batches hybrid particulates
Difference is less to be caused.Table 2 shows, the enzymolysis experiment of two batches, hydrophobic GMA-Trypsin identifies 574 protein altogether
With 2248 peptide fragments；Hydrophilic GMA-G-Trypsin identifies 623 protein and 2115 peptide fragments altogether, and the two identifies scale
It is closer to, illustrates that the enzymolysis efficiency of two kinds of immobilization proteinase reagent is close, be used equally to the fast of complex proteins group sample
Enzymolysis fast, efficient.Use conventional soln enzymolysis, under the conditions of same qualification, identify 602 protein and 2488 altogether
Peptide fragment.The immobilization proteinase reagent enzymolysis of the data above explanation single carrier of use 1 minute, just can reach and in-solution digestion 12
Hour similar effect, the polymer hybrid magnetic-particle immobilization proteinase again illustrating to prepare based on SI-ATRP method
Enzymolysis high efficiency.But between hydrophobic GMA-Trypsin and two kinds of immobilization proteinase reagent enzymolysis results of hydrophilic GMA-G-Trypsin
Mutual coverage rate the lowest, only 77.9%(albumen), 60.1%(peptide fragment), hence it is evident that less than immobilization proteinase reagent of the same race not
With the enzymolysis repeatability between batch.Data above explanation has the carrier material of different hydrophilic, hydrophobic character and has protein substrate
There is different affinitys, therefore cause two kinds of immobilization proteinase reagent to have certain substrate selective and enzymolysis is complementary.
Enzymatic hydrolysate to two kinds of immobilization proteinase reagent (hydrophobic GMA-Trypsin and hydrophilic GMA-G-Trypsin) identifies
Protein carry out the analysis of hydrophilic, hydrophobic property and demonstrate this supposition further, result is as shown in Figure 7.
Fig. 7 shows, in strongly hydrophilic region (GRAVY value <-0.75), GMA-G-Trypsin has clear superiority, identifies
Protein amounts accounts for it and all identifies about the 30% of protein amounts, hence it is evident that higher than the 10% of GMA-Trypsin；And at hydrophobicity relatively
Strong region (-0.35 < GRAVY value < 0), GMA-Trypsin has clear superiority, identifies that protein amounts accounts for it and all identifies
About the 50% of protein amounts, hence it is evident that higher than the 30% of GMA-G-Trypsin.
By hydrophobic GMA-Trypsin and two kinds of immobilization proteinase reagent combinations of hydrophilic GMA-G-Trypsin, mix
Enzymolysis (CITD), identifies 765 protein and 3505 peptide fragments as shown in Figure 8 from yeast whole protein extracting solution altogether.
In Fig. 8, GMA is hydrophobic GMA-Trypsin, and GMA-G is hydrophilic GMA-G-Trypsin.
Fig. 8 (a) be whole protein extract respectively through hydrophobic GMA-Trypsin, hydrophilic GMA-G-Trypsin and hybrid particles
(CITD) after enzymolysis, Mass Spectrometric Identification arrives protein and peptide fragment；B () and (c) is respectively GMA-Trypsin and GMA-G-Trypsin
The mutual coverage rate of protein of qualification result summation and CITD qualification result and the mutual coverage rate of peptide fragment.
Fig. 8 shows, compared with hydrophobic GMA-Trypsin, hydrophilic GMA-G-Trypsin, protein and peptide fragment are reflected by CITD
Determined number the most at least improves 20% and 50%, and effectively covers qualification result when two kinds of immobilized enzyme are used alone.Say
Bright immobilization proteinase based on double carrier, can effectively reduce the enzymolysis bias of single carrier immobilized protease, improves enzyme
Solve is comprehensive, thus dramatically increases the qualification quantity of protein and peptide fragment.
Mixed enzymolysis (CITD) and in-solution digestion result are as shown in Figure 9.
The protein of the Mass Spectrometric Identification result that Fig. 9 (a) and (b) are respectively CITD and conventional soln enzymatic hydrolysate mutually covers
Rate and the mutual coverage rate of peptide fragment.
Fig. 9 shows, compared with in-solution digestion effect, CITD can provide more albumen and peptide fragment to identify quantity, but two
Plant enzyme solution and there is certain complementarity.The qualification result of the two is merged, the peptide fragment that average each qualification protein is corresponding
Identify that quantity is increased to 5.18 by the 4.13 of in-solution digestion.Therefore, CITD is except improving the mirror of protein and peptide fragment
Beyond set pattern mould, it is also possible to increase the reliability of qualification result.
Claims (13)
1. an immobilized enzyme mixture, by the immobilized enzyme that hydrophobic carrier material is substrate with hydrophilic carrier material be
The immobilized enzyme composition of substrate；
Described hydrophobic carrier material is the immobilized enzyme of substrate and the described immobilized enzyme with hydrophilic carrier material as substrate
Mass ratio be 1: 1；
Described hydrophobic carrier material is that the immobilized enzyme of substrate by hydrophobic carrier material and is fixed on described hydrophobic carrier
Protease composition on material；
Described hydrophobic carrier material be hydrophobic monomer with surface Atom Transfer Radical Polymerization initiator
There is atom transfer radical polymerization and the product that obtains in the surface of grain；Wherein, described hydrophobic monomer aggregates into polymeric chain；Institute
State the polymeric chain that hydrophobic monomer is polymerized bonded by C-Si-C with described granule；
Protease on described hydrophobic carrier material is linked by carbonnitrogen bond with the polymeric chain on described hydrophobic carrier material；
The described granule with surface Atom Transfer Radical Polymerization initiator is for cause atom transferred free radical by surface
Polymerization initiator is fixedly connected on the surface of described granule and makes；
One end of described surface Atom Transfer Radical Polymerization initiator is the coupling agent being combined with silicone hydroxyl, the other end
For atom transfer radical polymerization initiator；
Described hydrophobic monomer is glycidyl methacrylate；
The method of described fixing connection is described surface Atom Transfer Radical Polymerization initiator and described particle surface
It is covalently bound that silicone hydroxyl generation dehydration generates siliconoxygen bond；
The described immobilized enzyme with hydrophilic carrier material as substrate is by hydrophilic carrier material and is fixed on described hydrophilic load
Protease composition on body material；
Protease on described hydrophilic carrier material is linked by carbonnitrogen bond with described hydrophilic carrier material；
Described hydrophilic carrier material be hydrophilic monomer described with surface Atom Transfer Radical Polymerization initiator
The surface of granule there is atom transfer radical polymerization and the product that obtains；Wherein, described hydrophilic monomer aggregates into polymerization
Chain；The polymeric chain that described hydrophilic monomer is polymerized and described granule are bonded by C-Si-O；
One end of the surface Atom Transfer Radical Polymerization initiator of described granule is the coupling agent being combined with silicone hydroxyl,
The other end is atom transfer radical polymerization initiator；
Described coupling agent is silane coupler；
Described silane coupler is APTES, 3-TSL 8330 or γ-mercapto propyl group
Triethoxysilane；
The initiator of described atom transfer radical polymerization is 2-bromine isobutyl acylbromide, alpha-brominated isoamyl acylbromide or α-bromopropionyl bromide；
Described silane coupler is connected by amido link with the initiator of described atom transfer radical polymerization；
Described hydrophilic monomer is the hydrophilic monomer containing monosaccharide group, acrylic monomer, butylene acrylic monomer or amylene
Acrylic monomer；
Described monosaccharide is glucosamine or epichitosamine；
Described granule is magnetic nanoparticle；
Described magnetic nanoparticle is the magnetic nanoparticle of Silica-coated；
Protease on described hydrophobicity and hydrophilic carrier material be trypsin, protein incision enzyme, intracellular protein enzyme or
Chymase.
Immobilized enzyme mixture the most according to claim 1, it is characterised in that: described hydrophilic monomer is 2-metering system
Acid 3-Glucoamino propyl ester monomer.
Immobilized enzyme mixture the most according to claim 2, it is characterised in that: described 2-methacrylic acid 3-glucosamine
Base propyl ester monomer is prepared as follows: glycidyl methacrylate aoxidized, and obtains the metering system of oxidation
Acid glycidyl ester；Glucosamine is mixed with sodium cyanoborohydride in methanol, obtains the methanol solution of glucosamine；
The methanol solution of the glycidyl methacrylate of oxidation Yu glucosamine is carried out schiff base reaction and obtains reactant liquor；Will
Reactant liquor noble gas dries up to paste, to obtain final product；
Described noble gas is nitrogen.
Immobilized enzyme mixture the most according to claim 1, it is characterised in that: atom transferred free radical is caused on described surface
The preparation method of polymerization initiator is as follows: silane coupler and Bronsted acid agent for capturing is reacted in ice bath, adds former
Sub-transfer radical polymerization initiator, reacts, and to obtain final product；
The molar ratio of described silane coupler, atom transfer radical polymerization initiator and Bronsted acid agent for capturing is 0.5-1:
1∶1。
Immobilized enzyme mixture the most according to claim 4, it is characterised in that: described silane coupler, atom transfer are certainly
It is 0.8: 1: 1 by the molar ratio of base polymerization initiator and Bronsted acid agent for capturing.
6. according to the arbitrary described immobilized enzyme mixture of claim 1-5, it is characterised in that: described with surface initiation atom
The preparation method of granules of transfer radical polymerization initiator is as follows: the magnetic nanoparticle of described Silica-coated is carried out acid
Alkali processes and exposes silicone hydroxyl, it is mixed in solvent with surface Atom Transfer Radical Polymerization initiator and is dehydrated
Reaction generates siliconoxygen bond, to obtain final product；
Described solvent be in ethanol, methanol and Hexalin any one；
The temperature of described dehydration is 20-30 DEG C；
The time of described dehydration is 1-24 hour；
Described magnetic nanoparticle is 1: 0.37-with the mass ratio of the initiator of described surface Atom Transfer Radical Polymerization
7.4。
Immobilized enzyme mixture the most according to claim 6, it is characterised in that: the time of described dehydration is 10 little
Time；Described magnetic nanoparticle is 1: 0.74 with the mass ratio of the initiator of described surface Atom Transfer Radical Polymerization.
Immobilized enzyme mixture the most according to claim 1, it is characterised in that: the preparation side of described hydrophobic carrier material
Method is as follows: hydrophobic monomer, catalyst, part and solvent is mixed, obtains mixed liquor 1；By mixed liquor 1 with described with surface
The granule mixing of Atom Transfer Radical Polymerization initiator, obtains mixed liquor 2；Remove the oxygen in mixed liquor 2, cause atom
Transition free radical polymerization reaction, to obtain final product；
Described catalyst is the halogenide of any one metal following: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and Pb；
Described part be following any one: N, N, N ', N ", N "-PMDETA, 2,2 '-bipyridyl, tetramethyl
Ethylenediamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl triens and three (2-dimethylaminoethyl) amine；
Solvent for use be following any one: methanol, ethanol, Hexalin, dimethyl sulfoxide, oxolane；
Described hydrophobic monomer, catalyst, the mol ratio of part are 200: 0.5-5: 0.75-7.5；
The method that described protease is fixed on described hydrophobic carrier material is as follows:
(1) by hydrophobic carrier material functional, the carrier material of functionalization is obtained；
(2) carry out the fixing of protease on the carrier material of the functionalization obtained in step (1), obtain securing the merit of protease
Carrier can be changed；
(3) the residue functional group on the functional supports securing protease is closed, obtain purpose immobilization proteinase；
Method fixing described in described step (2) is that described protease is dissolved in the ammonium bicarbonate soln of pH 8.0, adds cyanogen
Base sodium borohydride obtains mixed liquor；Mixed liquor is joined in the carrier material of the functionalization described in step (1), reaction, then general
Grain takes out and get final product；
The method closed described in described step (3) be with ethylaminoethanol fixing of obtaining of phosphate buffer and step (2)
The functional supports mixing of protease, reacts and get final product；
Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be 5%-20%；
The method of described functionalization is aldehyde radical and/or chemical modification；
Described aldehyde radical turns to mix described hydrophobic carrier material with the dilute sulfuric acid of 0.2M, and at 25 DEG C, lucifuge is reacted and get final product；
Described protease is connected by carbonnitrogen bond with the carrier material of described functionalization；
Described protease is 1: 10-10: 1 with the mass ratio of the carrier material of described functionalization；
Described functional group is the group being combined with described protease；
Described functional group is positioned on the polymeric chain of described carrier material.
Immobilized enzyme mixture the most according to claim 8, it is characterised in that: described hydrophobic monomer, catalyst, part
Mol ratio be 200: 1: 1.5；Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be
10%；Described protease is 2: 1 with the mass ratio of the carrier material of described functionalization.
Immobilized enzyme mixture the most according to claim 1 and 2, it is characterised in that: the system of described hydrophilic carrier material
Preparation Method is as follows: hydrophilic monomer, catalyst, part and solvent is mixed, obtains mixed liquor 1；By mixed liquor 1 with described with
The granule mixing of surface Atom Transfer Radical Polymerization initiator, obtains mixed liquor 2；Remove the oxygen in mixed liquor 2, cause
Atom transition free radical polymerization reaction, obtains reactant liquor；Residual reactant in reactant liquor is removed, obtains immobilization proteinase
Hydrophilic carrier material；
Described catalyst is the halogenide of any one metal following: Cu, Mo (IV), Ru, Rh, Fe, Re, Ni, Pd and Pb；
Described part be following any one: N, N, N ', N ", N "-PMDETA, 2,2 '-bipyridyl, tetramethyl
Ethylenediamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl triens and three (2-dimethylaminoethyl) amine；
Solvent for use be following any one: methanol, ethanol, Hexalin, dimethyl sulfoxide, oxolane；
Described hydrophilic monomer, catalyst, the mol ratio of part are 200: 0.5-5: 0.75-7.5；
The synthetic method of described immobilization proteinase is as follows:
(1) by carrier material functionalization, the carrier material of functionalization is obtained；
(2) carry out the fixing of protease on the carrier material of the functionalization obtained in step (1), obtain securing the merit of protease
Carrier can be changed；
(3) the residue functional group on the functional supports securing protease is closed, obtain purpose immobilization proteinase；
Method fixing described in described step (2) is that described protease is dissolved in the ammonium bicarbonate soln of pH8.0, adds cyano group
Sodium borohydride obtains mixed liquor；Mixed liquor is joined in the carrier material of the functionalization described in step (1), reaction, then by granule
Take out and get final product；
The method closed described in described step (3) be with ethylaminoethanol fixing of obtaining of phosphate buffer and step (2)
The functional supports mixing of protease, reacts and get final product；
Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be 5%-20%；
Described carrier material is to be polymerized with polymeric chain or the hydrophobic monomer being polymerized by hydrophilic monomer by surface
The granule of polymeric chain；
The method of described functionalization is aldehyde radical and/or chemical modification；
Described aldehyde radical is the vicinal diamines thaumatropy of the monosaccharide group on the polymeric chain being polymerized by described hydrophilic monomer
Carry out aldehyde radical for aldehyde radical, obtain aldehyde radical carrier；
Or,
Described aldehyde radical is that the cycloalkyl groups on the polymeric chain being polymerized by hydrophobic monomer is converted into aldehyde radical, obtains aldehyde radical
Change carrier；
Described protease is 1: 10-10: 1 with the mass ratio of the carrier material of described functionalization；
Described protease is connected by carbonnitrogen bond with the carrier material of described functionalization；
Described functional group is the group being combined with described protease；
Described functional group is positioned on the polymeric chain of described carrier material.
11. immobilized enzyme mixture according to claim 10, it is characterised in that: described hydrophilic monomer, catalyst, join
The mol ratio of body is 200: 1: 1.5；Described ethylaminoethanol ethylaminoethanol volumn concentration in phosphate buffer be
10%；Described protease is 2: 1 with the mass ratio of the carrier material of described functionalization.
The method of 12. 1 kinds of enzymolysis proteins, comprises the steps: the albumen treating enzymolysis is carried out degeneration, and obtain degeneration treats enzyme
Solve albumen；Arbitrary for claim 1-11 described immobilized enzyme mixture and degeneration treated that enzymolysis protein is at pH=8.0 25-
Mix homogeneously in the phosphate buffer of 100mM ammonium bicarbonate soln or pH=7.8 25-100mM, hatches, and obtains enzymolysis solution；
The method that the described albumen treating enzymolysis carries out degeneration is: add DTT in the described albumen treating enzymolysis, in 37 DEG C of water-baths also
Former, placement is alkylated in dark place to add IAA.
The application in proteolysis of the 13. claim 1-11 arbitrary described immobilized enzyme mixture；
Described albumen is yeast whole protein.
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| CN104911162B (en) * | 2015-06-24 | 2018-08-17 | 中国科学院广州能源研究所 | A kind of controlled pore carrier-free immobilized lipase and preparation method thereof |
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