CN103882002A - Preparation and application of immobilized protease reagent - Google Patents
Preparation and application of immobilized protease reagent Download PDFInfo
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- CN103882002A CN103882002A CN201410062221.1A CN201410062221A CN103882002A CN 103882002 A CN103882002 A CN 103882002A CN 201410062221 A CN201410062221 A CN 201410062221A CN 103882002 A CN103882002 A CN 103882002A
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Abstract
The invention discloses preparation and application of an immobilized protease reagent. An immobilized enzyme disclosed by the invention is composed of a hydrophobic carrier material and a protease immobilized on the hydrophobic carrier material. Two different-property, namely one hydrophilic and one hydrophobic, polymer chain modified magnetic nanoparticle immobilized trypsins prepared independently by using a surface initiate atom transfer radical polymerization (SI-ATRP) realize quick, efficient and complete enzymolysis of a protein, and the two, one hydrophilic and one hydrophobic, immobilized proteases are combined to use so that complementary enzymolysis is realized, and therefore, enzymatic bias caused by the selectivity of the carrier can be effectively reduced, the comprehensiveness of protein enzymolysis can be improved, and then the identification number of proteins and peptide fragments can be remarkably increased.
Description
Technical field
The present invention relates to a kind of preparation and application thereof of immobilization proteinase reagent.
Background technology
As the priority research areas of genome times afterwards comprehensively, proteomics research can not only be provided fundamental basis for illustrating of life mechanics, also can play a role at aspects such as the diagnosis of disease, treatment, prevention, infective pathogen pathogenesis and new drug developments.Be chosen as behind the six large hot research fields of 21 century by " science " magazine from calendar year 2001, proteomics research has been subject to scientist's extensive concern.Being most widely used at present proteomics research strategy is " shotgun " (shot-gun method) strategy.In this strategy, the quantitative and qualitative analysis information of protein is to obtain by corresponding enzymolysis product peptide section is analyzed, therefore fast, efficiently, completely protein digestion just become protein group accurately, extremely important link in high throughput identification and quantitative examination.But, in traditional in-solution digestion for fear of proteolytic enzyme self enzymolysis, often must use lower proteolytic enzyme and substrate protein white matter ratio (proteolytic enzyme: protein substrate=1:50), therefore the incubation time (12-20 hour) that enzyme digestion reaction conventionally need to be longer, and be difficult to realize complete enzymolysis for the sample of high complexity, thereby limit the analysis throughput of sample, the sensitivity of identification of proteins and the accuracy of quantitative examination, cannot meet that protein science is extensive, the requirement of high throughput analysis.
Based on above reason, develop protein digestion technology fast and efficiently, be very necessary for the qualification of complex proteins group sample.Enzyme immobilization technology has been avoided the problem of proteolytic enzyme self enzymolysis, therefore can use higher enzyme/substrate ratio, thereby significantly accelerate the process of enzyme digestion reaction, have recyclable simultaneously and the plurality of advantages such as reuse. in numerous Carrier Materials of Immobilized Enzyme, as silicon dioxide microsphere, polymeric film, Monolithic Columns, (Xu in mesoporous material matrix, F.et 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 its larger specific surface area and be easy to realize the characteristics such as magnetic resolution and highlight advantage (Lin, S.et al.J.Proteome Res.2008, 7, 1297-1307.).But what generally adopt at present carries out the fixing method of individual layer enzyme at substrate material surface, makes the enzyme supported quantity of unit mass immobilized enzyme material be subject to the long-pending restriction of substrate material surface, thereby has limited the further raising of enzymolysis efficiency.Existing immobilized enzyme reagent all adopts single solid support material simultaneously, and the enzymolysis bias that inevitable carrier itself causes the affinity difference of protein, causes protein digestion comprehensive not, has affected the fraction of coverage of protein and the qualification of peptide section.
Transfer Radical Polymerization (Atom Transfer Radical Polymerization, ATRP) is a kind of controllable free radical polymerization process that MatyjaszewkiK. professor group proposed first in nineteen ninety-five.Since more than ten years, obtain the extensive concern of international academic community and industry member.ATRP method has the features such as product polymer architecture controllability is good, narrow molecular weight distribution, suitable monomers scope is wide, reaction conditions requirement is moderate.Surface Atom Transfer Radical Polymerization method (SI-ATRP) is a kind of method that ATRP initiator is fixed on to original position initiated polymerization thing growth after material surface, has now been widely used in finishing and the functionalization of material.
Summary of the invention
The object of this invention is to provide a kind of preparation and application thereof of immobilization proteinase reagent.
A kind of immobilized enzyme provided by the invention, this immobilized enzyme is made up of hydrophobic carrier material and the proteolytic enzyme being fixed on described hydrophobic carrier material;
Described hydrophobic carrier material is that hydrophobic monomer, on the surface of the particle with surperficial Atom Transfer Radical Polymerization initiator, the product that atom transfer radical polymerization obtains occurs; Wherein, described hydrophobic monomer aggregates into polymeric chain; The polymeric chain that described hydrophobic monomer is polymerized is connected by C-Si-O key with described particle;
Described proteolytic enzyme links by carbonnitrogen bond with the polymeric chain on described hydrophobic carrier material.
In above-mentioned immobilized enzyme, described particle is magnetic nanoparticle;
Described magnetic nanoparticle is specially the magnetic nanoparticle of Silica-coated;
The described particle with surperficial Atom Transfer Radical Polymerization initiator is that make on the surface that surperficial Atom Transfer Radical Polymerization initiator is fixedly connected on to described particle;
One end of described surperficial Atom Transfer Radical Polymerization initiator be can with the coupling agent of silicon hydroxy combining, the other end is atom transfer radical polymerization initiator;
Described hydrophobic monomer is the Racemic glycidol esters monomer that contains epoxy group(ing), is specially glycidyl methacrylate;
Described proteolytic enzyme is specially trypsinase, protein incision enzyme, intracellular protein enzyme or Quimotrase.
In above-mentioned arbitrary described immobilized enzyme, described in the method that is fixedly connected be that the silicon hydroxyl generation dehydration reaction generation siloxane bond of described surperficial Atom Transfer Radical Polymerization initiator and described particle surface is covalently bound;
Described coupling agent is silane coupling agent;
Described silane coupling agent is 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-bromopropionyl bromide;
Described silane coupling agent is connected by amido linkage with described atom transfer radical polymerization initiator.
In above-mentioned arbitrary described immobilized enzyme, the preparation method of described surperficial Atom Transfer Radical Polymerization initiator is as follows: silane coupling agent and protonic acid trapping agent are reacted in ice bath, add again atom transfer radical polymerization initiator, react, to obtain final product;
The molar ratio of described silane coupling agent, atom transfer radical polymerization initiator and protonic acid trapping agent is 0.5-1:1:1, is specially 0.8:1:1.
In above-mentioned arbitrary described immobilized enzyme, the described preparation method of granules with surperficial Atom Transfer Radical Polymerization initiator is as follows: the magnetic nanoparticle of described Silica-coated is carried out to acid-alkali treatment and expose silicon hydroxyl, itself and surperficial Atom Transfer Radical Polymerization initiator are mixed to generation dehydration reaction generation siloxane bond in solvent, to obtain final product;
Described solvent be in ethanol, methyl alcohol and hexalin any one;
The temperature of described dehydration reaction is 20-30 DEG C;
The time of described dehydration reaction is 1-24 hour, is specially 10 hours;
The mass ratio of the initiator of described magnetic nanoparticle and described surperficial Atom Transfer Radical Polymerization is 1:0.37-7.4, is specially 1:0.74.
In above-mentioned arbitrary described immobilized enzyme, the preparation method of described hydrophobic carrier material is as follows: by hydrophobic monomer, catalyzer, part and solvent, obtain mixed solution 1; Mixed solution 1 is mixed with the described particle with surperficial Atom Transfer Radical Polymerization initiator, obtain mixed solution 2; Remove the oxygen in mixed solution 2, Atom Transfer Radical Polymerization reaction, to obtain final product;
Described catalyzer is the halogenide of following any one metal: 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'-dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (2-dimethylaminoethyl) amine;
Solvent for use be following any one: methyl alcohol, ethanol, hexalin, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF);
The mol ratio of described hydrophobic monomer, catalyzer, part is 200:0.5-5:0.75-7.5, is specially 200:1:1.5;
The method that described proteolytic enzyme is fixed on described hydrophobic carrier material is as follows:
(1), by hydrophobic carrier material functional, obtain the solid support material of functionalization;
(2) on the solid support material of the functionalization obtaining in step (1), carry out the fixing of proteolytic enzyme, be fixed the functional supports of proteolytic enzyme;
(3) by the residue functional group sealing of having fixed on the functional supports of proteolytic enzyme, obtain object immobilization proteinase;
Described in described step (2), fixing method is the ammonium bicarbonate soln that described proteolytic enzyme is dissolved in to pH8.0, adds sodium cyanoborohydride to obtain mixed solution; Mixed solution is joined in the solid support material of the described functionalization of step (1), reaction, then particle is taken out and be get final product;
Described in described step (3) sealing method be with monoethanolamine phosphate buffered saline buffer mix with the functional supports of having fixed proteolytic enzyme that step (2) obtains, react and get final product;
Described monoethanolamine monoethanolamine volumn concentration in phosphate buffered saline buffer be 5%-20%, be specially 10%;
The method of described functionalization is aldehyde radical and/or chemically modified;
Described aldehyde radical is specially described hydrophobic carrier material is mixed with the dilute sulphuric acid of 0.2M, and lucifuge is reacted and get final product at 25 DEG C;
Described proteolytic enzyme is connected by carbonnitrogen bond with the solid support material of described functionalization;
The mass ratio of the solid support material of described proteolytic enzyme and described functionalization is 1:10-10:1, is specially 2:1;
Described functional group is the group that can be combined with described proteolytic enzyme;
Described functional group is positioned on the polymeric chain of described solid support material.
A kind of immobilized enzyme mixture also belongs to protection scope of the present invention, is made up of above-mentioned arbitrary described immobilized enzyme and the immobilized enzyme taking hydrophilic carrier material as substrate;
Above-mentioned arbitrary described immobilized enzyme and the mass ratio of the described immobilized enzyme taking hydrophilic carrier material as substrate are specially 1:1;
The described immobilized enzyme taking hydrophilic carrier material as substrate is made up of hydrophilic carrier material and the proteolytic enzyme being fixed on described hydrophilic carrier material;
Described proteolytic enzyme is specially trypsinase, protein incision enzyme, intracellular protein enzyme or Quimotrase;
Described proteolytic enzyme links by carbonnitrogen bond with described hydrophilic carrier material;
Described hydrophilic carrier material is that hydrophilic monomer, on the surface of the described particle with surperficial Atom Transfer Radical Polymerization initiator, the product that atom transfer radical polymerization obtains occurs; Wherein, described hydrophilic monomer aggregates into polymeric chain; The polymeric chain that described hydrophilic monomer is polymerized is connected by C-Si-O key with described particle;
One end of the surperficial Atom Transfer Radical Polymerization initiator of described particle be can with the coupling agent of silicon hydroxy combining, the other end is atom transfer radical polymerization initiator;
Described coupling agent is silane coupling agent;
Described silane coupling agent is 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-bromopropionyl bromide;
Described silane coupling agent is connected by amido linkage with the initiator of described atom transfer radical polymerization;
Described hydrophilic monomer is specially the hydrophilic monomer, acrylic monomer, butylene acids monomer or the amylene acids monomer that contain monose group;
Described monose is specially glucosamine or epichitosamine;
Described hydrophilic monomer is specially 2-methacrylic acid 3-Glucoamino propyl ester monomer;
Described particle 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: glycidyl methacrylate is oxidized, obtains the glycidyl methacrylate of oxidation; Glucosamine is mixed in methyl alcohol with sodium cyanoborohydride, obtain the methanol solution of glucosamine; The methanol solution of the glycidyl methacrylate of oxidation and glucosamine is carried out to schiff base reaction and obtain reaction solution; Reaction solution is dried up to paste with rare gas element, to obtain final product;
Described rare gas element is specially nitrogen.
In above-mentioned immobilized enzyme mixture, the preparation method of described hydrophilic carrier material is as follows: by hydrophilic monomer, catalyzer, part and solvent, obtain mixed solution 1; Mixed solution 1 is mixed with the described particle with surperficial Atom Transfer Radical Polymerization initiator, obtain mixed solution 2; Remove the oxygen in mixed solution 2, Atom Transfer Radical Polymerization reaction, obtains reaction solution; Residual reactant in reaction solution is removed to the hydrophilic carrier material of being fixed proteolytic enzyme;
Described catalyzer is the halogenide of following any one metal: 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'-dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (2-dimethylaminoethyl) amine;
Solvent for use be following any one: methyl alcohol, ethanol, hexalin, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF);
The mol ratio of described hydrophilic monomer, catalyzer, part is 200:0.5-5:0.75-7.5, is specially 200:1:1.5;
The synthetic method of described immobilization proteinase is as follows:
(1), by solid support material functionalization, obtain the solid support material of functionalization;
(2) on the solid support material of the functionalization obtaining in step (1), carry out the fixing of proteolytic enzyme, be fixed the functional supports of proteolytic enzyme;
(3) by the residue functional group sealing of having fixed on the functional supports of proteolytic enzyme, obtain object immobilization proteinase;
Described in described step (2), fixing method is the ammonium bicarbonate soln that described proteolytic enzyme is dissolved in to pH8.0, adds sodium cyanoborohydride to obtain mixed solution; Mixed solution is joined in the solid support material of the described functionalization of step (1), reaction, then particle is taken out and be get final product;
Described in described step (3) sealing method be with monoethanolamine phosphate buffered saline buffer mix with the functional supports of having fixed proteolytic enzyme that step (2) obtains, react and get final product;
Described monoethanolamine monoethanolamine volumn concentration in phosphate buffered saline buffer be 5%-20%, be specially 10%;
The serve as reasons particle of the polymeric chain that surface is polymerized with the polymeric chain being polymerized by hydrophilic monomer or hydrophobic monomer of described solid support material;
The method of described functionalization is aldehyde radical and/or chemically modified;
The adjacent diol structure of the monose group on the polymeric chain that described aldehyde radicalization is specifically polymerized described hydrophilic monomer is converted into aldehyde radical and carries out aldehyde radical, obtains aldehyde radical carrier;
Or,
Cycloalkyl groups on the polymeric chain that described aldehyde radicalization is specifically polymerized hydrophobic monomer is converted into aldehyde radical, obtains aldehyde radical carrier;
The mass ratio of the solid support material of described proteolytic enzyme and described functionalization is 1:10-10:1, is specially 2:1;
Described proteolytic enzyme is connected by carbonnitrogen bond with the solid support material of described functionalization;
Described functional group is the group that can be combined with described proteolytic enzyme;
Described functional group is positioned on the polymeric chain of described solid support material.
The method of enzymolysis protein also belongs to a protection scope of the present invention, comprises the steps: the albumen for the treatment of enzymolysis to carry out sex change, obtains the enzymolysis protein for the treatment of of sex change; Above-mentioned arbitrary described immobilized enzyme mixture is mixed in the phosphate buffered saline buffer of pH=8.025-100mM ammonium bicarbonate soln or pH=7.825-100mM with the enzymolysis protein for the treatment of of sex change, hatch, obtain enzymolysis solution;
The method that the described albumen for the treatment of enzymolysis carries out sex change is specific as follows: in the described albumen for the treatment of enzymolysis, add DTT, in 37 DEG C of water-baths, reduce, then add IAA to place and carry out alkylation in dark place.
The application of above-mentioned arbitrary described immobilized enzyme in proteolysis also belongs to protection scope of the present invention;
Or,
The application of above-mentioned arbitrary described immobilized enzyme mixture in proteolysis also belongs to protection scope of the present invention;
Described albumen is specially yeast whole protein.
The present invention utilizes surperficial Atom Transfer Radical Polymerization method (SI-ATRP) to prepare respectively hydrophilic, hydrophobic two kinds of magnetic nano particle immobilized trypsinase that polymer chain of different nature is modified, realize quick, efficient, comprehensive enzymolysis of albumen, and the immobilization proteinase of two kinds of hydrophilic, hydrophobic different in kinds is combined use by the present invention, realize complementary enzymolysis, can effectively reduce the enzymolysis bias that carrier selectivity causes, improve the comprehensive of protein digestion, thereby significantly increase the qualification quantity of protein and peptide section.Method provided by the invention can be applicable to before utilization " shotgun " protein group qualification strategy carries out Analysis and Identification to protein.
Technical scheme provided by the invention has the following advantages:
One, the magnetic nanoparticle surface of Silica-coated is modified by the standby polymer chain of SI-ATRP legal system, polymer lateral chain is with a large amount of aldehyde radical functional group, and noncrosslinking polymer chain also can be used as timbering material and supports 3 D stereo, multilayer proteolytic enzyme to fix.This special immobilization way has ensured that the proteolytic enzyme that is positioned at internal layer also can occur effectively to contact with protein substrate.Therefore, compared with the immobilized enzyme that adopts conventional monolayers replace mode to prepare, this polymer-modified magnetic nanoparticle not only can obviously increase the supported quantity of the proteolytic enzyme of unit mass immobilized enzyme reagent, and contribute to improve the probability of collision of immobilization proteinase and protein substrate, improve the availability of proteolytic enzyme;
Two, with by trypsinase being fixed on compared with immobilized enzyme prepared by hard substrate material surface, trypsinase in this polymer-modified immobilized enzyme is fixed on soft polymer chain, there is larger degree of freedom, be conducive to fully contacting of enzyme and protein substrate, thereby promote the carrying out of enzymolysis;
Three, compared with needing just can complete for 12-20 hour enzymolysis with traditional in-solution digestion, polymer-modified magnetic nano particle immobilized enzyme completes protein digestion required time extremely short (1 minute), has greatly accelerated sample preparation speed;
Four, this immobilization proteinase convenience easy to use.After enzymolysis completes, enzymolysis product can be separated with magnetic-particle immobilization proteinase by a step magnetic resolution;
Five, compared with the immobilization proteinase of preparing with the single carrier of existing employing, this double carrier immobilization proteinase has hydrophilic and hydrophobic two kinds of character concurrently, hydrophilic and hydrophobic protein are all had to better affinity, thereby can effectively improve the comprehensive of protein digestion, significantly increase the qualification quantity of protein and peptide section, be therefore specially adapted to the analyzing and processing of complex proteins group sample;
Six, because this immobilization proteinase reagent has very high enzymolysis efficiency and comprehensive, the qualification overburden depth when therefore contributing to improve complex proteins group sample and carrying out qualitative analysis with " shotgun " qualification strategy and the reliability of result;
Seven, these two kinds of immobilized enzyme can be repeatedly used.After each use, can be reclaimed by magnetic resolution.Through being repeatedly used, enzymolysis efficiency has no decline.
Brief description of the drawings
Fig. 1 is the schema of SI-ATRP legal system for technical scheme and the double carrier immobilizing trypsinase enzymolysis protein matter of hydrophilic, hydrophobic double carrier immobilizing trypsinase.
Fig. 2 is the MALDI-TOF-MS spectrogram of GMA-G.
Fig. 3 is the thermogravimetric analysis of the magnetic nanoparticle of different surfaces modification.
Fig. 4 is the static contact angle analysis of the magnetic nanoparticle of different surfaces modification.
Fig. 5 is that water and the organic phase of the magnetic nanoparticle of different surfaces modification disperseed photo.
Fig. 6 is the non-specific adsorption research of magnetic nanoparticle to protein that different surfaces is modified.
Fig. 7 is the hydrophobicity GRAVY value distribution plan of hydrophobic GMA-Trypsin and two kinds of immobilization proteinase enzymolysis products of hydrophilic GMA-G-Trypsin.
Fig. 8 is the enzymolysis qualification result of hydrophobic GMA-Trypsin, hydrophilic GMA-G-Trypsin and CITD.
Fig. 9 is the enzymolysis qualification result of CITD and solution.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Material therefor, as preparation method is not provided, is and can be obtained by commercial sources.
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: 2.31g ferroferric oxide magnetic nanoparticle is joined to ultrasonic 60min in 40ml dehydrated alcohol, obtain suspension, be 25% ammoniacal liquor to adding 1.5ml volumn concentration in this suspension, 6ml water, 1.5ml tetraethoxy, in 40 DEG C of water-baths after vigorous stirring 2h, after ultrasonic 60min, with 40ml ethanol rinsing particle 3 times, Eddy diffusion is in 40mL dehydrated alcohol again, 60 DEG C of reflux 12h, the particle finally obtaining is the ferroferric oxide magnetic nanoparticle of Silica-coated, it is suspended from 60mL dehydrated alcohol to room temperature preservation again for subsequent use.
Synthesizing of the solid support material of embodiment 1, immobilization proteinase
SI-ATRP legal system for the technical scheme of hydrophilic, hydrophobic double carrier immobilizing trypsinase and the schema of double carrier immobilized enzyme enzymolysis protein matter as shown in Figure 1.
One, SI-ATRP initiator is synthetic
Use the synthetic SI-ATRP initiator of APTES and 2-bromine isobutyryl bromine reaction, one end of this initiator is and the silane coupling agent of Silica-coated magnetic nanoparticle surface bonding that the other end is ATRP initiator.Concrete steps are as follows: 8mmol3-aminopropyltriethoxywerene werene and 10mmol triethylamine are joined in 12.5ml tetrahydrofuran (THF), after mixing, pass into nitrogen deoxygenation ice bath 30min simultaneously, obtain silane coupling agent with this, afterwards 10mmol2-bromine isobutyl acylbromide (ATRP initiator) is slowly added drop-wise in mixed solution, 25 DEG C of vigorous stirring 4h(continue logical nitrogen), finally solution filter final vacuum is dried to initial volume 1/3 to remove tetrahydrofuran (THF) and triethylamine, centrifugal rear removal precipitation, after drying up, nitrogen can obtain yellow thick SI-ATRP initiator, airtight 4 DEG C of preservations after inflated with nitrogen.
Two, SI-ATRP initiator is fixing
Utilize the silicon hydroxyl generation dehydration reaction on the ferroferric oxide magnetic nanoparticle surface of silane coupling agent on SI-ATRP initiator and Silica-coated to generate siloxane bond covalently bound, thereby SI-ATRP initiator is fixed on magnetic-particle surface.Concrete steps are as follows: the silicon hydroxyl that first exposes ferroferric oxide magnetic nanoparticle (the following abbreviation magnetic nanoparticle) surface of Silica-coated by acid-alkali treatment, use 0.1M HCl solution soaking magnetic nanoparticle, after 30 minutes by HCl solution removal, it is 7 left and right that water cleans magnetic nanoparticle to pH value of solution, re-use 0.1M NaOH solution soaking magnetic nanoparticle, after 30 minutes by NaOH solution removal, water clean magnetic nanoparticle to pH be 7 left and right.The ethanolic soln that contains 500 μ M SI-ATRP initiators of afterwards being prepared by step 1 mixes (mass ratio of the magnetic nanoparticle after SI-ATRP initiator and acid-alkali treatment is 0.74:1) with the magnetic nanoparticle after acid-alkali treatment; at 20-30 DEG C, react after 10 hours and remove and remain SI-ATRP initiator; and cleaning magnetic nanoparticle with methanol solution, nitrogen dries up.
Three, 2-methacrylic acid 3-Glucoamino propyl ester (GMA-G) monomer is synthetic
In 674.53 μ l glycidyl methacrylate (GMA), add 0.2M sulfuric acid, be positioned over heating in 50 DEG C of water-baths and carry out oxidizing reaction after 4 hours, obtain the GMA of oxidation.Separately measure 20 ml methanol, progressively add 1.1 grams of glucosamines, be stirred to dissolving, and add the sodium cyanoborohydride of 5mg/ml.The GMA of gained oxidation is dropwise joined in the methanol solution that continues the glucosamine stirring, 20-30 DEG C of stirring carried out schiff base reaction and within 4 hours, is yellow transparent solution, and the solution nitrogen preparing is dried up to evaporate into be paste, obtain GMA-G monomer, after airtight inflated with nitrogen, place 4 DEG C of preservations, the MALDI-TOF-MS spectrogram of GMA-G as shown in Figure 2, Fig. 2 shows that the molecular weight of GMA-G hydrophilic monomer is 303, on MALDI-TOF-MS spectrogram, show as hydrogenation peak, m/z=304, coincide with theoretical molecular.
Four, cause SI-ATRP reaction and generate hydrophobic or hydrophilic polymer chains at magnetic nanoparticle surface in situ
Monomer used be the synthetic one end of hydrophobicity GMA or step 3 with two key the other ends the wetting ability GMA-G with glucose.
By GMA or GMA-G monomer, catalyzer cuprous bromide, part N, N, N', N'', N''-PMDETA adds (GMA uses hexalin, and GMA-G uses methyl alcohol) in solvent according to the ratio of mol ratio 200/200:1:1.5, and supersound process makes its dissolving and mixes.The magnetic nanoparticle that SI-ATRP initiator has been fixed on the surface that above-mentioned mixed solution is prepared with step 2 mixes; adopt freezing-vacuumize-thaw-method of rushing nitrogen removes the oxygen in system; so circulation 3 times; with sealed membrane sealing, cause 6 hours (GMA) or 24 hours (GMA-G) of ST-ATRP reaction in the 20-30 DEG C of surface in situ at silica filler afterwards.After having reacted, repeatedly with washed with methanol, removal residual reactant, obtain hydrophobicity GMA or the polymer-modified magnetic nanoparticle of wetting ability GMA-G, the i.e. solid support material of immobilization proteinase provided by the invention.
Five, the property analysis of three kinds of magnetic-particles
(1) take respectively the polymer-modified magnetic nanoparticle of 500mgGMA, the magnetic nanoparticle of the magnetic nanoparticle that 500mgGMA-G is polymer-modified and 500mg parcel silicon-dioxide carries out thermogravimetric analysis, and result as shown in Figure 3.
In Fig. 3, (a) be the thermogravimetric analysis curve of the magnetic nanoparticle of Silica-coated; (b) be the thermogravimetric analysis curve of the polymer-modified magnetic nanoparticle of GMA-G; (c) be the thermogravimetric analysis curve of the polymer-modified magnetic nanoparticle of GMA.
Thereby the polymer-modified magnetic nanoparticle of GMA, GMA-G by thermal decomposition cause mass loss polymer layer and not heat decomposable magnetic-particle core two portions form.As can be seen from Figure 3, the magnetic nanoparticle of Silica-coated with the raising of thermal treatment temp without obvious mass loss, and the polymer-modified magnetic nanoparticle of GMA-G, with the raising of thermal treatment temp, show obvious mass loss (approximately 25%), the magnetic nanoparticle that GMA is polymer-modified, with the raising of thermal treatment temp, shows obvious mass loss (approximately 25%) equally.Prove that be highly effective by SI-ATRP method at magnetic nanoparticle in situ Polymerization generation GMA or GMA-G polymkeric substance.Meanwhile, due to the high controllability of SI-ATRP method, the amount that magnetic-particle surface aggregate generates GMA or GMA-G polymkeric substance can be effectively controlled by changing polymerization time, thereby realizes the effective control to proteolytic enzyme supported quantity.
(2) take respectively the magnetic nanoparticle that 500mg GMA polymer chain is modified, the magnetic nanoparticle of the magnetic nanoparticle that 500mg GMA-G polymer chain is modified and 500mg parcel silicon-dioxide, carries out static contact angle experiment.Result as shown in Figure 4.
In Fig. 4, (a) be the static contact angle photo of the magnetic nanoparticle of Silica-coated; (b) the static contact angle photo of magnetic nanoparticle of modifying for GMA polymer chain; (c) the static contact angle photo of magnetic nanoparticle of modifying for GMA-G polymer chain.
Fig. 4 shows, static contact angle after the magnetic nanoparticle press mold of Silica-coated is 78.6 °, static contact angle after the polymer-modified magnetic nanoparticle press mold of GMA is 50.5 °, and the static contact angle after the polymer-modified magnetic nanoparticle press mold of GMA-G is 0 °.Above data declaration, silica sphere has extremely strong hydrophobicity, and GMA polymkeric substance has moderate hydrophobicity, and GMA-G polymkeric substance has extremely strong wetting ability.
(3) the hydrophilic and hydrophobic difference of different polymer-modified magnetic nanoparticles can further prove by the difference of its degree of scatter in different solutions system.Take respectively the magnetic nanoparticle of 100mg Silica-coated, the magnetic nanoparticle of 100mgGMA polymer chain modification and the magnetic nanoparticle that 100mg GMA-G polymer chain is modified and be scattered in respectively in acetonitrile or water, result as shown in Figure 5.
In Fig. 5, the water that (a) is respectively the magnetic nanoparticle of Silica-coated with (b) disperses photo mutually with acetonitrile; (c) be respectively with (d) water of magnetic nanoparticle that GMA polymer chain modifies and disperse mutually photo with acetonitrile; (e) be respectively with (f) water of magnetic nanoparticle that GMA-G polymer chain modifies and disperse mutually photo with acetonitrile.
Fig. 5 shows, the magnetic nanoparticle of Silica-coated is reunited completely in water, is deposited in EP pipe bottom, and dispersed in mutually at acetonitrile; The magnetic nanoparticle that GMA polymer chain is modified can disperse in water, but the dispersiveness in is mutually better at acetonitrile, and degree of scatter is higher; The magnetic nanoparticle that GMA-G polymer chain is modified disperses at water camber, and obviously reunites in mutually at acetonitrile, is deposited in EP pipe bottom.The magnetic nanoparticle of above data proving again Silica-coated has stronger hydrophobicity, and the magnetic nanoparticle that GMA polymer chain is modified has moderate hydrophobicity, and the magnetic nanoparticle that GMA-G polymer chain is modified has extremely strong wetting ability.
(4) adopt dynamic light scattering to carry out diameter characterization to three kinds of particles.Result shows, the magnetic nanoparticle particle diameter of Silica-coated is 57.4nm, and the magnetic nanoparticle particle diameter that GMA polymer chain is modified is 73.7nm, and the particle diameter of the magnetic nanoparticle that GMA-G polymer chain is modified is 85.9nm.
(5) take respectively the magnetic nanoparticle that 500mg GMA polymer chain is modified, the magnetic nanoparticle that 500mg GMA-G polymer chain is modified and the magnetic nanoparticle of 500mg Silica-coated, carry out the experiment of fluoroscopic examination albumen non-specific adsorption, result as shown in Figure 6.
In Fig. 6, (a) be the fluorescent microscope photo of the magnetic nanoparticle of fluorescent protein B SA-FITC and Silica-coated after hatching, (d) for PBS(pH=7.4) fluorescent microscope photo after cleaning; (b) the fluorescent microscope photo after the magnetic nanoparticle of modifying for fluorescent protein B SA-FITC and GMA polymer chain is hatched, (e) for PBS(pH=7.4) fluorescent microscope photo after cleaning; (c) the fluorescent microscope photo after the magnetic nanoparticle of modifying for fluorescent protein B SA-FITC and GMA-G polymer chain is hatched, (f) for PBS(pH=7.4) fluorescent microscope photo after cleaning.
Fig. 6 shows, the magnetic nanoparticle of parcel silicon-dioxide has strong non-specific adsorption to fluorescin, after cleaning, PBS still have a large amount of fluorescins to remain in particle surface, illustrate that silica sphere hydrophobicity is excessively strong, easily cause the non-specific adsorption of proteins/peptides section, cause sample loss, the magnetic nanoparticle of parcel silicon-dioxide should not be served as the solid support material of immobilization proteinase.Although the magnetic nanoparticle that hydrophobic GMA polymer chain is modified also can adsorb fluorescin, but after PBS cleans, on particle, substantially do not have fluorescin residual, illustrate that it has appropriate hydrophobicity, can be used as hydrophobic carrier material and be applied to immobilization proteinase.The magnetic nanoparticle that hydrophilic GMA-G polymer chain is modified, without any non-specific adsorption, illustrates that it has good wetting ability to fluorescin, can be used as hydrophilic carrier material for the preparation of immobilized enzyme reagent.
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 the 10mM sodium periodate aqueous solution is modified with GMA-G polymer chain mixes, lucifuge reaction 2 hours at 20-30 DEG C.React the methanol aqueous solution that rear use volumn concentration is 50% and repeatedly cleaned, removed residual reactant.
The aldehyde radical functionalization of polymer lateral chain of the magnetic nanoparticle that GMA polymer chain is modified, concrete grammar is as follows: mix with the magnetic nanoparticle that the dilute sulphuric acid of 0.2M is modified GMA polymer chain, at room temperature lucifuge is reacted 2 hours.React the methanol aqueous solution that rear use volumn concentration is 50% and repeatedly cleaned, removed residual reactant.
Two, on the magnetic-particle of modifying at the GMA of aldehyde radical, GMA-G polymer chain, carrying out trypsinase fixes
Concrete grammar is as follows, trypsinase is dissolved in to 50mM ammonium bicarbonate soln (pH=8.0), and adds 5mg/ml sodium cyanoborohydride, obtains mixed solution, and wherein tryptic final concentration is 2mg/ml.Solution after 1ml is mixed joins in the magnetic-particle solution that the GMA that contains 1mg aldehyde radical or GMA-G polymer chain modify, and 4 DEG C of reactions were used magnet that magnetic nanoparticle is adsorbed in to tube wall after 12 hours, removed supernatant liquor.By trypsin solution before and after the immobilization in the change calculations supernatant liquor of the ultraviolet absorption value at 280nm place residual trypsinase amount, thereby show that the supported quantity of trypsinase on the magnetic nanoparticle of GMA or the modification of GMA-G polymer chain is respectively 183.1 μ g/mg, 170.4 μ g/mg.Use afterwards 50mM ammonium bicarbonate soln to clean magnetic nanoparticle, remove residual reactant.
The sealing of the polymer lateral chain residue aldehyde radical of the magnetic-particle that three, GMA, GMA-G polymer chain are modified
Dose volume percentage composition be 10% monoethanolamine phosphate buffered saline buffer, it is mixed with the magnetic nanoparticle of step 2, react 4 hours at 4 DEG C.Repeatedly clean particle with 50mM ammonium bicarbonate soln afterwards, remove residual reactant and obtain the magnetic-particle immobilized enzyme (GMA-Trypsin) of GMA polymer chain modification and the magnetic-particle immobilized enzyme (GMA-G-Trypsin) that GMA-G polymer chain is modified.
The functional analysis of the magnetic-particle immobilized enzyme (GMA-G-Trypsin) that the magnetic-particle immobilized enzyme (GMA-Trypsin) that embodiment 3, GMA polymer chain are modified and GMA-G polymer chain are modified
One, the extraction of yeast whole protein
(1) whole protein extracting solution: 50mM Tris-HCl (pH=8.0), 8M urea, 2mM EDTA.
(2) to " cocktail " formula proteinase inhibitor (purchased from Roche Germany) that adds 10 μ l in 500 μ l whole protein extracting solutions, after mixing, join and be equipped with in saccharomycetic test tube, ultrasonication cell.Centrifugal 20 minutes of 20000g at 4 DEG C, removes not smudge cells and fragment afterwards, and solution is yeast whole protein.
(3) in yeast whole protein solution, add the cold acetone solution (20 DEG C of precoolings) of 4-5 times of volume, after-20 DEG C of placements are greater than 2 hours, centrifugal 10 minutes of 4 DEG C of 12000g, carefully draw supernatant, retain precipitation.
(4) precipitation is positioned in stink cupboard, makes to remain acetone and fully volatilize.The albumen that acetone precipitation is obtained is again dissolved in 8M urea buffer solution and adopts Bradford method to measure protein concn.Yeast whole protein immobilized enzyme enzyme is cut
Two, preparation 2mg/ml yeast whole protein solution, adds DTT, in 37 DEG C of water-baths, reduces 4 hours, adds afterwards IAA to place and within 1 hour, carry out alkylation in dark place.In the protein soln of handling well, add 50mM ammonium bicarbonate soln, after mixing, be divided into 4 parts, first part of 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) that adds 10 μ l GMA polymer chains to modify, second part of magnetic nano particle immobilized enzyme (GMA-G-Trypsin) (the magnetic nano particle immobilized enzyme that in second part, GMA-G polymer chain is modified is 10:1 with the mass ratio of albumen to be degraded) that adds 10 μ l GMA-G polymer chains to modify, the 3rd part adds mixture (each 5 μ l of GMA-Trypsin and two kinds of immobilized enzyme of GMA-G-Trypsin, mixed enzymolysis (CITD)) (two kinds of immobilized enzyme in the 3rd part, the mass ratio of albumen to be degraded is 5:5:1), in 37 DEG C of water-baths, hatch after 1 minute respectively, use magnet that magnetic nano particle immobilized enzyme is adsorbed in to tube wall, draw supernatant.The 4th part according to conventional enzymatic hydrolysis condition, and adding mass ratio is 1:50(trypsinase: treat degrade proteins) trypsinase, be placed in 37 DEG C of water-baths and hatch that after 12 hours, to add volumn concentration be that 0.1% TFA is by trypsinase deactivation.Get respectively three kinds of immobilized enzyme enzymolysis and conventional soln enzymolysis product and use chromatography-electrospray-ionization/mass spectrometry coupling to identify, and the data that qualification is obtained use MASCOT software to search storehouse, result as shown in Table 1 and Table 2.
The circulation ratio analysis of table 1 hydrolysis result
The mutual coverage rate analysis of table 2 hydrolysis result
Table 1 shows, uses the hydrophobic GMA-Trypsin enzymolysis yeast whole protein of two batches, under same experiment condition, can identify respectively 527 and 510 protein, 1860 and 1785 peptide sections, and circulation ratio can reach respectively 89.1% and 83.9; Use the hydrophilic GMA-G-Trypsin enzymolysis yeast whole protein of two batches, under same experiment condition, can identify respectively 560 and 533 protein, 1847 and 1744 peptide sections, circulation ratio can reach respectively 86.0% and 76.3%.Above data declaration, GMA-Trypsin, the GMA-G-Trypsin of two batches all has good enzymolysis circulation ratio, identification of proteins circulation ratio can reach more than 85%, and peptide section qualification circulation ratio can reach 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 be controlled by polymerization time, less the causing of supported quantity difference of nucleocapsid structure, surface aggregate thing content and proteolytic enzyme between different batches hydridization particle.Table 2 shows, the enzymolysis experiment of two batches, and hydrophobic GMA-Trypsin identifies 574 protein and 2248 peptide sections altogether; Hydrophilic GMA-G-Trypsin identifies 623 protein and 2115 peptide sections altogether, and the two qualification scale is comparatively approaching, illustrates that the enzymolysis efficiency of two kinds of immobilization proteinase reagent approaches, and all can be used for quick, the efficient enzymolysis of complex proteins group sample.Use conventional soln enzymolysis, under same qualification condition, identify altogether 602 protein and 2488 peptide sections.Above data declaration uses the immobilization proteinase reagent enzymolysis 1 minute of single carrier, just can reach and the in-solution digestion similar effect of 12 hours, the enzymolysis high efficiency of the polymer hybrid magnetic-particle immobilization proteinase standby based on SI-ATRP legal system is described again.But the mutual fraction of coverage between two kinds of immobilization proteinase reagent enzymolysis results of hydrophobic GMA-Trypsin and hydrophilic GMA-G-Trypsin is very low, only has 77.9%(albumen), 60.1%(peptide section), be starkly lower than the enzymolysis circulation ratio between immobilization proteinase reagent different batches of the same race.The solid support material that above data declaration has different hydrophilic, hydrophobic character has different affinities to protein substrate, therefore causes two kinds of immobilization proteinase reagent to have certain substrate selective and enzymolysis complementarity.The protein identifying in the enzymolysis product of two kinds of immobilization proteinase reagent (hydrophobic GMA-Trypsin and hydrophilic GMA-G-Trypsin) is carried out to the analysis of hydrophilic, hydrophobic property and further verified this supposition, result as shown in Figure 7.
Fig. 7 shows, in strongly hydrophilic region (GRAVY value <-0.75), GMA-G-Trypsin has clear superiority, and identification of protein quantity accounts for approximately 30% of its whole identification of protein quantity, apparently higher than 10% of GMA-Trypsin; And in the stronger region of hydrophobicity (0.35<GRAVY value <0), GMA-Trypsin has clear superiority, identification of protein quantity accounts for approximately 50% of its whole identification of protein quantity, apparently higher than 30% of GMA-G-Trypsin.
By hydrophobic GMA-Trypsin and the coupling of two kinds of immobilization proteinase reagent of hydrophilic GMA-G-Trypsin, carry out mixed enzymolysis (CITD), from yeast whole protein extracting solution, identify altogether 765 protein and 3505 peptide sections as shown in Figure 8.
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, protein and peptide section that after hydrophilic GMA-G-Trypsin and composite grain (CITD) enzymolysis, Mass Spectrometric Identification arrives; And (c) be respectively the qualification result summation of GMA-Trypsin and GMA-G-Trypsin and the mutual fraction of coverage of protein of CITD qualification result and the mutual fraction of coverage of peptide section (b).
Fig. 8 shows, compared with hydrophobic GMA-Trypsin, hydrophilic GMA-G-Trypsin, CITD has at least improved respectively 20% and 50% by protein and peptide section qualification quantity, and has effectively covered qualification result when two kinds of immobilized enzyme use separately.Immobilization proteinase based on double carrier is described, can effectively reduces the enzymolysis bias of single carrier immobilized proteolytic enzyme, improve the comprehensive of enzymolysis, thereby significantly increase the qualification quantity of protein and peptide section.
Mixed enzymolysis (CITD) and in-solution digestion result are as shown in Figure 9.
Fig. 9 (a) and (b) be respectively the mutual fraction of coverage of protein and the mutual fraction of coverage of peptide section of the Mass Spectrometric Identification result of CITD and conventional soln enzymolysis product.
Fig. 9 shows, compared with in-solution digestion effect, CITD can provide more albumen and peptide section qualification quantity, but two kinds of enzyme solutions have certain complementarity.The qualification result of the two is merged, and peptide section corresponding to average each identification of protein qualification quantity is increased to 5.18 by 4.13 of in-solution digestion.Therefore, CITD, except can improving the qualification scale of protein and peptide section, can also increase the reliability of qualification result.
Claims (10)
1. an immobilized enzyme, this immobilized enzyme is made up of hydrophobic carrier material and the proteolytic enzyme being fixed on described hydrophobic carrier material;
Described hydrophobic carrier material is that hydrophobic monomer, on the surface of the particle with surperficial Atom Transfer Radical Polymerization initiator, the product that atom transfer radical polymerization obtains occurs; Wherein, described hydrophobic monomer aggregates into polymeric chain; The polymeric chain that described hydrophobic monomer aggregates into is connected by C-Si-O key with described particle;
Described proteolytic enzyme links by carbonnitrogen bond with the polymeric chain on described hydrophobic carrier material.
2. immobilized enzyme according to claim 1, is characterized in that: described particle is magnetic nanoparticle;
Described magnetic nanoparticle is specially the magnetic nanoparticle of Silica-coated;
The described particle with surperficial Atom Transfer Radical Polymerization initiator is that make on the surface that surperficial Atom Transfer Radical Polymerization initiator is fixedly connected on to described particle;
One end of described surperficial Atom Transfer Radical Polymerization initiator be can with the coupling agent of silicon hydroxy combining, the other end is atom transfer radical polymerization initiator;
Described hydrophobic monomer is the Racemic glycidol esters monomer that contains epoxy group(ing), is specially glycidyl methacrylate;
Described proteolytic enzyme is specially trypsinase, protein incision enzyme, intracellular protein enzyme or Quimotrase.
3. immobilized enzyme according to claim 1 and 2, is characterized in that: described in the method that is fixedly connected with to be that the silicon hydroxyl generation dehydration reaction of described surperficial Atom Transfer Radical Polymerization initiator and described particle surface generates siloxane bond covalently bound;
Described coupling agent is silane coupling agent;
Described silane coupling agent is 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-bromopropionyl bromide;
Described silane coupling agent is connected by amido linkage with described atom transfer radical polymerization initiator.
4. according to the arbitrary described immobilized enzyme of claim 1-3, it is characterized in that: the preparation method of described surperficial Atom Transfer Radical Polymerization initiator is as follows: silane coupling agent and protonic acid trapping agent are reacted in ice bath, add again atom transfer radical polymerization initiator, react, to obtain final product;
The molar ratio of described silane coupling agent, atom transfer radical polymerization initiator and protonic acid trapping agent is 0.5-1:1:1, is specially 0.8:1:1.
5. according to the arbitrary described immobilized enzyme of claim 1-4, it is characterized in that: the described preparation method of granules with surperficial Atom Transfer Radical Polymerization initiator is as follows: the magnetic nanoparticle of described Silica-coated is carried out to acid-alkali treatment and expose silicon hydroxyl, itself and surperficial Atom Transfer Radical Polymerization initiator are mixed to generation dehydration reaction generation siloxane bond in solvent, to obtain final product;
Described solvent be in ethanol, methyl alcohol and hexalin any one;
The temperature of described dehydration reaction is 20-30 DEG C;
The time of described dehydration reaction is 1-24 hour, is specially 10 hours;
The mass ratio of the initiator of described magnetic nanoparticle and described surperficial Atom Transfer Radical Polymerization is 1:0.37-7.4, is specially 1:0.74.
6. according to the arbitrary described immobilized enzyme of claim 1-5, it is characterized in that: the preparation method of described hydrophobic carrier material is as follows: by hydrophobic monomer, catalyzer, part and solvent, obtain mixed solution 1; Mixed solution 1 is mixed with the described particle with surperficial Atom Transfer Radical Polymerization initiator, obtain mixed solution 2; Remove the oxygen in mixed solution 2, Atom Transfer Radical Polymerization reaction, to obtain final product;
Described catalyzer is the halogenide of following any one metal: 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'-dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (2-dimethylaminoethyl) amine;
Solvent for use be following any one: methyl alcohol, ethanol, hexalin, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF);
The mol ratio of described hydrophobic monomer, catalyzer, part is 200:0.5-5:0.75-7.5, is specially 200:1:1.5;
The method that described proteolytic enzyme is fixed on described hydrophobic carrier material is as follows:
(1), by hydrophobic carrier material functional, obtain the solid support material of functionalization;
(2) on the solid support material of the functionalization obtaining in step (1), carry out the fixing of proteolytic enzyme, be fixed the functional supports of proteolytic enzyme;
(3) by the residue functional group sealing of having fixed on the functional supports of proteolytic enzyme, obtain object immobilization proteinase;
Described in described step (2), fixing method is the ammonium bicarbonate soln that described proteolytic enzyme is dissolved in to pH8.0, adds sodium cyanoborohydride to obtain mixed solution; Mixed solution is joined in the solid support material of the described functionalization of step (1), reaction, then particle is taken out and be get final product;
Described in described step (3) sealing method be with monoethanolamine phosphate buffered saline buffer mix with the functional supports of having fixed proteolytic enzyme that step (2) obtains, react and get final product;
Described monoethanolamine monoethanolamine volumn concentration in phosphate buffered saline buffer be 5%-20%, be specially 10%;
The method of described functionalization is aldehyde radical and/or chemically modified;
Described aldehyde radical is specially described hydrophobic carrier material is mixed with the dilute sulphuric acid of 0.2M, and lucifuge is reacted and get final product at 25 DEG C;
Described proteolytic enzyme is connected by carbonnitrogen bond with the solid support material of described functionalization;
The mass ratio of the solid support material of described proteolytic enzyme and described functionalization is 1:10-10:1, is specially 2:1;
Described functional group is the group that can be combined with described proteolytic enzyme;
Described functional group is positioned on the polymeric chain of described solid support material.
7. an immobilized enzyme mixture, is made up of the arbitrary described immobilized enzyme of claim 1-6 and the immobilized enzyme taking hydrophilic carrier material as substrate;
The arbitrary described immobilized enzyme of described claim 1-6 and the mass ratio of the described immobilized enzyme taking hydrophilic carrier material as substrate are specially 1:1;
The described immobilized enzyme taking hydrophilic carrier material as substrate is made up of hydrophilic carrier material and the proteolytic enzyme being fixed on described hydrophilic carrier material;
Described proteolytic enzyme is specially trypsinase, protein incision enzyme, intracellular protein enzyme or Quimotrase;
Described proteolytic enzyme links by carbonnitrogen bond with described hydrophilic carrier material;
Described hydrophilic carrier material is that hydrophilic monomer, on the surface of the described particle with surperficial Atom Transfer Radical Polymerization initiator, the product that atom transfer radical polymerization obtains occurs; Wherein, described hydrophilic monomer aggregates into polymeric chain; The polymeric chain that described hydrophilic monomer is polymerized is connected by C-Si-O key with described particle;
One end of the surperficial Atom Transfer Radical Polymerization initiator of described particle be can with the coupling agent of silicon hydroxy combining, the other end is atom transfer radical polymerization initiator;
Described coupling agent is silane coupling agent;
Described silane coupling agent is 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-bromopropionyl bromide;
Described silane coupling agent is connected by amido linkage with the initiator of described atom transfer radical polymerization;
Described hydrophilic monomer is specially the hydrophilic monomer, acrylic monomer, butylene acids monomer or the amylene acids monomer that contain monose group;
Described monose is specially glucosamine or epichitosamine;
Described hydrophilic monomer is specially 2-methacrylic acid 3-Glucoamino propyl ester monomer;
Described particle 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: glycidyl methacrylate is oxidized, obtains the glycidyl methacrylate of oxidation; Glucosamine is mixed in methyl alcohol with sodium cyanoborohydride, obtain the methanol solution of glucosamine; The methanol solution of the glycidyl methacrylate of oxidation and glucosamine is carried out to schiff base reaction and obtain reaction solution; Reaction solution is dried up to paste with rare gas element, to obtain final product;
Described rare gas element is specially nitrogen.
8. immobilized enzyme mixture according to claim 7, is characterized in that: the preparation method of described hydrophilic carrier material is as follows: by hydrophilic monomer, catalyzer, part and solvent, obtain mixed solution 1; Mixed solution 1 is mixed with the described particle with surperficial Atom Transfer Radical Polymerization initiator, obtain mixed solution 2; Remove the oxygen in mixed solution 2, Atom Transfer Radical Polymerization reaction, obtains reaction solution; Residual reactant in reaction solution is removed to the hydrophilic carrier material of being fixed proteolytic enzyme;
Described catalyzer is the halogenide of following any one metal: 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'-dipyridyl, Tetramethyl Ethylene Diamine, 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) and three (2-dimethylaminoethyl) amine;
Solvent for use be following any one: methyl alcohol, ethanol, hexalin, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF);
The mol ratio of described hydrophilic monomer, catalyzer, part is 200:0.5-5:0.75-7.5, is specially 200:1:1.5;
The synthetic method of described immobilization proteinase is as follows:
(1), by solid support material functionalization, obtain the solid support material of functionalization;
(2) on the solid support material of the functionalization obtaining in step (1), carry out the fixing of proteolytic enzyme, be fixed the functional supports of proteolytic enzyme;
(3) by the residue functional group sealing of having fixed on the functional supports of proteolytic enzyme, obtain object immobilization proteinase;
Described in described step (2), fixing method is the ammonium bicarbonate soln that described proteolytic enzyme is dissolved in to pH8.0, adds sodium cyanoborohydride to obtain mixed solution; Mixed solution is joined in the solid support material of the described functionalization of step (1), reaction, then particle is taken out and be get final product;
Described in described step (3) sealing method be with monoethanolamine phosphate buffered saline buffer mix with the functional supports of having fixed proteolytic enzyme that step (2) obtains, react and get final product;
Described monoethanolamine monoethanolamine volumn concentration in phosphate buffered saline buffer be 5%-20%, be specially 10%;
The serve as reasons particle of the polymeric chain that surface is polymerized with the polymeric chain being polymerized by hydrophilic monomer or hydrophobic monomer of described solid support material;
The method of described functionalization is aldehyde radical and/or chemically modified;
The adjacent diol structure of the monose group on the polymeric chain that described aldehyde radicalization is specifically polymerized described hydrophilic monomer is converted into aldehyde radical and carries out aldehyde radical, obtains aldehyde radical carrier;
Or,
Cycloalkyl groups on the polymeric chain that described aldehyde radicalization is specifically polymerized hydrophobic monomer is converted into aldehyde radical, obtains aldehyde radical carrier;
The mass ratio of the solid support material of described proteolytic enzyme and described functionalization is 1:10-10:1, is specially 2:1;
Described proteolytic enzyme is connected by carbonnitrogen bond with the solid support material of described functionalization;
Described functional group is the group that can be combined with described proteolytic enzyme;
Described functional group is positioned on the polymeric chain of described solid support material.
9. a method for enzymolysis protein, comprises the steps: the albumen for the treatment of enzymolysis to carry out sex change, obtains the enzymolysis protein for the treatment of of sex change; Immobilized enzyme mixture described in claim 7 or 8 is mixed in the phosphate buffered saline buffer of pH=8.025-100mM ammonium bicarbonate soln or pH=7.825-100mM with the enzymolysis protein for the treatment of of sex change, hatch, obtain enzymolysis solution;
The method that the described albumen for the treatment of enzymolysis carries out sex change is specific as follows: in the described albumen for the treatment of enzymolysis, add DTT, in 37 DEG C of water-baths, reduce, then add IAA to place and carry out alkylation in dark place.
10. the application of the arbitrary described immobilized enzyme of claim 1-6 in proteolysis;
Or,
The application of immobilized enzyme mixture described in claim 7 or 8 in proteolysis;
Described albumen is specially yeast whole protein.
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| CN105289558A (en) * | 2014-07-28 | 2016-02-03 | 北京蛋白质组研究中心 | Preparation and application of multi-component segmented hydrophilic copolymer-silica gel hybrid chromatographic packing material |
| CN107406841A (en) * | 2015-03-09 | 2017-11-28 | 株式会社岛津制作所 | The immobilization proteinase improved to the patience of outside environmental change |
| CN105001376A (en) * | 2015-06-23 | 2015-10-28 | 天津大学 | Method for preparing high-capacity protein chromatographic medium through atom transfer radical polymerization |
| CN104911162A (en) * | 2015-06-24 | 2015-09-16 | 中国科学院广州能源研究所 | Controllable porous vector-free immobilized lipase and preparation method thereof |
| CN105039302A (en) * | 2015-09-08 | 2015-11-11 | 北京蛋白质组研究中心 | Immobilized glycosidase reagent and preparing method thereof |
| CN105087542A (en) * | 2015-09-17 | 2015-11-25 | 李爱冰 | Environmental stress sensitive type immobilized enzyme porous micro-sphere carrier material and preparing method of porous micro-sphere carrier material |
| CN105289759A (en) * | 2015-11-13 | 2016-02-03 | 西北大学 | Strong cation exchanger based on a waste immobilized enzyme, and preparation method and application thereof |
| CN107460186A (en) * | 2017-08-01 | 2017-12-12 | 东南大学 | A kind of immobilized chymotrypsin material of silicon rubber and its preparation method and application |
| CN107460186B (en) * | 2017-08-01 | 2021-01-05 | 东南大学 | Silicone rubber immobilized chymotrypsin material and preparation method and application thereof |
| CN110982810A (en) * | 2019-12-27 | 2020-04-10 | 广东希普生物科技股份有限公司 | Method for producing fish peptide by using immobilized compound protease |
| CN110982810B (en) * | 2019-12-27 | 2021-05-14 | 广东希普生物科技股份有限公司 | Method for producing fish peptide by using immobilized compound protease |
| CN112679701A (en) * | 2020-12-28 | 2021-04-20 | 重庆宸安生物制药有限公司 | Immobilized lysine endopeptidase and preparation method and application thereof |
| CN114113287A (en) * | 2022-01-25 | 2022-03-01 | 北京青莲百奥生物科技有限公司 | Serum protein preparation method and serum proteome mass spectrum detection method |
| CN118146358A (en) * | 2024-05-09 | 2024-06-07 | 成都维德医疗器械有限责任公司 | Process for extracting collagen from beef tendon |
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