CN103464062B - A kind of total silicon colloid body microcapsules encapsulating biology enzyme and preparation method thereof - Google Patents
A kind of total silicon colloid body microcapsules encapsulating biology enzyme and preparation method thereof Download PDFInfo
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- CN103464062B CN103464062B CN201310401109.1A CN201310401109A CN103464062B CN 103464062 B CN103464062 B CN 103464062B CN 201310401109 A CN201310401109 A CN 201310401109A CN 103464062 B CN103464062 B CN 103464062B
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 103
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 103
- 239000000084 colloidal system Substances 0.000 title claims abstract description 99
- 239000003094 microcapsule Substances 0.000 title claims abstract description 80
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 229910052710 silicon Inorganic materials 0.000 title claims abstract description 68
- 239000010703 silicon Substances 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 328
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 164
- 239000002105 nanoparticle Substances 0.000 claims abstract description 133
- 239000007788 liquid Substances 0.000 claims abstract description 75
- 239000003960 organic solvent Substances 0.000 claims abstract description 42
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229910000077 silane Inorganic materials 0.000 claims abstract description 30
- 239000000839 emulsion Substances 0.000 claims abstract description 15
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 137
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- 239000006185 dispersion Substances 0.000 claims description 56
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 55
- 239000006228 supernatant Substances 0.000 claims description 53
- 235000012239 silicon dioxide Nutrition 0.000 claims description 48
- 238000005119 centrifugation Methods 0.000 claims description 40
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 35
- 239000011259 mixed solution Substances 0.000 claims description 34
- 239000002245 particle Substances 0.000 claims description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 31
- 239000003607 modifier Substances 0.000 claims description 26
- 230000008569 process Effects 0.000 claims description 26
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical group ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 16
- 230000007062 hydrolysis Effects 0.000 claims description 15
- 238000006460 hydrolysis reaction Methods 0.000 claims description 15
- 238000004945 emulsification Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 239000007762 w/o emulsion Substances 0.000 claims description 12
- 108010029541 Laccase Proteins 0.000 claims description 11
- 239000004367 Lipase Substances 0.000 claims description 11
- 102000004882 Lipase Human genes 0.000 claims description 11
- 108090001060 Lipase Proteins 0.000 claims description 11
- 235000019421 lipase Nutrition 0.000 claims description 11
- 238000000967 suction filtration Methods 0.000 claims description 11
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 10
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 claims description 10
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 9
- 108091005804 Peptidases Proteins 0.000 claims description 9
- 239000004365 Protease Substances 0.000 claims description 9
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 230000001476 alcoholic effect Effects 0.000 claims description 8
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007979 citrate buffer Substances 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 238000006068 polycondensation reaction Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- -1 carrene Substances 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 8
- 238000006555 catalytic reaction Methods 0.000 abstract description 4
- 230000035699 permeability Effects 0.000 abstract description 2
- 230000001804 emulsifying effect Effects 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 10
- 108091005508 Acid proteases Proteins 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 4
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical compound [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000001338 self-assembly Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 241000723655 Cowpea mosaic virus Species 0.000 description 1
- 229910004298 SiO 2 Inorganic materials 0.000 description 1
- 229910010413 TiO 2 Inorganic materials 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
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- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
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- 238000004132 cross linking Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- XJKVPKYVPCWHFO-UHFFFAOYSA-N silicon;hydrate Chemical group O.[Si] XJKVPKYVPCWHFO-UHFFFAOYSA-N 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Silicon Compounds (AREA)
Abstract
The present invention relates to a kind of total silicon colloid body microcapsules encapsulating biology enzyme and preparation method thereof, first will containing the cushioning liquid of biology enzyme with containing the organic solvent emulsifying of improved silica nano particle, formed by nano particle at the stable water/oily emulsion of two-phase interface; Then add silane, silane hydrolyzate, by fixing for nano particle bonding, namely prepares the water core total silicon shell colloid body microcapsules of encapsulating biology enzyme.Preparation condition of the present invention is gentle, achieves biology enzyme activity stability in organic solvent.Complete silicon structure colloid body microcapsules prepared by the present invention have good physics and chemistry stability, solve the scattering problem of biology enzyme in organic media, substantially increase biology enzyme activity in organic solvent; And be the controllable shell wall structure with certain permeability, the controlled activity release of biology enzyme in organic media can be realized and can reuse.This invention has a wide range of applications to the catalytic reaction of biology enzyme in organic media.
Description
Technical field
The present invention relates to a kind of total silicon colloid body microcapsules encapsulating biology enzyme and preparation method thereof, particularly relate to a kind of total silicon water core colloid body microcapsules and preparation method thereof of encapsulating biology enzyme of the controllable shell wall structure that one or more layers Nano particles of silicon dioxide formed of being bondd by silane hydrolyzate.
Background technology
Colloid body is a kind of novel microcapsules with semipermeable shell wall structure of novelty, and its shell wall is dispersed in the colloidal particle interphase interaction of pickering emulsion oil-water interface by self assembly and solidify to form.Colloid body encapsulating core has been subject to showing great attention to of the fields such as medicine, cosmetics, food, but is only in the starting stage.At present, the colloidal particle that can be used for preparing colloid body microcapsules reported has polymer microsphere (PS colloidal particle, PMMA colloidal particle, PNiPAAm colloidal particle, P (NiPAAm-co-AAc) colloidal particle, the micro-rod of epoxy resin, the micro-rod of nano-cellulose, CPMV particle etc.) and inorganic particulate (SiO
2nano particle, TiO
2nano particle, Fe
3o
4nano particle, clay nanoparticle etc.).
Biology enzyme is the protein macromolecule with biocatalytic Activity, and enzymatic has the distinguishing features such as catalytic efficiency is high, selectivity is strong.Biological enzyme has become current focus that is that pay close attention to and research, but finds in research process that the many important substrate of biology enzyme is water-soluble poor; And there is the problem such as main reaction and hydrolysis competition in the catalytic action of part biological enzyme, therefore need with an organic solvent to solve the problems referred to above as reaction medium under water system condition.But be also faced with biology enzyme poor stability, mutability, active low in organic media simultaneously, and be not easily scattered in organic media.Problems limit the extensive use of biology enzyme in each field, the catalytic applications especially in organic media system.
Colloid body encapsulating biology enzyme prevents extraneous poor environment to the destruction of biology enzyme conformation, the method with potentiality and feasibility improving biology enzyme stability and recyclable recycling performance.The preparation of current colloid body forms organic, the hybrid colloid body microcapsules with certain permeability mainly through the colloidal particle being cemented in two-phase interface self assembly from methods such as bonding (utilizing particle swellability, glass transition etc.), physical gel method (colloidal particle is fixed by gelled aqueous phase), polymerization and photo-crosslinkings.Above-mentioned preparation method's condition violent (high temperature, UV-irradiation), easily causes biology enzyme to lose activity; The colloid body of preparation is mostly organic, hybrid structure; its physics and chemistry stability is lower; easily break in the process of delivery, storage core, thus the encapsulating protection lost core and delivery effect, be not more suitable for and realize catalysis in encapsulating biology enzyme organic media.
The complete silicon structure colloid body that the Nano particles of silicon dioxide that silane hydrolyzate bonding is self-assembled to oil-water interface is formed has good biocompatibility, and there is excellent mechanical performances and chemical stability, can long time be scattered in organic media and keep good shape characteristic.Be with a wide range of applications with the catalysis in organic media of this total silicon colloid body encapsulating biology enzyme.
Summary of the invention
The object of this invention is to provide a kind of total silicon colloid body microcapsules encapsulating biology enzyme and preparation method thereof, propose and be a kind ofly self-assembled to the Nano particles of silicon dioxide of oil-water interface with silane hydrolyzate bonding and form total silicon colloid body and preparation method thereof of encapsulating biology enzyme, method of the present invention is simple to operate, mild condition, is applicable to encapsulating and has bioactive biology enzyme.The controllable shell wall structure adopting this preparation method to prepare is the total silicon colloid body of the encapsulating biology enzyme of one or more layers Nano particles of silicon dioxide shell wall, can realize biology enzyme controlled activity in organic solvent and discharge and recycling.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme of the present invention, described colloid body microcapsules are have shell wall structure that one or more layers Nano particles of silicon dioxide formed through silane hydrolyzate bonding and encapsulate the spherical nucleocapsid structure microcapsules of biology enzyme.The self assembly under ultrasonic emulsification of improved silica nano particle is arranged in water (cushioning liquid containing biology enzyme)-oil (organic solvent) two-phase interface place, formed by the stable Water-In-Oil pickering emulsion of Nano particles of silicon dioxide, add the silane as binding agent subsequently, by fixing for Nano particles of silicon dioxide bonding under hydrolysis, form the total silicon colloid body microcapsules of stable encapsulating biology enzyme.Because this colloid body microcapsules shell wall is rearranged by Nano particles of silicon dioxide, therefore this colloid body is that capsule has certain semipermeable, and this shell wall semipermeable can be relevant to the thickness of the Nano particles of silicon dioxide layer forming shell wall.This colloid body be the semipermeable of capsule shell wall impart that the biology enzyme that is encapsulated in colloid body inside can contact with external substance may, thus the catalysis of the biology enzyme that can realize being encapsulated in colloid body inside material to external world, and the catalytic activity of the biology enzyme of encapsulating realizes controlled by adjustment shell wall structure.This colloid body is total silicon shell wall structure simultaneously, has good physics and chemical stability, can be scattered in organic solvent for a long time and keep good shape characteristic and performance.Micro-water environment of colloid body microcapsules inside substantially increases biology enzyme activity in organic solvent; and be encapsulated in the biology enzyme of colloid body inside; under the shielding protection of colloid body microcapsules shell wall; be scattered in organic solvent for a long time and can keep higher activity stability to allow; and be beneficial to from organic solvent separation, realize recycling.
As preferred technical scheme:
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described silane refers to the hyperbranched silane formed for raw material and acetic anhydride polycondensation reaction with ethyl orthosilicate or methyl silicate.Hyperbranched silane has more active group, is conducive to hydrolysis bonding Nano particles of silicon dioxide, forms rock-steady structure.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described biology enzyme is laccase, lipase or protease; Described biology enzyme is scattered in cushioning liquid, and the concentration range of biology enzyme is 0.5 ~ 50mg/mL; Described cushioning liquid is Lin acid hydrogen Er Na – citrate buffer solution, Lin acid hydrogen Er Na – sodium dihydrogen phosphate buffer or Tris-hydrochloric acid buffer solution, and the pH value of described cushioning liquid is 2 ~ 10.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described colloid body Microcapsules Size distribution is between 0.5 ~ 2 μm; Described colloid body microcapsules shell wall has semipermeable, and thickness and the density of its semipermeable and colloid body microcapsules shell wall are relevant.
Present invention also offers a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme, comprise the following steps:
A) be dissolved in alcoholic solution by organosilicon source, controlling solution ph is 8 ~ 12;
B) at above-mentioned steps A) add modifier in gained solution, and continue to stir, obtain the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, centrifugal treating at ambient temperature, after removing supernatant liquor, carries out exchange of solvent process to precipitated silica; Repeated centrifugation process also carries out the operation 3 ~ 5 times of exchange of solvent process after removing supernatant liquor, the improved silica precipitated the most at last is scattered in organic solvent, is mixed with the stand-by liquid of improved silica nano particle organic solvent that concentration is 1 ~ 10wt%;
D) mixed with the cushioning liquid containing biology enzyme by the stand-by liquid of described improved silica nano particle organic solvent, both mass ratioes are 5 ~ 15:1, are prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification;
E) in described emulsion, add silane, the mass ratio of addition and the stand-by liquid of improved silica nano particle organic solvent is 1:50 ~ 100, and stirred at ambient temperature hydrolysis 24 ~ 120h, namely obtains the total silicon colloid body microcapsules of the encapsulating biology enzyme being scattered in organic solvent.
A kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as above, steps A) in: organosilicon source and alcoholic solution mass ratio are 1:10 ~ 100; When organosilicon source is dissolved in alcoholic solution, at room temperature stir 3 ~ 48h; Described organosilicon source is ethyl orthosilicate or methyl silicate; Described alcohol is methyl alcohol or ethanol; Control solution ph and adopt sodium hydroxide solution, sodium carbonate liquor, sodium bicarbonate solution or ammoniacal liquor.
A kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as above, step B) in: the molecular formula of described modifier is C
nh
2n-Si (OC
mh
2m+1)
3, wherein n is 5 ~ 18, m is 1 ~ 4; Between described modifier addition and organosilicon source, mass ratio is 1:50 ~ 100; The described time of continuing to stir is 24 ~ 120h.
A kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as above, step C) in: the described centrifugal treating time is 15 ~ 60min.
A kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as above, described exchange of solvent process refers to and realizes improved silica nanoparticle dispersion system is replaced into organic solvent completely process by alcoholic solution: first, first the improved silica nano particle of centrifugation is scattered in alcohol and organic solvent volume than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after alcohol and organic solvent volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in organic solvent to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, improved silica nano particle is replaced completely and to be scattered in organic solvent to naked-eye observation without bulky grain aggregate, form the stand-by liquid of improved silica nano particle organic solvent; Described organic solvent is carbon tetrachloride, oxolane, toluene, benzene, carrene, chloroform or ethyl acetate.
A kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as above, further, by described step e) the total silicon colloid body microcapsules of encapsulating biology enzyme that obtain carry out suction filtration process or centrifugal treating;
Described suction filtration process is by described step e) the total silicon colloid body microcapsules organic solvent dispersion system of encapsulating biology enzyme that obtains, suction filtration process under room temperature, collects and is deposited on total silicon colloid body filter membrane being encapsulated biology enzyme, and be scattered in organic solvent; Repetition like this 3 ~ 5 suction filtrations and dispersion, to remove not encapsulated biology enzyme;
Described centrifugal treating is by described step e) the total silicon colloid body microcapsules organic solvent dispersion system of encapsulating biology enzyme that obtains, centrifugal 5 ~ 30min under 3000 ~ 5000rpm rotating speed, the total silicon colloid body microcapsules getting the encapsulating biology enzyme of precipitation are scattered in organic solvent again; Repeated centrifugation like this and dispersion 3 ~ 5 times, to remove not encapsulated biology enzyme.
Beneficial effect:
The present invention proposes the total silicon colloid body method based on Nano particles of silicon dioxide by silane hydrolyzate bonding mode preparation encapsulating biology enzyme first;
Preparation technology of the present invention is simple, preparation condition is gentle, and the complete silicon structure colloid body of encapsulating biology enzyme has good mechanical property and chemical stability, can be scattered in organic media for a long time;
The present invention can realize the reusing of biology enzyme in organic media with the total silicon colloid body of silane hydrolyzate consolidation Nano particles of silicon dioxide preparation encapsulating biology enzyme, and preparation method's controllable shell wall of the present invention is one deck and one deck above Nano particles of silicon dioxide layer shell wall, biology enzyme can be realized in the controlled activity release in organic media.
Detailed description of the invention
Below in conjunction with detailed description of the invention, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme of the present invention, described colloid body microcapsules are have shell wall structure that one or more layers Nano particles of silicon dioxide formed through silane hydrolyzate bonding and encapsulate the spherical nucleocapsid structure microcapsules of biology enzyme.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described silane refers to the hyperbranched silane formed for raw material and acetic anhydride polycondensation reaction with ethyl orthosilicate or methyl silicate.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described biology enzyme is laccase, lipase or protease; Described biology enzyme is scattered in cushioning liquid, and the concentration range of biology enzyme is 0.5 ~ 50mg/mL; Described cushioning liquid is Lin acid hydrogen Er Na – citrate buffer solution, Lin acid hydrogen Er Na – sodium dihydrogen phosphate buffer or Tris-hydrochloric acid buffer solution, and the pH value of described cushioning liquid is 2 ~ 10.
A kind of total silicon colloid body microcapsules encapsulating biology enzyme as above, described colloid body Microcapsules Size distribution is between 0.5 ~ 2 μm; Described colloid body microcapsules shell wall has semipermeable, and its semipermeable is relevant to the thickness of colloid body microcapsules shell wall.
Embodiment 1
A) ethyl orthosilicate mixes with the ratio of 1:10 with methyl alcohol, and adds ammoniacal liquor and regulate this mixed solution pH value to 12, stirred at ambient temperature 3h;
B) at above-mentioned steps A) add modifier C in gained solution
10h
20-Si (OC
4h
9)
3, between modifier addition and organosilicon source, mass ratio is 1:50, continues to stir 24h, namely obtains the methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle, 5000rpm centrifugal treating 60min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in methyl alcohol and chloroform volume ratio is that after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after methyl alcohol and chloroform volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in chloroform to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in chloroform to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle chloroform that concentration is 1wt%;
D) by C) the stand-by liquid of improved silica nano particle chloroform of step preparation with contain the pH2 sodium hydrogen phosphate-citric acid solution of 0.5mg/ml acid protease and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 15:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle chloroform is the silane of 1:50, stirred at ambient temperature hydrolysis 120h, namely prepares the total silicon colloid body microcapsules of the encapsulating acid protease being scattered in chloroform; For being further purified, remove not encapsulated acid protease, centrifugal 30min under 3000rpm condition, after removing supernatant, again be scattered in chloroform, repetition aforesaid operations like this 3 times, obtains the total silicon colloid body microcapsules being made up of the encapsulating acid protease of shell wall layer of silicon dioxide nano particle that average grain diameter is 2 μm.
Embodiment 2
A) ethyl orthosilicate mixes with the ratio of 1:100 with ethanol, and adds sodium acid carbonate and regulate this mixed solution pH value to 8, stirred at ambient temperature 36h;
B) at above-mentioned steps A) add modifier C in gained solution
5h
10-Si (OCH
3)
3, between modifier addition and organosilicon source, mass ratio is 1:100, continues to stir 48h, namely obtains the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 7000rpm centrifugal treating 30min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and volume of toluene than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol and volume of toluene ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in toluene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in toluene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle toluene that concentration is 3wt%;
D) by C) the stand-by liquid of improved silica nano particle toluene of step preparation with contain the pH5 sodium hydrogen phosphate-citric acid solution of 50mg/ml laccase and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle toluene is the silane of 1:100, and stirred at ambient temperature hydrolysis 48h, namely prepares the total silicon colloid body microcapsules of the encapsulating laccase being scattered in toluene; For being further purified, remove not encapsulated laccase, centrifugal 10min under 5000rpm condition, after removing supernatant, again be scattered in toluene, repetition aforesaid operations like this 5 times, obtains the total silicon colloid body microcapsules being made up of the encapsulating laccase of shell wall layer of silicon dioxide nano particle that average grain diameter is 1 μm.
Embodiment 3
A) methyl silicate mixes with the ratio of 1:50 with ethanol, and adds ammoniacal liquor and regulate this mixed solution pH value to 11, stirred at ambient temperature 10h;
B) at above-mentioned steps A) add modifier C18H36-Si (OC2H5) 3 in gained solution, between modifier addition and organosilicon source, mass ratio is 1:60, continue to stir 120h, namely obtain the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 13000rpm centrifugal treating 15min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and oxolane volume ratio is that after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol alcohol and oxolane volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in oxolane to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in oxolane to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle oxolane that concentration is 5wt%;
D) by C) the stand-by liquid of improved silica nano particle oxolane of step preparation with contain the pH7.5 sodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 30mg/ml lipase and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle oxolane is the silane of 1:60, stirred at ambient temperature hydrolysis 96h, namely prepares the total silicon colloid body microcapsules of the encapsulating lipase being scattered in oxolane; For being further purified, remove not encapsulated lipase, centrifugal 20min under 4000rpm condition, after removing supernatant, again be scattered in oxolane, repetition aforesaid operations like this 3 times, obtains the total silicon colloid body microcapsules being made up of the encapsulating lipase of shell wall two layers of Nano particles of silicon dioxide that average grain diameter is 0.7 μm.
Embodiment 4
A) ethyl orthosilicate mixes with the ratio of 1:30 with methyl alcohol, and adds NaOH and regulate this mixed solution pH value to 12, stirred at ambient temperature 24h;
B) at above-mentioned steps A) add modifier C in gained solution
10h
20-Si (OCH
3)
3, between modifier addition and organosilicon source, mass ratio is 1:70, continues to stir 96h, namely obtains the methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle, 10000rpm centrifugal treating 20min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in methyl alcohol and benzene volume ratio is that after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after methyl alcohol and benzene volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in benzene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in benzene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle benzene that concentration is 8wt%;
D) by C) the stand-by liquid of improved silica nano particle benzene of step preparation with contain the pH10Trish-hydrochloric acid buffer solution of 40mg/ml protease and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 5:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle benzene is the silane of 1:70, and stirred at ambient temperature hydrolysis 72h, namely prepares the total silicon colloid body microcapsules of the encapsulating protein enzyme being scattered in benzene; For being further purified, remove not encapsulated protease, after under room temperature condition, suction filtration is scattered in the total silicon colloid body microcapsules of the encapsulating biology enzyme of benzene, collect the total silicon colloid body of the encapsulating protein enzyme be deposited on filter membrane, again be scattered in benzene, repetition aforesaid operations like this 3 times, obtains the total silicon colloid body microcapsules being made up of the encapsulating protein enzyme of shell wall two layers of Nano particles of silicon dioxide that average grain diameter is 0.8 μm.
Embodiment 5
A) methyl silicate mixes with the ratio of 1:70 with methyl alcohol, and adds NaOH and regulate this mixed solution pH value to 12, stirred at ambient temperature 48h;
B) at above-mentioned steps A) add modifier C in gained solution
10h
20-Si (OC
2h
5)
3, between modifier addition and organosilicon source, mass ratio is 1:70, continues to stir 96h, namely obtains the methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle, 10000rpm centrifugal treating 60min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in methyl alcohol and ethyl acetate volume ratio is that after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after methyl alcohol and ethyl acetate volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in ethyl acetate to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in ethyl acetate to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle ethyl acetate that concentration is 10wt%;
D) by C) the stand-by liquid of improved silica nano particle ethyl acetate of step preparation with contain the pH5 sodium hydrogen phosphate-citric acid solution of 10mg/ml protease and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 6:1
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle ethyl acetate is the silane of 1:70, stirred at ambient temperature hydrolysis 72h, namely prepares the total silicon colloid body microcapsules of the encapsulating protein enzyme being scattered in ethyl acetate; For being further purified, remove not encapsulated protease, after under room temperature condition, suction filtration is scattered in the total silicon colloid body microcapsules of the encapsulating biology enzyme of benzene, collect the total silicon colloid body of the encapsulating protein enzyme be deposited on filter membrane, again be scattered in ethyl acetate, repetition aforesaid operations like this 5 times, obtain average grain diameter be 0.5 μm by three layer by layer Nano particles of silicon dioxide form the total silicon colloid body microcapsules of the encapsulating protein enzyme of shell wall.
Embodiment 6
A) ethyl orthosilicate mixes with the ratio of 1:80 with ethanol, and adds ammoniacal liquor and regulate this mixed solution pH value to 10, stirred at ambient temperature 12h;
B) at above-mentioned steps A) add modifier C in gained solution
18h
36-Si (OCH
3)
3, between modifier addition and organosilicon source, mass ratio is 1:80, continues to stir 100h, namely obtains the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 9000rpm centrifugal treating 30min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and volume of toluene than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol and volume of toluene ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in toluene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in toluene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle toluene that concentration is 2wt%;
D) by C) the stand-by liquid of improved silica nano particle toluene of step preparation with contain the pH5 sodium hydrogen phosphate-citric acid solution of 10mg/ml laccase and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle toluene is the silane of 1:80, and stirred at ambient temperature hydrolysis 120h, namely prepares the total silicon colloid body microcapsules of the encapsulating laccase being scattered in toluene; For being further purified, remove not encapsulated laccase, after under room temperature condition, suction filtration is scattered in the total silicon colloid body microcapsules of the encapsulating laccase of toluene, collect the total silicon colloid body of the encapsulating protein enzyme be deposited on filter membrane, again be scattered in toluene, repetition aforesaid operations like this 3 times, obtains the total silicon colloid body microcapsules being made up of the encapsulating paint of shell wall layer of silicon dioxide nano particle that average grain diameter is 1.5 μm.
Embodiment 7
A) methyl silicate mixes with the ratio of 1:90 with ethanol, and adds ammoniacal liquor and regulate this mixed solution pH value to 11, stirred at ambient temperature 10h;
B) at above-mentioned steps A) add modifier C12H24-Si (OC2H5) 3 in gained solution, between modifier addition and organosilicon source, mass ratio is 1:60, continue to stir 120h, namely obtain the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 13000rpm centrifugal treating 15min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and carbon tetrachloride volume ratio is that after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol alcohol and carbon tetrachloride volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in carbon tetrachloride to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in oxolane to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle carbon tetrachloride that concentration is 4wt%;
D) by C) the stand-by liquid of improved silica nano particle carbon tetrachloride of step preparation with contain the pH7.5 sodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 10mg/ml lipase and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle carbon tetrachloride is the silane of 1:100, stirred at ambient temperature hydrolysis 100h, namely the total silicon colloid body microcapsules of the encapsulating lipase being scattered in carbon tetrachloride are prepared, average grain diameter 0.8 μm, shell wall is made up of two layers of Nano particles of silicon dioxide.
Embodiment 8
A) methyl silicate mixes with the ratio of 1:30 with ethanol, and adds ammoniacal liquor and regulate this mixed solution pH value to 11, stirred at ambient temperature 10h;
B) at above-mentioned steps A) add modifier C in gained solution
12h
24-Si (OC
2h
5)
3, between modifier addition and organosilicon source, mass ratio is 1:70, continues to stir 24h, namely obtains the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 13000rpm centrifugal treating 15min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and volume of toluene than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol alcohol and volume of toluene ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in toluene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in toluene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle toluene that concentration is 5wt%;
D) by C) the stand-by liquid of improved silica nano particle toluene of step preparation with contain the pH7.5 sodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 50mg/ml lipase and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle toluene is the silane of 1:100, stirred at ambient temperature hydrolysis 100h, namely the total silicon colloid body microcapsules of the encapsulating lipase being scattered in toluene are prepared, average grain diameter 0.6 μm, shell wall is made up of two layers of Nano particles of silicon dioxide.
Embodiment 9
A) ethyl orthosilicate mixes with the ratio of 1:30 with ethanol, and adds ammoniacal liquor and regulate this mixed solution pH value to 11, stirred at ambient temperature 24h;
B) at above-mentioned steps A) add modifier C in gained solution
12h
24-Si (OCH
3)
3, between modifier addition and organosilicon source, mass ratio is 1:70, continues to stir 24h, namely obtains the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, 13000rpm centrifugal treating 15min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in ethanol and volume of toluene than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after ethanol alcohol and volume of toluene ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in toluene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in toluene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle toluene that concentration is 3wt%;
D) by C) the stand-by liquid of improved silica nano particle toluene of step preparation with contain the pH10Tris-hydrochloric acid buffer solution of 10mg/ml protease and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle toluene is the silane of 1:80, and stirred at ambient temperature hydrolysis 72h, namely prepares the total silicon colloid body microcapsules of the encapsulating protein enzyme being scattered in toluene; Average grain diameter 1 μm, shell wall is made up of layer of silicon dioxide nano particle
Embodiment 10
A) methyl silicate mixes with the ratio of 1:70 with methyl alcohol, and adds ammoniacal liquor and regulate this mixed solution pH value to 11, stirred at ambient temperature 3h;
B) at above-mentioned steps A) add modifier C in gained solution
10h
20-Si (OCH
3)
3, between modifier addition and organosilicon source, mass ratio is 1:70, continues to stir 24h, namely obtains the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described methyl alcohol dispersion liquid containing modified manometer silicon dioxide particle, 13000rpm centrifugal treating 15min at ambient temperature, after removing supernatant liquor, exchange of solvent process is carried out to precipitated silica: first, first the improved silica nano particle of centrifugation is scattered in methyl alcohol and volume of toluene than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after methyl alcohol and volume of toluene ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in toluene to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, by improved silica nanoparticle dispersion in toluene to naked-eye observation without bulky grain aggregate, be mixed with the stand-by liquid of improved silica nano particle toluene that concentration is 7wt%;
D) by C) the stand-by liquid of improved silica nano particle toluene of step preparation with contain the pH10Tris-hydrochloric acid buffer solution of 20mg/ml protease and mix, be prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification, wherein both mass ratioes are 10:1;
E) to step D) in the emulsion prepared, adding with the mass ratio of the stand-by liquid of improved silica nano particle toluene is the silane of 1:80, and stirred at ambient temperature hydrolysis 72h, namely prepares the total silicon colloid body microcapsules of the encapsulating biology enzyme being scattered in toluene; Average grain diameter 0.5 μm, shell wall is made up of three layers of Nano particles of silicon dioxide.
Claims (7)
1. encapsulate total silicon colloid body microcapsules for biology enzyme, it is characterized in that: described colloid body microcapsules are have shell wall structure that one or more layers Nano particles of silicon dioxide formed through silane hydrolyzate bonding and encapsulate the spherical nucleocapsid structure microcapsules of biology enzyme; Described silane refers to the hyperbranched silane formed for raw material and acetic anhydride polycondensation reaction with ethyl orthosilicate or methyl silicate; Described biology enzyme is laccase, lipase or protease; Described biology enzyme is scattered in cushioning liquid, and the concentration range of biology enzyme is 0.5 ~ 50mg/mL; Described cushioning liquid is Lin acid hydrogen Er Na – citrate buffer solution, Lin acid hydrogen Er Na – sodium dihydrogen phosphate buffer or Tris-hydrochloric acid buffer solution, and the pH value of described cushioning liquid is 2 ~ 10; Described colloid body Microcapsules Size distribution is between 0.5 ~ 2 μm; Described colloid body microcapsules shell wall has semipermeable, and its semipermeable is relevant to the thickness of colloid body microcapsules shell wall.
2. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme as claimed in claim 1, is characterized in that comprising the following steps:
A) be dissolved in alcoholic solution by organosilicon source, controlling solution ph is 8 ~ 12;
B) at above-mentioned steps A) add modifier in gained solution, and continue to stir, obtain the alcohol dispersion liquid containing modified manometer silicon dioxide particle;
C) by the described alcohol dispersion liquid containing modified manometer silicon dioxide particle, centrifugal treating at ambient temperature, after removing supernatant liquor, carries out exchange of solvent process to precipitated silica; Repeated centrifugation process also carries out the operation 3 ~ 5 times of exchange of solvent process after removing supernatant liquor, the improved silica precipitated the most at last is scattered in organic solvent, is mixed with the stand-by liquid of improved silica nano particle organic solvent that concentration is 1 ~ 10wt%;
D) mixed with the cushioning liquid containing biology enzyme by the stand-by liquid of described improved silica nano particle organic solvent, both mass ratioes are 5 ~ 15:1, are prepared into the stable water in oil emulsion of improved silica nano particle through ultrasonic emulsification;
E) in described emulsion, add silane, the mass ratio of addition and the stand-by liquid of improved silica nano particle organic solvent is 1:50 ~ 100, and stirred at ambient temperature hydrolysis 24 ~ 120h, namely obtains the total silicon colloid body microcapsules of the encapsulating biology enzyme being scattered in organic solvent.
3. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme according to claim 2, is characterized in that, steps A) in: organosilicon source and alcoholic solution mass ratio are 1:10 ~ 100; When organosilicon source is dissolved in alcoholic solution, at room temperature stir 3 ~ 48h; Described organosilicon source is ethyl orthosilicate or methyl silicate; Described alcohol is methyl alcohol or ethanol; Control solution ph and adopt sodium hydroxide solution, sodium carbonate liquor, sodium bicarbonate solution or ammoniacal liquor.
4. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme according to claim 2, is characterized in that, step B) in: the molecular formula of described modifier is C
nh
2n-Si (OC
mh
2m+1)
3, wherein n is 5 ~ 18, m is 1 ~ 4; Between described modifier addition and organosilicon source, mass ratio is 1:50 ~ 100; The described time of continuing to stir is 24 ~ 120h.
5. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme according to claim 2, is characterized in that, step C) in: the described centrifugal treating time is 15 ~ 60min, and centrifugal reporting is 5000 ~ 13000rpm.
6. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme according to claim 2, it is characterized in that, described exchange of solvent process refers to that realizing improved silica nanoparticle dispersion system by alcoholic solution complete exchange is the process of organic solvent: first, first the improved silica nano particle of centrifugation is scattered in alcohol and organic solvent volume than for after existing to naked-eye observation without bulky grain aggregate in the mixed solution of 7:3, centrifugal treating also removes supernatant liquor; Secondly, by the improved silica nanoparticle dispersion of above-mentioned centrifugation after alcohol and organic solvent volume ratio are exist to naked-eye observation without bulky grain aggregate in the mixed solution of 3:7, centrifugal treating also removes supernatant liquor; Again, the improved silica nano particle of centrifugation is replaced completely be scattered in organic solvent to naked-eye observation without bulky grain aggregate exist after, centrifugal treating also removes supernatant liquor; Finally, improved silica nano particle is replaced completely and to be scattered in organic solvent to naked-eye observation without bulky grain aggregate, form the stand-by liquid of improved silica nano particle organic solvent; Described organic solvent is carbon tetrachloride, oxolane, toluene, benzene, carrene, chloroform or ethyl acetate.
7. a kind of preparation method encapsulating the total silicon colloid body microcapsules of biology enzyme according to claim 2, is characterized in that, further, by described step e) the total silicon colloid body microcapsules of encapsulating biology enzyme that obtain carry out suction filtration process or centrifugal treating;
Described suction filtration process is by described step e) the total silicon colloid body microcapsules organic solvent dispersion system of encapsulating biology enzyme that obtains, suction filtration process under room temperature, collects and is deposited on total silicon colloid body filter membrane being encapsulated biology enzyme, and be scattered in organic solvent; Repetition like this 3 ~ 5 suction filtrations and dispersion, to remove not encapsulated biology enzyme;
Described centrifugal treating is by described step e) the total silicon colloid body microcapsules organic solvent dispersion system of encapsulating biology enzyme that obtains, centrifugal 5 ~ 30min under 3000 ~ 5000rpm rotating speed, the total silicon colloid body microcapsules getting the encapsulating biology enzyme of precipitation are scattered in organic solvent again; Repeated centrifugation like this and dispersion 3 ~ 5 times, to remove not encapsulated biology enzyme.
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