CN108130321A - A kind of hydrogel containing protease-inorganic hybrid nano flower and its preparation method and application - Google Patents
A kind of hydrogel containing protease-inorganic hybrid nano flower and its preparation method and application Download PDFInfo
- Publication number
- CN108130321A CN108130321A CN201711407474.8A CN201711407474A CN108130321A CN 108130321 A CN108130321 A CN 108130321A CN 201711407474 A CN201711407474 A CN 201711407474A CN 108130321 A CN108130321 A CN 108130321A
- Authority
- CN
- China
- Prior art keywords
- protease
- inorganic hybrid
- nano flower
- hybrid nano
- hydrogel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000002057 nanoflower Substances 0.000 title claims description 106
- 102000004190 Enzymes Human genes 0.000 claims abstract description 57
- 108090000790 Enzymes Proteins 0.000 claims abstract description 57
- 239000004365 Protease Substances 0.000 claims abstract description 51
- 239000000499 gel Substances 0.000 claims abstract description 45
- 108091005804 Peptidases Proteins 0.000 claims abstract description 27
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 23
- 229920001477 hydrophilic polymer Polymers 0.000 claims abstract description 13
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 11
- 108090000526 Papain Proteins 0.000 claims description 28
- 235000019834 papain Nutrition 0.000 claims description 28
- 229940055729 papain Drugs 0.000 claims description 27
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 23
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 21
- 235000019419 proteases Nutrition 0.000 claims description 20
- 235000014101 wine Nutrition 0.000 claims description 17
- 229920001661 Chitosan Polymers 0.000 claims description 14
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 10
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 10
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 8
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000000661 sodium alginate Substances 0.000 claims description 7
- 235000010413 sodium alginate Nutrition 0.000 claims description 7
- 229940005550 sodium alginate Drugs 0.000 claims description 7
- 229920000936 Agarose Polymers 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 239000000648 calcium alginate Substances 0.000 claims description 4
- 235000010410 calcium alginate Nutrition 0.000 claims description 4
- 229960002681 calcium alginate Drugs 0.000 claims description 4
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000006116 polymerization reaction Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 3
- -1 polyethylene Polymers 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 108091005508 Acid proteases Proteins 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 2
- 239000004698 Polyethylene Substances 0.000 claims 2
- 229920000573 polyethylene Polymers 0.000 claims 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 235000019441 ethanol Nutrition 0.000 claims 1
- 235000015110 jellies Nutrition 0.000 claims 1
- 239000008274 jelly Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 17
- 230000002255 enzymatic effect Effects 0.000 abstract description 8
- 238000006555 catalytic reaction Methods 0.000 abstract description 7
- 239000000758 substrate Substances 0.000 abstract description 7
- 239000003054 catalyst Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000000376 reactant Substances 0.000 abstract description 2
- 238000001338 self-assembly Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 49
- 230000000694 effects Effects 0.000 description 21
- 229940068984 polyvinyl alcohol Drugs 0.000 description 16
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 16
- 229920000642 polymer Polymers 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 11
- 241000219095 Vitis Species 0.000 description 9
- 230000003197 catalytic effect Effects 0.000 description 8
- 235000020097 white wine Nutrition 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000017854 proteolysis Effects 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000001354 calcination Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 240000006432 Carica papaya Species 0.000 description 3
- 235000009467 Carica papaya Nutrition 0.000 description 3
- 238000002441 X-ray diffraction Methods 0.000 description 3
- XMPISTNRBUJNNR-UHFFFAOYSA-L [Cu+2].O.[PH2](=O)[O-].[PH2](=O)[O-] Chemical compound [Cu+2].O.[PH2](=O)[O-].[PH2](=O)[O-] XMPISTNRBUJNNR-UHFFFAOYSA-L 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229940045110 chitosan Drugs 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 235000006264 Asimina triloba Nutrition 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- YQDHCCVUYCIGSW-LBPRGKRZSA-N ethyl (2s)-2-benzamido-5-(diaminomethylideneamino)pentanoate Chemical compound NC(=N)NCCC[C@@H](C(=O)OCC)NC(=O)C1=CC=CC=C1 YQDHCCVUYCIGSW-LBPRGKRZSA-N 0.000 description 2
- 229960004279 formaldehyde Drugs 0.000 description 2
- 235000019256 formaldehyde Nutrition 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000009941 weaving Methods 0.000 description 2
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical class [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000012424 Freeze-thaw process Methods 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229910000153 copper(II) phosphate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011806 microball Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/0052—Preparation of gels
- B01J13/0065—Preparation of gels containing an organic phase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/02—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Aquogel type enzymatic material of the flower of inorganic hybrid nano containing protease being combined to the invention discloses a kind of secondary fixation and its preparation method and application.By the use of protease as organic component and inorganic metal ion, the synthetic proteins enzyme inorganic hybrid nano by way of self assembly is spent, then it is embedded into gel carrier system of the biocompatibility preferably containing at least two hydrophilic polymers, and the hydrogel spent containing protease inorganic hybrid nano is prepared in the method for passing through repeatedly freeze-thaw, its unique tridimensional network makes enzyme be stable in the presence of in catalyst system and catalyzing, so as to achieve the purpose that secondary immobilization.The present invention makes protease from the influence of external environment, so as to which the stability for making enzyme is improved, further improves the reusing of enzyme by secondary immobilization.The step of catalysis material with reactant without being detached, eliminating enzyme-to-substrate separation, will have in enzyme immobilizatio field and be widely applied very much prospect.
Description
Technical field
The invention belongs to field of enzyme immobilization, be related to that a kind of secondary fixation is combined to containing protease-inorganic hybrid nano
Colored aquogel type enzymatic material and its preparation method and application.
Background technology
The enzyme that protease is hydrolyzed as catalytic proteins has been widely used for biochemistry, biological medicine, food
The fields such as product, weaving, chemical industry.But since most of protease is under the environmental condition of strong acid, highly basic, high temperature and heavy metal ion
It is easily destroyed, the production and application for making enzyme are extremely limited, thus how to be improved the stability of enzyme and expanded enzyme
Utilization scope become the focus of people's research.
In recent years, domestic and international each side scholar had having for three-dimensional flower-shape structure using the method for Supramolecular Assembling in synthesis
Machine-inorganic hybrid nano, which is taken, achieves breakthrough progress, and this method is using enzyme as organic component, and metal ion is as nothing
Machine component not only realizes enzyme immobilizatio, also greatly improves the Activity and stabill of enzyme, so as to expand it in biology
The application potential in the fields such as catalysis, bio-sensing, energy storage, gas detection, proteome analysis.However, this nanometer
The mechanical strength of flower pattern catalysis material is very low, easily is influenced to drop so as to cause stability by external environment or external force factor
It is low, and need to centrifuge and nano flower could be detached with substrate, it is quickly carried out so as to be unfavorable for industrialized production.
Meanwhile have many domestic and foreign scholars by the method for embedding by enzyme immobilization to biocompatibility preferably, thermostabilization
Preferably, in porous polyalcohol hydrogel, such as:Sodium alginate gel microsphere, graphene polymer composite hydrogel, chitosan-
Clay nano composite membrane, silicon polymer gel micro-ball etc., so as to improve the mechanical property of enzymatic material, improve enzyme
Stability, Unfortunately the diffusion of enzyme is greatly limited after immobilization, while also with a small amount of enzyme from polymer
The phenomenon that being dissolved out in gel, so as to make its loss of enzyme activity more serious.Therefore, it looks for one kind and can improve enzyme activity and can guarantee again
The fixing means of enzyme stability is particularly important.
Invention content
Protease is consolidated as industrial catalyst in actual application there are still many defects, and in nanoscale at present
Though enzyme activity is improved after fixedization, easily it is affected by the external environment and the phenomenon that stability reduction occurs, so as to significantly limit it
Further industrial applications, therefore in order to solve this problem, the present invention provides a kind of secondary process for fixation, by albumen
The excellent performance of both hydrogels that enzyme-hydrophilic polymers such as inorganic hybrid nano flower and chitosan-PVA are formed adequately is tied
Be combined, so as to obtain there is some strength and have both higher catalytic activity and stability containing enzyme-inorganic hybrid nano flower
Aquogel type enzymatic material.
Technical scheme is as follows:
A kind of hydrogel containing protease-inorganic hybrid nano flower, including:
(a) protease that protease and inorganic metal ion are formed by self assembly-inorganic hybrid nano flower;And
(b) the gel carrier system containing at least two hydrophilic polymers.
In the above-mentioned technical solutions, the protease-inorganic hybrid nano flower is embedded into the gel carrier
It is prepared in system.
In the above-mentioned technical solutions, the hydrophilic polymer is chitosan, polyvinyl alcohol, sodium alginate, alginic acid
Calcium or agarose.
In the above-mentioned technical solutions, the one kind of the inorganic metal ion in copper chloride, copper sulphate, copper nitrate
It is or a variety of.
In the above-mentioned technical solutions, protease is papain, in trypsase, alkali protease, acid protease
One or more.
The present invention also provides the preparation methods of the hydrogel containing protease-inorganic hybrid nano flower, include the following steps:
(1) it is prepared to obtain the protein enzyme solution of a concentration of 0.1~2mg/mL with aqueous solution, it is molten to add in inorganic metal ion
Liquid stands 24~96 hours at 20~40 DEG C, collects precipitation, by washing, centrifuging, being freeze-dried and obtain protease-inorganic
Hybridized nanometer is spent;
(2) at least two hydrophilic polymers are dissolved in aqueous solution to the gel carrier system that is prepared, add in step
(1) protease obtained-inorganic hybrid nano flower, is stirred at room temperature mixing;
(3) mixed solution of step (2) through circulating frozen and the process thawed, is obtained containing protease-inorganic hybrid nano flower
Hydrogel.
In above-mentioned technical solution, in step (2), the protease-inorganic hybrid nano flower is coagulated in described
Mass percentage in glue carrier system is 1~5%.Protease-matter of the inorganic hybrid nano flower in gel carrier system
Measuring percentage composition influences dispersion degree of the nano flower in carrier system, and excessive concentration, dispersibility of the nano flower in gel will
It is deteriorated, so as to influence the abundant combination of enzyme-to-substrate, and then influences the activity of enzymatic material.
In above-mentioned technical solution, in step (2), may be used at least two hydrophilic polymers be dissolved in it is aqueous molten
In liquid, stable gel carrier system is prepared.The hydrophilic polymer be selected from chitosan, polyvinyl alcohol, sodium alginate,
Calcium alginate or agarose.Preferably, at least two hydrophilic polymers are selected from chitosan and polyvinyl alcohol or sodium alginate
With polyvinyl alcohol or calcium alginate and polyvinyl alcohol or agarose and polyvinyl alcohol or polyvinyl alcohol and other are two or more poly-
Close the composition polymer of object combination.In the combination of polymers of described two polymer composition, polyvinyl alcohol is in the gel
The mass percentage added in carrier system is 10~20%, residual polymer such as chitosan, sodium alginate, alginic acid
The mass percentage that calcium or agarose are added in the gel carrier system is 4~6%.More than described two
In the composition polymer of combination of polymers, quality percentage that the polyvinyl alcohol is added in the gel carrier system
Content is 10~20%, the mass percentage that remaining other polymers are added in the gel carrier system for 4~
6%, wherein not limiting the ratio shared by the remaining other polymers, this can be realized when summation is in the range of described 4~6%
Invention.Preferably, the viscosity average molecular weigh of the chitosan is 620000 ± 75000, and the degree of polymerization of the polyvinyl alcohol is
1750 ± 50, the sodium alginate viscosity average molecular weigh is 150000 ± 35000, and the molecular weight of calcium alginate is 584.45, institute
The agarose molecules amount stated is 630.55.
In the above-mentioned technical solutions, the aqueous solution for phosphate buffer (PBS), water or contains acidic materials
Or the aqueous solution of alkaline matter, the acidic materials are acetic acid, hydrochloric acid etc., the alkaline matter is sodium hydroxide, hydroxide
Potassium etc..
In above-mentioned technical solution, in step (3), the condition of the freezing be at -20~-30 DEG C freezing 12~
For 24 hours, the condition of the defrosting is the 6~12h that thaws at 4~25 DEG C, and the circulating frozen and the process thawed are 2~7 times.Contain
There is the polymer solution of lipase-inorganic hybrid nano flower, the hydrogen bond action due to polymer during freeze-thaw is formed
Close three-dimensional crosslinked network makes lipase nano flower successfully be embedded into the hydrogel with higher-strength, protects nanometer
Colored structural stability, so as to keep its catalytic activity.
In above-mentioned technical solution, in step (1), inorganic metal ion in the inorganic metal ion solution
A concentration of 100~140mM.
The swelling ratio of the hydrogel of fatty enzyme of the present invention-inorganic hybrid nano flower is 20~60.
Hydrogel the present invention also provides the above-mentioned flower containing protease-inorganic hybrid nano is in grape wine of degrading in albumen
Application.The stability of grape wine determines the quality of wine, and the unstable protein contained in grape wine can be under certain condition
Aggregation is generated, grape wine is made to become muddy, this greatly affected the quality of wine, and egg unstable in conventional process grape wine
White method can together be removed albumen is stablized in wine, this not only affects the mouthfeel of wine, and also reduces the nutriture value of wine
Value.The present invention spends labile protein in catalytic degradation grape wine using the protease in hydrogel-inorganic hybrid nano is embedded in
Matter improves the quality and nutritive value of wine.
The present invention using protease as organic component and inorganic metal ion by way of self assembly synthetic proteins enzyme-
Inorganic hybrid nano is spent, which has the larger property of nano material specific surface area, avoids the mass transfer resistance between enzyme-to-substrate
Hinder, so as to which the protease after immobilization be made to improve compared with the enzyme activity of resolvase.The present invention has high enzyme by above-mentioned simultaneously
The nano flower of catalytic activity is embedded into gel carrier system of the biocompatibility preferably containing at least two hydrophilic polymers
In, and the hydrogel material containing protease-inorganic hybrid nano flower is prepared in the method for passing through repeatedly freeze-thaw,
Unique tridimensional network makes enzyme be stable in the presence of in catalyst system and catalyzing, so as to achieve the purpose that secondary immobilization and be applied
In practical catalysis reaction.
Beneficial effects of the present invention:
The present invention provides through the hydrogels containing protease-inorganic hybrid nano flower synthesized by the method for secondary immobilization
Type enzymatic material.Compared with floating preteins enzyme, protease nano flower obtains the catalytic activity of enzyme because of its larger specific surface area
To improve, by secondary immobilization, make protease nano flower from the influence of external environment, so as to which the stability for making enzyme obtains
Improve, further improve the reusing of enzyme.The material obtains protease-inorganic hybrid nano flower and hydrophilic polymer
To the excellent performance of both hydrogels (such as chitosan-PVA hydrogels) be adequately combined together, so as to living things catalysis,
The every field such as biological medicine, food industry will all have a wide range of applications.Compared with traditional process for fixation, the catalysis
The step of material with reactant without being detached, eliminating enzyme-to-substrate separation, and the hydrogel of the present invention can be as needed
It is processed into variously-shaped.
Description of the drawings
Fig. 1 is the infrared spectrogram for preparing papain-inorganic hybrid nano flower each stage product of gel.
Fig. 2 is the fixed X-ray diffraction spectrogram (XRD with unlocked papain-inorganic hybrid nano flower hydrogel
Figure).
Fig. 3 is papain-inorganic hybrid nano flower, Blank gel (without nano flower), papain-inorganic miscellaneous
Change the stereoscan photograph (SEM figures) of nano flower gel.
Fig. 4 is the energy spectrum diagram (EDS) of papain-inorganic hybrid nano flower hydrogel.
Fig. 5 is papain-inorganic hybrid nano flower hydrogel mechanical curves figure.
Specific embodiment
Following embodiments can make those of ordinary skill in the art that the present invention be more fully understood, but not in any way
The limitation present invention.
Following embodiments can make those of ordinary skill in the art that the present invention be more fully understood, but not in any way
The limitation present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, reagent used
Deng can chemically or biological reagent company purchase.
The material and papain enzyme activity determination method that following embodiments use:
1st, material
Papain:Purchased from Shanghai Yuan Ye bio tech ltd
Papain is also known as papain, is primarily present in root, stem, leaf and the fruit of papaya, and molecular weight is
23.5kD, containing 212 amino acid residues, activated centre position is mainly by 158 3 kinds of Cys 25, His 159, Asp amino
Acid composition.Papain has good hydrolysis for multiple proteins, being capable of catalyzing hydrolysis peptide bond, ester bond, amido bond.
In recent years, papain because have many advantages, such as high catalytic efficiency, good thermal stability due to be widely used in bioid
The fields such as, biological medicine, food, weaving, chemical industry.
Chitosan:Viscosity be 50~800mPas, viscosity average molecular weigh about 620,000;
Polyvinyl alcohol (PVA):The degree of polymerization is 1750 ± 50;
Phosphate buffer (PBS):0.01M, pH 7.4, is conventionally prepared.
2nd, proteinase activity assay method:
0.02mg/mL protease enzyme solutions are configured, according to each reagent of data addition in table, concussion reaction 5min after mixing,
Light absorption value is surveyed under 253nm.BAEE is acyl-L-arginine ethyl ester hydrochloride in following table, and DTT is dithiothreitol (DTT).
Protease activity unit of force (U) is defined as:Using 2mmol/L BAEE as substrate, at 25 DEG C, measurement volume is 6mL, often
Minute makes△A253It is an enzyme activity unit to increase by 0.001.
3rd, the assay method of protein content and amino acid
White wine examines horse with the protein content before and after proteasome degradation using the method for conventional measure protein content
This brilliant blue method measures, and amino acid content is measured using the formol titration of conventional measure amino acid content.
4th, papain-inorganic hybrid nano spends the measure of middle enzyme content:By calcination method (RSC Advances,
2015,5,96997-97002) it measures, using the organic principle in calcining removal nano flower in proper temperature, accurately obtains and receive
Practical enzyme content in popped rice.Specially:It weighs in 150~200mg nano flowers to crucible, 700 DEG C of calcination temperature, calcines 2h.
It weighs to powder remaining after calcining, the enzyme content in nano flower is calculated according to formula (1).
Enzyme content:
In formula:GNWeight for nano flower;G0For Cu3(PO4)2·3H2The weight of O
Embodiment 1~16
(1) pawpaw that initial enzyme concentration is respectively 0.1mg/mL, 0.4mg/mL, 0.8mg/mL, 1.2mg/mL is prepared with PBS
Protein enzyme solution takes the 6mL papain solutions respectively, adds in the 40 μ L of copper chloride solution of a concentration of 120mM, after shaking up
Quiescent culture collects precipitation after 72 hours under room temperature (25 DEG C), and precipitation is washed with deionized three times, and centrifuged with low-temperature and high-speed
Machine centrifuges 5min under the conditions of 4 DEG C, 3500rpm, by sediment fraction freeze-drying for 24 hours, obtains blue powder, obtains initial enzyme concentration
The respectively papain of 0.1mg/mL, 0.4mg/mL, 0.8mg/mL, 1.2mg/mL-inorganic hybrid nano flower;
(2) a certain amount of polyvinyl alcohol and chitosan is taken to be dissolved in a certain amount of phosphate buffer, is uniformly mixed, obtains solidifying
Glue carrier system adds in papain-inorganic hybrid nano flower of a certain amount of step (1), dissolves, be uniformly mixed;Institute
State the mass percentage such as table of papain in gel carrier system-inorganic hybrid nano flower, polyvinyl alcohol and chitosan
1。
(3) uniformly mixed solution carries out circulating frozen-defrosting, specially:At -20~-30 DEG C freezing 12~for 24 hours, 4
Thaw 6~12h at~25 DEG C, and freeze-thaw process repeats 3-6 cycle, obtains containing papain-inorganic hybrid nano
Colored hydrogel.Polymer solution containing papain-inorganic hybrid nano flower, due to polymerization during freeze-thaw
The hydrogen bond action of object forms close three-dimensional crosslinked network, and papain nano flower is made successfully to be embedded into higher-strength
Hydrogel in, the structural stability of nano flower is protected, so as to keep its catalytic activity.Freezing and defrosting temperature and time, can
Suitably to be adjusted according to actual conditions, the three-dimensional cross-linked net that can be formed by polyme in the range, Encapsulated Enzyme-
Inorganic hybrid nano is spent, and the intensity of hydrogel is also maintained at good state.In the following embodiments, what step (2) obtained contains
There is the mixed solution of nano flower and polymer to freeze 6h, thaw at RT 2h at -20 DEG C, 6 cycles are repeated, are contained
The hydrogel of papain-inorganic hybrid nano flower.
The component used of the hydrogel containing papain-inorganic hybrid nano flower is prepared in 1. embodiment 1~16 of table
And content
According to the constituent content of table 1, different enzyme initial concentrations and various concentration papain-inorganic is prepared
The hydrogel containing papain-inorganic nano flower of nano flower, chitosan and polyvinyl alcohol, obtains secondary fixation and is combined to
The flower containing protease-inorganic hybrid nano aquogel type enzymatic material.Meanwhile after the same method, identical housings are prepared
Glycan and the Blank gel without nano flower of polyvinyl alcohol ratio are as blank control.
In the hydrogel that is prepared of the present invention, chitosan mainly serve it is antibacterial, content 4 in system~
6% preferably.
The above-mentioned performance test results of the hydrogel containing papain-inorganic hybrid nano flower being prepared:
Fig. 1 is that the infrared spectrum that papain-inorganic hybrid nano spends each stage product of gel is prepared in embodiment 1
Figure, respectively three hypophosphite monohydrate copper (a), papain (b), papain-inorganic hybrid nano spend (c), without nanometer
Colored chitosan-PVA hydrogels, that is, blank control (d) and hydrogel (e) containing papain-inorganic hybrid nano flower
Infrared figure.As schemed shown in a, 1042cm-1, 988cm-1, 623cm-1The absorption peak at place is the characteristic absorption peak of phosphate group.Scheme b
In, 2800-3000cm-1For-CH2With-CH3Stretching vibration, in 3300cm-1For the characteristic absorption peak of-OH, scheme simultaneously to have in c
There is the characteristic absorption peak of papain and three hypophosphite monohydrate copper.Scheme that there is papain-inorganic hybrid nano flower and sky in e
The absorption peak of gel, this illustrates that nano flower is successfully introduced in hydrogel.
Fig. 2 is the fixed X-ray diffraction spectrogram (XRD with unlocked papain-inorganic hybrid nano flower hydrogel
Figure), i.e. in Fig. 2, a is the chitosan-PVA hydrogels without nano flower, and b is contains papain-inorganic hybrid nano flower
Chitosan-PVA hydrogels.As seen from the figure, when assembled fixed nano flower is again after gel immobilized, diffraction maximum position
Similar to former empty gel, illustrate papain-inorganic hybrid nano flower introduces the structure for not influencing former gel rubber system.
Fig. 3 is papain-inorganic hybrid nano flower (a and b), the blank hydrogel (c) without nano flower, containing pawpaw
The stereoscan photograph (SEM figures) of protease-inorganic hybrid nano flower hydrogel (d).It can be seen from the figure that assembled fixation
Nano flower into petal-shaped and be evenly distributed, gel network obtained is clear, be conducive to it is gel immobilized, and can be in figure
Nanometer flower structure is observed in the network of gel, illustrates that nano flower is successfully introduced in hydrogel, and be evenly distributed, shape
Looks remain intact.
Fig. 4 is the Energy dispersive x-ray diffraction pattern (EDS) of papain-inorganic hybrid nano flower hydrogel.By scheming
It is found that Cu and P signal in nano flower hydrogel are strongly.It further proves as a result, fatty enzyme-inorganic hybrid nano
The inorganic constituents of colored hydrogel is three hypophosphite monohydrate copper crystals.
Fig. 5 is that the papain-inorganic hybrid nano that contains that embodiment 15 is prepared spends hydrogel mechanical property bent
Line chart, Fig. 5 a are stretching-stress curve, and Fig. 5 b are compression-stress curve.It can be seen from the figure that, papain-inorganic hybridization
Nano flower hydrogel has excellent tensile property, and elongation at break is up to 300%, in compression height than the condition for 80%
Under, stress is up to 4.0MPa.Papain-excellent mechanical property of inorganic hybrid nano flower gel is its repetitive cycling profit
Ensured with providing.
The mechanical property containing papain-inorganic hybrid nano flower hydrogel that above-described embodiment 1~16 is prepared
Can be good, elongation at break is about in the range of 250-400%, and stress is about in the range of 2.0-4.0MPa.
Embodiment 17
Under different pH value, the enzyme activity containing papain-inorganic hybrid nano flower hydrogel of the present invention is measured, specifically
Method is:Weigh with the papain of 0.4mg enzyme equivalent-inorganic hybrid nano flower hydrogel (in hydrogel containing enzyme amount with
It is equal containing enzyme amount in nano flower), make its be scattered in 2mL PBS (0.01mol/L, pH value is adjusted to 6.5 respectively with NaOH,
7.5th, in 8.0 and 9.0, gel aqueous fluid is made.According to above-mentioned proteinase activity assay method, each reagent is added to hydrogel
In liquid, concussion reaction 5min after mixing surveys light absorption value under 253nm, calculates enzyme activity.As a result such as table 2~5.As control, in phase
With the enzyme activity that papain-inorganic hybrid nano flower (0.4mg enzymes equivalent) is determined under the conditions of pH, as a result such as table 6.
The enzyme activity of the papain that under 2. difference pH of table prepared by embodiment 13-inorganic hybrid nano flower hydrogel
The enzyme activity of the papain that under 3. difference pH of table prepared by embodiment 14-inorganic hybrid nano flower hydrogel
The enzyme activity of the papain that under 4. difference pH of table prepared by embodiment 15-inorganic hybrid nano flower hydrogel
The enzyme activity of the papain that under 5. difference pH of table prepared by embodiment 16-inorganic hybrid nano flower hydrogel
The enzyme activity of papain-inorganic hybrid nano flower under 6. difference pH of table
Gel is spent in pH by the papain-inorganic hybrid nano for understanding to obtain through dual process for fixation in table 2~5
Higher activity is respectively provided under the conditions of=6-9, compared with table 6, the enzyme activity of papain-inorganic hybrid nano flower gel is slightly lower
In papain-inorganic hybrid nano flower, this is because the embedding of gel can make the specific surface area that nano flower is contacted with substrate
It reduces, but simultaneously after the secondary fixation of gel, the stability higher of nano flower, is more advantageous to progress and the nanometer of catalysis reaction
Colored recycling.Meanwhile from table 2~5 it can be seen that papain is by fitting the pH of reaction solution after dual immobilization
Answering property enhances.
Embodiment 18
Papain-inorganic hybrid nano flower hydrogel protein degradation performance of different nano flower contents is measured, specifically
For:Weigh the papain of different nano flower contents-inorganic hybrid nano flower water-setting that 2g embodiments 13-16 is prepared
Glue is added it in 20g white wines, room temperature concussion reaction 24 hours.Then to protein in grape wine and amino acid into
Row detection.
In embodiment 13~16, in papain-inorganic hybrid nano flower hydrogel nano flower content be respectively 1%,
2%th, 3% and 4%.As a result such as table 7.
The papain of the different enzyme contents of table 7.-inorganic hybrid nano flower hydrogel protein degradation performance
By, it is found that with the increase of enzyme content in gel, the protein in sample wine is gradually degraded in table 7.Work as gel
When nano flower content is 2% in system, the protein content in whole system has minimum 0.63g/L.It is to receive to illustrate the concentration
Popped rice introduces the most suitable enzyme concentration of gel.
Embodiment 19
Papain under condition of different pH-inorganic hybrid nano flower hydrogel protein degradation performance is measured, specially:Claim
The papain of different nano flower contents-inorganic hybrid nano flower hydrogel that 2g embodiments 13-16 is prepared is taken, by it
It is added in 20g white wines.Respectively pH value under conditions of 6.5,7.5,8 and 9, room temperature concussion reaction 24 hours.So
Protein in grape wine and amino acid are detected afterwards.As a result such as table 8~11.
The papain that under 8. condition of different pH of table prepared by embodiment 13-inorganic hybrid nano flower hydrogel protein degradation
Performance
The papain that under 9. condition of different pH of table prepared by embodiment 14-inorganic hybrid nano flower hydrogel protein degradation
Performance
10. condition of different pH of table descends papain-inorganic hybrid nano flower hydrogel degradation egg prepared by embodiment 15
Bai Xingneng
11. condition of different pH of table descends papain-inorganic hybrid nano flower hydrogel degradation egg prepared by embodiment 16
Bai Xingneng
By table 7~11 it is found that solution ph is at 7.5 or so, the protein content in white wine is relatively low, amino acid content
It is higher, illustrate the albumen optimum pH in fixed papain degradation grape wine near 7.5.
Embodiment 20
Weigh papain-inorganic hybrid nano flower hydrogel that 30g obtains by the above embodiment of the present invention, by itself plus
Enter into the white wine of 100mL, be respectively sealing oscillation under conditions of 6.5,7.5,8 and 9 in pH value, room temperature reaction 40
After~72h, the content of protein and ammonia nitrogen before and after reacting is measured respectively using Coomassie brilliant blue and formol titration, and then really
Before and after fixed reaction in white wine protein degradation situation, be measured by the protein of the product to being obtained after enzymolysis,
So as to evaluate the degree of its enzymolysis, when protein content is 0.1g/L, it is believed that enzymolysis is completed.Water-setting is detached from enzymolysis product
Glue adds white wine, is digested under these conditions, realizes recycling for protease.
The specific method of separation hydrogel is from enzymolysis product:By two layers of sterilized clean gauze mistake of enzymolysis product
Filter, makes enzymolysis product be detached with hydrogel, so as to collect hydrogel.
The papain that table 12 is prepared for the embodiment of the present invention 3,7,11 and 15-inorganic hybrid nano spends hydrogel
Under condition of different pH digest white wine in albumen when hydrogel recycle number and last time recycle
Conversion ratio (recycling the ratio between the amount of enzymolysis protein matter and the amount of first time enzymolysis protein matter for the last time).
12. papains of table-inorganic hybrid nano flower hydrogel recycles number and conversion ratio
Embodiment 21
The obtained hydrogel containing papain-inorganic hybrid nano flower of embodiment 5 is put into aqueous beaker
It is middle immersion 48h after, in beaker there is no be precipitated substance occur, the intact immersion of hydrogel in water, as Fig. 3 is presented
Equally, papain-inorganic hybrid nano flower is steadily present in the three-dimensional network-like structure of hydrogel, in addition, passing through
This enzymatic material is carried out after recycling 40 times, gel is still intact, is dissolved out from hydrogel without any substance, therefore
The method of this immobilised enzymes shows preferable structural stability.
Claims (10)
1. a kind of hydrogel containing protease-inorganic hybrid nano flower, including:
(a) protease that protease and inorganic metal ion are formed by self assembly-inorganic hybrid nano flower;And
(b) the gel carrier system containing at least two hydrophilic polymers.
2. hydrogel according to claim 1, which is characterized in that it wraps the protease-inorganic hybrid nano flower
It buries and is prepared in the gel carrier system.
3. hydrogel according to claim 1, which is characterized in that the hydrophilic polymer is chitosan, polyethylene
Alcohol, sodium alginate, calcium alginate or agarose.
4. hydrogel according to claim 1, which is characterized in that the inorganic metal ion is from copper chloride, sulphur
It is one or more in sour copper, copper nitrate.
5. hydrogel according to claim 1, which is characterized in that the protease for papain, trypsase,
One or more of alkali protease, acid protease.
6. the preparation method of hydrogel of any one of them of Claims 1 to 5 containing protease-inorganic hybrid nano flower, packet
Include following steps:
(1) it is prepared to obtain the protein enzyme solution of a concentration of 0.1~2mg/mL with aqueous solution, adds in inorganic metal ion solution,
24~96 hours are stood at 20~40 DEG C, collects precipitation, by washing, centrifuging, being freeze-dried and obtain protease-inorganic hybridization
Nano flower;
(2) at least two hydrophilic polymers are dissolved in aqueous solution to the gel carrier system that is prepared, step (1) is added in and obtains
The protease arrived-inorganic hybrid nano flower, is stirred at room temperature mixing;
(3) mixed solution of step (2) through circulating frozen and the process thawed, obtains the water containing protease-inorganic hybrid nano flower
Gel.
7. preparation method according to claim 6, which is characterized in that in step (2), the protease-inorganic miscellaneous
It is 1~5% to change mass percentage of the nano flower in the gel carrier system.
8. preparation method according to claim 6, which is characterized in that in step (2), chitosan and polyvinyl alcohol is molten
Gel carrier system is prepared in aqueous solution, the chitosan and polyethylene added in the gel carrier system
The mass percentage of alcohol is respectively 4~6% and 10~20%, the viscosity average molecular weigh of the chitosan for 620000 ±
75000, the degree of polymerization of the polyvinyl alcohol is 1750 ± 50.
9. preparation method according to claim 6, which is characterized in that in step (3), the condition of the freezing be -
Freezing 12 at 20~-30 DEG C~for 24 hours, the condition of the defrosting is the 6~12h that thaws at 4~25 DEG C, the circulating frozen reconciliation
The process of jelly is 2~7 times.
10. application of the hydrogel containing protease-inorganic hybrid nano flower in grape wine of degrading in albumen.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711407474.8A CN108130321B (en) | 2017-12-22 | 2017-12-22 | Hydrogel containing protease-inorganic hybrid nanoflower as well as preparation method and application of hydrogel |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711407474.8A CN108130321B (en) | 2017-12-22 | 2017-12-22 | Hydrogel containing protease-inorganic hybrid nanoflower as well as preparation method and application of hydrogel |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108130321A true CN108130321A (en) | 2018-06-08 |
CN108130321B CN108130321B (en) | 2020-10-13 |
Family
ID=62391618
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711407474.8A Active CN108130321B (en) | 2017-12-22 | 2017-12-22 | Hydrogel containing protease-inorganic hybrid nanoflower as well as preparation method and application of hydrogel |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108130321B (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108918864A (en) * | 2018-08-03 | 2018-11-30 | 军事科学院军事医学研究院军事兽医研究所 | A kind of MnO2Hybridized nanometer flower and its preparation method and application |
CN109270061A (en) * | 2018-10-31 | 2019-01-25 | 青岛农业大学 | A kind of device and the application of quick detection and degrading organic phosphor pesticides |
CN109852603A (en) * | 2018-12-29 | 2019-06-07 | 大连工业大学 | A kind of iron/copper composite magnetic nano flower and its preparation method and application containing papain |
CN110055288A (en) * | 2019-03-15 | 2019-07-26 | 深圳大学 | A method of polyphenol compound is synthesized with novel enzyme immobilization technology |
CN111172571A (en) * | 2020-01-04 | 2020-05-19 | 桂林理工大学 | Method for preparing organic-inorganic hybrid nanoflower by electrodeposition |
CN112552914A (en) * | 2019-11-28 | 2021-03-26 | 兰州大学 | Rare earth fluoride hybrid nanoflower and preparation method thereof |
CN112924508A (en) * | 2021-01-28 | 2021-06-08 | 合肥工业大学 | Uricase protein inorganic hybrid nanoflower material, electrode, electrochemical sensor, preparation method and application in uric acid monitoring |
CN113041354A (en) * | 2021-03-30 | 2021-06-29 | 广州中医药大学(广州中医药研究院) | Nanoparticles of specific hydrolyzed template protein molecules and preparation method and application thereof |
CN113943725A (en) * | 2021-10-18 | 2022-01-18 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
CN114505070A (en) * | 2022-04-02 | 2022-05-17 | 陕西师范大学 | Porous nanoenzyme, porous nanoenzyme crystal, and preparation method and application thereof |
CN114686467A (en) * | 2022-04-07 | 2022-07-01 | 湖北大学 | Nano-immobilization method based on protein trans-splicing, application and immobilized enzyme |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101914519A (en) * | 2010-07-08 | 2010-12-15 | 东南大学 | Method for preparing hybridizing gel carrier immobilized enzyme |
CN103267786A (en) * | 2013-04-10 | 2013-08-28 | 太原理工大学 | Micro-needle enzyme-free glucose sensor electrode and preparation method thereof |
CN104386663A (en) * | 2014-11-19 | 2015-03-04 | 西北大学 | Preparation method of three-dimensional spherical dicalcium phosphate dihydrate nanoflower |
CN105950604A (en) * | 2016-06-03 | 2016-09-21 | 中国科学院青岛生物能源与过程研究所 | Enzyme immobilization method |
-
2017
- 2017-12-22 CN CN201711407474.8A patent/CN108130321B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101914519A (en) * | 2010-07-08 | 2010-12-15 | 东南大学 | Method for preparing hybridizing gel carrier immobilized enzyme |
CN103267786A (en) * | 2013-04-10 | 2013-08-28 | 太原理工大学 | Micro-needle enzyme-free glucose sensor electrode and preparation method thereof |
CN104386663A (en) * | 2014-11-19 | 2015-03-04 | 西北大学 | Preparation method of three-dimensional spherical dicalcium phosphate dihydrate nanoflower |
CN105950604A (en) * | 2016-06-03 | 2016-09-21 | 中国科学院青岛生物能源与过程研究所 | Enzyme immobilization method |
Non-Patent Citations (4)
Title |
---|
张宝亮等: "蛋白/磷酸锌杂化纳米花的制备及应用研究", 《中国化学会第30届学术年会-第三十六分会:纳米材料合成与组装》 * |
梁丽雯: "利用组装方法制备木瓜蛋白酶—无机杂化纳米材料", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
汪多仁: "《绿色净水处理剂》", 30 November 2006, 科学技术文献出版社 * |
薛巍等: "《生物医用水凝胶》", 31 December 2012 * |
Cited By (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108918864A (en) * | 2018-08-03 | 2018-11-30 | 军事科学院军事医学研究院军事兽医研究所 | A kind of MnO2Hybridized nanometer flower and its preparation method and application |
CN109270061B (en) * | 2018-10-31 | 2022-01-11 | 青岛农业大学 | Device for rapidly detecting and degrading organophosphorus pesticide and application |
CN109270061A (en) * | 2018-10-31 | 2019-01-25 | 青岛农业大学 | A kind of device and the application of quick detection and degrading organic phosphor pesticides |
CN109852603A (en) * | 2018-12-29 | 2019-06-07 | 大连工业大学 | A kind of iron/copper composite magnetic nano flower and its preparation method and application containing papain |
CN109852603B (en) * | 2018-12-29 | 2022-06-28 | 大连工业大学 | Papain-containing iron-copper composite magnetic nanoflower as well as preparation method and application thereof |
CN110055288A (en) * | 2019-03-15 | 2019-07-26 | 深圳大学 | A method of polyphenol compound is synthesized with novel enzyme immobilization technology |
CN112552914A (en) * | 2019-11-28 | 2021-03-26 | 兰州大学 | Rare earth fluoride hybrid nanoflower and preparation method thereof |
CN112552914B (en) * | 2019-11-28 | 2021-10-29 | 兰州大学 | Rare earth fluoride hybrid nanoflower and preparation method thereof |
CN111172571A (en) * | 2020-01-04 | 2020-05-19 | 桂林理工大学 | Method for preparing organic-inorganic hybrid nanoflower by electrodeposition |
WO2021135998A1 (en) * | 2020-01-04 | 2021-07-08 | 桂林理工大学 | Method for preparing organic-inorganic hybrid nanoflower by means of electrodeposition |
US11891712B2 (en) | 2020-01-04 | 2024-02-06 | Guilin University Of Technology | Method for preparing organic-inorganic hybrid nanoflower by electrodeposition |
CN112924508A (en) * | 2021-01-28 | 2021-06-08 | 合肥工业大学 | Uricase protein inorganic hybrid nanoflower material, electrode, electrochemical sensor, preparation method and application in uric acid monitoring |
CN113041354B (en) * | 2021-03-30 | 2022-12-23 | 广州中医药大学(广州中医药研究院) | Nanoparticles of specific hydrolyzed template protein molecules and preparation method and application thereof |
CN113041354A (en) * | 2021-03-30 | 2021-06-29 | 广州中医药大学(广州中医药研究院) | Nanoparticles of specific hydrolyzed template protein molecules and preparation method and application thereof |
CN113943725A (en) * | 2021-10-18 | 2022-01-18 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
CN113943725B (en) * | 2021-10-18 | 2023-08-11 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
CN114505070A (en) * | 2022-04-02 | 2022-05-17 | 陕西师范大学 | Porous nanoenzyme, porous nanoenzyme crystal, and preparation method and application thereof |
CN114505070B (en) * | 2022-04-02 | 2024-02-02 | 陕西师范大学 | Porous nano-enzyme, porous nano-enzyme crystal, preparation method and application thereof |
CN114686467A (en) * | 2022-04-07 | 2022-07-01 | 湖北大学 | Nano-immobilization method based on protein trans-splicing, application and immobilized enzyme |
CN114686467B (en) * | 2022-04-07 | 2023-11-24 | 湖北大学 | Nanometer immobilization method based on protein trans-splicing, application and immobilized enzyme |
Also Published As
Publication number | Publication date |
---|---|
CN108130321B (en) | 2020-10-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108130321A (en) | A kind of hydrogel containing protease-inorganic hybrid nano flower and its preparation method and application | |
CN108048446A (en) | A kind of hydrogel of fatty enzyme-inorganic hybrid nano flower and its preparation method and application | |
Lv et al. | Immobilization of urease onto cellulose spheres for the selective removal of urea | |
CA2833695C (en) | Cross-linked poly-e-lysine particles | |
Xu et al. | Immobilization of cellulase proteins on zeolitic imidazolate framework (ZIF-8)/polyvinylidene fluoride hybrid membranes | |
Liao et al. | Green magnetic hydrogels synthesis, characterization and flavourzyme immobilization based on chitin from Hericium erinaceus residue and polyvinyl alcohol | |
Ren et al. | Multiscale immobilized lipase for rapid separation and continuous catalysis | |
CN101716494B (en) | Magnetic compatible microsphere for purifying thrombin and preparation method and application thereof | |
CN107254460A (en) | A kind of carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and its application | |
CN109158089A (en) | A kind of sulfhydryl modified cellulose aerogels of ultrasonic wave added and preparation method | |
Metin | Immobilization studies and biochemical properties of free and immobilized Candida rugosa lipase onto hydrophobic group carrying polymeric support | |
CN111808840B (en) | Magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity and preparation method thereof | |
Babaei et al. | Use of mesoporous MnO2 as a support for immobilization of lipase from Candida rugosa | |
CN113308457A (en) | Preparation method of covalent organic framework encapsulated enzyme | |
CN101157914B (en) | Bentonite, biological polysaccharide plural gel immobilized cell carrier and preparation method thereof | |
CN107446910A (en) | A kind of preparation method of fibroin fiber immobilization chitosanase | |
CN110746636B (en) | Temperature-sensitive sodium alginate/cellulose ether composite hydrogel and preparation method and application thereof | |
JPS6331538A (en) | Immobilizing carrier | |
CN114317513A (en) | Chitosan-carboxymethyl cellulose immobilized enzyme and preparation method thereof | |
CN106701729A (en) | Immobilized enzyme taking polypeptide-modified amino resin as carrier and preparation method thereof | |
Miguezb et al. | Preparation and scanning electronic microscopy study of chitosan/polyvinyl (alcohol)-encapsulated crude urease extract | |
CN106519050A (en) | Preparing technology of adipic acid porous crosslinked starch | |
Sangeetha et al. | Investigation on the development of sturdy bioactive hydrogel beads | |
Mulko et al. | Smart Hydrogels: Application in bioethanol production | |
CN107988196A (en) | A kind of preparation method of the carboxyl carbon nanotubes immobilization laccase coated with polymethyl methacrylate |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20220314 Address after: 261200 Room 101, workshop building 6, Beidou science and Technology Innovation Industrial Park, Fenghuang street, Fangzi District, Weifang City, Shandong Province Patentee after: Shandong Tongyi photoresist material technology Co.,Ltd. Address before: 116034 No. 1 light industry garden, Ganjingzi District, Liaoning, Dalian Patentee before: DALIAN POLYTECHNIC University |