CN110373405A - A kind of biological enzyme of graft polymers and preparation method thereof and fixing means - Google Patents
A kind of biological enzyme of graft polymers and preparation method thereof and fixing means Download PDFInfo
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- CN110373405A CN110373405A CN201910671566.XA CN201910671566A CN110373405A CN 110373405 A CN110373405 A CN 110373405A CN 201910671566 A CN201910671566 A CN 201910671566A CN 110373405 A CN110373405 A CN 110373405A
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- biological enzyme
- polymer
- enzyme
- graft polymers
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 155
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 155
- 229920000578 graft copolymer Polymers 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 58
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229920000642 polymer Polymers 0.000 claims abstract description 38
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 238000010521 absorption reaction Methods 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- 125000000524 functional group Chemical group 0.000 claims abstract description 8
- 238000004132 cross linking Methods 0.000 claims abstract description 7
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 7
- 239000011570 nicotinamide Substances 0.000 claims abstract description 7
- JIQXKYSNGXUDJU-UHFFFAOYSA-N propan-2-ylidenehydrazine Chemical compound CC(C)=NN JIQXKYSNGXUDJU-UHFFFAOYSA-N 0.000 claims abstract description 7
- HCXJFMDOHDNDCC-UHFFFAOYSA-N 5-$l^{1}-oxidanyl-3,4-dihydropyrrol-2-one Chemical group O=C1CCC(=O)[N]1 HCXJFMDOHDNDCC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 64
- 150000001412 amines Chemical class 0.000 claims description 33
- 238000006116 polymerization reaction Methods 0.000 claims description 32
- 239000003607 modifier Substances 0.000 claims description 31
- 239000007993 MOPS buffer Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 27
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 24
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 23
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 22
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 22
- 239000011521 glass Substances 0.000 claims description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000008055 phosphate buffer solution Substances 0.000 claims description 16
- 230000015572 biosynthetic process Effects 0.000 claims description 15
- 238000003786 synthesis reaction Methods 0.000 claims description 14
- HBBSDZXXUIHKJE-UHFFFAOYSA-N 6-hydrazinylpyridine-3-carboxylic acid Chemical compound NNC1=CC=C(C(O)=O)C=N1 HBBSDZXXUIHKJE-UHFFFAOYSA-N 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 13
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 11
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 11
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Substances NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 11
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 claims description 10
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 10
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 6
- 229910052786 argon Inorganic materials 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- HUMNYLRZRPPJDN-KWCOIAHCSA-N benzaldehyde Chemical group O=[11CH]C1=CC=CC=C1 HUMNYLRZRPPJDN-KWCOIAHCSA-N 0.000 claims description 5
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 229960001124 trientine Drugs 0.000 claims description 4
- 239000004697 Polyetherimide Substances 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 229920001601 polyetherimide Polymers 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 230000003252 repetitive effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000009736 wetting Methods 0.000 claims description 3
- 125000003700 epoxy group Chemical group 0.000 abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
- 239000000178 monomer Substances 0.000 abstract description 2
- 238000010382 chemical cross-linking Methods 0.000 abstract 1
- 229920001059 synthetic polymer Polymers 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 21
- 238000002835 absorbance Methods 0.000 description 12
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000000460 chlorine Substances 0.000 description 8
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 150000001299 aldehydes Chemical class 0.000 description 6
- 150000001336 alkenes Chemical class 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 239000004367 Lipase Substances 0.000 description 5
- 102000004882 Lipase Human genes 0.000 description 5
- 108090001060 Lipase Proteins 0.000 description 5
- 235000019421 lipase Nutrition 0.000 description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 3
- ITPDZLBALZCKIB-UHFFFAOYSA-N 6-hydrazinylpyridine-3-carboxylic acid;hydrochloride Chemical compound Cl.N[NH2+]C1=CC=C(C([O-])=O)C=N1 ITPDZLBALZCKIB-UHFFFAOYSA-N 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000001953 recrystallisation Methods 0.000 description 3
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 3
- 206010015856 Extrasystoles Diseases 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- ATHHXGZTWNVVOU-UHFFFAOYSA-N N-methylformamide Chemical compound CNC=O ATHHXGZTWNVVOU-UHFFFAOYSA-N 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- WDAXFOBOLVPGLV-UHFFFAOYSA-N ethyl isobutyrate Chemical compound CCOC(=O)C(C)C WDAXFOBOLVPGLV-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002077 nanosphere Substances 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- -1 amino, sulfydryl Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001768 cations Chemical group 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000035943 smell Effects 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals
- C08F220/325—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals containing glycidyl radical, e.g. glycidyl (meth)acrylate
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
A kind of biological enzyme of graft polymers disclosed by the invention, biological enzyme and polymer are connected with double aryl hydrazone bonds that succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generates by bridging agent benzaldehyde, polymer surfaces have the functional group that covalent cross-linking and physical absorption are easily carried out with carrier.The invention also discloses the preparation method of the biological enzyme of graft polymers and fixing means.The present invention generates double aryl hydrazone bonds by two kinds of bridging agent groups, connects biological enzyme and polymer；The functional group on carrier remarkably promotes the physical absorption of enzyme by the physical absorption affinity interaction to polymer simultaneously；The functional group chemical crosslinking of epoxy group, amino and substrate surface, promotes enzyme and is chemically bonded on carrier on polymer；Furthermore the monomer that selects of the present invention contains epoxy group after synthetic polymer, is easy to happen and react with a variety of groups on biological enzyme, fix enzyme more stable；Reaction process is mild, easy to operate.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of biological enzyme of graft polymers, the present invention also relate to
And a kind of preparation method of the biological enzyme of graft polymers, a kind of fixing means of the biological enzyme of graft polymers.
Background technique
Enzyme is a kind of important biocatalyst, has the characteristics that high catalytic efficiency, specificity are strong, reaction condition is mild,
Have in food, brewing, medicine and other fields and is widely applied.Due to the special nature of enzyme, higher structure is very sensitive to environment,
Such as to temperature, pH, organic solvent, heavy metal ion factor, and can not be recycled after reacting, make enzyme in industrial application by
Limitation.
Enzyme immobilizatio method mainly has: absorption method, investment, cross-linking method and covalent method.Absorption method refers to that organized enzyme exists
Absorption on carrier surface, including physical absorption and ionic adsorption, solidification process are not required to chemical reagent, to the activity influence of enzyme
It is small, have many advantages, such as that easy to operate, step is few, physical absorption has enzyme degradation seldom occurs, but the enzyme after fixing is easily from carrier
On fall off, the problems such as there are stability and poor reproducibility, and the biological enzyme time-to-live is short, therefore cannot be widely used.Embedding
Method is the fixing means being embedded in enzyme in high-polymer three-dimensional space net structure.Mild experiment can be used in this method
Condition, but this immobilization technology is there is also limitation, if the free radical that generates in polymer forming process is to the active group of enzyme
Group has an impact, and reduces the activity of enzyme.Cross-linking method refers to through double-functional group reagent, between enzyme molecule, enzyme molecule and solidifying
The method for being cross-linked to form reticular structure between glue, polymer and keeping enzyme fixed, most common crosslinking agent is glutaraldehyde, this method
The disadvantage is that the formation condition of film is not easy to determine, the conditions such as pH, ionic strength, temperature and reaction time must be carefully controlled.Covalently
Combined techniques refer to the enzyme molecule method fixed in conjunction with carrier surface by covalent bond, although enhancing the binding force of enzyme,
But there are problems that the active group of enzyme causes enzymatic activity to be substantially reduced because participating in covalent reaction.
Chinese patent " a kind of can be in the preparation method of magnetic immobilized lipase used in organic media " (applying date:
20160912；Application number: CN201610817706.6；Publication date: 20181012；Publication number: CN106148320B) it is open with magnetic
Property Fe3O4/ P (St-AA) nanosphere or magnetic Fe3O4/PS-CH2Cl nanosphere consolidates lipase as carrier, by covalent bond
It is scheduled on magnetic Fe3O4/ P (St-AA) nanometer ball surface or magnetic Fe3O4/PS-CH2Cl nanometers of ball surfaces, the fixed reaction item of this method
Part is harsher, and biological enzyme activity decreases after immobilization, and immobilization process is more complex.
Chinese patent " method of lipase immobilization carrier and its fixed fat the enzyme " (applying date: 20140828；Application
Number: CN201410433204.4；Publication date: 20171226；Publication number: CN104293763B), it discloses a kind of lipase and fixes
Changing carrier after being successively crosslinked under the conditions of acid condition and middle meta-alkalescence with crosslinking agent, is obtained using collagen as substrate
Fatty zymophore, also provide it is a kind of using above-mentioned lipase immobilization carrier come the method for fixed fat enzyme, but this method due to
Cross-linking reaction condition is more violent, and preparation process is complicated, is not suitable for industrial production.
Since there are the above problem, this field, which needs one kind, can make enzyme keep higher activity, while having again well
Stability and simply and easily enzyme immobilization method.
Summary of the invention
The first purpose of the invention is to provide a kind of biological enzyme of graft polymers, solve existing fixed biological enzyme
Afterwards, bioenzyme activity reduce, the problem of stability difference.
A second object of the present invention is to provide a kind of preparation methods of the biological enzyme of graft polymers, solve existing system
React violent during standby, process is complicated, condition harshness problem.
Third object of the present invention is to provide a kind of fixing means of the biological enzyme of graft polymers.
First technical solution of the present invention is that a kind of biological enzyme of graft polymers passes through bridging agent benzene first
Aldehyde connects biological enzyme with double aryl hydrazone bonds that succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generates and gathers
Close object.
It is of the invention to be further characterized in that,
Polymer surfaces have the functional group that covalent cross-linking and physical absorption are easily carried out with carrier.
Second technical solution of the present invention is a kind of preparation method of the biological enzyme of graft polymers, including
Following steps:
Step 1, the synthesis of polymer and bridging agent:
Step 1.1, by styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and bigeminy
Pyridine Bpy is placed in polymerization pipe, freezes under liquid nitrogen environment, vacuumizes 15-30min to polymerization pipe using oil pump, is added
The CuBr of 0.352mmol；Continuation is freezed and is vacuumized under liquid nitrogen environment, then to high-purity argon gas is filled in polymerization pipe, is repeated
Operation no less than three times after, by polymerization pipe tube sealing go forward side by side 50-60 DEG C of trip temperature oil bath react 0.5-6.0h；After reaction,
Solution in polymerization pipe is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, it is inhaled using aluminum oxide column
Attached Cu2+, obtain P (St-r-GMA)；
Step 1.2, amine modifiers are dissolved in anhydrous n,N-Dimethylformamide DMF, obtain amine modifiers solution, it will
The P (St-r-GMA) of step 1.1 is dissolved in anhydrous n,N-Dimethylformamide DMF, and is added dropwise in amine modifiers solution anti-
It answers, obtains mixed liquor, after reaction, mixed liquor is by dialysis, and wherein molecule interception is 3500, is contained-NH2It is poly-
Close object；
Step 1.3,6- hydrazinonicotinic acid is dissolved in anhydrous n,N-Dimethylformamide DMF, sequentially add triethylamine and
Acetone, after reaction is stirred at room temperature, be added n-hydroxysuccinimide, EDCl stirring, the reaction was continued, product through extraction, washing, do
It is dry, concentration, obtain succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic after purification；
Step 2, the biological enzyme of synthesis modification and modified polymer:
Using benzaldehyde modified biological enzyme, modified biological enzyme is obtained；
S-HyNic that step 1.3 is obtained with contain-NH2Polymer carry out forerunner's precursor reactant, obtain modified polymerization
Object；
Step 3, the biological enzymatic synthesis of graft polymers:
By the modified biological enzyme of step 2 and modified polymer reaction to get the biological enzyme for arriving graft polymers.
It is of the invention to be further characterized in that,
2- isobutyl bromide ethyl ester EBIB, second bipyridine Bpy, glycidyl methacrylate GMA, benzene in step 1.1
The molar ratio of ethylene St is 1:2:50-100:100-200.
Amine modifiers in step 1.2 are specially ethylenediamine EDA or diethylenetriamine DETA or triethylene tetramine
Any one in TETA or polyetherimide PEI400；
In amine modifiers solution, the volume ratio of anhydrous n,N-Dimethylformamide DMF and amine modifiers is 10:1-5,
The molar concentration of obtained amine modifiers solution are as follows: 1mmol/1.2-1.5ml；
The volume ratio of the P (St-r-GMA) and anhydrous n,N-Dimethylformamide DMF of step 1.2 are 1-5:15.
6- hydrazinonicotinic acid in step 1.3, acetone, n-hydroxysuccinimide and EDCL molar ratio be 1:1-1.3:1.1-
1.5:1.1-1.4。
The synthesis process of modified biological enzyme in step 2 are as follows:
Biological enzyme is dissolved in phosphate buffer solution, the volume ratio of biological enzyme and phosphate buffer solution is 1:2-5, is obtained
Benzaldehyde solution is added dropwise in the phosphate buffer solution of biological enzyme and reacts 2- at normal temperature by the phosphate buffer solution of biological enzyme
3h, wherein the phosphate buffer solution volume ratio 1:35-40 of benzaldehyde solution and biological enzyme, ultrafiltration obtain modified biological enzyme；
The synthesis process of modified polymer:
- NH will be contained obtained in step 1.22Polymer be dissolved in MOPS buffer, with the S- being dissolved in DMF
HyNic normal-temperature reaction 2-3h, control-NH in reaction process2The ratio of group and-HyNic group is not more than 1:2, ultrafiltration to get
Modified polymer.
Benzaldehyde group and-HyNic group ratio are 1:15-20 in step 3 reaction process.
Third technical solution of the present invention is a kind of fixing means of the biological enzyme of graft polymers, substrate
The fixing means on surface are as follows: the then clean carrier surface MOPS buffer wetting being placed in centrifuge tube is gone with suction pipe
Fall MOPS buffer, the phosphate buffer of biological enzyme graft polymers is added in centrifuge tube；After standing 1-2h, biological enzyme is removed
Graft polymer solution, and coverslip is washed with MOPS buffer, MOPS buffer, which is added, is totally submerged glass cover-slip, cold
Hiding saves, i.e. the fixation of the biological enzyme of completion graft polymers.
It is of the invention to be further characterized in that,
Biological enzyme fixing means inside substrate are as follows: glass tube is cleaned and dried through EtOH Sonicate, is moistened with MOPS buffer solution
Its wet inner surface fills biological enzyme graft polymers buffer solution；After standing 1-2h, biological enzyme graft polymer solution is removed,
And glass tube is rinsed with MOPS buffer solution, MOPS buffer solution is refilled, glass tube both ends connection rubber tube is simultaneously sealed with rubber band
Mouthful, it is stored in refrigerator.
The beneficial effects of the present invention are: the present invention generates double aryl hydrazone bonds by two kinds of bridging agent groups, biological enzyme is connected
And polymer；The functional group (anionic groups such as hydroxyl) on carrier passes through to polymer (cation groups such as amino) simultaneously
Physical absorption affinity interaction, the physical absorption of enzyme can be remarkably promoted；And epoxy group, amino and substrate table on polymer
The functional group in face is chemically crosslinked, and is promoted enzyme and is chemically bonded on carrier；Furthermore the monomer that the present invention selects, synthesis are poly-
Hold very much after closing object containing a variety of groups (amino, sulfydryl, phenolic hydroxyl group etc.) in epoxy group, with biological enzyme protein macromolecule chain
It easily reacts, keeps the fixation of enzyme more stable；Reaction process is mildly simple, easy to operate.
Detailed description of the invention
Fig. 1 is a kind of chemical structural drawing of the biological enzyme of graft polymers of the present invention；
Fig. 2 is the fixed HRP enzymatic activity measurement figure of conventional surface of the present invention；
Fig. 3 is that HRP enzymatic activity measurement figure is fixed in glass tube of the present invention；
Fig. 4 is the modified HRP enzyme stability measurement figure of the present invention.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of biological enzyme of graft polymers of the present invention, as shown in Figure 1, passing through bridging agent benzaldehyde and succinimide
The double aryl hydrazone bonds connection biological enzyme and polymer that base -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generates.Pass through polymerization
Bridging agent group on object and enzyme, which reacts, generates BAH key, and polymer chain is connected with enzyme molecule, and BAH key is that aldehyde hydrazine is anti-
The double aryl hydrazone bonds that should be generated.
Polymer surfaces have the functional group that covalent cross-linking and physical absorption are easily carried out with carrier.
A kind of preparation method of the biological enzyme of graft polymers, comprising the following steps:
Step 1, the synthesis of polymer and bridging agent:
Step 1.1, by styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and bigeminy
Pyridine Bpy is placed in polymerization pipe (wherein 2- isobutyl bromide ethyl ester EBIB, second bipyridine Bpy, glycidyl methacrylate
GMA, styrene St molar ratio be 1:2:50-100:100-200), freezed under liquid nitrogen environment, using oil pump to polymerization pipe take out
The CuBr of 0.352mmol is added in vacuum 15-30min；Continuation is freezed and is vacuumized under liquid nitrogen environment, then in polymerization pipe
Fill high-purity argon gas, repetitive operation no less than three times after, by polymerization pipe tube sealing go forward side by side 50-60 DEG C of trip temperature oil bath react 0.5-
6.0h；After reaction, solution in polymerization pipe is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, make
With aluminum oxide column Adsorption of Cu2+, obtain P (St-r-GMA)；
Step 1.2, amine modifiers are dissolved in anhydrous n,N-Dimethylformamide DMF (anhydrous n,N-Dimethylformamide
The volume ratio of DMF and amine modifiers is 10:1-5, the molar concentration of obtained amine modifiers solution are as follows: 1mmol/1.2-
1.5ml), amine modifiers solution is obtained, the P (St-r-GMA) of step 1.1 is dissolved in anhydrous n,N-Dimethylformamide DMF
(volume ratio of the P (St-r-GMA) and anhydrous n,N-Dimethylformamide DMF of addition are 1-5:15), and it is modified to be added dropwise to amine
It is reacted in agent solution, obtains mixed liquor, after reaction, mixed liquor is by dialysis, and wherein molecule interception is 3500, is contained
There is-NH2Polymer；
Wherein, amine modifiers are specially ethylenediamine EDA or diethylenetriamine DETA or triethylene tetramine TETA or gather
Any one in etherimide PEI400.
Step 1.3,6- hydrazinonicotinic acid is dissolved in anhydrous n,N-Dimethylformamide DMF, sequentially add triethylamine and
Acetone, after reaction is stirred at room temperature, be added n-hydroxysuccinimide, EDCl stirring, the reaction was continued, product through extraction, washing, do
It is dry, concentration, obtain succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic after purification；
Wherein the molar ratio of 6- hydrazinonicotinic acid, acetone, n-hydroxysuccinimide and EDCL are 1:1-1.3:1.1-1.5:
1.1-1.4；The purpose of general excessive addition of triethylamine, triethylamine is to essentially drop out reaction to improve alkaline environment.
Step 2, the biological enzyme of synthesis modification and modified polymer:
Using benzaldehyde modified biological enzyme, modified biological enzyme is obtained, specifically:
Biological enzyme is dissolved in phosphate buffer solution (PB1,0.1M dibastic sodium phosphate, 0.15M NaCl, PH=7.2), it is raw
The volume ratio of object enzyme and phosphate buffer solution is 1:2-5, obtains the phosphate buffer solution of biological enzyme, benzaldehyde solution is added dropwise to
2-3h is reacted in the phosphate buffer solution of biological enzyme and at normal temperature, wherein the phosphate buffer solution of benzaldehyde solution and biological enzyme
Volume ratio 1:35-40, ultrafiltration (Mcutoff=100kDa) obtain modified biological enzyme.
S-HyNic that step 1.3 is obtained with contain-NH2Polymer carry out forerunner's precursor reactant, obtain modified polymerization
Object, specifically:
- NH will be contained obtained in step 1.22Polymer be dissolved in MOPS buffer (0.1M MOPS, 0.15M
NaCl, pH=7.6) in, and the S-HyNic normal-temperature reaction 2-3h being dissolved in DMF ,-NH in reaction process2Group and-HyNic
The ratio of group is not more than 1:2, and ultrafiltration (Mcutoff=100kDa) is to get modified polymer.
Step 3, the biological enzymatic synthesis of graft polymers:
By the modified biological enzyme of step 2 and modified polymer reaction to get the biological enzyme for arriving graft polymers, specifically
Are as follows: HRP and P after benzaldehyde modified are measured from the buffer solution of MesB1 (0.1M MES, 0.15NaCl, PH=4.7)
(St-r-GMA) for-HyNic using aldehyde hydrazine reaction in room temperature reaction 5h, repeated ultrafiltration purification obtains biological enzyme graft polymers.
Wherein benzaldehyde group and-HyNic group ratio are 1:15-20 in reaction process.
The fixing means of substrate surface are as follows: the clean carrier surface MOPS buffer wetting being placed in centrifuge tube,
Then remove MOPS buffer with suction pipe, the phosphate buffer of biological enzyme graft polymers is added in centrifuge tube；Stand 1-2h
Afterwards, biological enzyme graft polymer solution is removed, and washs coverslip with MOPS buffer, MOPS buffer, which is added, makes glass cover glass
Piece is totally submerged, stored refrigerated, i.e. the fixation of the biological enzyme of completion graft polymers.
Biological enzyme fixing means inside substrate are as follows: glass tube is cleaned and dried through EtOH Sonicate, is moistened with MOPS buffer solution
Its wet inner surface fills biological enzyme graft polymers buffer solution；After standing 1-2h, biological enzyme graft polymer solution is removed,
And glass tube is rinsed with MOPS buffer solution, MOPS buffer solution is refilled, glass tube both ends connection rubber tube is simultaneously sealed with rubber band
Mouthful, it is stored in refrigerator.
Embodiment 1
A kind of preparation method of immobilised enzymes, comprising the following steps:
By styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and second bipyridine Bpy
(2- isobutyl bromide ethyl ester, second bipyridine, glycidyl methacrylate, the quality of styrene be respectively 0.069g,
0.109g,3.413g,5.00g；Molar ratio 1:2:70:150) it is placed in polymerization pipe, it is freezed under liquid nitrogen environment, using oil pump to poly-
It closes pipe and vacuumizes 20min, the CuBr of 0.352mmol is added；Continuation is freezed and is vacuumized under liquid nitrogen environment, then to polymerization pipe
Middle filling high-purity argon gas, repetitive operation no less than three times after, by polymerization pipe tube sealing go forward side by side 60 DEG C of trip temperature oil bath react 5h；Instead
After answering, solution in polymerization pipe is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, use three oxygen
Change two aluminium column Adsorption of Cu2+, obtain P (St-r-GMA)；
12.0g amine modifiers polyetherimide PEI400 is dissolved in the anhydrous n,N-Dimethylformamide DMF (nothing of 25ml
The volume ratio of water n,N-Dimethylformamide DMF and amine modifiers is 10:1), amine modifiers solution is obtained, by P (St-r-
GMA (the volume of P (St-r-GMA) and anhydrous n,N-Dimethylformamide DMF) is dissolved in anhydrous n,N-Dimethylformamide DMF
It than for 5:15), and is added dropwise in amine modifiers solution and reacts, obtain mixed liquor, after 60 DEG C of reaction 10h, mixed liquor warp
Dialysis is crossed, wherein molecule interception is 3500, product P (St-r-GMA)-NH2It indicates.
The 6- chlorine apellagrin of 1.901g is dissolved into 10mL hydrazine hydrate, 6h is condensed back, vacuum concentration drying is re-dissolved in
It is acidified in 35%HCL solution, adjusts PH=5, precipitation to be precipitated can be obtained 6- hydrazinonicotinic acid hydrochloride, use ethyl alcohol after filtering
Washing, recrystallization (ethyl alcohol: water=2:1), obtain 1.245g 6- hydrazinonicotinic acid.Take above-mentioned made 6- hydrazinonicotinic acid 1.083g dissolution
It in 20mL anhydrous DMF, is added 2.5mL triethylamine (excessive, raising alkaline environment, be not involved in reaction), adds in Xiang Shangshu solution
Enter 0.5mL acetone, 1.023g n-hydroxysuccinimide 0.778g, EDCl 1.023g is then added in room temperature magnetic agitation 4h
It is stirred overnight, the extraction of 30mL chloroform is added, after purification, crude product purifies (methylene chloride: methanol=50:1) with column chromatography again,
Obtain 0.937g S-HyNic.
2.5mL is measured from 82.3 μM of HRP (206nmol, 1eq) stostes is dissolved in phosphate buffer solution (PB1,0.2M
NaCl, PH=7.2), it is mixed with the benzaldehyde solution of 72 μ L 20mM, obtains benzaldehyde base through multiple ultrafiltration after room temperature reaction 3h
The HRP of modification.
Take P (St-r-GMA) solution of 2mL be dissolved in MOPS buffer (MopsB1,0.1M MOPS, 0.15M NaCl,
PH=7.6 in), and anhydrous DMF (3.49 μm of ol, 20eq./100NH are dissolved in2) in 349 μ L 10mM S-HyNic in room
Temperature is lower to react 4h, by being centrifugated and obtaining polymer P (St-r-GMA)-by ultrafiltration (Mcutoff=100kDa)
HyNic。
250 μ L are measured from the buffer solution of MesB1 (0.1M MES, 0.15NaCl, PH=4.7) to modify through benzaldehyde base
HRP and 36 μ L P (St-r-GMA)-HyNic using aldehyde hydrazine reaction in room temperature reaction 5h, repeated ultrafiltration purification obtains biology
Enzyme graft polymers.
Surface enzyme activity measurement:
The bioenzyme activity of desorption measures: the buffer being sucked out in centrifuge tube with suction pipe (has absorption to give birth in the centrifuge tube
Object enzyme graft polymers glass cover-slip), it is added in cuvette, substrate A BTS/H is added2O2, which is recorded with the interval of 15s
The UV/vis spectrum 2min for determining mixture prepares each sample and measures three times, after the measurement linear recurrence of the data obtained
The initial rate of product ABTS- generation is obtained divided by the time.
It adsorbs the determination of activity of biological enzyme: removing the buffer in centrifuge tube, substrate A BTS/H is added2O2With buffer, often
Minute is gently inverted centrifuge tube, and after 15min, substrate solution is taken out from reaction tube, with UV/vis spectrum record product to ABTS-
Absorbance, with buffer washing centrifuge tube, and at 4 DEG C until further using.As shown in Fig. 2, fixed in conventional surface
Enzyme, absorbance of the ABTS- at 414nm are floated 2.0 or so.
Enzymatic activity measures in glass tube: catalysis substrate is flowed with certain speed, flows through the glass-micropipe two of absorption biological enzyme
Hold connection rubber tube, substrate A BTS/H2O2The glass-micropipe of attaching organism enzyme graft polymers is reacted by catalysis, the product of reaction
ABTS- is recorded in cuvette through real-time UV/vis spectrum.As shown in figure 3, enzyme fixed in glass tube, ABTS-
Absorbance at 414nm is floated 1.0 or so.
The activity stabilized Journal of Sex Research of biological enzyme graft polymers on the adsorbent material: load there is into biological enzyme graft polymers
Adsorbent material store the different time, study its bioactivity with storage using identical method is measured with bioenzyme activity
Deposit the stability of time.As shown in figure 4, the enzyme is still able to maintain after the biological enzyme graft polymer solution stores 30 days at 4 DEG C
Its activity, relative activity is up to 1.0.
Embodiment 2
A kind of preparation method of immobilised enzymes, comprising the following steps:
By styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and second bipyridine Bpy
(2- isobutyl bromide ethyl ester EBIB, second bipyridine Bpy, glycidyl methacrylate GMA, styrene St molar ratio be
It 1:2:50:100) is placed in polymerization pipe, is freezed under liquid nitrogen environment, 15min is vacuumized to polymerization pipe using oil pump, is added
The CuBr of 0.352mmol；Continuation is freezed and is vacuumized under liquid nitrogen environment, then to high-purity argon gas is filled in polymerization pipe, is repeated
Operation three times after, by polymerization pipe tube sealing go forward side by side 50 DEG C of trip temperature oil bath react 6h；After reaction, solution in polymerization pipe is existed
It is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, use aluminum oxide column Adsorption of Cu2+, obtain P (St-
r-GMA)；
2.3g amine modifiers ethylenediamine EDA is dissolved in anhydrous n,N-Dimethylformamide DMF (the anhydrous N, N- bis- of 25ml
The volume ratio of methylformamide DMF and amine modifiers is 10:5), amine modifiers solution is obtained, P (St-r-GMA) is dissolved in
(P (St-r-GMA) and the volume ratio of anhydrous n,N-Dimethylformamide DMF are in the anhydrous n,N-Dimethylformamide DMF of 5ml
1:15), it and is added dropwise in amine modifiers solution and reacts, obtain mixed liquor, after 60 DEG C of reaction 10h, mixed liquor is by saturating
Analysis, wherein molecule interception is 3500, product P (St-r-GMA)-NH2It indicates.
The 6- chlorine apellagrin of 1.901g is dissolved into 10mL hydrazine hydrate, 6h is condensed back, vacuum concentration drying is re-dissolved in
It is acidified in 35%HCL solution, adjusts PH=5, precipitation to be precipitated can be obtained 6- hydrazinonicotinic acid hydrochloride, use ethyl alcohol after filtering
Washing, recrystallization (ethyl alcohol: water=2:1), obtain 1.245g6- hydrazinonicotinic acid.
It takes above-mentioned made 6- hydrazinonicotinic acid to be dissolved in 20mL anhydrous DMF, 2.5mL triethylamine is added, in Xiang Shangshu solution
Acetone is added, room temperature magnetic agitation 4h is then added n-hydroxysuccinimide g, EDCl and is stirred overnight (6- hydrazinonicotinic acid, third
The molar ratio of ketone, n-hydroxysuccinimide and EDCL is 1:1:1.1:1.1), the extraction of 30mL chloroform is added, it is after purification, thick to produce
Object is purified (methylene chloride: methanol=50:1) with column chromatography again, obtains S-HyNic.
Prepare HRP and polymer P (St-r-GMA)-HyNic (- NH in reaction process of benzaldehyde base modification2Group with-
The ratio 1:2 of HyNic group)；
250 μ L are measured from the buffer solution of MesB1 (0.1M MES, 0.15NaCl, PH=4.7) to modify through benzaldehyde base
HRP and 36 μ L P (St-r-GMA)-HyNic using aldehyde hydrazine reaction in room temperature reaction 5h, repeated ultrafiltration purification obtains biology
Enzyme graft polymers, benzaldehyde group and-HyNic group ratio are 1:15 in reaction process.
The biological enzyme graft polymers substrate for enzymatic activity prepared using the feed ratio, as shown in Fig. 2, solid on conventional surface
Fixed enzyme, absorbance of the ABTS- at 414nm are floated 1.5 or so.As shown in figure 3, enzyme fixed in glass tube,
ABTS-Absorbance at 414nm is floated 0.9 or so.As shown in figure 4, the biological enzyme graft polymer solution is stored up at 4 DEG C
After depositing 30 days, which is still able to maintain its activity, and relative activity is up to 0.95.
Embodiment 3
A kind of preparation method of immobilised enzymes, comprising the following steps:
By styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and second bipyridine Bpy
(2- isobutyl bromide ethyl ester EBIB, second bipyridine Bpy, glycidyl methacrylate GMA, styrene St molar ratio be
It 1:2:100:200) is placed in polymerization pipe, is freezed under liquid nitrogen environment, 30min is vacuumized to polymerization pipe using oil pump, is added
The CuBr of 0.352mmol；Continuation is freezed and is vacuumized under liquid nitrogen environment, then to high-purity argon gas is filled in polymerization pipe, is repeated
Operation three times after, by polymerization pipe tube sealing go forward side by side 55 DEG C of trip temperature oil bath react 1h；After reaction, solution in polymerization pipe is existed
It is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, use aluminum oxide column Adsorption of Cu2+, obtain P (St-
r-GMA)；
3.0951g amine modifiers diethylenetriamine DETA is dissolved in the anhydrous n,N-Dimethylformamide DMF of 25ml, is obtained
To amine modifiers solution, P (St-r-GMA) is dissolved in the anhydrous n,N-Dimethylformamide DMF of 5ml, and is added dropwise to amine
It is reacted in modifier solution, obtains mixed liquor, after 60 DEG C of reaction 10h, mixed liquor is by dialysis, and wherein molecule interception is
3500, product P (St-r-GMA)-NH2It indicates.
The 6- chlorine apellagrin of 1.901g is dissolved into 10mL hydrazine hydrate, 6h is condensed back, vacuum concentration drying is re-dissolved in
It is acidified in 35%HCL solution, adjusts PH=5, precipitation to be precipitated can be obtained 6- hydrazinonicotinic acid hydrochloride, use ethyl alcohol after filtering
Washing, recrystallization (ethyl alcohol: water=2:1), obtain 1.245g 6- hydrazinonicotinic acid.
It takes above-mentioned made 6- hydrazinonicotinic acid to be dissolved in 20mL anhydrous DMF, 2.5mL triethylamine is added, in Xiang Shangshu solution
Acetone is added, room temperature magnetic agitation 4h is then added n-hydroxysuccinimide g, EDCl and is stirred overnight (6- hydrazinonicotinic acid, third
The molar ratio of ketone, n-hydroxysuccinimide and EDCL is 1:1.3:1.5:1.4), the extraction of 30mL chloroform is added, after purification, slightly
Product is purified (methylene chloride: methanol=50:1) with column chromatography again, obtains S-HyNic.
Prepare HRP and polymer P (St-r-GMA)-HyNic (- NH in reaction process of benzaldehyde base modification2Group with-
The ratio 1:1 of HyNic group)；
250 μ L are measured from the buffer solution of MesB1 (0.1M MES, 0.15NaCl, PH=4.7) to modify through benzaldehyde base
HRP and 36 μ L P (St-r-GMA)-HyNic using aldehyde hydrazine reaction in room temperature reaction 5h, repeated ultrafiltration purification obtains biology
Enzyme graft polymers, benzaldehyde group and-HyNic group ratio are 1:20 in reaction process.
The biological enzyme graft polymers substrate for enzymatic activity prepared using the feed ratio, as shown in Fig. 2, solid on conventional surface
Fixed enzyme, absorbance of the ABTS- at 414nm are floated 1.4 or so.As shown in figure 3, enzyme fixed in glass tube,
Absorbance of the ABTS- at 414nm is floated 0.8 or so.As shown in figure 4, the biological enzyme graft polymer solution is stored up at 4 DEG C
The enzyme is still able to maintain its activity after depositing 30 days, and relative activity is up to 0.95.
Embodiment 4
Referring to embodiment 1,2- smells isobutyric acid ethyl ester, second bipyridine, glycidyl methacrylate (GMA), benzene second
The molar ratio of alkene (St) is constant, obtains P (St-r-GMA).Remaining condition is constant, and amine modifiers are triethylene tetramine at this time
The TETA of 4.3869g is dissolved in the anhydrous DMF of 25ml by TETA, and P (St-r-GMA) is dissolved in 5mL anhydrous DMF, by polymer solution
It is slowly added dropwise in DETA solution.After being added dropwise, 60 DEG C of reaction 10h of mixed liquor.After completion of the reaction, mixed liquor is through dialysis three
It, removes unreacted DETA and solvent, is contained-NH2Polymer.
The biological enzyme graft polymers substrate for enzymatic activity prepared using the feed ratio, the fixed enzyme on conventional surface,
Absorbance of the ABTS- at 414nm is floated 1.3 or so.Fixed enzyme, extinction of the ABTS- at 414nm in glass tube
Degree floats 0.7 or so.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, phase
To activity up to 0.95.
Embodiment 5
Referring to embodiment 1, change horseradish peroxidase is protease, remaining condition is constant, which is graft-polymerized
After object solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is up to 0.95.It is fixed on conventional surface
Enzyme, absorbance of the ABTS- at 414nm 1.5 or so float.The fixed enzyme in glass tube, ABTS- is in 414nm
The absorbance at place is floated 0.9 or so.
Embodiment 6
Referring to embodiment 1, change horseradish peroxidase superoxide dismutase, remaining condition is constant, which connects
After branch polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is up to 0.95.In conventional surface
The enzyme of upper fixation, absorbance of the ABTS- at 414nm are floated 1.5 or so.Fixed enzyme, ABTS- exist in glass tube
Absorbance at 414nm is floated 1.2 or so.
Claims (10)
1. a kind of biological enzyme of graft polymers, which is characterized in that pass through bridging agent benzaldehyde and succinimido -6- hydrazine
The double aryl hydrazone bonds connection biological enzyme and polymer that base-niacinamide acetone hydrazone S-HyNic reaction generates.
2. a kind of biological enzyme of graft polymers according to claim 1, which is characterized in that the polymer surfaces have
The functional group of covalent cross-linking and physical absorption is easily carried out with carrier.
3. a kind of preparation method of the biological enzyme of graft polymers as claimed in claim 1 or 2, which is characterized in that including with
Lower step:
Step 1, the synthesis of polymer and bridging agent:
Step 1.1, by styrene St, glycidyl methacrylate GMA, 2- isobutyl bromide ethyl ester EBIB and second bipyridine
Bpy is placed in polymerization pipe, freezes under liquid nitrogen environment, vacuumizes 15-30min to the polymerization pipe using oil pump, is added
The CuBr of 0.352mmol；Continuation is freezed and is vacuumized under liquid nitrogen environment, then to filling high-purity argon gas in the polymerization pipe,
Repetitive operation no less than three times after, by the polymerization pipe tube sealing go forward side by side 50-60 DEG C of trip temperature oil bath react 0.5-6.0h；Reaction
After, solution in the polymerization pipe is precipitated in ether, sediment is simultaneously dissolved in CH by filtering precipitate2Cl2In, use three
Al 2 O column Adsorption of Cu2+, obtain P (St-r-GMA)；
Step 1.2, amine modifiers are dissolved in anhydrous n,N-Dimethylformamide DMF, obtain amine modifiers solution, by step
1.1 P (St-r-GMA) is dissolved in anhydrous n,N-Dimethylformamide DMF, and is added dropwise in amine modifiers solution and is reacted, and is obtained
To mixed liquor, after reaction, the mixed liquor is by dialysis, and wherein molecule interception is 3500, is contained-NH2It is poly-
Close object；
Step 1.3,6- hydrazinonicotinic acid is dissolved in anhydrous n,N-Dimethylformamide DMF, sequentially adds triethylamine and acetone,
After reaction is stirred at room temperature, n-hydroxysuccinimide, EDCl stirring is added, the reaction was continued, and product is through extraction, washing, drying, dense
Contracting obtains succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic after purification；
Step 2, the biological enzyme of synthesis modification and modified polymer:
Using benzaldehyde modified biological enzyme, modified biological enzyme is obtained；
S-HyNic that step 1.3 is obtained with contain-NH2Polymer carry out forerunner's precursor reactant, obtain modified polymer；
Step 3, the biological enzymatic synthesis of graft polymers:
By the modified biological enzyme of step 2 and modified polymer reaction to get the biological enzyme for arriving graft polymers.
4. a kind of preparation method of the biological enzyme of graft polymers according to claim 3, which is characterized in that the step
Mole of 2- isobutyl bromide ethyl ester EBIB, second bipyridine Bpy, glycidyl methacrylate GMA, styrene St in 1.1
Than for 1:2:50-100:100-200.
5. a kind of preparation method of the biological enzyme of graft polymers according to claim 3, which is characterized in that the step
Amine modifiers in 1.2 are specially ethylenediamine EDA or diethylenetriamine DETA or triethylene tetramine TETA or polyetherimide
Any one in amine PEI400；
In the amine modifiers solution, the volume ratio of anhydrous n,N-Dimethylformamide DMF and amine modifiers is 10:1-5,
The molar concentration of obtained amine modifiers solution are as follows: 1mmol/1.2-1.5ml；
The volume ratio of the P (St-r-GMA) and anhydrous n,N-Dimethylformamide DMF of the step 1.2 are 1-5:15.
6. a kind of preparation method of the biological enzyme of graft polymers according to claim 3, which is characterized in that the step
6- hydrazinonicotinic acid in 1.3, acetone, n-hydroxysuccinimide and EDCL molar ratio be 1:1-1.3:1.1-1.5:1.1-1.4.
7. a kind of preparation method of the biological enzyme of graft polymers according to claim 3, which is characterized in that the step
The synthesis process of modified biological enzyme in 2 are as follows:
Biological enzyme is dissolved in phosphate buffer solution, the volume ratio of biological enzyme and phosphate buffer solution is 1:2-5, obtains biology
Benzaldehyde solution is added dropwise in the phosphate buffer solution of biological enzyme and is reacted at normal temperature 2-3h by the phosphate buffer solution of enzyme,
The wherein phosphate buffer solution volume ratio 1:35-40 of benzaldehyde solution and biological enzyme, ultrafiltration obtain modified biological enzyme；
The synthesis process of the polymer of the modification:
- NH will be contained obtained in step 1.22Polymer be dissolved in MOPS buffer, with the S-HyNic being dissolved in DMF
Normal-temperature reaction 2-3h, control-NH in reaction process2The ratio of group and-HyNic group is not more than 1:2, and ultrafiltration is to get modification
Polymer.
8. a kind of preparation method of the biological enzyme of graft polymers according to claim 3, which is characterized in that the step
Benzaldehyde group and-HyNic group ratio are 1:15-20 in 3 reaction process.
9. a kind of fixing means of the biological enzyme of graft polymers as claimed in claim 1 or 2, which is characterized in that substrate table
The fixing means in face are as follows: the then clean carrier surface MOPS buffer wetting being placed in centrifuge tube is removed with suction pipe
The phosphate buffer of biological enzyme graft polymers is added in centrifuge tube MOPS buffer；After standing 1-2h, removes biological enzyme and connect
Branch polymer solution, and coverslip is washed with MOPS buffer, MOPS buffer, which is added, is totally submerged glass cover-slip, refrigerates
It saves, i.e. the fixation of the biological enzyme of completion graft polymers.
10. a kind of fixing means of the biological enzyme of graft polymers as claimed in claim 1 or 2, which is characterized in that in substrate
The biological enzyme fixing means in portion are as follows: glass tube is cleaned and dried through EtOH Sonicate, soaks its inner surface with MOPS buffer solution, filling
Biological enzyme graft polymers buffer solution；After standing 1-2h, biological enzyme graft polymer solution is removed, and with MOPS buffer solution
Glass tube is rinsed, refills MOPS buffer solution, glass tube both ends connection rubber tube is simultaneously sealed with rubber band, is stored in refrigerator.
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Citations (3)
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|---|---|---|---|---|
| WO2014082080A2 (en) * | 2012-11-26 | 2014-05-30 | Callidus Biopharma, Inc. | Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases |
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| CN109609487A (en) * | 2018-11-23 | 2019-04-12 | 西安理工大学 | A kind of biological enzyme of graft polymers and preparation method thereof and fixing means |
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| CN104160033A (en) * | 2011-05-27 | 2014-11-19 | 阿米库斯治疗学公司 | Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases |
| WO2014082080A2 (en) * | 2012-11-26 | 2014-05-30 | Callidus Biopharma, Inc. | Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases |
| CN109609487A (en) * | 2018-11-23 | 2019-04-12 | 西安理工大学 | A kind of biological enzyme of graft polymers and preparation method thereof and fixing means |
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