CN100417724C - Nanometer carbonic anhydrase grain for biological catalysis of polymer and its prepn process - Google Patents
Nanometer carbonic anhydrase grain for biological catalysis of polymer and its prepn process Download PDFInfo
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- CN100417724C CN100417724C CNB2006100897224A CN200610089722A CN100417724C CN 100417724 C CN100417724 C CN 100417724C CN B2006100897224 A CNB2006100897224 A CN B2006100897224A CN 200610089722 A CN200610089722 A CN 200610089722A CN 100417724 C CN100417724 C CN 100417724C
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- 229920000642 polymer Polymers 0.000 title claims abstract description 33
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 19
- 102000003846 Carbonic anhydrases Human genes 0.000 title claims description 72
- 108090000209 Carbonic anhydrases Proteins 0.000 title claims description 72
- 238000000034 method Methods 0.000 title claims description 13
- 230000008569 process Effects 0.000 title description 4
- 239000000178 monomer Substances 0.000 claims abstract description 37
- 238000002360 preparation method Methods 0.000 claims abstract description 32
- 239000000463 material Substances 0.000 claims abstract description 19
- 238000010526 radical polymerization reaction Methods 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 9
- 230000003197 catalytic effect Effects 0.000 claims abstract description 8
- 150000003254 radicals Chemical class 0.000 claims abstract description 5
- 125000003342 alkenyl group Chemical group 0.000 claims description 31
- 239000002245 particle Substances 0.000 claims description 26
- 239000003795 chemical substances by application Substances 0.000 claims description 19
- 230000009144 enzymatic modification Effects 0.000 claims description 19
- 239000003999 initiator Substances 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 10
- -1 persulfuric acid salt Chemical class 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims description 4
- 150000004291 polyenes Chemical class 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 claims description 3
- 238000010494 dissociation reaction Methods 0.000 claims description 3
- 230000005593 dissociations Effects 0.000 claims description 3
- 150000002978 peroxides Chemical class 0.000 claims description 3
- 239000001488 sodium phosphate Substances 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 235000011008 sodium phosphates Nutrition 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical class [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
- 229910021538 borax Inorganic materials 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- 235000019800 disodium phosphate Nutrition 0.000 claims description 2
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims description 2
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 235000007686 potassium Nutrition 0.000 claims description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 2
- 235000015320 potassium carbonate Nutrition 0.000 claims description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 229940093916 potassium phosphate Drugs 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 239000004328 sodium tetraborate Substances 0.000 claims description 2
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 2
- WUUHFRRPHJEEKV-UHFFFAOYSA-N tripotassium borate Chemical compound [K+].[K+].[K+].[O-]B([O-])[O-] WUUHFRRPHJEEKV-UHFFFAOYSA-N 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 26
- 108090000790 Enzymes Proteins 0.000 abstract description 26
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 229920006037 cross link polymer Polymers 0.000 abstract description 3
- 210000004072 lung Anatomy 0.000 abstract description 3
- 239000003607 modifier Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000004220 aggregation Methods 0.000 abstract 1
- 230000002776 aggregation Effects 0.000 abstract 1
- 150000001336 alkenes Chemical class 0.000 abstract 1
- 238000007385 chemical modification Methods 0.000 abstract 1
- 239000011258 core-shell material Substances 0.000 abstract 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 abstract 1
- 239000002861 polymer material Substances 0.000 abstract 1
- 238000011957 budget and coverage analysis Methods 0.000 description 18
- 239000011942 biocatalyst Substances 0.000 description 14
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 235000011089 carbon dioxide Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 6
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 5
- 239000000654 additive Substances 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 3
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical group CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 3
- 239000004159 Potassium persulphate Substances 0.000 description 3
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium peroxydisulfate Substances [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 3
- VAZSKTXWXKYQJF-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)OOS([O-])=O VAZSKTXWXKYQJF-UHFFFAOYSA-N 0.000 description 3
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 3
- 235000019394 potassium persulphate Nutrition 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- OMIGHNLMNHATMP-UHFFFAOYSA-N 2-hydroxyethyl prop-2-enoate Chemical compound OCCOC(=O)C=C OMIGHNLMNHATMP-UHFFFAOYSA-N 0.000 description 2
- PKZCRWFNSBIBEW-UHFFFAOYSA-N 2-n,2-n,2-trimethylpropane-1,2-diamine Chemical compound CN(C)C(C)(C)CN PKZCRWFNSBIBEW-UHFFFAOYSA-N 0.000 description 2
- OFNISBHGPNMTMS-UHFFFAOYSA-N 3-methylideneoxolane-2,5-dione Chemical compound C=C1CC(=O)OC1=O OFNISBHGPNMTMS-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 2
- GTTSNKDQDACYLV-UHFFFAOYSA-N Trihydroxybutane Chemical compound CCCC(O)(O)O GTTSNKDQDACYLV-UHFFFAOYSA-N 0.000 description 2
- 150000003926 acrylamides Chemical class 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 125000005587 carbonate group Chemical group 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- NPSSWQJHYLDCNV-UHFFFAOYSA-N prop-2-enoic acid;hydrochloride Chemical compound Cl.OC(=O)C=C NPSSWQJHYLDCNV-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 2
- 230000004304 visual acuity Effects 0.000 description 2
- ZMARGGQEAJXRFP-UHFFFAOYSA-N 1-hydroxypropan-2-yl 2-methylprop-2-enoate Chemical compound OCC(C)OC(=O)C(C)=C ZMARGGQEAJXRFP-UHFFFAOYSA-N 0.000 description 1
- GWZMWHWAWHPNHN-UHFFFAOYSA-N 2-hydroxypropyl prop-2-enoate Chemical compound CC(O)COC(=O)C=C GWZMWHWAWHPNHN-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000010750 Metalloproteins Human genes 0.000 description 1
- 108010063312 Metalloproteins Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003546 flue gas Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- VLCAYQIMSMPEBW-UHFFFAOYSA-N methyl 3-hydroxy-2-methylidenebutanoate Chemical compound COC(=O)C(=C)C(C)O VLCAYQIMSMPEBW-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000007348 radical reaction Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
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- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The nanometer carbonic anhydrse grain for biological catalysis of polymer and its preparation process belongs to the field of chemical modification of enzyme. The nanometer carbonic anhydrse grain with bioactivity is a core-shell structure with core of carbonic anhydrse, shell of crosslinked polymer material and chemical bond connection between the core and the shell, and possesses high catalytic activity, high heat stability and no aggregation. This kind of nanometer carbonic anhydrse grain has wide application foreground in CO2 absorption and separation and artificial lung system, etc. Its preparation process includes introducing C-C double bond radical with enzyme modifier to the surface of carbonic anhydrse and subsequent free radical polymerization with the olefin monomer with C-C double bond as material.
Description
Technical field
The present invention relates to a kind of core is nano-polymer biocatalyst particle of carbonic anhydrase and preparation method thereof, belongs to the chemically modified field of enzyme.
Background technology
Carbonic anhydrase (carbonic anhydrase) is extensively to have biological intravital a kind of zinciferous metalloprotein, and it is catalysis carbonic acid gas aquation and anhydrating process efficiently, is one of most important enzyme in the interior carbonic acid gas circulation of organism.Carbonic anhydrase mainly is that carbonic acid gas is converted into bicarbonate radical or the carbonate form is used for photosynthesis through hydration in plant or algae, mainly is that the carbonic acid gas that will exist with bicarbonate radical or carbonate form turns into being discharged in the air through anhydrating in lung in animal body.As a kind of biological catalyst efficiently, carbonic anhydrase can improve 2000 times of carbon dioxide absorption or desorption rates with, can realize that also extremely low concentration absorbs efficiently as 0.04% carbonic acid gas, has therefore obtained people's extensive concern.Carbonic anhydrase is at aerospace submarine enclosed space CO at present
2Gas absorption, flue gas percent of greenhouse gases CO
2Reduction of discharging, Sweet natural gas CO
2CO such as separation, artificial lung system
2Obtained in the treatment system using widely, for CO
2Important effect is played in the reduction of the dwindling of treatment system volume, running cost, the raising that improves operational efficiency.But the stability of the mammiferous carbonic anhydrase of natural extract is difficult to satisfy the demand of practical application at present, with the carbonic anhydrase that comes from the ox tissue extraction is example, when denaturation temperature is more than 63 ℃, carbonic anhydrase can lose activity immediately, assemble to form and do not have bioactive precipitation, at normal temperatures simultaneously, because also can be between the carbonic anhydrase owing to hydrophobic interaction and electrostatic interaction, assemble mutually and lose activity, under 37 ℃, muddy phenomenon, deactivated 30～50% will appear significantly assembling in 4～8 hours, under 25 ℃, in 3～7 days, also can assemble gradually and deactivated 40～60%.Therefore for the further application level of developing carbonic anhydrase, take the engineering means improve carbonic anhydrase stability, suppress carbonic anhydrase clustering phenomena significant.
In the transformation of carbonic anhydrase, methods such as additive, bio-orientation evolution are compared has clear superiority.The additive add-on height that additive method needs can significantly improve the viscosity of system, reduces CO greatly
2Uptake rate, present most of additive is merely able to adsorb the carbonic anhydrase of inactivation simultaneously, suppresses the carbonic anhydrase clustering phenomena, can not solve the inactivation of carbonic anhydrase and the problem of thermostability, uses less; And the method for rite-directed mutagenesis since stability improve effect limited, can't solve a large amount of cheap problems of producing of engineering carbonic anhydrase, do not paid attention to widely yet.Therefore develop and a kind ofly have high stability, high biology catalytic activity, easy to implement, nanometer carbonic anhydrase grain for biological catalysis of polymer that particle diameter is controlled and preparation method thereof such chemically modified remodeling method and have vast market and great value.
Summary of the invention
One of purpose of the present invention provides the nano-polymer biocatalyst particle that a kind of core is a carbonic anhydrase, and is to solve thermally-stabilised poor, the easy accumulative problem of existing carbonic anhydrase, significant for the application level of further raising carbonic anhydrase.
Another object of the present invention provides a kind of preparation method of nano-polymer biocatalyst particle of carbonic anhydrase, improves the problem not obvious and resistance to mass transfer that the introducing of immobilization carbonic anhydrase is higher to solve existing carbonic anhydrase chemically modified stabilizing effect.This preparation method can realize that the crosslinked polymer of enzyme and outer core is covalently bound, thermostability improve obviously and the anti-character of assembling remarkable, particle scale is below 100 nanometers, does not introduce remarkable mass transport affects.
The solution of the present invention is as follows:
The invention provides a kind of nanometer carbonic anhydrase grain for biological catalysis of polymer, this particle has the carbonic anhydrase biology catalytic activity, is nucleocapsid structure, and core is a carbonic anhydrase, and shell is crosslinked macromolecular material, has chemical bond to connect between the nucleocapsid; This particle is a raw material with following material, obtains by the radical polymerization preparation;
Carbonic anhydrase: be 10 parts by weight,
The enzyme modification agent: be 2～40 parts by weight,
Alkenyl monomer: be 20～200 parts by weight,
Initiator: be 0.5～20 part by weight.
The present invention also provides a kind of radical polymerization preparation method of nanometer carbonic anhydrase grain for biological catalysis of polymer, and this method is a raw material with carbonic anhydrase, enzyme modification agent, alkenyl monomer and initiator, and wherein the content of each composition is:
Carbonic anhydrase: be 10 parts by weight,
The enzyme modification agent: be 2～40 parts by weight,
Alkenyl monomer: be 5～100 parts by weight,
Initiator: be 0.5～20 part by weight;
Concrete processing step is as follows:
1) carbonic anhydrase of said ratio and enzyme modification agent being added pH in the buffered soln between 7～10, reacted 0.5～4 hour under 4～10 ℃ of conditions;
2) mixture that step 1) is obtained joins in the aqueous solutions of organic solvent, 10～50% of the alkenyl monomer amount of adding said ratio;
3) initiator of adding said ratio reacted 0.5～2 hour down at 10～25 ℃;
4) add remaining alkenyl monomer, continue reaction 1～6 hour down at 10～25 ℃;
5) product is through after dialysing, and it is carbonic anhydrase that freeze-drying obtains core, and shell is crosslinked macromolecular material, the nanometer carbonic anhydrase grain for biological catalysis of polymer that has chemical bond to connect between the nucleocapsid.
In the present invention, described alkenyl monomer is the mixture of at least a mono alkenyl monomer and at least a polyene-based monomer class material.Described mono alkenyl monomer can be selected from one or more the mixture in acrylamide, hydroxyethyl methylacrylate, Hydroxyethyl acrylate, Propylene glycol monoacrylate, the Rocryl 410.Described polyene-based monomer can be selected from N, the mixture of one or more in N '-methylene diacrylamide, the trihydroxy methyl propane trimethyl acrylic ester.
In the present invention, described enzyme modification agent is to contain a carbon carbon unsaturated double-bond in the molecular structure at least and can react with the amino acid of carbonic anhydrase to form the material of chemical bonding.Described enzyme modification agent can be selected from one or more mixture of vinylformic acid succinimide ester, acrylate chloride, itaconic anhydride.
In the present invention, described initiator is under 10～25 ℃ of conditions, has 20～35kcal/mol dissociation energy and can produce free radical to cause alkenyl monomer polymeric persulfuric acid salt material or peroxide material.Described initiator can be selected from one or more and ferrous salt, sulphite, the N of Potassium Persulphate, Ammonium Persulfate 98.5, hydrogen peroxide, N, N ', the composite initiation system of N '-Tetramethyl Ethylene Diamine, piperidines, one or more compositions of N-methylmorpholine.
In the present invention, described buffered soln is one or more the aqueous solution in sodium phosphate, sodium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, potassium hydrogen phosphate, potassium primary phosphate, boric acid, Sodium Tetraborate, potassium borate, yellow soda ash, sodium bicarbonate, salt of wormwood, the saleratus.
In the present invention, described aqueous solutions of organic solvent is the aqueous solution that contains one or more organic solvents in dimethyl sulfoxide (DMSO), methyl alcohol, dioxane, acetonitrile, ethanol, the acetone, and wherein, the weight percent of organic solvent is in 0.5～4% scope.
The radical polymerization preparation method's of nanometer carbonic anhydrase grain for biological catalysis of polymer of the present invention core is how preparation process keeps the activity of carbonic anhydrase, and higher preparation yield can be provided again simultaneously.Therefore the present invention has used temperature control on the carbonic anhydrase surface by enzyme modification agent introducing carbon-carbon double bond group stage and radical polymerization stage, guarantees that preparation temperature occurs in 4～10 ℃, can avoid the inactivation problem of carbonic anhydrase in preparation process as far as possible; In order to guarantee that radical polymerization can obtain higher yield under lower preparation temperature, used composite initiation system simultaneously; In order to guarantee that radical polymerization mainly occurs in the enzyme surface, the aqueous solutions of organic solvent that has used low levels guarantees that as the radical polymerization environment radical polymerization mainly occurs in the enzyme surface.All things considered, this preparation method is owing to mild condition, and the inactivation of the carbonic anhydrase that preparation process causes is less.And this preparation method can prepare the preparation nanometer carbonic anhydrase grain for biological catalysis of polymer of the following yardstick of 100 nanometers, avoided high resistance to mass transfer in the enzyme immobilization process in the past, had that particle diameter is little, specific surface area is high, convenient various practical applications of no mass transfer diffusional resistance characteristics.Simultaneously this preparation method easy, be easy to industrial implementation and amplification.
Adopt the nanometer carbonic anhydrase grain for biological catalysis of polymer of method for preparing to have higher biocatalysis carbonic acid gas aquation and anhydrating activity, simultaneously because enzyme and outer core polymer are covalently bound, so the polymer of the shell problem that can not occur coming off; Simultaneously outer shell has strengthened the rigidity of enzymatic structure, thus the inactivation problem that has effectively stoped the structure thermal vibration of the carbonic anhydrase under the high temperature to cause; Crosslinked polymer shell has been strengthened the structure of carbonic anhydrase greatly, therefore the thermostability of carbonic anhydrase obtains bigger enhancing, and hydrophilic shell can effectively suppress direct hydrophobic interaction and electrostatic interaction between the natural carbon acid anhydrides enzyme, thereby the clustering phenomena that can suppress carbonic anhydrase under all temps condition fully has the advantages that activity is high, thermostability is strong.The carbonic anhydrase of this nano-polymer biocatalyst particle form is with a wide range of applications in the carbon dioxide treatment system as a kind of high performance nano enzyme preparation.
Description of drawings
Fig. 1 is the BCA nano-polymer biocatalyst particle aspect graph that utilizes high resolving power transmission electron microscope observing the present invention preparation.
75 ℃ of heat inactivation curve comparison diagrams of Fig. 2 BCA nano-polymer biocatalyst particle of the present invention and natural BCA.
The BCA nano-polymer biocatalyst particle of Fig. 3 the present invention preparation and natural BCA solution solution turbidity (characterizing with visible light 500nm absorbancy) under 75 ℃ of conditions changes comparison diagram.
Embodiment
Below in conjunction with embodiment nanometer carbonic anhydrase grain for biological catalysis of polymer of the present invention and preparation method thereof is given further instruction, but do not limit the present invention.Carbonic anhydrase among the embodiment derives from commercial enzyme, animal extracts or microorganism extracts.
Embodiment 1: raw material is that BCA is 10 parts by weight, the enzyme modification agent is that the vinylformic acid succinimide ester is 2 parts by weight, alkenyl monomer is the methylene diacrylamide of 18 parts of acrylamides and 4.5 parts, add up to 22.5 parts, initiator is the N of 2 parts of Ammonium Persulfate 98.5s and 3 parts, N, N ', N '-Tetramethyl Ethylene Diamine adds up to 5 parts.It is that reaction is 2 hours under 4 ℃ of conditions in 8.5 the 20mM borate buffer solution that above-mentioned BCA and enzyme modification agent are added pH, and modifier and borate are removed in the water dialysis.The mixture that obtains joined in 1% the dimethyl sulphoxide aqueous solution, add 50% of alkenyl monomer amount, temperature remains 10 ℃, and magnetic agitation adds initiator, reacted 0.5 hour, add remaining alkenyl monomer, temperature remains 10 ℃, continues reaction 2 hours, utilize deionized water dialysis 12 hours then, freeze-drying can obtain the BCA nano-polymer biocatalyst particle.This particle has the carbonic anhydrase biology catalytic activity, is nucleocapsid structure, and core is a carbonic anhydrase, and shell is crosslinked macromolecular material, has chemical bond to connect between the nucleocapsid.Those skilled in the art will appreciate that described carbonic anhydrase biology catalytic activity is meant the ability of catalysis carbonic acid gas aquation and anhydrating process, reaction formula is
Measure the CO of nano-polymer biocatalyst particle based on the Wilbur-Anderson method
2Aquation catalytic activity total recovery is 73%.The polyreaction yield is 91%.
Utilize the form of high resolving power transmission electron microscope observing BCA nano-polymer biocatalyst particle, the result as shown in Figure 1, median size is 11nm, size distribution is comparatively even.Figure 2 shows that 75 ℃ of following natural BCA transformation period are 3 minutes, and the BCA nano-polymer biocatalyst particle transformation period 90 minutes have been reached.Fig. 3 utilizes nephelometry to characterize the gathering situation of 75 ℃ of following BCAs, natural BCA solution is muddy rapidly, and any absorbancy variation did not appear in BCA nano-polymer biocatalyst particle solution yet until 30 minutes, assembled and was suppressed fully.
Embodiment 2: raw material is that BCA is 10 parts by weight, the enzyme modification agent is that acrylate chloride is 5 parts by weight, alkenyl monomer is the methylene diacrylamide of 60 parts of acrylamides and 15 parts, add up to 75 parts, initiator is the N of 5 parts of Ammonium Persulfate 98.5s and 10 parts, N, N ', N '-Tetramethyl Ethylene Diamine adds up to 15 parts.It is that reaction is 2 hours under 4 ℃ of conditions in 7.5 the 200mM buffer solution of sodium phosphate that above-mentioned BCA and enzyme modification agent are added pH, and modifier and phosphoric acid salt are removed in the water dialysis.The mixture that obtains joined in 1% the acetonitrile solution, add 20% of alkenyl monomer amount, temperature remains 10 ℃, and magnetic agitation adds initiator, reacted 0.5 hour, add remaining alkenyl monomer, temperature remains 10 ℃, continues reaction 3 hours, utilize deionized water dialysis 24 hours then, freeze-drying obtains the BCA nano-polymer biocatalyst particle.Connecting the biology catalytic activity total recovery that aniline measures nano-polymer biocatalyst particle as substrate with tetramethyl-is 62%, and the polyreaction yield is 90%, and the product particle diameter is 30～35nm.
Embodiment 3: change the enzyme modification agent among the embodiment 2 into 10 parts of itaconic anhydrides, alkenyl monomer is 80 parts acrylamide and 20 parts trihydroxy methyl propane trimethyl acrylic ester, free radical reaction solution is 4% dioxane, temperature of reaction is 25 ℃, initiator is 10 parts Potassium Persulphate and 5 parts sodium bisulfite, all the other prescriptions are identical with embodiment 2 with step, stability, activity yield and the polyreaction yield that obtain product this moment are suitable with embodiment 2, and the product particle diameter is 45～55nm.
Embodiment 4: change the alkenyl monomer among the embodiment 1 into 40 parts of Hydroxyethyl acrylates and 10 parts of methylene diacrylamides, the Raolical polymerizable temperature is 20 ℃, for the first time adding the alkenyl monomer amount is 30%, initiator is 10 parts Potassium Persulphate and 5 parts sodium bisulfite, all the other prescriptions are identical with embodiment 1 with step, this moment, to obtain its lytic activity yield be 60%, and the stability of product, polyreaction yield are suitable with embodiment 1, and the product particle diameter is 20～25nm.
Embodiment 5: change the BCA among the embodiment 1 the people carbonic anhydrase of expression from the artificial recombination of intestinal bacteria into, all the other prescriptions are identical with embodiment 1 with step, activity yield, stability, the polyreaction yield that obtain product this moment are suitable with embodiment 1, and the product particle diameter is 14～18nm.
Comparative example 1: the preparation method is with embodiment 1, but radical polymerization is combined under 35 ℃ and carries out, the activity yield that obtains product this moment only 35%.
Comparative example 2: the preparation method is with embodiment 2, but with monomer acrylamide and the disposable adding of methylene diacrylamide, it is irregular to obtain the product particle diameter, and product polymerization yield is lower.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, therefore, suitable with claims of the present invention contain with scope in any change, all will be understood that it is to be included in the scope of claims.
Claims (10)
1. nanometer carbonic anhydrase grain for biological catalysis of polymer, it is characterized in that: this particle has the carbonic anhydrase biology catalytic activity, is nucleocapsid structure, and core is a carbonic anhydrase, and shell is crosslinked macromolecular material, has chemical bond to connect between the nucleocapsid; And prepare by following method:
Carbonic anhydrase: be 10 parts by weight,
The enzyme modification agent: be 2～40 parts by weight,
Alkenyl monomer: be 20～200 parts by weight,
Initiator: be 0.5～20 part by weight;
It is in the buffered soln between 7～10 that the carbonic anhydrase of said ratio and enzyme modification agent are added pH, reacts 0.5～4 hour under 4～10 ℃ of conditions; The mixture that obtains joins in the aqueous solutions of organic solvent, add again said ratio the alkenyl monomer amount 10～50%; The initiator that adds said ratio then reacted 0.5～2 hour down at 10～25 ℃, added remaining alkenyl monomer afterwards, continued down to react 1～6 hour at 10～25 ℃; Product is through dialysis, freeze-drying.
2. nanometer carbonic anhydrase grain for biological catalysis of polymer according to claim 1 is characterized in that: described alkenyl monomer is the mixture of at least a mono alkenyl monomer and at least a polyene-based monomer class material.
3. nanometer carbonic anhydrase grain for biological catalysis of polymer according to claim 1 is characterized in that: described enzyme modification agent is to contain a carbon carbon unsaturated double-bond in the molecular structure at least and can react with the amino acid of carbonic anhydrase to form the material of chemical bonding.
4. nanometer carbonic anhydrase grain for biological catalysis of polymer according to claim 1, it is characterized in that: described initiator is under 10～25 ℃ of conditions, has 20～35kcal/mol dissociation energy and can produce free radical to cause alkenyl monomer polymeric persulfuric acid salt material or peroxide material.
5. the radical polymerization preparation method of the described nanometer carbonic anhydrase grain for biological catalysis of polymer of claim 1, it is characterized in that: this method is a raw material with carbonic anhydrase, enzyme modification agent, alkenyl monomer and initiator, and wherein the content of each composition is:
Carbonic anhydrase: be 10 parts by weight,
The enzyme modification agent: be 2～40 parts by weight,
Alkenyl monomer: be 20～200 parts by weight,
Initiator: be 0.5～20 part by weight;
Concrete processing step is as follows:
1) carbonic anhydrase of said ratio and enzyme modification agent being added pH in the buffered soln between 7～10, reacted 0.5～4 hour under 4～10 ℃ of conditions;
2) mixture that step 1) is obtained joins in the aqueous solutions of organic solvent, 10～50% of the alkenyl monomer amount of adding said ratio;
3) initiator of adding said ratio reacted 0.5～2 hour down at 10～25 ℃;
4) add remaining alkenyl monomer, continue reaction 1～6 hour down at 10～25 ℃;
5) product is through after dialysing, and it is carbonic anhydrase that freeze-drying obtains core, and shell is crosslinked macromolecular material, the nanometer carbonic anhydrase grain for biological catalysis of polymer that has chemical bond to connect between the nucleocapsid.
6. preparation method according to claim 5 is characterized in that: described alkenyl monomer is the mixture of at least a mono alkenyl monomer and at least a polyene-based monomer class material.
7. preparation method according to claim 5 is characterized in that: described enzyme modification agent is to contain a carbon carbon unsaturated double-bond in the molecular structure at least and can react with the amino acid of carbonic anhydrase to form the material of chemical bonding.
8. preparation method according to claim 5, it is characterized in that: described initiator is under 10～25 ℃ of conditions, has 20～35kcal/mol dissociation energy and can produce free radical to cause alkenyl monomer polymeric persulfuric acid salt material or peroxide material.
9. preparation method according to claim 5 is characterized in that: described buffered soln is one or more the aqueous solution of mixture in sodium phosphate, sodium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, potassium hydrogen phosphate, potassium primary phosphate, boric acid, Sodium Tetraborate, potassium borate, yellow soda ash, sodium bicarbonate, salt of wormwood, the saleratus.
10. preparation method according to claim 5, it is characterized in that: described aqueous solutions of organic solvent is the aqueous solution that contains one or more organic solvents in dimethyl sulfoxide (DMSO), methyl alcohol, dioxane, acetonitrile, ethanol, the acetone, wherein, the weight percent of organic solvent is in 0.5～4% scope.
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| EP2212427A1 (en) | 2007-10-18 | 2010-08-04 | Novozymes A/S | Processes of producing fermentation products |
| CA2738358A1 (en) * | 2008-09-29 | 2010-04-01 | Richard T. Zvosec | Process for accelerated capture of carbon dioxide |
| CN101838640B (en) * | 2010-04-13 | 2012-05-02 | 浙江大学 | Unimolecule embedding method for enzyme |
| CN102229923B (en) * | 2011-04-27 | 2013-03-27 | 中国石油天然气股份有限公司 | Lipase nano-polymer biocatalytic particle and preparation method thereof |
| CN104437050B (en) * | 2014-11-14 | 2016-03-23 | 上海立足生物科技有限公司 | Thermostability carbonic anhydrase is at catalysis By Amine Solutions cyclic absorption-desorb CO 2in application |
| CN104437002B (en) * | 2014-11-14 | 2016-03-23 | 上海立足生物科技有限公司 | A kind of thermostability carbonic anhydrase is at catalysis By Amine Solutions desorb CO 2in application |
| CN107312767B (en) * | 2017-07-17 | 2020-11-06 | 安徽工程大学 | Combined immobilized beta-glucosidase particle and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5306501A (en) * | 1990-05-01 | 1994-04-26 | Mediventures, Inc. | Drug delivery by injection with thermoreversible gels containing polyoxyalkylene copolymers |
| CN1128764A (en) * | 1995-05-24 | 1996-08-14 | 复旦大学 | Regenerated silk protein and complex material thereof for fixation of enzyme and bio-active substance |
| CN1180102A (en) * | 1997-10-10 | 1998-04-29 | 中国科学院感光化学研究所 | Oxidase function compound sensitive film containing hydrophobic nanometre prill and its making method and use |
| CN1563374A (en) * | 2004-04-08 | 2005-01-12 | 合肥工业大学 | Carrier material in use for preparing dextran through method of enzyme immobilization |
-
2006
- 2006-07-14 CN CNB2006100897224A patent/CN100417724C/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5306501A (en) * | 1990-05-01 | 1994-04-26 | Mediventures, Inc. | Drug delivery by injection with thermoreversible gels containing polyoxyalkylene copolymers |
| CN1128764A (en) * | 1995-05-24 | 1996-08-14 | 复旦大学 | Regenerated silk protein and complex material thereof for fixation of enzyme and bio-active substance |
| CN1180102A (en) * | 1997-10-10 | 1998-04-29 | 中国科学院感光化学研究所 | Oxidase function compound sensitive film containing hydrophobic nanometre prill and its making method and use |
| CN1563374A (en) * | 2004-04-08 | 2005-01-12 | 合肥工业大学 | Carrier material in use for preparing dextran through method of enzyme immobilization |
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