CN107164358A - Preparation method and application of dopamine and derivative thereof rapid cross-linking surfactant-enzyme nano composite catalyst - Google Patents
Preparation method and application of dopamine and derivative thereof rapid cross-linking surfactant-enzyme nano composite catalyst Download PDFInfo
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- CN107164358A CN107164358A CN201710504209.5A CN201710504209A CN107164358A CN 107164358 A CN107164358 A CN 107164358A CN 201710504209 A CN201710504209 A CN 201710504209A CN 107164358 A CN107164358 A CN 107164358A
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- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 239000003054 catalyst Substances 0.000 title claims abstract description 60
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 56
- 229960003638 dopamine Drugs 0.000 title claims abstract description 43
- 238000004132 cross linking Methods 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 108090000790 Enzymes Proteins 0.000 claims abstract description 41
- 239000012074 organic phase Substances 0.000 claims abstract description 9
- 239000012071 phase Substances 0.000 claims abstract description 9
- 229940088598 enzyme Drugs 0.000 claims description 83
- 229920001690 polydopamine Polymers 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 27
- 239000007864 aqueous solution Substances 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 238000005119 centrifugation Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 12
- 108090001060 Lipase Proteins 0.000 claims description 10
- 102000004882 Lipase Human genes 0.000 claims description 10
- 108010091086 Recombinases Proteins 0.000 claims description 10
- 102000018120 Recombinases Human genes 0.000 claims description 10
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 claims description 8
- 229960001149 dopamine hydrochloride Drugs 0.000 claims description 8
- 239000004094 surface-active agent Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 7
- 239000004365 Protease Substances 0.000 claims description 7
- 239000008346 aqueous phase Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000006555 catalytic reaction Methods 0.000 claims description 7
- 238000000975 co-precipitation Methods 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 239000004367 Lipase Substances 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 6
- 229940040461 lipase Drugs 0.000 claims description 6
- 235000019421 lipase Nutrition 0.000 claims description 6
- 229940055729 papain Drugs 0.000 claims description 6
- 235000019834 papain Nutrition 0.000 claims description 6
- 239000003431 cross linking reagent Substances 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000002525 ultrasonication Methods 0.000 claims description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 229910021645 metal ion Inorganic materials 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 241000321538 Candidia Species 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 235000009508 confectionery Nutrition 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 101710098556 Lipase A Proteins 0.000 claims description 2
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 claims description 2
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 claims description 2
- 108050006759 Pancreatic lipases Proteins 0.000 claims description 2
- 102000019280 Pancreatic lipases Human genes 0.000 claims description 2
- 108090000284 Pepsin A Proteins 0.000 claims description 2
- 102000057297 Pepsin A Human genes 0.000 claims description 2
- 229960001091 chenodeoxycholic acid Drugs 0.000 claims description 2
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000003760 magnetic stirring Methods 0.000 claims description 2
- 229940116369 pancreatic lipase Drugs 0.000 claims description 2
- 229940111202 pepsin Drugs 0.000 claims description 2
- 238000005057 refrigeration Methods 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- 229940045946 sodium taurodeoxycholate Drugs 0.000 claims description 2
- YXHRQQJFKOHLAP-FVCKGWAHSA-M sodium;2-[[(4r)-4-[(3r,5r,8r,9s,10s,12s,13r,14s,17r)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 YXHRQQJFKOHLAP-FVCKGWAHSA-M 0.000 claims description 2
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims 1
- 108010031797 Candida antarctica lipase B Proteins 0.000 claims 1
- 239000004380 Cholic acid Substances 0.000 claims 1
- 108091005804 Peptidases Proteins 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 229960002471 cholic acid Drugs 0.000 claims 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims 1
- 235000019416 cholic acid Nutrition 0.000 claims 1
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims 1
- 210000000496 pancreas Anatomy 0.000 claims 1
- 235000019419 proteases Nutrition 0.000 claims 1
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 2
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 238000004064 recycling Methods 0.000 description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 9
- 238000012986 modification Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 108010084311 Novozyme 435 Proteins 0.000 description 4
- 238000013019 agitation Methods 0.000 description 4
- 239000011572 manganese Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229930003427 Vitamin E Natural products 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 238000005352 clarification Methods 0.000 description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000012798 spherical particle Substances 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical class OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 3
- 229940046009 vitamin E Drugs 0.000 description 3
- 235000019165 vitamin E Nutrition 0.000 description 3
- 239000011709 vitamin E Substances 0.000 description 3
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- BXRRQHBNBXJZBQ-UHFFFAOYSA-L dichloromanganese;hydrate Chemical compound O.Cl[Mn]Cl BXRRQHBNBXJZBQ-UHFFFAOYSA-L 0.000 description 2
- 239000008236 heating water Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940014800 succinic anhydride Drugs 0.000 description 2
- UYVVLXVBEQAATF-UHFFFAOYSA-N 4-(1,3,7,12-tetrahydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl)pentanoic acid Chemical compound OC1CC2CC(O)CC(O)C2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 UYVVLXVBEQAATF-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- -1 DOPA Amine Chemical class 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- 101710098554 Lipase B Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960004502 levodopa Drugs 0.000 description 1
- 150000002641 lithium Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a preparation method and application of a dopamine and dopamine derivative rapid cross-linking surfactant-enzyme nano composite catalyst, and belongs to the field of application of immobilized enzymes. The invention adopts dopamine to self-polymerize and cross-link and attach to the surface of the surfactant-enzyme nano composite catalyst, thereby embedding the surfactant-enzyme nano composite catalyst, leading the obtained surfactant-enzyme nano composite catalyst to have certain mechanical strength, enhancing the structural stability, the repeated utilization rate and the tolerance, greatly reducing the use cost of the enzyme, and being especially suitable for the application in pure organic phase or water-oil two-phase biocatalysis.
Description
Technical field
The invention belongs to fixation techniques for enzyme field, and in particular to a kind of dopamine and its derivative Quick cross-linking surface
The preparation method and applications of activating agent-enzyme nano-composite catalyst.
Background technology
Enzyme immobilization can not only significantly improve the stability of enzyme, and can realize the recycling of enzyme, drop significantly
The use cost of low enzyme.During enzyme immobilization, often use glutaraldehyde as crosslinking agent, however, there are some researches show：It is fixed
Change enzyme activity to be inversely proportional with glutaraldehyde consumption, excessive glutaraldehyde is crosslinked between causing unnecessary enzyme molecule, and excessive friendship
Joint conference distorts the structure of enzyme, causes enzyme activity to reduce, in addition, the bio-toxicity of glutaraldehyde, limits its application.Therefore,
It is badly in need of finding the efficient green immobilization that a kind of simple, nontoxic, gentle method carries out enzyme.
Dopamine is a kind of biological neural mediator, auto polymerization reaction can occur in alkaline aerobic solution, so as to form one
Poly-dopamine layer of the layer strongly adherent in solid material surface.Because dopamine has good dispersion, biocompatibility and temperature
And reaction condition, it is used for field of enzyme immobilization as a kind of preferable bonding material.
The Chinese patent of application number 201110460474.0 discloses a kind of using glutaraldehyde cross-linking immobilization modification fat
Enzyme Novozyme435 method, the preparation method is with glutaraldehyde as cross linker, in 20-50 by lipase Novozyme 435
DEG C, lipase Novozy after cross-linking reaction 15-120min, the secondary modification obtained after chemical crosslinking is carried out under 40-240rpm
435 stability is improved, and gained yield is more uncrosslinked after the Novozyme 435 after secondary modification is reused 5 times
The lipase Novozyme 435 of modification improves 49.3%.
On to address the main crosslinking agent of existing cross-linked immobilized enzyme catalyst be glutaraldehyde solution, preparation-obtained two
Immobilised enzymes after secondary modification, although recycling rate of waterused is improved, but immobilised enzymes is in the presence of glutaraldehyde, the knot of enzyme
Structure is distorted, and causes enzyme activity to reduce, in addition, the bio-toxicity of glutaraldehyde, limits its application, therefore, is badly in need of seeking
Look for a kind of efficient green secondary modification of simple, nontoxic, gentle method being fixed enzyme.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of dopamine and its derivative Quick cross-linking table
The preparation method and applications of face activating agent-enzyme nano-composite catalyst, this method is crosslinking agent with dopamine or derivatives thereof,
Dissolved oxygen is provided in the presence of magnetic agitation to be aoxidized from poly- crosslinking, be it is a kind of it is simple, nontoxic, gently, it is efficient
The secondary modification immobilised enzymes of green.
To solve prior art problem, the technical scheme that the present invention takes is：
A kind of preparation method of dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst, exists first
Room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst, is then with dopamine or derivatives thereof
Crosslinking agent, stirring is lower to be carried out from poly- crosslinking, when surfactant-enzyme nano-composite catalyst outer wrapping black poly-dopamine,
Centrifugation freeze-drying, produces the surfactant-enzyme nano-composite catalyst of " nucleocapsid " shape after dopamine crosslinking embedding.
It is that described dopamine or derivatives thereof is Dopamine hydrochloride as improved.
A kind of preparation side of above-mentioned dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst
Method, comprises the following steps：
Step 1, room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst
1）0.01-1 mmol/mL aqueous surfactant solutions are prepared, at room temperature, are dripped while stirring with 100 ~ 150 rpm speed
Plus free enzyme aqueous solution obtains mixed liquor to clarifying；
2）Mixed liquor is added dropwise to 0.01-1 mmol metal ion salt solutions while stirring, at room temperature with 100 ~ 150 rpm speed
Spend after stirring reaction 30-60 min and obtain the nano-composite catalyst aqueous solution of surfactant-enzyme；
3）The nano-composite catalyst aqueous solution of surfactant-enzyme is centrifuged, and with deionized water rinsing 1-3 times, washed away not
The resolvase of absorption, vacuum freeze drying to constant weight is that can obtain surfactant-enzyme nano-composite catalyst；
Step 2, dopamine is from poly- crosslinking
20-200mg surfactants-enzyme nano-composite catalyst is taken to be dispersed in 10-100 mL moles under ultrasonication
Concentration is in 10-100 mmol Tris-HCl buffer solution, addition 10-40 mg dopamine or derivatives thereof, at room temperature magnetic force
Stir and centrifuged after 24h, and be washed with deionized 1-3 times, vacuum freeze drying to constant weight is produced after dopamine crosslinking embedding
The surfactant of " nucleocapsid " shape-enzyme nano-composite catalyst.
Preferably, the 1 of step 1）Described in surfactant be NaTDC, sodium taurodeoxycholate, sweet ammonia take off
Oxycholic acid receive or chenodesoxycholic acid sodium in it is any；Resolvase is antarctic candidia lipase A, antarctic candidia lipase
B, fold lipase from candida sp, porcine pancreatic lipase, dredge cotton like thermophilic silk embrace bacterium lipase, papain, pepsin or
One or more combinations in trypsase, the free enzyme aqueous solution middle reaches are 0.1 ~ 1 from the mass ratio of enzyme and water:50.
Preferably, the 1 of step 1）In using aqueous surfactant solution as object of reference, the addition of the free enzyme aqueous solution
Measure as 0.005 ~ 1 mL/mL.
Preferably, the 2 of step 1）Described in metal ion be Co2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+
Or Mg2+In it is any.
Preferably, the 3 of step 1）5 ~ 15 min of middle centrifugation, rotating speed is 4000 ~ 8000 rpm during centrifugation, and vacuum refrigeration is done
Dry vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Preferably, the pH of Tris-HCl buffer solutions is 8 in step 2, and magnetic stirring speed is 100 ~ 150 rpm, stirring
Time is 1-36h, and rotating speed is that 4000 ~ 8000 rpm centrifuge 5-15min during centrifugation, and the vacuum of vacuum freeze drying is 1.3 ~ 13
Pa, temperature is -85 DEG C ~ -10 DEG C.
Above-mentioned dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst is in pure organic phase or water
Application in oily two-phase living things catalysis.
Compared with prior art, the present invention is attached to surfactant-enzyme nano combined using dopamine from poly- crosslinking
The surface of catalyst, so that the embedding of surfactant-enzyme nano-composite catalyst be got up, so as to get surfactant-enzyme
Nano-composite catalyst has certain mechanical strength, strengthens its structural stability, recycling rate of waterused and tolerance, substantially reduces
The use cost of enzyme, is particluarly suitable for the application in pure organic phase biological catalysis.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of embodiment 1, wherein,（a）For CRL-MSNC,（b）For PDA@CRL-MSNC；
Fig. 2 is the transmission electron microscope picture of embodiment 1, wherein,（a）For CRL-MSNC,（b）For PDA@CRL-MSNC；
The organic solvent-resistant stable that Fig. 3 is CRL, CRL-MSNC and PDA@CRL-MSNC in embodiment 1；
Fig. 4 is the scanning electron microscope (SEM) photograph of embodiment 2, wherein,（a）For Papain-MSNC,（b）For PDA@Papain-MSNC；
Fig. 5 is CRL-MSNC and the recycling rates of waterused of PDA@CRL-MSNC in aqueous in embodiment 1；
Fig. 6 catalyzes and synthesizes the conversion ratio of VE-succinate for CRL, CRL-MSNC and PDA@CRL-MSNC in embodiment 1；
Fig. 7 is recycling rates of waterused of the CRL-MSNC and PDA@CRL-MSNC in DMSO in embodiment 1.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1
A kind of Dopamine hydrochloride Quick cross-linking is modified deoxycholic acid-lipase from candida sp-Co2+The system of nano-composite catalyst
Preparation Method, comprises the following steps：
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1）Surfactant-modified resolvase
The mL of 0.1 mmol/mL deoxycholic acids sodium water solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1 mL/s
Drop rate be added dropwise the 20 mg/mL lipase from candida sp aqueous solution（CRL）2 mL, stirring obtains mixed liquor to solution clarification；
2）Mixed liquor is added into the 0.2 mmol cobalt chloride hexahydrate aqueous solution with 1 mL/s drop rate while stirring, at room temperature
To obtain deoxycholic acid-lipase from candida sp-Co after the 120 rpm min of speed stirring reaction 302+Nano-composite catalyst water
Solution；
3）By deoxycholic acid-lipase from candida sp-Co2+8000 rpm of nano-composite catalyst aqueous solution centrifugation,
And with deionized water rinsing 2 times, unadsorbed resolvase is washed away, vacuum freeze drying to constant weight is that can obtain deoxycholic acid-vacation
Silk Yeast-lipase-Co2+Nano-composite catalyst（CRL-MSNC）, wherein, vacuum be 1.3 ~ 13 Pa, temperature be -85 DEG C ~ -
10℃。
Step 2, dopamine is from poly- cross-linking modified
100 mg CRL-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM（pH 8.0）
In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature；
By centrifugation of the CRL-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted DOPA
Amine, vacuum freeze drying to constant weight produces cross-linking modified rear " nucleocapsid " shape deoxycholic acid-lipase from candida sp-Co of dopamine2+
Nano-composite catalyst（PDA@CRL-MSNC）, wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Scanning figures of the CRL-MSNC and PDA@CRL-MSNC manufactured in the present embodiment under Electronic Speculum as shown in Figure 1-2, from figure
In as can be seen that CRL-MSNC be a kind of spherical particle, its average grain diameter is in 80 nm or so.And after poly-dopamine modified lithium
Similar shape is also presented in the PDA@CRL-MSNC of " nucleocapsid " shape of generation, and its particle size range shows to gather in 100 nm or so
Dopamine successfully sticks to its surface, and thickness is about 20 nm.
As shown in figure 5, the present embodiment CRL-MSNC and PDA@CRL-MSNC are in aqueous by 10 recyclings
Afterwards, CRL-MSNC relative enzyme activity is less than 80%, and PDA@CRL-MSNC relative enzyme activity remains to maintain more than 93%.Natural CRL
Optimal pH with PDA CRL-MSNC is 7.0, but is due to protective effect of the nano-carrier to CRL so that PDA CRL-
MSNC is more extensive to pH accommodation, its in aqueous optimal catalytic pH range be 6.0-8.0.
As shown in figure 3, after ethanol, methanol and dmso treatment, CRL-MSNC and PDA@CRL-MSNC phase
68%, more than 88% can be maintained at respectively to vigor, and under identical treatment conditions, free CRL can only keep less vigor
（<10%）.
Embodiment 2
A kind of Dopamine hydrochloride Quick cross-linking is modified deoxycholic acid-papain-Mn2+The preparation side of nano-composite catalyst
Method, comprises the following steps：
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1）Surfactant-modified resolvase
The mL of 0.1 mmol/mL deoxycholic acids sodium water solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1 mL/s
Drop rate be added dropwise 20 mg/mL Papain enzyme aqueous solutions（Papain）2 mL, stirring obtains mixed liquor to solution clarification；
2）Mixed liquor is added into the chloride hydrate manganese aqueous solution of 0.2 mmol tetra- with 1 mL/s drop rate while stirring, at room temperature
To obtain deoxycholic acid-papain-Mn after the 120 rpm min of speed stirring reaction 302+The nano-composite catalyst aqueous solution；
3）By deoxycholic acid-papain-Mn2+The nano-composite catalyst aqueous solution 8000 rpm centrifugation, is used in combination
Deionized water rinsing 2 times, washes away unadsorbed resolvase, and vacuum freeze drying to constant weight is that can obtain deoxycholic acid-pawpaw egg
White enzyme-Mn2+Nano-composite catalyst（Papain-MSNC）, wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Step 2, dopamine is from poly- cross-linking modified
100 mg Papain-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM（pH
8.0）In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature；
By centrifugation of the Papain-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted
Dopamine, vacuum freeze drying to constant weight produces cross-linking modified rear " nucleocapsid " shape deoxycholic acid-papain-Mn of dopamine2+
Nano-composite catalyst（PDA@Papain-MSNC）, wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Scanning electron microscope (SEM) photograph such as Fig. 2 institutes of the Papain-MSNC and PDA@Papain-MSNC manufactured in the present embodiment under Electronic Speculum
Show, it can be seen that Papain-MSNC is the spherical particle that average grain diameter is 50 nm, PDA@Papain-MSNC are portions
Scattered besom shape is decomposed, in the aqueous solution after 10 times reuse, Papain-MSNC relative enzyme activity is less than 83%, and PDA@
Papain-MSNC relative enzyme activity remains to maintain more than 94%, and natural Papain and PDA@Papain-MSNC optimal pH is equal
For 5.0, PDA@Papain-MSNC, optimal catalytic pH range is 3.5-8.0 in aqueous.
Embodiment 3
A kind of Dopamine hydrochloride Quick cross-linking is modified glycodesoxycholic acid-papain-Mn2+The system of nano-composite catalyst
Preparation Method, comprises the following steps：
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1）Surfactant-modified resolvase
The mL of the 0.1 mmol/mL Glycodeoxrycholic acids aqueous solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1
The mL of 20 mg/mL Papains enzyme aqueous solution 2 is added dropwise in mL/s drop rate, and stirring obtains mixed liquor to solution clarification；
2）Mixed liquor is added into the chloride hydrate manganese aqueous solution of 0.2 mmol tetra- with 1 mL/s drop rate while stirring, at room temperature
To obtain glycodesoxycholic acid-papain-Mn after the 120 rpm min of speed stirring reaction 302+Nano-composite catalyst water
Solution；
3）By glycodesoxycholic acid-papain-Mn2+8000 rpm of nano-composite catalyst aqueous solution centrifugation,
And with deionized water rinsing 2 times, unadsorbed resolvase is washed away, vacuum freeze drying to constant weight is that can obtain sweet ammonia deoxidation courage
Acid-papain-Mn2+Nano-composite catalyst（Papain- glycodesoxycholic acids-Mn2+）.
Step 2, dopamine is from poly- cross-linking modified
100 mg Papain-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM（pH
8.0）In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature；
By centrifugation of the Papain-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted
Dopamine, vacuum freeze drying to constant weight can obtain cross-linking modified rear " nucleocapsid " shape glycodesoxycholic acid-Papain of dopamine
Enzyme-Mn2+Nano-composite catalyst（PDA@Papain- glycodesoxycholic acids-Mn2+）.
Papain- glycodesoxycholic acids-the Mn that the present embodiment is prepared2+With PDA@Papain- glycodesoxycholic acids-
Mn2+Scanning figure under Electronic Speculum shows, Papain- glycodesoxycholic acids-Mn2+It is the spherical particle that average grain diameter is 80 nm,
PDA@Papain- glycodesoxycholic acids-Mn2+It is the besom shape that part is dismissed, in the aqueous solution after 10 times reuse,
Papain- glycodesoxycholic acids-Mn2+Relative enzyme activity be less than 76%, and PDA@Papain- glycodesoxycholic acids-Mn2+It is relative
Enzyme activity remains to maintain more than 90%, and natural Papain and PDA@Papain- glycodesoxycholic acids-Mn2+Optimal pH be 5.0,
PDA@Papain- glycodesoxycholic acids-Mn2+Optimal catalytic pH range is 4.0-8.0 in aqueous.
Embodiment 4
Vitamin E amber is catalyzed and synthesized in pure organic phase with gained CRL-MSNC and PDA the@CRL-MSNC of embodiment 1 and natural CRL
The application of amber acid esters, comprises the following steps：
The first step, takes 10 mL plastic tube, sequentially adds 0.2 mmol vitamin E, 1 mmol succinic anhydride, 400 μ L
Natural CRL or CRL-MSNC containing equal protein content and PDA@CRL-MSNC, 5 mL organic solvent；
Second step, by the mixture obtained in step 1, closed processes are stirred after 55 °C of heating water baths with 120 rpm speed
Reaction；
After 3rd step, 72 h of reaction, the content of substrate and product is determined using U.S.'s Agilent highly effective liquid phase chromatographic system.
VE succinic acid is catalyzed and synthesized to calculate by the measure substrate of highly effective liquid phase chromatographic system and the content of product
Ester conversion rate, when reaction temperature be 55 °C, stir speed (S.S.) be 800 rpm when, under identical reaction conditions, CRL-MSNC with
PDA@CRL-MSNC, which catalyze and synthesize VE-succinate conversion ratio, can reach 55%（Fig. 6）, natural CRL catalyzes and synthesizes
More than 4 times, demonstrate the PDA@CRL-MSNC of the invention prepared and possess same with CRL-MSNC superior urge in pure organic phase
Change efficiency.
Wherein highly effective liquid phase chromatographic system, chromatographic column is C-18 reversed-phase columns（250 mm×4.6 mm,5 μm）, mobile phase is
Methanol and acetic acid solution（Volume ratio is 50:0.3）, flow velocity is 1.0 mL/min, and post case temperature is controlled at 36 °C, and detector is two
Pole pipe array and detector, Detection wavelength are 285 nm.
Embodiment 5
With CRL-MSNC the and PDA@CRL-MSNC of the gained of embodiment 1, determine the CRL-MSNC containing equal protein content with
PDA@CRL-MSNC catalyze and synthesize the recycling rate of waterused of VE-succinate in DMSO, comprise the following steps：
The first step, takes 10 mL plastic tube, sequentially adds 0.2 mmol vitamin E, 1 mmol succinic anhydride, contains phase
The organic solvent of CRL-MSNC and PDA@CRL-MSNC, 5 mL Deng protein content；
Second step, by the mixture obtained in step 1, closed processes are stirred after 55 °C of heating water baths with 120rpm speed
Reaction；
After 3rd step, the h of stirring reaction 4, the mixture reacted in step 2 is centrifuged into 10 min under 10000 rpm, height is used
The content of substrate and product in effect liquid phase chromatogram system measurement supernatant；
4th step, will centrifuge obtained precipitation, is washed with deionized 2 times, for next group enzyme reaction, enters repeatedly in step 3
Row 10 times catalysis calculates its recycling rate of waterused.
As a result closed as shown in fig. 7, calculating catalysis by the measure substrate of highly effective liquid phase chromatographic system and the content of product
Into VE-succinate conversion ratio, when reaction temperature is 55 °C, and stir speed (S.S.) is 800 rpm, in identical reaction condition
Under, after 10 times reuse, PDA@CRL-MSNC enzyme activity is reduced only by 55%, shows the PDA@in this reaction system
CRL-MSNC possesses good recycling performance.And by comparison, CRL-MSNC react 30 min after just by
DMSO is completely dissolved, and causing CRL-MSNC, only its enzyme activity after primary first-order equation is just greatly reduced.Further demonstrate
PDA@CRL-MSNC prepared by the present invention possess more superior catalytic efficiency in pure organic phase.
The present invention can be seen that using dopamine from poly- crosslinking by embodiment 1-3, be attached to surfactant-enzyme and receive
The surface of rice composite catalyst, so that the embedding of surfactant-enzyme nano-composite catalyst be got up, so as to get surface-active
Agent-enzyme nano-composite catalyst has certain mechanical strength, strengthens its structural stability, recycling rate of waterused and tolerance, greatly
The use cost of big reduction enzyme, is particluarly suitable for the application in pure organic phase biological catalysis.Pass through 10 repetitions in aqueous
After utilization, the relative enzyme activity that PDA@CRL-MSNC relative enzyme activity can maintain more than 93%, PDA@Papain-MSNC can be maintained
More than 94%, PDA@Papain- glycodesoxycholic acids-Mn2+Relative enzyme activity can maintain more than 90%；Pass through embodiment 4-5
As can be seen that " nucleocapsid " shape surfactant-enzyme is nano combined that the present invention is prepared using dopamine after cross-linking modified is urged
Agent has more superior catalytic efficiency in water-oil phase living things catalysis, before especially being had a wide range of applications in pure organic phase
Scape.
Claims (9)
1. the preparation method of a kind of dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst, it is special
Levy and be, surfactant-enzyme nano-composite catalyst is prepared into room temperature aqueous phase Rapid coprecipitation first, then with dopamine
Or derivatives thereof be crosslinking agent, stirring is lower to be carried out from poly- crosslinking, when surfactant-enzyme nano-composite catalyst outer wrapping is black
During color poly-dopamine, centrifugation freeze-drying, the surfactant-enzyme nanometer for producing " nucleocapsid " shape after dopamine crosslinking embedding is multiple
Close catalyst.
2. a kind of dopamine according to claim 1 and its derivative Quick cross-linking surfactant-enzyme is nano combined urges
The preparation method of agent, it is characterised in that described dopamine or derivatives thereof is Dopamine hydrochloride.
3. a kind of dopamine according to claim 1 and its derivative Quick cross-linking surfactant-enzyme is nano combined urges
The preparation method of agent, it is characterised in that comprise the following steps：
Step 1, room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst
0.01-1 mmol/mL aqueous surfactant solutions are prepared, at room temperature, are added dropwise while stirring with 100 ~ 150 rpm speed
Free enzyme aqueous solution obtains mixed liquor to clarifying；
Mixed liquor is added dropwise to 0.01-1 mmol metal ion salt solutions while stirring, at room temperature with 100 ~ 150 rpm speed
The nano-composite catalyst aqueous solution of surfactant-enzyme is obtained after stirring reaction 30-60 min；
The nano-composite catalyst aqueous solution of surfactant-enzyme is centrifuged, and with deionized water rinsing 1-3 times, washes away and does not inhale
Attached resolvase, vacuum freeze drying to constant weight is that can obtain surfactant-enzyme nano-composite catalyst；
Step 2, dopamine is from poly- crosslinking
20-200mg surfactants-enzyme nano-composite catalyst is taken to be dispersed in 10-100 mL moles under ultrasonication
Concentration is in 10-100 mmol Tris-HCl buffer solution, addition 10-40 mg dopamine or derivatives thereof, at room temperature magnetic force
Stir and centrifuged after 24h, and be washed with deionized 1-3 times, vacuum freeze drying to constant weight is produced after dopamine crosslinking embedding
The surfactant of " nucleocapsid " shape-enzyme nano-composite catalyst.
4. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3
Method, it is characterised in that the 1 of step 1）Described in surfactant be NaTDC, sodium taurodeoxycholate, sweet ammonia deoxidation
Cholic acid receive or chenodesoxycholic acid sodium in it is any；Resolvase be antarctic candidia lipase A, candida antarctica lipase B,
Fold lipase from candida sp, porcine pancreatic lipase, the thermophilic silk of thin cotton like embrace bacterium lipase, papain, pepsin or pancreas
One or more combinations in protease, the free enzyme aqueous solution middle reaches are 0.1 ~ 1 from the mass ratio of enzyme and water:50.
5. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3
Method, it is characterised in that the 1 of step 1）In using aqueous surfactant solution as object of reference, the addition of the free enzyme aqueous solution
Measure as 0.005 ~ 1 mL/mL.
6. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3
Method, it is characterised in that the 2 of step 1）Described in metal ion be Co2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+Or
Mg2+In it is any.
7. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3
Method, it is characterised in that the 3 of step 1）5 ~ 15 min of middle centrifugation, rotating speed is 4000 ~ 8000 rpm during centrifugation, and vacuum refrigeration is done
Dry vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
8. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3
Method, it is characterised in that the pH of Tris-HCl buffer solutions is 8 in step 2, and magnetic stirring speed is 100 ~ 150 rpm, during stirring
Between be 1-36h, rotating speed is that 4000 ~ 8000 rpm centrifuge 5-15min during centrifugation, and the vacuum of vacuum freeze drying is 1.3 ~ 13
Pa, temperature is -85 DEG C ~ -10 DEG C.
9. based on dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst obtained by claim 1
Application in pure organic phase or water phase and an oil phase living things catalysis.
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