CN106222157A - A kind of polystyrene resin modified with polyamino acid immobilized enzyme as carrier and preparation method thereof - Google Patents
A kind of polystyrene resin modified with polyamino acid immobilized enzyme as carrier and preparation method thereof Download PDFInfo
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- CN106222157A CN106222157A CN201610658506.0A CN201610658506A CN106222157A CN 106222157 A CN106222157 A CN 106222157A CN 201610658506 A CN201610658506 A CN 201610658506A CN 106222157 A CN106222157 A CN 106222157A
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- 239000002253 acid Substances 0.000 title claims abstract description 33
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 28
- 229920005990 polystyrene resin Polymers 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 75
- 108090000790 Enzymes Proteins 0.000 claims abstract description 75
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 24
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 24
- 235000001014 amino acid Nutrition 0.000 claims abstract description 21
- 150000001413 amino acids Chemical group 0.000 claims abstract description 21
- 229920005989 resin Polymers 0.000 claims abstract description 21
- 239000011347 resin Substances 0.000 claims abstract description 21
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims abstract description 16
- 230000005494 condensation Effects 0.000 claims abstract description 8
- 238000009833 condensation Methods 0.000 claims abstract description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 5
- 239000012508 resin bead Substances 0.000 claims abstract description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 8
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 150000003053 piperidines Chemical class 0.000 claims description 6
- 125000006239 protecting group Chemical group 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 230000008961 swelling Effects 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 3
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 2
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000003746 solid phase reaction Methods 0.000 claims description 2
- 238000010671 solid-state reaction Methods 0.000 claims description 2
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims 1
- 238000007334 copolymerization reaction Methods 0.000 claims 1
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims 1
- UIDUKLCLJMXFEO-UHFFFAOYSA-N propylsilane Chemical compound CCC[SiH3] UIDUKLCLJMXFEO-UHFFFAOYSA-N 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 238000009792 diffusion process Methods 0.000 abstract description 6
- 230000001681 protective effect Effects 0.000 abstract description 2
- OZRFYUJEXYKQDV-UHFFFAOYSA-N 2-[[2-[[2-[(2-amino-3-carboxypropanoyl)amino]-3-carboxypropanoyl]amino]-3-carboxypropanoyl]amino]butanedioic acid Chemical compound OC(=O)CC(N)C(=O)NC(CC(O)=O)C(=O)NC(CC(O)=O)C(=O)NC(CC(O)=O)C(O)=O OZRFYUJEXYKQDV-UHFFFAOYSA-N 0.000 abstract 1
- OZNSCVPYWZRQPY-CIUDSAMLSA-N Arg-Asp-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O OZNSCVPYWZRQPY-CIUDSAMLSA-N 0.000 abstract 1
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 abstract 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 abstract 1
- 102000018120 Recombinases Human genes 0.000 description 19
- 108010091086 Recombinases Proteins 0.000 description 19
- 230000000694 effects Effects 0.000 description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000376 reactant Substances 0.000 description 5
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N Alumina Chemical compound [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229920006389 polyphenyl polymer Polymers 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- NIPYQLPZPLBOLF-UHFFFAOYSA-N 3'-hydroxy-6'-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound OC1C(O)C(O)C(CO)OC1OC1=CC=C2C3(C4=CC=CC=C4C(=O)O3)C3=CC=C(O)C=C3OC2=C1 NIPYQLPZPLBOLF-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 1
- ZLFYGRYDKNWCTD-UHFFFAOYSA-N [Pb].C(C(=C)C)(=O)OCCOC(C(=C)C)=O Chemical compound [Pb].C(C(=C)C)(=O)OCCOC(C(=C)C)=O ZLFYGRYDKNWCTD-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229920005613 synthetic organic polymer Polymers 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
Abstract
A kind of polystyrene resin modified with polyamino acid immobilized enzyme as carrier, including enzyme and the carrier of fixing described enzyme；Wherein, described enzyme is β glucosidase；Described carrier is the aminomethyl polystyrene resin beads that polyamino acid is modified, described polyamino acid, its aminoacid sequence is: Arg Asp Glu Glu Lys Lys Asp Asp Asp Asp, the carboxy terminal amino acid Asp in aminoacid sequence form peptide bond with the aminomethyl dehydrating condensation on resin and be connected.The polystyrene resin that the present invention uses above-mentioned polyamino acid to modify is carrier immobilized β glucosidase; owing to the charge environment of carrier surface is close with the optimum pH of β glucosidase (about about 4.5); the diffusion electric double layer on its surface has protective effect to enzyme molecular structure, significantly improves the operational stability of immobilized enzyme.
Description
Technical field
The invention belongs to technical field of enzyme engineering, be specifically related to a kind of fixed enzyme vector and preparation method thereof.
Background technology
Enzyme immobilization is by the constraint of enzyme molecule or to be limited in certain area so that it is can still provide for distinctive catalytic reaction,
And recyclable and a kind of enzyme engineering technology of recycling.Compared with resolvase, immobilized enzyme is maintaining the distinctive catalysis of enzyme
While activity, also present stability high, the series of advantages such as separation and recovery easily, repeatable operates with, simple process.
The performance of immobilized enzyme depends primarily on process for fixation and carrier material used.Absorption method, covalent coupling method,
Cross-linking method and investment etc. are all the traditional methods of relatively common enzyme immobilizatio.Wherein absorption method is because of side simple to operate
Just, adsorption capacity compared with the advantage such as big, process costs is low, be immobilized enzyme most common method.Conventional carrier be divided into have airborne
Body and inorganic carrier, organic carrier includes the natural polymer carriers such as agarose, glucosan, cellulose, and polyethylene, polyphenyl second
The synthetic organic polymer carrier such as alkene and polyacrylate；Inorganic carrier includes silicon oxide, titanium oxide, aluminium oxide ceramics, diatom
Soil etc..The structure of carrier and performance have significant impact for catalysis activity and the stability of immobilized enzyme.
Research shows, the surface charge property (zero point charge value, pzc) of the enzyme immobilization carrier of different substrates is different, leads
The pH value of the surface microenvironment causing it produces difference, and the activity of enzyme and the stability of enzyme molecule are also had by the change of the height of pH value
Considerable influence.Therefore, when selecting enzyme immobilization carrier, the size of enzyme load capacity to be considered and the fastness of connection, simultaneously
Also need to take into account the charge property of carrier surface, make carrier surface charge environment match with the optimum pH of enzyme.But owing to lacking at present
The related data of weary all kinds of carrier surface charge property, and the regulation and control method to carrier surface charge property cannot be realized so that
People's blindness when selection enzyme immobilization carrier is bigger.
Aminoacid is a class ampholyte, has 1 α-NH in its basic structure2With 1 α-COOH, and acid or alkaline
Free-COOH and-NH is contained the most respectively on amino acid whose side chain2, these active groups can electricity under specific acid or alkali environment
From one-tenth-NH3 +With-COO-, there is certain buffer capacity.The polyamino acid of different polymerization degree, different aminoacids combination has not
Same isoelectric point, IP and buffering range.Therefore, by rational sequential design, and the method combining solid phase synthesis, different are gathered
Amino acid fragment grafts on carrier surface, just can get the various enzyme immobilization carrier with different surfaces charge property.This kind of
There is certain thickness in functional vector surface, and has the diffusion electric double layer of buffer capacity, and its isoelectric point, IP is the poly-ammonia of grafting
The pI value of base acid fragment, when the pI value of diffusion electric double layer matches with the optimum pH of target enzyme, can be that enzyme molecule provides one
Individual stable, suitable catalysis microenvironment, reduces the impact that enzyme is lived by external condition (such as pH value, temperature etc.), improves immobilized enzyme
Stability.But method, technology about this respect are not reported at present.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of polystyrene resin modified with polyamino acid as carrier
Immobilized β-glucosidase, to improve stability and the service efficiency of beta-glucosidase.
The present invention also to solve the technical problem that the preparation method being to provide above-mentioned immobilized enzyme.
For solving above-mentioned technical problem, the thinking of the present invention is, beta-glucosidase is that a kind of widely used industry is raw
Thing catalyst, but less stable.The present invention with beta-glucosidase as target enzyme, by isoelectric point, IP be 4.09, the degree of polymerization be 10
Polyamino acid fragment graft on aminomethyl polystyrene resin, form a kind of stable weak acid environment at carrier surface；Logical
Cross physical absorption to be incorporated on modified carrier by beta-glucosidase (GLU), owing to carrier surface charge environment is with natural
The optimum pH of enzyme is close, and has stronger buffer capacity, makes the stability of native enzyme be largely increased.
The technical solution used in the present invention is as follows:
A kind of polystyrene resin modified with polyamino acid immobilized enzyme as carrier, including enzyme and fixing described enzyme
Carrier；
Wherein,
Described enzyme is beta-glucosidase；
Described carrier is the aminomethyl polystyrene resin beads that polyamino acid is modified, described polyamino acid, its ammonia
Base acid sequence is: Arg-Asp-Glu-Glu-Lys-Lys-Asp-Asp-Asp-Asp, and isoelectric point, IP is 4.09, and expression formula is
RDEEKKDDDD, the carboxy terminal amino acid Asp in aminoacid sequence form peptide bond with the aminomethyl dehydrating condensation on resin and are connected.
Wherein, described aminomethyl polystyrene resin be aminomethyl, styrene and Ethylene glycol dimethacrylate lead to
Cross the cross-linked polystyrene resin that copolymerisation is formed；Grain diameter is 100-150 μm, and loading is 0.6-0.8mmol/g, table
Reaching formula is DEG-AM, and this resin can the most commercially obtain.
Polyamino acid trim grafts on polystyrene resin beads surface by the method for Solid-phase synthesis peptides, is formed weak
Acid, and there is the diffused electric doublet layer structure of certain buffer capacity.The present invention uses the polyphenyl second that above-mentioned polyamino acid is modified
Olefine resin is carrier immobilized beta-glucosidase, due to charge environment and the optimum pH of beta-glucosidase of carrier surface
(about about 4.5) are close, and the diffusion electric double layer on its surface has protective effect to enzyme molecular structure, significantly improves immobilization
The operational stability of enzyme.
The preparation method of the above-mentioned polystyrene resin modified with the polyamino acid immobilized enzyme as carrier, it includes as follows
Step:
Step one, the preparation of carrier:
Use polypeptide solid-state reaction method, polyamino acid trim and aminomethyl polystyrene resin coupling are obtained carrier；
Step 2, the preparation of immobilized enzyme:
Carrier is added in beta-glucosidase enzymatic solution, low temperature oscillating reactions 1～3h, liquid is leached, drenches with buffer
Wash, after draining and get final product.
Step one comprises the steps:
(1) in aminomethyl polystyrene resin, add solvent, after swelling 4-8h, take out solvent；
(2) according in aminoacid sequence from c-terminus to N-terminal order, successively each is had the ammonia of protection group
Base acid is coupled on resin；The described aminoacid with protection group is Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-
OH, Fmoc-Glu (OtBu)-OH and Fmoc-Arg (Pbf)-OH；Wherein ,-Fmoc is alpha-amido protection group, parenthetic protection
Base is the protection group of the acid and alkaline group in R base；
(3) Side chain protective group on excision aminoacid.
In step (1), described solvent is DMF (DMF).
In step (2), in coupling process, the condensation reagent of use is the compositions of HoBt and DIC, described aminoacid,
The ratio of the amount of the material of HoBt, DIC and resin is 5～2:6～2:6～2:1；The method of described removing amino protecting group is to make
With the DMF solution of 20%v/v piperidines, react 8-15min.
In step (3), the method for the side chain protected on excision aminoacid is: cutting liquid adds the carrier with protection group
In, after cleavage reaction 2-3h, filter out filtrate, respectively with methanol and dichloromethane alternately washing 4 times, it is ensured that last is all over using
Methanol washs, and is finally washed to neutral with water, drains and both obtain required carrier；Described cutting liquid includes such as lower body
The component of long-pending percentage ratio: 95% trifluoroacetic acid, 2.5% tri isopropyl silane, 2.5%H2O。
In step 2, in beta-glucosidase enzymatic solution, the concentration of beta-glucosidase is 0.3～30mg/mL, and solvent is
The citrate-phosphate disodium hydrogen buffer of 50mM pH5.0；For the carrier of every g dry weight, the consumption of beta-glucosidase enzymatic solution
It is 5～15mL.
In step 2, the enzyme of beta-glucosidase scope alive is 0.112～48.99U/mg.
Enzyme is lived and is defined as follows: with paranitrophenol-β-D-Glucose glycosides (pNPG) as substrate, hydrolysis per minute generates 1 μ
Enzyme amount required for mol paranitrophenol.
In step 2, described low temperature is 3-10 DEG C.
In step 2, described buffer is the citrate-phosphate disodium hydrogen buffer of 50mM pH5.0.
The technology path of the present invention is as shown in Figure 1.
Beneficial effect: the present invention utilizes with aminomethyl polystyrene resin as carrier, by solid phase synthesis process grafting etc.
Electricity point is the polyamino acid fragment of 4.09, it is thus achieved that the functional vector of a kind of surface faintly acid (zero point charge value pzc is 4.3).
Charge environment in this functional vector diffusion into the surface electric double layer is close with the optimum pH of beta-glucosidase, and beneficially enzyme divides
Son keeps stable catalysis activity；Meanwhile, diffusion electric double layer also can alleviate the change of external environment to the work of beta-glucosidase enzyme
Impact.The fixed enzyme vector DEG-AM-DDDDKKEEDR prepared the in the process of the present invention more unmodified tree of adsorbance to GLU
Fat DEG-AM significantly improves；The optimum temperature of immobilized enzyme DEG-AM-DDDDKKEEDR-GLU and optimum pH and free GLU
Close, and stability is greatly improved.
Accompanying drawing explanation
The Technology Roadmap of Fig. 1 present invention.
Fig. 2 is the mass spectrum of peptide chain RDEEKKDDDD, and its molecular weight is 1264.22, and in figure, 632.63 corresponding peptide chains is double
Electric charge peak [M+2]2+, it was demonstrated that the sequence of RDEEKKDDDD is correct.
Fig. 3 is that the Zeta potential of DEG-AM carrier compares before and after surface modification.Showing in figure, the pzc value of DEG-AM is
5.8, it is 4.3 through the pzc of the modified carrier of polyamino acid, illustrates that being grafted isoelectric point, IP is the peptide chain of 4.09 electricity to carrier surface
Lotus environment has significant impact.
Fig. 4 is the suitableeest action pH of resolvase and DEG-AM-DDDDKKEEDR-GLU.Showing in figure, resolvase is with fixing
The optimum pH changing enzyme is pH about 5.0.
Fig. 5 is the optimum temperature of resolvase and DEG-AM-DDDDKKEEDR-GLU.Figure shows, immobilized enzyme
Optimum temperature relatively resolvase improves 10 DEG C.
Fig. 6 is the operational stability of resolvase and DEG-AM-DDDDKKEEDR-GLU.Figure shows, DEG-AM-
The half-life of DDDDKKEEDR-GLU is up to more than 24 days, and the enzyme of resolvase lived the half-life less than 6 days.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that reality
Execute the content described by example and be merely to illustrate the present invention, and should be also without limitation on basis described in detail in claims
Invention.
In embodiment, enzyme activity detection method is as follows:
Enzyme is lived and is defined: with paranitrophenol-β-D-Glucose glycosides (pNPG) as substrate, hydrolysis per minute generates 1 μm ol pair
Enzyme amount required for nitrophenol.
Assay method: take the pNPG solution of enzyme liquid that 0.1ml suitably dilutes or appropriate immobilized enzyme and 5mmol/l, pH5.0
After 0.9ml (preheating 5min respectively) mixing, in 50 DEG C of shaking bath oscillating reactions 10min.Add 2ml, 1mol/lNa immediately2CO3
Solution terminates reaction, measures absorbance under 410nm.With 0.1ml distilled water or empty carrier as blank.
DEG-AM Resin described in following example is purchased from Tianjin Nankai Compositech Inc..
GLU described in following example is purchased from sigma company, and enzyme is lived as >=6U/mg.
The preparation of embodiment 1:DEG-AM-DDDDKKEEDR carrier
(1) weigh DEG-AM Resin (loading 0.7mmol/g) 3g (2.1mmol) and be placed in the solid phase with defecator
In synthetic reaction pipe, take out after adding DMF (DMF) swelling 4h；Weighing 4.2mmolFmoc-Asp (OtBu)-
OH, 4.2mmol HoBt and 4.2mmol DIC 10ml DMF puts in reactor after dissolving；N2Filter after advertising reaction 2.5h
Reactant liquor, adds DMF washing resin 3 times.
(2) take a small amount of resin that step (1) obtains in centrifuge tube, be sequentially added into ethanol solution, 1,2,3-indantrione monohydrate wherein molten
Liquid, is then placed in the oil cauldron of 105 DEG C reaction 5 minutes, after reaction terminates, observes the solution colour in centrifuge tube.
(3) if chromogenic reaction is bright yellow, then add 20%v/v piperidines/dimethyl formamide solution, react 10min, weight
It is added with 20% piperidines to wash after 3 times, removes amino protecting group.Wash 3 times with DMF again.
(4) weigh Fmoc-Asp (OtBu)-OH, each 4.2mmol of HoBt, DIC, after dissolving with 10ml DMF, put into reaction tube
In, carry out advertising reaction.Make protected amino acid activate, carry out coupled reaction and form peptide bond.
(5) according to the amino acid sequence of Asp-Asp-Asp-Asp-Lys-Lys-Glu-Glu-Asp-Arg, aminoacid and contracting
The consumption closing reagent is identical with Fmoc-Asp (OtBu)-OH, repeats condensation and deprotection reaction, the peptide chain needed for synthesis.
(6) by cutting liquid 10ml (95%v/v trifluoroacetic acid (TIF), 2.5%v/vTIS (tri isopropyl silane), 2.5%
v/v H2O) add in protection obtained above peptide-resin, after cleavage reaction 2h, filter out filtrate, respectively with methanol (MeOH),
Dichloromethane (DCM) alternately washing 4 times, it is ensured that last washs all over use MeOH.Finally washed to neutral with water,
Drain and both obtain required modified resin.The joint efficiency recording amino acid chain and resin is 78%.
The preparation of embodiment 2:DEG-AM-DDDDKKEEDR carrier
(1) weigh DEG-AM Resin (loading 0.7mmol/g) 5g (3.5mmol) and be placed in the polypeptide with defecator
In solid phase synthesis pipe, take out after adding dimethylformamide (DMF) swelling 8h.Feed intake by 5 times of loadings: weigh
17.5mmol Fmoc-Asp (OtBu)-OH, 17.5mmol HoBt and 17.5mmol DIC 20ml DMF put into anti-after dissolving
Answer in device, N2Advertise, filter out reactant liquor after reaction 3h, add DMF washing resin 3 times.
(2) take a small amount of resin in centrifuge tube, be sequentially added into ethanol solution, ninhydrin solution wherein, then put
React 5 minutes in the oil cauldron of 105 DEG C, after reaction terminates, observe the solution colour in centrifuge tube.
(3) if chromogenic reaction is bright yellow, add 20%v/v piperidines/dimethyl formamide solution, react 10min, repeat
Add 20% piperidines to wash after 4 times, remove amino protecting group.Wash 3 times with DMF again.
(4) condensation aminoacid: weigh Fmoc-Asp (OtBu)-OH, each 17.5mmol of HoBt, DIC, dissolves with 20ml DMF
After put in reaction tube, carry out advertising reaction.Make protected amino acid activate, carry out coupled reaction and form peptide bond.
(5) according to the amino acid sequence of Asp-Asp-Asp-Asp-Lys-Lys-Glu-Glu-Asp-Arg, aminoacid and contracting
The consumption closing reagent is identical with Fmoc-Asp (OtBu)-OH, repeats condensation and deprotection reaction, the peptide chain needed for synthesis.
(6) by cutting liquid 15ml (95%v/v trifluoroacetic acid (TIF), 2.5%v/vTIS (tri isopropyl silane), 2.5%
v/vH2O) add in protection obtained above peptide-resin, after cleavage reaction 3h, filter out filtrate, the most do not use methanol
(MeOH), dichloromethane (DCM) alternately washing 4 times, it is ensured that last washs all over use MeOH.Finally washed with water
To neutral, drain and both obtain required modified resin.The joint efficiency recording amino acid chain and resin is 85%.
The preparation of embodiment 3:DEG-AM-DDDDKKEEDR-GLU
The fixed enzyme vector obtained is applied in the immobilization of beta-glucosidase.Weigh with in embodiment 2 relatively
The DEG-AM-DDDDKKEEDR 0.5g that method for optimizing prepares is placed in reaction tube, adds GLU enzyme liquid by solid-to-liquid ratio 1g:15mL
(enzyme liquid concentration is 1.5mg/ml), in 4 DEG C, 150rpm shaking table vibration 1h；Reaction filters reactant liquor after terminating, and uses 50mM pH5.0
Citrate-phosphate disodium hydrogen buffer drip washing repeatedly 4 times, sucking filtration drain after both DEG-AM-DDDDKKEEDR-GLU.GLU inhales
Attached amount is 12mg/g resin.
The preparation of embodiment 4:DEG-AM-DDDDKKEEDR-GLU
The fixed enzyme vector obtained is applied in the immobilization of beta-glucosidase.Weigh with in embodiment 2 relatively
The DEG-AM-DDDDKKEEDR 1.0g that method for optimizing prepares is placed in reaction tube, adds GLU enzyme liquid by solid-to-liquid ratio 1g:5mL
(enzyme liquid concentration is 10mg/ml), in 4 DEG C, 150rpm shaking table vibration 1h；Reaction filters reactant liquor, with 50mM pH's 5 after terminating
Citrate-phosphate disodium hydrogen buffers drip washing repeatedly 4 times, and sucking filtration had both obtained DEG-AM-DDDDKKEEDR-GLU after draining.GLU adsorbs
Amount is 14.8mg/g resin.
The optimum pH of embodiment 5:DEG-AM-DDDDKKEEDR-GLU
Immobilized enzyme is prepared according to embodiment 4 method.To same volume, different pH value (pH value is respectively 3.0,4.0,
5.0,6.0,7.0) citrate-phosphate disodium hydrogen buffer adds pNPG to final concentration of 5mmol/l, then is separately added into
Measure immobilized enzyme or resolvase that enzyme is lived, measure resolvase and the immobilized enzyme enzyme under condition of different pH and live, live the highest with enzyme
Value is 100%, calculates the enzyme activity under condition of different pH.Result is as shown in Figure 4:
The maximum enzyme of resolvase and DEG-AM-DDDDKKEEDR-GLU is lived and is both present in about pH5.0, but the change of pH value
Impact on DEG-AM-DDDDKKEEDR-GLU is relatively small, under conditions of pH3.0, DEG-AM-DDDDKKEEDR-GLU's
Enzyme is lived relatively 37.65%, and the relative enzyme of resolvase is lived all less than 6%.
Embodiment 6: the optimum temperature of resolvase and DEG-AM-DDDDKKEEDR-GLU compares
Immobilized enzyme is prepared according to embodiment 4 method.The citrate-phosphate disodium hydrogen of 5.0 it is to same volume, pH value
Buffer adds pNPG to final concentration of 5mmol/l, then is separately added into immobilized enzyme or the resolvase that equivalent amounts of enzyme is lived；By above-mentioned
Reactant liquor at 35 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, measures enzyme and lives, with enzyme peak alive be respectively under the temperature conditions of 75 DEG C
100%, calculate the enzyme activity under different temperatures.Result is as it is shown in figure 5, the optimal reactive temperature of resolvase is 55 DEG C；Gu
Surely the enzyme variation with temperature trend alive changing enzyme is similar to resolvase, but the highest enzyme of DEG-AM-DDDDKKEEDR-GLU lives in
Present about 65 DEG C, relatively resolvase improves 10 DEG C.
Embodiment 7: resolvase and the operational stability of DEG-AM-DDDDKKEEDR-GLU
Immobilized enzyme is prepared according to embodiment 4 method.Take the DEG-AM-DDDDKKEEDR-GLU or free that a certain amount of enzyme is lived
Enzyme is placed in 4 DEG C of freezer storages, periodically takes out mensuration enzyme and lives, and initial enzyme is lived and is designated as 100%, calculates immobilized enzyme and resolvase through not
With the remnant enzyme activity after period of storage and different access times.As shown in Figure 6, resolvase is after depositing 3 days, and residual enzyme activity is
63.74%, after depositing 15 days, remnant enzyme activity drops to 11.2%, and after 24 days, remnant enzyme activity is 3.05%；And through amino acid modified
After carrier immobilized enzyme DEG-AM-DDDDKKEEDR-GLU, after storing 15 days, remnant enzyme activity is 91.17%, and after 24 days, it is residual
Remaining enzyme is lived still up to the 76.78% of initial value, demonstrates good stability.
Claims (10)
1. the polystyrene resin immobilized enzyme as carrier modified with polyamino acid, it is characterised in that include enzyme and solid
The carrier of fixed described enzyme；
Wherein,
Described enzyme is beta-glucosidase；
Described carrier is the aminomethyl polystyrene resin beads that polyamino acid is modified, described polyamino acid, its aminoacid
Sequence is: Arg-Asp-Glu-Glu-Lys-Lys-Asp-Asp-Asp-Asp, the carboxy terminal amino acid Asp in aminoacid sequence
Form peptide bond with the aminomethyl dehydrating condensation on resin to be connected.
The polystyrene resin modified with the polyamino acid the most according to claim 1 immobilized enzyme as carrier, its feature
Be, described aminomethyl polystyrene resin be aminomethyl, styrene and Ethylene glycol dimethacrylate made by copolymerization
With the cross-linked polystyrene resin formed；Grain diameter is 100-150 μm, and loading is 0.6-0.8mmol/g.
3. the preparation method of the polystyrene resin immobilized enzyme as carrier modified with polyamino acid described in claim 1,
It is characterized in that, it comprises the steps:
Step one, the preparation of carrier:
Use polypeptide solid-state reaction method, polyamino acid trim and aminomethyl polystyrene resin coupling are obtained carrier；
Step 2, the preparation of immobilized enzyme:
Carrier is added in beta-glucosidase enzymatic solution, low temperature oscillating reactions 1～3h, liquid is leached, uses buffer drip washing, take out
After Gan and get final product.
Preparation method the most according to claim 3, it is characterised in that step one comprises the steps:
(1) in aminomethyl polystyrene resin, add solvent, after swelling 4-8h, take out solvent；
(2) according in aminoacid sequence from c-terminus to N-terminal order, successively each is had the aminoacid of protection group
It is coupled on resin；The described aminoacid with protection group is Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH,
Fmoc-Glu (OtBu)-OH and Fmoc-Arg (Pbf)-OH；
(3) Side chain protective group on excision aminoacid.
Preparation method the most according to claim 4, it is characterised in that in step (1), described solvent is N, N-dimethyl
Methanamide.
Preparation method the most according to claim 4, it is characterised in that in step (2), in coupling process, the condensation of use
Reagent is the compositions of HoBt and DIC, and the ratio of the amount of the material of described aminoacid, HoBt, DIC and resin is 5～2:6～2:
6～2:1；The method of described removing amino protecting group is to use the DMF solution of 20%v/v piperidines, reaction
8-15min。
Preparation method the most according to claim 4, it is characterised in that in step (3), the side chain protected on excision aminoacid
Method be: cutting liquid is added and has in the carrier of protection group, after cleavage reaction 2-3h, filter out filtrate, use methanol respectively
With dichloromethane alternately washing 4 times, it is ensured that last, all over using methanol to wash, is finally washed to neutral with water, take out
Do and both obtain required carrier；Described cutting liquid includes the component of following percent by volume: 95% trifluoroacetic acid, 2.5% 3 is different
Propyl silane, 2.5%H2O。
Preparation method the most according to claim 3, it is characterised in that in step 2, in beta-glucosidase enzymatic solution, β-Portugal
The concentration of polyglycoside enzyme is 0.3～30mg/mL, and solvent is the citrate-phosphate disodium hydrogen buffer of 50mM pH5.0；For often
The carrier of g dry weight, the consumption of beta-glucosidase enzymatic solution is 5～15mL.
Preparation method the most according to claim 3, it is characterised in that in step 2, described low temperature is 3-10 DEG C.
Preparation method the most according to claim 3, it is characterised in that in step 2, described buffer is
The citrate-phosphate disodium hydrogen buffer of 50mMpH5.0.
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| CN106701729A (en) * | 2017-02-15 | 2017-05-24 | 南京工业大学 | Immobilized enzyme taking polypeptide-modified amino resin as carrier and preparation method thereof |
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