CN104911175A - Amino silanization magnetic nanoparticle co-immobilized laccase and mediator system and preparation method thereof - Google Patents
Amino silanization magnetic nanoparticle co-immobilized laccase and mediator system and preparation method thereof Download PDFInfo
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 103
- 239000002122 magnetic nanoparticle Substances 0.000 title claims abstract description 94
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000002444 silanisation Methods 0.000 title claims abstract description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 title abstract 2
- 238000006243 chemical reaction Methods 0.000 claims abstract description 48
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 27
- 239000002105 nanoparticle Substances 0.000 claims abstract description 19
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 125000003277 amino group Chemical group 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 17
- 239000008367 deionised water Substances 0.000 claims description 16
- 230000009514 concussion Effects 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 8
- 238000013019 agitation Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000002242 deionisation method Methods 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims 3
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims 3
- GDOPTJXRTPNYNR-UHFFFAOYSA-N methyl-cyclopentane Natural products CC1CCCC1 GDOPTJXRTPNYNR-UHFFFAOYSA-N 0.000 abstract description 9
- 238000004132 cross linking Methods 0.000 abstract description 5
- 238000000975 co-precipitation Methods 0.000 abstract 1
- 239000003431 cross linking reagent Substances 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 abstract 1
- 239000008351 acetate buffer Substances 0.000 description 15
- YBCVMFKXIKNREZ-UHFFFAOYSA-N acoh acetic acid Chemical compound CC(O)=O.CC(O)=O YBCVMFKXIKNREZ-UHFFFAOYSA-N 0.000 description 14
- 238000004042 decolorization Methods 0.000 description 13
- 230000031700 light absorption Effects 0.000 description 12
- 239000000975 dye Substances 0.000 description 8
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000010919 dye waste Substances 0.000 description 4
- 238000010907 mechanical stirring Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000007935 neutral effect Effects 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
- 238000006555 catalytic reaction Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- AXDJCCTWPBKUKL-UHFFFAOYSA-N 4-[(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]aniline;hydron;chloride Chemical compound Cl.C1=CC(=N)C(C)=CC1=C(C=1C=CC(N)=CC=1)C1=CC=C(N)C=C1 AXDJCCTWPBKUKL-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
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- 150000003384 small molecules Chemical class 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000003910 Baronia <angiosperm> Species 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 108010075016 Ceruloplasmin Proteins 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000043368 Multicopper oxidase Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- -1 aromatic amine compounds Chemical class 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 238000004065 wastewater treatment Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses an amino silanization magnetic nanoparticle co-immobilized laccase and mediator system and a preparation method thereof. In Fe2+And Fe3+Adding NaOH solution into the solution to carry out coprecipitation reaction to prepare Fe3O4Nanoparticles; modifying the magnetic nanoparticles by using 3-Aminopropyltriethoxysilane (APTES) to obtain magnetic nanoparticles with aminated surfaces; glutaraldehyde is used as a cross-linking agent to graft part of amino groups on the surface of the magnetic nanoparticles with 4-NH2TEMPO, obtaining nanoparticles grafted with the mediator TEMPO; and finally adding the immobilized laccase into a laccase solution, and crosslinking and immobilizing the laccase by glutaraldehyde. The laccase and the mediator are co-immobilized on the magnetic carrier for the first time, and the enzyme and the mediator can be conveniently recycled and reused through an external magnetic field, so that the utilization rate of the enzyme is improved, and the cost is reduced.
Description
Technical field
The invention belongs to and prepare immobilized enzyme field, be specifically related to a kind of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system and preparation method thereof.
Background technology
Laccase (EC 1.10.3.2) is a kind of polyphenoloxidase of cupric, and the Vitamin C oxidase in plant, mammiferous ceruloplasmin belong to blue blue multicopper oxidase family together.Laccase is distributed widely in fungal secretion, higher plant, a small amount of bacterium and insect.Rhus verniciferalaccase, fungal laccase and bacterial laccase is mainly divided into by its source laccase.Laccase molecule is generally containing 4 cupric ions, in the process of Laccase Catalyzed reaction, 4 cupric ions carry out electron transmission between enzyme-to-substrate, thus oxidation substrates is free radical, and free radical generates various product by the further catalyzed oxidation of laccase or non-enzymatic catalysis reaction subsequently.As a kind of special redox enzymes, laccase can utilize the multiple phenols of Oxygen Catalytic Oxidation and aromatic amine compounds, and substrate spectrum is quite wide.Therefore laccase has important using value in wastewater treatment, food-processing, aromatic compound conversion, biological medicine and sensor design etc.But free laccase in use easy with the change of environment deactivation, and be not easily separated from reaction system, this limits the industrial applications of laccase to a certain extent.
But immobilization laccase can improve the thermostability of enzyme, organic solvent tolerance and extreme environment stability to a certain extent, also have reusable simultaneously and be easy to the feature separated from reaction system.It is the effective means realizing laccase industrial applications to the investigation and application of immobilization laccase.Divide by laccase and carrier-bound mode, the process for fixation of laccase is mainly divided into two classes: physical method (physisorphtion and embedding are sent out) and chemical process (ionic adsorption method, covalent coupling method and crosslinking).But, although reported the research work of a large amount of Laccase Immobilization, but still there is various deficiency, up to the present, also do not have a kind of immobilization laccase to be applied to industrial production.On the other hand, because the redox potential of laccase is lower, cause its substrate spectrum narrower, in order to overcome this problem, people develop using small-molecule substances such as TEMPO, HBT as amboceptor and construct efficient laccase-mediator system, to expand the range of application of laccase.Although but consequent problem is that small molecules amboceptor addition is few, because price is generally higher and cannot recycle, the large-scale industrial application of laccase thus also cannot be promoted.
Magnetic Nano material is applied to the focus that enzyme immobilizatio has become research.The magnetic response itself possessed due to magnetic nano-carrier material and particle surface long-pending large, particle diameter is little, brings advantage to immobilized enzyme, as easily separated, not easily strong by burn into stability such as the soda acids in reaction environment, the rate of recovery is high, cost is low.So far, immobilization laccase and immobilized amboceptor all have relevant research report, but also do not find the report of covalent cross-linking co-immobilization laccase and amboceptor.Therefore, the present invention intends by magnetic nano-particle Fe
3o
4carry out amido modified, and covalent cross-linking fixing laccase and TEMPO amboceptor simultaneously.
Summary of the invention
The object of this invention is to provide a kind of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system and preparation method thereof, for solving free laccase and TEMPO amboceptor service efficiency is low, the not easily separated and problem that can not reuse.
To achieve these goals, the technique means that the present invention adopts is:
A kind of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, this system is jointly immobilized on Aminosilylation magnetic nano-particle by laccase and amboceptor, be fixed with 0.2-0.5 mmol amboceptor in every 1g co-immobilization laccase and mediator system, the laccase activity after immobilization is 80-200U/g.
The preparation method of described Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, comprises the steps:
1) Fe
3o
4the preparation of magnetic nano-particle: by FeCl
2and FeCl
3mixing, at N
2under the condition of protection, stirring, 70 ° of C water-baths, add NaOH solution, and after Keep agitation 1-2 h, separated by black product, deionized water is washed till neutrality, obtains Fe
3o
4magnetic nano-particle, then by Fe
3o
4magnetic nano-particle be placed in 7 (wt) % Tetramethylammonium hydroxide (TMAOH) aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use;
2) Fe
3o
4magnetic nano-particle finishing 3-aminopropyl triethoxysilane (APTES): the Fe that step 1) is prepared
3o
4magnetic nano-particle is placed in aqueous ethanolic solution, then adds APTES, 50 DEG C, stirring reaction 10h, and reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle;
3) immobilization of amboceptor: get step 2) the Aminosilylation magnetic nano-particle that obtains is placed in glutaraldehyde solution and leaves standstill 2h, and deionization washed several times with water, to remove unnecessary glutaraldehyde, adds 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor;
4) laccase is fixing: added by laccase in the acetic acid-acetate buffer of 50 mM pH 4.5, add the nanoparticle of grafting amboceptor again, after 35 ° of C concussion reaction 4h, again with the acetic acid of 50 mM pH 4.5-acetate buffer cleaning for several times, to wash away loose enzyme, obtain Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system.
FeCl in step 1)
2and FeCl
3mol ratio be 1:2-2:3.
In step 1), NaOH solution concentration is 3 mol/L.
Step 2) in the Fe for preparing of every 1 g
3o
4magnetic nano-particle is placed in 100-200 mL aqueous ethanolic solution, then adds 1-3 mL APTES.
Step 2) in aqueous ethanolic solution the volume ratio of ethanol and water be 1:1.
In step 3), the mass concentration of glutaraldehyde solution is 2%.
In step 3), every 1g Aminosilylation magnetic nano-particle adds the 4-NH of 1-3 mmol
2-TEMPO.
In step 4), the 1g grafting nanoparticle of amboceptor and the laccase of 200 U carry out crosslinked fixing.
Beneficial effect:
Laccase and amboceptor are fixed on magnetic carrier by the present invention first altogether, magnetic nanotechnology is used to prepare Aminosilylation magnetic nano-particle carrier, preparation technology is simple, with low cost, and simultaneously covalent cross-linking laccase and TEMPO amboceptor, construct efficient laccase-amboceptor catalysis system, the laccase-mediator system of co-immobilization can pass through the easy recovery in external magnetic field, Reusability, thus improve the utilization ratio of enzyme and amboceptor, reduce production cost.
Accompanying drawing explanation
The continuous degradation decolouring result figure of Fig. 1 Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system 10 times.
Embodiment
The laccase used in the present embodiment is purchased from SIGMA company, the 4-NH used
2-TEMPO is purchased from the uncommon love of ladder and changes into industrial development company limited.
Embodiment 1
The preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, comprises the steps:
(1) Fe
3o
4the preparation of magnetic nano-particle: by 0.02 mol FeCl
2with 0.04 mol FeCl
3be dissolved in 100mL deionized water, at N
2add the NaOH solution 250mL of 3 mol/L under protecting the condition of bad border, mechanical stirring, 70 ° of C water-baths, dropwise rear Keep agitation 1 h.Under the effect of externally-applied magnetic field, black product is separated, and by washed with de-ionized water until product is neutral, by the Fe obtained
3o
4magnetic nano-particle is placed in 7 %(mass percents) Tetramethylammonium hydroxide (TMAOH) aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use.
(2) Fe
3o
4magnetic nano-particle finishing APTES: get the Fe that 1 g prepares
3o
4magnetic nano-particle is placed in 150 mL aqueous ethanolic solutions (volume ratio 1:1), then adds 2 mL APTES, 50 DEG C, stirring reaction 10h.Reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle.
(3) immobilization of amboceptor: get Aminosilylation magnetic nano-particle that 1 g prepares be placed in mass concentration be 2% glutaraldehyde solution leave standstill 2h, deionization washed several times with water is to remove unnecessary glutaraldehyde.Add 2 mmol 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor.
(4) laccase is fixing: get 20 U laccases and add in the acetic acid-acetate buffer of 10 mL50 mM pH 4.5, add the 100 mg grafting nanoparticle of amboceptor again, after 35 ° of C concussion reaction 4h, acetic acid-acetate buffer the cleaning of 50 mM pH 4.5 for several times, to wash away loose enzyme, be placed in 4 DEG C of refrigerators for subsequent use, obtain Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system (i.e. the magnetic nano-particle of laccase and amboceptor co-immobilization).Be fixed with 0.4mmol amboceptor in the magnetic nano-particle of every 1g co-immobilization laccase and amboceptor, the laccase activity after immobilization is 120 U/g.
Investigate above-mentioned magnetic nano-particle co-immobilization laccase and the effect of mediator system in degradation of dye waste water, choose C.I. 42685 dyestuff, add the co-immobilization magnetic nano-particle of the above-mentioned preparation of C.I. 42685 dyestuff 5mL, 100mg of 500 mg/L and the acetic acid-acetate buffer of 95 mL 50 mM pH 4.5 in 250mL Erlenmeyer flask, total reaction system is 100mL.Be placed in 100rpm isothermal vibration instrument at 40 DEG C to react, interval 2 h measures the change of maximum absorption wave strong point light absorption value.Percent of decolourization=(1-A/A
0) * 100%, A
0for solution light absorption value before decoloring reaction, A is solution light absorption value after decoloring reaction, and all decoloring reactions all repeat 3 times and are averaged, and calculates percent of decolourization and reaches 84.55%.And in the presence not having TEMPO, only adding the free laccase of equal enzyme activity, percent of decolourization only has 2.3%.
Embodiment 2
The preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, comprises the steps:
(1) Fe
3o
4the preparation of magnetic nano-particle: by 0.02 mol FeCl
2with 0.04 mol FeCl
3be dissolved in 100mL deionized water, at N
2add the NaOH solution 250mL of 3 mol/L under protecting the condition of bad border, mechanical stirring, 70 ° of C water-baths, dropwise rear Keep agitation 1 h.Under the effect of externally-applied magnetic field, black product is separated, and by washed with de-ionized water until product is neutral, by the Fe obtained
3o
4nanoparticle be placed in 7 (wt) % Tetramethylammonium hydroxide (TMAOH) aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use.
(2) Fe
3o
4magnetic nano-particle finishing APTES: get the Fe that 1 g prepares
3o
4magnetic nano-particle is placed in 150 mL aqueous ethanolic solutions (the volume ratio 1:1 of aqueous ethanolic solution ethanol and water), then adds 2 mL APTES, 50 DEG C, stirring reaction 10h.Reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle.
(3) immobilization of amboceptor: get Aminosilylation magnetic nano-particle that 1 g prepares be placed in mass concentration be 2% glutaraldehyde solution leave standstill 2h, deionization washed several times with water is to remove unnecessary glutaraldehyde.Add 1 mmol 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor.
(4) laccase is fixing: get 25 U laccases and add in the acetic acid-acetate buffer of 10 mL 50 mM pH 4.5, add the 100 mg grafting nanoparticle of amboceptor again, after 35 ° of C concussion reaction 4h, acetic acid-acetate buffer the cleaning of 50 mM pH 4.5 for several times, to wash away loose enzyme, be placed in 4 DEG C of refrigerators for subsequent use, obtain the magnetic nano-particle of laccase and amboceptor co-immobilization, be i.e. Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system.Be fixed with 0.22mmol amboceptor in the magnetic nano-particle of every 1g co-immobilization laccase and amboceptor, the laccase activity after immobilization is 160 U/g.
Investigate the effect of magnetic nano-particle in degradation of dye waste water of above-mentioned co-immobilization laccase and amboceptor, choose C.I. 42685 dyestuff, add the co-immobilization magnetic nano-particle of the above-mentioned preparation of C.I. 42685 dyestuff 5mL, 100mg of 500 mg/L and the acetic acid-acetate buffer of 95 mL 50 mM pH4.5 in 250mL Erlenmeyer flask, total reaction system is 100mL.Be placed in 100rpm isothermal vibration instrument at 40 DEG C to react, interval 2 h measures the change of maximum absorption wave strong point light absorption value.Percent of decolourization=(1-A/A
0) * 100%, A
0for solution light absorption value before decoloring reaction, A is solution light absorption value after decoloring reaction, and all decoloring reactions all repeat 3 times and are averaged, and calculates percent of decolourization and reaches 70.05%.And in the presence not having TEMPO, only adding the free laccase of equal enzyme activity, percent of decolourization only has 3.13%.
Embodiment 3
The preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, comprises the steps:
(1) Fe
3o
4the preparation of magnetic nano-particle: by 0.02 mol FeCl
2with 0.04 mol FeCl
3be dissolved in 100mL deionized water, at N
2add the NaOH solution 250mL of 3 mol/L under protecting the condition of bad border, mechanical stirring, 70 ° of C water-baths, dropwise rear Keep agitation 1 h.Under the effect of externally-applied magnetic field, black product is separated, and by washed with de-ionized water until product is neutral, by the Fe obtained
3o
4nanoparticle be placed in 7 % Tetramethylammonium hydroxide (TMAOH) aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use.
(2) Fe
3o
4magnetic nano-particle finishing APTES: get the Fe that 1 g prepares
3o
4magnetic nano-particle is placed in 150 mL ethanol/water (volume ratio 1:1) solution, then adds 2 mL APTES, 50 DEG C, stirring reaction 10h.Reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle.
(3) immobilization of amboceptor: get Aminosilylation magnetic nano-particle that 1 g prepares be placed in mass concentration be 2% glutaraldehyde solution leave standstill 2h, deionization washed several times with water is to remove unnecessary glutaraldehyde.Add 2 mmol 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor.
(4) laccase is fixing: get 20 U laccases and add in the acetic acid-acetate buffer of 10 mL50 mM pH 4.5, add the 100 mg grafting nanoparticle of amboceptor again, after 35 ° of C concussion reaction 4h, acetic acid-acetate buffer the cleaning of 50 mM pH 4.5 for several times, to wash away loose enzyme, be placed in 4 DEG C of refrigerators for subsequent use, obtain the magnetic nano-particle of laccase and amboceptor co-immobilization, be i.e. Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system.Be fixed with 0.4mmol amboceptor in the magnetic nano-particle of every 1g co-immobilization laccase and amboceptor, the laccase activity after immobilization is 120 U/g.
Investigate the effect of magnetic nano-particle in degradation of dye waste water of above-mentioned co-immobilization laccase and amboceptor, choose alkaline fuchsin dye, add the co-immobilization magnetic nano-particle of the above-mentioned preparation of C.I. 42685 dyestuff 5mL, 100mg of 500 mg/L and the acetic acid-acetate buffer of 95 mL 50 mM pH4.5 in 250mL Erlenmeyer flask, total reaction system is 100mL.Be placed in 100rpm isothermal vibration instrument at 40 DEG C to react, interval 2 h measures the change of maximum absorption wave strong point light absorption value.Percent of decolourization=(1-A/A
0) * 100%, A
0for solution light absorption value before decoloring reaction, A is solution light absorption value after decoloring reaction, and all decoloring reactions all repeat 3 times and are averaged, and calculates percent of decolourization and reaches 75.05%.And in the presence not having TEMPO, only adding the free laccase of equal enzyme activity, percent of decolourization only has 4.43%.
Embodiment 4
The preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, comprises the steps:
(1) Fe
3o
4the preparation of magnetic nano-particle: by 0.02 mol FeCl
2with 0.04 mol FeCl
3be dissolved in 100mL deionized water, at N
2add the NaOH solution 250mL of 3 mol/L under protecting the condition of bad border, mechanical stirring, 70 ° of C water-baths, dropwise rear Keep agitation 1 h.Under the effect of externally-applied magnetic field, black product is separated, and by washed with de-ionized water until product is neutral, by the Fe obtained
3o
4nanoparticle be placed in 7 % Tetramethylammonium hydroxide (TMAOH) aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use.
(2) Fe
3o
4magnetic nano-particle finishing APTES: get the Fe that 1 g prepares
3o
4magnetic nano-particle is placed in 150 mL ethanol/water (volume ratio 1:1) solution, then adds 2 mL APTES, 50 DEG C, stirring reaction 10h.Reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle.
(3) immobilization of amboceptor: get Aminosilylation magnetic nano-particle that 1 g prepares be placed in mass concentration be 2% glutaraldehyde solution leave standstill 2h, deionization washed several times with water is to remove unnecessary glutaraldehyde.Add 1 mmol 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor.
(4) laccase is fixing: get 25 U laccases and add in the acetic acid-acetate buffer of 10 mL pH50 mM pH 4.5, add the 100 mg grafting nanoparticle of amboceptor again, after 35 ° of C concussion reaction 4h, acetic acid-acetate buffer the cleaning of 50 mM pH 4.5 for several times, to wash away loose enzyme, be placed in 4 DEG C of refrigerators for subsequent use, obtain the magnetic nano-particle of laccase and amboceptor co-immobilization, be i.e. Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system.Be fixed with 0.22mmol amboceptor in the magnetic nano-particle of every 1g co-immobilization laccase and amboceptor, the laccase activity after immobilization is 160 U/g.
Investigate the effect of magnetic nano-particle in degradation of dye waste water of above-mentioned co-immobilization laccase and amboceptor, choose alkaline fuchsin dye, add the co-immobilization magnetic nano-particle of the above-mentioned preparation of C.I. 42685 dyestuff 5mL, 100mg of 500 mg/L and the acetic acid-acetate buffer of 95 mL 50 mM pH4.5 in 250mL Erlenmeyer flask, total reaction system is 100mL.Be placed in 100rpm isothermal vibration instrument at 40 DEG C to react, interval 2 h measures the change of maximum absorption wave strong point light absorption value.Percent of decolourization=(1-A/A
0) * 100%, A
0for solution light absorption value before decoloring reaction, A is solution light absorption value after decoloring reaction, and all decoloring reactions all repeat 3 times and are averaged, and calculates percent of decolourization and reaches 62.23%.And in the presence not having TEMPO, only adding the free laccase of equal enzyme activity, percent of decolourization only has 4.82%.
Embodiment 5
Magnetic nano-particle embodiment 1 being participated in the co-immobilization laccase after decoloring reaction and amboceptor reclaims, adopt the fractionation by adsorption co-immobilization magnetic nano-particle of magnet, then 4-5 time is rinsed with the acetic acid-acetate buffer of 50 mM pH4.5, reuse the co-immobilization magnetic nano-particle of recovery, by the continuous degradation decoloring reaction of 10 times, the results are shown in Figure 1, as can be seen from Figure 1 reclaim through 7 times, co-immobilization magnetic nanoparticle still keeps satisfactory stability, and percent of decolourization is more than 50.55%.
Claims (10)
1. an Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system, it is characterized in that: this system is jointly immobilized on Aminosilylation magnetic nano-particle by laccase and amboceptor, be fixed with 0.2-0.5 mmol amboceptor in every 1g co-immobilization laccase and mediator system, the laccase activity after immobilization is 80-200U/g.
2. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system described in claim 1, is characterized in that comprising the steps:
1) Fe
3o
4the preparation of magnetic nano-particle: by FeCl
2and FeCl
3mixing, at N
2under the condition of protection, stirring, 70 ° of C water-baths, add NaOH solution, and after Keep agitation 1-2 h, separated by black product, deionized water is washed till neutrality, obtains Fe
3o
4magnetic nano-particle, then by Fe
3o
4magnetic nano-particle be placed in 7 (wt) % tetramethylammonium hydroxide aqueous solution leave standstill 2 h, wash with water 3-5 time for subsequent use;
2) Fe
3o
4magnetic nano-particle finishing APTES: the Fe that step 1) is prepared
3o
4magnetic nano-particle is placed in aqueous ethanolic solution, then adds APTES, 50 DEG C, stirring reaction 10h, and reaction product is with after washed with de-ionized water 3-5 time, and vacuum-drying, obtains surface amino groups silanization magnetic nano-particle;
3) immobilization of amboceptor: get step 2) the Aminosilylation magnetic nano-particle that obtains is placed in glutaraldehyde solution and leaves standstill 2h, and deionization washed several times with water, to remove unnecessary glutaraldehyde, adds 4-NH
2-TEMPO, concussion reaction 4h under room temperature, after reaction terminates, isolate product, washing, obtains the nanoparticle of grafting amboceptor;
4) laccase is fixing: added by laccase in acetic acid-sodium acetate buffer solution, add the nanoparticle of grafting amboceptor again, after 35 DEG C of concussion reaction 4h, again with acetic acid-sodium acetate buffer solution cleaning for several times, to wash away loose enzyme, obtain Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system.
3. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and mediator system according to claim 2, is characterized in that: FeCl in step 1)
2and FeCl
3mol ratio be 1:2-2:3.
4. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: in step 1), NaOH solution concentration is 3 mol/L.
5. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: step 2) in the Fe for preparing of every 1 g
3o
4magnetic nano-particle is placed in 100-200 mL aqueous ethanolic solution, then adds 1-3 mL APTES.
6. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: step 2) in aqueous ethanolic solution the volume ratio of ethanol and water be 1:1.
7. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: in step 3), the mass concentration of glutaraldehyde solution is 2%.
8. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: in step 3), every 1g Aminosilylation magnetic nano-particle adds the 4-NH of 1-3 mmol
2-TEMPO.
9. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: in step 4), the 1g grafting nanoparticle of amboceptor and the laccase of 200 U carry out crosslinked fixing.
10. the preparation method of Aminosilylation magnetic nano-particle co-immobilization laccase and amboceptor according to claim 2, is characterized in that: in step 4), the concentration of acetic acid-sodium acetate buffer solution is 50 mM, pH is 4.5.
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| CN111137988A (en) * | 2020-01-18 | 2020-05-12 | 安徽工程大学 | Method for decoloring dye wastewater |
| CN114525269A (en) * | 2022-03-21 | 2022-05-24 | 中国林业科学研究院林产化学工业研究所 | Method for immobilizing laccase based on covalent bonding of polyethylene glycol and superparamagnetic iron oxide nanoparticles |
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| CN105647902A (en) * | 2015-12-30 | 2016-06-08 | 江苏大学 | Carbon nano tube immobilized laccase and application thereof |
| CN105647902B (en) * | 2015-12-30 | 2019-07-05 | 江苏大学 | A kind of carbon nanotube immobilization laccase and application thereof |
| CN106434621A (en) * | 2016-10-28 | 2017-02-22 | 华北电力大学 | Method for immobilizing laccase on polyacrylamide-coated magnetic nano particles and application of method |
| CN106636056A (en) * | 2016-11-17 | 2017-05-10 | 南京工业大学 | Amino silanization magnetic graphene oxide nanoparticle co-immobilized laccase and mediator system and preparation method thereof |
| CN109234258A (en) * | 2018-09-11 | 2019-01-18 | 新乡医学院 | Laccase natural amboceptor immobilization magnetic nano-particle and its preparation method and application |
| CN109234258B (en) * | 2018-09-11 | 2022-01-18 | 新乡医学院 | Laccase natural mediator immobilized magnetic nanoparticles and preparation method and application thereof |
| CN111137988A (en) * | 2020-01-18 | 2020-05-12 | 安徽工程大学 | Method for decoloring dye wastewater |
| CN114525269A (en) * | 2022-03-21 | 2022-05-24 | 中国林业科学研究院林产化学工业研究所 | Method for immobilizing laccase based on covalent bonding of polyethylene glycol and superparamagnetic iron oxide nanoparticles |
| CN114525269B (en) * | 2022-03-21 | 2024-03-22 | 中国林业科学研究院林产化学工业研究所 | Method for covalently binding immobilized laccase based on polyethylene glycol and superparamagnetic iron oxide nanoparticles |
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