CN102649954A - Immobilized enzyme for magnetic nanometer clay carrier and regenerating method thereof - Google Patents
Immobilized enzyme for magnetic nanometer clay carrier and regenerating method thereof Download PDFInfo
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- CN102649954A CN102649954A CN2012101521079A CN201210152107A CN102649954A CN 102649954 A CN102649954 A CN 102649954A CN 2012101521079 A CN2012101521079 A CN 2012101521079A CN 201210152107 A CN201210152107 A CN 201210152107A CN 102649954 A CN102649954 A CN 102649954A
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Abstract
The invention relates to an immobilized enzyme for a magnetic nanometer clay carrier. The immobilized enzyme comprises a carrier with saccharifying enzyme and corresponding immobilized enzyme as immobilizing objects. The immobilized enzyme is prepared by taking a covalence coupling method as an immobilizing method and is characterized in that: the carrier is a magnetic nanometer Fe3O4@ clay compound material; the magnetic nanometer Fe3O4@ clay compound material is composed of magnetic Fe3O4 nanometer grains and a nanometer clay material; and the magnetic Fe3O4 nanometer grains are assembled on the nanometer clay in sequence. A superparamagnetic Fe3O4@ clay compound material is prepared by adopting a solvent heat method according to the invention; the surface of the Fe3O4@ clay compound material is modified by using gamma-aminopropyl triethoxy silane, and then glutaraldehyde is used as a coupling agent for covalently fixing the saccharifying enzyme on the surface of the carrier; two novel carrier regenerating strategies are designed; after the saccharifying enzyme is immobilized by the regenerated carrier, the excellent properties, such as original activity, thermal stability and reusability, of the immobilized enzyme are still maintained; and the immobilized enzyme is also suitable for the regeneration of the immobilized enzyme carrier according to the covalence coupling method.
Description
Technical field
The present invention relates to biological technical field, relate in particular to magnetic Nano clay carrier immobilized enzyme and regenerated method thereof.
Background technology
Immobilized enzyme allows the repetition of enzyme or uses continuously, be easy to substrate and product recovery, be easy to purifying, improved the performance of biological catalyst to a certain extent.In recent years, nano material such as nanoclay have caused the very big concern of people as the carrier of immobilized enzyme, and reason is that its cost is low, particle diameter is little, and have unique intercalation character; After the another one major reason is the silicon hydroxyl process different functional groups activation of surface of clay, can be used as the attachment site of biomacromolecule.Yet; The greatest problem that in these researchs, exists be exactly after immobilized enzyme is accomplished catalyzed reaction carrier be difficult to separated; And the heterogeneous catalyst of magneticsubstance can satisfy biological catalyst high efficiency separation and catalytic process continue carry out; Therefore, the clay of magnetic Nano material modification is expected to become the excellent carrier material of immobilized enzyme.
There has been bibliographical information to cross martial ethiops/clay nanocomposites; Yet; These results of study in the past show; Magnetic nanoparticle often is fixed on surface of clay, just may be covered as the silicon hydroxyl of immobilized enzyme attachment site so, and this has just limited the application of magnetic Nano clay material in the immobilized enzyme field.In addition, the magneticintensity of prepared these magnetic Nano materials is also very low, and is not easily separated, this also big limitations its application.
With regard to enzyme immobilization; In absorption, crosslinked, embedding and four kinds of main carrier immobilized enzyme methods of covalent coupling; Other three kinds of immobilized enzyme that method is prepared except that absorption method; Its carrier all can not be regenerated, and is dropped along with the inactivation of enzyme, and this has just caused environmental pollution, the wasting of resources and production cost to increase.Though prepared its carrier of immobilized enzyme of traditional absorption method can be regenerated, carrier combines loss insecure, enzyme more serious with enzyme, and this has also just limited its application.Therefore, designing, develop and preparing reproducible solid support material just becomes one of this important function for of research emphasis, the rare report of work of at present relevant this respect.
Summary of the invention
Technical problem to be solved by this invention provides a kind of magnetic nano-particle to the less magnetic Nano clay carrier immobilized enzyme of surface of clay influence.
Another technical problem to be solved by this invention provides the preparation method of this magnetic Nano clay carrier immobilized enzyme.
The 3rd technical problem to be solved by this invention provides the regenerated method of this magnetic Nano clay carrier immobilized enzyme.
For addressing the above problem, magnetic Nano clay carrier immobilized enzyme of the present invention comprises that fixed object is the carrier of saccharifying enzyme and respective fixation enzyme, and is that process for fixation is processed with the e, and it is characterized in that: said carrier is magnetic Nano Fe
3O
4Clay composite material, this magnetic Nano Fe
3O
4Clay composite material is by magnetic Fe
3O
4Nanoparticle and nanoclay material are formed, and magnetic Fe
3O
4Be assembled on the said nanoclay nano particle orderedly.
Said magnetic Nano Fe
3O
4Clay composite material is meant at first with FeCl
36H
2O is dissolved in the feed liquid weightmeasurement ratio of 1:20 ~ 1:50 and forms settled solution in the terepthaloyl moietie; Then NaAc, polyoxyethylene glycol are joined in the said settled solution, secondly the nanoclay with hydrochloric acid activation joins in the said settled solution, and ultra-sonic dispersion 1 ~ 5h obtains mixed solution; At last said mixed solution is joined in the stainless steel cauldron of teflon lined, keep 160 ~ 210 ℃ of reaction 5 ~ 24h and promptly get; The weightmeasurement ratio of wherein said NaAc and said settled solution is 1:7 ~ 1:18; The weightmeasurement ratio of said polyoxyethylene glycol and said settled solution is 1:20 ~ 1:50; The nanoclay of said hydrochloric acid activation and the weightmeasurement ratio of said settled solution are 1:60 ~ 1:150.
The nanoclay of said hydrochloric acid activation is meant puts into the HCl solution that concentration is 1 ~ 3 M with nanoclay by the feed liquid weightmeasurement ratio of 1:20 ~ 30; After ultrasonic 0.5 ~ 2 h carries out activation under 20 ~ 40 ℃ of temperature; Through spinning and wash to supernatant and be neutral; Obtain throw out after the spinning again, this throw out grinds, crosses 30 ~ 60 μ m sieves and promptly gets after vacuum-drying to constant weight.
Aforesaid magnetic Nano clay carrier immobilized enzyme is characterized in that: said nanoclay is any one in Attapulgite, kaolin, the polynite.
The preparation method of aforesaid magnetic Nano clay carrier immobilized enzyme may further comprise the steps:
⑴ with magnetic Nano Fe
3O
4Clay composite material is immersed in 2 ~ 4h in the xylene solution with the feed liquid weightmeasurement ratio of 1:40 ~ 1:80, makes its abundant swelling, under agitation γ-An Jibingjisanyiyangjiguiwan is joined in the said xylene solution then; 90 ~ 130 ℃ of following oil bath reflux, and N
2Protection is reaction 6 ~ 15h down, and reaction finishes back washing with alcohol three times, after magnetic separates, obtains throw out, and this throw out obtains the magnetic Nano Fe of modification through vacuum-drying to constant weight
3O
4Clay composite material is designated as Fe
3O
4Clay-NH
2The weightmeasurement ratio of wherein said γ-An Jibingjisanyiyangjiguiwan and said xylene solution is 1:8 ~ 1:12;
⑵ be to add said Fe in 2 ~ 10% the glutaraldehyde solution in mass concentration
3O
4Clay-NH
2, under room temperature, react 2 ~ 8h; Carry out magnetic then and separate, obtain throw out, this throw out is removed unreacted LUTARALDEHYDE for several times through distilled water wash; Using concentration at last is 5.5 hac buffer washing precipitate as 50mM, pH value, and this throw out note is made Fe
3O
4Clay-GA; Said Fe
3O
4Clay-NH
2With the weightmeasurement ratio of said glutaraldehyde solution be 1:30 ~ 60;
⑶ with said Fe
3O
4It is that 50mM, pH value are 5.5, saccharifying enzyme content is in the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg that clay-GA carrier joins concentration by the feed liquid weightmeasurement ratio of 1:10 ~ 20, carries out the immobilization of enzyme in 2 ~ 8 hours 20 ~ 40 ℃ of reactions; Immobilization is after spinning and be after 50mM, pH value are 5.5 hac buffer washed twice, promptly to get immobilized enzyme through concentration;
⑷ said immobilized enzyme is placed under 4 ℃, to preserve and gets final product.
The regenerated method of aforesaid magnetic Nano clay carrier immobilized enzyme may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation;
2. in the immobilized enzyme of said inactivation, add the ultrasonic half a hour of acetone of 5 ~ 10 times of its weight, under 80 ~ 100 ℃, boiled 20 ~ 60 minutes then, obtain pretreated carrier; After said pretreated carrier uses concentration to be the washing for several times of 5.5 hac buffer as 50mM, pH value; Joining mass concentration by the feed liquid weightmeasurement ratio of 1:30 ~ 60 is in 2 ~ 10% the glutaraldehyde solution or epoxy chloropropane solution; Under room temperature, react 2 ~ 8h, promptly get the carrier of regenerating;
3. after said regeneration carrier uses concentration to be the washing for several times of 5.5 hac buffer as 50mM, pH value; Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 20 ~ 40 ℃ of airbath immobilization 2 ~ 8 h, promptly accomplish the immobilization once more of enzyme; The weightmeasurement ratio of said regeneration carrier and said saccharifying enzyme acetate buffer solution is 1:10 ~ 20.
Replace said step 2. with following preparation method: 10 ~ 20 times of its weight of adding and concentration are the Cu of 0.5 ~ 10 mg/ml in the immobilized enzyme of said inactivation
2+/ Zn
2+After shaking 0.5 ~ 6h under the ion, room temperature, promptly get the carrier of regenerating through spinning.
The present invention compared with prior art has the following advantages:
1, because the present invention adopts solvent-thermal method to prepare the nano-magnetic clay, therefore, magnetic nano-particle is less to the surface of clay influence, and prepared magnetic Nano clay has higher magneticintensity.
2, because the present invention considers that enzyme is a kind of protein; It is made up of amino acid, and therefore, its side chain and end group have a large amount of reactive group (as: amino, carboxyl, sulfydryl and imidazolyl etc.); Thereby behind the immobilized enzyme inactivation; Carrier so that covalent linkage connects can also be regenerated, and carrier regenerating method is simple, and has the wide spectrum suitability.
3, because the present invention adopts solvent-thermal method to prepare the nano-magnetic clay, therefore, magnetic Nano Fe
3O
4Clay composite material can reduce extraneous material to the Effect on Performance of clay own at the specific site modified clay for research in the past, and has higher magneticintensity and make it be prone to separate with reaction solution.
4, because the fixed enzyme vector of using among the present invention has magnetic; And consider that enzyme is a kind of protein; It is made up of amino acid, and its side chain and end group have a large amount of reactive group (as: amino, carboxyl, sulfydryl and imidazolyl etc.), therefore; Immobilized enzyme of the present invention not only is and can regenerates to the carrier of covalent bonds, and be it be easy to reclaim and recycle.The testing method of immobilized enzyme repeat performance is following: magnetic Nano Fe
3O
4Be recovered behind the clay composite material and the carrier immobilized saccharifying enzyme catalytic hydrolysis starch of regenerating,, get into the catalyzed reaction of a new round once more with hac buffer (50 mM, pH=5.5) washing three times.After ten secondary response cycles accomplished, magnetic Nano Fe
3O
4Clay composite material all approaches 60% with the residual vigor of the carrier immobilized saccharifying enzyme of regeneration.The loss of its vigor mainly is because the part zymoprotein molecule that is adsorbed onto carrier surface in the use comes off, and in the catalytic process due to the sex change of part zymoprotein.Obviously, enzyme is fixed on magnetic Nano Fe
3O
4Increased its operational stability on the clay composite material.In addition, the good repeatability of renewable carrier immobilized enzyme has further proved the feasibility of carrier regeneration strategy.
5, pass through magnetic Nano Fe
3O
4Clay composite material carries out transmissioning electric mirror test (FEI Tecnai G20), can find out that median size is the Fe of 150 nm
3O
4Attached to the different positions of delaminated kaoline, conclude the Fe that gets up to have three kinds of different structures
3O
4The kaolin nano composite material that is to say, Fe
3O
4Nanoparticle can be fixed on the edge and the surface of clay laminate structure, or insert two-layer between (referring to Fig. 1).
6, pass through magnetic Nano Fe
3O
4Clay composite material carry out VSM test (LAKESHORE-7304, USA), Fe at room temperature
3O
4Clay composite material has the characteristic of superparamagnetism, because it has very high magnetic effect, thereby has very fast magnetic response for the external magnetic field, this explains that also the saturated magneticintensity of nano composite material has enough satisfied the demand of immobilized enzyme.In addition, can find out pure Fe
3O
4The saturated magneticintensity of nanoparticle appears at 88.97 emu/g.Yet since the existence of clay, Fe
3O
4The magnetic saturation intensity of kaolin nano composite material is reduced to 38.56 emu/g, Fe
3O
4ATP drops to 40.85 emu/g, this basically with Fe
3O
4Ratio in nano composite material is consistent.
Description of drawings
Do further detailed explanation below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is Fe among the present invention
3O
4The projection electromicroscopic photograph of kaolin nano composite material.
Embodiment
Embodiment 1Magnetic Nano clay carrier immobilized enzyme comprises that fixed object is the carrier of saccharifying enzyme and respective fixation enzyme, and is that process for fixation is processed with the e.Carrier is magnetic Nano Fe
3O
4Clay composite material, this magnetic Nano Fe
3O
4Clay composite material is by magnetic Fe
3O
4Nanoparticle and nanoclay material are formed, and magnetic Fe
3O
4Be assembled on the said nanoclay nano particle orderedly.
Wherein: magnetic Nano Fe
3O
4Clay composite material is meant at first with FeCl
36H
2O is dissolved in the feed liquid weightmeasurement ratio (kg/L) of 1:20 and forms settled solution in the terepthaloyl moietie; Then NaAc, polyoxyethylene glycol are joined in the settled solution, secondly the nanoclay with hydrochloric acid activation joins in the settled solution, and ultra-sonic dispersion (40kHz) 1h obtains mixed solution; At last mixed solution is joined in the stainless steel cauldron of teflon lined, keep 160 ℃ of reaction 24h and promptly get.
The weightmeasurement ratio of NaAc and settled solution (kg/L) is 1:7; The weightmeasurement ratio of polyoxyethylene glycol and settled solution (kg/L) is 1:20; The nanoclay of hydrochloric acid activation and the weightmeasurement ratio of settled solution (kg/L) are 1:60.
The nanoclay of hydrochloric acid activation is meant puts into the HCl solution that concentration is 1 M with nanoclay by the feed liquid weightmeasurement ratio of 1:20, and ultrasonic (40kHz) 2 h carry out activation under 20 ℃ of temperature, to remove residual carbon and the sodium ion of surface of clay.After activation is accomplished; Through spinning (4000 commentaries on classics/min) and with zero(ppm) water repetitive scrubbing to supernatant be neutral; (4000 change/obtain throw out after min), this throw out grinds after vacuum-drying (50 ℃, 24 hours) to constant weight, mistake 30 μ m sieves promptly get in spinning again.
Embodiment 2Magnetic Nano clay carrier immobilized enzyme comprises that fixed object is the carrier of saccharifying enzyme and respective fixation enzyme, and is that process for fixation is processed with the e.Carrier is magnetic Nano Fe
3O
4Clay composite material, this magnetic Nano Fe
3O
4Clay composite material is by magnetic Fe
3O
4Nanoparticle and nanoclay material are formed, and magnetic Fe
3O
4Be assembled on the said nanoclay nano particle orderedly.
Wherein: magnetic Nano Fe
3O
4Clay composite material is meant at first with FeCl
36H
2O is dissolved in the feed liquid weightmeasurement ratio (kg/L) of 1:50 and forms settled solution in the terepthaloyl moietie; Then NaAc, polyoxyethylene glycol are joined in the settled solution, secondly the nanoclay with hydrochloric acid activation joins in the settled solution, and ultra-sonic dispersion (40kHz) 5h obtains mixed solution; At last mixed solution is joined in the stainless steel cauldron of teflon lined, keep 210 ℃ of reaction 5h and promptly get.
The weightmeasurement ratio of NaAc and settled solution (kg/L) is 1:18; The weightmeasurement ratio of polyoxyethylene glycol and settled solution (kg/L) is 1:50; The nanoclay of hydrochloric acid activation and the weightmeasurement ratio of settled solution (kg/L) are 1:150.
The nanoclay of hydrochloric acid activation is meant puts into the HCl solution that concentration is 3 M with nanoclay by the feed liquid weightmeasurement ratio of 1:30, and ultrasonic (40kHz) 0.5 h carries out activation under 40 ℃ of temperature, to remove residual carbon and the sodium ion of surface of clay.After activation is accomplished; Through spinning (4000 commentaries on classics/min) and with zero(ppm) water repetitive scrubbing to supernatant be neutral; (4000 change/obtain throw out after min), this throw out grinds after vacuum-drying (50 ℃, 24 hours) to constant weight, mistake 60 μ m sieves promptly get in spinning again.
Embodiment 3Magnetic Nano clay carrier immobilized enzyme comprises that fixed object is the carrier of saccharifying enzyme and respective fixation enzyme, and is that process for fixation is processed with the e.Carrier is magnetic Nano Fe
3O
4Clay composite material, this magnetic Nano Fe
3O
4Clay composite material is by magnetic Fe
3O
4Nanoparticle and nanoclay material are formed, and magnetic Fe
3O
4Be assembled on the said nanoclay nano particle orderedly.
Wherein: magnetic Nano Fe
3O
4Clay composite material is meant at first with FeCl
36H
2O is dissolved in the feed liquid weightmeasurement ratio (kg/L) of 1:35 and forms settled solution in the terepthaloyl moietie; Then NaAc, polyoxyethylene glycol are joined in the settled solution, secondly the nanoclay with hydrochloric acid activation joins in the settled solution, and ultra-sonic dispersion (40kHz) 3 h obtain mixed solution; At last mixed solution is joined in the stainless steel cauldron of teflon lined, keep 185 ℃ of reaction 15h and promptly get.
The weightmeasurement ratio of NaAc and settled solution (kg/L) is 1:12; The weightmeasurement ratio of polyoxyethylene glycol and settled solution (kg/L) is 1:35; The nanoclay of hydrochloric acid activation and the weightmeasurement ratio of settled solution (kg/L) are 1:110.
The nanoclay of hydrochloric acid activation is meant puts into the HCl solution that concentration is 2 M with nanoclay by the feed liquid weightmeasurement ratio of 1:26, and ultra-sonic dispersion (40kHz) 1 h carries out activation under 30 ℃ of temperature, to remove residual carbon and the sodium ion of surface of clay.After activation is accomplished; Through spinning (4000 commentaries on classics/min) and with zero(ppm) water repetitive scrubbing to supernatant be neutral; (4000 change/obtain throw out after min), this throw out grinds after vacuum-drying (40 ℃, 48 hours) to constant weight, mistake 45 μ m sieves promptly get in spinning again.
Above-mentioned
Embodiment 1 ~ 3In nanoclay be in Attapulgite, kaolin, the polynite any one.
Embodiment 4Above-mentioned
Embodiment 1 ~ 3The preparation method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
⑴ with magnetic Nano Fe
3O
4Clay composite material is immersed in 2h in the xylene solution with the feed liquid weightmeasurement ratio (kg/L) of 1:40, makes its abundant swelling, under agitation γ-An Jibingjisanyiyangjiguiwan is joined in the xylene solution then; 90 ℃ of following oil bath reflux, and N
2Protection is reaction 6h down, and reaction finishes back washing with alcohol three times, after magnet separates it, obtains throw out, and this throw out obtains the magnetic Nano Fe of modification through vacuum-drying (40 ℃, 48 hours) to constant weight
3O
4Clay composite material is designated as Fe
3O
4Clay-NH
2This Fe
3O
4Clay-NH
2Measure the content of (C, H and N) element through Vario EL elemental analyser, the result obtains Fe
3O
4Clay and Fe
3O
4The Attapulgite surface connects amino content and is respectively 0.6 mmol/g and 0.9 mmol/g.
Wherein the weightmeasurement ratio of γ-An Jibingjisanyiyangjiguiwan and xylene solution (kg/L) is 1:8.
⑵ add Fe in mass concentration is 2% glutaraldehyde solution
3O
4Clay-NH
2, under room temperature, react 2h; Through magnet it is separated then, obtain throw out, this throw out is removed unreacted LUTARALDEHYDE for several times through distilled water wash; Using concentration at last is 5.5 hac buffer washing precipitate as 50mM, pH value, and this throw out note is made Fe
3O
4Clay-GA.
Fe
3O
4Clay-NH
2With the weightmeasurement ratio (kg/L) of glutaraldehyde solution be 1:30.
⑶ with Fe
3O
4It is that 50mM, pH value are 5.5, saccharifying enzyme content is in the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg that clay-GA carrier joins concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:10, carries out the immobilization of enzyme in 2 hours 20 ℃ of reactions; Immobilization is after magnet is after 50mM, pH value are 5.5 hac buffer washed twice, promptly to get immobilized enzyme with its separation and through concentration.Reacted solution and washings merge, and are used for measuring loose protein content.
⑷ immobilized enzyme is placed under 4 ℃, to preserve and gets final product.
Embodiment 5Above-mentioned
Embodiment 1 ~ 3The preparation method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
⑴ with magnetic Nano Fe
3O
4Clay composite material is immersed in 4h in the xylene solution with the feed liquid weightmeasurement ratio (kg/L) of 1:80, makes its abundant swelling, under agitation γ-An Jibingjisanyiyangjiguiwan is joined in the xylene solution then; 130 ℃ of following oil bath reflux, and N
2Protection is reaction 15h down, and reaction finishes back washing with alcohol three times, after magnet separates it, obtains throw out, and this throw out obtains the magnetic Nano Fe of modification through vacuum-drying (40 ℃, 48 hours) to constant weight
3O
4Clay composite material is designated as Fe
3O
4Clay-NH
2This Fe
3O
4Clay-NH
2Measure the content of (C, H and N) element through Vario EL elemental analyser, the result obtains Fe
3O
4Clay and Fe
3O
4The Attapulgite surface connects amino content and is respectively 0.8 mmol/g and 1.2 mmol/g.
Wherein the weightmeasurement ratio of γ-An Jibingjisanyiyangjiguiwan and xylene solution (kg/L) is 1:12.
⑵ add Fe in mass concentration is 10% glutaraldehyde solution
3O
4Clay-NH
2, under room temperature, react 8h; Through magnet it is separated then, obtain throw out, this throw out is removed unreacted LUTARALDEHYDE for several times through distilled water wash; Using concentration at last is 5.5 hac buffer washing precipitate as 50mM, pH value, and this throw out note is made Fe
3O
4Clay-GA.
Fe
3O
4Clay-NH
2With the weightmeasurement ratio (kg/L) of glutaraldehyde solution be 1:60.
⑶ with Fe
3O
4It is that 50mM, pH value are 5.5, saccharifying enzyme content is in the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg that clay-GA carrier joins concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:20, carries out the immobilization of enzyme in 8 hours 40 ℃ of reactions; Immobilization is after magnet is after 50mM, pH value are 5.5 hac buffer washed twice, promptly to get immobilized enzyme with its separation and through concentration.Reacted solution and washings merge, and are used for measuring loose protein content.
⑷ immobilized enzyme is placed under 4 ℃, to preserve and gets final product.
Embodiment 6Above-mentioned
Embodiment 1 ~ 3The preparation method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
⑴ with magnetic Nano Fe
3O
4Clay composite material is immersed in 3h in the xylene solution with the feed liquid weightmeasurement ratio (kg/L) of 1:60, makes its abundant swelling, under agitation γ-An Jibingjisanyiyangjiguiwan is joined in the xylene solution then; 110 ℃ of following oil bath reflux, and N
2Protection is reaction 10h down, and reaction finishes back washing with alcohol three times, after magnet separates it, obtains throw out, and this throw out obtains the magnetic Nano Fe of modification through vacuum-drying (40 ℃, 48 hours) to constant weight
3O
4Clay composite material is designated as Fe
3O
4Clay-NH
2This Fe
3O
4Clay-NH
2Measure the content of (C, H and N) element through Vario EL elemental analyser, the result obtains Fe
3O
4Clay and Fe
3O
4The Attapulgite surface connects amino content and is respectively 0.7 mmol/g and 1.1 mmol/g.
Wherein the weightmeasurement ratio of γ-An Jibingjisanyiyangjiguiwan and xylene solution (kg/L) is 1:10.
⑵ add Fe in mass concentration is 6% glutaraldehyde solution
3O
4Clay-NH
2, under room temperature, react 5h; Through magnet it is separated then, obtain throw out, this throw out is removed unreacted LUTARALDEHYDE for several times through distilled water wash; Using concentration at last is 5.5 hac buffer washing precipitate as 50mM, pH value, and this throw out note is made Fe
3O
4Clay-GA.
Fe
3O
4Clay-NH
2With the weightmeasurement ratio (kg/L) of glutaraldehyde solution be 1:40.
⑶ with Fe
3O
4It is that 50mM, pH value are 5.5, saccharifying enzyme content is in the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg that clay-GA carrier joins concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:15, carries out the immobilization of enzyme in 5 hours 30 ℃ of reactions; Immobilization is after magnet is after 50mM, pH value are 5.5 hac buffer washed twice, promptly to get immobilized enzyme with its separation and through concentration.Reacted solution and washings merge, and are used for measuring loose protein content.
⑷ immobilized enzyme is placed under 4 ℃, to preserve and gets final product.
Embodiment 7Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. in the immobilized enzyme of inactivation, add the ultrasonic half a hour of acetone of 5 times of its weight, under 80 ℃, boiled 60 minutes then, obtain pretreated carrier; After pretreated carrier washs for several times with hac buffer (50mM, pH=5.5); Joining mass concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:30 is in 2% the glutaraldehyde solution or epoxy chloropropane solution; Under room temperature, react 2h, promptly get the carrier of regenerating.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 20 ℃ of airbath immobilization 8h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:10.
Embodiment 8Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. in the immobilized enzyme of inactivation, add the ultrasonic half a hour of acetone of 10 times of its weight, under 100 ℃, boiled 20 minutes then, obtain pretreated carrier; After pretreated carrier washs for several times with hac buffer (50mM, pH=5.5); Joining mass concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:60 is in 10% the glutaraldehyde solution or epoxy chloropropane solution; Under room temperature, react 8h, promptly get the carrier of regenerating.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 40 ℃ of airbath immobilization 2h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:20.
Embodiment 9Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. in the immobilized enzyme of inactivation, add the ultrasonic half a hour of acetone of 8 times of its weight, under 90 ℃, boiled 40 minutes then, obtain pretreated carrier; After pretreated carrier washs for several times with hac buffer (50mM, pH=5.5); Joining mass concentration by the feed liquid weightmeasurement ratio (kg/L) of 1:40 is in 6% the glutaraldehyde solution or epoxy chloropropane solution; Under room temperature, react 5h, promptly get the carrier of regenerating.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 30 ℃ of airbath immobilization 5h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:15.
Embodiment 10Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. 10 times of its weight of adding and concentration are the Cu of 0.5mg/ml in the immobilized enzyme of inactivation
2+/ Zn
2+Ion, behind the concussion 0.5h, (4000 change/min) promptly get the carrier of regenerating through spinning under the room temperature.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 20 ℃ of airbath immobilization 2h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:10.
Embodiment 11Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. 20 times of its weight of adding and concentration are the Cu of 10 mg/ml in the immobilized enzyme of inactivation
2+/ Zn
2+Ion, behind the concussion 6h, (4000 change/min) promptly get the carrier of regenerating through spinning under the room temperature.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 40 ℃ of airbath immobilization 8 h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:20.
Embodiment 12Above-mentioned
Embodiment 1 ~ 3The regenerated method of magnetic Nano clay carrier immobilized enzyme, may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation.
2. 15 times of its weight of adding and concentration are the Cu of 5 mg/ml in the immobilized enzyme of inactivation
2+/ Zn
2+Ion, behind the concussion 3h, (4000 change/min) promptly get the carrier of regenerating through spinning under the room temperature.
3. after the carrier of regenerating washs for several times with hac buffer (50mM, pH=5.5); Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 30 ℃ of airbath immobilization 5h, promptly accomplish the immobilization once more of enzyme.The weightmeasurement ratio (kg/L) of regeneration carrier and saccharifying enzyme acetate buffer solution is 1:15.
Should be appreciated that the embodiment that discusses and embodiment can propose various improvement and variation just in order to explain to the people who is familiar with this field here, these improvement and variation will be included in the application's spirit and the scope and appended claim scope.
Claims (7)
1. magnetic Nano clay carrier immobilized enzyme comprises that fixed object is the carrier of saccharifying enzyme and respective fixation enzyme, and is that process for fixation is processed with the e, and it is characterized in that: said carrier is magnetic Nano Fe
3O
4Clay composite material, this magnetic Nano Fe
3O
4Clay composite material is by magnetic Fe
3O
4Nanoparticle and nanoclay material are formed, and magnetic Fe
3O
4Be assembled on the said nanoclay nano particle orderedly.
2. magnetic Nano clay carrier immobilized enzyme as claimed in claim 1 is characterized in that: said magnetic Nano Fe
3O
4Clay composite material is meant at first with FeCl
36H
2O is dissolved in the feed liquid weightmeasurement ratio of 1:20 ~ 1:50 and forms settled solution in the terepthaloyl moietie; Then NaAc, polyoxyethylene glycol are joined in the said settled solution, secondly the nanoclay with hydrochloric acid activation joins in the said settled solution, and ultra-sonic dispersion 1 ~ 5h obtains mixed solution; At last said mixed solution is joined in the stainless steel cauldron of teflon lined, keep 160 ~ 210 ℃ of reaction 5 ~ 24h and promptly get; The weightmeasurement ratio of wherein said NaAc and said settled solution is 1:7 ~ 1:18; The weightmeasurement ratio of said polyoxyethylene glycol and said settled solution is 1:20 ~ 1:50; The nanoclay of said hydrochloric acid activation and the weightmeasurement ratio of said settled solution are 1:60 ~ 1:150.
3. magnetic Nano clay carrier immobilized enzyme as claimed in claim 2; It is characterized in that: the nanoclay of said hydrochloric acid activation is meant puts into the HCl solution that concentration is 1 ~ 3 M with nanoclay by the feed liquid weightmeasurement ratio of 1:20 ~ 30; After ultrasonic 0.5 ~ 2 h carries out activation under 20 ~ 40 ℃ of temperature; Through spinning and wash to supernatant and be neutral, obtain throw out after the spinning again, this throw out grinds after vacuum-drying to constant weight, mistake 30 ~ 60 μ m sieves promptly get.
4. magnetic Nano clay carrier immobilized enzyme as claimed in claim 3 is characterized in that: said nanoclay is any one in Attapulgite, kaolin, the polynite.
5. the preparation method of magnetic Nano clay carrier immobilized enzyme as claimed in claim 1 may further comprise the steps:
⑴ with magnetic Nano Fe
3O
4Clay composite material is immersed in 2 ~ 4h in the xylene solution with the feed liquid weightmeasurement ratio of 1:40 ~ 1:80, makes its abundant swelling, under agitation γ-An Jibingjisanyiyangjiguiwan is joined in the said xylene solution then; 90 ~ 130 ℃ of following oil bath reflux, and N
2Protection is reaction 6 ~ 15h down, and reaction finishes back washing with alcohol three times, after magnetic separates, obtains throw out, and this throw out obtains the magnetic Nano Fe of modification through vacuum-drying to constant weight
3O
4Clay composite material is designated as Fe
3O
4Clay-NH
2The weightmeasurement ratio of wherein said γ-An Jibingjisanyiyangjiguiwan and said xylene solution is 1:8 ~ 1:12;
⑵ be to add said Fe in 2 ~ 10% the glutaraldehyde solution in mass concentration
3O
4Clay-NH
2, under room temperature, react 2 ~ 8h; Carry out magnetic then and separate, obtain throw out, this throw out is removed unreacted LUTARALDEHYDE for several times through distilled water wash; Using concentration at last is 5.5 hac buffer washing precipitate as 50mM, pH value, and this throw out note is made Fe
3O
4Clay-GA; Said Fe
3O
4Clay-NH
2With the weightmeasurement ratio of said glutaraldehyde solution be 1:30 ~ 60;
⑶ with said Fe
3O
4It is that 50mM, pH value are 5.5, saccharifying enzyme content is in the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg that clay-GA carrier joins concentration by the feed liquid weightmeasurement ratio of 1:10 ~ 20, carries out the immobilization of enzyme in 2 ~ 8 hours 20 ~ 40 ℃ of reactions; Immobilization is after spinning and be after 50mM, pH value are 5.5 hac buffer washed twice, promptly to get immobilized enzyme through concentration;
⑷ said immobilized enzyme is placed under 4 ℃, to preserve and gets final product.
6. the regenerated method of magnetic Nano clay carrier immobilized enzyme as claimed in claim 1 may further comprise the steps:
1. make the immobilized enzyme complete deactivation after using for several times, obtain the immobilized enzyme of inactivation;
2. in the immobilized enzyme of said inactivation, add the ultrasonic half a hour of acetone of 5 ~ 10 times of its weight, under 80 ~ 100 ℃, boiled 20 ~ 60 minutes then, obtain pretreated carrier; After said pretreated carrier uses concentration to be the washing for several times of 5.5 hac buffer as 50mM, pH value; Joining mass concentration by the feed liquid weightmeasurement ratio of 1:30 ~ 60 is in 2 ~ 10% the glutaraldehyde solution or epoxy chloropropane solution; Under room temperature, react 2 ~ 8h, promptly get the carrier of regenerating;
3. after said regeneration carrier uses concentration to be the washing for several times of 5.5 hac buffer as 50mM, pH value; Add concentration then and be 50mM, pH value and be 5.5, saccharifying enzyme content is the saccharifying enzyme acetate buffer solution of 0.1 ~ 0.6 mg; Adopt shaking table at 20 ~ 40 ℃ of airbath immobilization 2 ~ 8 h, promptly accomplish the immobilization once more of enzyme; The weightmeasurement ratio of said regeneration carrier and said saccharifying enzyme acetate buffer solution is 1:10 ~ 20.
7. the regenerated method of magnetic Nano clay carrier immobilized enzyme as claimed in claim 6 is characterized in that: replace step described in the claim 6 2. with following preparation method: 10 ~ 20 times of its weight of adding and concentration are the Cu of 0.5 ~ 10 mg/ml in the immobilized enzyme of said inactivation
2+/ Zn
2+After shaking 0.5 ~ 6h under the ion, room temperature, promptly get the carrier of regenerating through spinning.
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