CN104498472A - Magnetic immobilized cellobiase nano mesoporous material and preparation method thereof - Google Patents
Magnetic immobilized cellobiase nano mesoporous material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a magnetic immobilized cellobiase nano mesoporous material, which comprises the following steps: a sol forming step: dilute acid is taken as a catalyst for hydrolyzing tetraethoxysilane to form transparent sol, ethanol is dissociated from the sol until mass content is less than or equal to 5%; and a gel forming step: a template solution, a cellobiase buffer and magnetic nanoparticles Fe3O4 in sol under stirring, adjusting a system to neutrality by a dilute alkali solution, and placing to generate the gel. The invention also discloses the magnetic immobilized cellobiase nano mesoporous material prepared by the method. aperture of a nano medium material obtained by the method is about 2-7 nm, so that specific surface area is high; the nano medium material can be gathered in short time under magnetic field effect, so that recovery and usage are convenient; and the nano medium material has the advantages of high biological activity and reutilization.
Description
Technical field
The present invention relates to a kind of mesoporous nano material and preparation method thereof, particularly relate to a kind of magnetic immobilized cellobiase mesoporous nano material and preparation method thereof.
Background technology
By the impact of the factors such as world oil supply, price, environmental protection and Global climate change, the development of renewable clear energy sources-biofuel receives many
countryattention, the especially U.S., Brazil and Chinese.Biofuel is based on alcohol fuel, and biotechnology enzyme process is that the recyclability carbon source material Mierocrystalline cellulose utilizing quantity on the earth maximum obtains one of important step of alcohol fuel.But cellulosic high-polymerization degree makes its difficult degradation, cellulosic enzymolysis speed will differ from more than 2 orders of magnitude compared with starch, make the high cost of cellulase, thus the industrial application of serious this technology of restriction, investigator takes many measure head it offs both at home and abroad for this reason, as being fixed cellulase, improving its stability, can reuse.
Increasing research shows, operability and repeatable immobilized cellulase can greatly reduce cellulase hydrolysis process costs, and this becomes domestic and international study hotspot.Carrier is considered to one of key factor determining immobilized enzyme effect.According to carriers Water-soluble, soluble carrier and the large class of insoluble carrier two can be divided into.Natural cellulose is all insoluble carrier, and many investigators think that can only design soluble carrier immobilized cellulase just has activity at first.But along with the fast development of insoluble carrier in recent years, the superiority of soluble carrier anchoring fiber element enzyme is no longer obvious.Be fixed on by cellulase in soluble carrier, its kinetic property does not change, and operational stability improves also not remarkable, on the contrary owing to there is the critical defect not easily reclaimed, departs from the visual field of investigator after the nineties in 20th century very soon.Insoluble carrier mainly comprises the chitosan etc. of low cost starch grafted propylene nitrile, the porous prilled carrier of acrylamide amphipathy macromolecule compound formation and modification.It is extremely convenient that insoluble carrier immobilized cellulase reclaims, and enzyme stability improves also fairly obvious, but and substrate, the resistance to mass transfer between especially insoluble substrate is large, therefore needs to make it have large specific surface area to insoluble carrier transformation.
Wherein, alcohol fuel mainly comprises grain alcohol and cellulosic ethanol.Under the overall background of world's food shortage, " do not strive grain with people; do not strive ground with grain ", with the grand strategy that the material production renewable energy sources (as: cellulosic ethanol) such as stalk, sawdust, bagasse, domestic refuse are countries in the world future source of energy development, therefore cellulosic ethanol industry obtains and greatly develops.The annual cellulosic ethanol yield of China 4,000,000 tons, China " 12 "
countrystrategic planning realizes low-cost cellulase and breaks through.According to the industrial scale of current cellulosic ethanol, the cellulase only required for U.S.'s cellulosic ethanol industrial market can reach more than 300,000 tons, and the demand of China's cellulase is also nearly at about 100,000 tons.But there is cellulase can not reclaim and reuse in cellulosic ethanol production process, production cost is high and cannot the problem such as scale operation.Immobilized cellulase is mainly for the production of cellulosic ethanol.Replace free-fiber element enzyme with immobilized cellulase, can reuse thus reduce the production cost of cellulosic ethanol, can greatly promote regenerated biological energy industry development.
At present, immobilized cellulase technology adopts geopolymer gel material to be carrier mostly, or adopts the gelation process of tensio-active agent.Employing geopolymer gel material is that the immobilized enzyme life cycle of carrier is short, cost is high, toxin immunity is poor, activity is low and it is few to reuse number of times; Adopt tenside gel method prepare the sol-gel process of immobilized enzyme and template remove process often need harsh condition (as
high temperature is highpressure, strong acid-base environment and use a large amount of organic solvent), these harsh conditions have great destruction to biologically active substance (as: protein and nucleic acid), thus significantly limit the application of conventional surfactant method at biological field, and the recovery of immobilized enzyme that the method prepares also has problems.
Summary of the invention
For this reason, the present invention proposes a kind of a kind of magnetic immobilized cellobiase mesoporous nano material that is that can solve the problem or that at least can partly solve the problem and preparation method thereof.
According to an aspect of the present invention, provide a kind of preparation method of magnetic immobilized cellobiase mesoporous nano material, the method comprises: colloidal sol forming step: be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, and detach to mass content≤5% by ethanol in colloidal sol; Gel formation step: under agitation add template solution, cellobiase damping fluid and magnetic Nano Fe successively in described colloidal sol
3o
4particle, is adjusted to neutrality with sig water by system, places and generates gel.
Alternatively, preparation in accordance with the present invention, wherein said diluted acid is the HCl solution of 0.1 ~ 0.3M, and the mass ratio of described HCl solution and described TEOS is 5:32 ~ 13:32.
Alternatively, preparation in accordance with the present invention, wherein said template is glucose, maltose, fructose, dibenzoyl-L-tartaric, cyclodextrin, urea, glycerine, Zulkovsky starch, citric acid, xitix or oligopeptides.
Alternatively, preparation in accordance with the present invention, the mass ratio of wherein said template solution and described TEOS is 2:32 ~ 6:32.
Alternatively, preparation in accordance with the present invention, wherein said cellobiase buffer concentration is 10 ~ 100mg/ml, and the mass ratio of described cellobiase damping fluid and described TEOS is 1:32 ~ 10:32.
Alternatively, preparation in accordance with the present invention, wherein said magnetic Nano Fe
3o
4the mass ratio of particle and described TEOS is 1:32 ~ 5:32.
Alternatively, preparation in accordance with the present invention, wherein said sig water is NaOH solution, KOH solution or NH
3.H
2o.
Alternatively, preparation in accordance with the present invention, the concentration of wherein said NaOH solution is 0.4 ~ 0.8M.
Alternatively, preparation in accordance with the present invention, comprises further:
Remove template drying step: ground by described gel, add water washing removing template suction filtration and obtain gel, dry 3-7 days in silica gel imbedded by the gel obtained by suction filtration.
According to a further aspect in the invention, a kind of magnetic immobilized cellobiase mesoporous nano material obtained according to above-mentioned preparation method is provided.
The Product Activity of the mesoporous material immobilized cellobiase obtained prepared according to the methods of the invention can reach more than more than 80%, reusable ten times of free-fiber Disacchridase Activities and keep activity not reduce; Described nano material realizes reclaiming by the magnetic nanoparticle included and uses, thus significantly reduces cellulosic ethanol production cost; Utilize mesoporous material anchoring fiber disaccharidase can improve stability in storage and the use temperature of cellobiase, extend its memory cycle in the presence of a harsh environment.The inventive method adopts nonsurfactant method, room temperature, normal pressure, close to neutral and be almost produce meso-hole structure in the environment of pure water phase, realize the synchronization of collosol and gel pore-creating and embedding in situ biologically active substance, technological process is simple, mild condition, environmental friendliness, is suitable for industrialization.
Accompanying drawing explanation
By reading hereafter detailed description of the preferred embodiment, various other advantage and benefit will become cheer and bright for those of ordinary skill in the art.Attached
figureonly for illustrating the object of preferred implementation, and do not think limitation of the present invention.Attached
figurein:
figure1 shows the preparation method's flow process according to magnetic immobilized cellobiase nano-medium material of the present invention
figure;
figure2 forms showing magnetic immobilized cellobiase nano-medium material;
figurethe 3 magnetic field enrichments showing magnetic immobilized cellobiase nano-medium material contrast
figure;
figurethe 4 preparation method's principles showing the magnetic immobilized cellobiase mesoporous nano material according to the inventive method
figure.
Embodiment
The invention provides many applicable creative concepts, this creative concept can be reflected in a large number of in concrete context.The specific embodiment described in following embodiments of the present invention only as the exemplary illustration of specific implementation of the present invention, and does not form limitation of the scope of the invention.
According to the preparation method of magnetic immobilized cellobiase mesoporous nano material of the present invention, principle
figureshown in 4.
According to
figure4 Hes
figureflow process shown in 1
figurecan find out, first the method enters colloidal sol forming step S1100: be that tetraethoxy (TEOS) is hydrolyzed into vitreosol by catalyzer under whipped state with diluted acid under normal temperature, and detach ethanol in colloidal sol to mass content≤5%.
Wherein said TEOS is tetraethoxy, take diluted acid as catalyzer at normal temperatures, generally adopts the HCl solution of 0.1 ~ 0.3M, and the mass ratio of HCl solution and TEOS is 5:32 ~ 13:32.In TEOS, dilute hydrochloric acid is added while stirring under normal temperature, vigorous stirring 10 ~ 20min, obtain transparent colloidal sol, by the vitreosol that obtains after hydrolysis at 0-10 DEG C of cooling (carrying out as being placed in refrigerator) 2 ~ 4h, the coproduct ethanol produced in removing system hydrolytic process is detached with high vacuum system, temperature is cooled to 0-10 DEG C for occurring that boiling phenomenon prevents colloidal sol from spilling because ethanol content in system is too high when preventing from vacuumizing, after detaching for some time, for ethanol in system is removed more thorough, sol system heating in water bath is extracted further to 37-50 DEG C of constant temperature, ethanol mass content≤5% to system, extract for some time post-heating to detach further, ethanol is detached more thorough.Ethanol content in system is reduced to≤5%, it is the activity that can affect the magnetic immobilized cellobiase of follow-up generation owing to there is a large amount of ethanol in colloidal sol, and after adopting present method, the content of ethanol in system is controlled to impact the activity of magnetic immobilized cellobiase≤5% time, therefore the preparation condition of this preparation method is relatively more loose, easily realizes.Wherein, colloidal sol forming reactions equation is as follows:
According to method of the present invention and above-mentioned principle
figure, then the method carries out gel formation step: under agitation in the colloidal sol formed, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 48 ~ 72h generate gel at 4-10 DEG C.
Be glucose, maltose, fructose, dibenzoyl-L-tartaric, cyclodextrin, urea, glycerine, Zulkovsky starch, citric acid, xitix or oligopeptides in the template of gel formation step.The mass ratio of template and TEOS is 2:32 ~ 6:32, becomes solution to add in system Template preparation.Cellobiase buffer concentration is 10 ~ 100mg/ml, and the mass ratio of cellobiase damping fluid and TEOS is 1:32 ~ 10:32.Magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 1:32 ~ 5:32.Sig water is NaOH solution, KOH solution or NH
3.H
2o.Wherein the concentration of NaOH solution is 0.4 ~ 0.8M.Polycondensation and alcoholysis occur colloidal sol further and to form cross-linked network gelation principle as follows:
Preparation in accordance with the present invention and above-mentioned principle
figure, the method enters further removes template drying step S1300: ground by described gel, add water washing removing template suction filtration and obtain gel, dry 3-7 days in silica gel imbedded by the gel obtained by suction filtration, and it is for subsequent use dried gel abrasive to be crossed 40 mesh sieve packs.
The mesoporous material immobilized cellobiase Product Activity obtained produced according to the present invention can reach more than more than 80%, reusable ten times of free-fiber Disacchridase Activities and keep activity not reduce; Described nano material realizes reclaiming by the magnetic nanoparticle included and uses, thus significantly reduces cellulosic ethanol production cost; Utilize mesoporous material anchoring fiber disaccharidase can improve stability in storage and the use temperature of cellobiase, extend its memory cycle in the presence of a harsh environment.The inventive method adopts nonsurfactant method, room temperature, normal pressure, close to neutral and be almost produce meso-hole structure in the environment of pure water phase, realize the synchronization of collosol and gel pore-creating and embedding in situ biologically active substance, technological process is simple, mild condition, environmental friendliness, is suitable for industrialization.
According to the preparation method of magnetic immobilized cellobiase mesoporous nano material of the present invention, condition, the amount adding template and the kind that can be formed according to collosol and gel, the concentration of cellobiase damping fluid and add-on, magnetic Nano Fe
3o
4the factors such as the amount of particle are carried out specific design and are gone out different embodiments, below by with the preparation method of magnetic immobilized cellobiase mesoporous nano material for embodiment is entered preparation method of the present invention
row showsexample explanation.
Use raw material as follows in embodiment 1-6:
Embodiment 1
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 5 grams of 0.1M, the mass ratio of hydrochloric acid soln and TEOS is 5:32, after vigorous stirring 10min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 0 DEG C of 2h that cools with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 37 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 48h and generate gel at 4 DEG C.Template solution is that 2 grams of glucose are dissolved in the solution (the mass ratio 2:32 of glucose and TEOS) made in 10 grams of water.Add 10mg/ml cellobiase damping fluid 1 gram (mass ratio of cellobiase damping fluid and TEOS is 1:32).The NaOH solution of sig water to be concentration be 0.4M.Add 1 gram of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 1:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water washing and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration to be embedded in dry silica gel dry 3 days, ground 40 mesh sieves, pack for subsequent use.
Embodiment 2
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 13 grams of 0.3M, the mass ratio of hydrochloric acid soln and TEOS is 13:32, after vigorous stirring 20min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 10 DEG C of 4h that cool with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 50 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 72h and generate gel at 10 DEG C.Template solution is that 6 grams of fructose are dissolved in the solution (the mass ratio 6:32 of fructose and TEOS) made in 10 grams of water.Add 100mg/ml cellobiase damping fluid 10 grams (mass ratio of cellobiase damping fluid and TEOS is 10:32).The NaOH solution of sig water to be concentration be 0.8M.Add 5 grams of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 5:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water washing and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration to be embedded in dry silica gel dry 7 days, cross 40 mesh sieves after grinding, pack for subsequent use.
Embodiment 3
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 9 grams of 0.2M, the mass ratio of hydrochloric acid soln and TEOS is 9:32, after vigorous stirring 15min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 5 DEG C of 3h that cool with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 45 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 60h and generate gel at 8 DEG C.Template solution is that 4 grams of maltose are dissolved in the solution (the mass ratio 4:32 of maltose and TEOS) made in 10 grams of water.Add 60mg/ml cellobiase damping fluid 5 grams (mass ratio of cellobiase damping fluid and TEOS is 5:32).The NaOH solution of sig water to be concentration be 0.6M.Add 3 grams of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 3:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water washing and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration to be embedded in dry silica gel dry 4 days, ground 40 mesh sieves, pack for subsequent use.
Embodiment 4
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 7 grams of 0.3M, the mass ratio of hydrochloric acid soln and TEOS is 7:32, after vigorous stirring 16min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 7 DEG C of 3.5h that cool with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 48 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 68h and generate gel at 8 DEG C.Template solution is that 3 grams of dibenzoyl-L-tartaric are dissolved in the solution (the mass ratio 3:32 of dibenzoyl-L-tartaric and TEOS) made in 10 grams of water.Add 50mg/ml cellobiase damping fluid 6 grams (mass ratio of cellobiase damping fluid and TEOS is 6:32).The NaOH solution of sig water to be concentration be 0.7M.Add 4 grams of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 4:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water washing and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration to be embedded in dry silica gel dry 4 days, ground 40 mesh sieves, pack for subsequent use.
Embodiment 5
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 12 grams of 0.2M, the mass ratio of hydrochloric acid soln and TEOS is 12:32, after vigorous stirring 13min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 6 DEG C of 3h that cool with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 42 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 55h and generate gel at 7 DEG C.Template solution is that 3 grams of glucose are dissolved in the solution (the mass ratio 3:32 of glucose and TEOS) made in 10 grams of water.Add 70mg/ml cellobiase damping fluid 8 grams (mass ratio of cellobiase damping fluid and TEOS is 8:32).The NaOH solution of sig water to be concentration be 0.6M.Add 2 grams of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 2:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water washing and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration to be embedded in dry silica gel dry 4 days, ground 40 mesh sieves, pack for subsequent use.
Embodiment 6
According to
figurethe preparation method of the magnetic immobilized cellobiase mesoporous nano material of the present invention shown in 1, first colloidal sol forming step S1100 is entered: take tetraethoxy (TEOS) 32 grams, under agitation add rare HCl solution of 12.5 grams of 0.1M, the mass ratio of hydrochloric acid soln and TEOS is 12.5:32, after vigorous stirring 15min, be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, vitreosol after hydrolysis is detached the coproduct ethanol produced in system hydrolytic process after 4 DEG C of 2h that cool with high vacuum system, after detaching for some time, colloidal sol constant temperature water bath to 37 DEG C is detached, until ethanol mass content≤5% in colloidal sol.
Then gel formation step S1200 is entered: under agitation in described colloidal sol, add template solution, cellobiase damping fluid and magnetic Nano Fe successively
3o
4particle, is adjusted to neutrality with sig water by system, places 48h and generate gel at 4 DEG C.Template solution is that 2 grams of fructose are dissolved in the solution (the mass ratio 2:32 of fructose and TEOS) made in 10 grams of water.Add 10mg/ml cellobiase damping fluid 1 gram (mass ratio of cellobiase damping fluid and TEOS is 1:32).The NaOH solution of sig water to be concentration be 0.4M.Add 1 gram of magnetic Nano Fe
3o
4particle (magnetic Nano Fe
3o
4the mass ratio of particle and TEOS is 1:32).
Finally enter and remove template drying step S1300: the gel of formation is ground, adds 150ml water agitator treating and remove glucose suction filtrations for 2 times and obtain white gels, the gel filter paper obtained by suction filtration is embedded in dry 3 days (72h) in dry silica gel, ground 40 mesh sieves, pack for subsequent use.
According to the mesoporous nano material of the magnetic immobilized cellobiase that embodiment 1-6 prepares, immobilized cellobiase Product Activity can reach more than 80% of free-fiber Disacchridase Activities; Described material more than reusable ten times and keep activity not reduce; Described mesoporous nano material realizes reclaiming by the magnetic nanoparticle enrichment at short notice (about 10min) included and uses, thus significantly reduces cellulosic ethanol production cost; Utilize mesoporous material anchoring fiber disaccharidase can improve stability in storage and the use temperature of cellobiase, extend its memory cycle in the presence of a harsh environment.The inventive method adopts nonsurfactant method, room temperature, normal pressure, close to neutral and be almost produce meso-hole structure in the environment of pure water phase, realize the synchronization of collosol and gel pore-creating and embedding in situ biologically active substance, technological process is simple, mild condition, environmental friendliness, is suitable for industrialization.
Serial performance characterization has been carried out to the mesoporous nano material of the magnetic immobilized cellobiase that preparation in accordance with the present invention obtains.
(1) determination of activity
Vigour-testing method: the standard method that cellobiose enzyme activity (cellobiase activity, CBA) is recommended according to IUPAC measures, and represents with international unit IU.Get 1.0mL cellobiose substrate (15mmol/L, pH=4.8) in test tube, add the enzyme liquid that 1mL suitably dilutes, isothermal reaction 30min at 50 DEG C.With the glucose that determination of glucose oxidase generates, and deduct blank test measured value.A cellobiose enzyme activity international unit (CBIU) equals per minute under standard enzymatic reaction condition and generates the enzyme amount of 2.0umol glucose.Then have:
Accurately take the magnetic immobilized cellobiase nano-medium material powder 1.000g that the drying and grinding for preparing in embodiment 6 is sieved, carry out determination of activity according to above-mentioned testing method, will vibrate in mensuration process enzymatic hydrolysis system in time.The activity recording magnetic immobilized cellobiase nanometer in embodiment 6 is 209IU/g, is 265IU/g with a batch free-fiber Disacchridase Activities.
(2) aperture
According to
figurethe electromicroscopic photograph of the meso-porous nano material utilizing embodiment 6 to prepare shown in 2 can find out that the aperture of meso-porous nano material is between 2 ~ 7nm, increase the specific surface area of meso-porous nano material, thus reduce the resistance to mass transfer when preparing cellulosic ethanol between carrier and substrate.
(3) enrichment time contrast test
According to
figurephoto shown in 3, wherein A is not for add magnetic Nano Fe
3o
4the sample of particle, B and C are the magnetic Nano Fe that embodiment 6 prepares
3o
4the sample of particle, adds long strip shape magnet and forms magnetic field in B, add magneton in C and form magnetic field, the magneticstrength of B is greater than the magneticstrength of C, and find the complete enrichment in 2-6min of B sample by timing, C sample is complete enrichment in 10min.And do not add magnetic Nano Fe
3o
4the sample A of particle then needs the enrichment of 5 hours ability, and this shows that the enrichment time under the action of a magnetic field having added magnetic nano-particle greatly shortens, and magneticstrength is stronger, enrichment time is shorter
(4) activity system being remained the magnetic immobilized cellobiase that different content ethanol prepares has carried out comparative determination.
Activity determination method is with reference to the method for determination of activity in (), and measurement result is as follows:
As can be seen here, residual ethanol content in system is controlled≤5%, the preparation of magnetic immobilized cellobiase can be realized, and make it keep higher activity.
It should be noted, the present invention will be described instead of limit the invention for above-described embodiment, and those skilled in the art can design alternative embodiment when not departing from claims scope.In the claims, any reference symbol between bracket should be configured to limitations on claims.Word " comprises " not to be got rid of existence and does not arrange element in the claims or step.
Claims (10)
1. a preparation method for magnetic immobilized cellobiase mesoporous nano material, the method comprises:
Colloidal sol forming step S1100: be that TEOS is hydrolyzed into formation vitreosol by catalyzer at normal temperatures with diluted acid, and the ethanol in colloidal sol is detached to mass content≤5%;
Gel formation step S1200: under agitation add template solution, cellobiase damping fluid and magnetic Nano Fe successively in described colloidal sol
3o
4particle, is adjusted to neutrality with sig water by system, places and generates gel.
2. preparation method according to claim 1, wherein said diluted acid is the HCl solution of 0.1 ~ 0.3M, and the mass ratio of described HCl solution and described TEOS is 5:32 ~ 13:32.
3. preparation method according to claim 1, wherein said template is glucose, maltose, fructose, dibenzoyl-L-tartaric, cyclodextrin, urea, glycerine, Zulkovsky starch, citric acid, xitix or oligopeptides.
4. preparation method according to claim 1, the mass ratio of wherein said template and described TEOS is 2:32 ~ 6:32.
5. preparation method according to claim 1, wherein said cellobiase buffer concentration is 10 ~ 100mg/ml, and the mass ratio of described cellobiase damping fluid and described TEOS is 1:32 ~ 10:32.
6. preparation method according to claim 1, wherein said magnetic Nano Fe
3o
4the mass ratio of particle and described TEOS is 1:32 ~ 5:32.
7. preparation method according to claim 1, wherein said sig water is NaOH solution, KOH solution or NH
3.H
2o.
8. preparation method according to claim 7, the concentration of wherein said NaOH solution is 0.4 ~ 0.8M.
9., according to the arbitrary described preparation method of claim 1-8, the method comprises further:
Remove template drying step S1300: ground by described gel, add water washing removing template suction filtration and obtain gel, dry 3-7 days in silica gel imbedded by the gel obtained by suction filtration.
10. the magnetic immobilized cellobiase mesoporous nano material obtained according to the arbitrary described preparation method of claim 1-9.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434793A (en) * | 2016-12-09 | 2017-02-22 | 厦门庚能新材料技术有限公司 | Method for producing fuel ethanol from household garbage |
| CN106480130A (en) * | 2016-12-09 | 2017-03-08 | 厦门庚能新材料技术有限公司 | A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw |
| CN106480108A (en) * | 2016-12-09 | 2017-03-08 | 厦门庚能新材料技术有限公司 | A kind of method that employing hollow palm fruit string produces alcohol fuel |
| CN106480000A (en) * | 2016-12-09 | 2017-03-08 | 厦门庚能新材料技术有限公司 | A kind of production method of magnetic immobilized enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106434793A (en) * | 2016-12-09 | 2017-02-22 | 厦门庚能新材料技术有限公司 | Method for producing fuel ethanol from household garbage |
| CN106480130A (en) * | 2016-12-09 | 2017-03-08 | 厦门庚能新材料技术有限公司 | A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw |
| CN106480108A (en) * | 2016-12-09 | 2017-03-08 | 厦门庚能新材料技术有限公司 | A kind of method that employing hollow palm fruit string produces alcohol fuel |
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| CN106480130B (en) * | 2016-12-09 | 2019-08-30 | 厦门庚能新材料技术有限公司 | A kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application |
| CN108165592A (en) * | 2017-11-27 | 2018-06-15 | 凡秘能特种糖业有限公司 | A kind of method using mesoporous material immobilization multiplex-enzyme extraction inulin |
| CN108165592B (en) * | 2017-11-27 | 2020-08-07 | 深圳市华利康纤生物科技有限公司 | Method for extracting inulin by using mesoporous material immobilized complex enzyme |
| CN111621494A (en) * | 2020-07-07 | 2020-09-04 | 厦门庚能新材料技术有限公司 | Preparation method of magnetic immobilized lipase |
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