CN101974509B - Preparation method of immobilized enzyme with multilevel structure - Google Patents
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- 239000000377 silicon dioxide Substances 0.000 claims abstract description 13
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- 238000001179 sorption measurement Methods 0.000 claims abstract description 7
- VIJYFGMFEVJQHU-UHFFFAOYSA-N aluminum oxosilicon(2+) oxygen(2-) Chemical compound [O-2].[Al+3].[Si+2]=O VIJYFGMFEVJQHU-UHFFFAOYSA-N 0.000 claims description 26
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 7
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- 229940061607 dibasic sodium phosphate Drugs 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to a preparation method of a multi-level structure immobilized enzyme, belonging to the technical field of bioengineering. The preparation method comprises the following steps: (1) preparing an alumina-silica composite membrane with a mesoporous structure; (2) fixing enzyme on the alumina-silica composite membrane by filtration and adsorption; (3) modifying a layer of mesoporous nano particle film on the surface of the alumina-silica composite film to form the multi-stage structure immobilized enzyme. The method has mild preparation conditions, the enzyme and the carrier are adsorbed by physical action, and the enzyme can keep high activity; the mesoporous nano particle membrane prevents the loss of enzyme and does not influence the passing of a substrate, so that the immobilized enzyme has good operation stability; meanwhile, the preparation method realizes the continuous operation of the immobilized enzyme and the direct separation of the product.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly a kind of preparation method of multilevel hierarchy immobilized enzyme.This method is fixed in enzyme in aluminium oxide-silicon oxide composite package mesoporous, further stops enzyme to run off with the film modified aluminium oxide-silicon oxide composite package of medium hole nano particles surface again.This preparation method can obtain the immobilized enzyme of high reactivity and high stability, has universality.
Background technology
Immobilized enzyme all has important use in food, medicine, chemical industry and biosensor manufacturing.Through 50 years of researches and development, enzyme immobilization technology has been obtained significant progress, has successively developed various fixed method and the various solid support material of performance.It can not only stabilized enzyme, realize the enzyme recycling, and can change enzyme specificity, improve enzyme activity, make it more to meet people's generic request.
The enzyme fixing means of laboratory study at present and industrial application mainly contains absorption method, e, four big types of crosslinking and entrapping methods.Absorption method is to reach fixedly purpose method through the weak interaction between carrier surface and enzyme molecular surface.Carrier commonly used has inorganic adsorbent and organic adsorbents such as Mierocrystalline cellulose, collagen such as kaolin, silica gel, aluminum oxide, micropore glass.It is simple to operate that absorption method prepares immobilized enzyme, can fully select different electric charges and difform carrier, and adsorption process possibly reach purifying and immobilization simultaneously.Weak point is that under the industrial application condition of high concentration of substrate and high ionic strength, the enzyme losing issue is serious owing to bonding force between enzyme and the carrier is not strong, can cause the forfeiture of catalysis activity and stain reaction product; E is by covalent linkage the active nonessential side-chain radical of enzyme and the functional group of carrier to be carried out the method that coupling prepares immobilized enzyme.The covalent method advantage is that the connecting key between enzyme and the carrier is very firm, coming off of enzyme can not taken place in the use, and stability better.Weak point is the activation or the immobilization operation more complicated of carrier, and reaction conditions is also relatively more violent, so often need the higher immobilized enzyme of strict control condition ability acquisition vigor; Crosslinking promptly utilizes difunctional or poly functional reagent is intermolecular at enzyme, between enzyme molecule and inert protein or carry out crosslinking reaction between enzyme molecule and carrier, prepare the method for immobilized enzyme with covalent linkage.Linking agent commonly used has LUTARALDEHYDE, adipoyl imido acid dimethyl ester etc.Crosslinking is simple to operate, but crosslinking reaction is often relatively fiercer, and many enzymes are prone in immobilization process, lose efficacy, and the enzyme recovery is not high; Entrapping method is with after the monomer of polymkeric substance and the enzyme solution mixing, relends the effect that helps polymerization promotor and carries out polymerization, and enzyme is embedded in the polymkeric substance to reach immobilized purpose.Embedded material commonly used has agar, alginate calcium and acrylamide gel etc.Entrapping method all require substrate can freely get into interior contact of gel with enzyme react, the product gel that can dissociate, while enzyme molecule is retained in the gel.This method weak point is in the chemical polymerization process owing between generation, heat release and the enzyme of radical and reagent chemical reaction etc. possibly take place, and often causes enzyme deactivation.
This shows that various immobilized enzyme methods have advantage and the application system that is fit to separately, but they still there is very big limitation.How the contradiction between the high reactivity of enzyme equilibrium and the firm immobilization is immobilized enzyme one of insoluble problem in science in catalytic applications always.To this science difficult point; We have proposed a kind of preparation method of multilevel hierarchy immobilized enzyme; Utilize multilevel hierarchy that aluminium oxide-silicon oxide-nanoparticle is compounded to form that enzyme is limited in carrier mesoporous, allow substrate freely to pass through simultaneously, the activity of enzyme and stability obtain maximum the reservation; And all-in-one-piece membrane structure handled easily and control are applicable to the immobilization and the catalytic applications of various enzymes.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of multilevel hierarchy immobilized enzyme, solved the high reactivity of enzyme in the immobilized enzyme system and the problem that held stationaryization is difficult to get both.
The preparation method of multilevel hierarchy immobilized enzyme of the present invention is following:
(1) preparation of aluminium oxide-silicon oxide composite package
Polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer (P123) is dissolved in 10～60 ℃ hydrochloric acid, the mixing solutions of ethanol (EtOH) and water.Add toluene, stirred 0.5～5 hour, dropwise add tetraethoxy (TEOS) again, continue to stir 0.5～5 hour, obtain silica precursors solution.Each material molar ratio of solution is 1TEOS: 0.006～0.06P123: 4～12H
2O: 3～103EtOH: 0.001～0.05HCl: 0.01～0.5 toluene.With anodic alumina films, impregnated in silica precursors solution, vacuum condition volatilized 0.2～2 hour down, and solution forms sticking shape colloidal sol; Take out pellumina, room temperature nature crystallization.Segmented copolymer P123 in the alcohol reflux spe membrane, drying at room temperature.
(2) Filtration Adsorption of enzyme
The Sodium phosphate, dibasic of preparation 0.1mol/L and the potassium dihydrogen phosphate of 0.1mol/L; Prepare the buffered soln of pH 6～8 with this; Perhaps prepare tris solution and the 0.1mol/L hydrochloric acid soln of 0.1mol/L, prepare the buffered soln of pH 8～9 with this; Enzyme is dissolved in one of above-mentioned two kinds of buffered soln, the enzyme solution of preparation 0.5～5mg/mL; The aluminium oxide-silicon oxide composite package is placed membrane filter appts, and 1～5mL enzyme solution passes through the aluminium oxide-silicon oxide composite package under 50～1000Pa pressure;
(3) preparation of aluminium oxide-silicon oxide-enzyme-medium hole nano particles film multilevel hierarchy
A, cetyl trimethylammonium bromide CTAB is dissolved in 40～80 ℃ of deionized waters, adds ammoniacal liquor, dropwise add tetraethoxy again, vigorous stirring 0.5～3 hour is transferred in the hydrothermal reaction kettle 80～150 ℃ of crystallization 10～24 hours again; The molar ratio of each material of solution is 1TEOS: 0.004～0.08CTAB: 10～40NH3: 500～2000H
2O; The gained solid filtering separates, and is dry down at 60～140 ℃ behind the deionized water wash; Through 400～600 ℃, CTAB is removed in roasting in 2～6 hours, obtains medium hole nano particles;
B, with medium hole nano particles earlier with Virahol ultra-sonic dispersion 0.5～2 hour, dilute with water again, continuation ultra-sonic dispersion 0.5～2 hour; Medium hole nano particles suspension liquid concentration is 0.4～4wt%, and Virahol and quality ratio are 1: 1～1: 5;
C, general aluminium oxide-silicon oxide composite package vertical dipping in the medium hole nano particles suspension liquid of adsorptive enzyme lift 1～5 time, flood 1～10 minute at every turn, at the uniform velocity lift, and lift pull rate 0.01～0.3cm/s behind the natural drying at room temperature next time.
The aluminium oxide-silicon oxide composite package of step (1), mesoporous aperture homogeneous, 5～12nm is adjustable for its size.In the step (2), the size of selected enzyme should be less than the size in mesoporous aperture, and the immobilization amount of enzyme is 0.5～3mg; Step (3) forms the medium hole nano particles film of 1～5 μ m at the upper and lower surfaces of aluminium oxide-silicon oxide composite package.
The invention has the advantages that: utilize the high-ratio surface and the hole dimension that is fit to of mesopore silicon oxide, have higher enzyme charge capacity; The weak interaction of enzyme and silicon oxide surface does not influence the activity of enzyme, and the modification of medium hole nano particles film can stop the loss of enzyme; This immobilized enzyme not only has good activity and operational stability, the operate continuously of all-in-one-piece membrane structure realization response simultaneously and the direct separation of product.
Embodiment
Embodiment 1
(1) preparation of aluminium oxide-silicon oxide composite package
With being dissolved in 5g ethanol and 1.2g under 1g polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer (P123) room temperature, in the mixed solution of 0.2mol/L hydrochloric acid, add 0.03g toluene, stirred 2 hours.2.08g tetraethoxy dropwise adds in the solution, stirs to obtain silica precursors solution in 1 hour.With the 47mm diameter, the anodic alumina films in 200nm aperture impregnated in silica precursors solution, and vacuum condition volatilized 1 hour down.Take out pellumina, natural crystallization 1 day.180mL ethanol divides the 24 hours extraction segmented copolymer P123 that reflux for three times.Drying at room temperature.
(2) Filtration Adsorption of oxyphorase
Get 5mL, the Sodium phosphate, dibasic of 0.1mol/L and 90mL, the potassium dihydrogen phosphate of 0.1mol/L, the buffered soln of preparation pH pH 6 is prepared the hemoglobin solutions of 2mg/mL with this.The aluminium oxide-silicon oxide composite package is placed membrane filter appts, and the 3mL hemoglobin solutions passes through the aluminium oxide-silicon oxide composite package under 300Pa pressure.The fixed amount of hemoglobinase is 3.0mg.
(3) preparation of aluminium oxide-silicon oxide-oxyphorase-medium hole nano particles film multilevel hierarchy
A, be dissolved in the 250mL deionized water with under 80 ℃ of the 0.25g cetyl trimethylammonium bromides (CTAB), add 17mL ammoniacal liquor, dropwise add the 2.97g tetraethoxy again, vigorous stirring 1 hour is transferred in the hydrothermal reaction kettle 100 ℃ of crystallization 12 hours again.The gained solid filtering separates, and is dry down at 100 ℃ behind the deionized water wash.Through 550 ℃, CTAB is removed in roasting in 4 hours, obtains medium hole nano particles.
B, with the 300mg medium hole nano particles earlier with 3.6g Virahol ultra-sonic dispersion 1 hour, dilute continuation ultra-sonic dispersion 1 hour again with 14.4g water.
C, general aluminium oxide-silicon oxide composite package vertical dipping in the medium hole nano particles suspension liquid of adsorptive enzyme lift 2 times, flood 5 minutes at every turn, are at the uniform velocity lifted by computer control, lift pull rate 0.15cm/s behind the natural drying at room temperature next time.
The film of nanoparticles of d, formation is about 2.6 μ m.
(4) performance test
Multilevel hierarchy immobilization oxyphorase is carried out the test of leachability.Get 60mL, the Sodium phosphate, dibasic of 0.1mol/L and 40mL, the potassium dihydrogen phosphate of 0.1mol/L, the buffered soln of preparation pH 7.Get the 3mL damping fluid at 25 ℃, 1kPa pressure through multilevel hierarchy immobilization oxyphorase, circulates and does not find any loss ten times down.As a comparison, do not pass through the film modified aluminium oxide-silicon oxide composite package of medium hole nano particles, the oxyphorase of ten circulation backs about 40% runs off.Multilevel hierarchy immobilization oxyphorase is carried out active testing, i.e. catalyzed oxidation O-Phenylene Diamine is with 3mL; The 6mmol/L substrate solution is at 25 ℃, and through multilevel hierarchy immobilization oxyphorase, activity is 0.06 μ mol/min to 1kPa pressure down; Circulate ten times, activity remains unchanged basically.
Embodiment 2
(1) preparation of aluminium oxide-silicon oxide composite package
With being dissolved in 5g ethanol and 1g under 1g polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer (P123) room temperature, in the mixed solution of 0.2mol/L hydrochloric acid, add 0.15g toluene, stirred 2 hours.2.08g tetraethoxy dropwise adds in the solution, stirs to obtain silica precursors solution in 1 hour.With the 47mm diameter, the anodic alumina films in 200nm aperture impregnated in silica precursors solution, and vacuum condition volatilized 1 hour down.Take out pellumina, natural crystallization 1 day.180mL ethanol divides the 24 hours extraction segmented copolymer P123 that reflux for three times.Drying at room temperature.
(2) Filtration Adsorption of myohaemoglobin
Get 10mL, the Sodium phosphate, dibasic of 0.1mol/L and 90mL, the potassium dihydrogen phosphate of 0.1mol/L, the buffered soln of preparation pH 6 is prepared the myohaemoglobin solution of 2mg/mL with this.The aluminium oxide-silicon oxide composite package is placed membrane filter appts, and 3mL myohaemoglobin solution passes through the aluminium oxide-silicon oxide composite package under 300Pa pressure.The fixed amount of myohaemoglobin is 0.7mg.
(3) preparation of aluminium oxide-silicon oxide-myohaemoglobin-medium hole nano particles film multilevel hierarchy
A, be dissolved in the 250mL deionized water with under 70 ℃ of the 0.25g cetyl trimethylammonium bromides (CTAB); Add 20mL ammoniacal liquor, temperature rises to 80 ℃, dropwise adds the 2.97g tetraethoxy; Vigorous stirring 1 hour is transferred in the hydrothermal reaction kettle 100 ℃ of crystallization 12 hours again.The gained solid filtering separates, and is dry down at 100 ℃ behind the deionized water wash.Through 550 ℃, CTAB is removed in roasting in 4 hours, obtains medium hole nano particles.
B, with the 400mg medium hole nano particles earlier with 3.6g Virahol ultra-sonic dispersion 1 hour, dilute continuation ultra-sonic dispersion 1 hour again with 14.4g water.
C, general aluminium oxide-silicon oxide composite package vertical dipping in the medium hole nano particles suspension liquid of adsorptive enzyme lift 3 times, flood 5 minutes at every turn, are at the uniform velocity lifted by computer control, lift pull rate 0.05cm/s behind the natural drying at room temperature next time.
The film of nanoparticles of d, formation is about 4 μ m.
(4) performance test
Multilevel hierarchy immobilization myohaemoglobin is carried out the test of leachability.Get 60mL, the Sodium phosphate, dibasic of 0.1mol/L and 40mL, the potassium dihydrogen phosphate of 0.1mol/L, the buffered soln of preparation pH 7.As a comparison, do not pass through the film modified aluminium oxide-silicon oxide composite package of medium hole nano particles, the myohaemoglobin of ten circulation backs about 40% runs off.Multilevel hierarchy immobilization myohaemoglobin is carried out active testing, i.e. catalyzed oxidation O-Phenylene Diamine is with 3mL; The 6mmol/L substrate solution is at 25 ℃, and through multilevel hierarchy immobilization oxyphorase, activity is 0.02 μ mol/min to 1kPa pressure down; Circulate ten times, activity remains unchanged basically.
Embodiment 3
(1) preparation of aluminium oxide-silicon oxide composite package
With being dissolved in 4.8g ethanol and 1.2g under 0.687g polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer (P123) room temperature, in the mixed solution of 0.2mol/L hydrochloric acid, add 0.01g toluene, stirred 2 hours.2.08g tetraethoxy dropwise adds in the solution, stirs to obtain silica precursors solution in 1 hour.With the 47mm diameter, the anodic alumina films in 200nm aperture impregnated in silica precursors solution, and vacuum condition volatilized 1 hour down.Take out pellumina, natural crystallization 1 day.180mL ethanol divides the 24 hours extraction segmented copolymer P123 that reflux for three times.Drying at room temperature.
(2) tryptic Filtration Adsorption
Get 100mL, trihydroxy-aminomethane and the 11.4mL of 0.1mol/L, the hydrochloric acid soln of 0.1mol/L, the buffered soln of preparation pH 9 is prepared the trypsin solution of 1mg/mL with this.The aluminium oxide-silicon oxide composite package is placed membrane filter appts, and the 5mL trypsin solution passes through the aluminium oxide-silicon oxide composite package under 200Pa pressure.Tryptic fixed amount is 2.3mg.
(3) preparation of aluminium oxide-silicon oxide-trypsinase-medium hole nano particles film multilevel hierarchy
A, be dissolved in the 250mL deionized water with under 60 ℃ of the 0.25g cetyl trimethylammonium bromides (CTAB); Add 17mL ammoniacal liquor, temperature rises to 80 ℃, dropwise adds the 2.97g tetraethoxy; Vigorous stirring 1 hour is transferred in the hydrothermal reaction kettle 100 ℃ of crystallization 12 hours again.The gained solid filtering separates, and is dry down at 100 ℃ behind the deionized water wash.Through 550 ℃, CTAB is removed in roasting in 4 hours, obtains medium hole nano particles.
B, with the 200mg medium hole nano particles earlier with 3.6g Virahol ultra-sonic dispersion 1 hour, dilute continuation ultra-sonic dispersion 1 hour again with 14.4g water.
C, general aluminium oxide-silicon oxide composite package vertical dipping in the medium hole nano particles suspension liquid of adsorptive enzyme lift 3 times, flood 5 minutes at every turn, are at the uniform velocity lifted by computer control, lift pull rate 0.1cm/s behind the natural drying at room temperature next time.
The film of nanoparticles of d, formation is about 4 μ m.
(4) performance test
With the multilevel hierarchy immobilizing trypsinase, carry out activity and stability test, i.e. hydrolysis is to toluene sulfo group-L-arginine methyl esters hydrochloride (TAME).Get 100mL, trihydroxy-aminomethane and the 58.4mL of 0.1mol/L, the hydrochloric acid soln of 0.1mol/L, the buffered soln of preparation pH 8 is prepared the TAME solution of 5mmol/L with this.The TAME solution of getting 3mL is at 25 ℃, and 1kPa pressure through the multilevel hierarchy immobilizing trypsinase, circulates ten times down, and activity remains unchanged basically, explains that this immobilized enzyme has good operational stability.When concentration of substrate during less than 1 μ mol/min substrate transform fully, realized the direct separation of product.
Claims (1)
1. the preparation method of a multilevel hierarchy immobilized enzyme is characterized in that, preparation process is:
(1) preparation of aluminium oxide-silicon oxide composite package
Polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer P123 is dissolved in the hydrochloric acid under the room temperature, the mixing solutions of ethanol EtOH and water; Add toluene, stirred 2 hours, dropwise add tetraethoxy TEOS again, continue to stir 1 hour, obtain silica precursors solution; Each material molar ratio of solution is 1TEOS: 0.006～0.06P123: 4～12H
2O: 3～103EtOH: 0.001～0.05HCl: 0.01～0.5 toluene, and HCl concentration is 0.2mol/L; With the 47mm diameter, the anodic alumina films in 200nm aperture impregnated in silica precursors solution, and vacuum condition volatilized 1 hour down, and solution forms sticking shape colloidal sol; Take out pellumina, room temperature nature crystallization 1 day; Segmented copolymer P123 in the alcohol reflux spe membrane, drying at room temperature;
(2) Filtration Adsorption of enzyme
When enzyme was trypsinase, trihydroxy-aminomethane solution and the 0.1mol/L hydrochloric acid soln of preparation 0.1mol/L were prepared the buffered soln of pH 8～9 with this; When enzyme was myohaemoglobin or oxyphorase, the Sodium phosphate, dibasic of preparation 0.1mol/L and the potassium dihydrogen phosphate of 0.1mol/L were prepared the buffered soln of pH 6～8 with this;
Enzyme is dissolved in one of above-mentioned two kinds of buffered soln, the enzyme solution of preparation 0.5～5mg/mL; The aluminium oxide-silicon oxide composite package is placed membrane filter appts, and 1～5mL enzyme solution passes through the aluminium oxide-silicon oxide composite package under 200～300Pa pressure;
(3) preparation of aluminium oxide-silicon oxide-enzyme-medium hole nano particles film multilevel hierarchy
A, cetyl trimethylammonium bromide CTAB is dissolved in 40～80 ℃ of deionized waters, adds ammoniacal liquor, dropwise add tetraethoxy again, vigorous stirring 1 hour is transferred in the hydrothermal reaction kettle 100 ℃ of crystallization 12 hours again; The molar ratio of each material of solution is 1TEOS: 0.004～0.08CTAB: 10～40NH
3: 500～2000H
2O; The gained solid filtering separates, and is dry down at 60～140 ℃ behind the deionized water wash; Through 400～600 ℃, CTAB is removed in roasting in 2～6 hours, obtains medium hole nano particles;
B, with medium hole nano particles earlier with Virahol ultra-sonic dispersion 0.5～2 hour, dilute with water again, continuation ultra-sonic dispersion 0.5～2 hour; Medium hole nano particles suspension liquid concentration is 0.4～4wt%, and Virahol and quality ratio are 1: 1～1: 5;
C, general aluminium oxide-silicon oxide composite package vertical dipping in the medium hole nano particles suspension liquid of adsorptive enzyme lift 1～5 time, flood 5 minutes at every turn, at the uniform velocity lift, and lift pull rate 0.01～0.3cm/s behind the natural drying at room temperature next time.
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| CN101451133A (en) * | 2009-01-04 | 2009-06-10 | 上海大学 | Method for preparing silicon base mesoporous molecular sieve SBA-15 immobilized enzyme biocatalyst |
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