CN101724619A - Enzyme immobilization application of mesoporous molecular sieve modified by functionalized ion liquid in - Google Patents
Enzyme immobilization application of mesoporous molecular sieve modified by functionalized ion liquid in Download PDFInfo
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- CN101724619A CN101724619A CN200910264822A CN200910264822A CN101724619A CN 101724619 A CN101724619 A CN 101724619A CN 200910264822 A CN200910264822 A CN 200910264822A CN 200910264822 A CN200910264822 A CN 200910264822A CN 101724619 A CN101724619 A CN 101724619A
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- molecular sieve
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- 150000002500 ions Chemical class 0.000 title claims abstract description 96
- 239000002808 molecular sieve Substances 0.000 title claims abstract description 91
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- 239000007788 liquid Substances 0.000 title claims abstract description 90
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 85
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 85
- -1 amnio group Chemical group 0.000 claims abstract description 16
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- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 6
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 8
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- 238000001291 vacuum drying Methods 0.000 description 4
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- MCMFEZDRQOJKMN-UHFFFAOYSA-N 1-butylimidazole Chemical compound CCCCN1C=CN=C1 MCMFEZDRQOJKMN-UHFFFAOYSA-N 0.000 description 2
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 2
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of enzyme immobilization vectors and discloses the enzyme immobilization application of a mesoporous molecular sieve modified by a functionalized ion liquid. The surface of the mesoporous molecular sieve is provided with an organic functional group from the functionalized ion liquid, which can be combined with the action of an amino acid residue. By utilizing designability of the ion liquid, the invention respectively introduces the functionalized ion liquid with alkyl, amnio group, carboxyl, hydroxyl and other functional groups to the surface of a mesoporous material; and by the actions of static, hydrogen bond, hydrophobic bond and the like of the groups and enzyme molecule amino acid residue, the invention enhances the bonding force of the vector and enzyme, changes the micro environment in which the enzyme is positioned and improves the catalytic performance of immobilized enzyme. The invention uses the mesoporous material modified by the functionalized ion liquid for the immobilization of the enzyme, not only provides a brand-new concept and enriches the method for modifying the mesoporous material, but also expands the application of the ion liquid and the mesoporous material in the field of enzyme engineering.
Description
Technical field
The invention belongs to the support of the catalyst field, relate to a kind of application of mesopore molecular sieve in enzyme immobilization of modified by functionalized ion liquid.
Background technology
Enzyme has characteristics such as high efficiency, specificity, mildness, environmental friendliness as catalyzer, is widely used in industries such as food, medicine and chemical industry.Wherein the relative resolvase of immobilized enzyme has good stability, easily separates, can reuse, is convenient to advantages such as the serialization of transporting and storing, helping producing and automatization from reactive system, therefore has more wide application space.And solid support material is as the part of immobilized enzyme, and its structure and performance have very big influence to the performance of immobilized enzyme.All can influence the residing microenvironment of enzyme as the specific surface area of carrier, pore size distribution, pore volume, topological framework, surface properties, stability etc., thereby change the catalysis characteristics of enzyme, so the selection of carrier is very important.
Mesoporous material (aperture: 2-50nm) have characteristics such as bigger specific surface area, orderly nano pore structure, bigger pore volume, narrower pore size distribution, good thermostability and chemical stability as a kind of carrier, activity hydroxy is rich on what is more important mesoporous material surface, can carry out finishing to it easily, change carrier characteristics.Therefore, mesoporous material has advantageous advantage and broad prospect of application in the immobilization of enzyme.By using enzyme-carrying silicon-based mesoporous material SBA-15, successful catalyzed oxidation has been separated out aldehydes matter in the water as Chinese patent CN101575135.Japanese Patent JP2009153448 has successfully realized the hydrolysis of starch by lytic enzyme is fixed on the mesoporous material.Chinese patent CN101451133 has also successfully loaded on horseradish peroxidase on the mesoporous material SBA-15.
Yet enzyme directly is fixed on the mesoporous material by physical adsorption way, and enzyme molecule and carrier bonding force are not strong, causes the immobilization efficiency of enzyme and stability to reduce easily, and enzyme is come off when wash-out and reaction easily.In addition, its katalysis is transferred to heterogeneously behind the enzyme immobilization by homogeneous phase, and the diffusion limitation effect of bringing thus, sterically hindered, partition effect etc. have a significant impact the catalysis characteristics of enzyme.(Enzyme?immobilization?using?SBA-15mesoporous?molecular?sieveswith?functionalised?surfaces.Journal?of?Molecular?Catalysis.B,Enzymatic,2001.15(1-3):81-92)。
Functionalized ion liquid is meant the salt of only being made up of zwitterion that at room temperature is in a liquid state, it have very strong solvability, almost do not have vapour pressure, characteristics such as thermostability, chemical stability are preferably arranged; In addition, can also change character such as ion liquid wetting ability, hydrophobicity, stickiness, fusing point, density by the structure that changes zwitterion, the synthetic ionic liquid of design with suitable certain reaction requirement, therefore, functionalized ion liquid is also claimed " designability solvent "; Simultaneously, ionic liquid is because environment amenable characteristic " green " solvent that is otherwise known as, and is used widely in fields such as synthetic in organic synthesis, sepn process, material, catalysis.And functionalized ion liquid has also obtained using widely in biocatalysis field in recent years, plurality of enzymes such as lipase, proteolytic enzyme, oxydo-reductase have all demonstrated good stability in functionalized ion liquid, and enzymatic zone, enantio-selectivity often all increase in functionalized ion liquid.(On?thebackground?of?enhanced?stability?and?reusability?of?enzymes?in?ionic?liquids.BiochemicalSociety?transactions.(2007),35(Pt?6),1624-1627.)
But, functionalized ion liquid all is to use as reaction medium basically in enzymic catalytic reaction at present, and its usage quantity is big, the cost height, and functionalized ion liquid also existence and product separation difficulty in some enzymic catalytic reaction, the viscosity height makes problems such as mass transfer is restricted.In the last few years, patent JP2009207492, JP2009203454, WO2008090156, CN101225427 etc. utilize " green " solvent and " designability solvent " character of functionalized ion liquid, simplified the product separating process, improved the stable or active of enzyme, but it is excessive to have run into the ionic liquid consumption equally, be not suitable for the problem of suitability for industrialized production, though and the stable stability that raising but still too late immobilized enzyme are arranged.
Summary of the invention
The objective of the invention is in order to overcome the deficiency that existing enzyme immobilization technology exists, the mesopore molecular sieve that provides a kind of ionic liquid to modify gets carrier as enzyme immobilization, improves the zymologic property of enzyme.
A kind of mesopore molecular sieve of modified by functionalized ion liquid, its surperficial existence can come from the organo-functional group of functionalized ion liquid with enzyme amino-acid residue effect bonded.
Wherein, described mesopore molecular sieve has SBA-15, SBA-16, MCM-41 or MCM-48 etc.
The positively charged ion of described functionalized ion liquid is the imidazole-like ionic that contains different organo-functional groups.Wherein, organo-functional group is alkyl, hydroxyl, carboxyl or amino, effects such as the static of the mesopore molecular sieve of modified by functionalized ion liquid provided by the invention by organo-functional group and enzyme, hydrogen bond, hydrophobic or chemical bonding strengthen the bonding force of enzyme and carrier, improve the catalysis characteristics of enzyme.
The negatively charged ion of described functionalized ion liquid is to have different hydrophilic or electronegative polarity ion, the preferred hydrophilic negatively charged ion of tetrafluoroborate and hydrophobic negatively charged ion of phosphofluoric acid, the mesopore molecular sieve of modified by functionalized ion liquid provided by the invention is by the zymologic property of the difference regulatory enzyme of negatively charged ion hydrophilic and hydrophobic matter.
The mean pore size of the former powder of described mesopore molecular sieve is 2-50nm, and specific surface area is at 300-1500m
2Between/the g, help the immobilization and the high dispersing of enzyme.
The mol ratio of the organo-functional group of described mesopore molecular sieve and functionalized ion liquid is 3-100: 1.
The preparation method of the mesopore molecular sieve of described modified by functionalized ion liquid comprises the steps (principle is seen Fig. 1 (a)):
In the reaction flask of packing into after the former powder vacuum outgas processing of mesopore molecular sieve, add chloro propyl group methoxy silane then and under chloroform refluxes, carry out linked reaction, add imidazoles again and replace, then add the organo-functional group donor and introduce different organo-functional group modifications, add KPF then
6Perhaps KBF
4Carry out ion-exchange, obtain modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector, after above-mentioned material is purified, carry out vacuum-drying at 50-150 ℃ and obtain the functionalization molecular sieve mesoporous material that modified by functionalized ion liquid is crossed; The wherein former powder of mesopore molecular sieve, solvent chloroform, chloro propyl group methoxy silane, imidazoles, organo-functional group donor and KPF
6Perhaps KBF
4Mol ratio be 3-100: 30: 1: 1: 1: 1.
Wherein, described organo-functional group is alkyl, hydroxyl, carboxyl or amino, and the organo-functional group donor is a halogenated alkane, the halogenated alkane of hydroxyl, the halogenated alkane of hydroxyl or the halogenated alkane of hydroxyl.
Organo-functional group is that the mesopore molecular sieve of the modified by functionalized ion liquid of alkyl also can prepare (principle is seen Fig. 1 (b)) as follows:
Alkyl imidazole compounds and chloropropyltrimethoxy silane are reacted backflow 10-40 hour under 50-150 ℃ of following nitrogen atmosphere, with product and the KPF that obtains
6Perhaps KBF
423~30 ℃ of stirrings obtained the thick liquid of ionic liquid in three days in acetone, and the thick liquid of ionic liquid is purified obtains ionic liquid; In the reaction flask of packing into after the former powder vacuum outgas processing of mesopore molecular sieve, add functionalized ion liquid then, under 50-100 ℃ of temperature, 20-40h refluxes in chloroform, obtain modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector, after above-mentioned material purification, carry out vacuum-drying at 50-150 ℃ and obtain the functionalization molecular sieve mesoporous material that modified by functionalized ion liquid is crossed, the wherein former powder of mesopore molecular sieve, solvent chloroform, alkyl imidazole, chloropropyltrimethoxy silane and KPF
6Perhaps KBF
4Mol ratio be 3-100: 30: 1: 1: 1.
The mesopore molecular sieve of modified by functionalized ion liquid of the present invention can be used as the fixation support of enzyme; be applied to the immobilization of enzyme, can be widely used in the immobilization of biological enzyme agent such as trypsinase, amylase, penicillin acylase, lipase or horseradish peroxidase.
Beneficial effect of the present invention:
The present invention is used to modify mesopore molecular sieve with functionalized ion liquid, given full play to mesoporous material and the functionalized ion liquid advantage in catalyzed reaction, and it is big to have overcome when functionalized ion liquid is as reaction medium in the prior art consumption, defectives such as production cost is high provide a kind of novel meso-porous molecular sieve material.This molecular sieve has improved the zymologic property of enzyme on fixation support, solved that the ubiquitous carrier loaded amount efficiency in current immobilized enzyme field is low, activity of the immobilized enzyme is not high enough, and when finishing group and enzyme during, often owing to the three-dimensional structure picture of enzyme and flexibility thereof are restricted the enzyme problem such as live that influences with the chemical bond covalent attachment.
The present invention by the ionic liquid grafting on mesoporous material, reduce make full use of that ionic liquid has in the ionic liquid consumption than the strong solubility energy, make the heterogeneous reaction of immobilized enzyme catalysis have the characteristics of accurate homogeneous reaction, alleviate the influence of diffusional limitation, developed a kind of novelty, enzyme immobilization mesoporous material efficiently enzyme catalysis efficient.
The present invention modifies the immobilization that mesoporous material is used for enzyme with functional ionic liquids, is not only to have proposed a completely new concept, has also expanded ionic liquid and mesoporous material in the enzyme engineering Application for Field.
The mesopore molecular sieve of modified by functionalized ion liquid provided by the invention, its surface exist can and the enzyme amino-acid residue between produce the ionic liquid organo-functional group of static, hydrogen bond, effect such as hydrophobic, can effectively combine with enzyme and improve zymologic property, can not cause big destruction to enzymatic structure again, synoptic diagram is seen Fig. 2.
The mesopore molecular sieve of modified by functionalized ion liquid provided by the invention has the duct of aperture 2-50nm distribution homogeneous, and it is close with the size of enzyme molecule that the size in duct can be tuned to, and makes the enzyme molecule can enter into its nano-scale duct.
The mesopore molecular sieve specific surface area of modified by functionalized ion liquid provided by the invention is at 300-1500m
2/ g, enzyme supported quantity height, the activity of the immobilized enzyme of preparation and stability are high.
Utilize the mesopore molecular sieve immobilized enzyme of modified by functionalized ion liquid provided by the invention, the immobilized reactant mild condition can be carried out under the neutrallty condition about 30-80 ℃, and operating process is simple, is suitable for industrial applications.
The mesopore molecular sieve surface of modified by functionalized ion liquid provided by the invention shows inertia in enzyme-catalyzed reaction, and carrier itself has chemical stability and heatproof thermostabilitys such as good acid resistance, organic solvent resistance.
Utilize the immobilized enzyme of mesopore molecular sieve of modified by functionalized ion liquid provided by the invention to separate with product and recovery and immobilized enzyme apparent activity and repeated use number of times height by simple filtration.
Description of drawings
Synthetic and the grafting of Fig. 1 functionalized ion liquid is at the schematic diagram on mesopore molecular sieve surface;
Wherein, figure (a) synthesize for functionalized ion liquid and grafting at the procedure chart on mesopore molecular sieve surface, wherein R is an alkyl, or the organic group of functional groups such as hydroxyl, carboxyl, amido; Figure (b) is the preparation process figure of mesopore molecular sieve of the modified by functionalized ion liquid of alkyl for organo-functional group, and wherein R is alkyl such as methyl, ethyl, propyl group, butyl.
The mimic diagram of Fig. 2 modified by functionalized ion liquid mesoporous material immobilized enzyme catalysis agent.
The modified by functionalized ion liquid mesopore molecular sieve infared spectrum of Fig. 3 embodiment 1 preparation
Fig. 4 temperature is to the figure that influences of immobilized porcine pancreatic lipase and free porcine pancreatic lipase.
The operational stability figure of Fig. 5 immobilized porcine pancreatic lipase.
Embodiment
1) mesoporous molecular sieve SBA-15 is synthetic: according to people such as Zhao Dongyuan at (Triblock Copolymer Syntheses ofMesoporous Silica with Periodic 50to 300Angstrom Pores.Science, 1998, narration 279:548-553), prepare the SBA-15 molecular screen primary powder, the aperture is 10nm, and specific surface area is 350m
2/ g.
2) functionalized ion liquid is synthetic: 1-Methylimidazole and chloropropyltrimethoxy silane were 120 ℃ of following back flow reaction 40 hours, be cooled to room temperature, the product and the KPF6 stirring at room in acetone that obtain were obtained the thick liquid of functionalized ion liquid in three days, the thick liquid of ionic liquid is filtered, washing, separatory, underpressure distillation steams acetone and water, obtain functionalized ion liquid, wherein 1-Methylimidazole, chloropropyltrimethoxy silane and KPF
6Mol ratio is 1: 1: 1.
3) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: 180 ℃ of vacuum-drying activatory 1g molecular screen primary powders are dispersed in the 50ml chloroform, add above-mentioned ionic liquid, with the ratio of the former powder molar mass of mesopore molecular sieve be 1: 50, at 85 ℃ of backflow 26h, filter and washing, in vacuum drying oven, be dried to constant weight, obtain the modified by functionalized ion liquid mesoporous molecular sieve enzyme immobilized carrier.Through infrared detection, infared spectrum shows that ionic liquid success grafting on the surface of molecular sieve, sees Fig. 3; Results of elemental analyses shows that the ionic liquid scale of construction of load on the 1g mesoporous material is 0.2mmol.
4) enzyme immobilization: the Tris-HCl damping fluid of getting 25ml PH=7.5 adds in the Erlenmeyer flask, adds mesoporous carrier of 0.5g and 25ml deionized water again; Stir evenly in room temperature in the weighing 0.125g porcine pancreatic lipase adding Erlenmeyer flask; 10h vibrates in the vibrator of 30 ℃ of temperature; Suction filtration, being fixed enzyme.
5) enzyme activity determination:
Carrier immobilized porcine pancreatic lipase hydrolysis glycerine triacetate emulsion is a probe reaction.30 ℃ of temperature, PH is 7.5 o'clock, adopts the vigor of this immobilized enzyme of determination of acid-basetitration, and apparent activity is a 450IU/g lipase.
Wherein enzyme is lived and to be defined as: 30 ℃ of temperature, PH is 7.5 o'clock, the enzyme amount that needs when per minute enzyme catalysis glycerine triacetate generates 1 micromole's acetic acid, and calculation formula is:
U=(V-V
0)×50/(10×0.25)=20(V-V
0)
Wherein: V---the volume of the NaOH solution that sample consumes, ml;
V
0---the volume of the NaOH solution that blank sample consumes, ml;
50---micromole's number of the NaOH that every milliliter of NaOH solution contains;
10---reaction times, min;
0.25---resolvase or immobilized enzyme amount, g..
Investigate the ordinary method of enzyme temperature stability according to this area and investigated this immobilized enzyme and the temperature stability of free porcine pancreatic lipase in 28~40 ℃ of scopes respectively, the results are shown in Figure 4.As seen from Figure 4, the optimal reaction temperature of resolvase is 30 ℃, not modified corresponding mesopore molecular sieve the optimal reaction temperature of immobilized porcine pancreatic lipase at 36 ℃, and the mesopore molecular sieve of the prepared modified by functionalized ion liquid of the present invention the optimal reaction temperature of immobilized porcine pancreatic lipase be 38 ℃, and apparent enzyme work significantly improves, as seen by the mesopore molecular sieve of the prepared modified by functionalized ion liquid of the present invention as fixation support fixed porcine pancreatic lipase, its temperature stability obviously improves than resolvase and the not modified immobilized porcine pancreatic lipase of mesopore molecular sieve.
The mesopore molecular sieve of having investigated this modified by functionalized ion liquid according to the ordinary method of this area investigation enzyme operational stability reaches the not modified immobilized porcine pancreatic lipase of corresponding mesopore molecular sieve, result such as Fig. 5.Mesopore molecular sieve through modified by functionalized ion liquid provided by the present invention as shown in Figure 5 immobilized porcine pancreatic lipase than not modified corresponding mesopore molecular sieve immobilized porcine pancreatic lipase have more superior operational stability, activity was still initial apparent activity 80% or more (and wherein free-fat enzyme enzyme live be 190IU/g lipase) after the former reused 5 times.
1) mesopore molecular sieve MCM-48's is synthetic: according to Mobil company at (Ordered mesoporous molecular sievessynthesized by a liquid-crystal template mechanism.Nature, 1992.359 (6397): the narration 710-712), prepare the MCM-41 molecular screen primary powder, the aperture is 5nm, and specific surface area is 1500m
2/ g.
2) functionalized ion liquid is synthetic: 1-butyl imidazole and chloropropyltrimethoxy silane were cooled to room temperature and obtain product in 40 hours (adding drying tube) of 120 ℃ of following back flow reaction. with product and the KPF that obtains
6Stirring at room obtained ionic liquid in three days in acetone. and obtain product and filter, washing, separatory, underpressure distillation steams acetone and water, obtains ionic liquid, wherein 1-butyl imidazole, chloropropyltrimethoxy silane and KPF
6Mol ratio is 1: 1: 1.
3) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: the operational condition and the process of modifying with embodiment 1 are identical, and the ratio that adds ion liquid amount and the former powder molar mass of mesopore molecular sieve is 1: 4.Results of elemental analyses shows that the raw powder supported ionic liquid scale of construction of 1g mesopore molecular sieve is 0.3mmol.
4) operational condition with embodiment 1 preparation immobilized enzyme is identical, the apparent activity that records the immobilized porcine pancreatic lipase of this preparing carriers is a 650IU/g lipase, and the work of the enzyme of resolvase is 190IU/g lipase, and the apparent activity of the immobilized porcine pancreatic lipase of unmodified is a 250IU/g lipase.
5) 1) mesopore molecular sieve MCM-48's is synthetic: according to Mobil company at (Ordered mesoporous molecular sievessynthesized by a liquid-crystal template mechanism.Nature, 1992.359 (6397): the narration 710-712), prepare the MCM-48 molecular screen primary powder, the aperture is 8nm, and specific surface area is 1400m
2/ g.
2) functionalized ion liquid is synthetic: 1-ethyl imidazol(e) and chloropropyltrimethoxy silane were cooled to room temperature and obtain product in 40 hours (adding drying tube) of 120 ℃ of following back flow reaction.With product and the KPF that obtains
6Stirring at room obtained ionic liquid in three days in acetone. and obtain product and filter, washing, separatory, underpressure distillation steams acetone and water, obtains ionic liquid. wherein 1-ethyl imidazol(e), chloropropyltrimethoxy silane and KPF
6Mol ratio is 1: 1: 1.
3) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: the operational condition and the process of modifying with embodiment 1 are identical, and the ratio that adds ion liquid amount and molecular sieve molar mass is 1: 10.Results of elemental analyses shows that the raw powder supported ionic liquid scale of construction of 1g mesopore molecular sieve is 1mmol.
4) enzyme immobilization: with the operational condition identical (using penicillin acylase instead) of embodiment 1 preparation immobilized enzyme; 35 ℃ of temperature; PH is in 7.5 the phosphate buffer soln; hydrolyzing penicillin G reacts 10min; the apparent activity that records the immobilized penicillin acylated enzyme of this preparing carriers with acid base titration (seeing embodiment 1) is the 1600IU/g penicillin acylase; and the work of the enzyme of resolvase is the 400IU/g penicillin acylase, and the apparent activity of the immobilized penicillin acylated enzyme of unmodified is the 850IU/g penicillin acylase.
1) mesopore molecular sieve MCM-41's is synthetic: according to Mobil Res.And Dev.Corp company at (ANew Familyof Mesoporous Molecular Sieves Prepared with Liquid Crystal Templates.J.Am.Chem.Sot.1992,114,10834-10843.) in narration, prepare the MCM-41 molecular screen primary powder, the aperture is 5nm, and specific surface area is 1200m
2/ g.
2) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: in the reaction flask of packing into after the former powder vacuum outgas processing of 1g mesopore molecular sieve, add chloro propyl group methoxy silane and under the 50ml chloroform refluxes, carry out linked reaction, adding imidazoles again replaces, then introduce functional group's material Mono Chloro Acetic Acid, add KBF then
4Carry out ion-exchange, obtain modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector, results of elemental analyses shows that the raw powder supported ionic liquid scale of construction of 1g mesopore molecular sieve is 0.3mmol.The wherein former powder of mesopore molecular sieve, chloro propyl group methoxy silane, imidazoles, Mono Chloro Acetic Acid and KBF
4Mol ratio be 4: 1: 1: 1: 1.
3) enzyme immobilization: with the operational condition identical (using penicillin acylase instead) of embodiment 1 preparation immobilized enzyme; 35 ℃ of temperature; PH is in 7.5 the phosphate buffer soln; hydrolyzing penicillin G reacts 10min; the apparent activity that records the immobilized penicillin acylated enzyme of this preparing carriers with acid base titration (seeing embodiment 1) is the 1800IU/g penicillin acylase; and the work of the enzyme of resolvase is the 400IU/g penicillin acylase, and the apparent activity of the immobilized penicillin acylated enzyme of unmodified is the 850IU/g penicillin acylase.
1) mesopore molecular sieve MCM-41's is synthetic: according to Mobil Res.And Dev.Corp company at (A New Family ofMesoporous Molecular Sieves Prepared with Liquid Crystal Templates.J.Am.Chem.Sot.1992,114,10834-10843.) in narration, prepare the MCM-41 molecular screen primary powder, the aperture is 6nm, and specific surface area is 1400m
2/ g.
2) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: in the reaction flask of packing into after the former powder vacuum outgas processing of 1g mesopore molecular sieve, add chloro propyl group methoxy silane then and under the 75ml chloroform refluxes, carry out linked reaction, adding imidazoles again replaces, then introduce functional group's material bromoethanol, add KPF then
6Carry out ion-exchange, obtain modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector, results of elemental analyses shows that the raw powder supported ion liquid amount of 1g mesopore molecular sieve is 0.15mmol.Wherein molecular sieve, chloro propyl group methoxy silane, imidazoles, bromoethanol and KPF
6Mol ratio be 50: 1: 1: 1: 1.
3) enzyme immobilization: with the operational condition identical (using amylase instead) of embodiment 1 preparation immobilized enzyme), 65 ℃ of temperature, PH is in 6.5 the buffered soln, hydrolyzed starch reaction 5min, with spectrophotometry (discussion of determination of amylase method. the Jiangsu food fermentation, 1996) apparent activity that records the immobilization diastase of this preparing carriers is 6U/ml, and the enzyme of resolvase is lived and is 2U/ml, and the apparent activity of the immobilization diastase of unmodified is a 3.5U/ml amylase.
Embodiment 6
1) mesoporous molecular sieve SBA-15 is synthetic: according to people such as Zhao Dongyuan at (Triblock Copolymer Syntheses ofMesoporous Silica with Periodic 50to 300Angstrom Pores.Science, 1998, narration 279:548-553), prepare the SBA-15 molecular screen primary powder, the aperture is 15nm, and specific surface area is 300m
2/ g.
2) preparation of modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector: in the reaction flask of packing into after the former powder vacuum outgas processing of 1g mesopore molecular sieve, add chloro propyl group methoxy silane then and under the 50ml chloroform refluxes, carry out linked reaction, adding imidazoles again replaces, then introduce functional group's material chlorethamin, add KBF then
4Carry out ion-exchange, obtain modified by functionalized ion liquid mesopore molecular sieve fixed enzyme vector, results of elemental analyses shows that the raw powder supported ionic liquid scale of construction of 1g mesopore molecular sieve is 0.5mmol.Wherein molecular sieve, chloro propyl group methoxy silane, imidazoles, chloroethanol and KBF
4Mol ratio be 20: 1: 1: 1: 1.
3) enzyme immobilization: with the operational condition identical (using amylase instead) of embodiment 1 preparation immobilized enzyme), 65 ℃ of temperature, PH is in 6.5 the buffered soln, hydrolyzed starch reaction 5min, with spectrophotometry (discussion of determination of amylase method. the Jiangsu food fermentation, 1996) apparent activity that records the immobilization diastase of this preparing carriers is that 6U/ml and the enzyme of resolvase are lived and be 2U/ml, and the apparent activity of the immobilization diastase of unmodified is a 3.5U/ml amylase.
Claims (10)
1. the mesopore molecular sieve of a modified by functionalized ion liquid is characterized in that mesopore molecular sieve surface existence can come from the organo-functional group of functionalized ion liquid with enzyme amino-acid residue effect bonded.
2. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 1 is characterized in that described mesopore molecular sieve is SBA-15, MCM-41, MCM-48 or SBA-16.
3. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 1, the positively charged ion that it is characterized in that described functionalized ion liquid is the imidazole-like ionic that contains different organo-functional groups.
4. according to the mesopore molecular sieve of claim 1 or 3 described modified by functionalized ion liquid, it is characterized in that described organo-functional group is alkyl, hydroxyl, carboxyl or amino.
5. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 1, the negatively charged ion that it is characterized in that described functionalized ion liquid is to have different hydrophilic or electronegative polarity ion.
6. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 5, the negatively charged ion that it is characterized in that described functionalized ion liquid is the hydrophilic negatively charged ion of tetrafluoroborate and hydrophobic negatively charged ion of phosphofluoric acid.
7. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 1 is characterized in that the mean pore size of described mesopore molecular sieve is 2-50nm, and specific surface area is at 300-1500m
2Between/the g.
8. the mesopore molecular sieve of modified by functionalized ion liquid according to claim 1, the mol ratio that it is characterized in that the organo-functional group of described mesopore molecular sieve and functionalized ion liquid is 3-100: 1.
9. the application of mesopore molecular sieve in enzyme immobilization of the described modified by functionalized ion liquid of claim 1.
10. the application of the mesopore molecular sieve of the described modified by functionalized ion liquid of claim 1 in trypsinase, amylase, penicillin acylase, lipase or horseradish peroxidase immobilization.
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