CN102703412A - Aramagnetic aldehyde group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof - Google Patents
Aramagnetic aldehyde group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses an aramagnetic aldehyde group mesoporous molecular sieve for immobilizing biological enzymes, and a preparation method thereof. According to the invention, gamma aldehyde glycidoxy propyl trimethoxy silane is utilized to introduce aldehyde groups onto the surface of the mesoporous molecular sieve, and then a covalent bond is utilized to immobilize aramagnetic Fe3O4 nano particles and biological enzymes onto the outer surface and the inner surface of the mesoporous molecular sieve respectively, wherein the aramagnetic Fe3O4 nano particles are subjected to gamma-amino propyl triethoxy silane surface modification and have the particle sizes larger than the pore diameter of the mesoporous molecular sieve, as a result, specially-structured aramagnetic immobilized enzymes can be prepared and are separated from the liquid phase system easily under the action of an external magnetic field, and further, the property and the separation efficiency of the immobilized enzymes are improved. The aramagnetic aldehyde group mesoporous molecular sieve can be used for immobilization of water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucosylase, trypsin and amylase, the activity of the prepared aramagnetic immobilized penicillin acylase is 9901 U/g, and 91.2% of the initial activity is retained after ten times of cycle use.
Description
Technical field
The present invention relates to a kind of paramagnetic aldehyde group mesoporous molecular sieve that is used for bio-enzyme immobilization and preparation method thereof, belong to biological technical field.
Background technology
Semacylase (EC 3.5.1.11; Enzyme molecular dimension:
) is the enzyme of most critical during semi-synthetic β-Nei Xiananleikangshengsu is produced; It can catalysis penicillium mould and the ring expansion acid hydrolysis remove side chain; Produce the important intermediate 6-amino-penicillanic acid (6-APA) and the 7-amino-3-deacetoxy cefaeicosanoic acid (7-ADCA) of semi-synthetic β-Nei Xiananleikangshengsu; Again can catalysis 6-APA and 7-ADCA and side chain condensation; Produce multiple semi-synthetic β-Nei Xiananleikangshengsu (like Ampicillin, Amoxicillin, Cephalexin and Cefadroxil etc.).
China is populous; Demand to semi-synthetic β-Nei Xiananleikangshengsu is very big; With immobilized penicillin acylated enzyme explained hereafter semisynthetic penicillin is a green production process cheaply, has been classified as one of six big emphasis directions of China's biological medicine development from now on.Because engineered fast development; Recombinant penicillin acylase can efficiently express and mass production; Chinese Academy of Sciences's Shanghai school of life and health sciences has obtained the Semacylase engineering strain of high yield with the DNA operative technique, greatly reduces the production cost of resolvase.But the whole dependence on import of used carrier are gone up by China's industry at present, and the production cost of immobilized enzyme is also too high.Therefore; The synthetic technology of the fixation support of excellent performance has become the technical bottleneck of China's immobilized penicillin amidase technical development; Development has the fixation support synthetic route of China's independent intellectual property right; Fundamentally break away from the passive situation that the long-term dependence on import carrier of China is produced immobilized enzyme; Exploitation has the semi-synthetic β-Nei Xiananleikangshengsu enzyme method technique of independent intellectual property right, satisfying growing domestic needs and the international competitiveness that strengthens product, thereby makes positive contribution for the Sustainable development of the progress of China's enzyme immobilization technology and microbiotic industry.
Resolvase directly is used for catalytic process has many deficiencies, as unstable in high temperature, strong acid, highly basic and organic solvent, easy loss of catalytic activity; Resolvase reclaims difficulty, and is unreasonable economically, also causes product to be difficult to separate and purifies, and has a strong impact on quality product; Production process is difficult to realize operate continuously, can only disposable periodical operation etc.Immobilized enzyme has overcome the above-mentioned deficiency of resolvase, has not only kept the distinctive catalysis characteristics of resolvase, has also improved operational stability; Production process is easy to realize operate continuously; Reaction is easy to separate with product and can reuse after accomplishing, and the product purity of gained is high, and production cost is low.Therefore, the immobilization of enzyme is hot research fields such as catalytic chemistry, biological chemistry and materials chemistry always.
The bio-enzyme immobilization carrier can be divided into two types: inorganic carrier and organic carrier.Compare with widely used organic carrier; Inorganic carrier has higher physical strength and better chemical stability, and its structure and surface properties are controlled easily, and its outstanding advantage is that the enzyme of load just can be removed through simple process such as roastings; Carrier can be reused; This just greatly reduces the cost of immobilized enzyme, has also avoided the aftertreatment problem of the immobilized enzyme of inactivation, has alleviated the pressure to bad border.Novel mesopore molecular sieve has in bigger continuously adjustable aperture, high specific surface area, bigger loading capacity and the duct and is rich in the slightly acidic hydroxyl; Can make the bigger enzyme molecule of volume be fixed in the mesoporous neutralization reaction product of molecular sieve and in time diffuse out the duct; Keep the suitable microenvironment of immobilized enzyme; Thereby the immobilized enzyme that makes has advantages of high catalytic activity, and the use temperature of immobilized enzyme is lower simultaneously, can avoid the problem of the ubiquitous hydrothermal stability difference of mesopore molecular sieve; Therefore, mesopore molecular sieve is one type of rising enzyme immobilization novel inorganic carrier.
We discover (Micropor.Mesopor.Mater., 2008,114 (1-3): 507-510; J Mol.Catal.B-Enzym., 2004,30 (2): 75-81), the performance of immobilized penicillin acylated enzyme and the structure of mesopore molecular sieve (crystalline phase, aperture, pore volume and specific surface area) are closely related.The aperture of mesopore molecular sieve is the key factor that influences activity of the immobilized enzyme; When the aperture of mesopore molecular sieve during greater than the molecular dimension of Semacylase; In the immobilization process of enzyme; The enzyme molecule just gets into easily in the mesopore molecular sieve duct and combines with the functional groups on surface, makes full use of the pore volume of mesopore molecular sieve, and the immobilized enzyme that obtains has greater activity.The crystalline phase of mesopore molecular sieve, pore volume and specific surface area all have considerable influence to activity of the immobilized enzyme.SBA-15 mesopore molecular sieve and MCM-41 mesopore molecular sieve (activity of the immobilized enzyme is 402U/g) are similar, all are the one-dimensional tunnel structure of six side's phase p6mm, are the immobilized enzyme poor-performing (activity of the immobilized enzyme is 1343U/g) of preparing carriers with it; And the KIT-6 mesopore molecular sieve with cube phase Ia3d structure of larger aperture has and the similar three-dimensional open-framework of specific surface area, pore volume and co-continuous of MCM-48 mesopore molecular sieve (activity of the immobilized enzyme is 1509U/g); But the former has bigger aperture than the latter; The diffusion of enzyme, substrate and product molecule is had excellent transmission performance, is that the immobilized enzyme of preparing carriers has higher activity (activity of the immobilized enzyme is 3522U/g) with the former.Spumescence mesopore molecular sieve (Mesostructured Cellular Foams; MCFs) (J.Am.Chem.Soc., 1999,121 (1): 254-255); It is a kind of mesopore silicon oxide with ultra-large aperture and 3D foamy structure; Its spherical pore chamber becomes three-dimensional open-framework by the window connection of homogeneous, and the aperture is 16～42nm, and pore volume is 1.0～2.4cm
3/ g, owing to have bigger window diameter and spherical pore chamber, the transmission that makes more enzyme molecule can get into spherical pore chamber interior and substrate and product is more prone to.We discover, are that the activity of the immobilized penicillin acylated enzyme of preparing carriers can reach 9104U/g with the MCFs mesopore molecular sieve.Therefore, the MCFs mesopore molecular sieve is expected to become the enzyme immobilization inorganic carrier of excellent performance.
The mesopore molecular sieve particle diameter is more little, helps the immobilization and the activity that improve immobilized enzyme of Semacylase on the mesopore molecular sieve surface more, but causes immobilized enzyme in the repeated use process, to be difficult to effectively separated.Owing to normally combining with more weak hydrogen bond action power between the surperficial slightly acidic hydroxyl of Semacylase molecule and mesopore molecular sieve, in use a part of enzyme can come off, and operational stability remains further to be improved.The covalent attachment method is by covalent linkage the active nonessential side-chain radical of enzyme and the functional groups of fixation support to be carried out the method that coupling prepares immobilized enzyme; Combine with covalent linkage because of between enzyme and the carrier; Presenting good operational stability, is that widely used enzyme immobilization method is gone up in industry at present.Therefore, the present invention will be through the MCFs mesopore molecular sieve and the paramagnetic Fe of surface graft aldehyde radical functional group
3O
4Nanoparticle effectively combines to prepare paramagnetic aldehyde group mesoporous molecular sieve fixation support, and immobilized enzyme can be separated rapidly under the effect of externally-applied magnetic field, improves the separation efficiency of immobilized enzyme.
The magnetic mesoporous molecular sieve of bibliographical information all is with paramagnetic Fe at present
3O
4Or Fe
3O
4SiO
2Be nuclear, the magnetic mesoporous molecular sieve of synthetic kernel shell structure (Adv.Funct.Mater., 2004,14 (4): 345-351 under alkaline condition then; J.Am.Chem.Soc., 2005,127 (25): 8916-8917; J.Am.Chem.Soc., 2006,128 (22): 7130-7131; J.Am.Chem.Soc., 2008,130 (1): 28-29).And the MCFs mesopore molecular sieve is under strong acidic condition, to be the template synthetic with the P123 nonionogenic tenside, Fe
3O
4Under strong acidic condition, be difficult to be adsorbed on Fe by acid etching and P123 easily
3O
4SiO
2The surface just can't prepare paramagnetic MCFs mesopore molecular sieve with conventional magnetic mesoporous molecular sieve preparation method like this.People such as Lu Anhui (J.Am.Chem.Soc.; 2004; 126 (28): 8616-8617) with the polymethylmethacrylate be the hole-blocking agent of SBA-15 mesopore molecular sieve, then will coat the outside surface of the paramagnetism Co nanoparticle deposition of carbon-coating, remove hole-blocking agent with 850 ℃ of high-temperature roastings then at molecular sieve; Prepared paramagnetic SBA-15 mesopore molecular sieve, but should the preparation process comparatively loaded down with trivial details.
Chinese patent CN101139127A discloses a kind of functionalized silicon coating mesoporous magnetic carrier.This carrier is the composite magnetic carrier with the nucleocapsid structure of coated with silica magnetic ferroferric oxide formation; Thickness and uniformity coefficient through twice bag silicon technology increase silicon fiml; Effectively the protection magnetic carrier is avoided in application by acid and alkali corrosion; And this magnetic carrier surface bond has active amino or sulfydryl, can carry out selective chemical absorption to target substance.The used template of this carrier is a cetyl trimethylammonium bromide, and the carrier aperture that obtains is smaller, is not suitable for macromole enzyme and absorption of proteins.
Chinese patent CN102286455A discloses a kind of immobilization laccase and preparation method thereof.This material is to be carrier with magnetic mesoporous carbon, and laccase is fixed on the magnetic mesoporous carbon through physisorption, and the duct of magnetic mesoporous carbon is embedded in magnetic nanoparticle; Laccase adsorptive capacity on the magnetic mesoporous carbon is more than 140mg/g; The enzymic activity recovery is 60%～95%, and the material aperture is 4-18nm, saturation magnetisation value lower (4.1emu/g); The magnetic separating power is more weak, so that disengaging time is long.
Chinese patent CN101256864A discloses a kind of Superparamagnetism mesoporous silicon dioxide complex microsphere and preparation method thereof.The kernel of this complex microsphere is magnetic ferrite nanoparticle group bunch, the spheroid of the coating mesoporous silicon-dioxide of shell, and the quality percentage composition of ferrite nano particles is 40～80% in the particulate.This material has bigger specific surface area (300-1000m
2/ g) and stronger magnetic separating power (20-80emu/g), and good dispersivity in water, the surface is easily through modifying a laggard one-step functional, but the maximum diameter of hole of this material has only 3.8nm, and a lot of macromole enzymes are difficult to get into the duct, and adsorptive capacity is lower.
Chinese patent CN101752048A discloses a kind of orderly short duct magnetic mesoporous material.This mesoporous material in the alkaline system of traditional synthesize meso-porous material, add that the surface is coated with silicon oxide with Fe
3O
4Also pass through the pH value of conditioned reaction solution for the nano magnetic particle of magnetic kernel; Prepared a kind of ordered short-channel and pattern magnetic mesoporous material clearly; May be used in bioseparation, pharmaceutical carrier and the catalyzed reaction; But the aperture ratio of this material is less, is about 3nm, is difficult to the macromolecular enzyme of absorption it is got in the duct.
Chinese patent CN101966344A discloses a kind of hollow nucleocapsid mesoporous nano medicine-carried system and preparation and application with magnetic and luminescent properties.This mesoporous carrier is to be kernel with the magnetic ferroferric oxide Capsules, is shell with the mesoporous silicon oxide, and the shell outside surface is modified luminous organic material and biocompatible polymer; Have nucleocapsid structure and cavity structure; Size is even, good biocompatibility, and have good magnetic; For co-focusing imaging, zeugmatography and pharmaceutical carrier good application is arranged, but be not suitable for adsorbing the macromole enzyme.
Chinese patent CN102247803A discloses magnetic mesoporous Nano microsphere of a kind of core-shell type and preparation method thereof and application.With FeOxSiO
2MSiO
2Template place the solution of carbon source wetting and solidify after carry out carbonization, obtain mesoporous carbon/FeOxSiO
2MSiO
2Mixture with this mixture and sodium hydroxide solution reaction, promptly gets the magnetic mesoporous Nano microsphere of core-shell type; Diameter of micro ball is 100-800nm; The aperture is 2-3nm, for the good effect of having purified waste water, but has certain limitation for absorption macromole enzyme.
Chinese patent CN102389771A discloses the magnetic mesoporous silicon ball of a kind of small bell type preparation of adsorbent method.Through the selective etch process, utilize mesoporous silicon with macro nanometer γ-Fe
2O
3Particle coats, and obtains the magnetic mesoporous silicon microballoon of hollow matrix material, and as tsiklomitsin and 2 kinds of microbiotic of sulphamethazine in the sorbent material sharp separation aqueous systems, this synthetic material aperture is about 3nm, is not suitable for the absorption of macromole enzyme equally.
Summary of the invention
The object of the invention is exactly for the defective that overcomes above-mentioned prior art existence a kind of preparation method who improves the paramagnetic aldehyde group mesoporous molecular sieve that is used for bio-enzyme immobilization of immobilized enzyme performance and separation efficiency to be provided.
The object of the invention can be realized through following technical scheme: introduce aldehyde radical functional group with γ-aldehyde propyl trimethoxy silicane at the surfaces externally and internally of mesopore molecular sieve earlier, do not need further activation just can use the covalent attachment mode will pass through the paramagnetism Fe of γ-An Bingjisanyiyangjiguiwan finishing
3O
4Nanoparticle (Fe
3O
4Particle diameter is difficult to get in the duct of mesopore molecular sieve greater than the aperture of mesopore molecular sieve) the grafting internal surface that is fixed on mesopore molecular sieve at the outside surface and the enzyme of mesopore molecular sieve, thus improve the performance and the separation efficiency of immobilized enzyme.
Described γ-aldehyde propyl trimethoxy silicane is to the functional modification on mesopore molecular sieve surface; Be that mesopore molecular sieve and γ-aldehyde propyl trimethoxy silicane are added in the toluene solution; 100～120 ℃ of reflux 6～18 hours; Then mesopore molecular sieve is filtered with after the washing with alcohol, drying is 6～18 hours in 60～120 ℃ of vacuum drying ovens, obtains aldehyde group mesoporous molecular sieve.
The mass ratio of described γ-aldehyde propyl trimethoxy silicane and mesopore molecular sieve is 0.5～1.6: 1.
Described γ-An Bingjisanyiyangjiguiwan is to paramagnetism Fe
3O
4The finishing of nanoparticle is with usefulness coprecipitation method synthetic Fe
3O
4Nanoparticle is scattered in the ethanolic soln, adds γ-An Bingjisanyiyangjiguiwan then, and normal-temperature reaction is 10 hours under vigorous stirring, under externally-applied magnetic field, removes ethanolic soln at last, obtains the Fe of aminopropyl finishing
3O
4Nanoparticle.
Described γ-An Bingjisanyiyangjiguiwan and paramagnetism Fe
3O
4The mass ratio of nanoparticle is 0.5～2.0: 1.
The preparation method of described paramagnetic aldehyde group mesoporous molecular sieve is the paramagnetism Fe with the aminopropyl finishing
3O
4The aqueous suspension of nanoparticle is added drop-wise in the aqueous suspension of aldehyde group mesoporous molecular sieve; Normal-temperature reaction was removed the aqueous solution with the separation of magnet magnetic after 3～10 hours; In 40～80 ℃ of vacuum drying ovens dry 6～18 hours then, obtain paramagnetic aldehyde group mesoporous molecular sieve.
The paramagnetism Fe of described aminopropyl finishing
3O
4The mass ratio of nanoparticle and aldehyde group mesoporous molecular sieve is 0.1～0.6: 1.
Using the mean pore size of the paramagnetic aldehyde group mesoporous molecular sieve of method for preparing is 15～30nm, and specific surface area is 350～600m
2/ g, pore volume is at 1.5～2.0cm
3/ g, saturation magnetization is 10～25emu/g, can be used for the immobilization of water-soluble biological enzymes such as Semacylase, glucose isomerase, glucose transglucosidase, trypsinase and glycase, is specially adapted to the immobilization of Semacylase.
Compared with prior art, the preparing method's of paramagnetism aldehyde group mesoporous molecular sieve of the present invention remarkable advantage is, the particle diameter through will passing through finishing is greater than the paramagnetism Fe in mesopore molecular sieve aperture
3O
4Nanometer particle load is to the outside surface of aldehyde group mesoporous molecular sieve; Reduce of the obstruction of paramagnetism nanoparticle for the mesopore molecular sieve duct as far as possible; Reduction is to the influence of aperture, specific surface area and the pore volume of mesopore molecular sieve; Do not need further activation just can directly make enzyme be fixed on the internal surface of mesopore molecular sieve, prepare the paramagnetism immobilized enzyme of special construction, can make immobilized enzyme under the effect of externally-applied magnetic field with the covalent attachment mode; Be easy to from liquid-phase system, separate, thus the performance and the separation efficiency of raising immobilized enzyme.
One of remarkable advantage of paramagnetism aldehyde group mesoporous molecular sieve of the present invention is; The molecular dimension of the aperture ratio Semacylase of this mesopore molecular sieve is much bigger; And have bigger specific surface area and a pore volume; Help the immobilization of enzyme molecule and the diffusion of substrate and product molecule, thereby improve the activity of immobilized enzyme.
Two of the remarkable advantage of paramagnetism aldehyde group mesoporous molecular sieve of the present invention is that this mesopore molecular sieve will pass through the paramagnetism Fe of aminopropyl finishing through the aldehyde radical on surface with the covalent attachment mode
3O
4The internal surface that the nanoparticle grafting is fixed on mesopore molecular sieve at the outside surface and the enzyme of mesopore molecular sieve, thus the operational stability of immobilized enzyme improved.
Three of the remarkable advantage of paramagnetism aldehyde group mesoporous molecular sieve of the present invention is; This mesopore molecular sieve has paramagnetism, can under the effect of externally-applied magnetic field, be easy to from liquid-phase system, separate; When removing externally-applied magnetic field; Immobilized enzyme can be dispersed in the liquid-phase system again again, and is easy and simple to handle, easy for industrialized.
Description of drawings
Fig. 1 is preparation process and the immobilization synoptic diagram of enzyme of the paramagnetic aldehyde group mesoporous molecular sieve of special construction.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated.
In following examples, adopt following method to carry out the immobilization of Semacylase, and activity and the operational stability of measuring immobilized enzyme:
The immobilization of Semacylase: take by weighing 0.020g carrier and 6.0mL Semacylase solution (V through the phosphate buffer soln dilution of pH=7.8
Buffer/ V
Enzyme=9: 1) mix, put into 30 ℃ shaking bath immobilization and carry out magnetic after 12 hours and separate, the gained solid carries out determination of activity after repeatedly washing with the phosphate buffer soln of pH=7.8.
The determination of activity of immobilized enzyme (the penicilline g potassium salt hydrolysis prepares 6-APA): under 37 ℃ temperature; Penicilline g potassium salt brine solution (with the dilution of 0.1mol/L pH=7.8 phosphate buffer soln) uniform mixing with said fixing enzyme and 20mL 4wt%; Use the NaOH solution titration of concentration then as 0.1mol/L; Make the pH value of mixing solutions remain on 7.8, write down the consumption of NaOH in 10 minutes.Calculate the activity of immobilized enzyme then with following formula:
A(U/g)＝V
NaOH×C
NaOH×10
3/(m×t)
Wherein A represents the activity of immobilized enzyme; V
NaOHRepresent NaOH consumption (ml); C
NaOHRepresent NaOH concentration (mol/L); M represents carrier dry weight (g); The t representative test used time (min).
The operational stability of immobilized enzyme is measured: used immobilized enzyme solution is carried out magnetic separate, then immobilized enzyme is transferred in the reactor drum, adopt above-mentioned activity determination method to measure the activity of used immobilized enzyme.After recycling through 10 times, the activity of immobilized enzyme and the per-cent of initial activity are high more, explain that then the operational stability of immobilized enzyme is good more.
Comparative Examples
At ambient temperature, with 2.0g Pluronic P123 (EO
20PO
70EO
20, M
Av=5800) be dissolved in the HCl solution of 75mL 1.6mol/L; Treat that P123 dissolves the back fully and adds 0.023g Neutral ammonium fluoride and 3.0g 1,3, the 5-trimethylbenzene is warming up to 37 ℃ with solution then and also continues stirring after 1 hour, adds the 4.4g tetraethoxy, and 37 ℃ of lasting down stirrings 20 hours; Solution changed over to have in the teflon-lined hydrothermal crystallizing still, in 100 ℃ of hydrothermal crystallizings 24 hours; Treat that solution is cooled to the room temperature after-filtration and obtains white solid, dried overnight in 100 ℃ baking oven, program is warming up to 500 ℃ of roastings and obtained mesopore molecular sieve in 8 hours in retort furnace then.1.0g mesopore molecular sieve and 1.1g γ-aldehyde propyl trimethoxy silicane are joined in the 50mL toluene solution; 110 ℃ of reflux 12 hours; Mesopore molecular sieve is filtered the back use washing with alcohol, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 90 ℃ of vacuum drying ovens.Above-mentioned mesopore molecular sieve is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9901U/g; Through recycling back (carry out the separation of immobilized enzyme with whizzer, operating process is comparatively complicated) for 10 times, immobilized enzyme has kept 91.2% initial activity.
Embodiment 1
With 0.365g FeCl
36H
2O and 0.208g FeSO
47H
2O (Fe
3+/ Fe
2+Mol ratio is 1.8: 1) be dissolved in the 10mL water and be warming up to 80 ℃, regulate the pH value with strong aqua and be about 11, ageing is 2 hours under this temperature, is washed with water to the Fe that neutrality obtains 0.15g after being cooled to room temperature
3O
4Nanoparticle is scattered in the ethanolic soln of 150mL then, adds the γ-An Bingjisanyiyangjiguiwan of 0.290g, and normal-temperature reaction is 10 hours under intense agitation, under externally-applied magnetic field, removes ethanolic soln at last, obtains the Fe of aminopropyl finishing
3O
4Nanoparticle.
Fe with aldehyde group mesoporous molecular sieve for preparing in the 0.85g Comparative Examples and the finishing of 0.15g aminopropyl
3O
4Nanoparticle is scattered in respectively in the 25mL water, mixes normal-temperature reaction then 10 hours, and reaction is accomplished to separate with magnet magnetic and removed the aqueous solution, and drying 12 hours in 60 ℃ of vacuum drying ovens obtains paramagnetic aldehyde group mesoporous molecular sieve then.Above-mentioned paramagnetic aldehyde group mesoporous molecular sieve is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9563U/g; Through recycling back (carrying out the separation of immobilized enzyme with magnet, easy and simple to handle) for 10 times, immobilized enzyme has kept 89.1% initial activity.
Embodiment 2
Change Weight To 0.45g with aldehyde group mesoporous molecular sieve among the embodiment 1; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9621U/g, and after recycling through 10 times, immobilized enzyme has kept 87.6% initial activity.
With the Change Weight To 0.55g of aldehyde group mesoporous molecular sieve among the embodiment 1 and the Fe of aminopropyl finishing
3O
4The Change Weight To 0.30g of nanoparticle; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 8733U/g, and after recycling through 10 times, immobilized enzyme has kept 86.6% initial activity.
Embodiment 4
Change Weight To 0.074g with γ-An Bingjisanyiyangjiguiwan among the embodiment 1; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9822U/g, and after recycling through 10 times, immobilized enzyme has kept 85.8% initial activity.
Embodiment 5
Change Weight To 0.147g with γ-An Bingjisanyiyangjiguiwan among the embodiment 1; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9324U/g, and after recycling through 10 times, immobilized enzyme has kept 87.9% initial activity.
Embodiment 6
Fe with aldehyde group mesoporous molecular sieve among the embodiment 1 and aminopropyl finishing
3O
4The normal-temperature reaction time of nanoparticle changes 3 hours into; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9711U/g, and after recycling through 10 times, immobilized enzyme has kept 86.9% initial activity.
Embodiment 7
Fe with aldehyde group mesoporous molecular sieve among the embodiment 1 and aminopropyl finishing
3O
4The normal-temperature reaction time of nanoparticle changes 5 hours into; Other preparation processes are identical with embodiment 1; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9500U/g, and after recycling through 10 times, immobilized enzyme has kept 88.9% initial activity.
Embodiment 8
With the γ in the Comparative Examples-aldehyde propyl trimethoxy silicane Change Weight To 0.53g; Other preparation processes are identical with embodiment 1 with Comparative Examples; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9834U/g, and after recycling through 10 times, immobilized enzyme has kept 86.1% initial activity.
Embodiment 9
With the γ in the Comparative Examples-aldehyde propyl trimethoxy silicane Change Weight To 1.6g; Other preparation processes are identical with embodiment 1 with Comparative Examples; The paramagnetic aldehyde group mesoporous molecular sieve that makes is used for the immobilization of Semacylase; The activity of the immobilized enzyme that obtains is 9138U/g, and after recycling through 10 times, immobilized enzyme has kept 88.3% initial activity.
Claims (8)
1. preparation method who is used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization; It is characterized in that; This method is introduced aldehyde radical functional group with γ-aldehyde propyl trimethoxy silicane at the surfaces externally and internally of mesopore molecular sieve earlier, will pass through the paramagnetism Fe of the particle diameter of γ-An Bingjisanyiyangjiguiwan finishing greater than the mesopore molecular sieve aperture with the covalent attachment mode then
3O
4The nanoparticle grafting prepares the paramagnetism immobilized enzyme of special construction at the internal surface that the outside surface and the enzyme of mesopore molecular sieve is fixed on mesopore molecular sieve.
2. the preparation method who is used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1; It is characterized in that; Described γ-aldehyde propyl trimethoxy silicane is to the functional modification on mesopore molecular sieve surface; Be that mesopore molecular sieve and γ-aldehyde propyl trimethoxy silicane is added in the toluene solution,, then mesopore molecular sieve filtered with after the washing with alcohol 100～120 ℃ of reflux 6～18 hours; Drying is 6～18 hours in 60～120 ℃ of vacuum drying ovens, obtains aldehyde group mesoporous molecular sieve.
3. according to claim 1 and the 2 described preparing methods that are used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization, it is characterized in that the mass ratio of described γ-aldehyde propyl trimethoxy silicane and mesopore molecular sieve is 0.5～1.6: 1.
4. the preparation method who is used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1 is characterized in that described γ-An Bingjisanyiyangjiguiwan is to paramagnetism Fe
3O
4The finishing of nanoparticle is with usefulness coprecipitation method synthetic Fe
3O
4Nanoparticle is scattered in the ethanolic soln, adds γ-An Bingjisanyiyangjiguiwan then, and normal-temperature reaction is 10 hours under vigorous stirring, under externally-applied magnetic field, removes ethanolic soln at last, obtains the Fe of aminopropyl finishing
3O
4Nanoparticle.
5. according to claim 1 and the 4 described preparing methods that are used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization, it is characterized in that described γ-An Bingjisanyiyangjiguiwan and paramagnetism Fe
3O
4The mass ratio of nanoparticle is 0.5～2.0: 1.
6. the preparation method who is used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1 is characterized in that, the preparation method of described paramagnetic aldehyde group mesoporous molecular sieve is the paramagnetism Fe with the aminopropyl finishing
3O
4The aqueous suspension of nanoparticle is added drop-wise in the aqueous suspension of aldehyde group mesoporous molecular sieve; Normal-temperature reaction was removed the aqueous solution with the separation of magnet magnetic after 3～10 hours; In 40～80 ℃ of vacuum drying ovens dry 6～18 hours then, obtain paramagnetic aldehyde group mesoporous molecular sieve.
7. according to claim 1 and the 6 described preparing methods that are used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization, it is characterized in that the paramagnetism Fe of described aminopropyl finishing
3O
4The mass ratio of nanoparticle and aldehyde group mesoporous molecular sieve is 0.1～0.6: 1.
8. the preparation method who is used for the paramagnetic aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1; It is characterized in that described paramagnetic aldehyde group mesoporous molecular sieve is used for Semacylase, glucose isomerase, glucose transglucosidase, trypsinase or diastatic immobilization.
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| CN114797945A (en) * | 2022-04-11 | 2022-07-29 | 中国石油大学胜利学院 | Bonded phase Fenton-like catalyst and preparation method thereof |
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