CN101864410A - Epoxy-based mesoporous molecular sieve for biological enzyme immobilization and preparation method thereof - Google Patents
Epoxy-based mesoporous molecular sieve for biological enzyme immobilization and preparation method thereof Download PDFInfo
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Abstract
本发明公开了用于生物酶固定化的环氧基介孔分子筛及其制备方法。本发明用间氯过氧苯甲酸对含有乙烯基的立方相Ia3d结构的介孔分子筛和表面乙烯基功能化的介孔分子筛的环氧化，在介孔分子筛表面引入链长较短的环氧基官能团，尽量减少表面功能化过程对于介孔分子筛的孔径、比表面积和孔容的影响，不需要进一步活化就可以直接使生物酶以共价结合方式固定在介孔分子筛表面，提高固定化酶的性能。将上述环氧基介孔分子筛可用于青霉素酰化酶、葡萄糖异构酶、葡萄糖转苷酶、胰蛋白酶和淀粉酶等水溶性生物酶的固定化，特别适用于青霉素酰化酶的固定化，得到的固定化酶活性为8416U/g，经过10次循环使用后，固定化酶保留了90％的初始活性。
The invention discloses an epoxy-based mesoporous molecular sieve for biological enzyme immobilization and a preparation method thereof. The present invention uses m-chloroperoxybenzoic acid to epoxidize the mesoporous molecular sieve with the cubic phase Ia3d structure containing vinyl and the mesoporous molecular sieve with vinyl functionalization on the surface, and introduces epoxy with shorter chain length on the surface of the mesoporous molecular sieve. functional groups to minimize the impact of the surface functionalization process on the pore size, specific surface area, and pore volume of mesoporous molecular sieves. Without further activation, biological enzymes can be directly immobilized on the surface of mesoporous molecular sieves in a covalent manner, improving the efficiency of immobilized enzymes. performance. The above-mentioned epoxy-based mesoporous molecular sieves can be used for the immobilization of water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transsidase, trypsin and amylase, especially for the immobilization of penicillin acylase, The activity of the obtained immobilized enzyme was 8416U/g, and after 10 cycles of use, the immobilized enzyme retained 90% of its initial activity.
Description
Technical field
The present invention relates to a kind of epoxy mesoporous molecular sieve that is used for bio-enzyme immobilization and preparation method thereof, specifically, by with the epoxidation of metachloroperbenzoic acid to the mesopore molecular sieve of the mesopore molecular sieve of cube phase Ia3d structure that contains vinyl and surperficial vinyl functionalization, introduce the short epoxy functionality of chain length on the mesopore molecular sieve surface, make biological enzyme be fixed on the mesopore molecular sieve surface then, improve the performance of immobilized enzyme in the covalent attachment mode.
Background technology
Resolvase is directly used in 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, after finishing, reaction is easy to separate with product and can reuse, the product purity height of gained, and production cost is low.Therefore, the immobilization of enzyme is the research focus in fields such as catalytic chemistry, biological chemistry and materials chemistry always.
The preparation method commonly used of immobilized enzyme can be divided into four kinds: entrapping method, crosslinking, absorption method and covalent attachment method.Entrapping method is divided into grid type and microcapsule-type two classes, and can obtain higher enzyme activity and reclaim, but necessary ingenious design reaction conditions, the immobilized enzyme that makes is not suitable for macromolecule substrate, generally is used to prepare immobilized cell.Crosslinking is meant and earlier enzyme is adsorbed on the insoluble carrier, use bifunctional or more function group linking agent to make then to carry out between the enzyme molecule crosslinked, form reticulated structure, can obtain the higher immobilized enzyme of zymoprotein unit's concentration, reaction conditions is violent, enzyme is lived deficiencies such as loss but exist.Absorption method comprises physical adsorption and ionic adsorption method, have enzyme active center and be difficult for destroyed and enzyme higher structure and change advantages such as less, but owing to be to be connected with more weak reactive force such as ionic linkage, Van der Waals force and hydrogen bond between enzyme and the carrier, enzyme runs off easily.The covalent attachment method is by covalent linkage the active nonessential side-chain radical of enzyme and the functional groups of carrier 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 present industrial widely used enzyme immobilization method.
By the preparation method of said fixing enzyme as can be known, expect the immobilized enzyme of excellent performance, carrier must satisfy following requirement: carrier surface has and can interactional functional groups take place with the enzyme molecule, maybe can carry out functionalization by surface modification; The functional groups quantity of carrier surface and distribution thereof are suitable, and approaching easily; Carrier must be a porous mass, has higher specific surface area; Carrier has bigger aperture, to reduce the diffusional resistance that exists in the enzyme-catalyzed reaction; Carrier has enough physical strengths and chemical stability.
The bio-enzyme immobilization carrier can be divided into two classes: inorganic carrier and organic carrier.Compare with widely used organic carrier, inorganic carrier has higher physical strength and better chemical stability, its structure and surface properties are controlled easily, 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 environment.Novel mesopore molecular sieve has bigger continuously adjustable aperture, high specific surface area, be rich in the slightly acidic hydroxyl in bigger loading capacity and the duct, 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, the microenvironment that being maintained fixed enzyme is suitable, thereby the immobilized enzyme that makes has advantages of high catalytic activity, 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 the rising enzyme immobilization novel inorganic carrier of a class.
The performance of immobilized enzyme and the structure of mesopore molecular sieve (crystal formation, 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 biological enzyme, in the immobilization process of enzyme, the enzyme molecule just enters easily in the mesopore molecular sieve duct and combines with the functional groups on surface, make full use of the pore volume of mesopore molecular sieve, the immobilized enzyme that obtains has greater activity.The crystal formation of mesopore molecular sieve, pore volume and specific surface area all have considerable influence to activity of the immobilized enzyme.Owing to normally combining between the surperficial slightly acidic hydroxyl of biological enzyme molecule and mesopore molecular sieve with more weak hydrogen bond action power, in use a part of enzyme can come off, operational stability remains further to be improved, how under the prerequisite of being maintained fixed enzymic activity substantially, improve the operational stability of immobilized enzyme, still need to do further research.
Immobilized penicillin acylated enzyme (EC 3.5.1.11, the enzyme molecular dimension:
) be the enzyme of most critical during semi-synthetic β-Nei Xiananleikangshengsu is produced, it can catalysis penicillin 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 (as 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 listed in 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 penicillin acylase engineering strain of high yield with the DNA operative technique, greatly reduces the production cost of resolvase.But the whole dependence on import of the present industrial used carrier of China, 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 to strengthen the international competitiveness of product, thereby make positive contribution for the Sustainable development of the progress of China's enzyme immobilization technology and microbiotic industry.
Chinese patent CN1320688A discloses a kind of mesoporous material that is used for immobilized penicillin amidase.The maximum diameter of hole of this mesoporous material is 3.3nm; the highest enzyme of immobilized enzyme is lived and is 511U/g; but because the molecular dimension of the aperture ratio penicillin acylase of this mesoporous material is much smaller; and the more weak hydrogen bond action power between slightly acidic hydroxyl by utilizing carrier surface and the enzyme molecule realizes the immobilization of enzyme; therefore, the immobilized enzyme poor-performing that obtains.
Chinese patent CN1935994A discloses a kind of organic group functionized mesoporous molecular sieve enzyme immobilized carrier and preparation method thereof.By contain oxygen glycidyl propyl trimethoxy silicane or γ-An Bingjisanyiyangjiguiwan and glutaraldehyde functional modification with 3-to the mesopore molecular sieve surface; introduce organo-functional groups such as epoxy group(ing) or aldehyde radical (account for carrier total mass 2～9%); then under mild conditions with covalent attachment mode fixed biologically enzyme molecule; the immobilized penicillin acylated enzyme that obtains has high catalytic activity and stability in use; the apparent activity of immobilized enzyme is 1000～2000IU/g; after reusing 10 times, immobilized enzyme keeps the initial apparent activity more than 90%.But because the chain length of the organo-functional group that above-mentioned two kinds of methods are introduced is long, through after surface-functionalized, the aperture of mesopore molecular sieve, specific surface area and pore volume all obviously diminish, and greatly reduce the activity of immobilized enzyme.
Chinese patent CN1580233A discloses a kind of mesoporous reactor that is used for enzyme immobilization and preparation method thereof.This mesoporous reactor interacts by the oh group and the enzyme molecule carboxyl oxygen atom on mesoporous material surface, the enzyme molecule is fixed in the mesoporous material duct, utilize crosslinking technology silane coupling agent to be grafted on the place, aperture of mesoporous material again, utilize the double-bond polymerization of polymerization end of silane at last, the place forms reticulated structure in the aperture.Zhi Bei mesoporous reactor can be effectively dwindles the aperture of carrier by this method, also can not cause resistance to mass transfer to substrate and product when the inhibitory enzyme molecule runs off, thereby obtain having the immobilized enzyme of high catalytic activity and high stability of operation.Mesoporous material selects for use a kind of among SBA-15, MCM-41, MCM-48 and the FSM, enzyme to select a kind of in porcine pancreatic lipase, horseradish peroxidase, sphaeroprotein enzyme, the Regular Insulin for use.But this method has been used a large amount of organic solvents in preparation immobilized enzyme process, make the resolvase loss of catalytic activity easily.
Summary of the invention
The object of the invention is exactly the preparation method that the epoxy mesoporous molecular sieve that is used for bio-enzyme immobilization of a kind of activity that improves immobilized enzyme and operational stability is provided in order to overcome the defective that above-mentioned prior art exists.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of preparation method who is used for the epoxy mesoporous molecular sieve of bio-enzyme immobilization, it is characterized in that, this method is with the epoxidation of metachloroperbenzoic acid to the mesopore molecular sieve of the mesopore molecular sieve of cube phase Ia3d structure that contains vinyl and surperficial vinyl functionalization, introduces the short epoxy functionality of chain length on the mesopore molecular sieve surface.
Described method specifically comprises the steps: the vinyl mesopore molecular sieve is joined in the dichloromethane solution of metachloroperbenzoic acid (m-CPBA), 30～50 ℃ of lasting stirring and refluxing after 48～96 hours, filter, with filter cake methylene dichloride thorough washing three times, the gained pressed powder promptly obtains epoxy mesoporous molecular sieve 50～100 ℃ of vacuum-dryings 12～24 hours.
The mass ratio of described metachloroperbenzoic acid and vinyl mesopore molecular sieve is 1～3: 1, and the mass ratio of the volume of described methylene dichloride and vinyl mesopore molecular sieve is 50～150: 1.
Described epoxy mesoporous molecular sieve can be used for the immobilization of water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transglucosidase, trypsinase and amylase, is specially adapted to the immobilization of penicillin acylase.
The preparation method who is used for the epoxy mesoporous molecular sieve of bio-enzyme immobilization forms synthetic the comprising the steps: of therein ethylene base mesopore molecular sieve by synthetic and 2 processes of surperficial epoxy group(ing) functionalization of vinyl mesopore molecular sieve
Directly copolymerization method is synthetic: at ambient temperature, and with 5.68g Pluronic P123 (EO
20PO
70EO
20, M
Av=5800) and 11.36g Na
2SO
4Be dissolved in the HCl solution of 160mL 1.0mol/L; After treating to dissolve fully, solution is warming up to 35 ℃ and continue stirred for several hour, the tetraethoxy (TEOS) that the difference of slow Dropwise 5 3.2mmol is formed and the mixture of vinyltriethoxysilane (TEVS), and under 35 ℃ of conditions, continue to stir 20 hours, wherein the molar percentage of TEVS is 5～20%; Solution changed over to have in the teflon-lined hydrothermal crystallizing still, and place 100 ℃ baking oven crystallization 24 hours, then solution is cooled to the solid that the room temperature after-filtration obtains, dried overnight in 100 ℃ of baking ovens; With 1.0g not the mesopore molecular sieve of template agent removing place round-bottomed flask, add 50ml concentration and be 60% sulfuric acid, be heated to 95 ℃ and continue to stir 24 hours; Filtration is also extremely neutral with deionized water wash, filter cake is reclaimed the back add the 50mi dehydrated alcohol, continue to stir 12 hours after-filtration, triplicate at ambient temperature, in 50 ℃ of vacuum drying ovens dry 24 hours then, obtain containing the mesopore molecular sieve of cube phase Ia3d structure of vinyl.
Back grafting is synthetic: at room temperature with 2.0g Pluronic P123 (EO
20PO
70EO
20, M
Av=5800) be dissolved in the HCl solution of 75mL1.6mol/L; Treat that P123 dissolves the back fully and adds 0.023g Neutral ammonium fluoride and 1.5～3.5g 1,3, the 5-trimethylbenzene, then solution is warming up to 35 ℃ and continue to stir 45min after, add the 4.4g tetraethoxy, and continue down to stir 20 hours at 35 ℃; 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 4 hours in retort furnace then; Mesopore molecular sieve and vinyltriethoxysilane are joined in the toluene solution, 100～120 ℃ of reflux 5～15 hours, the mass ratio of therein ethylene ethyl triethoxy silicane alkane and mesopore molecular sieve is 0.5～1.5: 1, and the volume of toluene and the mass ratio of mesopore molecular sieve are 20～70: 1; With the mesopore molecular sieve filtration, with after the washing with alcohol three times, drying is 24 hours in 50 ℃ of vacuum drying ovens, obtains the mesopore molecular sieve of surperficial vinyl functionalization.
With the epoxy mesoporous molecular sieve of method for preparing, can be used for the immobilization of water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transglucosidase, trypsinase and amylase, be specially adapted to the immobilization of penicillin acylase.
Compared with prior art, the preparation method's of epoxy mesoporous molecular sieve of the present invention remarkable advantage is, with the epoxidation of metachloroperbenzoic acid to the mesopore molecular sieve of the mesopore molecular sieve of cube phase Ia3d structure that contains vinyl and surperficial vinyl functionalization, introduce the short epoxy functionality of chain length on the mesopore molecular sieve surface, reduce the aperture of surface-functionalized process for mesopore molecular sieve as far as possible, the influence of specific surface area and pore volume, do not need further to activate just directly to make biological enzyme be fixed on the mesopore molecular sieve surface, improve the performance of immobilized enzyme in the covalent attachment mode.
One of remarkable advantage of epoxy mesoporous molecular sieve of the present invention is; the molecular dimension of the aperture ratio penicillin acylase of this epoxy mesoporous molecular sieve is much bigger; help more enzyme molecule and enter in the mesopore molecular sieve duct and be immobilized, improve the activity of immobilized enzyme.
Two of the remarkable advantage of epoxy mesoporous molecular sieve of the present invention is, this epoxy mesoporous molecular sieve by the surface epoxy group(ing) and the enzyme molecule in the covalent linkage that forms between the amino realize the immobilization of enzyme, improve the operational stability of immobilized enzyme.
Description of drawings
Fig. 1 is for using vinyltrimethoxy silane and m-CPBA to the epoxy group(ing) functionalization on mesopore molecular sieve surface and the immobilization synoptic diagram of enzyme;
Fig. 2 for m-CPBA to the epoxy group(ing) functionalization on the mesopore molecular sieve surface of containing vinyl and the immobilization synoptic diagram of enzyme.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
In following examples, adopt following method to carrying out the immobilization of penicillin acylase, and activity and the operational stability of measuring immobilized enzyme:
The immobilization of penicillin acylase: take by weighing 0.05g carrier and 6.0mL penicillin acylase solution (V through the phosphate buffer soln dilution of pH=7.8
Buffer/ V
Enzyme=4: 1) mix, put into 30 ℃ shaking bath immobilization and carry out centrifugation after 24 hours, 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/LpH=7.8 phosphate buffer soln) uniform mixing with said fixing enzyme and 50mL 4wt%, be the NaOH solution titration of 0.1mol/L then with concentration, 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(IU/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 centrifugation, then immobilized enzyme is transferred in the reactor, 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, illustrate that then the operational stability of immobilized enzyme is good more.
Embodiment 1
With vinyltrimethoxy silane and m-CPBA as shown in Figure 1 to the immobilization synoptic diagram of the epoxy group(ing) functionalization on mesopore molecular sieve surface and enzyme, at ambient temperature, with 5.68g Pluronic P123 (EO
20PO
70EO
20, M
Av=5800) and 11.36gNa
2SO
4Be dissolved in the HCl solution of 160mL 1.0mol/L; After treating to dissolve fully, solution is warming up to 35 ℃ and continue stirred for several hour, the tetraethoxy (TEOS) of slow Dropwise 5 3.2mmol and the mixture (molar percentage of TEVS is 5%) of vinyltriethoxysilane (TEVS), and under 35 ℃ of conditions, continue to stir 20 hours; Solution changed over to have in the teflon-lined hydrothermal crystallizing still, and place 100 ℃ baking oven crystallization 24 hours, then solution is cooled to the solid that the room temperature after-filtration obtains, dried overnight in 100 ℃ of baking ovens; With 1.0g not the mesopore molecular sieve of template agent removing place round-bottomed flask, add 50ml concentration and be 60% sulfuric acid, be heated to 95 ℃ and continue to stir 24 hours; Filtration is also extremely neutral with deionized water wash, filter cake is reclaimed the back add the 50ml dehydrated alcohol, continue to stir 12 hours after-filtration, triplicate at ambient temperature, in 50 ℃ of vacuum drying ovens dry 24 hours then, obtain containing the mesopore molecular sieve of cube phase Ia3d structure of vinyl.
With the above-mentioned vinyl mesopore molecular sieve of 0.5g, join in the dichloromethane solution of 50mL metachloroperbenzoic acid (concentration is 1.0g/50mL), 35 ℃ of lasting stirring and refluxing after 72 hours, filter, with filter cake methylene dichloride thorough washing three times, the gained pressed powder promptly obtains epoxy mesoporous molecular sieve 50 ℃ of vacuum-dryings 24 hours.Above-mentioned epoxy mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 3611U/g, and after recycling through 10 times, immobilized enzyme has kept 77% initial activity.
Embodiment 2
Change the molar percentage of TEVS among the embodiment 1 into 10% by 5%; other conditions are identical with embodiment 1; obtain epoxy mesoporous molecular sieve; be used for the immobilization of penicillin acylase; the activity of the immobilized enzyme that obtains is 2262U/g; after recycling through 10 times, immobilized enzyme has kept 80% initial activity.
Embodiment 3
Change the molar percentage of TEVS among the embodiment 1 into 15% by 5%; other conditions are identical with embodiment 1; obtain epoxy mesoporous molecular sieve; be used for the immobilization of penicillin acylase; the activity of the immobilized enzyme that obtains is 1563U/g; after recycling through 10 times, immobilized enzyme has kept 66% initial activity.
Embodiment 4
Change the molar percentage of TEVS among the embodiment 1 into 20% by 5%; other conditions are identical with embodiment 1; obtain epoxy mesoporous molecular sieve; be used for the immobilization of penicillin acylase; the activity of the immobilized enzyme that obtains is 1308U/g; after recycling through 10 times, immobilized enzyme has kept 64% initial activity.
Embodiment 5
At room 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 1.5g 1,3, the 5-trimethylbenzene, then solution is warming up to 35 ℃ and continue to stir 45min after, add the 4.4g tetraethoxy, and continue down to stir 20 hours at 35 ℃; 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 4 hours in retort furnace then; 1.0g mesopore molecular sieve and 1.08g vinyltriethoxysilane are joined in the 55mL toluene solution, 110 ℃ of reflux 10 hours; With the mesopore molecular sieve filtration, with after the washing with alcohol three times, drying is 24 hours in 50 ℃ of vacuum drying ovens, obtains the mesopore molecular sieve of surperficial vinyl functionalization.
With m-CPBA as shown in Figure 2 to the immobilization synoptic diagram of the epoxy group(ing) functionalization on the mesopore molecular sieve surface of containing vinyl and enzyme, with the above-mentioned vinyl mesopore molecular sieve of 0.5g, join in the dichloromethane solution of 50mL metachloroperbenzoic acid (concentration is 1.0g/50mL), 35 ℃ of lasting stirring and refluxing after 75 hours, filter, with filter cake methylene dichloride thorough washing three times, the gained pressed powder promptly obtains epoxy mesoporous molecular sieve 50 ℃ of vacuum-dryings 24 hours.Above-mentioned epoxy mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8383U/g, and after recycling through 10 times, immobilized enzyme has kept 69% initial activity.
Embodiment 6
With among the embodiment 51; 3; the quality of 5-trimethylbenzene changes 2.5g into by 1.5g; other conditions are identical with embodiment 1; obtain epoxy mesoporous molecular sieve, be used for the immobilization of penicillin acylase, the activity of the immobilized enzyme that obtains is 8416U/g; after recycling through 10 times, immobilized enzyme has kept 90% initial activity.
Embodiment 7
With among the embodiment 51; 3; the quality of 5-trimethylbenzene changes 3.5g into by 1.5g; other conditions are identical with embodiment 1; obtain epoxy mesoporous molecular sieve, be used for the immobilization of penicillin acylase, the activity of the immobilized enzyme that obtains is 8016U/g; after recycling through 10 times, immobilized enzyme has kept 84% initial activity.
Embodiment 8
The preparation method of vinyl mesopore molecular sieve is with embodiment 5, with the above-mentioned vinyl mesopore molecular sieve of 0.5g, join in the dichloromethane solution of 25mL metachloroperbenzoic acid (concentration is 1.0g/50mL), 30 ℃ of lasting stirring and refluxing after 96 hours, filter, with filter cake methylene dichloride thorough washing three times, the gained pressed powder promptly obtains epoxy mesoporous molecular sieve 50 ℃ of vacuum-dryings 24 hours.Above-mentioned epoxy mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8543U/g, and after recycling through 10 times, immobilized enzyme has kept 66% initial activity.
Embodiment 9
The preparation method of vinyl mesopore molecular sieve is with embodiment 5, with the above-mentioned vinyl mesopore molecular sieve of 0.5g, join in the dichloromethane solution of 75mL metachloroperbenzoic acid (concentration is 1.0g/50mL), 40 ℃ of lasting stirring and refluxing after 72 hours, filter, with filter cake methylene dichloride thorough washing three times, the gained pressed powder promptly obtains epoxy mesoporous molecular sieve 100 ℃ of vacuum-dryings 12 hours.Above-mentioned epoxy mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8026U/g, and after recycling through 10 times, immobilized enzyme has kept 80% initial activity.
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| CN102286455A (en) * | 2011-08-26 | 2011-12-21 | 湖南大学 | Immobilized laccase and preparation method thereof |
| CN102329008A (en) * | 2011-08-26 | 2012-01-25 | 湖南大学 | Method for removing phenol pollutants in water body by using immobilized laccase based on magnetic mesoporous carbon |
| CN104014358A (en) * | 2014-05-28 | 2014-09-03 | 上海纳米技术及应用国家工程研究中心有限公司 | Supported catalyst eliminating carbon monoxide at room temperature, and preparation method and application thereof |
| CN115584634A (en) * | 2022-10-25 | 2023-01-10 | 江阴市沙江纺织科技有限公司 | Environment-friendly impregnated cord fabric for tire and production process thereof |
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| 《中国科技论文在线》 20090630 高振源等 MCFs介孔分子筛的环氧化及其固定化酶性能 第423-430页 1-4 第4卷, 第6期 * |
| 《中国科技论文在线》 20090930 吕勇军等 乙烯基Ia3d介孔分子筛的环氧化及固定化酶性能 第659-664页 1-4 第4卷, 第9期 2 * |
| 《石油学报（石油加工）》 20081031 彭洪根等 功能化介孔分子筛用于固定化青霉素酞化酶 第175-178，184页 1-4 , * |
| 《石油学报（石油加工）》 20081031 彭洪根等 功能化介孔分子筛用于固定化青霉素酞化酶 第175-178，184页 1-4 , 2 * |
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| CN102329008A (en) * | 2011-08-26 | 2012-01-25 | 湖南大学 | Method for removing phenol pollutants in water body by using immobilized laccase based on magnetic mesoporous carbon |
| CN102286455B (en) * | 2011-08-26 | 2013-05-29 | 湖南大学 | Immobilized laccase and its preparation method |
| CN104014358A (en) * | 2014-05-28 | 2014-09-03 | 上海纳米技术及应用国家工程研究中心有限公司 | Supported catalyst eliminating carbon monoxide at room temperature, and preparation method and application thereof |
| CN104014358B (en) * | 2014-05-28 | 2016-04-27 | 上海纳米技术及应用国家工程研究中心有限公司 | Room temperature eliminates loaded catalyst of carbon monoxide and its preparation method and application |
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