CN101851616A - Aldehyde group mesoporous molecular sieve used for immobilization of biological enzyme and preparation method thereof - Google Patents
Aldehyde group mesoporous molecular sieve used for immobilization of biological enzyme and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an aldehyde group mesoporous molecular sieve used for immobilization of a biological enzyme and a preparation method thereof. In the preparation method, a 60 Co-gamma ray pre-irradiation grafting acrolein technique or gamma-aldehyde propyl trimethoxy silane is used for performing functionalized modification on the surface of the mesoporous molecular sieve, an aldehyde propyl functional group with a shorter chain length is introduced to the surface of the mesoporous molecular sieve, the influences, caused by the surface functionalizing process, on the aperture, the specific area and the pore volume of the mesoporous molecular sieve are reduced as much as possible, and further activation is not needed to ensure that the biological enzyme is directly immobilized on the surface of the mesoporous molecular sieve in a covalent bonding mode, and the performance of the immobilized enzyme is improved. The aldehyde group mesoporous molecular sieve can be applied to the immobilization of water-soluble enzymes such as penicillin acylase, glucose isomerase, transglucosidase, trypsase, amylase and the like, and is particularly suitable for the immobilization of the penicillin acylase; and the obtained immobilized enzyme has the activity of 8895 U/g, and after 10 times of recycling, the immobilized enzyme maintains 92 percent of the initial activity.
Description
Technical field
The present invention relates to a kind of aldehyde group mesoporous molecular sieve that is used for bio-enzyme immobilization and preparation method thereof, specifically, use
60Co-gamma-rays pre-irradiation grafting acrolein technique or γ-aldehyde propyl trimethoxy silicane is introduced the short aldehyde propyl group (CH of functional group of chain length to the functional modification on mesopore molecular sieve surface on the mesopore molecular sieve surface
2-CH
2-CHO), make biological enzyme be fixed on the mesopore molecular sieve surface then in the covalent attachment mode, improve the performance of immobilized enzyme.
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 aldehyde group 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 aldehyde group mesoporous molecular sieve of bio-enzyme immobilization is characterized in that this method is to use
60Co-gamma-rays pre-irradiation grafting acrolein technique or γ-aldehyde propyl trimethoxy silicane is introduced the short aldehyde propyl group (CH of functional group of chain length to the functional modification on mesopore molecular sieve surface on the mesopore molecular sieve surface
2-CH
2-CHO).
Described
60Co-gamma-rays pre-irradiation grafting acrolein technique is that mesopore molecular sieve is placed
60Pre-irradiation in the Co-gamma-rays, irradiation dose is 24～120kGy, sample is transferred to volumn concentration behind pre-irradiation be in 10～90% the aqueous acrolein solution, in 30～80 ℃ of water-baths, reacted 10～30 hours, after then mesopore molecular sieve being filtered, remove the propenal polymkeric substance with the ethanol extracting in apparatus,Soxhlet's, drying obtained aldehyde group mesoporous molecular sieve in 12～24 hours in 50～100 ℃ of vacuum drying ovens again.
The volume of described aqueous acrolein solution and the mass ratio of mesopore molecular sieve are 50～200: 1.
Described γ-aldehyde propyl trimethoxy silicane is that mesopore molecular sieve and γ-aldehyde propyl trimethoxy silicane are added in the toluene solution to the functional modification on mesopore molecular sieve surface, 100～120 ℃ of reflux 5～15 hours, after then mesopore molecular sieve being filtered, remove unreacted γ-aldehyde propyl trimethoxy silicane with the ethanol extracting in apparatus,Soxhlet's, drying obtained aldehyde group mesoporous molecular sieve in 12～24 hours in 50～100 ℃ of vacuum drying ovens again.
The mass ratio of described γ-aldehyde propyl trimethoxy silicane and mesopore molecular sieve is 0.3～2: 1, and the volume of described toluene and the mass ratio of mesopore molecular sieve are 20～100: 1.
Described aldehyde group 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.
Mean pore size with the aldehyde group mesoporous molecular sieve of method for preparing is 10～35nm, and specific surface area is 300～600m
2/ g, pore volume is at 1.0～2.5cm
3Between/the g; aldehyde propyl group functional group accounts for 9.0～15.0% of mesopore molecular sieve total mass; 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 aldehyde group mesoporous molecular sieve of the present invention remarkable advantage is, by using
60Co-gamma-rays pre-irradiation grafting acrolein technique or γ-aldehyde propyl trimethoxy silicane is to the functional modification on mesopore molecular sieve surface, introduce the short aldehyde propyl group functional group of chain length on the mesopore molecular sieve surface, reduce of the influence of surface-functionalized process for aperture, specific surface area and the pore volume of mesopore molecular sieve as far as possible, 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 aldehyde group mesoporous molecular sieve of the present invention is; the molecular dimension of the aperture ratio penicillin acylase of this aldehyde group 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 aldehyde group mesoporous molecular sieve of the present invention is, this aldehyde group mesoporous molecular sieve by the surface the aldehyde propyl group 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
60Co-gamma-rays pre-irradiation grafting technology is to the aldehyde radical functionalization on mesopore molecular sieve surface and the immobilization synoptic diagram of enzyme;
Fig. 2 is for using γ-aldehyde propyl trimethoxy silicane to the aldehyde radical functionalization on mesopore molecular sieve surface 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 carry out the immobilization of penicillin acylase, and activity and the operational stability of measuring immobilized enzyme:
The immobilization of penicillin acylase: take by weighing 0.1g carrier and 6.0mL penicillin acylase solution (V through the phosphate buffer soln dilution of pH=7.8
Buffer/ V
Enzyme=2: 1) mix, put into 30 ℃ shaking bath immobilization and carry out centrifugation after 18 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/L pH=7.8 phosphate buffer soln) uniform mixing with said fixing enzyme and 100mL 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.
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, 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 550 ℃ of roastings and obtained mesopore molecular sieve in 8 hours in retort furnace then.Above-mentioned mesopore molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 10465U/g, and after recycling through 10 times, immobilized enzyme has kept 77% initial activity.
Embodiment 1
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, 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 550 ℃ of roastings and obtained mesopore molecular sieve in 8 hours in retort furnace then.
With
60Co-gamma-rays pre-irradiation grafting technology places the above-mentioned mesopore molecular sieve of 1.0g to the immobilized synoptic diagram of the aldehyde radical functionalization on mesopore molecular sieve surface and enzyme as shown in Figure 1
60Pre-irradiation in the Co-gamma-rays, irradiation dose are 72kGy, and sample is transferred to behind pre-irradiation in the aqueous acrolein solution of 160mL 50%, places ice-water bath, vacuumize, and inflated with nitrogen reacted 20 hours in 60 ℃ of water-baths then; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove the propenal polymkeric substance, drying obtained aldehyde group mesoporous molecular sieve in 24 hours in 50 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8580U/g, and after recycling through 10 times, immobilized enzyme has kept 83% initial activity.
Embodiment 2
The preparation method of mesopore molecular sieve is identical with embodiment 1.The above-mentioned mesopore molecular sieve of 1.0g is placed
60Pre-irradiation in the Co-gamma-rays, irradiation dose are 24kGy, and sample is transferred to behind pre-irradiation in the aqueous acrolein solution of 50mL 90%, places ice-water bath, vacuumize, and inflated with nitrogen reacted 30 hours in 30 ℃ of water-baths then; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove the propenal polymkeric substance, drying obtained aldehyde group mesoporous molecular sieve in 18 hours in 75 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8760U/g, and after recycling through 10 times, immobilized enzyme has kept 81% initial activity.
Embodiment 3
The preparation method of mesopore molecular sieve is identical with embodiment 1.The above-mentioned mesopore molecular sieve of 1.0g is placed
60Pre-irradiation in the Co-gamma-rays, irradiation dose are 120kGy, and sample is transferred to behind pre-irradiation in the aqueous acrolein solution of 200mL 10%, places ice-water bath, vacuumize, and inflated with nitrogen reacted 10 hours in 80 ℃ of water-baths then; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove the propenal polymkeric substance, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 100 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8920U/g, and after recycling through 10 times, immobilized enzyme has kept 80% initial activity.
Embodiment 4
The preparation method of mesopore molecular sieve is identical with embodiment 1.With γ-aldehyde propyl trimethoxy silicane as shown in Figure 2 to the immobilization synoptic diagram of the aldehyde radical functionalization on mesopore molecular sieve surface and enzyme, 1.0g mesopore molecular sieve and 1.78g γ-aldehyde propyl trimethoxy silicane are joined in the 50mL toluene solution, 110 ℃ of reflux 10 hours; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove unreacted γ-aldehyde propyl trimethoxy silicane, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 50 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8895U/g, and after recycling through 10 times, immobilized enzyme has kept 92% initial activity.
Embodiment 5
The preparation method of mesopore molecular sieve is identical with embodiment 1.1.0g mesopore molecular sieve and 1.07g γ-aldehyde propyl trimethoxy silicane are joined in the 50mL toluene solution, 110 ℃ of reflux 10 hours; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove unreacted γ-aldehyde propyl trimethoxy silicane, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 50 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 9240U/g, and after recycling through 10 times, immobilized enzyme has kept 89% initial activity.
Embodiment 6
The preparation method of mesopore molecular sieve is identical with embodiment 1.1.0g mesopore molecular sieve and 0.36g γ-aldehyde propyl trimethoxy silicane are joined in the 50mL toluene solution, 110 ℃ of reflux 10 hours; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove unreacted γ-aldehyde propyl trimethoxy silicane, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 50 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 9626U/g, and after recycling through 10 times, immobilized enzyme has kept 86% initial activity.
Embodiment 7
The preparation method of mesopore molecular sieve is identical with embodiment 1.1.0g mesopore molecular sieve and 0.3g γ-aldehyde propyl trimethoxy silicane are joined in the 20mL toluene solution, 100 ℃ of reflux 5 hours; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove unreacted γ-aldehyde propyl trimethoxy silicane, drying obtained aldehyde group mesoporous molecular sieve in 12 hours in 50 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 9650U/g, and after recycling through 10 times, immobilized enzyme has kept 85% initial activity.
Embodiment 8
The preparation method of mesopore molecular sieve is identical with embodiment 1.1.0g mesopore molecular sieve and 2.0g γ-aldehyde propyl trimethoxy silicane are joined in the 100mL toluene solution, 120 ℃ of reflux 15 hours; After mesopore molecular sieve filtered, in apparatus,Soxhlet's with the ethanol extracting to remove unreacted γ-aldehyde propyl trimethoxy silicane, drying obtained aldehyde group mesoporous molecular sieve in 24 hours in 100 ℃ of vacuum drying ovens then.Above-mentioned aldehyde group mesoporous molecular sieve is used for the immobilization of penicillin acylase, and the activity of the immobilized enzyme that obtains is 8775U/g, and after recycling through 10 times, immobilized enzyme has kept 93% initial activity.
Claims (6)
1. a preparation method who is used for the aldehyde group mesoporous molecular sieve of bio-enzyme immobilization is characterized in that, this method is to use
60Co-gamma-rays pre-irradiation grafting acrolein technique or γ-aldehyde propyl trimethoxy silicane is introduced the short aldehyde propyl group (CH of functional group of chain length to the functional modification on mesopore molecular sieve surface on the mesopore molecular sieve surface
2-CH
2-CHO).
2. the preparation method who is used for the aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1 is characterized in that, and is described
60Co-gamma-rays pre-irradiation grafting acrolein technique is that mesopore molecular sieve is placed
60Pre-irradiation in the Co-gamma-rays, irradiation dose is 24～120kGy, sample is transferred to volumn concentration behind pre-irradiation be in 10～90% the aqueous acrolein solution, in 30～80 ℃ of water-baths, reacted 10～30 hours, after then mesopore molecular sieve being filtered, remove the propenal polymkeric substance with the ethanol extracting in apparatus,Soxhlet's, drying obtained aldehyde group mesoporous molecular sieve in 12～24 hours in 50～100 ℃ of vacuum drying ovens again.
3. the preparation method who is used for the aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 2 is characterized in that, the volume of described aqueous acrolein solution and the mass ratio of mesopore molecular sieve are 50～200: 1.
4. the preparation method who is used for the 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 that mesopore molecular sieve and γ-aldehyde propyl trimethoxy silicane are added in the toluene solution to the functional modification on mesopore molecular sieve surface, 100～120 ℃ of reflux 5～15 hours, after then mesopore molecular sieve being filtered, remove unreacted γ-aldehyde propyl trimethoxy silicane with the ethanol extracting in apparatus,Soxhlet's, drying obtained aldehyde group mesoporous molecular sieve in 12～24 hours in 50～100 ℃ of vacuum drying ovens again.
5. the preparation method who is used for the aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 4, it is characterized in that, the mass ratio of described γ-aldehyde propyl trimethoxy silicane and mesopore molecular sieve is 0.3～2: 1, and the volume of described toluene and the mass ratio of mesopore molecular sieve are 20～100: 1.
6. the preparation method who is used for the aldehyde group mesoporous molecular sieve of bio-enzyme immobilization according to claim 1; it is characterized in that; described aldehyde group 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.
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| CN102703412A (en) * | 2012-06-06 | 2012-10-03 | 华东理工大学 | Aramagnetic aldehyde group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof |
| CN103232992A (en) * | 2013-06-05 | 2013-08-07 | 南京工业大学 | Immobilized lipase and preparation method and application thereof |
| CN112939016A (en) * | 2021-03-10 | 2021-06-11 | 成都理工大学 | Chain-shaped ZSM-5 micro mesoporous molecular sieve formed by egg protein induction and synthesis method thereof |
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|---|---|---|---|---|
| CN1935994A (en) * | 2006-08-23 | 2007-03-28 | 宁夏大学 | Organic group functionized mesoporous molecular sieve enzyme immobilized carrier, and its preparing method |
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| CN1935994A (en) * | 2006-08-23 | 2007-03-28 | 宁夏大学 | Organic group functionized mesoporous molecular sieve enzyme immobilized carrier, and its preparing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703412A (en) * | 2012-06-06 | 2012-10-03 | 华东理工大学 | Aramagnetic aldehyde group mesoporous molecular sieve for immobilized biological enzymes, and preparation method thereof |
| CN103232992A (en) * | 2013-06-05 | 2013-08-07 | 南京工业大学 | Immobilized lipase and preparation method and application thereof |
| CN103232992B (en) * | 2013-06-05 | 2014-09-24 | 南京工业大学 | Immobilized lipase and preparation method and application thereof |
| CN112939016A (en) * | 2021-03-10 | 2021-06-11 | 成都理工大学 | Chain-shaped ZSM-5 micro mesoporous molecular sieve formed by egg protein induction and synthesis method thereof |
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| CN101851616B (en) | 2012-07-25 |
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