CN103451175A - Nanometer magnetic Fe3O4 immobilized lipoxygenase as well as preparation method and application thereof - Google Patents
Nanometer magnetic Fe3O4 immobilized lipoxygenase as well as preparation method and application thereof Download PDFInfo
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- CN103451175A CN103451175A CN2013104133633A CN201310413363A CN103451175A CN 103451175 A CN103451175 A CN 103451175A CN 2013104133633 A CN2013104133633 A CN 2013104133633A CN 201310413363 A CN201310413363 A CN 201310413363A CN 103451175 A CN103451175 A CN 103451175A
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- oxygenase
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Abstract
The invention relates to nanometer magnetic Fe3O4 immobilized lipoxygenase as well as a preparation method and an application thereof. Nanometer magnetic Fe3O4 is prepared by adopting a sol-gel method, is wrapped by using Na2SiO3 and then is modified by using a silane coupling agent (KH550), and finally lipoxygenase is immobilized on a carrier so as to form immobilized enzyme. The prepared immobilized enzyme is used for oxidizing chlorpromazine and has good activity and high catalytic efficiency.
Description
Technical field
The present invention relates to a kind of nano-magnetic Fe
3o
4fixed fat oxygenase and its preparation method and application, belong to the biocatalysis technology field.
Background technology
Nano magnetic material has been subject to extensive concern as a kind of new Carrier Materials of Immobilized Enzyme.Fe
3o
4the magnetic nanoparticle method for making is simple, and human body is had no side effect, and its application is relatively more extensive.By nano-magnetic Fe
3o
4surface make it be with functional group after being modified, get final product ligase enzyme, antibody or antigen etc., thereby in the fields such as biological, medicine and foodstuffs industry, important Research Significance and using value arranged.Enzyme being fixed on to the magnetic that can utilize carrier on nano magnetic material separates enzyme rapidly, leads, be applicable to large-scale industrial operate continuously, also can utilize external magnetic field to control mode of motion and the direction of immobilized enzyme, improve the catalytic efficiency of enzyme, simultaneously, also be conducive to the recovery of enzyme and reuse, reaching the purpose that reduces production costs, reduces pollution.
Lipoxygenase is a kind of oxydo-reductase that contains nonheme iron, can oxidation contain suitable, cis-1, the polybasic unsaturated fatty acid of 4-pentadiene structure, as linolic acid, linolenic acid and arachidonic acid etc., obtain the intermediate of the synthetic or chemosynthesis of medicine, can further by scission reaction, obtain the fragrance matters such as aldehydes; On the other hand, lipoxygenase can also have H
2o
2condition under, the organic compound such as chlorine monoxide promazine, aminocarb, promote the degraded of these materials, eliminates its toxicity, thereby remove pollution and the harm of these materials to environment.
Summary of the invention
The object of the present invention is to provide a kind of nano-magnetic Fe
3o
4fixed fat oxygenase and its preparation method and application.
The technical scheme that the present invention takes is:
A kind of nano-magnetic Fe
3o
4the preparation method of fixed fat oxygenase comprises that step is as follows:
(1) preparation of Nanoscale Iron magnetic particle
By KNO
3be dissolved in the water with KOH, in 80-95 ℃ of water-bath, under stirring, drip FeSO
4the aqueous solution, after dropwising, then stop after stirring one section stirring, then that suspension ageing taking-up is cooling, after the washing drying, obtains nano-magnetic Fe
3o
4;
(2) SiO
2parcel modified Nano ferromagnetic particles
Dehydrated alcohol, water and tetraethoxy are mixed and fully stir into mixed solution, the nano-magnetic Fe that then adds step (1) to prepare
3o
4, adding appropriate strong aqua after fully stirring, stirring reaction, obtain surperficial parcel SiO after finally getting solids wash, drying
2nanometer Fe
3o
4;
(3) silane coupling agent modification Nanoscale Iron magnetic particle
Dehydrated alcohol, deionized water and KH550 are added in small beaker and be hydrolyzed, then the surface that adds step (2) to prepare parcel SiO
2nanometer Fe
3o
4, stirring reaction under nitrogen protection, standing, finally get solid and fully wash drying, obtain amidized nano level Fe
3o
4complex carrier;
(4) magnetic Nano Iron oxide support immobilized lipase oxygenase
Get the amidized nano level Fe of preparation in step (3)
3o
4the crude fat oxygenase liquid of complex carrier and certain volume, add in flask stirring at normal temperature absorption, then the enzyme after absorption is taken out washing, dry, obtain the fixing lipase oxygenase of absorption method;
Perhaps get step (3) and prepare amidized nano level Fe
3o
4crude fat oxygenase liquid and the glutaraldehyde of complex carrier, certain volume join in flask, and whip attachment under normal temperature is standing, by product washing, drying, obtain the fixing lipoxygenase of crosslinking.
KNO in above-mentioned steps (1)
3concentration is 15-25wt%, and KOH concentration is 0.4-0.5moL/L, in system, adds FeSO
47H
2the 0.5-0.8 that the molar weight of O is the KOH molar weight doubly.
In the described mixed solution of step (2), the volumetric concentration of dehydrated alcohol is 85-95%(v/v), the volumetric concentration of tetraethoxy is 3-6%, Fe
3o
4the quality added (g) is 1:15-25 with volume ratio (mL) example of mixed solution.Doubly, ammonia concn is 25-28wt% to the 0.005-0.01 that the consumption of ammoniacal liquor is mixeding liquid volume, described stirring reaction time 8-12 hour.
In step (3), the volume ratio of dehydrated alcohol and deionized water is 93-96:4-7, the 3-7% that the add-on of KH550 is water and total ethanol quality; The surface parcel SiO that adds step (2) to prepare
2nanometer Fe
3o
4the amount 6-12% that is system liquid total mass, the stirring reaction time is 1-4h, time of repose 8-12h.
Amidized nano level Fe in step (4)
3o
4the quality of complex carrier (g) is 1:15-25 with volume (mL) ratio of crude fat oxygenase liquid, and the consumption of glutaraldehyde is the long-pending 0.5-1.5% of crude fat oxygenase liquid, and the enzymic activity of crude fat oxygenase liquid is 6 * 10
5more than U/mL, the time 1-3h of whip attachment.
Nano-magnetic Fe prepared by aforesaid method
3o
4the fixed fat oxygenase.
Described nano-magnetic Fe
3o
4the application of fixed fat oxygenase in the catalyzed oxidation chlorpromazine.
Application method is: at chlorpromazine solution, add H
2o
2, make H
2o
2concentration in reaction system is 0.1-5mmoL/L, regulates pH to 2.5-4.5, adds the nano-magnetic Fe of liquid mass 1-3%
3o
4the fixed fat oxygenase, at 20-45 ℃ of temperature, reaction is 30 minutes.The concentration of chlorpromazine solution is 0.5-2.5mmoL/L.
Material purity grade of the present invention can be technical grade, chemical pure, analytical pure etc.
The present invention adopts sol-gel method to prepare nano-magnetic Fe
3o
4, and use Na
2siO
3parcel, recycle silicon alkane coupling agent (KH550) is modified, and to improve dispersion of particles and stability, then lipoxygenase is fixed on carrier and forms immobilized enzyme, thereby utilize immobilized enzyme to carry out the chlorine monoxide promazine.The innovative point of this problem is, first lipoxygenase is fixed on to nano-magnetic Fe
3o
4on complex carrier, utilize the immobilized enzyme chlorine monoxide promazine made, active good, catalytic efficiency is high.
Lipoxygenase is fixed on carrier and can improves its stability, with the method for the immobilized enzyme such as entrapping method relatively, nanometer magnetic carrier has dispersiveness and the very little characteristics of particle diameter preferably in liquid, these characteristics can be so that utilize the contact rate of the fixing enzyme-to-substrate of nanometer magnetic carrier to be improved, and be conducive to mass transfer and heat transfer, thereby can further promote the carrying out of reaction, the this patent innovative point is first lipoxygenase to be fixed on nanometer magnetic carrier, and uses this immobilized enzyme to carry out the organism such as catalyzed oxidation chlorpromazine.
The accompanying drawing explanation
Fig. 1 nano-magnetic F
3o
4carrier TEM photo;
Fig. 2 nano-magnetic F
3o
4the hot weightless picture of carrier and immobilized enzyme.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be described in detail.
Embodiment 1:
By 60g KNO
3be dissolved in 280mL water with 8.5g KOH, then solution be transferred in there-necked flask, and flask is put into to 90 ℃ of water-baths, pass into nitrogen protection, take 26g FeSO
47H
2o, after fully dissolving, slowly be added drop-wise in flask with the constant voltage dropper, and constantly stirring, after dropwising, then stop after stirring 10min simultaneously, the suspension ageing taken out cooling after 4 hours, gets wherein solid, obtains nano-magnetic Fe after fully washing drying
3o
4;
The dehydrated alcohol, 10mL water and the 5mL tetraethoxy that add 100mL in flask, after fully stirring, add above-mentioned 6g nano-magnetic Fe
3o
4, after fully stirring, then add the strong aqua of 2.0mL, on agitator, stir 10 hours, obtain surperficial parcel SiO after getting solids wash, drying
2nano-magnetic Fe
3o
4;
After 54g dehydrated alcohol, 3.0g deionized water and 2.38g KH550 are added in small beaker to hydrolysis 10min, then add the modified Nano level magnetic Fe prepared
3o
4in, strong stirring, react 2h under nitrogen protection.Standing 10h, after getting solid and fully washing drying, obtain amidized nano-scale magnetic Fe
3o
4complex carrier.
Get amidized nano-scale magnetic Fe
3o
4complex carrier 5.0g, homemade crude fat oxygenase liquid 100mL, add in flask stirring at normal temperature absorption 2h, then by the enzyme after absorption take out washing, dry, obtain the fixing lipase oxygenase of absorption method.
Get the chlorpromazine solution 3mL of 0.05mmoL/L, add 30mmoL/L H
2o
20.1mL, pH is adjusted to 3.5, then adds the 0.05g immobilized enzyme, under 30 ℃, reaction is 30 minutes, and by detecting production concentration, the rate of oxidation that obtains the substrate chlorpromazine is 0.78 μ mol/ (Lmin).
Embodiment 2:
By 60g KNO
3be dissolved in 280mL water with 8.5g KOH, then solution be transferred in there-necked flask, and flask is put into to 90 ℃ of water-baths, pass into nitrogen protection, take 26g FeSO
47H
2o, after fully dissolving, slowly be added drop-wise in flask with the constant voltage dropper, and constantly stirring, after dropwising, then stop after stirring 10min simultaneously, the suspension ageing taken out cooling after 4 hours, gets wherein solid, obtains nano-magnetic Fe after fully washing drying
3o
4;
The dehydrated alcohol, 10mL water and the 5mL tetraethoxy that add 100mL in flask, after fully stirring, add above-mentioned 6g nano-magnetic Fe
3o
4, after fully stirring, then add the strong aqua of 2.0mL, on agitator, stir 10 hours, obtain surperficial parcel SiO after getting solids wash, drying
2nano-magnetic Fe
3o
4;
After 54g dehydrated alcohol, 3.0g deionized water and 2.38g KH550 are added in small beaker to hydrolysis 10min, then add the modified Nano level magnetic Fe prepared
3o
4in, strong stirring, react 2h under nitrogen protection.Standing 10h, after getting solid and fully washing drying, obtain amidized nano-scale magnetic Fe
3o
4complex carrier.
Take the nano-magnetic Fe after parcel
3o
42.5g the glutaraldehyde solution 1mL that gets 50mL crude enzyme liquid and volumetric concentration 50% joins in flask, after stirring 2h under normal temperature, standing, by product washing, drying, obtains the fixing lipoxygenase of crosslinking.
Get the chlorpromazine solution 3mL of 2.5mmoL/L, add 30mmoL/L H
2o
20.1mL, pH is adjusted to 3.5, then adds the 0.05g immobilized enzyme, under 30 ℃, reaction is 30 minutes, and by detecting production concentration, the rate of oxidation that obtains the substrate chlorpromazine is 1.24 μ mol/ (Lming).
Claims (10)
1. a nano-magnetic Fe
3o
4the preparation method of fixed fat oxygenase, is characterized in that, comprises that step is as follows:
(1) preparation of Nanoscale Iron magnetic particle
By KNO
3be dissolved in the water with KOH, in 80-95 ℃ of water-bath, under stirring, drip FeSO
4the aqueous solution, after dropwising, then stop after stirring one section stirring, then that suspension ageing taking-up is cooling, after the washing drying, obtains nano-magnetic Fe
3o
4;
(2) SiO
2parcel modified Nano ferromagnetic particles
Dehydrated alcohol, water and tetraethoxy are mixed and fully stir into mixed solution, the nano-magnetic Fe that then adds step (1) to prepare
3o
4, adding appropriate strong aqua after fully stirring, stirring reaction, obtain surperficial parcel SiO after finally getting solids wash, drying
2nanometer Fe
3o
4;
(3) silane coupling agent modification Nanoscale Iron magnetic particle
Dehydrated alcohol, deionized water and KH550 are added in small beaker and be hydrolyzed, then the surface that adds step (2) to prepare parcel SiO
2nanometer Fe
3o
4, stirring reaction under nitrogen protection, standing, finally get solid and fully wash drying, obtain amidized nano level Fe
3o
4complex carrier;
(4) magnetic Nano Iron oxide support immobilized lipase oxygenase
Get the amidized nano level Fe of preparation in step (3)
3o
4the crude fat oxygenase liquid of complex carrier and certain volume, add in flask stirring at normal temperature absorption, then the enzyme after absorption is taken out washing, dry, obtain the fixing lipoxygenase of absorption method;
Perhaps get step (3) and prepare amidized nano level Fe
3o
4crude fat oxygenase liquid and the glutaraldehyde of complex carrier, certain volume join in flask, and whip attachment under normal temperature is standing, by product washing, drying, obtain the fixing lipoxygenase of crosslinking.
2. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, KNO in step (1)
3concentration is 15-25wt%, and KOH concentration is 0.4-0.5moL/L, in system, adds FeSO
47H
2the 0.5-0.8 that the molar weight of O is the KOH molar weight doubly.
3. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, in the described mixed solution of step (2), the volumetric concentration of dehydrated alcohol is 85-95%, and the volumetric concentration of tetraethoxy is 3-6%, Fe
3o
4the volume mL ratio of the quality g added and mixed solution is 1:15-25, and doubly, ammonia concn is 25-28% to the 0.005-0.01 that the consumption of ammoniacal liquor is total liquid volume.
4. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, step (2) 8-12 hour of described stirring reaction time.
5. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, in step (3), the volume ratio of dehydrated alcohol and deionized water is 93-96:4-7, the 3-7% that the add-on of KH550 is water and total ethanol quality; The surface parcel SiO that adds step (2) to prepare
2nanometer Fe
3o
4the amount 6-12% that is system liquid total mass.
6. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, the described stirring reaction of step (3) is 1-4h, time of repose 8-12h.
7. a kind of nano-magnetic Fe according to claim 1
3o
4the preparation method of fixed fat oxygenase, is characterized in that, amidized nano level Fe in step (4)
3o
4the quality g of complex carrier is 1:15-25 with the volume mL ratio of crude fat oxygenase liquid, and the consumption of glutaraldehyde is the long-pending 0.5-1.5% of crude fat oxygenase liquid, and the enzymic activity of crude fat oxygenase liquid is 6 * 10
5more than U/mL, the time 1-3h of whip attachment.
8. the nano-magnetic Fe that prepared by method claimed in claim 1
3o
4the fixed fat oxygenase.
9. nano-magnetic Fe claimed in claim 8
3o
4the application of fixed fat oxygenase in the catalyzed oxidation chlorpromazine.
10. application according to claim 9, is characterized in that, method is: at chlorpromazine solution, add H
2o
2, make H
2o
2concentration in reaction system is 0.1-5mmoL/L, regulates pH to 2.5-4.5, adds the nano-magnetic Fe of liquid mass 1-3%
3o
4the fixed fat oxygenase, at 20-45 ℃ of temperature, reaction is 30 minutes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
| CN108753761A (en) * | 2018-05-30 | 2018-11-06 | 北京理工大学珠海学院 | A kind of preparation method of amination modified composite magnetic nano-carrier for lipase immobilization |
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|---|---|---|---|---|
| CN101613694A (en) * | 2009-05-31 | 2009-12-30 | 华东理工大学 | A kind of magnetic/functionalized SiO 2 composite microsphere immobilized enzyme and preparation method thereof |
| CN102061293A (en) * | 2010-11-10 | 2011-05-18 | 东北农业大学 | Method for preparing magnetic immobilized phospholipase A1 and method for refining crude oil by using magnetic immobilized phospholipase A1 |
| CN102489343A (en) * | 2011-12-08 | 2012-06-13 | 山东师范大学 | Ferromagnetic ion exchange material and preparation method thereof |
-
2013
- 2013-09-11 CN CN201310413363.3A patent/CN103451175B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613694A (en) * | 2009-05-31 | 2009-12-30 | 华东理工大学 | A kind of magnetic/functionalized SiO 2 composite microsphere immobilized enzyme and preparation method thereof |
| CN102061293A (en) * | 2010-11-10 | 2011-05-18 | 东北农业大学 | Method for preparing magnetic immobilized phospholipase A1 and method for refining crude oil by using magnetic immobilized phospholipase A1 |
| CN102489343A (en) * | 2011-12-08 | 2012-06-13 | 山东师范大学 | Ferromagnetic ion exchange material and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 周洁等: "不同尺寸Fe3O4 磁性颗粒的制备和表征", 《东南大学学报》 * |
| 杨勇: "功能化磁性载体的制备及其固定化脂肪酶研究", 《中国优秀硕士学位论文全文数据库-工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
| CN108753761A (en) * | 2018-05-30 | 2018-11-06 | 北京理工大学珠海学院 | A kind of preparation method of amination modified composite magnetic nano-carrier for lipase immobilization |
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| CN103451175B (en) | 2015-08-12 |
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