CN103451175B - A kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application - Google Patents

A kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application Download PDF

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CN103451175B
CN103451175B CN201310413363.3A CN201310413363A CN103451175B CN 103451175 B CN103451175 B CN 103451175B CN 201310413363 A CN201310413363 A CN 201310413363A CN 103451175 B CN103451175 B CN 103451175B
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nano
magnetic
liquid
stirring
oxygenase
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CN103451175A (en
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丁文武
张其坤
雷步增
侯昭升
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Shandong Normal University
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Abstract

The present invention relates to a kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application, 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 then lipoxygenase is fixed on carrier and forms immobilized enzyme.Utilize obtained immobilized enzyme chlorine monoxide promazine, active good, catalytic efficiency is high.

Description

A kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application
Technical field
The present invention relates to a kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application, belongs to biocatalysis technology field.
Background technology
Nano magnetic material receives extensive concern as a kind of new Carrier Materials of Immobilized Enzyme.Fe 3o 4magnetic nanoparticle method for making is simple, and to human non-toxic's side effect, its application is relatively more extensive.By nano-magnetic Fe 3o 4surface modify after make it bring functional group, get final product ligase enzyme, antibody or antigen etc., thus have important Research Significance and using value in the field such as biological, medicine and foodstuffs industry.Being fixed on by enzyme on nano magnetic material to utilize the magnetic of carrier to be separated rapidly by enzyme, to lead, be applicable to large-scale industrial operate continuously, also external magnetic field can be utilized 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 object reducing production cost, decreasing pollution.
Lipoxygenase is a kind of oxydo-reductase containing nonheme iron, can be oxidized containing 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 pharmaceutical synthesis or chemosynthesis, the fragrance matters such as aldehydes can be obtained further by scission reaction; 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, eliminate its toxicity, thus remove these materials to the pollution of environment and harm.
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 step as follows:
(1) preparation of Nanoscale Iron magnetic particle
By KNO 3be dissolved in the water with KOH, in 80-95 DEG C of water-bath, under stirring, drip FeSO 4the aqueous solution, after dropwising, then stops after stirring one section stirring, then cooling is taken out in suspension ageing, namely obtain nano-magnetic Fe after washing drying 3o 4;
(2) SiO 2parcel modified Nano ferromagnetic particles
The mixing of dehydrated alcohol, water and tetraethoxy is fully stirred into mixed solution, then adds nano-magnetic Fe prepared by step (1) 3o 4, add appropriate strong aqua, stirring reaction after fully stirring, after finally getting solids wash, drying, namely obtain surperficial parcel SiO 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 is hydrolyzed, then add surface parcel SiO prepared by step (2) 2nanometer Fe 3o 4, stirred under nitrogen atmosphere reacts, and leaves standstill, finally gets 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, adds stirring at normal temperature absorption in flask, then the enzyme after absorption is taken out washing, drying, namely obtains the fixing lipase oxygenase of absorption method;
Or get step (3) and prepare amidized nano level Fe 3o 4the crude fat oxygenase liquid of complex carrier, certain volume and glutaraldehyde join in flask, whip attachment under normal temperature, leave standstill, and by product washing, drying, obtain the lipoxygenase that crosslinking is fixing.
KNO in above-mentioned steps (1) 3concentration is 15-25wt%, KOH concentration is 0.4-0.5moL/L, in system, add FeSO 47H 2the molar weight of O is 0.5-0.8 times of KOH molar weight.
In mixed solution described in 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 (g) added is 1:15-25 with volume ratio (mL) example of mixed solution.The consumption of ammoniacal liquor is 0.005-0.01 times of mixeding liquid volume, and ammonia concn is 25-28wt%, described stirring reaction time 8-12 hour.
The 3-7% of the volume ratio of dehydrated alcohol and deionized water to be the add-on of 93-96:4-7, KH550 be water and total ethanol quality in step (3); Add surface parcel SiO prepared by step (2) 2nanometer Fe 3o 4amount be the 6-12% of 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 (g) of complex carrier and the volume (mL) of crude fat oxygenase liquid are than being 1:15-25, and the consumption of glutaraldehyde is the 0.5-1.5% that crude fat oxygenase liquid amasss, 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 4fixed fat oxygenase.
Described nano-magnetic Fe 3o 4the application of fixed fat oxygenase in catalyzed oxidation chlorpromazine.
Application method is: add H at chlorpromazine solution 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 4fixed fat oxygenase, reacts 30 minutes at 20-45 DEG C of temperature.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, thus utilize immobilized enzyme to carry out chlorine monoxide promazine.The innovative point of this problem is, first lipoxygenase is fixed on nano-magnetic Fe 3o 4on complex carrier, utilize obtained immobilized enzyme chlorine monoxide promazine, active good, catalytic efficiency is high.
Lipoxygenase is fixed on carrier and can improves its stability, with the Measures compare of the immobilized enzyme such as entrapping method, nanometer magnetic carrier has preferably dispersed and feature that particle diameter is very little in a liquid, these features can make the contact rate of the enzyme-to-substrate utilizing nanometer magnetic carrier to fix be improved, and be conducive to mass transfer and heat transfer, thus the carrying out of reaction can be promoted further, this patent innovative point lipoxygenase is fixed on nanometer magnetic carrier first, and use this immobilized enzyme to carry out the organism such as catalyzed oxidation chlorpromazine.
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 90 DEG C of water-baths, pass into nitrogen protection, take 26g FeSO 47H 2o, after fully dissolving, is slowly added drop-wise in flask with constant voltage dropper, constantly stirs simultaneously, after dropwising, then stops after stirring 10min, suspension ageing was taken out cooling after 4 hours, gets wherein solid, fully namely obtains nano-magnetic Fe after washing drying 3o 4;
In flask, add the dehydrated alcohol of 100mL, 10mL water and 5mL tetraethoxy, after fully stirring, add above-mentioned 6g nano-magnetic Fe 3o 4, after fully stirring, then add the strong aqua of 2.0mL, agitator stir 10 hours, after getting solids wash, drying, namely obtains surperficial parcel SiO 2nano-magnetic Fe 3o 4;
54g dehydrated alcohol, 3.0g deionized water and 2.38g KH550 are added after being hydrolyzed 10min in small beaker, then add the modified Nano level magnetic Fe prepared 3o 4in, strong stirring, reacts 2h under nitrogen protection.Leave standstill 10h, get after solid fully washs 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 stirring at normal temperature absorption 2h in flask, then the enzyme after adsorbing is taken out washing, drying, namely obtains the fixing lipase oxygenase of absorption method.
Get the chlorpromazine solution 3mL of 0.05mmoL/L, add 30mmoL/L H 2o 20.1mL, is adjusted to 3.5 by pH, then adds 0.05g immobilized enzyme, reacts 30 minutes at 30 DEG C, and by detecting production concentration, the rate of oxidation obtaining substrate chlorpromazine is 0.78 μm of ol/ (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 90 DEG C of water-baths, pass into nitrogen protection, take 26g FeSO 47H 2o, after fully dissolving, is slowly added drop-wise in flask with constant voltage dropper, constantly stirs simultaneously, after dropwising, then stops after stirring 10min, suspension ageing was taken out cooling after 4 hours, gets wherein solid, fully namely obtains nano-magnetic Fe after washing drying 3o 4;
In flask, add the dehydrated alcohol of 100mL, 10mL water and 5mL tetraethoxy, after fully stirring, add above-mentioned 6g nano-magnetic Fe 3o 4, after fully stirring, then add the strong aqua of 2.0mL, agitator stir 10 hours, after getting solids wash, drying, namely obtains surperficial parcel SiO 2nano-magnetic Fe 3o 4;
54g dehydrated alcohol, 3.0g deionized water and 2.38g KH550 are added after being hydrolyzed 10min in small beaker, then add the modified Nano level magnetic Fe prepared 3o 4in, strong stirring, reacts 2h under nitrogen protection.Leave standstill 10h, get after solid fully washs drying, obtain amidized nano-scale magnetic Fe 3o 4complex carrier.
Take the nano-magnetic Fe after parcel 3o 42.5g, the glutaraldehyde solution 1mL getting 50mL crude enzyme liquid and volumetric concentration 50% joins in flask, after stirring 2h, leaves standstill under normal temperature, by product washing, drying, obtains the lipoxygenase that crosslinking is fixing.
Get the chlorpromazine solution 3mL of 2.5mmoL/L, add 30mmoL/L H 2o 20.1mL, is adjusted to 3.5 by pH, then adds 0.05g immobilized enzyme, reacts 30 minutes at 30 DEG C, and by detecting production concentration, the rate of oxidation obtaining substrate chlorpromazine is 1.24 μm of ol/ (Lming).

Claims (4)

1. a nano-magnetic Fe 3o 4the preparation method of fixed fat oxygenase, is characterized in that, comprises step as follows:
(1) preparation of Nanoscale Iron magnetic particle
By KNO 3be dissolved in the water with KOH, in 80-95 DEG C of water-bath, under stirring, drip FeSO 4the aqueous solution, after dropwising, then stops after stirring one section stirring, then cooling is taken out in suspension ageing, namely obtain nano-magnetic Fe after washing drying 3o 4; Wherein, KNO 3concentration is 15-25wt%, KOH concentration is 0.4-0.5moL/L, in system, add FeSO 4molar weight be the 0.5-0.8 of KOH molar weight doubly;
(2) SiO 2parcel modified Nano ferromagnetic particles
The mixing of dehydrated alcohol, water and tetraethoxy is fully stirred into mixed solution, then adds nano-magnetic Fe prepared by step (1) 3o 4, add appropriate strong aqua, stirring reaction after fully stirring, after finally getting solids wash, drying, namely obtain surperficial parcel SiO 2nanometer Fe 3o 4; In described mixed solution, 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 the consumption of ammoniacal liquor is 0.005-0.01 times of total liquid volume, and ammonia concn is 25-28%, the time 8-12 hour of stirring reaction;
(3) silane coupling agent modification Nanoscale Iron magnetic particle
Dehydrated alcohol, deionized water and KH550 are added in small beaker and is hydrolyzed, then add surface parcel SiO prepared by step (2) 2nanometer Fe 3o 4, stirred under nitrogen atmosphere reacts, and leaves standstill, finally gets solid and fully wash drying, obtain amidized nano level Fe 3o 4complex carrier, the 3-7% of the volume ratio of dehydrated alcohol and deionized water to be the add-on of 93-96:4-7, KH550 be water and total ethanol quality; Add surface parcel SiO prepared by step (2) 2nanometer Fe 3o 4amount be the 6-12% of system liquid total mass, stirring reaction is 1-4h, time of repose 8-12h;
(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, adds stirring at normal temperature absorption in flask, then the enzyme after absorption is taken out washing, drying, namely obtains the fixing lipoxygenase of absorption method; Or get step (3) and prepare amidized nano level Fe 3o 4the crude fat oxygenase liquid of complex carrier, certain volume and glutaraldehyde join in flask, whip attachment under normal temperature, leave standstill, and by product washing, drying, obtain the lipoxygenase that crosslinking is fixing; Amidized nano level Fe 3o 4the quality g of complex carrier and the volume mL of crude fat oxygenase liquid is than being 1:15-25, and the consumption of glutaraldehyde is the 0.5-1.5% that crude fat oxygenase liquid amasss, and the enzymic activity of crude fat oxygenase liquid is 6 × more than 105U/mL, the time 1-3h of whip attachment.
2. the nano-magnetic Fe for preparing of method according to claim 1 3o 4fixed fat oxygenase.
3. nano-magnetic Fe according to claim 2 3o 4the application of fixed fat oxygenase in catalyzed oxidation chlorpromazine.
4. application according to claim 3, is characterized in that, method is: add H at chlorpromazine solution 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 4fixed fat oxygenase, reacts 30 minutes at 20-45 DEG C of temperature.
CN201310413363.3A 2013-09-11 2013-09-11 A kind of nano-magnetic Fe 3o 4fixed fat oxygenase and its preparation method and application Expired - Fee Related CN103451175B (en)

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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
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