CN102250869B - Preparation method of magnetic immobilized enzyme for producing plant sterol ester - Google Patents
Preparation method of magnetic immobilized enzyme for producing plant sterol ester Download PDFInfo
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- CN102250869B CN102250869B CN201110138426XA CN201110138426A CN102250869B CN 102250869 B CN102250869 B CN 102250869B CN 201110138426X A CN201110138426X A CN 201110138426XA CN 201110138426 A CN201110138426 A CN 201110138426A CN 102250869 B CN102250869 B CN 102250869B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention relates to a preparation method of a magnetic immobilized enzyme for producing a plant sterol ester, belonging to the field of catalytic materials and chemical synthesis. The preparation method of the magnetic immobilized enzyme for producing the plant sterol ester is characterized by comprising the following steps: 1) carrying out chemical modification on a magnetic substrate with vinylphosphonic acid to introduce a carbon-carbon double bond with reactivity onto the surface of the magnetic substrate; 2) wrapping the surfaces of the modified magnetic particles with polymer layers containing epoxy or sulfonate radical groups to obtain magnetic polymer microspheres; and 3) in a buffer solution, immobilizing an enzyme on the surfaces of the magnetic polymer microspheres by an epoxy ring-opening or electrostatic adsorption method to obtain the magnetic immobilized enzyme for producing the plant sterol ester. The obtained magnetic immobilized enzyme has the characteristics of high catalytic activity, good reuse performance and is simple in separation and recovery.
Description
Technical field
The present invention relates to a kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester, belong to catalytic material and the field of chemical synthesis.
Background technology
Plant sterol has the reduction serum cholesterol level, the effect of preventing cardiovascular disease.But the low solubility of free plant sterol in water and oil limited it in Application in Food.Research shows that the plant sterol ester by plant sterol and lipid acid make through esterification can increase the fat-soluble of plant sterol greatly, can add grease more easily to or contains in the fatty foods.In addition, the absorption rate of plant sterol ester is 5 times of plant sterol, and has the decreasing cholesterol effect of better lipotropy and Geng Jia, is a kind of novel functional food ingredient.2010, plant sterol ester was listed in new food resource by China Ministry of Health.
At present, plant sterol ester synthetic mainly contains two kinds of chemical method and enzyme process.The approach of chemical method synthesizing phytosterol ester has: direct esterification method, esterification of acyl chloride method, anhydride esterifying method, ester-interchange method etc.Catalyzer commonly used mainly contains alkoxy base metallic compounds such as sodium methylate, sodium ethylate.These chemical catalysts are easy to industriallization but easy etching apparatus, and are prone to produce by product, also exist aftertreatment complicated, be prone to cause critical defect such as three-waste pollution environment.Enzyme catalysis synthesis method mild condition, the side reaction of having avoided high temperature to produce, and product separate easily purifying, but the stability of resolvase and recovery, repeated use problem are still restricting the application in the actual production.
The immobilized enzyme that with the magnetic nanoparticle is preparing carriers is easy to from reaction system, separate and reclaim, and is easy to operate; The mode of motion of external magnetic field control magnetic immobilized enzyme capable of using substitutes traditional mechanical stirring, improves the catalytic efficiency (of immobilized enzyme.In recent years, be that the research of preparing carriers immobilized enzyme emerges in an endless stream with the magnetic-particle, prepared 3 kinds of magnetic immobilized lipases that contain epoxy matrix like Li Yanfeng etc., this type of immobilized enzyme stability obviously is superior to resolvase; But the surface-treated of magneticsubstance adopts the method for silylanization usually, needs to use a large amount of deleterious toluene to make solvent, the not enough environmental protection of preparation process.CN101280298A adopts the amino Fe of surface adsorption
3O
4Powder has been preparing carriers immobilized enzyme, it actively obviously descends along with the increase of reusing number of times.Yet the research of magnetic immobilized enzyme mainly concentrates on preparation method and the preliminary activity rating, the magnetic immobilized enzyme that can be used for the actual chemical reaction of catalysis also seldom, the magnetic immobilized enzyme that is used for plant sterol ester production does not also appear in the newspapers.Therefore, activity, stability, repeated use number of times and the high efficiente callback utilization of magnetic immobilized enzyme in catalyzed chemical reaction is still urgent problem in the present magnetic immobilized enzyme technology of preparing.
Summary of the invention
The object of the present invention is to provide a kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester.This magnetic immobilized enzyme has catalytic activity height, use properties is good repeatedly, Separation and Recovery is easy characteristics.
Be to realize above-mentioned purpose, the technical scheme that the present invention adopted is: a kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester is characterized in that it comprises the steps:
1) press the magnetic matrix: vinyl phosphonic aqueous acid=20-30g: 200mL, choose magnetic matrix and vinyl phosphonic aqueous acid, the concentration of the therein ethylene base phosphonic acids aqueous solution is 20-35mg/mL, the pH of vinyl phosphonic aqueous acid is 6.0 (transferring pH with NaOH); The magnetic matrix is scattered in the vinyl phosphonic aqueous acid; Nitrogen protection refluxed reaction 8-12h; (zero(ppm) water, methyl alcohol respectively clean 3-5 time to adopt zero(ppm) water, methyl alcohol alternately to clean 3-5 time after reaction finishes; Hocket), vacuum-drying obtains modification magnetic matrix (or claiming the magnetic nanoparticle that two keys are modified);
Utilize the electron pair attack magnetic matrix (Fe of oxygen on the vinyl phosphonate
3O
4, Fe
2O
3Magnetic nanoparticle) surperficial enrich, electron deficiency Fe is adsorbed on the magnetic matrix vinyl phosphonate, the uniformly two keys of whole magnetic matrix surface band last layer;
2) with in the modification magnetic matrix suspension organic solvent, obtain the suspension-s that concentration is 10-20mg/mL; Press monomer: organic solvent=20-50mmol: 1L, the mol ratio of monomer and linking agent is 1: 1-1: 4, the consumption of initiator is the 1-2% of monomer and linking agent total mass, prepares monomer, linking agent and initiator; Monomer, linking agent and initiator are mixed with above-mentioned suspension-s, obtain mixture; Mixture heating up keeps 10-30min to 60-80 ℃, and elevated temperature is controlled at and steams half the organic solvent in the 60-120min to 92-96 ℃ then, keeps between the reaction period stirring; After reaction finishes, adopt externally-applied magnetic field to isolate granular substance, granular substance alternately cleans (zero(ppm) water, methyl alcohol respectively clean 3-5 time, hocket) 3-5 time with zero(ppm) water and methyl alcohol, and vacuum-drying obtains magnetic polymer microsphere;
Concentration and ratio that can be through monomer and linking agent in the control solution and the speed that steams solvent are come the thickness of controlling polymers layer;
3) be 99.9%: 0.1% by the phosphate buffer soln (concentration is 0.05M-0.1M) of pH 5.0-7.0 and the mass percent of tween 20, tween 20 is joined in the phosphate buffer soln, obtain containing the phosphate buffer soln of tween 20;
By lypase: the phosphate buffer soln of pH 5.0-7.0 (concentration is 0.05M-0.1M)=2-5mg: 1mL, lypase is joined in the phosphate buffer soln, obtain lipase solution;
By magnetic polymer microsphere: the phosphate buffer soln that contains tween 20: lipase solution=20-50mg: 1mL: 1mL, choose magnetic polymer microsphere, contain the phosphate buffer soln and the lipase solution of tween 20; Magnetic polymer microsphere is soaked into 1h in containing the phosphate buffer soln of tween 20, remove the phosphate buffer soln that this contains tween 20 then, change lipase solution (this contains the phosphate buffer soln of tween 20 by the lipase solution replacement) into; Carry out lipase immobilization; Temperature of reaction 20-30 ℃, shaking speed 150-200rpm, reaction times 6-10h; Reaction obtains the magnetic immobilized enzyme after finishing; The magnetic immobilized enzyme is cleaned 3-5 time through zero(ppm) water, after the lyophilize, promptly obtain being used to produce the magnetic immobilized enzyme of plant sterol ester.
Described magnetic matrix is that particle diameter is the Fe of 10-50nm
3O
4Or particle diameter is the Fe of 10-50nm
2O
3Nano particle.
Described organic solvent is acetonitrile or Virahol etc.
Described monomer comprises SY-Monomer G or the glycidyl allyl ether that contains epoxy matrix; Or contain 2-acrylic amide-2-methyl isophthalic acid-propanesulfonic acid of sulfonate group etc.
Described linking agent is an ethylene glycol dimethacrylate.
Described initiator is a Diisopropyl azodicarboxylate.
Described lypase; Comprise the pig pancreas enzyme that derives from animal; Or derive from fold candida, Candida lipolytica, antarctic candida, pseudomonas cepacia lypase or the Rhizopus oryzae etc. of microbial fermentation, preferably derive from the lypase that microbial fermentation obtains.
The particle diameter of the prepared magnetic immobilized enzyme of the present invention is 1-5 μ m.
The condition that said magnetic immobilized enzyme is used for the plant sterol catalytic esterification is: the octane-iso of selection low-pole, normal hexane, hexanaphthene etc. are solvent; Immobilized enzyme add-on 5-40mg/mL; The molecular sieve add-on is 20-60mg/mL, temperature of reaction 40-60 ℃, and reaction times 6-24h.Esterification can be carried out in the reaction unit that band stirs or in magnetic field fluidized bed; After reaction finishes, with permanent magnet or EM field the absorption of magnetic immobilized enzyme is reclaimed, the magnetic immobilized enzyme of recovery is alternately reusable more than 5 times after the cleaning through the trimethyl carbinol and octane-iso (or normal hexane, hexanaphthene).
The magnetic immobilized enzyme raw material suitability of the present invention's preparation is wide, can be respectively applied for esterification, the transesterification of fatty acid methyl (second) ester and plant sterol and the transesterification of triglyceride level and plant sterol of catalysis lipid acid and plant sterol.
Lipid acid of the present invention be palmitinic acid, Triple Pressed Stearic Acid, oleic acid, linolic acid, linolenic acid, etc. saturated or undersaturated lipid acid, also can be mixed fatty acid; Fatty ester is first (second) ester of above-mentioned lipid acid, also can be that vegetables oil is through methyl esters or the reacted mixed aliphatic ester of ethyl esterization; Triglyceride level is animal-plant oil such as rapeseed oil, tea-seed oil, sunflower seed oil, VT 18, oleum lini, fish oil.
Plant sterol of the present invention can be the miscellany of multiple sterols such as Stigmasterol, Sitosterol, brassicasterol, campesterol, pure article that also can above-mentioned single sterol.
The present invention at first adopts vinyl phosphonate that magnetic matrix is carried out chemical modification, introduces the carbon-to-carbon double bond with reactive behavior on its surface; Wrap up the polymer layer that contains epoxy group(ing), sulfonic group isoreactivity group on modification magnetic matrix (magnetic-particle of modification) surface then, make magnetic polymer microsphere; Next in buffered soln, realize the immobilization of enzyme, make the magnetic immobilized enzyme on the magnetic polymer microsphere surface through methods such as epoxy ring-opening or electrostatic adhesion.At last, this magnetic immobilized enzyme is used for the esterification of catalysis lipid acid and plant sterol or the transesterification of fatty ester and plant sterol.
The invention has the beneficial effects as follows:
1) at first adopts vinyl phosphonate that magnetic matrix is carried out chemical modification, can introduce carbon-to-carbon double bond easily on its surface with reactive behavior; Contain the polymer layer of epoxy group(ing), sulfonic group isoreactivity group through suspension polymerization at modification magnetic matrix surface parcel then, thereby make multiple magnetic polymer microsphere immobilized enzyme.
2) the present invention has simple and easy to doly, and particle size is controlled, and the magnetic immobilized enzyme catalysis is active high; Transformation efficiency keeps more than 80%; Use properties good (reuse often, can reach more than 5 times) can adopt externally-applied magnetic field to realize the characteristics such as efficient sharp separation of catalyzer and product repeatedly.
3) the magnetic immobilized enzyme raw material suitability of the present invention's preparation is wide.Can distinguish the esterification of catalysis lipid acid and plant sterol, the transesterification of fatty acid methyl (second) ester and plant sterol and the transesterification of triglyceride level and plant sterol are to satisfy multiple different needs.
4) the plant sterol ester route of synthesis of setting up based on this magnetic immobilized enzyme has been avoided poisonous and use corrosive reagents, and by product is few, and reaction conditions is gentle, and the product that makes has lighter color, characteristics that quality is high.
5) preparation process environmental protection.
Embodiment
Preferred version of the present invention is described below; It is included within the scope of the present invention's protection, but does not limit the present invention.
Embodiment 1:
A kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester, it comprises the steps:
1), the 4g vinyl phosphonate is dissolved in the 200mL zero(ppm) water, using NaOH to transfer pH is 6.0, obtains the vinyl phosphonic aqueous acid; Take by weighing Fe
3O
4Nano particle 20g (particle diameter is 10-50nm); The vinyl phosphonic aqueous acid that adds above-mentioned 200mL is under the mechanical stirring, at nitrogen protection refluxed 8h; (zero(ppm) water, methyl alcohol respectively clean 5 times to adopt zero(ppm) water and methyl alcohol alternately to clean 5 times after reaction finishes; Hocket), vacuum-drying obtains modification magnetic matrix.
2), 4g modification magnetic matrix is suspended in the 400mL acetonitrile, obtain suspension-s; Add monomer SY-Monomer G 2.4g (17mmol) in the suspension-s, linking agent ethylene glycol dimethacrylate 6.5g (33mmol), initiator Diisopropyl azodicarboxylate 0.178g is heated to 80 ℃, keeps 20min; Elevated temperature is controlled at the acetonitrile (organic solvent) that steams 200mL in the 80min to 92-96 ℃ then, keeps vigorous stirring between the reaction period; After reaction finishes, adopt externally-applied magnetic field to isolate granular substance (being about to magnetic polymer particles shifts out), granular substance alternately cleans 5 times with zero(ppm) water and methyl alcohol, and vacuum-drying makes the magnetic polymer microsphere that contains epoxy group(ing), and diameter is 1-3 μ m.
3), be 99.9%: 0.1% by the mass percent of the phosphate buffer soln (0.1M) of pH 7.0 and tween 20, tween 20 is joined in the phosphate buffer soln, obtain containing the phosphate buffer soln (preparation 50mL) of tween 20;
Press lypase: the phosphate buffer soln of pH 7.0 (0.1M)=2mg: 1mL, lypase is joined in the phosphate buffer soln, obtain lipase solution (preparation 50mL); Lypase derives from fold candida;
The 1g magnetic polymer microsphere after soaking into 1h in the phosphate buffer soln that contains tween 20 (0.1M, pH 7.0 contain mass percent 0.1% tween 20) of 50mL, is removed the phosphate buffer soln that this contains tween 20; Change the lipase solution of 50mL into, hatch 6h in 20 ℃ of shaking tables, rotating speed 160rpm; After reaction finishes; Clean 5 times with zero(ppm) water then, after the lyophilize, promptly obtain being used to produce the magnetic immobilized enzyme (4 ℃ of stored refrigerated) of plant sterol ester.
Use: sterol ester is combined in vegetables oil and sterol transesterification: take by weighing plant sterol 4.2g; By rapeseed oil and plant sterol mol ratio is to take by weighing triglyceride level at 1.5: 1, and 2g magnetic immobilized enzyme (embodiment 1) adds the 100mL octane-iso; Place on 55 ℃ of constant temperature shaking tables and react 12h, rotating speed is 160rpm.Remove the magnetic immobilized enzyme by magnetic field after reaction finishes, decompression steams solvent.The absolute ethyl alcohol that adds reactant quality 5% stirs and is placed on 4 ℃ of refrigerator overnight, the centrifugal unreacted plant sterol completely of removing, and short-path distillation is removed unreacted rapeseed oil, and esterification yield is 85.2%.
The magnetic immobilized enzyme reclaims after after octane-iso cleans 3 times, repeat above-mentioned reaction 5 times after, esterification yield is still greater than 80%.
Embodiment 2:
A kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester, it comprises the steps:
1), the 6g vinyl phosphonate is dissolved in the 200mL zero(ppm) water, using NaOH to transfer pH is 6.0, obtains the vinyl phosphonic aqueous acid; Take by weighing Fe
2O
3Nano particle 25g (particle diameter is 10-50nm) adds the vinyl phosphonic aqueous acid of above-mentioned 200mL, and under the mechanical stirring, at nitrogen protection refluxed 8h, the reaction back that finishes adopts zero(ppm) water and methyl alcohol alternately to clean 5 times, and vacuum-drying obtains modification magnetic matrix.
2), 6g modification magnetic matrix is suspended in the 400mL Virahol, obtain suspension-s; Add monomer 2-acrylic amide-2-methyl isophthalic acid-propanesulfonic acid 2.1g (10mmol) in the suspension-s, linking agent ethylene glycol dimethacrylate 7.9g (40mmol), initiator Diisopropyl azodicarboxylate 0.1g is heated to 80 ℃, keeps 30min; Elevated temperature is to 92-96 ℃ then; Be controlled at the Virahol (organic solvent) that steams 200mL in the 100min, after the reaction of maintenance vigorous stirring finishes between the reaction period, adopt externally-applied magnetic field to isolate granular substance (being about to magnetic polymer particles shifts out); Granular substance alternately cleans 5 times with zero(ppm) water and methyl alcohol; Vacuum-drying, the magnetic polymer microsphere that contains sulfonate radical that makes, diameter are 2-4 μ m.
3), be 99.9%: 0.1% by the mass percent of the phosphate buffer soln (0.05M) of pH 5.0 and tween 20, tween 20 is joined in the phosphate buffer soln, obtain containing the phosphate buffer soln (preparation 50mL) of tween 20;
Press lypase: the phosphate buffer soln of pH 5.0 (0.05M)=3mg: 1mL, lypase is joined in the phosphate buffer soln, obtain lipase solution (preparation 50mL); Lypase derives from Candida lipolytica;
The 2g magnetic polymer microsphere after soaking into 1h in the phosphate buffer soln that contains tween 20 (0.05M, pH 5.0 contain mass percent 0.1% tween 20) of 50mL, is removed the phosphate buffer soln that this contains tween 20; Change the lipase solution of 50mL into, hatch 6h in 25 ℃ of shaking tables, rotating speed 150rpm; After reaction finishes; Clean 5 times with zero(ppm) water then, after the lyophilize, promptly obtain being used to produce the magnetic immobilized enzyme (4 ℃ of stored refrigerated) of plant sterol ester.
Use: lipid acid and sterol synthesizing phytosterol ester: take by weighing plant sterol 4.2g; By alpha-linolenic acid and plant sterol mol ratio is to take by weighing alpha-linolenic acid at 2: 1; 3g magnetic immobilized enzyme (embodiment 2) adds the 100mL octane-iso, places on 55 ℃ of constant temperature shaking tables and reacts 8h; Rotating speed is 160rpm;
type molecular sieve 5g removes the magnetic immobilized enzyme by magnetic field after reaction finishes, and decompression steams solvent.The absolute ethyl alcohol that adds reactant quality 5% stirs and is placed on 4 ℃ of refrigerator overnight, the centrifugal unreacted plant sterol completely of removing; The Na that adds reaction solution volume 1-1.5 mass concentration 3% doubly
2CO
3Solution, 40 ℃ were stirred 10 minutes down, discarded water layer, removed unreacted linolenic acid, with zero(ppm) water product were washed till neutrality again, promptly obtained plant sterol after the vacuum-drying at product, and esterification yield is 93.2%.Immobilized enzyme reclaims after after octane-iso cleans 3 times, repeat above-mentioned reaction 5 times after, esterification yield is still greater than 80%.
Embodiment 3:
A kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester, it comprises the steps:
1), the 7g vinyl phosphonate is dissolved in the 200mL zero(ppm) water, transfers pH 6.0, obtain the vinyl phosphonic aqueous acid with NaOH; Take by weighing Fe
3O
4Nano particle 30g (particle diameter is 10-50nm) adds the vinyl phosphonic aqueous acid of above-mentioned 200mL, and under the mechanical stirring, at nitrogen protection refluxed 12h, the reaction back that finishes adopts zero(ppm) water and methyl alcohol alternately to clean 3 times, and vacuum-drying obtains modification magnetic matrix.
2), 8g modification magnetic matrix is suspended in the 400mL acetonitrile, add monomer glycidyl allyl ether 1.9g (17mmol), linking agent ethylene glycol dimethacrylate 6.5g (33mmol), initiator azo-bis-isobutyl cyanide 0.09g is heated to 60 ℃, keeps 10min; Elevated temperature is controlled at the organic solvent (acetonitrile) that steams 200mL in the 120min to 92-96 ℃ then, keeps vigorous stirring between the reaction period; After reaction finishes, magnetic polymer particles is shifted out (adopting externally-applied magnetic field separating particles shape thing), alternately clean 3 times with zero(ppm) water and methyl alcohol, vacuum-drying, the magnetic polymer microsphere that contains epoxy group(ing) that makes, diameter are 3-5 μ m.
3), be 99.9%: 0.1% by the mass percent of the phosphate buffer soln (0.1M) of pH7.0 and tween 20, tween 20 is joined in the phosphate buffer soln, obtain containing the phosphate buffer soln (preparation 50mL) of tween 20;
Press lypase: the phosphate buffer soln of pH7.0 (0.1M)=5mg: 1mL, lypase is joined in the phosphate buffer soln, obtain lipase solution (preparation 50mL); Lypase derives from fold candida;
The 2.5g magnetic polymer microsphere after soaking into 1h in the phosphate buffer soln that contains tween 20 (0.1M, pH 7.0 contain mass percent 0.1% tween 20) of 50mL, is removed the phosphate buffer soln that this contains tween 20; Change the lipase solution of 50mL into, hatch 10h in 30 ℃ of shaking tables, rotating speed 200rpm; After reaction finishes; Clean 3 times with zero(ppm) water then, after the lyophilize, promptly obtain being used to produce the magnetic immobilized enzyme (4 ℃ of stored refrigerated) of plant sterol ester.
Use: sterol ester is combined in fatty acid methyl ester and sterol transesterification: take by weighing plant sterol 4.2g; By fatty acid methyl ester (product of oleum lini esterification) and plant sterol mol ratio is to take by weighing fatty acid methyl ester at 8: 1; 2g magnetic immobilized enzyme (embodiment 3); Reaction 10h under the 50Pa vacuum condition, temperature of reaction is 55 ℃, the mechanical stirring rotating speed is 200rpm.Remove the magnetic immobilized enzyme by magnetic field after reaction finishes, adopt short-path distillation to reclaim excess fats acid methyl esters.The absolute ethyl alcohol that adds reactant quality 5% stirs and is placed on 4 ℃ of refrigerator overnight, and the centrifugal unreacted plant sterol completely of removing promptly obtains the plant sterol ester product, and esterification yield is 84.5%.Immobilized enzyme reclaims after after octane-iso cleans 3 times, repeat above-mentioned reaction 5 times after, esterification yield is still greater than 80%.
Each raw material (like each concrete raw material of lypase) that the present invention is cited; And the bound of each raw material of the present invention, interval value; And the bound of processing parameter (like temperature, time etc.), interval value can both realize the present invention, do not enumerate embodiment one by one at this.
Claims (2)
1. a preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester is characterized in that it comprises the steps:
1) press the magnetic matrix: vinyl phosphonic aqueous acid=20-30g: 200mL, choose magnetic matrix and vinyl phosphonic aqueous acid, the concentration of the therein ethylene base phosphonic acids aqueous solution is 20-35mg/mL, the pH of vinyl phosphonic aqueous acid is 6.0; The magnetic matrix is scattered in the vinyl phosphonic aqueous acid, and nitrogen protection refluxed reaction 8-12h adopts zero(ppm) water, methyl alcohol alternately to clean 3-5 time after reaction finishes, and vacuum-drying obtains modification magnetic matrix;
Described magnetic matrix is that particle diameter is the Fe of 10-50nm
3O
4Or particle diameter is the Fe of 10-50nm
2O
3Nano particle;
2) with in the modification magnetic matrix suspension organic solvent, obtain the suspension-s that concentration is 10-20mg/mL; Press monomer: organic solvent=20-50mmol: 1L, the mol ratio of monomer and linking agent is 1: 1-1: 4, the consumption of initiator is the 1-2% of monomer and linking agent total mass, prepares monomer, linking agent and initiator; Monomer, linking agent and initiator are mixed with above-mentioned suspension-s, obtain mixture; Mixture heating up keeps 10-30min to 60-80 ℃, and elevated temperature is controlled at and steams half the organic solvent in the 60-120min to 92-96 ℃ then, keeps between the reaction period stirring; After reaction finishes, adopt externally-applied magnetic field to isolate granular substance, granular substance alternately cleans 3-5 time with zero(ppm) water and methyl alcohol, and vacuum-drying obtains magnetic polymer microsphere;
Described organic solvent is acetonitrile or Virahol;
Described monomer is SY-Monomer G or glycidyl allyl ether; Or be 2-acrylic amide-2-methyl isophthalic acid-propanesulfonic acid;
Described linking agent is an ethylene glycol dimethacrylate;
Described initiator is a Diisopropyl azodicarboxylate;
3) be 99.9%: 0.1% by the phosphate buffer soln of pH 5.0-7.0 and the mass percent of tween 20, tween 20 is joined in the phosphate buffer soln, obtain containing the phosphate buffer soln of tween 20;
Press lypase: phosphate buffer soln=2-5mg of pH5.0-7.0: 1mL, lypase is joined in the phosphate buffer soln, obtain lipase solution;
By magnetic polymer microsphere: the phosphate buffer soln that contains tween 20: lipase solution=20-50mg: 1mL: 1mL, choose magnetic polymer microsphere, contain the phosphate buffer soln and the lipase solution of tween 20; Magnetic polymer microsphere is soaked into 1h in containing the phosphate buffer soln of tween 20, remove the phosphate buffer soln that this contains tween 20 then, change lipase solution into; Carry out lipase immobilization, temperature of reaction 20-30 ℃, shaking speed 150-200rpm; Reaction times 6-10h after reaction finishes, cleans 3-5 time through zero(ppm) water; After the lyophilize, promptly obtain being used to produce the magnetic immobilized enzyme of plant sterol ester.
2. a kind of preparation method who is used to produce the magnetic immobilized enzyme of plant sterol ester according to claim 1; It is characterized in that: described lypase is the pig pancreas enzyme that derives from animal, or derives from fold candida, Candida lipolytica, antarctic candida, pseudomonas cepacia lypase or the Rhizopus oryzae of microbial fermentation.
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| CN103993063A (en) * | 2014-05-29 | 2014-08-20 | 江苏大学 | Preparation method of ergosterol ester |
| CN105483111A (en) * | 2014-09-26 | 2016-04-13 | 丰益(上海)生物技术研发中心有限公司 | Immobilization lipase and preparation method thereof |
| CN107532110A (en) * | 2015-08-03 | 2018-01-02 | 于殿宇 | The method of phosphatide biological synthetic functional lipid in nano-magnetic immobilised enzymes continuously-directional catalysis crude oil of soybean |
| CN106244514B (en) * | 2016-08-02 | 2019-09-03 | 宝健（北京）生物技术有限公司 | A kind of cultural method of the soybean stem cell culture rich in phytosterol |
| CN109374605A (en) * | 2018-09-30 | 2019-02-22 | 东北农业大学 | A kind of method that nanogold colorimetric method detects lipase active in rice bran |
| CN112029756B (en) * | 2020-07-21 | 2022-11-25 | 南京工业大学 | Method for catalytically synthesizing phytosterol ester compound by using magnetic immobilized lipase |
| WO2022187115A1 (en) * | 2021-03-02 | 2022-09-09 | Idexx Laboratories, Inc. | Biochemical probes attached to epoxy-based resins |
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