CN106434621A - Method for immobilizing laccase on polyacrylamide-coated magnetic nano particles and application of method - Google Patents
Method for immobilizing laccase on polyacrylamide-coated magnetic nano particles and application of method Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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Abstract
The invention discloses a method for immobilizing laccase on polyacrylamide-coated magnetic nano particles and application of the method. The method comprises the following steps: taking an iron-containing compound, alkali and a surfactant as raw materials and preparing ferroferric oxide magnetic nano particles by using a hydrothermal method; modifying the magnetic nano particles with oleic acid; taking polyacrylamide as a main raw material and preparing the polyacrylamide-coated magnetic nano particles through suspension and polymerization; taking glutaraldehyde as a bonding agent and immobilizing the laccase on the polyacrylamide-coated magnetic nano particles; after being immobilized, the laccase is used for degrading phenolic pollutants in water. The method and the application thereof disclosed by the invention have the advantages that 1, the material preparation method is simple, and the operation is easy; the raw material cost is low, and the popularization and the application are convenient; 2, as the laccase is immobilized by using the raw materials, the laccase activity is better ensured, and the activity recovery rate reaches 93-98 percent; 3, after the laccase is immobilized by using the raw materials, the phenolic pollutants in the water are degraded, the removal rate reaches 97.5-99.5 percent, and the immobilized laccase still has high activity after repeated degradation for 10-20 times.
Description
Technical field
The invention belongs to magnetic material enzyme immobilization technology field is and in particular to one kind polyacrylamide coated magnetic is received
The methods and applications of rice grain immobilization laccase.
Background technology
Immobilization laccase extends the service life of laccase, reduces the use cost of laccase, improves stablizing of laccase
Property and its toleration to environment, make laccase more wide in the application prospect of the aspects such as Industrial Wastewater Treatment, biosensor.
The access times of immobilization laccase and efficiency are relevant with the product of enzymic catalytic reaction.When product is coloured oxygen
When the property changed material or insoluble substance, the access times of enzyme and efficiency are greatly reduced.Because the catalytic reaction process of laccase
In often produce the quinones substance of some oxidisability, their chemical property is very active it is easy to absorption, on carrier, makes fixation
Change enzyme inactivation.Therefore, it can the buffer solution flush vehicle high by using ionic strength and make this phenomenon obtain part and disappear
Remove.And will thoroughly eliminate this phenomenon and be necessary for selecting suitable fixation support.These will not be adsorbed in the surface of this carrier
Coloured oxidizing substance, such that it is able to the long-term activity keeping immobilization laccase.Although for example glass ceramic material is to laccase
Immobilization efficiency not high (only 50%), but it adsorbs very low to coloured oxidizing substance, is therefore processing textile mills
Can keep active the long period during with the sewage in paper mill.
On the other hand, when the product of catalytic reaction is insoluble substance, it can precipitate in the reactor, and easily
Deposition at reactor outlet filter membrane is thus lead to flows decrease even completely plugged outlet.This phenomenon typically can not
Avoid, because polyphenol oxidase can produce free radical and benzoquinone compound in catalytic reaction, these materials are easy to produce
Raw polymer is thus produce flocculent deposit.This problem can by using immobilized enzyme while, wadded a quilt with cotton with macromolecule cation
Solidifying agent such as shitosan etc. come cleaning reaction generation polymer or precipitation and solve.
From now on, the developing direction of Laccase Immobilization will be production and the application expanding immobilization laccase product.First have to solve
Determine is the industrial problems of the free laccase of funguses large-scale production；Next to that will be in the industrial metaplasia of immobilization laccase product
Product condition, equipment and the best processing route aspect are further studied.Immobilization laccase technology is as a kind of efficient
Biological enzyme technology will produce huge using value in Industrial Wastewater Treatment from now on.
Content of the invention
It is an object of the invention to provide a kind of method of the nano particle immobilized laccase of polyacrylamide coated magnetic and
Application.
The present invention utilizes hydro-thermal method and suspension polymerization, just can be prepared for immobilization laccase by shirtsleeve operation
Polyacrylamide coated magnetic nano-particle.
A kind of methods and applications of the nano particle immobilized laccase of polyacrylamide coated magnetic that the present invention provides, comprise
Processing step as follows：
（1）With iron containing compoundses（As ferric chloride, iron sulfate etc.）, alkali（As sodium acetate, ammonia etc.）, surfactant（As gathered
Ethylene glycol, sodium lauryl sulphate etc.）For raw material, prepare ferroferric oxide magnetic nanoparticle with hydro-thermal method；
（2）Using Oleic acid improved ferroferric oxide magnetic nanoparticle；
（3）With acrylamide as primary raw material, former for assisting with styrene, divinylbenzene, azodiisobutyronitrile, normal heptane
Material, prepares the ferroferric oxide magnetic nanoparticle of polyacrylamide cladding with suspension polymerisation；
（4）With glutaraldehyde for joint agent, the ferroferric oxide magnetic nanoparticle that Laccase Immobilization is coated in polyacrylamide
On；
（5）With the immobilized laccase of magnetic carrier, can repeatedly reclaim use, carry out the phenolic comp ' ds pollution in degradation water.
Step of the present invention（1）Described in hydro-thermal method synthesize ferroso-ferric oxide raw material be：Iron containing compoundses（As tri-chlorination
Ferrum, iron sulfate etc.）, alkali（As sodium acetate, ammonia etc.）, surfactant（As Polyethylene Glycol, sodium lauryl sulphate etc.）, solvent
For ethylene glycol, water, diethylene glycol etc. or its mixture, synthesis temperature is 60-300 DEG C, and generated time is 6-72 hour.
Step of the present invention（2）Described in Oleic acid be modified as：By step（1）The ferroferric oxide nano granules of middle synthesis add
In distilled water containing Oleic acid, reaction condition adjusts PH=10, reaction temperature 65-85 DEG C, mechanical agitation 1-3 hour for ammonia.
Step of the present invention（3）Described in the reaction solution of suspension polymerisation be：By step（2）In modified ferroso-ferric oxide
Nano-particle adds containing in acrylamide, styrene, divinylbenzene, azodiisobutyronitrile, the aqueous solution of normal heptane, and ice is molten
Ultrasonic 15-30 minute, 0-4 DEG C of standing 10-24 hour；Above-mentioned solution adds saturated sodium-chloride and amidin, reaction temperature
60-80 DEG C of degree, mechanical agitation 2-4 hour.
Step of the present invention（4）Described in immobilization laccase process be：By step（3）Poly- third preparing after middle suspension polymerisation
The ferroferric oxide magnetic nanoparticle of acrylamide cladding adds in the aqueous solution containing glutaraldehyde, 15-30 DEG C of mechanical agitation 5-8
Hour；The ferroferric oxide magnetic nanoparticle washes clean coating polyacrylamide again, adds in laccase solution, 20-30
DEG C Turning motion 5-7 hour.
Step of the present invention（5）Described in degraded water pollutant process be：Four oxidations that polyacrylamide is coated
The magnetic nano particle immobilized laccase of three-iron adds and contains phenolic comp ' ds pollution（As bisphenol-A, acetaminophen etc.）Water in,
15-30 DEG C of Turning motion 2-4 hour.
Specific embodiment
With reference to being embodied as example, the present invention is expanded on further.These embodiments be merely to illustrate the present invention rather than
Limit the scope of the present invention.In addition, it is to be understood that after having read the content of present invention instruction, those skilled in the art can be right
The present invention makes various changes or changes, and these equivalent form of values equally fall within the model that the application institute cum-rightss claim is limited
Enclose.
Embodiment 1：
Weigh 1g ferric chloride 2.7g sodium acetate 0.75g PEG6000, three kinds of solids be dissolved completely in 30ml ethylene glycol,
Ultrasonic 15 minutes, stir 30 minutes, obtain the reaction mixture of buff.Mixed solution is loaded 100ml reactor, puts into
React in Muffle furnace, 200 DEG C of reaction temperature, 8 hours response time.After the completion of reaction, it is naturally cooling to room temperature, use attraction
Supernatant liquid is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Ferroferric oxide nano granules.
The ferriferrous oxide nano weighing the above-mentioned synthesis of 0.5g adds in 10ml distilled water, adds 0.5g Oleic acid, uses ammonia
Adjust PH and be about 10,80 DEG C of reaction temperature, mechanical agitation 1 hour.After the completion of reaction, it is naturally cooling to room temperature, use attraction
The supernatant is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Lipophilic ferroferric oxide nano particles.
Weigh in the lipophilic ferroferric oxide nano particles addition 50ml distilled water that the above-mentioned reaction of 0.15g obtains, add
1.5g acrylamide, adds 0.36ml styrene, adds 0.38ml divinylbenzene, adds 0.022g azodiisobutyronitrile, plus
Enter 0.6ml normal heptane, after mix homogeneously, molten ultrasonic 20 minutes of ice, 4 DEG C stand 12 hours.30ml is added to satisfy in above-mentioned solution
And sodium chloride, add 0.69g starch, 70 DEG C of reaction temperature, mechanical agitation 2 hours.After the completion of reaction, it is naturally cooling to room temperature,
With attraction, supernatant liquid is poured out, solid water and ethanol replace washing, and again with methanol extracts 3 days, vacuum drying is cold
But, collect product and obtain black solid particle, be the magnetic ferroferric oxide nanometer granule with polyacrylamide cladding.
Weigh the magnetic ferroferric oxide nanometer granule that 0.1g polyacrylamide coats, add the glutaraldehyde of 20ml 10%
Aqueous solution, room temperature mechanical stirs 6 hours, after the completion of reaction, is replaced with water and ethanol and washs, be then added in 30ml distilled water,
Add laccase powder 0.02g, be then placed in the shaking table that rotating speed is 120rpm, 30 DEG C are reacted 6 hours.After immobilization terminates, use
Attraction separates, and uses PH=7.0, and the phosphate buffer of 0.1mol/l removes and fails with reference to resolvase up.
Use Bradford- Coomassie Brilliant Blue, measure the load enzyme amount of carrier, calculate the load enzyme rate of carrier, carry enzyme rate and exist
65%-75%.Use ABTS- UV-VIS spectrophotometry, the laccase activity after the Laccase activity of mensure free state and immobilization
Power, calculates enzymatic activity recovery, enzymatic activity recovery is in 93%-98%.
By immobilization, the ferroferric oxide magnetic nanoparticle 0.1g of the polyacrylamide cladding of laccase adds and contains
In the aqueous solution 20ml of 20mg/L bisphenol-A, room temperature Turning motion 2 hours, more than 99% bisphenol-A is decomposed.
Embodiment 2
Weigh 5g ferric chloride 13.5g sodium acetate 3.75g PEG6000, three kinds of solids are dissolved completely in 150ml ethylene glycol
In, ultrasonic 15 minutes, stir 30 minutes, obtain the reaction mixture of buff.Mixed solution is loaded 250ml reactor,
Put into reaction, 200 DEG C of reaction temperature, 8 hours response time in Muffle furnace.After the completion of reaction, it is naturally cooling to room temperature, use Magnet
Attract to pour out supernatant liquid, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder,
It is ferroferric oxide nano granules.
The ferriferrous oxide nano weighing the above-mentioned synthesis of 2.5g adds in 100ml distilled water, adds 2.5g Oleic acid, uses ammonia
Adjust PH and be about 10,80 DEG C of reaction temperature, mechanical agitation 1 hour.After the completion of reaction, it is naturally cooling to room temperature, use attraction
The supernatant is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Lipophilic ferroferric oxide nano particles.
Weigh in the lipophilic ferroferric oxide nano particles addition 250ml distilled water that the above-mentioned reaction of 0.75g obtains, add
7.5g acrylamide, adds 1.8ml styrene, adds 1.9ml divinylbenzene, adds 0.11g azodiisobutyronitrile, adds
3ml normal heptane, after mix homogeneously, molten ultrasonic 20 minutes of ice, 4 DEG C stand 12 hours.150ml saturation chlorine is added in above-mentioned solution
Change sodium, add 3.45g starch, 70 DEG C of reaction temperature, mechanical agitation 2 hours.After the completion of reaction, it is naturally cooling to room temperature, use magnetic
Ferrum attracts to pour out supernatant liquid, and solid water and ethanol replace washing, and again with methanol extracts 3 days, vacuum drying, and cooling is received
Collection product obtains black solid particle, is the magnetic ferroferric oxide nanometer granule with polyacrylamide cladding.
Weigh the magnetic ferroferric oxide nanometer granule that 0.5g polyacrylamide coats, add the penta 2 of 100ml 10%
Aldehyde aqueous solution, room temperature mechanical stirs 6 hours, after the completion of reaction, is replaced with water and ethanol and washs, be then added to 150ml distilled water
In, add laccase powder 0.1g, be then placed in the shaking table that rotating speed is 120rpm, 30 DEG C are reacted 6 hours.After immobilization terminates,
Separated with attraction, and use PH=7.0, the phosphate buffer of 0.1mol/l removes and fails with reference to resolvase up.
Use Bradford- Coomassie Brilliant Blue, measure the load enzyme amount of carrier, calculate the load enzyme rate of carrier, carry enzyme rate and exist
65%-75%.Use ABTS- UV-VIS spectrophotometry, the laccase activity after the Laccase activity of mensure free state and immobilization
Power, calculates enzymatic activity recovery, enzymatic activity recovery is in 93%-98%.
By immobilization, the ferroferric oxide magnetic nanoparticle 0.5g of the polyacrylamide cladding of laccase adds and contains
In the aqueous solution 100ml of 20mg/L bisphenol-A, room temperature Turning motion 2 hours, more than 99% bisphenol-A is decomposed.
Comparative example 1, and the present embodiment 2 is all to amplify reaction volume and quantity of solvent etc. in step each in embodiment 1
5 times, specific preparation method and operational approach in the same manner as in Example 1, the result obtaining is basically identical with embodiment 1.
Embodiment 3：
Weigh 1g ferric chloride 2.7g sodium acetate 0.75g PEG6000, three kinds of solids be dissolved completely in 30ml ethylene glycol,
Ultrasonic 15 minutes, stir 30 minutes, obtain the reaction mixture of buff.Mixed solution is loaded 100ml reactor, puts into
React in Muffle furnace, 180 DEG C of reaction temperature, 12 hours response time.After the completion of reaction, it is naturally cooling to room temperature, inhaled with Magnet
Draw and supernatant liquid is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, that is,
It is ferroferric oxide nano granules.
The ferriferrous oxide nano weighing the above-mentioned synthesis of 0.5g adds in 10ml distilled water, adds 0.5g Oleic acid, uses ammonia
Adjust PH and be about 10,80 DEG C of reaction temperature, mechanical agitation 1 hour.After the completion of reaction, it is naturally cooling to room temperature, use attraction
The supernatant is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Lipophilic ferroferric oxide nano particles.
Weigh in the lipophilic ferroferric oxide nano particles addition 50ml distilled water that the above-mentioned reaction of 0.15g obtains, add
1.5g acrylamide, adds 0.36ml styrene, adds 0.38ml divinylbenzene, adds 0.022g azodiisobutyronitrile, plus
Enter 0.6ml normal heptane, after mix homogeneously, molten ultrasonic 20 minutes of ice, 4 DEG C stand 12 hours.30ml is added to satisfy in above-mentioned solution
And sodium chloride, add 0.69g starch, 70 DEG C of reaction temperature, mechanical agitation 2 hours.After the completion of reaction, it is naturally cooling to room temperature,
With attraction, supernatant liquid is poured out, solid water and ethanol replace washing, and again with methanol extracts 3 days, vacuum drying is cold
But, collect product and obtain black solid particle, be the magnetic ferroferric oxide nanometer granule with polyacrylamide cladding.
Weigh the magnetic ferroferric oxide nanometer granule that 0.1g polyacrylamide coats, add the glutaraldehyde of 20ml 10%
Aqueous solution, room temperature mechanical stirs 6 hours, after the completion of reaction, is replaced with water and ethanol and washs, be then added in 30ml distilled water,
Add laccase powder 0.02g, be then placed in the shaking table that rotating speed is 120rpm, 30 DEG C are reacted 6 hours.After immobilization terminates, use
Attraction separates, and uses PH=7.0, and the phosphate buffer of 0.1mol/l removes and fails with reference to resolvase up.
Use Bradford- Coomassie Brilliant Blue, measure the load enzyme amount of carrier, calculate the load enzyme rate of carrier, carry enzyme rate and exist
65%-75%.Use ABTS- UV-VIS spectrophotometry, the laccase activity after the Laccase activity of mensure free state and immobilization
Power, calculates enzymatic activity recovery, enzymatic activity recovery is in 93%-98%.
By immobilization, the ferroferric oxide magnetic nanoparticle 0.1g of the polyacrylamide cladding of laccase adds and contains
In the aqueous solution 20ml of 20mg/L bisphenol-A, room temperature Turning motion 2 hours, more than 99% bisphenol-A is decomposed.
Comparative example 1, and the present embodiment 3 is that prepared by hydro-thermal method in embodiment 1 ferroferric oxide magnetic nanoparticle
Reaction temperature is changed to 180 degree, and the response time is changed to 12 hours, and specific preparation method and operational approach are in the same manner as in Example 1,
The result obtaining is basically identical with embodiment 1.
Embodiment 4：
Weigh 1g ferric chloride 2.7g sodium acetate 0.75g PEG6000, three kinds of solids be dissolved completely in 30ml ethylene glycol,
Ultrasonic 15 minutes, stir 30 minutes, obtain the reaction mixture of buff.Mixed solution is loaded 100ml reactor, puts into
React in Muffle furnace, 200 DEG C of reaction temperature, 8 hours response time.After the completion of reaction, it is naturally cooling to room temperature, use attraction
Supernatant liquid is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Ferroferric oxide nano granules.
The ferriferrous oxide nano weighing the above-mentioned synthesis of 0.5g adds in 10ml distilled water, adds 0.5g Oleic acid, uses ammonia
Adjust PH and be about 10,80 DEG C of reaction temperature, mechanical agitation 1 hour.After the completion of reaction, it is naturally cooling to room temperature, use attraction
The supernatant is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Lipophilic ferroferric oxide nano particles.
Weigh in the lipophilic ferroferric oxide nano particles addition 50ml distilled water that the above-mentioned reaction of 0.15g obtains, add
1.5g acrylamide, adds 0.36ml styrene, adds 0.38ml divinylbenzene, adds 0.022g azodiisobutyronitrile, plus
Enter 0.6ml normal heptane, after mix homogeneously, molten ultrasonic 20 minutes of ice, 4 DEG C stand 12 hours.30ml is added to satisfy in above-mentioned solution
And sodium chloride, add 0.69g starch, 80 DEG C of reaction temperature, mechanical agitation 3 hours.After the completion of reaction, it is naturally cooling to room temperature,
With attraction, supernatant liquid is poured out, solid water and ethanol replace washing, and again with methanol extracts 3 days, vacuum drying is cold
But, collect product and obtain black solid particle, be the magnetic ferroferric oxide nanometer granule with polyacrylamide cladding.
Weigh the magnetic ferroferric oxide nanometer granule that 0.1g polyacrylamide coats, add the glutaraldehyde of 20ml 10%
Aqueous solution, room temperature mechanical stirs 6 hours, after the completion of reaction, is replaced with water and ethanol and washs, be then added in 30ml distilled water,
Add laccase powder 0.02g, be then placed in the shaking table that rotating speed is 120rpm, 30 DEG C are reacted 6 hours.After immobilization terminates, use
Attraction separates, and uses PH=7.0, and the phosphate buffer of 0.1mol/l removes and fails with reference to resolvase up.
Use Bradford- Coomassie Brilliant Blue, measure the load enzyme amount of carrier, calculate the load enzyme rate of carrier, carry enzyme rate and exist
65%-75%.Use ABTS- UV-VIS spectrophotometry, the laccase activity after the Laccase activity of mensure free state and immobilization
Power, calculates enzymatic activity recovery, enzymatic activity recovery is in 93%-98%.
By immobilization, the ferroferric oxide magnetic nanoparticle 0.1g of the polyacrylamide cladding of laccase adds and contains
In the aqueous solution 20ml of 20mg/L bisphenol-A, room temperature Turning motion 2 hours, more than 99% bisphenol-A is decomposed.
Comparative example 1, and the present embodiment 4 is will to prepare four oxygen of polyacrylamide cladding in embodiment 1 with suspension polymerisation
Change the reaction temperature during three ferromagnetic nanoparticle and be changed to 80 DEG C, the mechanical agitation time is changed to 3 hours, specific preparation side
Method and operational approach are in the same manner as in Example 1, and the result obtaining is basically identical with embodiment 1.
Embodiment 5：
Weigh 1g ferric chloride 2.7g sodium acetate 0.75g PEG6000, three kinds of solids be dissolved completely in 30ml ethylene glycol,
Ultrasonic 15 minutes, stir 30 minutes, obtain the reaction mixture of buff.Mixed solution is loaded 100ml reactor, puts into
React in Muffle furnace, 200 DEG C of reaction temperature, 8 hours response time.After the completion of reaction, it is naturally cooling to room temperature, use attraction
Supernatant liquid is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Ferroferric oxide nano granules.
The ferriferrous oxide nano weighing the above-mentioned synthesis of 0.5g adds in 10ml distilled water, adds 0.5g Oleic acid, uses ammonia
Adjust PH and be about 10,80 DEG C of reaction temperature, mechanical agitation 1 hour.After the completion of reaction, it is naturally cooling to room temperature, use attraction
The supernatant is poured out, solid water and ethanol replace washing, vacuum drying, cooling, collect product and obtain black powder, are
Lipophilic ferroferric oxide nano particles.
Weigh in the lipophilic ferroferric oxide nano particles addition 50ml distilled water that the above-mentioned reaction of 0.15g obtains, add
1.5g acrylamide, adds 0.36ml styrene, adds 0.38ml divinylbenzene, adds 0.022g azodiisobutyronitrile, plus
Enter 0.6ml normal heptane, after mix homogeneously, molten ultrasonic 20 minutes of ice, 4 DEG C stand 12 hours.30ml is added to satisfy in above-mentioned solution
And sodium chloride, add 0.69g starch, 70 DEG C of reaction temperature, mechanical agitation 2 hours.After the completion of reaction, it is naturally cooling to room temperature,
With attraction, supernatant liquid is poured out, solid water and ethanol replace washing, and again with methanol extracts 3 days, vacuum drying is cold
But, collect product and obtain black solid particle, be the magnetic ferroferric oxide nanometer granule with polyacrylamide cladding.
Weigh the magnetic ferroferric oxide nanometer granule that 0.1g polyacrylamide coats, add the glutaraldehyde of 20ml 10%
Aqueous solution, room temperature mechanical stirs 6 hours, after the completion of reaction, is replaced with water and ethanol and washs, be then added in 30ml distilled water,
Add laccase powder 0.02g, be then placed in the shaking table that rotating speed is 120rpm, 30 DEG C are reacted 6 hours.After immobilization terminates, use
Attraction separates, and uses PH=7.0, and the phosphate buffer of 0.1mol/l removes and fails with reference to resolvase up.
Use Bradford- Coomassie Brilliant Blue, measure the load enzyme amount of carrier, calculate the load enzyme rate of carrier, carry enzyme rate and exist
65%-75%.Use ABTS- UV-VIS spectrophotometry, the laccase activity after the Laccase activity of mensure free state and immobilization
Power, calculates enzymatic activity recovery, enzymatic activity recovery is in 93%-98%.
By immobilization, the ferroferric oxide magnetic nanoparticle 0.1g of the polyacrylamide cladding of laccase adds and contains
In the aqueous solution 20ml of 20mg/L bisphenol-A, room temperature Turning motion 2 hours, more than 99% bisphenol-A is decomposed.
Reclaim the ferroferric oxide magnetic nanoparticle of the polyacrylamide cladding of immobilization laccase, repeat recovery and be used for
Degradation experiment 5 times, is all thoroughly washed to the immobilization laccase reclaiming after often processing primary pollution, still can be made 99%
Above bisphenol-A is decomposed.
Comparative example 1, and the present embodiment 5 is to repeat back to take in row 5 by the process of degrading phenol pollutant in embodiment 1
Secondary, specific preparation method and operational approach in the same manner as in Example 1, the result obtaining is basically identical with embodiment 1.
Claims (7)
1. a kind of methods and applications of the nano particle immobilized laccase of polyacrylamide coated magnetic, comprise following processing step：
（1）With iron containing compoundses（As ferric chloride, iron sulfate etc.）, alkali（As sodium acetate, ammonia etc.）, surfactant（As gathered
Ethylene glycol, sodium lauryl sulphate etc.）For raw material, prepare ferroferric oxide magnetic nanoparticle with hydro-thermal method；
（2）Using Oleic acid improved ferroferric oxide magnetic nanoparticle；
（3）With acrylamide as primary raw material, former for assisting with styrene, divinylbenzene, azodiisobutyronitrile, normal heptane
Material, prepares the ferroferric oxide magnetic nanoparticle of polyacrylamide cladding with suspension polymerisation；
（4）With glutaraldehyde for joint agent, the ferroferric oxide magnetic nanoparticle that Laccase Immobilization is coated in polyacrylamide
On；
（5）With the immobilized laccase of magnetic carrier, can repeatedly reclaim use, carry out the phenolic comp ' ds pollution in degradation water.
2. preparation method according to claim 1 is it is characterised in that step（1）Described in hydro-thermal method synthesis ferroso-ferric oxide
Raw material be：Iron containing compoundses（As ferric chloride, iron sulfate etc.）, alkali（As sodium acetate, ammonia etc.）, surfactant（As gathered
Ethylene glycol, sodium lauryl sulphate etc.）, solvent is ethylene glycol, water, diethylene glycol etc. or its mixture, and synthesis temperature is 60-
300 DEG C, generated time is 6-72 hour.
3. preparation method according to claim 1 is it is characterised in that step（2）Described in Oleic acid be modified as：By step（1）
The ferroferric oxide nano granules of middle synthesis add in the distilled water containing Oleic acid, and reaction condition adjusts PH=10, reaction for ammonia
Temperature 65-85 DEG C, mechanical agitation 1-3 hour.
4. preparation method according to claim 1 is it is characterised in that step（3）Described in suspension polymerisation reaction solution
For：By step（2）In modified ferroferric oxide nano granules add and contain acrylamide, styrene, divinylbenzene, idol
In nitrogen bis-isobutyronitrile, the aqueous solution of normal heptane, the molten ultrasonic 15-30 minute of ice, 0-4 DEG C of standing 10-24 hour；Above-mentioned solution adds again
Enter saturated sodium-chloride and amidin, reaction temperature 60-80 DEG C, mechanical agitation 2-4 hour.
5. preparation method according to claim 1 is it is characterised in that step（4）Described in immobilization laccase process be：Will
Step（3）The ferroferric oxide magnetic nanoparticle of the polyacrylamide cladding prepared after middle suspension polymerisation adds and contains penta 2
In the aqueous solution of aldehyde, 15-30 DEG C of mechanical agitation 5-8 hour；The ferroferric oxide magnetic nano again polyacrylamide being coated
Grain washes clean, adds in laccase solution, 20-30 DEG C of Turning motion 5-7 hour.
6. preparation method according to claim 1 is it is characterised in that step（5）Described in degraded water pollutant mistake
Cheng Wei：The ferroferric oxide magnetic nanoparticle immobilization laccase that polyacrylamide is coated adds and contains phenolic comp ' ds pollution（As
Bisphenol-A, acetaminophen etc.）Water in, 15-30 DEG C of Turning motion 2-4 hour.
7. preparation method according to claim 1 is it is characterised in that step（5）Described in verify the immobilization laccase of preparation
Can repeat to reclaim nonexpondable process and be：The immobilization laccase of preparation is polluted according in the degradation water in claim 6
The process of thing, repeats 5-10 time, all the immobilization laccase reclaiming thoroughly is washed after often processing primary pollution.
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