CN106701728B - It is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method and application - Google Patents
It is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method and application Download PDFInfo
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- CN106701728B CN106701728B CN201611073633.0A CN201611073633A CN106701728B CN 106701728 B CN106701728 B CN 106701728B CN 201611073633 A CN201611073633 A CN 201611073633A CN 106701728 B CN106701728 B CN 106701728B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- 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)
- C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C—CHEMISTRY; METALLURGY
- 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
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
Abstract
It is the method and application of carrier immobilized tyrosinase that the present invention provides a kind of with polyacrylonitrile microballoon.Using modified polyacrylonitrile microballoon as carrier, tyrosinase is combined as fixed enzyme by glutaraldehyde covalent cross-linking the method, and tyrosinase is fixed in polyacrylonitrile microballoon, achievees the purpose that degrading phenol compound.The method of the present invention is simple, low raw-material cost, reaction condition is easily achieved, it is good that immobilization tyrosinase after synthesis recycles effect, pH, temperature tolerance are high, by thermostabilization, the tyrosinase being incorporated in polyacrylonitrile microballoon can effectively remove the phenolic substances present invention in waste water by meeting the development trend of current international green decontamination, environmental protection decontamination by bioanalysis degrading phenol compound under oxygen effect.
Description
Technical field
The present invention relates to biological chemical fields, and in particular to one kind is with polyacrylonitrile microballoon for carrier immobilized tyrosinase
Method and application.
Technical background
Phenolic compound category aromatics species.Wherein common phenol is the industries such as dyestuff, papermaking, medicine, pesticide
Raw materials for production and intermediate, but it is also very important to the toxic action of human body.Phenol tissue any to human body has significant corruption
Erosion effect, and can chemically react when protein contacts in cell magma, form insoluble protein, and cell is made to lose work
Power.In recent years, with the fast development of China's chemical industry, the demand of Pyrogentisinic Acid is increasing, the discharge amount of phenol wastewater
Also it is being continuously increased.In the U.S., phenol is classified as excellent control pollutant by national security agency, for China, at phenol wastewater
Manage one of the normal work to do of also sewage treatment.
It is a kind of biodegradation method of rising in recent years using immobilised enzymes degrading phenol substance.Compared to physics or change
Method degrading phenol compound, such method are more green non-poisonous.Enzyme is fixed in respective carrier, greatly solves circulation
Utilizing question reduces costs and improves its thermal stability, storage stability.However due to carrier, enzyme and crosslinking
Mode it is different, in the field, corresponding activity of the immobilized enzyme and its temperature, PH tolerance are also not quite similar, most immobilizations
Method can not take into account above three condition completely.Therefore, suitable enzyme, fixed matrix how to be selected and is used accordingly
Cross-linking method becomes a great problem in enzyme immobilization degrading phenol compound field.
Summary of the invention
It for the method for carrier immobilized tyrosinase and is answered the purpose of the present invention is to provide a kind of with polyacrylonitrile microballoon
With, tyrosinase is fixed in polyacrylonitrile microballoon using this method, the micro phenolic substances in sewage can be effectively removed,
And this method can guarantee the activity of immobilization tyrosinase, at the same its temperature, PH tolerance be kept at one it is relatively high
It is horizontal.
To achieve the goals above, the invention adopts the following technical scheme:
A method of with polyacrylonitrile microballoon for carrier immobilized tyrosinase, modifiable polyacrylonitrile is prepared first
Then microballoon carries out microsphere surface modification and forms schiff bases and introduce aldehyde radical, can be combined to greatest extent with tyrosinase, will
Tyrosinase is fixed on polyacrylonitrile microballoon surface.
Polyacrylonitrile microballoon contains can be with the itrile group functional group of highly basic, strong acid successive reaction and then carboxylated.Pass through hydrogen-oxygen
After changing the processing of the series reactions reagents such as sodium, concentrated hydrochloric acid, ethylenediamine, glutaraldehyde, polyacrylonitrile microballoon be can be changed to containing azomethine
The schiff bases of characteristic group.
It is of the present invention with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, specifically includes the following steps:
1) polyacrylonitrile is dissolved in N-N dimethylformamide and forms homogeneous phase solution, peristaltic pump is utilized using phase inversion
Prepare polyacrylonitrile microballoon；
2) at 45-50 DEG C, polyacrylonitrile microballoon is soaked in 1-1.5h in the sodium hydroxide solution of 8-10wt%, then
It takes out polyacrylonitrile microballoon and is washed with distilled water immersion, then use 0.1M salt acid soak 2-2.5h carboxylation polypropylene at room temperature
Nitrile microballoon, the polyacrylonitrile microballoon of part carboxylation, which is then soaked in 1-1.5h in the ethylenediamine solution of 8-10wt%, makes its amide
Change, obtains modified polyacrylonitrile microballoon；
3) glutaraldehyde solution of above-mentioned modified polyacrylonitrile microballoon and 8-10wt% are protected from light to contact 1h at 4 DEG C；
4) finally polyacrylonitrile microballoon and tyrosinase-phosphate buffer haptoreaction are immobilized, is fixed
In the immobilization tyrosinase in polyacrylonitrile microballoon.
The mass ratio of the step 1), polyacrylonitrile and N-N dimethylformamide is 1:4.
The step 2), the temperature of amidation process are 20-40 DEG C.
Tyrosinase-phosphate buffer pH=8 of the step 4).
The immobilization time of the step 4) is 8-24h, and immobilization temperature is 20-40 DEG C.
Preferably, the immobilization time of the step 4) is 16h, and immobilization temperature is 25 DEG C.
The immobilization process of the step 4), the pH for controlling reaction system is 4-9, to adjust the catalytic activity of enzyme, thus
Further removal effect of the control immobilization tyrosinase to phenolic compound.
Preferably, the immobilization pH of the step 4) is 8.
Further, the step 4) passes through control immobilization time and immobilization temperature, adjustable immobilization tyrosine
The concentration of enzyme makes its concentration 0.5-5mg/ml.
Immobilization tyrosinase of the present invention is used for degrading phenol compound, under oxygen effect, using being fixed on
Tyrosinase catalysis phenolic compound in polyacrylonitrile microballoon forms high polymer not soluble in water, effectively removes phenol to reach
The purpose of substance.
In use, the polyacrylonitrile microballoon for being fixed with tyrosinase is immersed in the aqueous solution containing phenolic compound, and
Contaminant degradation is carried out under 30-50 DEG C of environmental condition.Immobilization tyrosinase of the invention can be completed under oxygen effect
Degradation to phenolic compound, and general peroxidase then needs to additionally introduce H2O2It is reacted as oxidizer catalytic.
The invention adopts the above technical scheme, and polyacrylonitrile microballoon tentatively modification is made its carboxylated first, is then passed through
Series reaction, which introduces, and to be fixed in polyacrylonitrile microballoon, reach by tyrosinase with the covalently bound aldehyde radical of tyrosinase
To the purpose of degrading phenol compound.The present invention is the aldehyde radical and tyrosinase surface using modified polyacrylonitrile microballoon surface
On amino acid residue carry out covalent bond, this mode do not act on the active site majority of enzyme itself, but to the sky of enzyme
Between structure make irreversible modification so that its vigor, stability, selectivity all have a distinct increment, and in conjunction with carrier
Degree on it is also relatively close.The tyrosinase being incorporated in polyacrylonitrile microballoon can urge phenolic compound under oxygen effect
Change becomes quinones, and the latter is unstable in aqueous solution, reacts through a series of enzymatics and non-enzymatic catalysis, self-polymerization or
Macromolecular substances not soluble in water are formed with other substance (organic amine compound etc.) polymerization reactions and are precipitated.
Remarkable advantage of the invention is that the immobilization tyrosinase of preparation has efficient class compound removal rate.With
For phenol, immobilization tyrosinase of the invention can reach 90% or more in the phenol degrading rate of a certain range concentration.It is common
Peroxidase in degrading phenol substance, generally require introduce H2O2As oxidant, and immobilization tyrosine of the invention
Enzyme only needs to increase nature dissolved oxygen, can complete to react.Immobilised enzymes system thermal stability prepared by the present invention is good, temperature, PH
Tolerance is strong, and the degradation demand to phenolic compound can be met in wider operating condition.Calculate through experiment, tyrosine
Enzyme-polyacrylonitrile system has good reusable property, and for Large Scale Biology technical application, the present invention can be brought can
The economic benefit of sight, while also complying with the development trend of current international green decontamination, environmental protection decontamination.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, present invention will be explained in further detail:
Fig. 1 is the schematic illustration of polyacrylonitrile microballoon immobilization tyrosinase；
Fig. 2 be polyacrylonitrile microballoon before modified after Fourier transform infrared spectroscopy figure (FTIR)；A is polypropylene before modified
Nitrile microballoon, B are the modified polyacrylonitrile microballoon of glutaraldehyde；
Fig. 3 is that tyrosinase is fixed on the X-ray energy dispersion spectrogram (EDS) before and after polyacrylonitrile microballoon；Before A is fixed
Polyacrylonitrile microballoon；B is the polyacrylonitrile microballoon after fixing；
Fig. 4 is the Scanning Electron micro-image (SEM) that tyrosinase is fixed on before and after polyacrylonitrile microballoon: before A is fixed
Polyacrylonitrile microballoon；B is the polyacrylonitrile microballoon after fixing, fixed concentration 1.79mg/cm2；
Fig. 5 is the tolerance comparison diagram of free tyrosine enzyme and immobilization tyrosinase to temperature；
Fig. 6 is the tolerance comparison diagram of free tyrosine enzyme and immobilization tyrosinase to PH；
Fig. 7 is the thermal stability comparison diagram of free tyrosine enzyme and immobilization tyrosinase；
Fig. 8 is the operational stability schematic diagram of free tyrosine enzyme and immobilization tyrosinase.
Specific embodiment
Several specific examples of the invention below, further describe the present invention, but the present invention is not limited only to this.
Embodiment 1
It take polyacrylonitrile microballoon as the method for carrier immobilized tyrosinase, comprising the following steps:
1) 2g polyacrylonitrile is dissolved in 8g N-N dimethylformamide and forms homogeneous phase solution, utilized using phase inversion compacted
Dynamic pump preparation polyacrylonitrile microballoon；
2) at 50 DEG C, polyacrylonitrile microballoon is soaked in 1h in the sodium hydroxide solution of 10wt%, then takes out poly- third
Alkene nitrile microballoon is simultaneously washed with distilled water immersion, then uses 0.1M salt acid soak 2h carboxylation polyacrylonitrile microballoon at room temperature, then
The polyacrylonitrile microballoon of part carboxylation, which is soaked in 1h in the ethylenediamine solution of 10wt%, makes its amidation (reaction temperature 25
DEG C), obtain modified polyacrylonitrile microballoon；
3) glutaraldehyde solution of above-mentioned modified polyacrylonitrile microballoon and 10wt% are protected from light to contact 1h at 4 DEG C；
4) finally the tyrosinase of polyacrylonitrile microballoon and configured pH=8-phosphate buffer haptoreaction are carried out
Immobilization, immobilization time 16h, immobilization temperature are 25 DEG C, obtain being fixed on the immobilization tyrosine in polyacrylonitrile microballoon
Enzyme.
What is be prepared is fixed with the polyacrylonitrile microballoon of tyrosinase, is directly immersed in the aqueous solution containing phenol, and degradation is dirty
Object is contaminated, optimal concentration of substrate is 2g/ml.
Embodiment 2
It take polyacrylonitrile microballoon as the method for carrier immobilized tyrosinase, comprising the following steps:
1) polyacrylonitrile is dissolved in N-N dimethylformamide and forms homogeneous phase solution, peristaltic pump is utilized using phase inversion
Prepare polyacrylonitrile microballoon；
2) at 45 DEG C, polyacrylonitrile microballoon is soaked in 1.5h in the sodium hydroxide solution of 8wt%, then takes out poly- third
Alkene nitrile microballoon is simultaneously washed with distilled water immersion, then uses 0.1M salt acid soak 2.5h carboxylation polyacrylonitrile microballoon at room temperature, so
The polyacrylonitrile microballoon of part carboxylation, which is soaked in 1.5h in 8% ethylenediamine solution, afterwards makes its amidation (reaction temperature 20
DEG C), obtain modified polyacrylonitrile microballoon；
3) glutaraldehyde solution of above-mentioned modified polyacrylonitrile microballoon and 8wt% are protected from light to contact 1h at 4 DEG C；
4) finally the tyrosinase of polyacrylonitrile microballoon and configured pH=4-phosphate buffer haptoreaction are carried out
Immobilization, immobilization time are 8h, and immobilization temperature is 40 DEG C, obtain being fixed on the immobilization tyrosine in polyacrylonitrile microballoon
Enzyme.
What is be prepared is fixed with the polyacrylonitrile microballoon of tyrosinase, is directly immersed in the aqueous solution containing phenol, and degradation is dirty
Contaminate object.
Embodiment 3
It take polyacrylonitrile microballoon as the method for carrier immobilized tyrosinase, comprising the following steps:
1) polyacrylonitrile is dissolved in N-N dimethylformamide and forms homogeneous phase solution, peristaltic pump is utilized using phase inversion
Prepare polyacrylonitrile microballoon；
2) at 50 DEG C, polyacrylonitrile microballoon is soaked in 1.2h in the sodium hydroxide solution of 10wt%, is then taken out poly-
Acrylonitrile microspheres are simultaneously washed with distilled water immersion, then use 0.1M salt acid soak 2.2h carboxylation polyacrylonitrile microballoon at room temperature,
Then the polyacrylonitrile microballoon of part carboxylation is soaked in 1.2h in the ethylenediamine solution of 8wt% makes its amidation (reaction temperature
It is 40 DEG C), obtain modified polyacrylonitrile microballoon；
3) glutaraldehyde solution of above-mentioned modified polyacrylonitrile microballoon and 10wt% are protected from light to contact 1h at 4 DEG C；
4) finally the tyrosinase of polyacrylonitrile microballoon and configured pH=9-phosphate buffer haptoreaction are carried out
Immobilization, immobilization time are that for 24 hours, immobilization temperature is 20 DEG C, obtain being fixed on the immobilization junket ammonia in polyacrylonitrile microballoon
Sour enzyme.
What is be prepared is fixed with the polyacrylonitrile microballoon of tyrosinase, is directly immersed in the aqueous solution containing phenol, and degradation is dirty
Contaminate object.
Embodiment 4
The measurement of immobilization tyrosinase Degradation of Phenol rate
To the measurements of different phenolic compound degradation rates by taking phenol as an example, phenol is measured using 4-APP spectrophotometry
Concentration, and degradation rate is determined according to the variation of immobilization tyrosinase phenol concentration before and after the processing.
Concrete operation method are as follows: draw 700 μ l sodium bicarbonate solutions (0.25M) with liquid-transfering gun, the phenol sample of 100 μ l is molten
Liquid, 100 μ l 4-AA solution (20.8mM is dissolved in the sodium bicarbonate solution of 0.25M), the iron cyaniding of 100 μ l
Potassium solution (83.4mM is dissolved in the sodium bicarbonate solution of 0.25M) is uniformly mixed under the conditions of being protected from light in test tube and reacts
After 10min, with the absorbance value A of spectrophotometric determination solution510.Using phenol concentration as abscissa, A510For ordinate, draw
A510- [C] standard curve.
The polyacrylonitrile microballoon prepared by the present invention for being fixed with tyrosinase is immersed in phenol solution (0.1M, PBS).In
Contaminant degradation 12h is carried out in 35 DEG C of shaken cultivation casees, is taken supernatant after 10000r/min centrifugation 10min, is measured immobilization junket
Absorbance value at 510nm of sample solution before and after propylhomoserin enzymatic treatment and compared with standard curve, can calculate phenol
Concentration.And the degradation rate (η) of phenol is calculated according to following publicity:
Wherein: C0: the phenol concentration (mg/L) before reacting in solution；
Cs: the phenol concentration (mg/L) after reaction in solution.
It is computed, under the conditions of experiment determining optimum control, phenol degrading rate can reach 92%, very efficiently.
Embodiment 5
The temperature of immobilization tyrosinase and free tyrosine enzyme, PH tolerance
Condition of different temperatures, temperature, influence of the pH to immobilization tyrosinase enzymatic activity are measured using relative activity.
Calculation formula is as follows:
Wherein: μp: the relative activity % of enzyme；
vmax: it is v that absolute enzyme activity maximum value under different temperatures and pH condition is chosen in experimentmax；
vp: the absolute enzyme activity under operating condition.
Within the scope of 20-60 DEG C, investigate before and after tyrosinase immobilization to the tolerance of temperature and its optimum temperature, with
Temperature is abscissa, and the relative activity of enzyme is ordinate.As shown in figure 5, the experimental results showed that, in 20-60 DEG C, dissociate junket ammonia
Sour enzyme relative activity is more significant with temperature change, peak value is reached at 35 DEG C then and dramatic decrease；Though and immobilization tyrosinase
Overall trend is similar to the former, but within the scope of 30-50 °C, the relative activity of enzyme remain unchanged substantially and be maintained at 90% with
On, illustrate that temperature tolerance can be significantly improved by doing the tyrosinase after carrier is fixed through polyacrylonitrile, therefore in practical operation
Cheng Zhong need to only maintain operation temperature in 30-50 DEG C, it is not necessary to make excessive demands an absolute specific temperature, reduce cost
Consumption.
And in pH 4-9 operating range, using pH as abscissa, the relative activity of enzyme is ordinate.As shown in fig. 6, experiment
The result shows that the optimal pH of free tyrosine enzyme and fixed tyrosinase is 7, but in the condition for deflecting away from optimal pH by a small margin
Under, the relative activity range of decrease of immobilization tyrosinase is significantly less than free tyrosine enzyme, this illustrates immobilization tyrosinase
PH tolerance is better than free tyrosine enzyme, and in practical operation, system pH allows to have slight fluctuation and will not seriously weaken
The activity of enzyme.
Embodiment 6
The thermal stability of immobilization tyrosinase and free tyrosine enzyme
The thermal stability of immobilization and free tyrosine enzyme is investigated under 50 DEG C and 60 DEG C, two temperature gradient conditions.With
Time is abscissa, and residual activity is ordinate, drawing.As shown in fig. 7, the enzyme of two kinds of forms is heat-treated at 50 DEG C
After 120min, the residual activity of immobilization tyrosinase remains unchanged substantially, still close to 100%, and the work of free tyrosine enzyme
Property but decline up to 30%；At 60 DEG C, residual activity remains at 50% or more after immobilization tyrosinase 12 0min, and dissociates
Tyrosinase 75min or so just complete deactivation.Due to can substantially reduce its Conformational flexibility, immobilised enzymes after enzyme immobilization
Thermal stability be often better than resolvase, for specific tyrosinase, be fixed in polyacrylonitrile carrier modified
Behind surface, this phenomenon is particularly evident, and the thermal stability of enzyme is greatly improved.
Embodiment 7:
The operational stability of immobilization tyrosinase
To measure whether immobilized enzyme is easily isolated and recycled, the recycling of enzyme is evaluated in experiment by calculating its operational stability
The quality of performance.After tyrosinase catalysis substrate reactions, polyacrylonitrile bead is recycled from reaction system.With 0.1M,
Make enzyme activity recovery after the PBS cleaning of pH7.0, is then added to new reaction system and carries out enzymatic experiment, measure the residual of enzyme
Stay activity, repetitive operation 10 times.Residual activity (%) calculation formula of immobilised enzymes is as follows:
Wherein: μn: enzyme residual activity: %；
ν0: the later initial activity of enzyme immobilization；
vn: the activity of immobilised enzymes after n times operation.
As shown in figure 8, experiment shows that after carrying out 5 degradations using immobilization tyrosinase, the residual activity of enzyme still may be used
70% or more is maintained, and after operation processing 10 times, residual activity is still maintained at 40% or so, illustrates the method for the present invention
The polyacrylonitrile immobilization tyrosinase microballoon reusing of preparation is very high.
Claims (9)
1. it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, it is characterised in that: prepare polypropylene first
Then nitrile microballoon carries out microsphere surface modification and forms schiff bases and introduce aldehyde radical, tyrosinase is fixed on polyacrylonitrile microballoon
Surface, the specific steps are as follows:
1) polyacrylonitrile is dissolved in N-N dimethylformamide and forms homogeneous phase solution, prepared using phase inversion using peristaltic pump
Polyacrylonitrile microballoon；
2) under 45-50 °C, polyacrylonitrile microballoon is soaked in 1-1.5h in the sodium hydroxide solution of 8-10wt%, is then taken out
Polyacrylonitrile microballoon is simultaneously washed with distilled water immersion, then micro- with 0.1M salt acid soak 2-2.5h carboxylation polyacrylonitrile at room temperature
Ball, the polyacrylonitrile microballoon of part carboxylation, which is then soaked in 1-1.5h in the ethylenediamine solution of 8-10wt%, makes its amidation, obtains
To modified polyacrylonitrile microballoon；
3) glutaraldehyde solution of above-mentioned modified polyacrylonitrile microballoon and 8-10wt% are protected from light to contact 1h under 4 °C；
4) finally polyacrylonitrile microballoon and tyrosinase-phosphate buffer haptoreaction are immobilized, obtains being fixed on poly-
Immobilization tyrosinase on acrylonitrile microspheres.
2. it is according to claim 1 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the mass ratio of the step 1), polyacrylonitrile and N-N dimethylformamide is 1:4.
3. it is according to claim 1 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the step 2, the temperature of amidation process are 20-40 DEG C.
4. it is according to claim 1 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
It is: tyrosinase-phosphate buffer pH=8 of the step 4).
5. it is according to claim 1 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the immobilization time of the step 4) is 8-24h, and immobilization temperature is 20-40 °C.
6. it is according to claim 5 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the immobilization time of the step 4) is 16h, and immobilization temperature is 25 °C.
7. it is according to claim 5 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the immobilization process of the step 4), the pH for controlling reaction system is 4-9.
8. it is according to claim 1 it is a kind of with polyacrylonitrile microballoon be carrier immobilized tyrosinase method, feature
Be: the step 4), by control immobilization time and immobilization temperature, the concentration for adjusting immobilization tyrosinase is 0.5-
5mg/ml。
9. the application for the immobilization tyrosinase that -8 either method obtain according to claim 1, it is characterised in that: the immobilization
Tyrosinase is used for degrading phenol compound, under oxygen effect, is urged using the tyrosinase being fixed in polyacrylonitrile microballoon
Change phenolic compound and form high polymer not soluble in water, to remove phenolic substances.
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| CN110804604B (en) * | 2019-05-07 | 2023-03-21 | 宁波大学 | Co-crosslinking immobilization method of tyrosinase |
| CN113429606B (en) * | 2021-07-05 | 2023-06-23 | 河南城建学院 | Polymer waterproof coiled material and preparation method and application thereof |
| CN114395551B (en) * | 2021-12-29 | 2024-01-02 | 深圳大学 | Preparation of magnetic material-metal organic framework-tyrosinase complex and method for dephenolization |
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