CN108977433A - A kind of preparation method and application of immobilization lignin peroxidase - Google Patents
A kind of preparation method and application of immobilization lignin peroxidase Download PDFInfo
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Abstract
A kind of preparation method and application of immobilization lignin peroxidase, preparation method is the following steps are included: (1) prepares ferriferrous oxide nano-particle by ferric trichloride, sodium citrate and sodium acetate；(2) magnetic silica nanoparticle is prepared；(3) dopamine polymerization embedded magnetic Nano particles of silicon dioxide is prepared；(4) embedding in situ is polymerize by dopamine and prepares magnetic silica immobilization lignin peroxidase；The immobilization lignin peroxidase and hydrogen peroxide of above method preparation are added into the waste water containing organic pollutant, reacts at room temperature, to remove removal organic polluter.Preparation process of the present invention is easy, the fixation support of use is nano-scale particle, safe and non-toxic, stability is good, large specific surface area, high with the Percentage bound of enzyme, dispersibility in catalyst system is preferably, risk higher to the removal efficiency of pollutant, without secondary pollution, the fixation support can recycle and reuse immobilised enzymes by Magnetic Isolation.
Description
Technical field
The present invention relates to a kind of preparation method of immobilization lignin peroxidase and its organic dirts in processing waste water
The application for contaminating object space face, belongs to Organic Pollutants in Wastewater processing technology field.
Background technique
Main organic pollutant has phenolic compound, benzene-like compounds, halohydrocarbon in industry and livestock breeding wastewater
Close object and antibiotic etc..These organic pollutant toxicity are big and are not easy by the microbial degradation in environment.Currently, processing waste water
In the method for organic pollutant mainly have chemical method, physical method and bioanalysis etc..Wherein physical method general device is more high
Expensive, the condition of processing requires high；Chemical method is quick during the treatment, and the period is short, but is easy to produce secondary pollution；Biology
Rule needs mass propgation microorganism, the period is longer and waste water in pollutant harm is generated to the growth of microorganism.
Enzyme is the green catalyst for being widely used in the fields such as medicine, food and environmental protection, has reaction condition mild, anti-
Answer the feature that speed is fast and by-product is few.In recent years, become sewage disposal technology using the organic pollutant in enzyme removal waste water
One hot spot in field.But after resolvase is poor, very sensitive to outside environmental elements with stability, price is high and reaction
The defects of can not recycling, therefore enzyme is limited in the application of every field.And by free biological enzyme in conjunction with fixation support
Afterwards, there is broader temperature and soda acid tolerance range, the stability and operability of enzyme are reinforced.
Research about the organic pollutant in immobilization laccase processing waste water is than wide.Immobilization laccase can be direct
Catalytic degradation phenolic compound, but because the redox potential of laccase is relatively low, the non-phenolic compound of catalytic degradation is then needed
Want the compound of some lower oxidation reduction gesture as efficient mediator.However, efficiently mediator is mostly artificial synthesized, it is expensive and deposit
In potential environmental toxicity.Lignin peroxidase (EC1.11.1.14) is the peroxidase containing heme, is had
Wide Substratspezifitaet, and only in the presence of hydrogen peroxide, so that it may catalytic degradation phenol, aromatic amine, polycyclic aromatization
Close the Some Organic Pollutants such as object.Compared to efficient mediator, hydrogen peroxide it is cheap, and toxicity will not be generated.Therefore, Gu
Surely changing lignin peroxidase processing Organic Pollutants in Wastewater has extremely wide industrial research and application prospect.
Currently, the material of enzyme immobilization carrier mainly has chitosan, charcoal, silica gel and macroreticular resin etc., but it is existing
Immobilization material it is different degrees of there are complicated for operation, higher cost in conjunction with enzyme, it is low to repeat recovery utilization rate, can generate two
The problems such as secondary pollution.It is " a kind of to remove in livestock breeding wastewater antibiotic as disclosed in Chinese patent literature CN107117719A
Method " it is to be fixed on lipase to be used to handle tetracycline antibiotics in fowl breeding wastewater on charcoal；CN103289982B
Disclosed " a kind of preparation of novel fixed enzyme vector " is with macroporous absorbent resin for basic carrier, using glutaraldehyde to its table
Face is modified, obtains a kind of novel fixed enzyme vector.The above immobilization technology raw material are easy to get, cheap, but inconvenient
Recycling is reused.
And " a kind of preparation method of immobilization laccase carrier and micro quantity organic pollutant of degrading disclosed in CN106811458A
Method " be by amido modified silica immobilization laccase by glutaraldehyde cross-linking for degradable organic pollutant；
Disclosed in CN105110482B " a method of with paracetamol in immobilised enzymes removal industrial wastewater " it is to be repaired by amination
Decorations, glutaraldehyde activated nano carbon microsphere are carrier, and immobilized HRP removes paracetamol in industrial wastewater；
" multi-arm magnetic composite microsphere immobilized HRP and its preparation method and application " disclosed in CN107418950A is
By six arm polyethylene glycol amino-magnetic polymer microballoon fixing Beta glucuroides, glutaraldehyde cross-linking reaction modification, immobilization is peppery
Root peroxidase removes phenol in industrial wastewater.The above immobilization technology has the advantages that easy to operate and treatment effeciency is high, but
It is that the glutaraldehyde that treatment process uses has toxicity, harm is all had to ecological environment and human body.
Therefore, need to develop it is a kind of for handle the cleaning of organic pollutant in waste water, efficiently, it is low cost, repeatable
Using and method easy to operate.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of simple processs, the fixation of safe and non-toxic
Change the preparation method of lignin peroxidase, while providing organic in immobilization lignin peroxidase processing waste water
The application of pollutant
The preparation method of immobilization lignin peroxidase of the invention is passed through using magnetic nanoparticle as carrier
Dopamine polymerization embedding in situ prepares magnetic silica immobilization lignin peroxidase, specifically includes the following steps:
(1) ferriferrous oxide nano-particle is prepared:
In the ratio of 2-5g:0.5-2g:5-10g:100mL, by ferric trichloride, sodium citrate and sodium acetate in lasting stirring
Under be successively dissolved in ethylene glycol, be then transferred in autoclave, react 8-16h at 120-200 DEG C；After cooling, it will consolidate
Body precipitating water and ethanol washing, are finally dried in vacuo to get ferriferrous oxide nano-particle is arrived；
(2) magnetic silica nanoparticle is prepared:
The ferriferrous oxide nano-particle and ethyl orthosilicate of step (1) preparation are weighed in the ratio of 600mg:2-5mL, it will
Ferriferrous oxide nano-particle is dispersed in the mixed system of water, ethyl alcohol and ammonium hydroxide, then adds dropwise positive silicic acid second
Ester vibrates 6-12h at 15-35 DEG C, and with magnet separation product, gained precipitating, which is washed with distilled water, removes unreacted raw material,
It is finally dried in vacuo to get magnetic silica nanoparticle is arrived；
(3) dopamine polymerization embedded magnetic Nano particles of silicon dioxide is prepared:
It is in the ratio of 600mg:200-500mL that the magnetic silica nanoparticle prepared in step (2) is evenly dispersed
Dopamine hydrochloride is added in TRIS buffer, then with the concentration of 1.2-2.0mg/mL, is shaken at 15-35 DEG C
Swing 12-24h；With magnet separation product, gained precipitating, which is washed with distilled water, removes unreacted raw material, is finally dried in vacuo, i.e.,
It obtains dopamine and polymerize embedded magnetic Nano particles of silicon dioxide, as immobilization lignin peroxidase carrier；
(4) immobilization lignin peroxidase is prepared:
In the ratio of 1g:5-50mL, by the immobilization lignin peroxidase carrier obtained in step (3), (dopamine is poly-
Close embedded magnetic Nano particles of silicon dioxide) to be dispersed in tartaric acid-sodium tartrate containing lignin peroxidase slow
In fliud flushing, lignin peroxidase protein concentration is 0.39mg/mL, vibrates 6-24h at 15-35 DEG C；It is separated and is produced with magnet
Object, gained precipitating is washed with tartaric acid-sodium tartrate buffer, until being finally freeze-dried without resolvase in supernatant, that is, obtaining
Obtain immobilization lignin peroxidase.
The partial size of the ferriferrous oxide nano-particle prepared in the step (1) is 50-200nm.
The volume ratio of water, ethyl alcohol and ammonium hydroxide is 15-20 in the mixed system of water, ethyl alcohol and ammonium hydroxide in the step (2):
70-80:1。
The concentration of step (4) mesotartaric acid-sodium tartrate buffer is 0.1mol/L, pH 2.5-3.5.
The partial size of immobilization lignin peroxidase is 200-400nm in the step (4), and enzyme supported quantity is 3-6mg/
G, enzyme activity 100-200U/g.
The step (2), (3), the dispersing method in (4) are mechanical stirring or ultrasonic method.
Organic pollutant in the immobilization lignin peroxidase processing waste water of above method preparation, detailed process
It is:
Immobilization lignin peroxidase and hydrogen peroxide are added into the waste water containing organic pollutant, reacts at room temperature
1-24h, to remove removal organic polluter.In practical operation can timing sampling, the concentration variation of pollutant is analyzed, until pollutant goes
Except rate reaches discharge standard.
The additional amount of the immobilization lignin peroxidase and the mass ratio of Organic Pollutants in Wastewater are 1g:1mg-
10mg。
Mass concentration of the hydrogen peroxide in waste water is 1.7-6.8mg/L.
The above process can be used for by polycyclic aromatic hydrocarbon in immobilization lignin peroxidase catalytic degradation waste water, phenols,
The organic pollutants such as tetracycline antibiotics.
Preparation process of the present invention is easy, and the fixation support of use is nano-scale particle, and safe and non-toxic, stability is good,
The immobilization lignin peroxidase of preparation has 36.5484m2The bigger serface of/g, Percentage bound 56.3-65.6%,
Preferably, higher to the removal efficiency of pollutant, which can pass through Magnetic Isolation to dispersibility in catalyst system
Recycle and reuse immobilised enzymes.
Detailed description of the invention
Fig. 1 is the electron microscope of immobilization lignin peroxidase prepared by embodiment 1.
Fig. 2 is influence signal of the enzyme concentration to the Percentage bound of immobilization lignin peroxidase enzyme activity and immobilised enzymes
Figure.
Fig. 3 is influence schematic diagram of the Dopamine hydrochloride concentration to immobilization lignin peroxidase enzyme activity.
Fig. 4 is influence schematic diagram of the immobilization time to immobilization lignin peroxidase enzyme activity.
Fig. 5 is influence of the temperature to free lignin peroxidase and immobilization lignin peroxidase relative activity
Schematic diagram.
Fig. 6 is that influence of the pH to free lignin peroxidase and immobilization lignin peroxidase relative activity is shown
It is intended to.
Fig. 7 is the stability schematic diagram that immobilization lignin peroxidase stores in 25 DEG C of systems.
Fig. 8 is the stability schematic diagram that immobilization lignin peroxidase stores in 4 DEG C of systems.
Fig. 9 is the reuse stability curve figure of immobilization lignin peroxidase.
Specific embodiment
Embodiment 1
The present embodiment prepares immobilization lignin peroxidase, the specific steps are as follows:
(1) preparation of ferriferrous oxide nano-particle
3g ferric trichloride, 0.8g sodium citrate and 6g sodium acetate are weighed, is successively dissolved in 100mL second two under continuous stirring
In alcohol, be then transferred in 100mL autoclave, reacted at 200 DEG C 10h. it is cooling after, black solid precipitating successively with water and
For several times, vacuum drying for 24 hours, obtains ferriferrous oxide nano-particle to ethanol washing.
(2) preparation of magnetic silica nanoparticle
The 600mg ferriferrous oxide nano-particle prepared is dispersed in the mixed system of water, ethyl alcohol and ammonium hydroxide.It is mixed
The volume of water, ethyl alcohol and ammonium hydroxide is respectively then the positive silicic acid second of 3.6mL is added dropwise in 40mL, 160mL and 2mL in zoarium system
Ester, in 25 DEG C of oscillation 12h, with magnet separation product, gained precipitating is washed with distilled water removes unreacted raw material, vacuum for several times
It is dry for 24 hours to get arriving magnetic silica nanoparticle.
(3) preparation of dopamine polymerization embedded magnetic Nano particles of silicon dioxide
600mg magnetic silica nanoparticle is dispersed in 300mL TRIS buffer,
The Dopamine hydrochloride for adding 1.6mg/mL, in 25 DEG C of oscillation 12h.With magnet separation product, gained precipitating is washed with distilled water number
Secondary to remove unreacted raw material, for 24 hours, i.e. acquisition dopamine polymerize embedded magnetic Nano particles of silicon dioxide, i.e., for drying under vacuum
Immobilization lignin peroxidase carrier.
(4) immobilization lignin peroxidase
By 1g immobilization lignin peroxidase carrier (dopamine polymerize embedded magnetic Nano particles of silicon dioxide)
It is even to be dispersed in tartaric acid-sodium tartrate buffer that 30mL contains lignin peroxidase, lignin peroxidase egg
White concentration is 0.39mg/mL, in 25 DEG C of oscillation 12h.With magnet separation product, gained precipitating tartaric acid-sodium tartrate buffering
Liquid washs number, until without resolvase in supernatant, freeze-drying for 24 hours, that is, obtains immobilization lignin peroxidase.Winestone
Acid-sodium tartrate buffer concentration is 0.1mol/L, and pH 3, the mass concentration of tartaric acid is 15.1g/L, sodium tartrate
Mass concentration is 19.4g/L.
Fig. 1 gives the electron microscope of immobilization lignin peroxidase manufactured in the present embodiment.
Embodiment 2
The present embodiment provides enzyme concentration to the enzyme activity of immobilization lignin peroxidase and the Percentage bound of immobilised enzymes
It influences.
Step (1), (2), (3) in the present embodiment and embodiment 1 unanimously,
By " 30mL contains tartaric acid-sodium tartrate buffering of lignin peroxidase in the step (4) in embodiment 1
Liquid " is changed to " 5mL, 10mL, 20mL, 40mL and 50mL " respectively, then measures the immobilization lignin peroxidase of acquisition respectively
The enzyme activity of enzyme.
Fig. 2 can be seen that enzyme concentration directly influences immobilization enzyme activity.Other conditions under the same conditions, with enzyme
The enzyme activity of the increase of amount, immobilization gradually increases.When enzyme concentration is more than 30mL, immobilization enzyme activity increases slow.On the other hand, with
The increase of enzyme concentration, the Percentage bound of immobilised enzymes be gradually reduced.Its enzyme concentration is bigger, and loss amount is also bigger.
Comprehensively consider, select enzyme concentration for 30mL, immobilization enzyme activity is 180.1U/g, and the Percentage bound of immobilised enzymes is
56.3%.
Embodiment 3
The present embodiment provides influence of the Dopamine hydrochloride concentration to the enzyme activity of immobilization lignin peroxidase.
Step (1), (2), (4) in the present embodiment and embodiment 1 unanimously,
In step (3) respectively according to mass concentration be 1.2mg/mL, 1.4mg/mL, 1.6mg/mL, 1.8mg/mL and
2.0mg/mL adds Dopamine hydrochloride, then measures the enzyme activity of the immobilization lignin peroxidase of acquisition respectively.
Fig. 3 gives influence of the Dopamine hydrochloride concentration to the enzyme activity of immobilization lignin peroxidase, with hydrochloric acid
The enzyme activity of the increase of dopamine concentration, immobilization gradually increases.When Dopamine hydrochloride concentration is more than 1.6mg/mL, immobilization enzyme activity
It is almost unchanged.
Comprehensively consider, select Dopamine hydrochloride concentration for 1.6mg/mL, immobilization enzyme activity is 182.8U/g.
Embodiment 4
The present embodiment provides influence of the set time to the enzyme activity of immobilization lignin peroxidase.
Step (1), (2), (3) in the present embodiment and embodiment 1 unanimously,
Duration of oscillation in step (4) is changed to 1h, 3h, 6h, 8h and for 24 hours respectively, then measures the fixation of acquisition respectively
Change the enzyme activity of lignin peroxidase.
Fig. 4 gives the influence living to immobilization lignin peroxidase of immobilization time, with the immobilization time
Extend, the enzyme activity of immobilization gradually increases.It is more than 12h when the immobilization time, immobilized enzyme is almost unchanged.Select immobilization
Time 12h, immobilization enzyme activity are 188.2U/g.
In summary embodiment is stated, enzyme concentration 30mL, Dopamine hydrochloride concentration is 1.6mg/mL, and the immobilization time is
12h is the optimum condition for preparing immobilization lignin peroxidase.The enzyme activity average value of immobilization lignin peroxidase
For 183.4U/g.
Embodiment 5
The present embodiment is a difference in that with embodiment 1:
2g ferric trichloride, 0.5g sodium citrate and 10g sodium acetate are weighed in step (1), are successively dissolved under continuous stirring
It in 100mL ethylene glycol, is then transferred in 100mL autoclave, reacts 16h at 120 DEG C.
2mL ethyl orthosilicate is added in step (2), in 15 DEG C of oscillation 9h.The volume of water, ethyl alcohol and ammonium hydroxide in mixed system
Respectively 30mL, 140mL and 2mL.
It is that 600mg magnetic silica nanoparticle is dispersed in 200mL trihydroxy methyl amino first in step (3)
In alkane buffer, the Dopamine hydrochloride of 1.6mg/mL is added, is vibrated for 24 hours at 15 DEG C.
It is to vibrate 6h at 35 DEG C in step (4).
Embodiment 6
The present embodiment is a difference in that with embodiment 1:
5g ferric trichloride, 2g sodium citrate and 5g sodium acetate are weighed in step (1), are successively dissolved under continuous stirring
It in 100mL ethylene glycol, is then transferred in 100mL autoclave, reacts 8h at 160 DEG C.
5mL ethyl orthosilicate is added in step (2), in 35 DEG C of oscillation 6h.The volume of water, ethyl alcohol and ammonium hydroxide in mixed system
Respectively 35mL, 150mL and 2mL.
It is that 600mg magnetic silica nanoparticle is dispersed in 500mL trihydroxy methyl amino first in step (3)
In alkane buffer, the Dopamine hydrochloride of 1.6mg/mL is added, in 35 DEG C of oscillation 18h.
It in step (4) is vibrated for 24 hours at 15 DEG C.
The partial size of the immobilization lignin peroxidase of the various embodiments described above preparation is 200-400nm, and enzyme supported quantity is
3-6mg/g, enzyme activity 100-200U/g.
Embodiment 7
The present embodiment can be carried out test to the zymology of immobilization lignin peroxidase prepared by embodiment 1.
(1) best catalytic temperature
Within the temperature range of 20-80 DEG C, minimum unit is 10 DEG C, the environment that tartaric acid-sodium tartrate pH of buffer is 3
In, measure the vigor of immobilization lignin peroxidase at each temperature.Highest enzyme activity is the optimum temperature of the enzyme, i.e., relatively
Enzyme activity is 100%.
As shown in Figure 5, the lignin peroxidase that dissociates is gradually increased with respect to enzyme activity with temperature rising, is reached most at 30 DEG C
Big value, hereafter with the rising of temperature, enzyme activity declines rapidly.Temperature be greater than 40 DEG C under the conditions of react when, enzyme activity by compared with
The opposite enzyme activity loss of the inhibition of big degree, lignin peroxidase is greater than 50%.To after 60 DEG C, enzyme activity almost loses
It loses.And the optimum temperature of immobilization lignin peroxidase of the invention is 40 DEG C, is showed in 20-60 DEG C of temperature range
Higher enzyme activity keeps 60% or more initial vigor, this illustrates that immobilization is conducive to improve the heat resistance of enzyme.
(2) best catalytic pH
Temperature of reaction system is 30 DEG C, in the range of pH is 2-6, minimum unit 0.5, and winestone when measuring different pH respectively
Lignin peroxidase vigor in acid-sodium tartrate buffer, highest enzyme activity are the optimum pH of the enzyme, i.e., are with respect to enzyme activity
100%.
It will be appreciated from fig. 6 that the optimal pH of free lignin peroxidase is that 3, pH shows high enzyme within the scope of 2.5-3.5
Living, enzyme activity is heavily suppressed when pH is greater than 5.The optimal pH of immobilization lignin peroxidase of the invention is that 3.5, pH exists
Within the scope of 2.5-4, enzyme activity is held in 70% or more.For free lignin peroxidase, immobilization
It is all expanded before and after the stable pH range of lignin peroxidase, opposite enzyme activity is also higher.Immobilization lignin peroxidase
Enzyme has broader soda acid tolerance range, this is beneficial to lignin peroxidase and widely applies.
(3) stability stored
Fig. 7 in 25 DEG C of tartaric acid-sodium tartrate buffer it is found that store, the activity for the lignin peroxidase that dissociates
Decrease speed is obviously faster than immobilization lignin peroxidase.After storing 14 days in buffer solution system, immobilization is wooden
Plain peroxidase is able to maintain about 43% initial activity, and free lignin peroxidase only keeps 8.6% initial live
Property.
After Fig. 8 in 4 DEG C of tartaric acid-sodium tartrate buffer it is found that store 60 days, immobilization lignin peroxidase
67.5% initial activity is kept, and free lignin peroxidase almost inactivates.It is stored 30 days in buffer solution system, Gu
Surely change lignin peroxidase it is living keep 80.3% initial activity, and free lignin peroxidase living only 38.1%
Initial activity, its structure is more stable after this illustrates lignin peroxidase immobilization, and more conducively long term storage uses.
Embodiment 8
The present embodiment is the immobilization lignin peroxidase reusability test prepared to embodiment 1.
Enzyme activity determination is carried out under the optimum temperature of immobilization lignin peroxidase, after recycling catalysis reaction every time
Immobilised enzymes after 3.0 tartaric acid of pH-sodium tartrate buffer solution for cleaning, rejoins new catalysis reaction solution with 0.1mol/L
In, its enzymatic activity is measured, METHOD FOR CONTINUOUS DETERMINATION 8 times, investigates the residual activity of immobilised enzymes with the situation of change of access times.It is fixed
After changing the reuse stability curve of lignin peroxidase as shown in figure 9, reusing 4 times, immobilization lignin mistake
Oxide enzyme has 70% activity, and after using 8 times, immobilised enzymes still has 30% vigor.
Embodiment 9
The present embodiment is that immobilization lignin peroxidase prepared by embodiment 1 is used for catalytic degradation polycyclic aromatic hydrocarbon,
Detailed process are as follows:
Phenanthrene (1mg), the fluoranthene (1mg) of 5mg/L or the benzo [a] of 5mg/L for being respectively 5mg/L containing concentration in 200mL
In the tartaric acid of the pH=3.0 of pyrene (1mg)-sodium tartrate buffer, immobilization lignin peroxide prepared by 1g embodiment 1 is added
Compound enzyme (mass ratio of the additional amount of immobilization lignin peroxidase and Organic Pollutants in Wastewater is 1g:1mg), and add
Enter hydrogen peroxide, makes the concentration of hydrogen peroxide 1.7mg/L in system, 25 DEG C of temperature of reaction system, the reaction time is for 24 hours.Body
In system the removal rate (%) of polycyclic aromatic hydrocarbon pollutant as shown in table 1, table 1 the result shows that, in the presence of hydrogen peroxide, immobilization
Lignin peroxidase can effectively remove the polycyclic aromatic hydrocarbon in water body.
1 immobilization lignin peroxidase catalytic degradation polycyclic aromatic hydrocarbon of table
Embodiment 10
The present embodiment is that immobilization lignin peroxidase prepared by embodiment 1 is used for the pollution of catalytic degradation phenols
Object, detailed process are as follows:
In 5- chlorophenol (2mg) or 10mg/L of the 200mL respectively containing phenol (2mg), 10mg/L that concentration is 10mg/L
2,4- Dichlorophenol (2mg) pH=3.0 tartaric acid-sodium tartrate buffer in, be added 0.5g embodiment 1 prepare fixation
Changing lignin peroxidase, (additional amount of immobilization lignin peroxidase and the mass ratio of Organic Pollutants in Wastewater are
1g:4mg), and hydrogen peroxide is added, makes the concentration of hydrogen peroxide 3.4mg/L in system, 25 DEG C of temperature of reaction system, reacts
Time is 3h.In system the removal rate (%) of tetracycline antibiotics as shown in table 2, table 2 the result shows that, peroxide exist
Under, immobilization lignin peroxidase can quickly and effectively remove the phenolic comp ' ds pollution in water body.
2 immobilization lignin peroxidase catalytic degradation phenolic comp ' ds pollution of table
Embodiment 11
The present embodiment is that immobilization lignin peroxidase prepared by embodiment 1 is used to catalytic degradation Tetracyclines to resist
Raw element pollutant, detailed process are as follows:
In aureomycin (10mg) or 50mg/ of the 200mL respectively containing tetracycline (10mg), 50mg/L that concentration is 50mg/L
In tartaric acid-sodium tartrate buffer of the pH=3.0 of the terramycin (10mg) of L, immobilization wood prepared by 1g embodiment 1 is added
Lignin peroxidase (mass ratio of the additional amount of immobilization lignin peroxidase and Organic Pollutants in Wastewater is 1g:
10mg), and hydrogen peroxide is added, makes the concentration of hydrogen peroxide 6.8mg/L in system, 25 DEG C of temperature of reaction system, when reaction
Between be 1h.In system the removal rate (%) of tetracycline antibiotics as shown in table 3, table 3 the result shows that, peroxide exist
Under, immobilization lignin peroxidase can quickly and effectively remove the tetracycline antibiotics in water body.
3 immobilization lignin peroxidase catalytic degradation tetracycline antibiotics of table
Claims (8)
1. a kind of preparation method of immobilization lignin peroxidase, characterized in that the following steps are included:
(1) ferriferrous oxide nano-particle is prepared:
In the ratio of 2-5g:0.5-2g:5-10g:100mL, by ferric trichloride, sodium citrate and sodium acetate under continuous stirring according to
It is secondary to be dissolved in ethylene glycol, it is then transferred in autoclave, reacts 8-16h at 120-200 DEG C；After cooling, solid is sunk
It forms sediment and uses water and ethanol washing, be finally dried in vacuo to get ferriferrous oxide nano-particle is arrived；
(2) magnetic silica nanoparticle is prepared:
The ferriferrous oxide nano-particle and ethyl orthosilicate that step (1) preparation is weighed in the ratio of 600mg:2-5mL, by four oxygen
Change three Fe nanometer particles to be dispersed in the mixed system of water, ethyl alcohol and ammonium hydroxide, then adds dropwise ethyl orthosilicate,
6-12h is vibrated at 15-35 DEG C, with magnet separation product, gained precipitating, which is washed with distilled water, removes unreacted raw material, last true
Sky is dried to arrive magnetic silica nanoparticle；
(3) dopamine polymerization embedded magnetic Nano particles of silicon dioxide is prepared
The magnetic silica nanoparticle prepared in step (2) is dispersed in three in the ratio of 600mg:200-500mL
In hydroxymethyl aminomethane buffer, then with the concentration addition Dopamine hydrochloride of 1.2-2.0mg/mL, vibrated at 15-35 DEG C
12-24h；With magnet separation product, gained precipitating, which is washed with distilled water, removes unreacted raw material, is finally dried in vacuo, that is, obtains
It obtains dopamine and polymerize embedded magnetic Nano particles of silicon dioxide, as immobilization lignin peroxidase carrier；
(4) immobilization lignin peroxidase is prepared:
The immobilization lignin peroxidase carrier obtained in step (3) is dispersed in the ratio of 1g:5-50mL and is contained
Have in tartaric acid-sodium tartrate buffer of lignin peroxidase, lignin peroxidase protein concentration is 0.39mg/
ML vibrates 6-24h at 15-35 DEG C；With magnet separation product, gained precipitating is washed with tartaric acid-sodium tartrate buffer, directly
It into supernatant without resolvase, is finally freeze-dried, i.e. acquisition immobilization lignin peroxidase.
2. the preparation method of immobilization lignin peroxidase according to claim 1, characterized in that the step (1)
The partial size of the ferriferrous oxide nano-particle of middle preparation is 50-200nm.
3. the preparation method of immobilization lignin peroxidase according to claim 1, characterized in that the step (2)
The volume ratio of water, ethyl alcohol and ammonium hydroxide is 15-20:70-80:1 in the mixed system of middle water, ethyl alcohol and ammonium hydroxide.
4. the preparation method of immobilization lignin peroxidase according to claim 1, characterized in that the step (4)
Mesotartaric acid-sodium tartrate buffer concentration is 0.1mol/L, pH 2.5-3.5.
5. the preparation method of immobilization lignin peroxidase according to claim 1, characterized in that the step (4)
The partial size of middle immobilization lignin peroxidase is 200-400nm, and enzyme supported quantity is 3-6mg/g, enzyme activity 100-200U/g.
6. the organic pollutant in immobilization lignin peroxidase processing waste water prepared by claim 1, detailed process is:
Immobilization lignin peroxidase and hydrogen peroxide are added into the waste water containing organic pollutant, reacts 1-24h at room temperature, with
Remove removal organic polluter.
7. the organic pollutant in immobilization lignin peroxidase processing waste water according to claim 6, feature
It is that the additional amount of the immobilization lignin peroxidase and the mass ratio of Organic Pollutants in Wastewater are 1g:1mg-10mg.
8. the organic pollutant in immobilization lignin peroxidase processing waste water according to claim 6, feature
It is that mass concentration of the hydrogen peroxide in waste water is 1.7-6.8mg/L.
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