CN105836896B - The method of immobilization laccase processing water pollutant based on protein mediator - Google Patents
The method of immobilization laccase processing water pollutant based on protein mediator Download PDFInfo
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Abstract
A kind of preparation method of the immobilization laccase for handling organic pollutants in biotic environment improvement field instills CuSO by the way that laccase is added into sodium alginate soln and is used as after the hemoglobin of mediator mixes4Precipitation i.e. immobilization laccase is obtained by the reaction in solution.The present invention is fixed on hemoglobin as protein mediator and laccase on copper alginate carrier jointly, can improve the activity of laccase, while the level for handling Organic substance in water pollutant is also improved.The concentration for the treatment of that the present invention is directed to pollutant in water sample is 0.1mM, can apply to the processing of contaminated areas water sample.
Description
Technical field
The present invention relates to the technology that a kind of biotic environment administers field, specifically a kind of use based on protein mediator
In the preparation method of the immobilization laccase of processing organic pollutants.
Background technology
The toxic chemical substance environmental pollution that " three wastes " and chemical pesticide in industry generate during decomposing is very big,
Wherein phenol, chlorophenol and aniline are main organic pollutions.The pollutant largely accumulates in the environment, is caused to human health
Great harm.Common processing method mainly has absorption method, ultrafiltration, Ozone, extraction, Catalyzed by Ultrasonic Wave degradation
Method, chemical deposition method, activated sludge process etc., but face a series of problems, such as toxicity is remaining, energy consumption is higher, secondary pollution.With
It is increasingly mature biotechnology, there is very strong superiority using biotechnology processing organic pollutants.Wherein laccase has
There is the toxic phenolic compounds of oxidation removal, therefore many researchers attempt laccase for handling the industrial wastewater containing phenol.
Laccase is a kind of polyphenol oxidase of cupric, can be catalyzed a series of phenols and non-phenols aromatic compound and
The oxidation of its derivative.As a kind of biological enzyme of great foreground, laccase can be applied to environmental protection, paper pulp papermaking, timber add
Work etc..
Laccase is found in lacquer tree for the first time, is widely present in plant, bacterium, insect.In addition to phenols and its derivative,
The substrate of laccase further includes arylamine, carboxylic acid, pigment, lignin etc..The active site of laccase contains there are four copper atom, studies table
Bright, metal copper ion is improved the activity of laccase.Since oxygen molecule is reduced to hydrone, Laccase Catalyzed using electronics transfer
Oxidation reaction is mild, it is considered to be ideal green catalyst, therefore laccase also has become the research of domestic and foreign scholars in recent years
Hot spot.
The major defect of cellulase treatment pollutant is price height at present, and easy in inactivation, product can not detach, it is difficult to repeat profit
With.Drawbacks described above can be overcome to a certain extent by enzyme immobilization technology, improves the stability of laccase, be easily isolated operation,
And it can reuse.The method of immobilised enzymes has very much, common for absorption, embedding, crosslinking, covalent bond etc..Utilize sea
Alginate molecules chain forms gel network with metal copper ion, can be by laccase molecule embedding wherein, and keeps higher work
Property.
Laccase can improve redox ability in the case where transmitting the mediation of mediator of electronics.Currently used mediator is divided into people
Work synthesizes mediator (ABTS, HBT etc.) and natural amboceptor (syringaldehyde, NSC 611398 etc.).But since these small molecules are situated between
Body there are stability it is poor, expensive, there may be toxic products the shortcomings of, laccase mediator systems are at present in industrial production
It is not yet received application, and the macromolecular mediator of protein types and has not been reported precedent.
By the retrieval discovery to the prior art, Chinese patent literature CN101914515A discloses (bulletin) day
2010.12.15, a kind of preparation method of immobilization laccase is disclosed, it is to carry that a kind of immobilization laccase, which is with chelating resin,
Laccase is fixed on this carrier by absorption, chelation, carboxylic work is bonded in the structure of the chelating resin by body
Sexual function group.The immobilization laccase of the technology can be applied in the sewage disposal containing phenolic comp ' ds pollution, have processing technology
Simply, the features such as of low cost, laccase adsorption rate is high, and laccase activity loss is small, property stabilization.But the technology is in practical applications
Need the mediator small molecule that character of use is unstable, of high cost and there may be toxic product.
Invention content
The present invention is directed to deficiencies of the prior art, proposes at a kind of immobilization laccase based on protein mediator
The method for managing water pollutant is fixed on using hemoglobin as protein mediator and laccase on copper alginate carrier jointly, can be with
The activity of laccase is improved, while the level for handling Organic substance in water pollutant is also improved.The present invention is directed to dirty in water sample
The concentration for the treatment of for contaminating object is 0.1mM, can apply to the processing of contaminated areas water sample.
The present invention is achieved by the following technical solutions：
The present invention relates to a kind of preparation methods for handling the immobilization laccase of organic pollutants, by seaweed
CuSO is instilled after laccase and mediator mixing are added in acid sodium solution4Precipitation i.e. immobilization laccase is obtained by the reaction in solution.
The laccase is whiterot fungi (T.versicolor) laccase, and the concentration ratio of concentration and mediator is 1:1.
The mediator is by hemoglobin and its minor structure for including and its derivative, such as heme group.
The mixing refers to：It weighs 0.037g laccases to be dissolved in 10mL distilled water, supernatant is taken after standing, is in addition weighed
0.037g hemoglobin solids are dissolved in 10mL distilled water, supernatant are taken after standing, and sodium alginate is added in two kinds of supernatants together
It is stirred until homogeneous in solution.
A concentration of 20g/L of the sodium alginate soln.
The precipitation does not preferably generate blue precipitate after being washed repeatedly into cleaning solution and NaOH is added.
The CuSO4A concentration of 0.05mol/L of solution.
The organic pollution includes but not limited to：Phenol, chlorophenol and aniline etc..
The processing refers to：The immobilization laccase is placed in the buffer solution containing organic pollution, in aerobic ring
It is stirred to react under border.
The pH of the buffer solution is 4.6.
Technique effect
Compared with prior art, technique effect of the invention includes：
1, hemoglobin improves the catalytic activity of laccase, and the organic contamination in applied to water as protein mediator
The reaction time is shortened during object processing；
2, hemoglobin is relatively stable in catalytic reaction process, does not generate secondary pollution；
3 compare with other artificial synthesized mediators, and hemoglobin cost is relatively low, can pass through the method and paint of biotechnology
Enzyme produces simultaneously；
4, laccase and hemoglobin are fixed jointly, mediator can not only be reused, and mediator and production can be made
It is detached between object, it is easy to operation.
Description of the drawings
Fig. 1 is that immobilization laccase handles 2-TBP result figure in embodiment；
Fig. 2 is that immobilization laccase handles 2,4- Dichlorophenol result figures in embodiment.
Specific implementation mode
It elaborates below to the embodiment of the present invention, the present embodiment is carried out lower based on the technical solution of the present invention
Implement, gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
It is prepared by immobilization laccase
Step 1：It weighs 2g sodium alginates and adds water to 100mL, be heated to being completely dissolved.
Step 2：A certain amount of laccase and hemoglobin are added after cooling, stirring 5min makes it uniformly.
The laccase is whiterot fungi (T.versicolor) laccase, is purchased from Sigma-Aldrich companies.
Step 3：Aforesaid liquid is instilled to the CuSO of 0.05mol/L with syringe4In solution, 0.5h is stood at room temperature and is waited for
It is solidified completely.
Step 4：The immobilised enzymes prepared is washed repeatedly with distilled water, until cleaning solution does not generate after NaOH is added
Blue precipitate prepares gained immobilization laccase and is light blue green spherical, diameter about 2mm, weight about 12.5mg.
Embodiment 2
The determination of activity of immobilization laccase
Select o-tolidine solution as immobilization laccase activity assay substrate.
Step 1：It measures 150mL concentrated hydrochloric acids and is diluted to 500mL with distilled water.1.00g o-tolidines are weighed, and are drawn
The prepared hydrochloric acid of 5mL, which is put into together in glass mortar device, is ground into paste.Then add 150mL distillations in the container of merging 1000mL
Water dilutes, while 495mL dilute hydrochloric acid is added and is sufficiently stirred with glass bar, is finally diluted to 1000mL with distilled water.
Step 2：It takes 0.2mL o-tolidine solution to be added in 1.8mL NaAc-HAc buffer solutions (pH=4.6), is added
Immobilization laccase 72.5mg is constantly passed through air and stirs, and reacts 5min.
Step 3：Changed using product assay after ultraviolet spectrometer (Agilent 8453) detection o-tolidine catalysis,
The average detection per 30s is primary.The relative reaction rate of immobilization laccase is obtained using spectral results, immobilization laccase is calculated and lives
Property.
O-tolidine characteristic wavelength is 365nm, 630nm and 850nm, selects at 365nm as test point.As shown in table 1,
By comparing the reaction rate of immobilization laccase and immobilized hemogolobin-laccase, immobilized hemogolobin-laccase relative response
Rate is 0.0469 ± 0.0017A/min, and the relative reaction rate of immobilization laccase is 0.0421 ± 0.0035A/min, fixed
Change hemoglobin-laccase and improves 11.5% relative to immobilization laccase activity.
Embodiment 3
Immobilization laccase handles benzidine hydrochloride
Step 1：Benzidine hydrochloride 0.026g is weighed, is added in 100mL water, is configured to a concentration of 1mM solution.
Step 2：It takes 0.2mL benzidine hydrochloride solution to be added in 1.8mL NaAc-HAc buffer solutions (pH=4.6), is added
Immobilization laccase 0.25g is constantly passed through air and stirs, and reacts 5min.
Step 3：Changed using product assay after ultraviolet spectrometer (Agilent 8453) detection benzidine hydrochloride catalysis,
The average detection per 30s is primary.The relative reaction rate of immobilization laccase is obtained using spectral results.
Benzidine hydrochloride characteristic wavelength is 360nm, 617nm and 850nm, selects at 360nm as test point.Such as Fig. 1 and table 1
It is shown, by comparing the reaction rate of immobilization laccase and immobilized hemogolobin-laccase, immobilized hemogolobin-laccase phase
It is 0.0323 ± 0.0018A/min to reaction rate, the relative reaction rate of immobilization laccase is 0.0230 ± 0.00033A/
Min, immobilized hemogolobin-laccase improve 40.4% relative to immobilization laccase activity.
Embodiment 4
Immobilization laccase handles 2-TBP
Step 1：2-TBP 0.015g is weighed, is added in 100mL water, is configured to a concentration of 1mM solution.
Step 2：It takes 0.2mL 2-TBP solution to be added in 1.8mL NaAc-HAc buffer solutions (pH=4.6), adds
Enter immobilization laccase 0.25g, be constantly passed through air and stir, reacts 2min.
Step 3：Become using product assay after ultraviolet spectrometer (Agilent 8453) detection 2-TBP catalysis
Change, the average detection per 0.5h is primary.The relative reaction rate of immobilization laccase is obtained using spectral results.
As shown in table 1, by comparing the reaction rate of immobilization laccase and immobilized hemogolobin-laccase, immobilization blood
Lactoferrin-laccase product assay is 5.16 ± 0.36A, and immobilization laccase product assay is 4.18 ± 0.09A, the blood red egg of immobilization
In vain-laccase improves 23.6% relative to immobilization laccase activity.
Embodiment 5
Immobilization laccase handles 2,4- Dichlorophenols
Step 1：2,4- Dichlorophenol 0.016g are weighed, is added in 100mL water, is configured to a concentration of 1mM solution.
Step 2：It takes 2mL 2,4- dichloro phenol solutions to be added in 8mL NaAc-HAc buffer solutions (pH=4.6), is added and fixes
Change laccase 0.25g, be constantly passed through air and stir, reacts 2h.
Step 3：0.5mL mixed liquors are taken, 2.5mL is diluted to.The NH of 300 a concentration of 0.5mol/L of μ L is added3·H2O, then
225 μ L disodium hydrogen phosphates-sodium dihydrogen phosphate buffer (pH=6.7) is added and adjusts pH to 7.9 ± 0.1.Sequentially add 50 μ L
The potassium ferricyanide aqueous solution of 2% 4-AA aqueous solution and 50 a concentration of 80g/L of μ L is stood after stir about 30s
15min, with the concentration of chlorophenol in ultraviolet spectrometer (Agilent 8453) measurement system.
As shown in Figure 2 and Table 1, fixed by comparing the reaction rate of immobilization laccase and immobilized hemogolobin-laccase
Change hemoglobin-laccase 2h 2,4- of degradation Dichlorophenols 29.8 ± 1.2%, immobilization laccase 2h 2,4- of degradation Dichlorophenols 19.4 ±
0.9%, immobilized hemogolobin-laccase improves 12.8% relative to immobilization laccase activity.
Table 1
Claims (7)
1. a kind of preparation method for handling the immobilization laccase of organic pollutants, which is characterized in that by seaweed
Addition laccase and the hemoglobin as mediator instill CuSO after mixing in acid sodium solution4Precipitation is obtained by the reaction in solution to fix
Change laccase；
The mediator is by hemoglobin and its minor structure for including and its derivative；
The laccase is whiterot fungi (T.versicolor) laccase, and the concentration ratio of concentration and mediator is 1:1；
The mixing refers to：It weighs 0.037g laccases to be dissolved in 10mL distilled water, supernatant is taken after standing, in addition weighs 0.037g
Hemoglobin solid is dissolved in 10mL distilled water, supernatant is taken after standing, and two kinds of supernatants are added in sodium alginate soln together
It is stirred until homogeneous.
2. according to the method described in claim 1, it is characterized in that, a concentration of 20g/L of the sodium alginate soln.
3. according to the method described in claim 1, it is characterized in that, NaOH is added into cleaning solution through washing repeatedly for the precipitation
Blue precipitate is not generated afterwards.
4. according to the method described in claim 1, it is characterized in that, the CuSO4A concentration of 0.05mol/L of solution.
5. according to the method described in claim 1, it is characterized in that, the processing refers to：The immobilization laccase is placed in and is contained
Have in the buffer solution of organic pollution, is stirred to react under aerobic environment.
6. according to the method described in claim 5, it is characterized in that, the pH of the buffer solution is 4.6.
7. according to the method described in claim 5, it is characterized in that, a concentration of 0.1mM of the organic pollution.
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| CN112877320A (en) * | 2021-01-26 | 2021-06-01 | 武汉纺织大学 | Preparation method of copper alginate loaded laccase and mediator ABTS composite biocatalyst |
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| Influence of counteranions on catalytic ability of immobilized laccase in Cu-alginate matrices: Inhibition of chloride and activation of acetate;Ting Pan等;<Chinese Chemical Letters>;20141231(第25期);第984页第2.1节至第986页第3.5节 * |
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