CN113832135A - Method for immobilizing laccase by modified polypropylene microporous membrane - Google Patents
Method for immobilizing laccase by modified polypropylene microporous membrane Download PDFInfo
- Publication number
- CN113832135A CN113832135A CN202111232308.5A CN202111232308A CN113832135A CN 113832135 A CN113832135 A CN 113832135A CN 202111232308 A CN202111232308 A CN 202111232308A CN 113832135 A CN113832135 A CN 113832135A
- Authority
- CN
- China
- Prior art keywords
- microporous membrane
- laccase
- polypropylene microporous
- immobilizing
- soaking
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0088—Physical treatment with compounds, e.g. swelling, coating or impregnation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
-
- 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
-
- 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
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
-
- 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)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- 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)
- C12Y110/03002—Laccase (1.10.3.2)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2323/00—Details relating to membrane preparation
- B01D2323/02—Hydrophilization
-
- 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
- C02F2101/345—Phenols
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Manufacturing & Machinery (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Hydrology & Water Resources (AREA)
- Inorganic Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
Abstract
The invention discloses a method for immobilizing laccase by a modified polypropylene microporous membrane. In the method, the polypropylene microporous membrane is soaked in n-butyl alcohol to initially improve the surface hydrophilicity; soaking the membrane by using a mixed solvent, so that the hydrophilicity of the membrane is improved again, and the surface smoothness of the membrane is improved at the same time; then coating with aluminum hydroxide colloid to ensure that the aluminum hydroxide colloid is firmly and uniformly coated on the surface of the membrane; and finally, the dopamine is tightly bonded with the colloid, so that the high-efficiency deposition of the dopamine is realized, and the effect of immobilizing the laccase is achieved. The modified polypropylene microporous membrane prepared by the method has small contact angle and good hydrophilicity; the immobilized laccase obtained by taking the modified polypropylene microporous membrane as the carrier has good stability, high enzyme curing amount, good degradation and adsorption effects on the phenol-containing wastewater and good application prospect.
Description
Technical Field
The invention belongs to the technical field of laccase immobilization, and particularly relates to a method for immobilizing laccase by using a modified polypropylene microporous membrane.
Background
Laccase belongs to blue oxidase, and has been widely applied to the technical fields of biochemical ring materials and the like since the discovery of the 19 th century. Because the free laccase has the defects of no acid and alkali resistance, no high temperature resistance and the like in the using process, the laccase immobilization technology is widely developed and applied.
Through long-term development, the immobilization methods of laccase at present are various and comprise a chemical method, a physical method, an embedding method, a self-immobilization method and the like. In various methods, the use of immobilization supports is generally not isolated; due to the structural and functional adjustability, membranes have been studied as carriers for laccase immobilization. For example, patent document CN101671665A reports a method for immobilizing laccase by using an electrodischarge fiber membrane, which comprises two steps of preparation of an enzyme-loaded electrospun fiber membrane and membrane-immobilized cross-linked laccase, the whole process is complex, and the enzyme activity recovery rate fluctuates greatly, and is only 82.8% at most; patent document CCN107034207A prepares an inorganic hybrid membrane for fixing laccase, and the decolorization of Congo red dye can reach more than 90%, but the treatment effect of phenol-containing wastewater with more complex components is not considered; patent document CN109012219A reports a preparation method of an in-situ immobilized laccase ultrafiltration membrane, wherein the relative enzyme activity of the immobilized laccase is only 64.7% at 20-60 ℃, and the removal rate of phenol-containing wastewater is only 78.4-81.5%. Therefore, it is necessary to develop a laccase membrane carrier with simple preparation method, good immobilized enzyme stability and high activity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for immobilizing laccase by using a modified polypropylene microporous membrane, which improves the hydrophilicity and the smoothness of the membrane surface by modifying the microporous membrane, efficiently grafts dopamine, realizes efficient immobilization of the laccase, and has good stability and good degradation and adsorption capacity on phenolic wastewater.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) immobilizing laccase;
the preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in a mixed solvent for soaking, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and coating by using aluminum hydroxide colloid;
s4, surface deposition of dopamine: and (5) placing the polypropylene microporous membrane coated in the step (S3) in a buffer solution containing dopamine, carrying out oscillation reaction, washing and drying after the reaction is finished, and thus obtaining the immobilized laccase carrier.
Preferably, the soaking time in step S1 is 3-10 min.
Preferably, the mixed solvent in the step S2 is prepared by mixing the following components in a mass ratio of 100: 5 deionized water and N, N-dimethylacetamide.
Preferably, the soaking temperature in step S2 is room temperature, and the soaking time is 0.5-2 d.
Preferably, the specific operation of coating in step S3 includes the following steps: and (3) placing the polypropylene microporous membrane subjected to solvent modification in a container, enabling the flow direction of the polypropylene microporous membrane to be parallel to the flow direction of the aluminum hydroxide colloid, and then coating the polypropylene microporous membrane by using the prepared aluminum hydroxide colloid.
Preferably, the pH of the aluminum hydroxide colloid in the step S3 is 7.0, and the concentration of aluminum ions is 0.035 mol/L.
Preferably, the coating temperature in step S3 is 25 ℃, and the coating time is 3-10 min.
Preferably, the buffer in step S4 is Tris buffer with pH =8.0, and the concentration of dopamine is 2 mg/mL.
Preferably, in the step S4, the oscillation reaction temperature is room temperature, and the oscillation reaction time is 8-24 h.
Preferably, the laccase immobilization in step (2) comprises the steps of: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use;
wherein the concentration of the laccase is 0.6 mg/mL.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method for immobilizing laccase by using the modified polypropylene microporous membrane provided by the invention comprises the steps of soaking the polypropylene microporous membrane in n-butyl alcohol to preliminarily improve the surface hydrophilicity; soaking the membrane by using a mixed solvent, so that the hydrophilicity of the membrane is improved again, and the surface smoothness of the membrane is improved at the same time; then coating with aluminum hydroxide colloid to ensure that the aluminum hydroxide colloid is firmly and uniformly coated on the surface of the membrane; and finally, the dopamine is tightly bonded with the colloid, so that the high-efficiency deposition of the dopamine is realized, and the effect of immobilizing the laccase is achieved.
(2) In the invention, the n-butyl alcohol is adopted for soaking, so that on one hand, the effect of removing impurities on the surface of the polypropylene microporous membrane can be achieved, and meanwhile, the hydrophilicity of the surface of the polypropylene microporous membrane can be preliminarily improved; the mixed solvent of deionized water and N, N-dimethylacetamide is used for modification, so that chain segments on the surface of the polypropylene microporous membrane are recombined, the hydrophilicity of the membrane is further improved, and the surface energy of the membrane is reduced; the modified polypropylene microporous membrane is coated with aluminum hydroxide colloid, so that the coating effect of the aluminum hydroxide can be improved, and the coating of the aluminum hydroxide can improve the internal and external hydrophilicity of the polypropylene microporous membrane and provide binding active sites for the subsequent deposition of dopamine; the dopamine can be firmly fixed on the surface of the polypropylene microporous membrane in a bonding mode through active point positions and aluminum hydroxide, so that the hydrophilicity of the surface of the membrane is improved again on one hand, and on the other hand, the dopamine surface has rich functional groups and can realize high-efficiency fixation of laccase.
(3) The modified polypropylene microporous membrane prepared by the method has small contact angle and good hydrophilicity; the immobilized laccase obtained by taking the modified polypropylene microporous membrane as a carrier has good stability and high enzyme curing amount, and the curing amount exceeds 160 mg/g; the degradation and adsorption effect on the phenol-containing wastewater is good, and the degradation and adsorption rate exceeds 96%.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly illustrated, the present invention will be further explained in detail with reference to the embodiments.
It is particularly emphasized that laccases are purchased from Aldrich; polypropylene microporous membranes were purchased from Membrana, germany, with specifications: the average pore diameter was 0.2 μm, and the porosity was 80%. Other materials or reagents in the present invention are also commercially available unless otherwise specified.
Example 1
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) and (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6 mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent for 4min, taking out, washing with deionized water for multiple times, and naturally drying;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in 30mL of mixed solvent, soaking for 1d at room temperature, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in a mass ratio of 100: 5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 5 min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step S3 in 25mL of dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Example 2
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) and (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6 mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent for 5min, taking out, washing with deionized water for multiple times, and naturally drying;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in 30mL of mixed solvent, soaking at room temperature for 1.5 days, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in a mass ratio of 100: 5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 6 min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step S3 in 25mL of dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 12h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Example 3
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) and (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6 mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent for 6min, taking out, washing with deionized water for multiple times, and naturally drying;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in 35mL of mixed solvent, soaking for 2d at room temperature, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in a mass ratio of 100: 5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 7 min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step S3 in 30mL of a Tris buffer solution containing dopamine (with the concentration of 2 mg/mL) and having the pH value of 8.0, carrying out oscillation reaction at room temperature for 16h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Comparative example 1
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) and (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6 mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent for 4min, taking out, washing with deionized water for multiple times, and naturally drying;
s2, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid having a pH =7 and a concentration of 0.035mol/L in a manner parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 5 min;
s3, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step S2 in 25mL of dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Comparative example 2
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) preparing an immobilized laccase carrier;
(2) and (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6 mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent for 4min, taking out, washing with deionized water for multiple times, and naturally drying;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in 30mL of mixed solvent, soaking for 1d at room temperature, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in a mass ratio of 100: 5, mixing the deionized water and the N, N-dimethylacetamide;
s3, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane subjected to solvent modification in the step S2 in 25mL of dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Carrying out a water contact angle test on the modified polypropylene microporous membranes prepared in the examples 1-3 and the comparative examples 1-2; the immobilized laccase prepared in the examples 1 to 3 and the comparative examples 1 to 2 are subjected to enzyme activity test, immobilized laccase amount test, degradation adsorption performance test and thermal stability test. The specific test method is as follows:
(1) water contact Angle test
The test was performed with reference to GB/T30447 and 2013.
Table 1 water contact angle test results of modified polypropylene microporous membranes
| Contact Angle/° | |
| Example 1 | 25 |
| Example 2 | 27 |
| Example 3 | 29 |
| Comparative example 1 | 34 |
| Comparative example 2 | 38 |
(2) Enzyme Activity assay
Adding 2mL of 1mg/mL free laccase or 2mg of obtained immobilized laccase into 1mmol/L ABTS solution, carrying out oscillation reaction at 25 ℃ for 15min, placing in an ice bath to terminate the reaction, carrying out centrifugal separation, and measuring the absorbance change of the supernatant at 420nm by using an ultraviolet-visible spectrophotometer to calculate the corresponding enzyme activity.
In this example, the model of the uv-vis spectrophotometer is new century T6.
(3) Immobilized laccase quantity test
a) Drawing a protein standard curve: respectively adding 1000 mug/mL of standard protein solution and distilled water according to different amounts, then respectively and sequentially adding 5mL of Coomassie brilliant blue G-250 reagent, measuring the absorbance after dyeing for 5min, and drawing a protein standard curve by taking the protein content as a horizontal coordinate and the absorbance as a vertical coordinate.
b) Determination of the amount of immobilized laccase: testing the laccase amount by adopting a Coomassie brilliant blue method; wherein, the enzyme amount calculation formula is as follows:
Q（%）=（Co-C）V/W*100；
in the above formula, Q represents the enzyme adsorption amount, CoConcentration of laccase in solution before immobilization; c is the concentration of laccase in the immobilized solution, V is the total volume of the immobilized solution, and W is the mass of the carrier. In the present case, Co=0.6 g/L，V=1.5mL=1.5*10-3L, W =0.5 g. The specific test and calculation results are shown in table 2.
TABLE 2 immobilized laccase quantity test results
| Amount of immobilized laccase/(mg/g) | |
| Example 1 | 163.5 |
| Example 2 | 164.1 |
| Example 3 | 162.9 |
| Comparative example 1 | 132.4 |
| Comparative example 2 | 129.3 |
As can be seen from Table 2, the immobilization amount of laccase in each example of the invention exceeds 160mg/g, and the immobilization efficiency is high.
(4) Degradation adsorption of 2, 4-dichlorophenol
Mixing 0.2g of immobilized laccase with 15mL of 10 mg/L2, 4-dichlorophenol, reacting for 5h at room temperature, centrifuging, diluting 10mL of supernatant, and sequentially adding 1.25mL of 0.5mol/L NH3·H2O, 0.5mL of 2% 4-aminoantipyrine, 0.5mL of 80g/L potassium ferricyanide solution, stirring and reacting for 15min, and measuring the absorbance change at the wavelength of 510nm by using an ultraviolet spectrophotometer to obtain the adsorption degradation rate of the immobilized laccase to the 2, 4-dichlorophenol.
In this example, HPLC is Shimadzu LC-20A.
TABLE 32, 4-dichlorophenol degradation adsorption test results
| Degradation adsorption rate/% | |
| Example 1 | 97.23 |
| Example 2 | 96.89 |
| Example 3 | 97.01 |
| Comparative example 1 | 83.46 |
| Comparative example 2 | 80.35 |
From the above table, it can be seen that the immobilized laccase prepared in the examples of the present application has a good adsorption degradation rate for 2, 4-dichlorophenol.
(5) Stability test
And (3) storing the immobilized laccase prepared in the embodiment 1-3 and the free enzyme at different temperatures for 1.5h, and calculating the residual activity of the enzyme according to the enzyme activity test method.
Test results show that the residual activity of the immobilized enzyme obtained in example 1 is still kept over 75% after the immobilized enzyme is stored for 2 hours at 60 ℃; the remaining activity of the enzyme in comparative example 1 was 69%; the remaining activity of the enzyme in comparative example 2 was 65%; while the residual activity of the free laccase is less than 60%.
The above description describes a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention as claimed. Any modification, equivalent replacement and improvement without departing from the principle and spirit of the present invention shall be considered to be within the protection scope of the present claims.
Claims (10)
1. A method for immobilizing laccase by a modified polypropylene microporous membrane is characterized by comprising the following steps:
(1) preparing an immobilized laccase carrier;
(2) immobilizing laccase;
the preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in a mixed solvent for soaking, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and coating by using aluminum hydroxide colloid;
s4, surface deposition of dopamine: and (5) placing the polypropylene microporous membrane coated in the step (S3) in a buffer solution containing dopamine, carrying out oscillation reaction, washing and drying after the reaction is finished, and thus obtaining the immobilized laccase carrier.
2. The method for immobilizing laccase by using the modified polypropylene microporous membrane according to claim 1, wherein the soaking time in step S1 is 3-10 min.
3. The method for immobilizing laccase on a modified polypropylene microporous membrane according to claim 1, wherein the mixed solvent in step S2 is prepared from 100: 5 deionized water and N, N-dimethylacetamide.
4. The method of claim 1, wherein the soaking temperature in the step S2 is room temperature, and the soaking time is 0.5-2 days.
5. The method for immobilizing laccase on a modified polypropylene microporous membrane according to claim 1, wherein the step S3 comprises the following steps: and (3) placing the polypropylene microporous membrane subjected to solvent modification in a container, enabling the flow direction of the polypropylene microporous membrane to be parallel to the flow direction of the aluminum hydroxide colloid, and then coating the polypropylene microporous membrane by using the prepared aluminum hydroxide colloid.
6. The method of claim 5, wherein the pH of the aluminum hydroxide colloid in the step S3 is 7.0, and the concentration of aluminum ions is 0.035 mol/L.
7. The method of claim 6, wherein the step S3 is carried out at a temperature of 25 ℃ for 3-10 min.
8. The method of claim 1, wherein the buffer in step S4 is Tris buffer with pH =8.0, and the concentration of dopamine is 2 mg/mL.
9. The method of claim 8, wherein the oscillation reaction temperature in step S4 is room temperature, and the oscillation reaction time is 8-24 h.
10. The method according to any one of claims 1 to 9, wherein the step (2) of immobilizing the laccase comprises the steps of: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5 mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use;
wherein the concentration of the laccase is 0.6 mg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111232308.5A CN113832135B (en) | 2021-10-22 | 2021-10-22 | Method for immobilizing laccase by modified polypropylene microporous membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111232308.5A CN113832135B (en) | 2021-10-22 | 2021-10-22 | Method for immobilizing laccase by modified polypropylene microporous membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113832135A true CN113832135A (en) | 2021-12-24 |
| CN113832135B CN113832135B (en) | 2022-11-01 |
Family
ID=78965765
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111232308.5A Active CN113832135B (en) | 2021-10-22 | 2021-10-22 | Method for immobilizing laccase by modified polypropylene microporous membrane |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113832135B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115124131A (en) * | 2022-07-29 | 2022-09-30 | 重庆工商大学 | Method for continuously treating bisphenol type wastewater |
| CN115286422A (en) * | 2022-08-30 | 2022-11-04 | 合润达源(湖北)科技有限公司 | Preparation method of selenium-rich ceramic teapot capable of flavoring tea water |
| CN115404225A (en) * | 2022-10-14 | 2022-11-29 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671665A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Method for preparing immobilized laccase in electrospun fiber membrane |
| CN101745327A (en) * | 2009-12-29 | 2010-06-23 | 浙江大学 | Method for fixing biological molecules on polymer microporous membrane surface |
| WO2014000167A1 (en) * | 2012-06-27 | 2014-01-03 | 新乡市中科科技有限公司 | Method for continuous hydrophilic modification of microporous polypropylene film |
| CN103585903A (en) * | 2013-10-25 | 2014-02-19 | 中国科学院广州化学研究所 | Chiral-separation solid membrane grafted by chiral identification body through dopamine pretreatment, and making method thereof |
| CN104524986A (en) * | 2014-12-08 | 2015-04-22 | 中国科学院宁波材料技术与工程研究所 | Preparation method of hydrophilic antimicrobial film of which surface is coated with dopamine and polyethyleneimine cations |
| CN105617883A (en) * | 2015-12-28 | 2016-06-01 | 山东省科学院海洋仪器仪表研究所 | Method for hydrophilic modification of polypropylene filtering membrane by using aluminum hydroxide gel |
| CN108410003A (en) * | 2018-02-09 | 2018-08-17 | 江南大学 | A kind of preparation of modified polyacrylonitrile film and its method applied to immobilized enzyme |
| CN108707244A (en) * | 2018-05-07 | 2018-10-26 | 武汉理工大学 | A kind of polyphenol compound deposition polypropylene surface hydrophilic modification method |
| CN109012219A (en) * | 2018-09-03 | 2018-12-18 | 安徽工程大学 | A kind of preparation method of fixation in situ laccase ultrafiltration membrane, a kind of processing method of phenol wastewater |
-
2021
- 2021-10-22 CN CN202111232308.5A patent/CN113832135B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671665A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Method for preparing immobilized laccase in electrospun fiber membrane |
| CN101745327A (en) * | 2009-12-29 | 2010-06-23 | 浙江大学 | Method for fixing biological molecules on polymer microporous membrane surface |
| WO2014000167A1 (en) * | 2012-06-27 | 2014-01-03 | 新乡市中科科技有限公司 | Method for continuous hydrophilic modification of microporous polypropylene film |
| CN103585903A (en) * | 2013-10-25 | 2014-02-19 | 中国科学院广州化学研究所 | Chiral-separation solid membrane grafted by chiral identification body through dopamine pretreatment, and making method thereof |
| CN104524986A (en) * | 2014-12-08 | 2015-04-22 | 中国科学院宁波材料技术与工程研究所 | Preparation method of hydrophilic antimicrobial film of which surface is coated with dopamine and polyethyleneimine cations |
| CN105617883A (en) * | 2015-12-28 | 2016-06-01 | 山东省科学院海洋仪器仪表研究所 | Method for hydrophilic modification of polypropylene filtering membrane by using aluminum hydroxide gel |
| CN108410003A (en) * | 2018-02-09 | 2018-08-17 | 江南大学 | A kind of preparation of modified polyacrylonitrile film and its method applied to immobilized enzyme |
| CN108707244A (en) * | 2018-05-07 | 2018-10-26 | 武汉理工大学 | A kind of polyphenol compound deposition polypropylene surface hydrophilic modification method |
| CN109012219A (en) * | 2018-09-03 | 2018-12-18 | 安徽工程大学 | A kind of preparation method of fixation in situ laccase ultrafiltration membrane, a kind of processing method of phenol wastewater |
Non-Patent Citations (4)
| Title |
|---|
| 佚名: "Preparation and Properties of Enzyme-Immobilized Porous Polypropylene Films", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE 》 * |
| 徐伟: "基于多巴胺修饰聚丙烯微孔膜表面的漆酶固定化研究", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 * |
| 杨谦: "聚丙烯微孔膜表面修饰的葡聚糖固定化研究", 《高等学校化学学报》 * |
| 胡惠敏: "聚丙烯微孔膜的表面亲水性能研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115124131A (en) * | 2022-07-29 | 2022-09-30 | 重庆工商大学 | Method for continuously treating bisphenol type wastewater |
| CN115286422A (en) * | 2022-08-30 | 2022-11-04 | 合润达源(湖北)科技有限公司 | Preparation method of selenium-rich ceramic teapot capable of flavoring tea water |
| CN115404225A (en) * | 2022-10-14 | 2022-11-29 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof |
| CN115404225B (en) * | 2022-10-14 | 2024-03-15 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF membrane immobilized laccase and method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113832135B (en) | 2022-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113832135B (en) | Method for immobilizing laccase by modified polypropylene microporous membrane | |
| CN106237869B (en) | A kind of polyphenol coating hydrophilic method of modified hydrophobic type polymeric membrane | |
| CN106310958B (en) | A kind of hydrophobic type polymeric membrane composite coated hydrophilic modification method | |
| CN102921315B (en) | Anti-pollution reverse osmosis membrane and preparation method thereof | |
| CN106669468A (en) | Metal doped g-C3N4 based visible light catalysis flat plate type ultra-filtration membrane and preparation method | |
| CN108654396A (en) | Polymer film and preparation method with absorption heavy metal and organic pollution function | |
| CN111790276A (en) | Preparation method of alkali-resistant nanofiltration composite membrane | |
| CN103263858B (en) | Cross-linking hyperbranched polymer composite nano filtration membrane prepared by one step method in water phase, preparation method and application | |
| CN104801206A (en) | Modified guanidyl film, and preparation and application thereof | |
| CN103203187B (en) | A preparation method for solid film dip-coated with a novel chiral recognition agent | |
| CN1296124C (en) | Method for preparing compound nanometer filtering membrane of chitosan sulfate-high molecule polymer | |
| CN102814126A (en) | Preparation method of high-flux antioxidant nanofiltration membrane | |
| CN113144912B (en) | Preparation method of high-flux covalent organic framework nanofiltration membrane based on TFC structure | |
| CN100556517C (en) | A kind of hydrophile organic-abio compound permeation vaporisation separation membrane and its production and application | |
| CN107551821A (en) | A kind of salt-resistant type anion exchange film chromatography medium and its production and use | |
| CN111921378B (en) | Polyamide membrane for separating dye and salt and preparation method thereof | |
| CN105561810A (en) | Method of using carbon nanofiber layer to modify ultrafiltration membrane | |
| CN112007526A (en) | Dopamine and taurine modified polysulfone membrane and preparation method thereof | |
| CN108939949B (en) | Polyimide affinity membrane with high adsorption performance on bilirubin and preparation method thereof | |
| CN111804162A (en) | Preparation method of high-flux polytetrafluoroethylene composite nanofiltration membrane | |
| CN101507904A (en) | Composite ultrafiltration membrane and preparation method thereof | |
| CN104548976B (en) | Reverse osmosis membrane as well as preparation method and application thereof | |
| CN108187506A (en) | A kind of waste water desalination forward osmosis membrane | |
| CN114832626B (en) | Composite nanofiltration membrane, preparation method and application | |
| CN110975637B (en) | Preparation method of gallic acid-chitosan/polysulfone composite nanofiltration membrane |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |