CN112877320A - Preparation method of copper alginate loaded laccase and mediator ABTS composite biocatalyst - Google Patents
Preparation method of copper alginate loaded laccase and mediator ABTS composite biocatalyst Download PDFInfo
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- CN112877320A CN112877320A CN202110106493.7A CN202110106493A CN112877320A CN 112877320 A CN112877320 A CN 112877320A CN 202110106493 A CN202110106493 A CN 202110106493A CN 112877320 A CN112877320 A CN 112877320A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- 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)
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- 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)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/30—Nature of the water, waste water, sewage or sludge to be treated from the textile industry
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Abstract
The invention discloses a preparation method of a composite biocatalyst system composed of copper alginate loaded laccase and a mediator ABTS. The composite biological enzyme catalyst is formed by respectively immobilizing free laccase protein molecules and mediator ABTS molecules by using natural biomass material sodium alginate as a carrier and utilizing the coordination complexation of a metal copper element. The immobilized macromolecular biological enzyme and the mediator ABTS can be recycled, the service life is long, more than 80% of degradation fraction of reactive brilliant blue KN-R and more than 75% of degradation rate of Congo red can be maintained after 5 times of repetition, the production cost is reduced, and the immobilized macromolecular biological enzyme and mediator ABTS have good industrial application prospects. The preparation method has the advantages of simple preparation process, mild reaction, easily obtained raw materials, low price and no pollution to the environment.
Description
Technical Field
The invention belongs to the technical field of enzyme catalysts, and particularly relates to a preparation method of a copper alginate loaded laccase and mediator ABTS composite biocatalyst.
Background
The laccase is an environment-friendly multi-copper oxidase, has the advantages of strong catalytic capability, high efficiency, wide range of acting substrates and the like, and is applied to the processes of printing and dyeing wastewater decolorization, paper pulp bleaching, sewage detoxification, bioremediation and the like. In the prior art, the preparation and recycling process of the composite catalyst with the biological enzyme-mediator ABTS (2, 2' -dinitrobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) is complex, the catalytic activity is low, the residual ABTS has certain environmental toxicity and is not beneficial to the green development of the surrounding environment, and the prepared biological enzyme-mediator catalyst cannot be recycled for multiple times and can not effectively catalyze substrates. However, the ABTS mediator is in a free state in the treatment process of the organic pollutants in the printing and dyeing wastewater, so the ABTS mediator needs to be added again in each repeated use process, the ABTS mediator cannot be recycled, and the production cost is increased.
Compared with free enzyme, the immobilized laccase improves the performances of heat stability, chemical stability, reusability, easy separation and the like of biological enzyme, and is widely applied to the fields of printing and dyeing wastewater decolorization, paper pulp bleaching, sewage detoxification, biological repair and the like. The chemical property and the cyclic utilization characteristic of the mediator substance ABTS play a key role in the catalytic activity and the application and popularization of the immobilized laccase. Since the mediator ABTS is a key intermediate substance for enhancing the catalytic activity of laccase, the selection of a proper material and method for immobilizing the mediator ABTS becomes a key problem.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a preparation method of a copper alginate loaded laccase and mediator ABTS composite biocatalyst. According to the invention, an immobilization technology of embedding and crosslinking is adopted, through the coordination crosslinking action of copper ions, after natural biomass alginic acid molecules are subjected to coordination bonding, biological enzymes are crosslinked with each other to form the immobilized biological enzyme catalyst, the anthraquinone dye active blue KN-R catalytic degradation decolorization reaction shows excellent catalytic activity in the printing and dyeing industry, and after macromolecular biological enzymes are immobilized, the immobilized biological enzyme catalyst can be recycled, has long service life and reduces the industrial application cost.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a preparation method of a copper alginate loaded laccase and mediator ABTS composite biocatalyst, which comprises the following steps:
s1, dissolving sodium alginate powder in deionized water to prepare a sodium alginate aqueous solution with the mass fraction of 1.5-3 wt%, and then carrying out ultrasonic treatment;
s2, dissolving anhydrous copper sulfate in deionized water to prepare a copper sulfate solution with the concentration of 0.1-0.2 mol/L;
s3, adding a dielectric substance ABTS into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution A, and then carrying out ultrasonic treatment;
s4, adding laccase into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution B, and then carrying out ultrasonic treatment;
s5, dropwise adding the solution A obtained in the step S3 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain immobilized mediator ABTS colloidal beads;
s6, dropwise adding the solution B obtained in the step S4 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain the immobilized laccase colloidal beads.
As a preferable technical scheme of the invention, the ultrasonic treatment in the step S1 is carried out at the temperature of 40-50 ℃ and the frequency of 100Hz for 10-20 min.
As a preferred technical scheme of the invention, the concentration of the mediator substance ABTS in the step S3 is 0.1mmol/L-5mmol/L, and the concentration of the laccase in the step S4 is 2.5g/L-10 g/L.
As a preferable technical scheme of the invention, the ultrasonic treatment in the step S3 and the step S4 is carried out at the temperature of 25 ℃ and the frequency of 50Hz for 10-20min, and the rubber tube is taken out for standby after the ultrasonic treatment is finished, and the gas in the rubber tube rises uniformly to form a solution which is mixed uniformly.
As a preferable technical scheme of the invention, the diameter of the composite catalyst is 1-3mm, and the sodium alginate loads laccase activity of 2.5-10 g/L.
Compared with the prior art, the invention has the following beneficial effects:
the prepared copper alginate-loaded laccase-mediator ABTS composite biological catalyst is a spherical composite biological enzyme catalyst formed by respectively using free laccase and mediator ABTS through the coordination complexation of copper ions by using a sodium alginate green biomass material as a template.
The prepared copper alginate-loaded laccase-mediator ABTS composite biocatalyst is prepared by complexing biomass copper alginate with enzyme molecules and ABTS molecules respectively, then carrying out biomass material loading catalysis, and shows excellent catalytic activity in environmental management of printing and dyeing wastewater degradation-resistant organic pollution treatment. After the macromolecular biological enzyme and the mediator ABTS are immobilized, the immobilized biological enzyme can be recycled, the service life is long, more than 80% of active brilliant blue KN-R degradation fraction and more than 75% of Congo red degradation rate can be maintained after 5 times of repetition, the production cost is reduced, and the immobilized biological enzyme has a good industrial application prospect.
The preparation method has the advantages of simple preparation process, mild reaction, easily obtained raw materials, low price and no pollution to the environment.
Drawings
FIG. 1 is a first schematic diagram of the decolorization rate of active brilliant blue by the cyclic reuse of a non-degradable dye in printing and dyeing wastewater by using a copper alginate-loaded laccase and a mediator ABTS composite biocatalyst prepared by the invention;
FIG. 2 is a schematic diagram II of the decolorization rate of active brilliant blue by the recycling of the refractory dye in printing and dyeing wastewater of the copper alginate-loaded laccase and mediator ABTS composite biocatalyst prepared by the invention;
FIG. 3 is a first SEM picture of the composite biocatalyst of copper alginate supported laccase and mediator ABTS prepared by the present invention;
FIG. 4 is a second SEM picture of the composite biocatalyst loaded with laccase and mediator ABTS from copper alginate prepared by the present invention;
FIG. 5 is a SEM picture III of the composite biocatalyst loaded with laccase and mediator ABTS from copper alginate prepared by the present invention;
FIG. 6 is a fourth SEM picture of the composite biocatalyst of copper alginate supported laccase and mediator ABTS prepared by the present invention;
FIG. 7 is a schematic illustration of an ABTS immobilization test for mediator substances;
FIG. 8 is a schematic diagram of the immobilization test of biological enzyme laccase.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
The invention provides a preparation method of a copper alginate loaded laccase and mediator ABTS composite biocatalyst, which comprises the following steps:
s1, dissolving sodium alginate powder in deionized water to prepare a sodium alginate aqueous solution with the mass fraction of 1.5-3 wt%, and then carrying out ultrasonic treatment; wherein the ultrasonic treatment is carried out at 40-50 deg.C and 100Hz for 10-20 min;
s2, dissolving anhydrous copper sulfate in deionized water to prepare a copper sulfate solution with the concentration of 0.1-0.2 mol/L;
s3, adding a mediator substance ABTS with the concentration of 0.1mmol/L-5mmol/L into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution A, and then carrying out ultrasonic treatment;
s4, adding laccase with the concentration of 2.5g/L-10g/L into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution B, and then carrying out ultrasonic treatment; performing ultrasonic treatment at 25 deg.C and 50Hz for 10-20min, taking out, and mixing with the gas in the rubber tube;
s5, dropwise adding the solution A obtained in the step S3 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain immobilized mediator ABTS colloidal beads; performing ultrasonic treatment at 25 deg.C and 50Hz for 10-20min, taking out, and mixing with the gas in the rubber tube;
s6, dropwise adding the solution B obtained in the step S4 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain the immobilized laccase colloidal beads.
Example 1
(1) Preparation of copper alginate carrier immobilized mediator substance ABTS
2.5g of sodium alginate powder was added to 100mL of deionized water and stirred vigorously at room temperature for about 30min to dissolve completely. Then, under the condition of the frequency of 100Hz, the ultrasonic treatment is carried out for about 10min at room temperature, and the sodium alginate water solution with the mass fraction of 2.5 wt% is prepared. Adding mediator ABTS (5mmol/L) into the solution, and performing normal temperature ultrasonic treatment at 50Hz for about 10min to obtain a uniform solution. The solution is added dropwise into 100mL of copper sulfate solution (0.1mol/L) and stirred until the reaction is finished, and the immobilized mediator ABTS colloidal beads are prepared.
(2) Preparation of copper alginate carrier immobilized laccase
2.5g of sodium alginate powder is added to 100mL of deionized water and stirred vigorously at room temperature for 30min until completely dissolved. Then, under the condition of the frequency of 100Hz, carrying out ultrasonic treatment at normal temperature for about 10min to prepare a sodium alginate aqueous solution with the mass fraction of 2.5%. Adding laccase (10g/L) into the solution, and performing normal temperature ultrasonic treatment at 50Hz for about 10min to obtain a uniform solution. And dropwise adding the solution into 100mL of copper sulfate solution (0.1mol/L), and simultaneously stirring until the reaction is finished to obtain the immobilized laccase glue beads.
Example 2
Enzyme activity determination of immobilized laccase
Mixing buffer solution and ABTS, taking 5g copper alginate carrier immobilized laccase, adding 10mL ABTS (0.1 mmol. L-1) and 40mL acetic acid-sodium acetate buffer solution (pH is 4.6), reacting for 20min, stopping ice bath, taking supernatant, filtering, and measuring absorbance at 420 nm.
The enzyme activity calculation formula of the immobilized laccase is as follows:
U/mL＝((ΔAV/εDt)/V0)×105。
ΔA＝1.43U＝U/L＝99.39μmol/mg/min99.39/422.23＝23.54％。
example 3
And (3) testing the performance of the catalyst:
the biological enzyme-immobilized ABTS composite catalyst obtained in the examples 1 and 2 is used for treating dye of organic matters difficult to degrade in printing and dyeing wastewater, and the specific using steps are as follows:
a dye catalytic degradation reaction experiment was carried out by adding 5g of immobilized mediator ABTS (0.1mmol/L) and 5g of immobilized laccase (10g/L) to 100mL of reactive brilliant blue KN-R (75mg/L) at a solution pH of about 6.5 to 7, and reacting at 35 to 45 ℃ for 1 hour with stirring. After the reaction is finished, filtering and washing with distilled water for three times to obtain the circularly regenerated composite catalyst, and then putting the composite catalyst into a second cycle of cyclic degradation reaction under the same conditions. The same procedure, 5 experiments were recycled. And detecting by using a Pujingyu ultraviolet-visible spectrophotometer TU-1901.
All activity data are subjected to more than three repeated experiments, and the error range is within 5%.
FIG. 1-FIG. 2 are schematic diagrams of the decolorization rate of the non-degradable dye in printing and dyeing wastewater of the composite biocatalyst loaded with copper alginate laccase and mediator ABTS, which is greater than or equal to 96%, on active brilliant blue, and the decolorization rate is recycled.
FIGS. 3-6 are SEM pictures of the composite biocatalyst of copper alginate supported laccase and mediator ABTS prepared by the present invention, and the obtained samples are shown in the shape of core-shell structure (20-200 μm).
The invention utilizes an Electron Probe Microanalyzer (EPMA) to respectively carry out material component test and structure characterization on the copper alginate loaded laccase-mediator ABTS composite biocatalyst, and supports the feasibility of the preparation method. FIG. 7 is a schematic diagram of the immobilization test of mediator substance ABTS (shown as the characteristic peak of element S in the figure). FIG. 8 is a schematic diagram of the immobilization test of biological enzyme laccase (shown as the characteristic peaks of elements N and O in the figure).
The biological enzyme-chemical composite catalyst prepared by the invention is used for the catalytic degradation reaction of anthraquinone dye active brilliant blue KN-R, shows excellent catalytic performance, has long catalytic life, and can be repeatedly used for more than 5 times and still keep higher dye decolorization rate. The immobilization and the recycling of the enzyme further reduce the production cost. The composite catalyst prepared by the invention has the environment-friendly characteristic of green natural biomass, and also has the advantages of immobilizing macromolecular enzyme for recycling, thereby showing good industrial application prospect.
The catalyst prepared by the invention is mainly synthesized into the biological enzyme catalyst by an embedding method, the biological enzyme catalyst is of a core-shell structure, the shell layer is copper alginate, and the inner core is of a cavity structure embedded with biological enzyme and a mediator. According to the invention, an immobilization technology of embedding and crosslinking is adopted, through the coordination crosslinking action of copper ions, after natural biomass alginic acid molecules are subjected to coordination bonding, biological enzymes are crosslinked with each other to form the immobilized biological enzyme catalyst, the anthraquinone dye active blue KN-R catalytic degradation decolorization reaction shows excellent catalytic activity in the printing and dyeing industry, and after macromolecular biological enzymes are immobilized, the immobilized biological enzyme catalyst can be recycled, has long service life and reduces the industrial application cost.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A preparation method of a copper alginate loaded laccase and mediator ABTS composite biocatalyst is characterized by comprising the following steps:
s1, dissolving sodium alginate powder in deionized water to prepare a sodium alginate aqueous solution with the mass fraction of 1.5-3 wt%, and then carrying out ultrasonic treatment;
s2, dissolving anhydrous copper sulfate in deionized water to prepare a copper sulfate solution with the concentration of 0.1-0.2 mol/L;
s3, adding a dielectric substance ABTS into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution A, and then carrying out ultrasonic treatment;
s4, adding laccase into the sodium alginate aqueous solution obtained in the step S1 to obtain a solution B, and then carrying out ultrasonic treatment;
s5, dropwise adding the solution A obtained in the step S3 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain immobilized mediator ABTS colloidal beads;
s6, dropwise adding the solution B obtained in the step S4 into the copper sulfate solution obtained in the step S2, and stirring until the reaction is finished to obtain the immobilized laccase colloidal beads.
2. The method for preparing the copper alginate-supported laccase and mediator ABTS composite biocatalyst as claimed in claim 1, wherein the ultrasonic treatment in step S1 is performed at 40-50 deg.C and 100Hz for 10-20 min.
3. The method of claim 1, wherein the concentration of mediator ABTS in step S3 is 0.1mmol/L-5mmol/L, and the concentration of laccase in step S4 is 2.5g/L-10 g/L.
4. The method for preparing the copper alginate-loaded laccase/mediator ABTS composite biocatalyst of claim 1, wherein the ultrasonic treatment in step S3 and step S4 is performed at 25 ℃ and 50Hz for 10-20min, and the mixture is taken out for use after the ultrasonic treatment is completed, and the gas in the rubber tube rises uniformly to form a solution and is mixed uniformly.
5. The preparation method of the copper alginate-loaded laccase/mediator ABTS composite biocatalyst of claim 1, wherein the diameter of the composite catalyst is 1-3mm, and the sodium alginate-loaded laccase activity is 2.5g/L-10 g/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943725A (en) * | 2021-10-18 | 2022-01-18 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1468968A1 (en) * | 2003-04-18 | 2004-10-20 | Technische Universitat, Institut fur Mikrobiologie und Abfalltechnologie | Biocatalyst containing a laccase |
| CN105836896A (en) * | 2015-01-16 | 2016-08-10 | 上海交通大学 | Method for treatment of water pollutants by protein mediator based immobilized laccase |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1468968A1 (en) * | 2003-04-18 | 2004-10-20 | Technische Universitat, Institut fur Mikrobiologie und Abfalltechnologie | Biocatalyst containing a laccase |
| CN105836896A (en) * | 2015-01-16 | 2016-08-10 | 上海交通大学 | Method for treatment of water pollutants by protein mediator based immobilized laccase |
Non-Patent Citations (4)
| Title |
|---|
| JUAN HUANG等: "Immobilization of a Laccase/2,2’-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic Acid System to Layered Double Hydroxide/Alginate Biohybrid Beads for Biodegradation of Malachite Green Dye", 《BIOMED RESEARCH INTERNATIONAL》 * |
| SONICA SONDHI等: "Immobilization of laccase-ABTS system for the development of a continuous flflow packed bed bioreactor for decolorization of textile efflfluent", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| YOUXUN LIU等: "Stable ABTS Immobilized in the MIL-100（Fe） Metal-Organic Framework as an Effificient Mediator for Laccase-Catalyzed Decolorization", 《MOLECULES》 * |
| 李飞等: "双孢菇漆酶的固定化及其对邻苯二甲酸二甲酯降解的研究", 《生物工程》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943725A (en) * | 2021-10-18 | 2022-01-18 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
| CN113943725B (en) * | 2021-10-18 | 2023-08-11 | 嘉兴学院 | Preparation method and application of alginate gel coated copper/zinc phosphate crystal flower immobilized laccase |
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