CN111057699B - Laccase-catalyzed preparation method for forming hydrogel in situ by utilizing ATRP - Google Patents
Laccase-catalyzed preparation method for forming hydrogel in situ by utilizing ATRP Download PDFInfo
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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Abstract
The invention discloses a laccase-catalyzed preparation method for forming hydrogel in situ by utilizing ATRP. According to the method, bromoisobutyric acid ethyl ester is used as an initiator, polyethylene glycol methyl ether acrylate is used as a monomer, poly (ethylene glycol) diacrylate is used as a cross-linking agent, and the hydrogel loaded with the protein is successfully synthesized in the presence of laccase, sodium ascorbate and a ligand. The preparation method disclosed by the invention has mild reaction conditions, the in-situ formed hydrogel is simple and quick to prepare, the immobilization of laccase is successfully realized, the laccase activity is effectively maintained, and the laccase can be repeatedly used without obvious reduction of the enzyme activity. The enzyme-loaded hydrogel has potential application prospect in the aspects of protein transmission and water treatment.
Description
Technical Field
The invention belongs to the fields of hydrogel and atom transfer radical polymerization, and relates to a laccase-catalyzed preparation method for forming hydrogel in situ by utilizing ATRP.
Background
Hydrogels are chemically or physically crosslinked networks of hydrophilic polymers having a porous three-dimensional structure. The most notable features of hydrogels are the similarity to natural extracellular matrices, including high water content, good biocompatibility, and controlled permeability, which makes hydrogels an ideal choice for loading and release of water-soluble biological macromolecules. Protein and polypeptide drugs have been loaded into numerous hydrogel or microgel delivery systems for sustained release testing. The hydrogel matrix may limit the migration of proteins during physical intercalation into the porous network, which is advantageous to preserve the fragile structure and natural activity of the protein drug. Protein release can also be well regulated by altering the crosslink density of the hydrogel or tailoring reversible protein-polymer interactions. All of these unique properties have led to the widespread use of hydrogels in protein storage systems, tissue engineering and diagnostic devices.
Atom Transfer Radical Polymerization (ATRP) has evolved to the most successful radical polymerization technique since more than twenty years ago. The biological macromolecules are used as catalysts, and are called ATRPase because of the catalytic activity of the enzyme under ATRP conditions. The biocatalytic ATRP avoids the pollution of metal to polymer products by virtue of the advantages of no toxicity and biodegradability, and is widely paid attention to people, and ATRPas are considered as ideal green catalysts. Laccase is a copper-containing oxidase which can catalyze a plurality of compounds to undergo oxidation reaction, and uses molecular oxygen as an electron acceptor to reduce the compounds into water, and is an environment-friendly enzyme. However, laccase is denatured and inactivated during use due to the change of environmental conditions, and especially, free enzyme is mixed with reaction products, so that recycling is difficult to realize, and the like, so that the industrial application of laccase preparations is limited. When laccase forms hydrogel, it has the advantage of (1) being convenient to separate from substrate and product; (2) improved laccase stability; (3) continuous reaction can be realized; and (4) recycling, so that the production cost is reduced.
Disclosure of Invention
The invention aims to disclose a laccase-catalyzed preparation method for forming hydrogel in situ by utilizing ATRP.
The technical scheme for realizing the invention is as follows:
the preparation method of the in-situ formed hydrogel by utilizing the ATRP under the catalysis of laccase takes bromoisobutyric acid ethyl ester as an initiator, polyethylene glycol methyl ether acrylate as a monomer, and poly (ethylene glycol) diacrylate as a cross-linking agent, and the hydrogel loaded with the protein is successfully synthesized by a one-pot method in the presence of laccase, sodium ascorbate and a ligand. The method has mild reaction conditions, the hydrogel formed in situ by the one-pot method is simple and quick to prepare, the immobilization of laccase is successfully realized, the laccase activity is effectively maintained, and the method can be repeatedly used for many times without obvious reduction of the enzyme activity.
Step 1, adding bromoisobutyric acid ethyl ester, tris [2- (dimethylamino) ethyl ] amine ligand, polyethylene glycol methyl ether acrylate, poly (ethylene glycol) diacrylate and water into a container I;
step 2, adding laccase, sodium ascorbate and water into a second container;
step 3, degassing the first container and the second container in the steps 1 and 2, and transferring the mixed liquid in the second container into the first container; the resulting hydrogel was stirred at 25℃for 6 hours, and the resulting hydrogel was thoroughly washed with distilled water.
Further, in step 1, ethyl bromoisobutyrate is used as an initiator in the polymerization system; tris [2- (dimethylamino) ethyl ] amine as a ligand in the polymerization system; polyethylene glycol methyl ether acrylate is used as a monomer in a polymerization system; poly (ethylene glycol) diacrylate as a polymeric crosslinker, with an average molecular weight of 400 or 1000; the water used is ultrapure water.
Further, in step 2, laccase is derived from Coriolus versicolor; sodium ascorbate acts as a reducing agent in the polymerization system. The mol ratio of bromoisobutyric acid ethyl ester, polyethylene glycol methyl ether acrylate, laccase, sodium ascorbate and tris [2- (dimethylamino) ethyl ] amine is 1:35:0.01:8:1.5.
Further, in the step 3, the nitrogen protection flow rate is 20mL/min, and the time is 15min.
Further, in the step 3, the reaction temperature is 25 ℃, the reaction time is 6 hours, and the stirring is continuously carried out during the reaction, and the rotating speed is 2000r/min.
Compared with the prior art, the invention has the following remarkable effects:
(1) Compared with the conventional heavy metal catalyst, the laccase is used as a biocatalyst, has obvious green pollution-free property, and avoids heavy metal pollution of post-treatment;
(2) The method has mild reaction conditions, the prepared hydrogel can be prepared rapidly by a one-pot method within 6 hours, and the preparation method does not need multi-step chemical reaction, but is synthesized in one step, so that the method is simple, convenient and rapid;
(3) The method realizes the immobilization of laccase through hydrogel, and the immobilized laccase has better pH, temperature and storage stability and better reusability.
Drawings
FIG. 1 shows rhodamine b labeled laccase (A) and hydrogel in different cross-linking agents (M n ＝400Da B&D；M n ＝1000Da，C&E) Optical and SEM scanning electron microscope images formed in situ. Wherein A is rhodamine-labeled laccase; b is hydrogel formed by selecting a poly (ethylene glycol) diacrylate crosslinking agent with a relative molecular weight of 400; c is hydrogel formed by selecting a poly (ethylene glycol) diacrylate crosslinking agent with the relative molecular weight of 1000; d is the sweep of the hydrogel in BAn electron microscope image is drawn; e is a scanning electron microscope image of the hydrogel in C.
FIG. 2 shows the free laccase and the immobilized laccase (Gel 400 ) Enzyme Activity at different pH (25 ℃) and temperature (pH 3.0). Wherein A is a free and immobilized laccase at 25℃and at different pH values (Gel 400 ) Is formed by a poly (ethylene glycol) diacrylate cross-linker having a relative molecular mass of 400; b is pH 3.0 and the free and immobilized laccase (Gel 400 ) Is formed from a poly (ethylene glycol) diacrylate crosslinker having a relative molecular mass of 400.
FIG. 3 shows the absorbance (420 nm) as a function of time for continuous oxidation of ATBS using recovered laccase-loaded hydrogels.
Detailed Description
The invention is further described in detail below with reference to examples and figures.
Example 1
Selecting a poly (ethylene glycol) diacrylate crosslinking agent with a relative molecular mass of 400, and preparing the hydrogel in situ by using ATRP under the catalysis of laccase, wherein the preparation method comprises the following steps:
step 1 Ethyl bromoisobutyrate, tris [2- (dimethylamino) ethyl ]]Amine ligands, polyethylene glycol methyl ether acrylate, poly (ethylene glycol) diacrylate (M n =400 Da), water was added to container one.
Step 2 laccase, sodium ascorbate and water are added to a second vessel.
And 3, degassing the containers in the steps 1 and 2, and transferring the mixed solution in the container II in the step 2 to the container I in the step 1. Stirring for 6h at 25 ℃ to obtain the hydrogel formed in situ by utilizing ATRP under laccase catalysis. The resulting hydrogel was thoroughly washed with distilled water.
FIGS. 1B and 1D are, respectively, an optical image of a hydrogel prepared using a poly (ethylene glycol) diacrylate having a relative molecular mass of 400 as a cross-linking agent and an SEM image. As can be seen from the figure, the laccase-supporting hydrogel is well formed.
Example 2
Selecting a poly (ethylene glycol) diacrylate crosslinking agent with a relative molecular mass of 1000, and performing laccase-catalyzed ATRP (atom transfer radical polymerization), wherein the preparation method for forming the hydrogel in situ comprises the following steps:
step 1 Ethyl bromoisobutyrate, tris [2- (dimethylamino) ethyl ]]Amine ligands, polyethylene glycol methyl ether acrylate, poly (ethylene glycol) diacrylate (M n =1000 Da), water was added to container one.
Step 2 laccase, sodium ascorbate and water are added to a second vessel.
And 3, degassing the containers in the steps (1) and (2), and transferring the mixed solution in the container II in the step 2 to the container I in the step 1. Stirring for 6h at 25 ℃ to obtain the hydrogel formed in situ by utilizing ATRP under laccase catalysis. The resulting hydrogel was thoroughly washed with distilled water.
FIGS. 1C and 1E are, respectively, an optical image of a hydrogel prepared using a poly (ethylene glycol) diacrylate of relative molecular mass 1000 as a crosslinker and an SEM image. As can be seen from the figure, the laccase-supporting hydrogel is well formed.
Laccase Activity assay
The enzyme activity of laccase is determined by ultraviolet/visible spectrophotometry using ABTS as substrate, usually after a defined time, the absorbance of a defined sample is measured at 420nm, and then the activity of laccase is deduced from kinetic parameters. Laccase activity was calculated according to the following formula.
Wherein r is the mass fraction of laccase in the hydrogel, and is calculated from the yield and coating rate of laccase. Δc is the concentration unit molar concentration of the sample, ΔE/Δt represents the activity of absorbance change (ΔE) at a specific time interval (Δt). ABTS has an oxidative extinction coefficient at 420nm of 36X 10 -3 M -1 cm -1 The optical element used had a path length of 1cm. One unit is defined as producing 1 millimeter of product per minute.
Exploring the optimal values of free laccase and immobilized enzyme under different pH values and temperatures
The hydrogel is used as a carrier to fix laccase, and the test experiments on the pH of a system are divided into 6 groups, wherein the pH values of each group are 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0. The experimental temperatures are all ensured to be 25 ℃. And (3) testing the absorbance of the corresponding system with different pH values by adopting an ultraviolet-visible spectrophotometer, and calculating the enzyme activity according to the formula (1).
The hydrogel is used as a carrier to fix laccase, and the test experiments on the system temperature are divided into 5 groups, and the temperatures of each group are 30, 40, 50, 60 and 70. The experimental pH is ensured to be 3.0. And (3) testing the absorbance of the corresponding systems with different temperatures by adopting an ultraviolet-visible spectrophotometer, and calculating the enzyme activity according to the formula (1).
The effect on the apparent enzyme activity was studied at different pH (2.0-7.0) and temperature (30-70 ℃). The optimal pH and temperature of the free and immobilized laccase are pH4.0 and 50℃respectively. These results indicate that hydrogels can effectively retain laccase activity during polymerization and storage.
FIG. 2 shows the enzymatic activity of the free laccase and the immobilized laccase at different pH (25 ℃) and temperature (pH 3.0). The cross-linking agent constituting the laccase-supporting hydrogel has a relative molecular mass of 400.
Laccase-loaded hydrogel reuse
The reusability of laccase loaded hydrogels was tested by continuous oxidation of ABTS. The hydrogel was first recovered by filtration and then immersed in excess water (1L) for 60 minutes in order to release the encapsulated small molecules, which were then reused. Then the hydrogel is directly added into a new ABTS solution for enzymolysis and oxidization. As can be seen from FIG. 3, the enzyme activity did not significantly decrease after the repeated use for 6 times, which can be seen from the slope value of the absorbance curve, indicating that the enzyme activity remained unchanged during the repeated use.
Claims (7)
1. A method for preparing a laccase-catalyzed ATRP in situ formed hydrogel, the method comprising the steps of:
step 1, adding bromoisobutyric acid ethyl ester, tris [2- (dimethylamino) ethyl ] amine ligand, polyethylene glycol methyl ether acrylate, poly (ethylene glycol) diacrylate and water into a container I;
step 2, adding laccase, sodium ascorbate and water into a second container;
step 3, degassing the first container and the second container in the steps 1 and 2, and transferring the mixed liquid in the second container into the first container; the resulting hydrogel was stirred at 25℃for 6 hours, and the resulting hydrogel was thoroughly washed with distilled water.
2. The method according to claim 1, wherein in step 1, the bromoisobutyric acid ethyl ester is used as an initiator in a polymerization system; tris [2- (dimethylamino) ethyl ] amine as a ligand in the polymerization system; polyethylene glycol methyl ether acrylate is used as a monomer in a polymerization system; poly (ethylene glycol) diacrylate was used as a polymeric crosslinker, with an average molecular weight of 400 or 1000.
3. The method of claim 1, wherein the water is ultrapure water.
4. The method of claim 1, wherein in step 2, the laccase is derived from coriolus versicolor; sodium ascorbate acts as a reducing agent in the polymerization system.
5. The method according to claim 1, wherein the molar ratio of bromoisobutyric acid ethyl ester, polyethylene glycol methyl ether acrylate, laccase, sodium ascorbate and tris [2- (dimethylamino) ethyl ] amine is 1:35:0.01:8:1.5.
6. The method according to claim 1, wherein in the step 3, the nitrogen protection flow rate is 20mL/min for 15min.
7. The process according to claim 1, wherein in step 3, the reaction temperature is 25 ℃, the reaction time is 6 hours, and stirring is continued at a rotation speed of 2000r/min.
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| CN104031904A (en) * | 2014-06-24 | 2014-09-10 | 南京大学 | Hydrogel immobilized laccase-mediator enzymatic reactor |
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| Yeap-Hung Ng 等.Metalloenzymatic radical polymerization using alkyl halides as initiators.《Polymer Chemistry》.2011,第2卷第589页右栏第2段. * |
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