CN114715951B - Magnetic core-shell structure porous silica carrier for laccase immobilization and preparation method and application thereof - Google Patents
Magnetic core-shell structure porous silica carrier for laccase immobilization and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a laccase immobilized magnetic core-shell structure porous silica carrier, a preparation method and application thereof, wherein the carrier is made of magnetic Fe 3 O 4 Microspheres as cores and then on their surface by sol-gel methodSurface-coated thin-layer compact SiO 2 (d‑SiO 2 ) Finally, coating a layer of porous SiO on the surface continuously by a template method 2 (p‑SiO 2 ) To obtain the magnetic porous silica (Fe) 3 O 4 @d‑SiO 2 @p‑SiO 2 ) And (3) microspheres. By porous Fe 3 O 4 @d‑SiO 2 @p‑SiO 2 As a carrier, laccase is immobilized by adsorption. In contrast to the free laccase, the immobilized laccase (Fe 3 O 4 @d‑SiO 2 @p‑SiO 2 Lac) has better stability and higher catalytic activity, can be reused, and has wide application prospect in the aspect of bioremediation of organic contaminated soil.
Description
Technical Field
The invention relates to the field of laccase immobilized carriers, in particular to a magnetic core-shell structure porous silica carrier for laccase immobilization, and a preparation method and application thereof.
Background
In recent years, with the movement and reconstruction of a large number of industrial enterprises, the remediation of organic contaminated soil has become a very important issue. Compared with the traditional physical and chemical repair technology, the microbial repair has the advantages of low cost, good effect, no harmful residue and the like. Enzyme repair is less limited by soil environmental factors than microbial repair, is insensitive to competition among microorganisms, and exhibits higher catalytic degradation activity even at lower contaminant concentrations.
The laccase is polyphenol oxidase containing copper ions, has excellent properties in the aspect of degrading various organic pollutants, and is a high-efficiency biocatalyst. However, the free laccase is unstable and difficult to recycle, and the enzyme immobilization technology uses a carrier material to bind or limit the enzyme in a certain space, so that the catalytic activity of the laccase can be reserved, the stability is enhanced, the storage and transportation are facilitated, the effect of recycling and continuously participating in the catalytic reaction for a long time is achieved, and the property of the carrier material is an important factor influencing the activity, the stability and the pollutant removal effect of the immobilized laccase.
Has very important significance for researching the enzyme immobilization carrier. In recent years, carriers for immobilized enzymes include porous glass, carbon nanotubes, biochar, cellulose, and the like. Although these supports are higher in loading and more stable after immobilization, there are also some disadvantages: (1) poor long-term storage; (2) the enzyme is easy to fall off after being fixed; (3) reuse is not possible.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the magnetic core-shell structure porous silica carrier for laccase immobilization, which has high load and strong stability after immobilization and can be reused for a long time, and the preparation method and the application thereof.
The aim of the invention can be achieved by the following technical scheme:
the inventor knows that the porous silica material has the advantages of large specific surface area, good biocompatibility, adjustable pore diameter, high mechanical strength, easy functionalization and the like, and is an ideal enzyme immobilization carrier material. In practical application, the porous silica has smaller particle size, so that recycling and reutilization become the current problem to be solved urgently. Magnetic nanoparticles, because of their particular magnetic properties, have attracted attention from the inventors. The magnetic porous silicon dioxide material with the core-shell structure has the characteristics of porous property and magnetic property, low toxicity, high stability, easy surface modification, convenient separation, low operation cost and the like, and is very suitable for serving as a carrier of immobilized enzyme. Therefore, the preparation of the carrier with the porous characteristic and the magnetic characteristic has very important significance for improving the stability and the catalytic activity of laccase, and the following scheme is further provided:
the invention adopts a hydrothermal method to synthesize magnetic Fe 3 O 4 The microsphere is used as a precursor, and then a sol-gel method and a template method are used for coating double-layer SiO on the surface of the microsphere 2 Obtaining the magnetic porous Fe with a core-shell structure 3 O 4 @d-SiO 2 @p-SiO 2 Microspheres and successfully applied to laccase immobilization. The prepared immobilized laccase has better stability and higher catalytic activity, and realizes the purpose of repeated use, and the specific scheme is as follows:
the preparation method of the magnetic core-shell structure porous silica carrier for laccase immobilization comprises the following steps:
Fe 3 O 4 @d-SiO 2 is prepared from the following steps: magnetic Fe 3 O 4 Dispersing in a mixed solvent, and carrying out ultrasonic treatment to obtain a suspension;
then adding tetraethyl orthosilicate (TEOS) into the suspension drop by drop, continuously stirring in a water bath for reaction, separating the product by a magnet after the reaction is finished, washing and drying to obtain Fe 3 O 4 @d-SiO 2 ；
Fe 3 O 4 @d-SiO 2 @p-SiO 2 Preparation of microspheres: fe is added to 3 O 4 @d-SiO 2 Dispersing in a mixed solution containing cetyltrimethylammonium bromide (CTAB), and stirring to form a uniform solution;
dripping TEOS into the uniform solution under continuous stirring, continuously stirring in a water bath for reaction, separating the product by using a magnet after the reaction is finished, and washing for multiple times to remove nonmagnetic products;
then re-dispersing the product into acetone, refluxing to remove CTAB, repeatedly extracting for 3 times, finally separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 Microsphere, namely porous silica carrier with magnetic core-shell structure for laccase immobilization.
Further, fe 3 O 4 @d-SiO 2 In the preparation process, TEOS and Fe 3 O 4 The mass ratio of (1) to (0.3) is (0.05-0.15);
the mixed solvent is ethanol, deionized water and concentrated ammonia water (28 wt%) with the volume ratio of (100-140) being (3-5).
Further, TEOS and Fe 3 O 4 The mass ratio of (2) is 0.2:0.1.
Further, fe 3 O 4 @d-SiO 2 @p-SiO 2 When the microsphere is prepared, TEOS and Fe are used for preparing 3 O 4 @d-SiO 2 The mass ratio of (1) to (0.2) is (0.3-0.5);
the mixed solution is prepared from CTAB (50-80 g), CTAB (70-90 mL), ethanol (0.5-2 mL), deionized water and concentrated ammonia water (28 wt%).
Further, TEOS and Fe 3 O 4 @d-SiO 2 The mass ratio of (2) is 0.4:0.15.
Further, the ultrasonic treatment is carried out for 20-40min; the stirring reaction time is 5-7h; the temperature of the reflux is 80-95 ℃ and the time is 40-50h.
The laccase immobilized magnetic core-shell structure porous silica carrier prepared by the method has a core-shell structure, the size is 380-400nm, and the specific surface area is not less than 270m 2 /g。
The application of the magnetic core-shell structured porous silica carrier for laccase immobilization, which is applied to laccase immobilization, comprises the following specific steps:
fe is added to 3 O 4 @d-SiO 2 @p-SiO 2 Dispersing the carrier in a citrate buffer solution containing laccase to obtain a mixed solution, and shaking and incubating in a biochemical incubator;
then washing with citrate buffer to remove unbound enzyme, and immobilizing laccase Fe 3 O 4 @d-SiO 2 @p-SiO 2 -Lac freeze-drying and then preserving at low temperature.
Further, in the mixed solution, the concentration of laccase is 0.5-2.0mg/mL, the concentration of citrate buffer solution is 0.1M, the pH is 3-10, and the incubation time is 4-12h.
Further, in the mixed solution, the concentration of laccase is 1.5mg/mL, the pH is 6, and the incubation time is 8h.
Compared with the prior art, the invention has the following advantages:
(1) The invention uses TEOS with low cost as silicon source by simple sol-gel method, and can be used for preparing Fe under room temperature condition 3 O 4 Surface coating of SiO successfully 2 The layer and the preparation method are simple. Fe obtained 3 O 4 @d-SiO 2 @p-SiO 2 Low toxicity, high stability and magnetismAnd porous nature, is very suitable as a carrier of immobilized laccase;
(2) The porous silica carrier with the magnetic core-shell structure has excellent performance in laccase immobilization. Compared with the free laccase, the immobilized laccase has wider pH and temperature application range, and the activity is still kept at 58% after being repeatedly used for 10 times, thus being expected to play an important role in bioremediation.
Drawings
FIG. 1 is an SEM image at a magnification of 1 μm of the magnetic core-shell structured porous silica support obtained in example 1;
FIG. 2 is a TEM image at 500nm magnification of the magnetic core-shell structured porous silica support obtained in example 1;
FIG. 3 is a FTIR chart of a magnetic core-shell structured porous silica support obtained in example 1;
FIG. 4 is an XRD pattern of the magnetic core-shell structured porous silica support obtained in example 1;
FIG. 5 is a graph showing the isothermal adsorption/desorption curves and pore size distribution of the porous silica carrier with a magnetic core-shell structure obtained in example 1;
FIG. 6 is a graph showing the comparison of the activities of the free laccase and the immobilized laccase obtained in example 1 at different pH values;
FIG. 7 is a graph showing the comparison of the activities of the free laccase and the immobilized laccase obtained in example 1 at different temperatures;
FIG. 8 is a graph showing the comparison of the activities of the immobilized laccase obtained in example 1 for 10 times.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection scope of the present invention is not limited to the following embodiments.
The preparation method of the magnetic silica microsphere with the core-shell structure comprises the following steps:
(1) Magnetic Fe 3 O 4 Dispersing in a mixed solution composed of ethanol, deionized water and concentrated ammonia water, and adding water to the mixed solutionThe suspension was obtained by sounding for 30 min. TEOS was then added dropwise to the suspension and stirring was continued for 6h in a 30℃water bath. After the reaction is finished, separating the product by using a magnet, washing and drying to obtain Fe 3 O 4 @d-SiO 2 . TEOS and Fe 3 O 4 The mass ratio of (1) to (0.3) g and (0.05-0.15) g.
(2) Fe is added to 3 O 4 @d-SiO 2 Dispersing in a mixed solution containing CTAB, ethanol, deionized water and concentrated ammonia water, and stirring for 30min to form a uniform solution. TEOS was added dropwise to the above solution under continuous stirring, and stirring was continued in a water bath at 30℃for 6 hours. After the reaction, the product was separated by a magnet, and the non-magnetic product was removed by washing several times. The product was then redispersed in acetone and refluxed for 48h at 90 ℃ to remove CTAB and the extraction repeated 3 times. Finally, separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 And (3) microspheres. TEOS and Fe 3 O 4 @d-SiO 2 The mass ratio of (1) is (0.3-0.5) g and (0.1-0.2) g. The carrier has a core-shell structure, a size of 380-400nm, and a specific surface area of no less than 270m 2 /g。
The application of the porous silica carrier with the magnetic core-shell structure is that the carrier is applied to the immobilization of laccase, and the specific steps are as follows:
fe is added to 3 O 4 @d-SiO 2 @p-SiO 2 The vector was dispersed in 0.1M citrate buffer containing laccase and incubated in a biochemical incubator with shaking at 200rpm for a period of time to reach adsorption equilibrium. And washing twice with citrate buffer to remove any unbound enzyme, immobilized laccase Fe 3 O 4 @d-SiO 2 @p-SiO 2 Lac is freeze-dried and stored at 4℃until use. The laccase concentration in the mixed solution is 0.5-2.0mg/mL, the pH of the citrate buffer solution is 3-10, and the incubation time is 4-12h.
ABTS was chosen as substrate for laccase activity assay. Free laccase or immobilized laccase was added to citrate buffer containing 1mM ABTS final concentration to make the total volume 1mL and mixed well. The absorbance of the mixed solution at the wavelength of 420nm is measured by an ultraviolet spectrophotometer, the absorbance is measured continuously for 10min, and the absorbance change value is recorded.
Example 1
The preparation method of the porous silica carrier with the magnetic core-shell structure comprises the following steps:
(1) 0.1g of magnetic Fe 3 O 4 Dispersing in a mixed solution composed of 120mL of ethanol, 40mL of deionized water and 4mL of concentrated ammonia water, and carrying out ultrasonic treatment for 30min to obtain a suspension. Then 0.2g TEOS was added dropwise to the suspension and stirred continuously in a water bath at 30℃for 6h. After the reaction is finished, separating the product by using a magnet, washing and drying to obtain Fe 3 O 4 @d-SiO 2 。
(2) 0.15g of Fe 3 O 4 @d-SiO 2 Dispersed in a mixed solution containing 0.3g CTAB,60mL ethanol, 80mL deionized water and 1mL concentrated ammonia, and stirred for 30min to form a homogeneous solution. 0.4g TEOS was added dropwise to the above solution under continuous stirring, and stirring was continued in a water bath at 30℃for 6 hours. After the reaction, the product was separated by a magnet, and the non-magnetic product was removed by washing several times. The product was then redispersed in acetone and refluxed for 48h at 90 ℃ to remove CTAB and the extraction repeated 3 times. Finally, separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 And (3) microspheres.
As can be seen from FIG. 1, the magnetic porous microsphere has good dispersibility and relatively uniform size, about 380nm. As can be seen from fig. 2, the carrier has a core-shell structure. As can be seen from FIG. 3, at 1090cm -1 An antisymmetric stretching vibration peak of Si-O-Si appears near the position, which indicates the successful coating of silicon dioxide. As can be seen from fig. 4, the spectral lines at 2θ=30.1°,35.4 °,43.1 °,57.0 °,62.6 ° all show obvious diffraction peaks, which can be attributed to Fe 3 O 4 The (220), (311), (400) (511), (440) crystal planes of (C) indicate Fe throughout the preparation process 3 O 4 The magnetic property of the material is ensured by the perfect reservation. A weak broad peak appears near 2θ=22° as amorphous SiO 2 (101) characteristic diffraction peaks indicative of SiO 2 Has been successfully coated with Fe 3 O 4 A surface. As can be seen in fig. 5, the support has a porous structure,calculated to have a specific surface area of 275.17m 2 /g。
The immobilization of laccase by the carrier comprises the following schemes:
the magnetic core-shell structured porous silica support obtained in example 1 was dispersed in 0.1M citrate buffer at pH 6 containing 1.5mg/mL laccase and incubated in a biochemical incubator with shaking at 200rpm for 8h to reach adsorption equilibrium. And washing twice with citrate buffer to remove any unbound enzyme, immobilized laccase Fe 3 O 4 @d-SiO 2 @p-SiO 2 Lac is freeze-dried and stored at 4℃until use.
Dispersing the immobilized laccase in 0.1M citrate buffer solution with pH of 3-10, preserving at 25deg.C for 12 hr, measuring activity, and repeating the above steps. As shown in FIG. 6, the activities of both the free laccase and the immobilized laccase increased and decreased with increasing pH, and the immobilized laccase activity was maximized at pH 5. In addition, the immobilized laccase has higher enzyme activity than the free laccase, and has wider pH application range, and particularly shows more obvious advantages under acidic conditions.
Dispersing the immobilized laccase in 0.1M citrate buffer solution with pH of 5, respectively maintaining at 20, 25, 30, 40, 50 and 60 ℃ for 30min, measuring the activity, and repeating the above operation with equal amount of free laccase for comparison. As shown in FIG. 7, the immobilized laccase and the free laccase show higher activity at 25 ℃ and 30 ℃ respectively, and the activity is reduced when the temperature is higher than 30 ℃, but the activity of the immobilized laccase is slightly higher than that of the free laccase, which indicates that the immobilized laccase has better heat resistance.
Dispersing the obtained immobilized laccase in a citrate buffer solution with the pH value of 5 and carrying out catalytic reaction for 10 times continuously by taking ABTS as a substrate. As shown in FIG. 8, the immobilized laccase activity remains 58% after 10 times of repeated use, and thus the operation stability of the immobilized laccase is high, although the core magnetic Fe 3 O 4 Is extremely unstable and is easily oxidized and corroded, but the shell layer d-SiO 2 @p-SiO 2 Not only improves the stability of the carrier materialThe specific surface area is also provided for adsorbing laccase and maintaining the conformation of laccase, so that the immobilized laccase maintains high stability in the repeated use process.
Example 2
The preparation method of the porous silica carrier with the magnetic core-shell structure comprises the following steps:
(1) 0.05g of magnetic Fe 3 O 4 Dispersing in a mixed solution composed of 120mL of ethanol, 40mL of deionized water and 4mL of concentrated ammonia water, and carrying out ultrasonic treatment for 30min to obtain a suspension. Then 0.1g TEOS was added dropwise to the suspension and stirred continuously in a water bath at 30℃for 6h. After the reaction is finished, separating the product by using a magnet, washing and drying to obtain Fe 3 O 4 @d-SiO 2 。
(2) 0.1g of Fe 3 O 4 @d-SiO 2 Dispersed in a mixed solution containing 0.3g CTAB,60mL ethanol, 80mL deionized water and 1mL concentrated ammonia, and stirred for 30min to form a homogeneous solution. 0.3g TEOS was added dropwise to the above solution under continuous stirring, and stirring was continued in a water bath at 30℃for 6 hours. After the reaction, the product was separated by a magnet, and the non-magnetic product was removed by washing several times. The product was then redispersed in acetone and refluxed for 48h at 90 ℃ to remove CTAB and the extraction repeated 3 times. Finally, separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 And (3) microspheres.
The magnetic porous microsphere has a core-shell structure and the size is about 380nm.
Example 3
The preparation method of the porous silica carrier with the magnetic core-shell structure comprises the following steps:
(1) 0.15g of magnetic Fe 3 O 4 Dispersing in a mixed solution composed of 120mL of ethanol, 40mL of deionized water and 4mL of concentrated ammonia water, and carrying out ultrasonic treatment for 30min to obtain a suspension. Then 0.3g TEOS was added dropwise to the suspension and stirred continuously in a water bath at 30℃for 6h. After the reaction is finished, separating the product by using a magnet, washing and drying to obtain Fe 3 O 4 @d-SiO 2 。
(2) 0.2g of Fe 3 O 4 @d-SiO 2 Dispersed in a mixed solution containing 0.3g CTAB,60mL ethanol, 80mL deionized water and 1mL concentrated ammonia, and stirred for 30min to form a homogeneous solution. 0.5g TEOS was added dropwise to the above solution under continuous stirring, and stirring was continued in a water bath at 30℃for 6 hours. After the reaction, the product was separated by a magnet, and the non-magnetic product was removed by washing several times. The product was then redispersed in acetone and refluxed for 48h at 90 ℃ to remove CTAB and the extraction repeated 3 times. Finally, separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 And (3) microspheres.
The magnetic porous microsphere has a core-shell structure and the size is about 400nm.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. The application of the magnetic core-shell structure porous silica carrier for laccase immobilization is characterized in that the carrier is applied to laccase immobilization, and the preparation method of the carrier comprises the following steps:
Fe 3 O 4 @d-SiO 2 is prepared from the following steps: magnetic Fe 3 O 4 Dispersing in a mixed solvent, and carrying out ultrasonic treatment to obtain a suspension; TEOS and Fe 3 O 4 The mass ratio of (1) to (0.3) is (0.05-0.15);
then adding tetraethyl orthosilicate (TEOS) into the suspension drop by drop, continuously stirring in a water bath for reaction, separating the product by a magnet after the reaction is finished, washing and drying to obtain Fe 3 O 4 @d-SiO 2 ；
Fe 3 O 4 @d-SiO 2 @p-SiO 2 Preparation of microspheres: fe is added to 3 O 4 @d-SiO 2 Dispersing in a mixed solution containing cetyltrimethylammonium bromide (CTAB), and stirring to form a uniform solution; TEOS and Fe 3 O 4 @d-SiO 2 The mass ratio of (1) to (0.2) is (0.3-0.5);
dripping TEOS into the uniform solution under continuous stirring, continuously stirring in a water bath for reaction, separating the product by using a magnet after the reaction is finished, and washing for multiple times to remove nonmagnetic products;
then re-dispersing the product into acetone, refluxing to remove CTAB, repeatedly extracting for 3 times, finally separating the product by using a magnet, washing and drying to obtain the magnetic porous Fe 3 O 4 @d-SiO 2 @p-SiO 2 Microsphere, namely porous silica carrier with magnetic core-shell structure for laccase immobilization.
2. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 1, wherein Fe 3 O 4 @d-SiO 2 The mixed solvent is ethanol, deionized water and concentrated ammonia water with the volume ratio of (100-140) being 40 (3-5).
3. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 2, wherein the porous silica carrier is characterized in that 3 O 4 The mass ratio of (2) is 0.2:0.1.
4. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 1, wherein Fe 3 O 4 @d-SiO 2 @p-SiO 2 When the microsphere is prepared, the mixed solution is prepared from CTAB, ethanol, deionized water and concentrated ammonia water with the dosage ratio of 0.3g (50-80) mL (70-90) mL (0.5-2) mL.
5. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 4, wherein TEOS and Fe are as follows 3 O 4 @d-SiO 2 Mass ratio of (2)0.4:0.15.
6. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 1, wherein the time of the ultrasound is 20-40min; the stirring reaction time is 5-7h; the temperature of the reflux is 80-95 ℃ and the time is 40-50h.
7. The use of a porous silica carrier with a magnetic core-shell structure for laccase immobilization according to claim 1, wherein the carrier has a core-shell structure, the size is 380-400nm, and the specific surface area is not less than 270m 2 /g。
8. The application of the magnetic core-shell structured porous silica carrier for laccase immobilization according to claim 1, wherein the specific steps of the application comprise:
fe is added to 3 O 4 @d-SiO 2 @p-SiO 2 Dispersing the carrier in a citrate buffer solution containing laccase to obtain a mixed solution, and shaking and incubating in a biochemical incubator;
then washing with citrate buffer to remove unbound enzyme, and immobilizing laccase Fe 3 O 4 @d-SiO 2 @p-SiO 2 -Lac freeze-drying and then preserving at low temperature.
9. The application of the magnetic core-shell structured porous silica carrier for laccase immobilization according to claim 8, wherein the concentration of laccase in the mixed solution is 0.5-2.0mg/mL, the concentration of citrate buffer is 0.1M, pH is 3-10, and incubation time is 4-12h.
10. The application of the magnetic core-shell structured porous silica carrier for laccase immobilization according to claim 9, wherein the concentration of laccase in the mixed solution is 1.5mg/mL, the pH is 6, and the incubation time is 8h.
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