Laccase-loaded porous microcarriers, preparation method and application

Abstract

The present disclosure provides a porous microcarrier loaded with laccase, a preparation method and an application. The porous microcarrier loaded with laccase includes: a hydrogel porous microcarrier; laccase, which is loaded on the hydrogel porous microcarrier by covalent bonding; wherein the hydrogel porous microcarrier is a methacrylated hydrogel microcarrier filled with chitosan, providing a loading site for laccase.

Description

Technical Field

[0001] The present disclosure relates to the technical field of water pollution treatment, and particularly to a porous microcarrier loaded with laccase, a preparation method and an application thereof. Specifically, it relates to a preparation method of a porous microcarrier for improving laccase activity and its application in removing bisphenol compounds in water. Background Art

[0002] In recent years, bisphenol compounds have been detected in different water environments. The widespread presence of bisphenol compounds in the environment and their potential ecotoxicological effects have attracted people's attention. Among them, bisphenol A (BPA) compounds are produced and used in large quantities and can be detected in various environments. They are particularly abundant in aquatic systems, causing reproductive dysfunction and developmental abnormalities in humans and other organisms. BPA is a typical endocrine disruptor. Due to the health risks of bisphenol A (BPA) compounds, bisphenol A (BPA) compounds have been continuously replaced by other bisphenol compounds with similar structures, such as bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bisphenol F (BPF), bisphenol Z (BPZ), bisphenol AF (BPAF), etc. Bisphenol compounds are a series of chemical substances containing two hydroxyphenyl groups in their structures. These bisphenol compounds have an endocrine disrupting effect similar to that of BPA. However, technologies for effectively removing them have not been fully studied. Summary of the Invention

[0003] In view of the above technical problems, the present disclosure provides a porous microcarrier loaded with laccase, a preparation method and an application thereof, in order to at least partially solve the above-mentioned technical problems.

[0004] In order to solve the above technical problems, the technical solutions provided by the present disclosure are as follows:

[0005] As one aspect of the present disclosure, there is provided a porous microcarrier loaded with laccase, comprising:

[0006] A hydrogel porous microcarrier;

[0007] Laccase, which is loaded on the hydrogel porous microcarrier by covalent binding;

[0008] Wherein, the hydrogel porous microcarrier is a methacrylated hydrogel microcarrier filled with chitosan, providing a loading site for laccase.

[0009] In one embodiment, the hydrogel porous microcarrier has an adjustable pore size;

[0010] The loading amount of laccase is 0.2 - 1%;

[0011] The concentration of chitosan is 0.5 - 3%;

[0012] The composition of the methacrylated hydrogel microcarrier includes: 15-30% methacrylated gelatin, 20-50% polyethylene glycol diacrylate, and 0.2-0.5% lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

[0013] In one embodiment, the composition of the methacrylated hydrogel microcarrier may further include 5-10% iron oxide nanoparticles to provide magnetism for the porous microcarrier.

[0014] As another aspect of the present disclosure, a method for preparing the above-mentioned porous microcarrier loaded with laccase is provided, including:

[0015] Using silica nanoparticles as the dispersed phase, cutting them into single emulsion droplets through a microfluidic device, drying to obtain silica photonic crystals, and calcining to form a microsphere template of silica photonic crystals;

[0016] Soaking the microsphere template of silica photonic crystals in the precursor solution of methacrylated hydrogel, and polymerizing under ultraviolet lamp irradiation to obtain composite photonic crystal microspheres;

[0017] Etching the composite photonic crystal microspheres with hydrofluoric acid solution to obtain methacrylated porous microcarriers;

[0018] After drying the methacrylated porous microcarriers, adding a chitosan hydrogel solution to fill them to form chitosan-methacrylated porous microcarriers;

[0019] Adding a glutaraldehyde solution to the chitosan-methacrylated porous microcarriers for activation, and then adding a laccase solution for covalent binding reaction to obtain the porous microcarrier loaded with laccase.

[0020] In one embodiment, the particle size of the silica nanoparticles includes 50-1000 nm;

[0021] The mass concentration of the dispersed phase is 15-30% (w / v).

[0022] In one embodiment, the calcination temperature is 600-1000 °C;

[0023] The calcination time is 3-6 h;

[0024] The soaking time is 2-8 h.

[0025] In one embodiment, the wavelength of the ultraviolet lamp irradiation is 330-450 nm;

[0026] The intensity of the ultraviolet lamp irradiation is 50-200 W;

[0027] The time of the ultraviolet lamp irradiation is 1-5 min.

[0028] In one embodiment, the concentration of the hydrofluoric acid solution is 2-8%;

[0029] The etching treatment time is 2-8 h;

[0030] The concentration of chitosan is 0.5-3%;

[0031] The filling time is 6-12 h.

[0032] In one embodiment, the concentration of glutaraldehyde is 0.5-5%;

[0033] The activation time is 1-6 h;

[0034] The concentration of laccase is 0.5-2 mg / mL;

[0035] The time for the covalent binding reaction is 2-10 h.

[0036] As another aspect of the present disclosure, a method for degrading bisphenol compounds is provided, including: adding the above-mentioned porous microcarriers loaded with laccase into a bisphenol compound solution to degrade the bisphenol compounds;

[0037] After the degradation reaction is completed, the porous microcarriers loaded with laccase are recovered and can be reused after being washed with a buffer solution.

[0038] Based on the above technical solutions, the porous microcarriers loaded with laccase, the preparation method and the application provided by the present disclosure have at least one of the following beneficial effects:

[0039] (1) According to the embodiments of the present disclosure, the hydrogel porous microcarriers in the porous microcarriers loaded with laccase have a three-dimensional porous structure and interconnected nanochannels, and under the filling effect of the chitosan hydrogel, more binding sites are provided for the immobilization of laccase. Laccase is loaded onto the hydrogel porous microcarriers by covalent binding. The hydrogel porous microcarriers provide functional active sites and the required microenvironment for laccase molecules, stabilizing the biological activity of laccase molecules and enabling them to effectively resist the interference of the external environment.

[0040] (2) In the embodiments of the present disclosure, the porous microcarriers loaded with laccase benefit from the protection of their biocompatible hydrogel shell and the confinement effect of the nanoholes, so that the numerous interconnected and interlaced network nanopores in the microcarriers can promote the mass transfer and diffusion of substrate molecules, thereby improving the effective contact between laccase and substrate molecules and promoting the enhancement of enzyme activity.

[0041] (3) According to an embodiment of the present disclosure, after calcining the silica photonic crystal and then immersing it in a methacrylated hydrogel precursor solution, a composite photonic crystal microsphere is prepared. The silica photonic crystal template is removed by etching, and then chitosan hydrogel is added for filling to obtain a chitosan-methacrylated porous microcarrier. After activation, it is covalently bonded with laccase to obtain a porous microcarrier loaded with laccase. This preparation method has adjustable pore size. Different pore-sized porous microcarriers can be obtained by using silica photonic crystals prepared from silica particles with different particle sizes as templates, and the laccase loading amount can be adjusted accordingly.

[0042] (4) According to an embodiment of the present disclosure, the porous microcarrier loaded with laccase can effectively degrade bisphenol A compounds, and its degradation performance for other bisphenol compounds is evaluated. Further, after the porous microcarrier loaded with laccase degrades bisphenol compounds, it can be recycled and reused. The introduction of iron oxide nanoparticles provides magnetic response performance for the microcarrier, enabling the porous microcarrier loaded with laccase to have the potential for recovery and reducing the cost in practical applications to a certain extent. Description of the Drawings

[0043] Figure 1 It is a step diagram for preparing a porous microcarrier loaded with laccase in an embodiment of the present disclosure;

[0044] Figure 2 It is a scanning electron microscope (SEM) image of microsphere templates of silica photonic crystals with different specifications in Example 1 of the present disclosure;

[0045] Figure 3 It is a scanning electron microscope (SEM) image of different methacrylated porous microcarriers in Example 2 of the present disclosure;

[0046] Figure 4 It is a laser confocal image of a porous microcarrier loaded with laccase in Example 4 of the present disclosure;

[0047] Figure 5 It is a removal efficiency diagram of a porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solutions with different concentrations in Example 5 of the present disclosure;

[0048] Figure 6 It is an efficiency diagram of a porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solutions at different pH values in Example 6 of the present disclosure;

[0049] Figure 7 It is an efficiency diagram of a porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solutions at different temperatures in Example 7 of the present disclosure;

[0050] Figure 8Removal efficiency diagram of laccase-loaded porous microcarriers and an equal amount of free enzyme for degrading different bisphenol compounds in Example 8 of the present disclosure;

[0051] Figure 9 Laccase activity test diagram stored at different temperatures in Example 9 of the present disclosure;

[0052] Figure 10 Relative activity change diagram after repeated use of laccase-loaded porous microcarriers in Example 10 of the present disclosure. Detailed implementation manners

[0053] To make the objectives, technical solutions and advantages of the present disclosure clearer and more understandable, the present disclosure will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0054] Currently, the methods for removing BPA from wastewater mainly include physical methods, chemical methods and biological methods. The physical and chemical treatment methods in the related technologies face challenges in sustainable wide application due to high energy consumption and costs. Therefore, there is an urgent need to develop micro-pollutant treatment technologies that are economical, effective and environmentally sustainable.

[0055] In the process of implementing the present disclosure, it is found that laccase is a copper-containing polyphenol oxidase with strong substrate specificity, which can catalyze a variety of phenols, anilines and aromatic compounds, and has been widely used in many fields such as industrial wastewater treatment, dye decolorization, textile printing and dyeing, analysis and detection, environmental pollutant degradation, and environmental remediation. However, the stability of free laccase is relatively poor. Compared with free laccase, immobilized enzymes enhance their tolerance to environmental conditions and thermal stability, and can be recycled, greatly broadening the scope of their practical applications. Many immobilized laccase materials in the related technologies have shown remarkable performance in degrading BPA, but it is not clear whether they can also effectively degrade other bisphenol compounds. Since bisphenol compounds have different chemical structures, which will affect the degradation efficiency and coexist in water, it is necessary to develop new carriers to immobilize laccase and evaluate its degradation performance for bisphenol compounds.

[0056] In view of the technical problems existing in the related art, the present disclosure provides a porous microcarrier loaded with laccase, a preparation method and an application thereof. The method comprises the following steps: calcining a silica photonic crystal to form a template, immersing the template in a methacrylated hydrogel precursor solution, preparing a composite photonic crystal microsphere, removing the template by etching, and then filling the microsphere with chitosan hydrogel to obtain a chitosan-methacrylated porous microcarrier. After activation, the microcarrier is covalently bound to the laccase to obtain a laccase-loaded porous microcarrier. The three-dimensional porous structure and interconnected nanochannels of the porous microcarrier, as well as the filling effect of the chitosan hydrogel, provide a large number of binding sites for the fixation of the laccase, provide a shell protection for the laccase, and stabilize the biological activity of the laccase. The obtained laccase-loaded porous microcarrier can be recycled and reused after degradation reaction, and has a high application development prospect.

[0057] Specifically, as one aspect of the present disclosure, the present disclosure provides a porous microcarrier loaded with laccase, comprising:

[0058] hydrogel porous microcarriers;

[0059] Laccase, loaded on hydrogel porous microcarriers by covalent binding;

[0060] The hydrogel porous microcarrier is a methacrylated hydrogel microcarrier filled with chitosan, which provides loading sites for laccase.

[0061] According to the embodiments of the present disclosure, the hydrogel porous microcarrier has a three-dimensional nanoporous structure and is composed of a methacrylated hydrogel microcarrier and chitosan filled into its microporous structure. Among them, chitosan is a natural organic polymer material with good biocompatibility and rich amino groups on its surface. The surface of the methacrylated hydrogel also contains a large number of amino groups. The amino groups provide more active sites for the fixation of laccase. Laccase can be loaded onto the hydrogel porous microcarrier through a covalent binding reaction using glutaraldehyde as a cross-linking agent to achieve enzyme immobilization. At the same time, the nanoporous structure of the hydrogel porous microcarrier protects the laccase and indirectly improves the stability of the enzyme activity.

[0062] According to the embodiments of the present disclosure, the hydrogel porous microcarrier has an adjustable pore size, and its pore size includes 50 to 1000 nm, for example, it can be 50 nm, 80 nm, 150 nm, 300 nm, 500 nm, 600 nm, 1000 nm, etc. Its pore size can be adjusted according to needs during the preparation process, and has size adjustability.

[0063] According to an embodiment of the present disclosure, the loading amount of laccase is 0.2-1%, for example, it can be 0.2%, 0.5%, 0.8%, 1%, etc.; the concentration of chitosan is 0.5-3%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, etc.

[0064] According to an embodiment of the present disclosure, the composition of the methacrylated hydrogel microcarrier includes: 15-30% methacrylated gelatin, which can be, for example, 15%, 20%, 25%, 30%, etc. Methacrylated gelatin provides amino groups for the porous microcarrier; 20-50% polyethylene glycol diacrylate, which can be, for example, 20%, 25%, 30%, 35%, 40%, 50%, etc. Polyethylene glycol diacrylate gives the porous microcarrier hardness, making the hydrogel porous structure have mechanical strength; and 0.2-0.5% lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, which can be, for example, 0.2%, 0.3%, 0.4%, 0.5%, etc. Lithium phenyl(2,4,6-trimethylbenzoyl)phosphate is a photosensitive material, and the precursor solution of the methacrylated hydrogel can be formed into a solid state by ultraviolet light irradiation. Deionized water is used to dissolve various substances. In addition, substances with the same or similar properties as methacrylated gelatin, polyethylene glycol diacrylate, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate can be selected as additives to the precursor solution of the methacrylated hydrogel according to actual needs, and reagents with other functions can also be added to prepare functional methacrylated hydrogel microcarriers.

[0065] According to an embodiment of the present disclosure, the composition of the methacrylated hydrogel microcarrier may further include 5-10% iron oxide nanoparticles, which provide magnetism for the porous microcarrier, making the porous microcarrier loaded with laccase have magnetic response performance. The porous microcarriers before and after use can be collected by magnetic separation technology. The addition amount of iron oxide nanoparticles can be 5%, 7%, 8%, 10%, etc.

[0066] As another aspect of the present disclosure, a method for preparing the above-mentioned porous microcarrier loaded with laccase is provided. Figure 1 It is a step diagram for preparing the porous microcarrier loaded with laccase in the embodiment of the present disclosure, including steps S101 to S105:

[0067] In step S101, the silica nanoparticles are used as the dispersed phase and cut into single emulsion droplets through a microfluidic device. After drying, silica photonic crystals are obtained, and microsphere templates of silica photonic crystals are formed by calcination.

[0068] In step S102, the microsphere template of the silica photonic crystal is immersed in the precursor solution of the methacrylated hydrogel and polymerized by ultraviolet lamp irradiation to obtain a composite photonic crystal microsphere.

[0069] In step S103, the composite photonic crystal microsphere is etched with a hydrofluoric acid solution to obtain a methacrylated porous microcarrier.

[0070] In step S104, after the methacrylated porous microcarriers are dried, a chitosan hydrogel solution is added for filling to form chitosan-methacrylated porous microcarriers;

[0071] In step S105, a glutaraldehyde solution is added to the chitosan-methacrylated porous microcarriers for activation, and then a laccase solution is added for a covalent binding reaction to obtain porous microcarriers loaded with laccase.

[0072] According to specific embodiments of the present disclosure, for the preparation method of the porous microcarriers loaded with laccase, the following is Figure 1 described in detail, and specifically may include:

[0073] In step S101, silica nanoparticles of different sizes are repeatedly washed with ultrapure water more than five times, and then adjusted to a silica solution with a certain concentration as the dispersed phase. Using methyl silicone oil as the continuous phase, the dispersed phase is cut into monodisperse single emulsion droplets by the methyl silicone oil continuous phase at the orifice of the microfluidic device. After drying, it is washed with a n-hexane solution to obtain silica photonic crystals of different specifications. The silica photonic crystals are calcined under high-temperature conditions to improve mechanical strength, forming microsphere templates of silica photonic crystals of different specifications.

[0074] In step S102, substances such as methacryloylated gelatin, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, polyethylene glycol diacrylate, and iron oxide nanoparticles are dissolved in an aqueous solution to prepare a precursor solution of methacrylated hydrogel. After the microsphere templates of silica photonic crystals are immersed in the precursor solution of methacrylated hydrogel for a period of time, due to capillary action, the precursor solution can enter the interior of the microsphere templates and fill the voids between the nanoparticles in the microsphere templates. Then, the precursor solution is irradiated with ultraviolet light for polymerization to obtain different composite photonic crystal microspheres.

[0075] In step S103, the composite photonic crystal microspheres are etched with a hydrofluoric acid solution, or other methods can also be selected for etching treatment to remove the microsphere templates of silica photonic crystals in the composite photonic crystal microspheres, thereby obtaining methacrylated porous microcarriers with different pore sizes.

[0076] In step S104, after the obtained methacrylated porous microcarriers with different pore sizes are dried, a chitosan hydrogel solution is added for shaking treatment for 6-12 h, so that chitosan fills into the porous structure of the methacrylated porous microcarriers to form chitosan-methacrylated porous microcarriers.

[0077] In step S105, a glutaraldehyde solution is added to the chitosan-methacrylated porous microcarrier, and the mixture is placed in a thermostatic oscillator for activation reaction. The activated microcarrier is collected by magnetic separation and washed with phosphate buffered saline to remove unreacted glutaraldehyde. Then, a laccase solution is added for covalent cross-linking reaction. After binding the laccase to the chitosan-methacrylated porous microcarrier, the reactants are collected by magnetic separation and washed with phosphate buffered saline to remove free laccase that has not bound to the chitosan-methacrylated porous microcarrier, finally obtaining porous microcarriers with different pore sizes loaded with laccase.

[0078] According to the embodiments of the present disclosure, the particle size of the silica nanoparticles ranges from 50 to 1000 nm, and they can have different sizes, such as 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1000 nm, etc. After repeatedly washing the silica nanoparticles with ultrapure water more than five times, they are adjusted to a certain concentration as the dispersed phase, and the mass concentration of the dispersed phase is 15 to 30% (w / v), such as 15% (w / v), 20% (w / v), 25% (w / v), 30% (w / v), etc.

[0079] According to the embodiments of the present disclosure, the calcination temperature is 600 to 1000 °C, such as 600 °C, 700 °C, 800 °C, 900 °C, 1000 °C, etc., and the calcination time is 3 to 6 h, such as 3 h, 4 h, 5 h, 6 h, etc. By high-temperature calcination, the silica photonic crystal shrinks, and the formed molecular structure is more closely linked, improving its mechanical strength.

[0080] According to the embodiments of the present disclosure, the soaking time of the microsphere template of the silica photonic crystal in the precursor solution of the methacrylated hydrogel is 2 to 8 h, such as 2 h, 4 h, 6 h, 8 h, etc.

[0081] According to the embodiments of the present disclosure, the polymerization reaction is carried out under the irradiation conditions of a UV lamp with a wavelength of 330 to 450 nm, an irradiation intensity of 50 to 200 W, and an irradiation time of 1 to 5 min. For example, the wavelength of the UV lamp irradiation can be 330 nm, 350 nm, 365 nm, 370 nm, 390 nm, 420 nm, etc., the irradiation intensity of the UV lamp can be 55 W, 60 W, 80 W, 100 W, 120 W, 150 W, 195 W, etc., and the irradiation time of the UV lamp can be 1 min, 2 min, 3 min, 3.5 min, 4 min, 5 min, etc.

[0082] According to an embodiment of the present disclosure, the concentration of the hydrofluoric acid solution is 2-8%, for example, it can be 2%, 3%, 4%, 5%, 6%, 8%, etc. The etching treatment time is 2-8h, for example, it can be 2h, 3h, 4h, 5h, 6h, 7h, 8h, etc. It can also be adjusted according to actual needs. Sodium hydroxide solution or other solutions and other methods can be selected to etch the composite photonic crystal microspheres to remove the microsphere template of the silica photonic crystal.

[0083] According to an embodiment of the present disclosure, the concentration of chitosan is 0.5-3%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc.; the filling time is 6-12h, for example, it can be 6h, 8h, 10h, 12h, etc.

[0084] According to an embodiment of the present disclosure, the concentration of glutaraldehyde is 0.5-5%, for example, it can be 0.5%, 1%, 2%, 3%, 4%, 5%, etc.; the activation time is 1-6h, for example, it can be 1h, 2h, 4h, 6h, etc.

[0085] According to an embodiment of the present disclosure, the concentration of laccase is 0.5-2 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, etc.; the time of the covalent binding reaction is 2-10h, for example, it can be 2h, 4h, 6h, 8h, 10h, etc.

[0086] As another aspect of the present disclosure, a method for degrading bisphenol compounds is provided, including: adding the above-mentioned porous microcarrier loaded with laccase into the bisphenol compound solution to degrade the bisphenol compound; after the degradation reaction is completed, the porous microcarrier loaded with laccase is recovered and can be reused after being washed with a buffer solution.

[0087] According to an embodiment of the present disclosure, the porous microcarrier loaded with laccase has the protection of a biocompatible hydrogel shell and the confinement effect of nano-pores. Numerous interconnected and interlaced network nano-pores can promote the mass transfer and diffusion of substrate molecules (pollutants, such as bisphenol compounds), which has a promoting effect on the enzyme-catalyzed reaction, thus showing an enhancement of enzyme activity. In addition, the introduction of iron oxide nanoparticles provides the porous microcarrier with magnetic ability, making it have the potential for recovery.

[0088] In order to make the purpose, technical solution and advantages of the present disclosure clearer and more definite, the following further elaborates and explains the technical solution and principle of the present disclosure through specific embodiments in combination with the accompanying drawings. It should be noted that the following specific embodiments are only for illustration, and the protection scope of the present disclosure is not limited thereto.

[0089] The test materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions. For those not specified in the examples in terms of specific techniques or conditions, they are all conventional methods and can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions.

[0090] Example 1

[0091] Prepare the microsphere template of silica photonic crystal:

[0092] Wash the silica nanoparticles with particle sizes of 100nm, 200nm, 300nm, 400nm, 500nm, and 600nm five times with ultrapure water respectively, and adjust their concentration to 22% (w / v) as the dispersed phase. Select 10cSt methyl silicone oil as the continuous phase. Cut the silica dispersed phase into monodisperse single emulsion droplets at the orifice of the microfluidic device. After drying the droplets at 72°C for 12h, wash them with n-hexane to obtain silica photonic crystals of different specifications. Then place the silica photonic crystals in a muffle furnace and calcine them at 800°C for 4h to improve their mechanical strength, and obtain the microsphere templates of silica photonic crystals of different specifications.

[0093] Characterize the morphological structure of the microsphere templates of silica photonic crystals of different specifications by electron microscopy, Figure 2 is the scanning electron microscope (SEM) image of the microsphere templates of silica photonic crystals of different specifications in Example 1 of the present disclosure, Figure 2 In (a) and (b) are the microsphere templates of 100nm silica photonic crystals, (c) and (d) are the microsphere templates of 200nm silica photonic crystals, (e) and (f) are the microsphere templates of 300nm silica photonic crystals, (g) and (h) are the microsphere templates of 400nm silica photonic crystals, (i) and (j) are the microsphere templates of 500nm silica photonic crystals, and (k) and (l) are the microsphere templates of 600nm silica photonic crystals.

[0094] From Figure 2 It can be seen that the silica nanoparticles with sizes of 100nm, 200nm, 300nm, 400nm, 500nm, and 600nm spontaneously and closely pack into a highly ordered hexagonal nanostructure on the surface and inside of the microsphere templates of silica photonic crystals.

[0095] Example 2

[0096] Prepare the methacrylated porous microcarrier:

[0097] Add 20% methacrylated gelatin, 20% polyethylene glycol diacrylate, 0.25% lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 5% iron oxide nanoparticles and 54.75% deionized water into a beaker to prepare a methacrylic hydrogel precursor solution. Immerse the six silica photonic crystal microsphere templates obtained in Example 1 into the methacrylic hydrogel precursor solution respectively. Due to capillary action, the precursor solution can enter the interior of the microsphere templates, filling the gaps between the nanoparticles in the silica photonic crystal microsphere templates. After soaking for 4 h, irradiate with an ultraviolet lamp for 2 min to polymerize each component in the precursor solution to obtain composite photonic crystal microspheres. Immerse the composite photonic crystal microspheres in 4% hydrofluoric acid to remove the template silica nanoparticles. After etching for 2 h, wash three times with deionized water to obtain six methacrylated porous microcarriers.

[0098] Characterize it using an electron microscope, Figure 3 which is the scanning electron microscope (SEM) image of different methacrylated porous microcarriers in Example 2 of the present disclosure.

[0099] As can be seen from Figure 3 it, the methacrylated porous microcarriers are replicated from silica photonic crystal microspheres, and they have an ordered three-dimensional pore structure and interconnected nanochannels, and these structures can increase the specific surface area of the porous microcarriers. From the electron microscope characterization results at the magnification shown in parts (c), (f), (i), (l), (o) and (r) of Figure 3 it, the pore diameters of the six methacrylated porous microcarriers are 76 nm, 148 nm, 216 nm, 302 nm, 443 nm and 518 nm respectively. Due to the shrinkage of the silica photonic crystal during high-temperature calcination, the pore diameters of the prepared porous microcarriers are significantly smaller than the particle size of silica on the microsphere templates.

[0100] Example 3

[0101] Prepare chitosan-methacrylated porous microcarriers:

[0102] After drying the six methacrylated porous microcarriers obtained in Example 2, add them into 1.5% chitosan gel solution respectively and shake for 8 h for filling treatment, so that chitosan fills into the porous structure of the methacrylated porous microcarriers to prepare chitosan-methacrylated porous microcarriers.

[0103] For different particle sizes of silica on the silica photonic crystal microsphere template, chitosan-methylacrylic acid porous microcarriers prepared using silica nanoparticles with particle sizes of 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, and 600 nm were respectively labeled as MGelMA-CS-1, MGelMA-CS-2, MGelMA-CS-3, MGelMA-CS-4, MGelMA-CS-5, and MGelMA-CS-6.

[0104] Example 4

[0105] Preparation of porous microcarriers loaded with laccase:

[0106] The six chitosan-methylacrylic acid porous microcarriers obtained in Example 3 were added to a 2% glutaraldehyde solution, placed in a thermostatic oscillator for activation, and the activation reaction was carried out for 6 h. After magnetic separation of the activated microcarriers, they were washed with a 0.01 M phosphate buffer solution with pH = 5 to remove unreacted glutaraldehyde; a laccase solution with a concentration of 1 mg / mL was prepared, and 2 mL of the laccase solution was added to the activated microcarriers and placed in a thermostatic oscillator for covalent cross-linking. After the reaction for 6 h, magnetic separation was carried out and then washed with a phosphate buffer solution to remove free laccase, obtaining six porous microcarriers loaded with laccase, which were respectively labeled as MGelMA-CS-Lac-1, MGelMA-CS-Lac-2, MGelMA-CS-Lac-3, MGelMA-CS-Lac-4, MGelMA-CS-Lac-5, and MGelMA-CS-Lac-6.

[0107] The porous microcarrier MGelMA-CS-Lac-1 loaded with laccase was characterized using a laser confocal microscope. Figure 4 This is the laser confocal image of the porous microcarrier loaded with laccase in Example 4 of the present disclosure. It can be Figure 4 proven that laccase was uniformly immobilized on the chitosan-methylacrylic acid porous microcarrier.

[0108] Furthermore, the activities of porous microcarriers loaded with laccase with different pore sizes were measured. Using 0.5 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as the substrate, the measurement was carried out in a 100 mM sodium acetate buffer solution (0.1 M, pH = 4.5). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute.

[0109] The absorbance of the mixture was measured at 420 nm using a UV-visible spectrophotometer, and the activity of laccase was calculated according to the following equation:

[0110] Laccase activity (U / g) = 65 × ΔA / W

[0111] Among them, ΔA represents the increased absorbance within a specific time interval, and W represents the mass of the sample.

[0112] Table 1 Enzyme immobilization amount and enzyme activity in porous microcarriers loaded with different amounts of laccase

[0113] Sample Name Enzyme Immobilization Amount (mg / g) Enzyme Activity (U / mg) MGelMA-CS-Lac-1 22.29 75.7 MGelMA-CS-Lac-2 17.85 48.07 MGelMA-CS-Lac-3 15.76 45.92 MGelMA-CS-Lac-4 14.83 43.95 MGelMA-CS-Lac-5 11.68 19.73 MGelMA-CS-Lac-6 10.85 11.30

[0114] As can be seen from Table 1, the enzyme immobilization amount of MGelMA-CS-1 is 22.29 mg / g, and the enzyme activity is 75.7 U / mg, showing the best performance in terms of loading capacity and maintaining enzyme activity. At the same volume, the hydrogel particles with smaller pore diameters have a larger specific surface area and a higher laccase loading amount than those with larger pore diameters.

[0115] Example 5

[0116] Degradation experiments of bisphenol A were carried out using the porous microcarrier MGelMA-CS-Lac-1 loaded with 1 mg of laccase and 1 mg of free laccase prepared in Example 4, respectively. Under the condition of 25 °C, MGelMA-CS-Lac-1 and free laccase were respectively put into bisphenol A (BPA) solutions with concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 50 mg / L. The pH of the BPA solution was adjusted to 5, and the reaction was continuously oscillated for 6.5 hours and samples were taken regularly. The supernatant was filtered through a 0.22 μm filter membrane, and the residual concentration of BPA was measured.

[0117] Figure 5 This is the removal efficiency diagram of the porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solutions with different concentrations in Example 5 of the present disclosure. It can be Figure 5 seen that the removal efficiency of free laccase for BPA decreases as the initial concentration of BPA increases from 1 mg / L to 50 mg / L, and it can degrade about 33.02% of 50 mg / L BPA. However, the porous microcarrier loaded with laccase effectively removes BPA with different concentrations, and the degradation efficiency for 50 mg / L BPA can reach 99.22%, which is 3 times higher than that of the free enzyme. Due to the protection of the biocompatible hydrogel microcarrier shell, the efficiency of laccase is enhanced. At the same time, the numerous interconnected and interlaced network nano-pores in the microcarrier can promote the mass transfer and diffusion of substrate molecules, thus promoting the enhancement of enzyme activity.

[0118] Example 6

[0119] Under the condition of 25 °C, 1 mg of MGelMA-CS-Lac-1 loaded with laccase and 1 mg of free laccase were put into a bisphenol A (BPA) solution with a concentration of 20 mg / L. The pH range of the bisphenol A (BPA) solution was adjusted to 2-9, and the reaction was continuously oscillated for 6.5 hours and samples were taken regularly. The supernatant was filtered through a 0.22 μm filter membrane, and the residual concentration of BPA was measured.

[0120] Figure 6 This is the efficiency diagram of the porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solution at different pH values in Example 6 of the present disclosure. The influence of pH on free laccase and the porous microcarrier loaded with laccase was measured. From Figure 6 It can be seen that as the pH value increases, the removal rates of free laccase and laccase immobilized on magnetic microcarriers for BPA both increase first and then decrease. Compared with other pH conditions, free laccase can remove BPA more effectively at pH = 5. When the pH is 2, 4, 6, 7, 8, and 9, the degradation rates are reduced by 86.7%, 47.6%, 6.6%, 14.8%, 30.7%, and 32.1% respectively compared with pH = 5. Free laccase has great limitations in application. The porous microcarrier loaded with laccase has a high removal efficiency for BPA in the range of pH 5-8, and the difference in removal efficiency is less than 4%. The porous microcarrier loaded with laccase is much less sensitive to pH than free enzyme. The microcarrier provides the microenvironment required to protect the functional active sites of laccase and stabilizes the bioactive conformation of enzyme molecules. Compared with free laccase, the porous microcarrier loaded with laccase can effectively resist the interference of the external environment.

[0121] Example 7

[0122] Under the condition of pH = 5, 1 mg of MGelMA-CS-Lac-1 loaded with laccase and 1 mg of free laccase were respectively put into a bisphenol A (BPA) solution with a concentration of 20 mg / L. The temperature of the bisphenol A (BPA) solution was adjusted to 5-55 °C, and the reaction was continuously oscillated for 6.5 hours and samples were taken regularly. The supernatant was filtered through a 0.22 μm filter membrane, and the residual concentration of BPA was measured to evaluate the influence of temperature on the activities of free laccase and the porous microcarrier loaded with laccase.

[0123] Figure 7 This is the efficiency diagram of the porous microcarrier loaded with laccase and an equal amount of free enzyme for degrading bisphenol A solution at different temperatures in Example 7 of the present disclosure. From Figure 7It can be seen that at different test temperatures, the BPA removal performance of the porous microcarriers loaded with laccase is higher than that of free laccase. For example, at 55 °C, the removal rates of BPA by the porous microcarriers loaded with laccase and free laccase are 99.4% and 63.3% respectively. At a lower temperature of 5 °C, the BPA removal performance decreases slightly, being 79.2% and 45.8% respectively. Laccase is immobilized on the microcarriers through adsorption-covalent bonding, which has a certain stability. The microcarrier shell protects the enzyme protein, preventing the change of the enzyme molecular conformation and indirectly improving the thermal stability of the laccase molecule.

[0124] Example 8

[0125] Prepare 20 mL of other bisphenol compound solutions with a concentration of 20 mg / L: BPB, BPC, BPE, BPF, BPZ, BPAF, and tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA). Add 1 mg of laccase-loaded porous microcarrier MGelMA-CS-Lac-1 and 1 mg of free enzyme respectively, and react with shaking at 25 °C for 6.5 h. Take the supernatant and filter it through a 0.22 μm filter membrane, and measure the residual concentrations of different bisphenol compounds after degradation by the laccase-loaded porous microcarriers and free laccase respectively.

[0126] Figure 8 This is the removal efficiency diagram of different bisphenol compounds degraded by the laccase-loaded porous microcarriers and the equivalent amount of free enzyme in Example 8 of the present disclosure. Figure 8 Analysis shows that the degradation efficiencies of free laccase for BPB, BPC, BPE, BPF, BPZ, BPAF, and TBBPA are 50.76%, 96.28%, 50.95%, 46.45%, 41.00%, 9.63%, and 25.70% respectively, while the degradation efficiencies of the laccase-loaded porous microcarriers reach 82.17%, 99.07%, 93.10%, 96.60%, 97.99%, 56.58%, and 67.55%. The degradation rate of the laccase-loaded porous microcarriers for bisphenol A analogs has been significantly improved compared to the equivalent amount of free enzyme, and it can be used as an effective biocatalyst to degrade typical bisphenol compounds widely present in the water environment.

[0127] Example 9

[0128] Store free laccase and the laccase-loaded porous microcarrier MGelMA-CS-Lac-1 at 4 °C and 25 °C for 30 days respectively, and measure the residual activity of laccase every three days. The initial laccase activity is defined as 100%.

[0129] Figure 9 This is the test diagram of laccase activity stored at different temperatures in Example 9 of the present disclosure. Figure 9Analysis shows that the porous microcarriers loaded with laccase can retain 83.3% of their initial activity after storage at 4°C for 15 days, 49.2% of their initial activity after storage at 25°C for 15 days, 65.2% and 33.9% of their initial activity after being placed at 4°C and 25°C for 30 days, respectively. For free laccase, after being placed at 4°C and 25°C for 30 days, the relative activities are only 39.3% and 8.2%, respectively. Different from free enzymes, microcarriers can provide a good hydrophilic environment for enzyme molecules, thus maintaining their original activity, which is convenient for storage in industrial practical applications and has great advantages.

[0130] Example 10

[0131] Continuously determine the removal efficiency of the porous microcarriers loaded with laccase for 10 mg / L BPA solution to evaluate the reusability of the porous microcarriers loaded with laccase. At 25°C, adjust the pH of the BPA solution to 5, and use the porous microcarrier MGelMA-CS-Lac-1 loaded with 1 mg of laccase to degrade 10 mg / L BPA solution. After continuously oscillating the reaction for 6.5 hours, take the supernatant and filter it through a 0.22 μm filter membrane, and measure the residual concentration of BPA. Collect the porous microcarriers loaded with laccase with a magnet, wash them three times with phosphate buffer solution, and then use the recovered porous microcarriers loaded with laccase for repeated degradation tests. Calculate the relative removal efficiency of the immobilized enzyme with the efficiency of the first reaction as 100%.

[0132] Figure 10 This is the graph of the relative activity change after repeated use of the porous microcarriers loaded with laccase in Example 10 of the present disclosure. According to Figure 10 the results analysis, after being reused 5 times, the removal rate of the porous microcarriers loaded with laccase for BPA still remains above 80%, and drops to 73.7% in the 10th cycle. This result fully demonstrates that the porous microcarriers endow laccase with reusability. The decrease in enzyme activity during continuous cycling may be due to the accumulation of reaction products blocking a part of the pores in the microcarriers and the shedding of a small amount of immobilized laccase. The reusability of the porous microcarriers loaded with laccase reduces the cost in practical applications.

[0133] Using the porous microcarriers loaded with laccase, preparation method and application provided by the embodiments of the present disclosure, a chitosan-methacrylated porous microcarrier with a three-dimensional porous structure and interconnected nanochannels is prepared by calcining a silica photonic crystal to form a template. The structure has a large number of amino groups, providing a large number of binding sites for the immobilization of laccase. In addition, the introduction of iron oxide nanoparticles endows the microcarriers with magnetic properties, enabling the synthesized biocatalyst to have the potential for recycling. The prepared porous microcarriers loaded with laccase are protected by a biocompatible hydrogel shell and the confinement effect of nano-pores. The numerous interconnected and interlaced network nano-pores in the porous microcarriers can promote the mass transfer and diffusion of substrate molecules, thus promoting the enhancement of enzyme activity, having high stability in a certain mild pH range, and showing high degradation efficiency for bisphenol compounds.

[0134] The specific embodiments described above further elaborate on the objectives, technical solutions and beneficial effects of the present disclosure. It should be understood that the above are only specific embodiments of the present disclosure and are not used to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. A method for degrading bisphenol compounds using a porous microcarrier loaded with laccase, the method comprising: Adding the porous microcarrier loaded with laccase into a bisphenol compound solution to degrade the bisphenol compound; After the degradation reaction is completed, recovering the porous microcarrier loaded with laccase, which can be reused after being washed with a buffer solution; Wherein, the porous microcarrier loaded with laccase comprises: a hydrogel porous microcarrier and laccase, and the laccase is loaded on the hydrogel porous microcarrier by covalent binding; The hydrogel porous microcarrier is a chitosan-filled methacrylated hydrogel microcarrier, which has a three-dimensional porous structure and interconnected nanochannels, providing loading sites for the laccase; The preparation method of the porous microcarrier loaded with laccase comprises: Using silica nanoparticles as the dispersed phase, cutting into single emulsion droplets through a microfluidic device, drying to obtain silica photonic crystals, and calcining to form a microsphere template of silica photonic crystals; Soaking the microsphere template of silica photonic crystals in the precursor solution of methacrylated hydrogel, and polymerizing by ultraviolet light irradiation to obtain composite photonic crystal microspheres; Etching the composite photonic crystal microspheres with a hydrofluoric acid solution to obtain a methacrylated porous microcarrier; After the methacrylated porous microcarrier is dried, adding a chitosan hydrogel solution to fill it to form a hydrogel porous microcarrier; Adding a glutaraldehyde solution to the hydrogel porous microcarrier for activation, and then adding a laccase solution for covalent binding reaction to obtain a porous microcarrier loaded with laccase; The hydrogel porous microcarrier has an adjustable pore size; The loading amount of the laccase is 0.2 - 1%; The concentration of the chitosan is 0.5 - 3%; The composition of the methacrylated hydrogel microcarrier includes: 15 - 30% methacrylated gelatin, 20 - 50% polyethylene glycol diacrylate, and 0.2 - 0.5% lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate.

2. According to the method of claim 1, wherein, The composition of the methacrylated hydrogel microcarrier may further include 5 - 10% iron oxide nanoparticles, providing magnetism for the porous microcarrier.

3. The method according to claim 1, wherein In the preparation method of the porous microcarrier loaded with laccase, The particle size of the silica nanoparticles includes 50 - 1000 nm; The mass concentration of the dispersed phase is 15 - 30%.

4. The method according to claim 1, wherein In the preparation method of the porous microcarrier loaded with laccase, the calcination temperature is 600 - 1000 °C; The calcination time is 3 - 6 h; The soaking time is 2 - 8 h.

5. The method according to claim 1, wherein In the preparation method of the porous microcarrier loaded with laccase, The wavelength of the ultraviolet light irradiation is 330 - 450 nm; The intensity of the ultraviolet light irradiation is 50 - 200 W; The time of the ultraviolet light irradiation is 1 - 5 min.

6. The method according to claim 1, wherein In the preparation method of the porous microcarrier loaded with laccase, The concentration of the hydrofluoric acid solution is 2 - 8%; The etching treatment time is 2 - 8 h; The concentration of the chitosan is 0.5 - 3%; The filling time is 6 - 12 h.

7. The method according to claim 1, wherein, In the preparation method of the porous microcarrier loaded with laccase, The concentration of the glutaraldehyde is 0.5 - 5%; The activation time is 1 - 6 h; The concentration of the laccase is 0.5 - 2 mg / mL; The time of the covalent binding reaction is 2 - 10 h.

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