CN116477770A

CN116477770A - Laccase-loaded porous microcarrier, preparation method and application

Info

Publication number: CN116477770A
Application number: CN202310486974.4A
Authority: CN
Inventors: 宋茂勇; 杜梅; 毕磊
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2023-05-04
Filing date: 2023-05-04
Publication date: 2023-07-25

Abstract

The present disclosure provides a laccase-loaded porous microcarrier, a preparation method and applications thereof, wherein the laccase-loaded porous microcarrier comprises: a hydrogel porous microcarrier; laccase supported on the hydrogel porous microcarrier by covalent bonding; the hydrogel porous microcarrier is a chitosan filled methacrylic acid hydrogel microcarrier, and provides a loading site for laccase.

Description

Laccase-loaded porous microcarrier, preparation method and application

Technical Field

The disclosure relates to the technical field of water pollution treatment, in particular to a porous microcarrier for carrying laccase, a preparation method and application thereof, and specifically relates to a preparation method of a porous microcarrier for improving laccase activity and application thereof in removing bisphenol compounds in water.

Background

In recent years, bisphenol compounds have been detected in different water environments, and the widespread presence of bisphenol compounds in the environment and the potential for ecotoxicological effects have attracted attention. Among them, bisphenol a (BPA) compounds are produced in large quantities and widely used, can be detected in various environments, and particularly exist in large quantities in aquatic systems, and cause reproductive dysfunction and dysplasia in humans and other organisms, and are a typical endocrine disruptor. Because of health risks of bisphenol a (BPA) compounds, bisphenol a (BPA) compounds are continually replaced with other bisphenol compounds similar to their structures, such as bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bisphenol F (BPF), bisphenol Z (BPZ), bisphenol AF (BPAF), etc., which are a series of chemical substances containing two hydroxyphenyl groups in their structures, which have similar endocrine disrupting effects as BPA, however, the technology for effectively removing them has not been fully studied yet.

Disclosure of Invention

Aiming at the technical problems, the disclosure provides a laccase-loaded porous microcarrier, a preparation method and application thereof, so as to at least partially solve the technical problems.

In order to solve the technical problems, the technical scheme provided by the disclosure is as follows:

as one aspect of the present disclosure, there is provided a laccase-supported porous microcarrier comprising:

a hydrogel porous microcarrier;

laccase supported on the hydrogel porous microcarrier by covalent bonding;

the hydrogel porous microcarrier is a chitosan filled methacrylic acid hydrogel microcarrier, and provides a loading site for laccase.

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

the load of laccase is 0.2-1%;

the concentration of the chitosan is 0.5-3%;

the composition of the methacrylated hydrogel microcarrier comprises: 15 to 30 percent of methacrylic acylated gelatin, 20 to 50 percent of polyethylene glycol diacrylate and 0.2 to 0.5 percent of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate.

In one embodiment, the composition of the methacrylated hydrogel microcarrier can also include 5-10% ferroferric oxide nanoparticles to provide magnetism to the porous microcarrier.

As another aspect of the present disclosure, there is provided a method for preparing the laccase-supporting porous microcarrier described above, comprising:

cutting the silica nanoparticles serving as a disperse phase into single emulsion droplets through a microfluidic device, drying to obtain silica photonic crystals, and calcining to form microsphere templates of the silica photonic crystals;

soaking a microsphere template of a silicon dioxide photonic crystal in a precursor solution of methacrylic acid hydrogel, and performing irradiation polymerization by an ultraviolet lamp to obtain a composite photonic crystal microsphere;

etching the composite photonic crystal microspheres by using hydrofluoric acid solution to obtain a methacrylic acid porous microcarrier;

drying the methacrylic acid porous microcarrier, and then adding chitosan hydrogel solution for filling to form a chitosan-methacrylic acid porous microcarrier;

and adding glutaraldehyde solution into the chitosan-methacrylic acid porous microcarrier for activation, and then adding laccase solution for covalent bonding reaction to obtain the laccase-loaded porous microcarrier.

In one embodiment, the particle size of the silica nanoparticles comprises 50 to 1000nm;

the mass concentration of the disperse phase is 15-30% (w/v).

In one embodiment, the calcination temperature is 600-1000 ℃;

the calcination time is 3-6 h;

the soaking time is 2-8 h.

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

the irradiation intensity of the ultraviolet lamp is 50-200W;

the irradiation time of the ultraviolet lamp is 1-5 min.

In one embodiment, 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.

In one embodiment, the glutaraldehyde concentration is 0.5-5%;

the activation time is 1-6 h;

the concentration of laccase is 0.5-2 mg/mL;

the time of covalent bonding reaction is 2-10 h.

As yet another aspect of the present disclosure, there is provided a method of degrading a bisphenol compound, comprising: adding the laccase-loaded porous microcarrier into bisphenol compound solution to degrade bisphenol compound;

after the degradation reaction is finished, recovering the porous microcarrier loaded with laccase, and washing the porous microcarrier with a buffer solution for reuse.

Based on the technical scheme, the laccase-loaded porous microcarrier, the preparation method and the application provided by the disclosure at least comprise one of the following beneficial effects:

(1) According to the embodiment of the disclosure, the hydrogel porous microcarrier in the porous microcarrier loaded with laccase has a three-dimensional porous structure and interconnected nano channels, more binding sites are provided for laccase fixation under the filling effect of chitosan hydrogel, the laccase is loaded on the hydrogel porous microcarrier through covalent bonding, the hydrogel porous microcarrier provides functional active sites and required microenvironment for laccase molecules, and the bioactivity of the laccase molecules is stabilized, so that the laccase molecules can effectively resist the interference of external environments.

(2) In the embodiment of the disclosure, the porous microcarrier loaded with laccase benefits from the protection of the biocompatible hydrogel shell and the domain-limiting effect of the nano holes, so that the mass transfer and diffusion of substrate molecules can be promoted by a plurality of interconnected and staggered network nano holes in the microcarrier, thereby improving the effective contact between laccase and substrate molecules and promoting the enhancement of enzyme activity.

(3) According to the embodiment of the disclosure, the silica photonic crystal is calcined and then soaked in a methacrylic acid hydrogel precursor solution to prepare composite photonic crystal microspheres, a silica photonic crystal template is removed through etching, chitosan hydrogel is added to fill the composite photonic crystal template to obtain a chitosan-methacrylic acid porous microcarrier, and the chitosan-methacrylic acid porous microcarrier is subjected to covalent bonding with laccase after activation to obtain a laccase-loaded porous microcarrier. The preparation method has adjustable pore size, and can prepare silica photonic crystals by using silica particles with different particle diameters as templates, so that porous microcarriers with different pore diameters are obtained, and the loading capacity of laccase can be adjusted.

(4) According to the embodiment of the disclosure, the laccase-loaded porous microcarrier can effectively degrade bisphenol A compounds, and the degradation performance of the laccase-loaded porous microcarrier on other bisphenol compounds is evaluated. Furthermore, after the porous microcarrier loaded with laccase degrades bisphenol compounds, the porous microcarrier can be recycled, and the introduction of the ferroferric oxide nano particles provides magnetic response performance for the microcarrier, so that the porous microcarrier loaded with laccase has recovery potential, and the cost in practical application is reduced to a certain extent.

Drawings

FIG. 1 is a step diagram of preparing a laccase-supporting porous microcarrier in an embodiment of the disclosure;

FIG. 2 is a Scanning Electron Microscope (SEM) image of microsphere templates of different specifications of silica photonic crystals in example 1 of the present disclosure;

FIG. 3 is a Scanning Electron Microscope (SEM) image of a different methacrylated porous microcarrier according to example 2 of the present disclosure;

FIG. 4 is a laser confocal plot of laccase-supported porous microcarriers in example 4 of the disclosure;

FIG. 5 is a graph showing the removal efficiency of laccase-supporting porous microcarriers and equal amounts of free enzyme to degrade bisphenol A solutions of different concentrations in example 5 of the present disclosure;

FIG. 6 is a graph showing the efficiency of laccase-supporting porous microcarriers and equal amounts of free enzyme in degrading bisphenol A solution at different pH's in example 6 of the present disclosure;

FIG. 7 is a graph showing the efficiency of laccase-supporting porous microcarriers and equal amounts of free enzyme in degrading bisphenol A solution at different temperatures in example 7 of the present disclosure;

FIG. 8 is a graph showing the removal efficiency of laccase-supporting porous microcarriers and equivalent amounts of free enzymes to degrade different bisphenol compounds in example 8 of the present disclosure;

FIG. 9 is a graph showing laccase activity assays stored at various temperatures in example 9 of the disclosure;

FIG. 10 is a graph showing the relative activity change after repeated use of laccase-supporting porous microcarriers according to example 10 of the disclosure.

Detailed Description

For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.

At present, the method for removing BPA in wastewater mainly comprises a physical method, a chemical method and a biological method. Physical and chemical treatment methods in the related art face challenges of sustainable wide application due to higher energy consumption and cost. Thus, there is an urgent need to develop an economical, efficient and environmentally sustainable micropollutant treatment technology.

In the process of realizing the disclosure, laccase is found to be a copper-containing polyphenol oxidase which has strong substrate specificity, can catalyze various phenols, anilines and aromatic compounds, and has been widely applied to various fields such as industrial wastewater treatment, dye decolorization, textile printing and dyeing, analysis and detection, environmental pollutant degradation, environmental remediation and the like. However, the stability of the free laccase is relatively poor, and compared with the free laccase, the immobilized enzyme enhances the tolerance and thermal stability of the immobilized enzyme to environmental conditions, can be recycled, and greatly widens the range of practical application. Many immobilized laccase materials in the related art exhibit remarkable properties in terms of degrading BPA, but it is unclear whether they can also degrade other bisphenol compounds effectively. Since bisphenol compounds have different chemical structures, affect degradation efficiency, and coexist in water, it is necessary to develop a novel vector to immobilize laccase and evaluate degradation performance on bisphenol compounds.

In view of the technical problems in the related art, the disclosure provides a laccase-loaded porous microcarrier, a preparation method and application thereof, which are characterized in that a template is formed by calcining a silicon dioxide photonic crystal, the silicon dioxide photonic crystal is soaked in a methacrylic acid hydrogel precursor solution to prepare a composite photonic crystal microsphere, the template is removed by etching, and then chitosan hydrogel is added to fill the composite photonic crystal microsphere to obtain the chitosan-methacrylic acid porous microcarrier, and the chitosan-methacrylic acid porous microcarrier is subjected to covalent bonding with laccase after activation to obtain the laccase-loaded porous microcarrier. The three-dimensional porous structure and the interconnected nano channels of the porous microcarrier and the filling effect of the chitosan hydrogel provide more binding sites for the immobilization of laccase, provide shell protection for laccase, stabilize the bioactivity of laccase, and the obtained porous microcarrier loaded with laccase can be recycled after degradation reaction, thus having higher application development prospect.

Specifically, as one aspect of the present disclosure, the present disclosure provides a laccase-loaded porous microcarrier comprising:

a hydrogel porous microcarrier;

laccase supported on the hydrogel porous microcarrier by covalent bonding;

According to embodiments of the present disclosure, the hydrogel porous microcarrier has a three-dimensional nano-porous structure, consisting of a methacrylated hydrogel microcarrier and chitosan filled into its microporous structure. The chitosan is a natural organic polymer material with good biocompatibility, the surface of the chitosan is rich in amino groups, the surface of the methacrylic acid hydrogel also contains more amino groups, the amino groups provide more active sites for the immobilization of laccase, glutaraldehyde can be used as a cross-linking agent to load laccase on the hydrogel porous microcarrier through covalent bonding reaction, so that the immobilization of enzyme is realized, meanwhile, the nano-pore structure of the hydrogel porous microcarrier plays a role in protecting laccase, and the stability of enzyme activity is indirectly improved.

According to embodiments of the present disclosure, the hydrogel porous microcarrier has an adjustable pore size, which includes 50-1000 nm, such as 50nm, 80nm, 150nm, 300nm, 500nm,600nm, 1000nm, etc., and which can be adjusted during the preparation process as needed, with size adjustability.

According to embodiments of the present disclosure, the loading of laccase is 0.2-1%, e.g., may be 0.2%, 0.5%, 0.8%, 1%, etc.; the concentration of chitosan is 0.5 to 3%, and may be, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc.

According to embodiments of the present disclosure, the composition of the methacrylated hydrogel microcarrier comprises: 15-30% of a methacryloylated gelatin, which may be, for example, 15%, 20%, 25%, 30%, etc., which provides amino groups to the porous microcarrier; 20-50% of polyethylene glycol diacrylate, for example, 20%, 25%, 30%, 35%, 40%, 50% and the like, wherein the polyethylene glycol diacrylate provides hardness for the porous microcarrier, so that the porous structure of the hydrogel has mechanical strength; and 0.2 to 0.5% of lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate, for example, 0.2%, 0.3%, 0.4%, 0.5% and the like, the lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate being a photosensitive material, and the precursor solution of the methacrylic hydrogel being formed into a coagulated state by irradiation with ultraviolet light. Deionized water was used to dissolve various substances. In addition, substances with the same or similar properties as those of the methacryloylated gelatin, the polyethylene glycol diacrylate and the phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate can be selected as additives of the precursor solution of the methacrylic acid hydrogel according to actual needs, and reagents with other functions can be added to prepare the functional methacrylic acid hydrogel microcarrier.

According to the embodiment of the disclosure, the composition of the methacrylic acid hydrogel microcarrier can also comprise 5-10% of ferroferric oxide nano particles, magnetism is provided for the porous microcarrier, so that the porous microcarrier loaded with laccase has magnetic response performance, the porous microcarrier before and after use can be collected by a magnetic separation technology, and the addition amount of the ferroferric oxide nano particles can be 5%, 7%, 8%, 10% and the like.

As another aspect of the present disclosure, there is provided a method for preparing the laccase-supporting porous microcarrier described above, fig. 1 is a step diagram of preparing the laccase-supporting porous microcarrier according to an embodiment of the present disclosure, including steps S101 to S105:

in step S101, silicon dioxide nano particles are used as a disperse phase, cut into single emulsion liquid drops through a microfluidic device, and the single emulsion liquid drops are dried to obtain silicon dioxide photonic crystals, and the silicon dioxide photonic crystals are calcined to form microsphere templates of the silicon dioxide photonic crystals;

in step S102, a microsphere template of a silicon dioxide photonic crystal is soaked in a precursor solution of methacrylic acid hydrogel, and is subjected to irradiation polymerization by an ultraviolet lamp to obtain composite photonic crystal microspheres;

in step S103, etching the composite photonic crystal microspheres by using hydrofluoric acid solution to obtain a methacrylic acid porous microcarrier;

in step S104, after the methacrylated porous microcarrier is dried, the chitosan hydrogel solution is added to fill to form a chitosan-methacrylated porous microcarrier;

in step S105, glutaraldehyde solution is added into the chitosan-methacrylic acid porous microcarrier for activation, and laccase solution is then added for covalent bonding reaction to obtain the laccase-loaded porous microcarrier.

According to a specific embodiment of the present disclosure, the preparation method of the laccase-loaded porous microcarrier is described in detail below in conjunction with fig. 1, and may specifically include:

in step S101, silica nanoparticles with different sizes are repeatedly washed with ultrapure water for more than five times, then a silica solution with a certain concentration is used as a disperse phase, methyl silicone oil is used as a continuous phase, the disperse phase is cut into monodisperse single emulsion droplets by the methyl silicone oil continuous phase at the orifice of the microfluidic device, after drying, silica photonic crystals with different specifications are obtained by washing with n-hexane solution, and the mechanical strength of the silica photonic crystals is improved by calcining at high temperature, so that microsphere templates of the silica photonic crystals with different specifications are formed.

In step S102, the precursor solution of the methacrylic acid hydrogel is prepared by dissolving the materials such as the methacrylic acid gelatin, the phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate, the polyethylene glycol diacrylate and the ferroferric oxide nano particles in the aqueous solution, the microsphere template of the silicon dioxide photonic crystal is soaked in the precursor solution of the methacrylic acid hydrogel for a period of time, and the precursor solution can enter the microsphere template due to capillary force to fill gaps among the nano particles in the microsphere template, and then the ultraviolet lamp is used for irradiating the polymerization precursor solution, so that different composite photonic crystal microspheres are obtained.

In step S103, etching treatment is performed on the composite photonic crystal microsphere by using a hydrofluoric acid solution, and other methods may be used to perform etching treatment to remove the microsphere template of the silica photonic crystal in the composite photonic crystal microsphere, thereby obtaining the methacrylic porous microcarrier with different pore sizes.

In step S104, the obtained methacrylic acid porous microcarriers with different pore sizes are dried, and then added with chitosan hydrogel solution for oscillation treatment for 6-12 hours, so that chitosan is filled into the porous structure of the methacrylic acid porous microcarriers to form the chitosan-methacrylic acid porous microcarriers.

In step S105, glutaraldehyde solution is added into the chitosan-methacrylic acid porous microcarrier, the mixture is placed in a constant temperature oscillator for activation reaction, the activated microcarrier is subjected to magnetic separation and collection, and the mixture is washed by phosphate buffer salt solution to remove unreacted glutaraldehyde; and then adding laccase solution to carry out covalent crosslinking reaction, combining laccase on the chitosan-methacrylic acid porous microcarrier, collecting reactants by using magnetic separation, and washing with phosphate buffer salt solution to remove free laccase which is not combined with the chitosan-methacrylic acid porous microcarrier, thus finally obtaining the porous microcarrier with different pore sizes and loaded with laccase.

According to embodiments of the present disclosure, the particle size of the silica nanoparticles includes 50 to 1000nm, and may have different sizes, for example, may be 50nm, 100nm, 200nm, 300nm,400nm,500nm,600nm, 800nm, 1000nm, etc.; the silica nanoparticles are repeatedly washed with ultrapure water for five or more times, and then adjusted to have a constant concentration as a dispersed phase, wherein the mass concentration of the dispersed phase is 15 to 30% (w/v), and may be, for example, 15% (w/v), 20% (w/v), 25% (w/v), 30% (w/v), or the like.

According to embodiments of the present disclosure, the calcination temperature is 600 to 1000 ℃, for example, 600 ℃, 700 ℃, 800 ℃, 900 ℃, 1000 ℃, etc., and the calcination time is 3 to 6 hours, for example, 3 hours, 4 hours, 5 hours, 6 hours, etc. The silicon dioxide photonic crystal is contracted by high-temperature calcination, so that the formed molecular structure is more tightly connected, and the mechanical strength of the silicon dioxide photonic crystal is improved.

According to embodiments of the present disclosure, the microsphere template of the silica photonic crystal is soaked in the precursor solution of the methacrylic acid hydrogel for 2 to 8 hours, for example, 2 hours, 4 hours, 6 hours, 8 hours, etc.

According to the embodiment of the present disclosure, the polymerization reaction is performed under irradiation conditions in which the wavelength of the ultraviolet lamp irradiation is 330 to 450nm, the intensity of the ultraviolet lamp irradiation is 50 to 200W, and the time of the ultraviolet lamp irradiation is 1 to 5min. For example, the wavelength of the ultraviolet lamp irradiation may be 330nm, 350nm, 365nm, 370nm, 390nm, 420nm, etc., the intensity of the ultraviolet lamp irradiation may be 55W, 60W, 80W, 100W, 120W, 150W, 195W, etc., and the time of the ultraviolet lamp irradiation may be 1min, 2min, 3min, 3.5min, 4min, 5min, etc.

According to the embodiment of the disclosure, the concentration of the hydrofluoric acid solution is 2-8%, for example, 2%, 3%, 4%, 5%, 6%, 8% and the like, and the etching treatment time is 2-8 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours and the like, and the etching treatment can be performed on the composite photonic crystal microspheres by selecting sodium hydroxide solution or other solutions and other methods according to actual needs, so as to remove the microsphere templates of the silicon dioxide photonic crystals.

According to embodiments of the present disclosure, the concentration of chitosan is 0.5-3%, for example, may be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc.; the filling time is 6 to 12 hours, and may be, for example, 6 hours, 8 hours, 10 hours, 12 hours, etc.

According to embodiments of the present disclosure, glutaraldehyde is present at a concentration of 0.5-5%, such as 0.5%, 1%, 2%, 3%, 4%, 5%, etc.; the activation time is 1 to 6 hours, and may be, for example, 1 hour, 2 hours, 4 hours, 6 hours, or the like.

According to embodiments of the present disclosure, the concentration of laccase is 0.5-2 mg/mL, e.g., may be 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, etc.; the time for the covalent bonding reaction is 2 to 10 hours, and may be, for example, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or the like.

As yet another aspect of the present disclosure, there is provided a method of degrading a bisphenol compound, comprising: adding the laccase-loaded porous microcarrier into bisphenol compound solution to degrade bisphenol compound; after the degradation reaction is finished, recovering the porous microcarrier loaded with laccase, and washing the porous microcarrier with a buffer solution for reuse.

According to embodiments of the present disclosure, laccase-loaded porous microcarriers have the protection of biocompatible hydrogel shells and the domain-limiting effect of nanopores, and numerous interconnected and staggered network nanopores can promote mass transfer and diffusion of substrate molecules (contaminants, such as bisphenols), which promote enzymatic reactions, thus exhibiting an enhancement of enzymatic activity. In addition, the introduction of ferroferric oxide nano-particles provides magnetic capability for the porous microcarrier, so that the porous microcarrier has recovery potential.

In order to make the objects, technical solutions and advantages of the present disclosure clearer, the technical solutions and principles of the present disclosure are further described below by specific embodiments with reference to the accompanying drawings. It should be noted that the following specific examples are given by way of illustration only and the scope of the present disclosure is not limited thereto.

The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The examples are not intended to identify specific techniques or conditions, but are conventional and may be carried out according to techniques or conditions described in the literature in this field or according to product specifications.

Example 1

Preparing a microsphere template of the silicon dioxide photonic crystal:

the silica nanoparticles with the particle diameters of 100nm, 200nm, 300nm,400nm,500nm and 600nm are respectively washed with ultrapure water for five times, the concentration of the silica nanoparticles is adjusted to 22% (w/v) as a disperse phase, 10cSt of methyl silicone oil is selected as a continuous phase, the silica disperse phase is cut into monodisperse single emulsion droplets at the orifice of a microfluidic device, the droplets are dried at the temperature of 72 ℃ for 12 hours, and then the silica photonic crystals with different specifications are obtained through washing with n-hexane. And then placing the silicon dioxide photonic crystal in a muffle furnace, and calcining for 4 hours at 800 ℃ to improve the mechanical strength of the silicon dioxide photonic crystal, thereby obtaining the microsphere templates of the silicon dioxide photonic crystals with different specifications.

Characterization of morphology structures of microsphere templates of silica photonic crystals of different specifications by electron microscopy, fig. 2 is a Scanning Electron Microscope (SEM) image of microsphere templates of silica photonic crystals of different specifications in example 1 of the present disclosure, and (a) (b) in fig. 2 is a microsphere template of silica photonic crystals of 100nm, (c) (d) is a microsphere template of silica photonic crystals of 200nm, (e) (f) is a microsphere template of silica photonic crystals of 300nm, (g) (h) is a microsphere template of silica photonic crystals of 400nm, (i) (j) is a microsphere template of silica photonic crystals of 500nm, (k) (l) is a microsphere template of silica photonic crystals of 600 nm.

As can be seen from fig. 2, silica nanoparticles of sizes 100nm, 200nm, 300nm,400nm,500nm and 600nm spontaneously close-packed into highly ordered hexagonal nanostructures both on the surface and inside the microsphere template of the silica photonic crystal.

Example 2

Preparation of a methacrylated porous microcarrier:

a methacryloylated hydrogel precursor solution was prepared by adding 20% of methacryloylated gelatin, 20% of polyethylene glycol diacrylate, 0.25% of lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate, 5% of ferroferric oxide nanoparticles, and 54.75% of deionized water in a beaker. The microsphere templates of the six silica photonic crystals obtained in the example 1 are respectively soaked in the precursor solution of the methacrylic acid hydrogel, the precursor solution can enter the microsphere templates due to capillary force, gaps among nano particles in the silica photonic crystal microsphere templates are filled, after soaking for 4 hours, each component in the precursor solution is polymerized by irradiating for 2 minutes by an ultraviolet lamp, and the composite photonic crystal microsphere is obtained. And immersing the composite photon crystal microsphere in 4% hydrofluoric acid to remove template silica nano particles, etching for 2 hours, and cleaning with deionized water for three times to obtain six methacrylic acid porous microcarriers.

This was characterized using an electron microscope and FIG. 3 is a Scanning Electron Microscope (SEM) image of a different methacrylated porous microcarrier according to example 2 of the present disclosure.

As can be seen from fig. 3, the methacrylic porous microcarrier is repeatedly prepared from silica photonic crystal microspheres, and has an ordered three-dimensional pore structure and interconnected nanochannels, and the structure can increase the specific surface area of the porous microcarrier. From the electron microscopy characterization results at the magnifications shown in FIG. 3, parts (c), (f), (i), (l), (o) and (r), the pore diameters of the six methacrylated porous microcarriers were 76nm,148nm,216nm,302nm, 447 nm and 518nm, respectively, and the pore diameters of the prepared porous microcarriers were significantly smaller than the particle size of the silica on the microsphere template due to shrinkage of the silica photonic crystal during high temperature calcination.

Example 3

Preparing a chitosan-methacrylic acid porous microcarrier:

and drying the six methacrylic acid porous microcarriers obtained in the example 2, respectively adding the dried six methacrylic acid porous microcarriers into 1.5% chitosan gel solution, and vibrating and filling for 8 hours to enable chitosan to be filled into the porous structure of the methacrylic acid porous microcarriers, so as to prepare the chitosan-methacrylic acid porous microcarriers.

The chitosan-methacrylic acid porous microcarrier prepared by using silica nanoparticles with the particle diameters of 100nm, 200nm, 300nm,400nm,500nm and 600nm is respectively marked as MGelMA-CS-1, MGelMA-CS-2, MGelMA-CS-3, MGelMA-CS-4, MGelMA-CS-5 and MGelMA-CS-6 corresponding to different particle diameters of silica on the silica photon crystal microsphere template.

Example 4

Preparing a laccase-supported porous microcarrier:

adding the 6 chitosan-methacrylic acid porous microcarriers obtained in the example 3 into glutaraldehyde solution with the concentration of 2%, placing the solution in a constant-temperature oscillator for activation, performing activation reaction for 6 hours, performing magnetic separation on the activated microcarriers, and then washing the microcarriers with 0.01M phosphate buffer solution with the pH of=5 to remove unreacted glutaraldehyde; preparing laccase solution with concentration of 1mg/mL, adding 2mL of laccase solution into the activated microcarrier, placing the microcarrier in a constant temperature oscillator for covalent crosslinking, performing magnetic separation after reaction for 6 hours, washing by using a phosphate buffer solution, and removing free laccase to obtain 6 porous microcarriers loaded with laccase, wherein the porous microcarriers are marked 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 respectively.

Characterization of laccase-loaded porous microcarrier MGelMA-CS-Lac-1 using a laser confocal microscope FIG. 4 is a laser confocal plot of laccase-loaded porous microcarrier in example 4 of the disclosure, from FIG. 4 it can be demonstrated that laccase is uniformly immobilized on chitosan-methacrylated porous microcarrier.

Further, the activity of porous microcarriers carrying laccase of different pore sizes was determined using 0.5mM 2, 2-diaza-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) as substrate in 100mM sodium acetate buffer (0.1M, pH=4.5), one laccase activity unit being defined as the amount of enzyme required to oxidize 1. Mu. Mol of ABTS per minute.

Absorbance of the mixture was measured at 420nm using an ultraviolet-visible spectrophotometer and the laccase activity was calculated according to the following equation:

laccase activity (U/g) =65×Δa/W

Where ΔA represents the increased absorbance over a particular time interval and W represents the mass of the sample.

TABLE 1 enzyme immobilization amount and enzyme Activity in different laccase-supporting porous microcarriers

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

As can be seen from Table 1, MGelMA-CS-1 has an enzyme immobilization of 22.29mg/g and an enzyme activity of 75.7U/mg, shows an optimal performance in terms of loading capacity and enzyme activity maintenance, and has a larger specific surface area and a higher laccase loading capacity for small-pore hydrogel particles than large-pore hydrogel particles in the same volume.

Example 5

A degradation test was performed on bisphenol A using the laccase-loaded porous microcarrier MGelMA-CS-Lac-1 prepared in example 4 and 1mg of the free laccase, respectively. MGelMA-CS-Lac-1 and free laccase are respectively put into bisphenol A (BPA) solution with the concentration of 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L at 25 ℃, the pH value of the bisphenol A solution is adjusted to 5, the reaction is continuously oscillated for 6.5 hours and periodically sampled, the supernatant is filtered by a 0.22 mu m filter membrane, and the residual concentration of the BPA is measured.

FIG. 5 is a graph showing the removal efficiency of laccase-loaded porous microcarriers and equal amounts of free enzyme in example 5 of the disclosure for degrading bisphenol A solution of different concentrations, and as can be seen from FIG. 5, the removal efficiency of free laccase for BPA decreases as the initial concentration of BPA increases from 1mg/L to 50mg/L, about 33.02% of 50mg/L of BPA can be degraded, and the laccase-loaded porous microcarriers can effectively remove BPA of different concentrations, and the degradation efficiency of 50mg/L of BPA can reach 99.22%, compared with the degradation rate of free enzyme, which is improved by 3 times, because the protection effect of the biocompatible hydrogel microcarrier shell is laccase efficiency enhancement, and meanwhile, numerous interconnected and staggered network nanopores in the microcarrier can promote mass transfer and diffusion of substrate molecules, thereby promoting enhancement of enzyme activity.

Example 6

MGelMA-CS-Lac-1 loaded with 1mg laccase and 1mg free laccase are put into bisphenol A (BPA) solution with concentration of 20mg/L at 25 ℃, the pH range of the bisphenol A (BPA) solution is respectively regulated to 2-9, the reaction is continuously oscillated for 6.5 hours and periodically sampled, the supernatant is filtered by a 0.22 mu m filter membrane, and the residual concentration of the BPA is measured.

FIG. 6 is a graph showing the efficiency of degrading bisphenol A solution with equal amounts of free enzyme at different pH values, and shows the effect of pH on the free laccase and the laccase-supporting porous microcarrier, wherein the removal rates of the free laccase and the magnetic microcarrier immobilized laccase are increased and then decreased with increasing pH value, as shown in FIG. 6. Free laccase is more effective at removing BPA at ph=5 than other pH conditions. At pH 2,4,6, 7, 8 and 9, degradation rates were reduced by 86.7%, 47.6%, 6.6%, 14.8%, 30.7% and 32.1% compared to ph=5, respectively, with free laccase having greater limitations in application. The porous microcarrier loaded with laccase has higher BPA removal efficiency in the pH range of 5-8, the difference of the removal efficiency is less than 4%, and the sensitivity of the porous microcarrier loaded with laccase to pH is far lower than that of free enzyme. The microcarrier provides the microenvironment needed for protecting the laccase functional active site, stabilizing the bioactive conformation of the enzyme molecule. Compared with free laccase, the porous microcarrier loaded with laccase can effectively resist the interference of external environment.

Example 7

Under the condition of pH=5, MGelMA-CS-Lac-1 loaded with 1mg laccase and 1mg free laccase are respectively put into bisphenol A (BPA) solution with concentration of 20mg/L, the temperature of the bisphenol A (BPA) solution is regulated to be 5-55 ℃, the reaction is continuously oscillated for 6.5 hours and periodically sampled, supernatant is taken and filtered by a 0.22 mu m filter membrane, the residual concentration of BPA is measured, and the influence of the temperature on the activities of the free laccase and the laccase-loaded porous microcarrier is evaluated.

FIG. 7 is a graph showing the efficiency of degrading bisphenol A solution with laccase-supporting porous microcarrier and equal amount of free enzyme at different temperatures in example 7 of the present disclosure. As can be seen from FIG. 7, the laccase-supporting porous microcarrier has higher BPA removal performance than the free laccase at different test temperatures. For example, at 55 ℃, the removal rate of BPA by laccase-loaded porous microcarriers and free laccase was 99.4% and 63.3%, respectively. At lower temperatures of 5 ℃, BPA removal performance was slightly reduced at 79.2% and 45.8%, respectively. Laccase is fixed on the microcarrier through adsorption-covalent bond, has certain stability, and the microcarrier shell plays a role in protecting enzyme protein, prevents the conformation of enzyme molecules from being changed, and indirectly improves the thermal stability of laccase molecules.

Example 8

20mL of other bisphenol compound solution with the concentration of 20mg/L is prepared: BPB, BPC, BPE, BPF, BPZ, BPAF and tetrabromobisphenol A-bis (2, 3-dibromopropyl ether) (TBBPA). Adding 1mg of laccase-loaded porous microcarrier MGelMA-CS-Lac-1 and 1mg of free enzyme respectively, carrying out oscillation reaction for 6.5h at 25 ℃, filtering the supernatant with a 0.22 μm filter membrane, and measuring the residual concentration of different bisphenol compounds after degradation by laccase-loaded porous microcarrier and free laccase respectively.

FIG. 8 is a graph showing the removal efficiency of laccase-loaded porous microcarriers and equivalent amounts of free enzymes for degrading different bisphenol compounds in example 8 of the present disclosure, and as can be seen from the analysis of FIG. 8, the degradation efficiencies of the free laccase on 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, whereas the laccase-loaded porous microcarriers have degradation efficiencies of 82.17%, 99.07%, 93.10%, 96.60%, 97.99%, 56.58% and 67.55%. Compared with the equivalent amount of free enzyme, the porous microcarrier loaded with laccase has obviously improved degradation rate of bisphenol A analogues, can be used as an effective biocatalyst, and can degrade typical bisphenol compounds widely existing in water environment.

Example 9

The free laccase and the laccase-loaded porous microcarrier MGelMA-CS-Lac-1 were stored at 4℃and 25℃for 30 days, respectively, and the residual activity of laccase was measured every three days. The initial laccase activity is defined as 100%.

FIG. 9 is a graph showing laccase activity assays stored at various temperatures in example 9 of the present disclosure, and from the analysis of FIG. 9, the laccase-loaded porous microcarriers retain 83.3% of their initial activity when stored at 4℃for 15 days, 49.2% of their initial activity when stored at 25℃for 15 days, 65.2% and 33.9% of their initial activity when left at 4℃and 25℃for 30 days, and the free laccase retain only 39.3% and 8.2% of their relative activity when left at 4℃and 25℃for 30 days, respectively. Unlike free enzyme, the microcarrier can provide a good hydrophilic environment for enzyme molecules, so that the original activity of the enzyme molecules is maintained, and the microcarrier is convenient to store in industrial practical application and has great advantages.

Example 10

The removal efficiency of the laccase-loaded porous microcarrier on 10mg/L BPA solution was continuously measured, the reusability of the laccase-loaded porous microcarrier was evaluated, the pH of the BPA solution was adjusted to 5 at 25℃and 10mg/LBPA solution was degraded by using 1mg of laccase-loaded porous microcarrier MGelMA-CS-Lac-1, the reaction was continued for 6.5 hours with shaking, and the supernatant was filtered through a 0.22 μm filter membrane to determine the residual concentration of BPA. And (3) collecting the laccase-loaded porous microcarrier by using a magnet, washing the laccase-loaded porous microcarrier with a phosphate buffer solution for three times, and performing a repeated degradation test by using the recovered laccase-loaded porous microcarrier, wherein the relative removal efficiency of the immobilized enzyme is calculated by taking the efficiency of the first reaction as 100%.

FIG. 10 is a graph showing changes in relative activity after repeated use of laccase-supporting porous microcarriers in example 10 of the present disclosure, and it is known from analysis of the results in FIG. 10 that after 5 times of repeated use, the removal rate of BPA by laccase-supporting porous microcarriers remains above 80%, and decreases to 73.7% in cycle 10, which fully proves that the porous microcarriers impart reusability to laccase, and that the decrease in enzyme activity in continuous cycles may be due to accumulation of reaction products blocking a part of holes in microcarriers and small amount of immobilized laccase. The reusability of the laccase-loaded porous microcarrier reduces the cost in practical applications.

According to the porous microcarrier loaded with laccase, the preparation method and the application, the porous microcarrier with the three-dimensional porous structure and the interconnected nano channels is prepared by calcining the silicon dioxide photonic crystal to form the template, more amino groups are contained in the structure of the porous microcarrier, a large number of binding sites are provided for laccase fixation, and in addition, the introduction of the ferroferric oxide nano particles provides magnetic capacity for the microcarrier, so that the synthesized biocatalyst has recovery potential. The prepared laccase-loaded porous microcarrier has the protection of a biocompatible hydrogel shell and the domain-limiting effect of nano holes, and a plurality of interconnected and staggered network nano holes in the porous microcarrier can promote mass transfer and diffusion of substrate molecules, so that the enhancement of enzyme activity is promoted, the laccase-loaded porous microcarrier has higher stability under a certain mild pH range, and the bisphenol compound is degraded more efficiently.

While the foregoing is directed to embodiments of the present disclosure, other and further details of the invention may be had by the present application, it is to be understood that the foregoing description is merely exemplary of the present disclosure and that no limitations are intended to the scope of the disclosure, except insofar as modifications, equivalents, improvements or modifications may be made without departing from the spirit and principles of the present disclosure.

Claims

1. A laccase-loaded porous microcarrier comprising:

a hydrogel porous microcarrier;

laccase supported on the hydrogel porous microcarrier by covalent binding;

2. The porous microcarrier of claim 1, wherein,

the hydrogel porous microcarrier has an adjustable pore size;

the load capacity of the laccase is 0.2-1%;

the concentration of the chitosan is 0.5-3%;

the composition of the methacrylic acid hydrogel microcarrier comprises: 15 to 30 percent of methacrylic acylated gelatin, 20 to 50 percent of polyethylene glycol diacrylate and 0.2 to 0.5 percent of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate.

3. The porous microcarrier of claim 2, wherein,

the composition of the methacrylic acid hydrogel microcarrier can also comprise 5-10% ferroferric oxide nano particles, so as to provide magnetism for the porous microcarrier.

4. A method of preparing the laccase loaded porous microcarrier of any one of claims 1-3, comprising:

soaking the microsphere template of the silicon dioxide photonic crystal in the precursor solution of the methacrylic acid hydrogel, and performing irradiation polymerization by an ultraviolet lamp to obtain composite photonic crystal microspheres;

after the methacrylic acid porous microcarrier is dried, adding chitosan hydrogel solution for filling to form a chitosan-methacrylic acid porous microcarrier;

5. The method of claim 4, wherein,

the particle size of the silica nano particles comprises 50-1000 nm;

the mass concentration of the disperse phase is 15-30% (w/v).

6. The method of claim 4, wherein,

the calcining temperature is 600-1000 ℃;

the calcination time is 3-6 hours;

the soaking time is 2-8 h.

7. The method of claim 4, wherein,

the irradiation wavelength of the ultraviolet lamp is 330-450 nm;

the irradiation intensity of the ultraviolet lamp is 50-200W;

the irradiation time of the ultraviolet lamp is 1-5 min.

8. The method of claim 4, wherein,

the concentration of the hydrofluoric acid solution is 2-8%;

the etching treatment time is 2-8 hours;

the concentration of the chitosan is 0.5-3%;

the filling time is 6-12 h.

9. The method of claim 4, wherein,

the concentration of glutaraldehyde is 0.5-5%;

the activation time is 1-6 h;

the concentration of laccase is 0.5-2 mg/mL;

the time of the covalent bonding reaction is 2-10 h.

10. A method of degrading bisphenol compounds comprising:

adding the laccase-loaded porous microcarrier of any one of claims 1-3 to a bisphenol compound solution to degrade the bisphenol compound;

and after the degradation reaction is finished, recovering the porous microcarrier loaded with laccase, and washing by using a buffer solution for repeated use.