Immobilized laccase based on inorganic porous material, its preparation method and application

Abstract

The present application relates to the field of biological enzyme catalyst, especially to a kind of immobilized laccase based on inorganic porous material and its preparation method and application, the immobilized laccase based on inorganic porous material designed in the present application, the immobilized laccase includes inorganic porous material and laccase adsorbed and fixed on the surface of inorganic porous material, the inorganic porous material is prepared by dynamic hydrothermal synthesis method with piperidine as template agent, with aluminum isopropylate as aluminum source, boric acid as boron source, fumed silica as silicon source. Laccase is adsorbed and immobilized by using the inorganic porous material, and the immobilized laccase catalyst obtained has good enzymatic performance, and the immobilized laccase has significant effect in removing quinolone antibiotics.

Description

Technical Field

[0001] This invention relates to the field of bio-enzyme catalysts, and more particularly to an immobilized laccase based on inorganic porous materials, its preparation method, and its application. Background Technology

[0002] Quinolone antibiotics (QNs) are widely used in healthcare and aquaculture due to their cost-effective antibacterial activity. Furthermore, because of their stable chemical structure and difficulty in biodegradation, quinolone antibiotics have also been detected in groundwater, lake water, and wastewater treatment plants. However, the release of incompletely metabolized QNs into water can lead to antibiotic resistance in microbial populations, reducing their efficacy and endangering human health. Currently, methods such as photodegradation, electrochemistry, and advanced ozone-mediated oxidation are commonly used to remove antibiotics from water. However, these methods are costly to operate and prone to generating secondary pollutants. Enzymatic methods, with their advantages of speed, efficiency, high specificity, and mild reaction conditions, have recently become a focus of research. Laccase (EC 1.10.3.2) has a strong oxidizing capacity and shows great potential in treating organic pollutants. Researchers have begun to study the biodegradation of antibiotics using laccase; however, laccase suffers from poor stability, easy inactivation, and difficulty in separating it from other components in the reaction system, resulting in low degradation efficiency. Currently, immobilizing laccase on a carrier is a common method to improve laccase degradation efficiency.

[0003] Commonly used enzyme immobilization materials include metal-organic frameworks (MOFs), magnetic nanomaterials (MNPs), and zeolite molecular sieves. MOFs offer advantages such as high designability and abundant functional groups, but their production cost is high, and the microporous channels can restrict enzyme binding, negatively impacting enzyme activity. Magnetic nanomaterials are easily separated from the reaction medium, but iron particles can leach out in acidic environments, causing secondary pollution. Zeolite molecular sieves are traditional inorganic materials, ideal for enzyme immobilization due to their excellent thermal stability, relatively low cost, high porosity, and environmental compatibility. However, the limited external surface area of ​​microporous zeolite molecular sieves restricts their application in enzyme immobilization. For example, in the Chinese invention patent application document with application number 202410697383.6 and invention title "A MOF-based immobilized laccase material and its preparation method and application", it is mentioned that "a novel LMS@ZIF-8 material was developed, which effectively immobilizes laccase and eugenol in ZIF-8. A co-immobilization strategy was adopted, using eugenol as a mediator to simultaneously immobilize laccase and eugenol; the immobilized laccase material obtained has a significantly higher degradation efficiency for antibiotics such as sulfamethoxazole compared with free laccase." The carrier used in this invention patent application is LMS@ZIF-8 material, which is used to immobilize laccase and eugenol by embedding polyvinylpyrrolidone and 2-methylimidazole. This embedding and immobilization method can easily affect the activity of laccase. As can be seen from the data in Table 1 of the examples, the enzyme activity of the immobilized material can only retain up to 72.97% of the original laccase activity. The 72.97% enzyme activity recovery rate still cannot meet the current industrial needs of immobilized enzymes, and the enzymatic performance of immobilized enzymes needs to be further improved. Summary of the Invention

[0004] To overcome the above problems, the purpose of this invention is to provide an immobilized laccase catalyst based on an inorganic porous material, its preparation method, and its application. The immobilized laccase catalyst obtained by adsorbing and immobilizing laccase using this inorganic porous material has good enzymatic properties, and the immobilized laccase has a significant effect on the removal of quinolone antibiotics.

[0005] To achieve the above objectives, the present invention designs an immobilized laccase based on an inorganic porous material. The immobilized laccase comprises an inorganic porous material and laccase adsorbed and immobilized on the surface of the inorganic porous material. The inorganic porous material is prepared by a dynamic hydrothermal synthesis method using piperidine as a template agent, aluminum isopropoxide as an aluminum source, boric acid as a boron source, and fumed silica as a silicon source. The inorganic porous material is in the form of microspheres, with a large number of irregularly arranged nanosheets distributed on its surface. Adjacent nanosheets enclose and form channels for accommodating laccase molecules. The inner walls of these channels are rough and have a groove-shaped angled structure. The roughness and groove-shaped angled structure of the inner walls of the channels form a gripping force for immobilizing the laccase. The surface of the inorganic porous material also has hydroxyl groups, which form hydrogen bonds with the NH2 of the laccase molecules. The laccase molecules are immobilized on the surface of the inorganic porous material by the hydrogen bonds and the gripping force of the inner walls of the channels. The specific explanation of the gripping force is as follows: the abundant cup-shaped pores on the surface of the nanosheets increase the surface roughness of the material, which can provide an effective gripping force for the adsorption of laccase molecules. Moreover, the angle formed by the irregularly staggered arrangement of nanosheets can also provide gripping force to trap some laccase molecules, making them less likely to fall off.

[0006] As a preferred embodiment, the pores on the surface of the inorganic porous material are multi-level pores, including micropores, mesopores and macropores, with an average pore diameter of 8~9 nm.

[0007] As a preferred embodiment, the laccase molecules are immobilized within the mesopores and macropores of the inorganic porous material.

[0008] As a preferred embodiment, the laccase is obtained through the recombinant strain X33 / pPICZαA- BpLac The recombinant strain X33 / pPICZαA- was obtained through exogenous expression. BpLac The recombinant plasmid pPICZαA- was obtained by inserting the laccase gene, as shown in SEQ ID NO: 1, into the pPICZαA plasmid. BpLac, Then the recombinant plasmid pPICZαA‐ BpLac It is formed by electroporation into Pichia pastoris X33.

[0009] A method for preparing immobilized laccase based on inorganic porous materials, characterized by comprising the following steps:

[0010] (1) Preparation of inorganic porous materials:

[0011] Inorganic porous materials were prepared by dynamic hydrothermal synthesis using piperidine as a template agent, aluminum isopropoxide as an aluminum source, boric acid as a boron source, and fumed silica as a silicon source. The surface of the inorganic porous materials is covered with a large number of irregularly arranged nanosheets. Adjacent nanosheets enclose pores to accommodate laccase molecules. The inner walls of these pores are rough and have a groove-shaped angle structure. The roughness and groove-shaped angle structure of the inner walls of the pores form a gripping force to fix the laccase. The surface of the inorganic porous materials also has hydroxyl groups that can form hydrogen bonds with NH2 laccase molecules.

[0012] (2) Adsorption and fixation

[0013] Inorganic porous materials are mixed with laccase to obtain a mixture. In the mixture, laccase molecules enter the pores of the inorganic porous materials under the adsorption of the inorganic porous materials. Furthermore, under the hydrogen bonding force of the inorganic porous materials and the gripping force of the inner wall of the pores, the laccase molecules are fixed on the surface of the inorganic porous materials.

[0014] (3) Collect immobilized laccase:

[0015] The mixture was centrifuged to collect the solids, which were then washed to remove unbound laccase molecules, resulting in immobilized laccase.

[0016] As a preferred embodiment, the specific process for preparing inorganic porous materials in step (1) is as follows: First, prepare the raw materials: the molar ratio of deionized water, piperidine, aluminum isopropoxide, boric acid, and fumed silica is 20~25:1~2:0.01~0.02:1~2:1~2; then add aluminum isopropoxide to the deionized water, and after complete hydrolysis, add piperidine, stir magnetically until uniform, add boric acid, stir again until uniform to obtain a first mixed solution, add fumed silica to the first mixed solution and stir until uniform to obtain a second mixed solution, place the second mixed solution in a homogeneous reactor for aging and crystallization, after crystallization, cool, clean and dry in sequence, and finally calcine in a muffle furnace to remove piperidine, and finally grind into powder to obtain inorganic porous materials.

[0017] As a preferred embodiment, the aging speed is 50~60 rpm, the temperature is 120~130℃, and the time is 8~12h; the crystallization temperature is 160~170℃ and the time is 2~3 days; and the muffle furnace calcination temperature is 500~600℃ and the calcination time is 10~12 hours.

[0018] As a preferred embodiment, in step (2), the adsorption and fixation parameters are: 5 mg of inorganic porous material, 0.5-2 ml of laccase, temperature of 20-30℃, rotation speed of 100-200 rpm, and time of 18-24 hours, wherein the enzyme activity of laccase is 20.35 U / mL and the concentration of laccase protein is 0.37 mg / ml.

[0019] An application of immobilized laccase in the removal of quinolone antibiotics from wastewater is characterized in that the catalytic system for removing quinolone antibiotics from wastewater comprises: quinolone antibiotics, immobilized laccase, and syringaldehyde.

[0020] As a preferred embodiment, the quinolone antibiotics include ciprofloxacin, enrofloxacin, norfloxacin, and moxifloxacin, and the catalytic system specifically includes: 30 mg / L quinolone antibiotics, 300 U / L immobilized laccase, and 1 mM syringaldehyde.

[0021] Compared with traditional methods for preparing immobilized laccase catalysts, the present invention has the following advantages:

[0022] (1) The present invention utilizes adsorption to fix laccase, which has less impact on the enzymatic properties of laccase compared to embedding. Moreover, the use of the inorganic porous material IPM of the present invention as a carrier improves the stability and catalytic efficiency of laccase.

[0023] (2) Immobilized laccase using the present invention Bp Lac@IPM significantly improves the degradation efficiency of quinolone antibiotics, including ciprofloxacin (CIP). Bp The removal rate of the Lac@IPM+SA system was 94.70±1.08%, compared to Bp The Lac+SA system improved by about 8% (86.15±2.27%) for norfloxacin (NOR). Bp The removal rate of the Lac@IPM+SA system was 92.11±2.55% for enrofloxacin (ENR). Bp The ENR removal rate of the Lac+SA system was only 15.85% ± 3.77%. Bp The Lac@IPM+SA system achieved a removal rate as high as 77.08±1.97% for moxifloxacin (MOX). Bp The removal rate of MOX in the Lac+SA system was only 10.37% ± 4.30%. Bp The removal rate of the Lac@IPM+SA system was as high as 81.77±5.69%. Attached Figure Description

[0024] Figure 1 Field emission scanning electron microscope image of inorganic porous material IPM;

[0025] Figure 2 This is a pore size distribution diagram of the inorganic porous material IPM;

[0026] Figure 3 The image of the recombinant plasmid pET28a-Lac;

[0027] Figure 4SDS-PAGE gel electrophoresis image of laccase BpLac obtained after purification and ultrafiltration.

[0028] Figure 5 Field emission scanning electron microscope image of immobilized laccase BpLac@IPM;

[0029] Figure 6 The XRD spectra of the IPM material before and after BpLac loading are shown.

[0030] Figure 7 shows the FTIR spectra of the IPM material before and after BpLac loading;

[0031] Figure 8 The BET pore size distribution before and after IPM immobilization is shown.

[0032] Figure 9 The graph shows the effect of pH on BpLac and BpLac@IPM.

[0033] Figure 10 The graph shows the effect of temperature on BpLac and BpLac@IPM.

[0034] Figure 11 The temperature stability curves of BpLac and BpLac@IPM at room temperature of 25℃ are shown.

[0035] Figure 12 The pH stability curves of BpLac and BpLac@IPM at pPH4 and pH7 are shown.

[0036] Figure 13 A bar chart showing the binding stability of BpLac@IPM;

[0037] Figure 14 A bar chart showing the reusability of BpLac@IPM;

[0038] Figure 15 A graph showing the removal effect of CIP;

[0039] Figure 16 This is a graph showing the removal effect of NOR;

[0040] Figure 17 A graph showing the removal effect of ENR;

[0041] Figure 18 The graph shows the removal effect of MOX.

[0042] Figure 19 Effects of ciprofloxacin catalytic products on the growth of Escherichia coli DH5α;

[0043] Figure 20A graph assessing the contributions of adsorption and catalysis to the removal of quinolone antibiotics;

[0044] Figure 21 Fourier transform infrared spectra of BpLac@IPM before and after binding with quinolone antibiotics;

[0045] Figure 22 This is a schematic diagram illustrating the removal mechanism of ciprofloxacin. Detailed Implementation

[0046] To better understand the present invention, the invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0047] Example 1: Preparation of Inorganic Porous Material IPM

[0048] Inorganic porous material IPM was prepared by dynamic hydrothermal synthesis using piperidine as a template agent, aluminum isopropoxide as the aluminum source, boric acid as the boron source, and fumed silica as the silicon source. The specific operation process is as follows: First, as shown in Table 1, the raw materials were prepared. Then, aluminum isopropoxide was added to deionized water and hydrolyzed for 20 h. Piperidine was then added, and the mixture was magnetically stirred for 30 min before adding boric acid to obtain the first mixed solution. After stirring the first mixed solution again for 1 h, fumed silica was added uniformly in three portions over 1 h and stirred for 1 h to obtain the second mixed solution. Then, the magnetic stir bar was removed using a magnet, and the second mixed solution was placed in a polytetrafluoroethylene liner and aged in a homogeneous reactor. The aging parameters were: rotation speed of 56 rpm, temperature of 130 °C, and time of 12 h. Crystallization was then carried out for 3 days at a temperature of 170 °C. After crystallization, the sample was cooled with tap water and the filtrate was washed with deionized water until neutral. After drying in an oven (100℃) for 12 h, it was calcined in a muffle furnace at 550℃ for 10 h to remove the template agent piperidine. Finally, the sample was ground into powder and named Inorganic Porous Material IPM. This powder was used as the carrier material IPM for subsequent laccase processing, as shown in Table 1. The prepared IPM has a large surface area and good adsorption performance.

[0049] Table 1. Raw materials for IPM preparation and IPM performance

[0050]

[0051] Example 2 Characteristic Detection of Inorganic Porous Materials IPM

[0052] Combination Figure 1As shown, electron microscopy was performed on the inorganic porous material IPM obtained in the first group of Example 1. The IPM exhibits a microsphere morphology with a cauliflower-like surface. Numerous irregularly arranged nanosheets are distributed on its surface, forming channels to accommodate laccase molecules. Unlike traditional regular geometric inner walls, the inner walls of these channels are rough and have irregular groove-like angles. This type of channel exhibits greater microscopic friction, making it difficult for protein molecules to escape after entering. Due to the varying tilt angles of the nanosheets, the pore sizes vary. These channels are hierarchical, including micropores, mesopores, and macropores. Micropores have a diameter less than 2 nm, mesopores (also called pores) have a diameter between 2 and 50 nm, and macropores have a diameter greater than 50 nm. Figure 2 As shown, the inorganic porous material IPM has microporous, mesoporous, and macroporous structures.

[0053] Furthermore, in hydrothermal synthesis, the terminal oxygen atoms of the silicon-oxygen tetrahedra formed by silicon and aluminum sources readily combine with water molecules to form silanol groups (Si-OH) or aluminumol groups (Al-OH). Therefore, the synthesized inorganic porous material IPM has a large number of hydroxyl groups on its surface. These numerous hydroxyl groups exhibit hydrogen bonding with the NH2 groups of laccase molecules, thereby promoting laccase formation. Bp The bonding between Lac and the inorganic porous material IPM is stable.

[0054] Example 3 Laccase Bp Lac Acquisition

[0055] Laccase in this embodiment Bp Lac from recombinant strain X33 / pPICZαA- Bp Lac expression was obtained. Bp The purification of Lac and the quantification of the protein were performed using Ni. 2+ Affinity chromatography and the Bradford method.

[0056] 1. Amplification of the laccase gene

[0057] The laccase gene sequence is shown in SEQ ID NO: 1. The laccase gene originates from... Bacillus pumilus strain ZB1laccase gene (GenBank: MW373470.1), laccase as shown in SEQ ID NO: 1 Bp The Lac gene was synthesized by a gene company and loaded into the pET28a plasmid, resulting in... Figure 3 Recombinant plasmid pET28a-Lac.

[0058] The laccase gene was amplified using the forward primer (Table 2) pPICZαA-Lac-F and the reverse primer pPICZαA-Lac-R. Bp The PCR amplification reaction system is shown in Table 3, and the reaction procedure is shown in Table 4. After the target gene amplification was completed, the PCR products were verified and recovered by nucleic acid gel electrophoresis.

[0059] Table 2 Primer sequence listing

[0060]

[0061] Table 3 Laccase PCR Amplification Reaction System

[0062]

[0063] Table 4 Laccase PCR Amplification Reaction Procedure

[0064]

[0065] 2. Recombinant plasmid pPICZαA- Bp Lac build

[0066] First, the pPICZαA plasmid was double-digested. The double digestion system of pPICZαA plasmid is shown in Table 5. The reaction was carried out at 37℃ for 15 min. The digestion products were recovered by nucleic acid gel electrophoresis.

[0067] Table 5. Double enzyme digestion system of plasmid pPICZαA

[0068]

[0069] Laccase gene ( Bp Lac) was ligated with double-digested pPICZαA enzyme. The ligation reaction system consisted of 1 µL linearized vector and 1 µL pPICZαA enzyme. Bp The Lac gene fragment, 2 µL Exnase II, 4 µL Exnase II buffer, and 12 µL deionized water were reacted at 37 °C for 30 min. Finally, DH5α was transformed using a standard transformation method. The transformation product was plated on LB agar plates (containing 100 µg / mL bleomycin) and incubated at 37 °C for 8–12 h.

[0070] Single colonies of the recombinant bacteria were picked for verification. After successful verification, the glycerol-containing bacteria were preserved, and pPICZαA- was extracted using a plasmid extraction kit. Bp Lac plasmid. Then the recombinant plasmid pPICZαA- BpLac was linearized with Sac I, verified by nucleic acid gel electrophoresis, and recovered. It was then transformed into Pichia pastoris X33 via electroporation, plated onto YPD solid plates (containing 100 µg / mL bleomycin), and incubated at 28°C for 48 h.

[0071] 3. Expression and purification of laccase

[0072] Transformants X33 / pPICZαA- grown on YPD plates containing bleomycin Bp Lac was inoculated into 50 mL of YPD medium, maintained, and then transferred at a 2% inoculum to BMGY medium supplemented with 0.5 mM CuSO4 and 0.2% biotin. The medium was incubated at 28°C with shaking at 200 rpm. When OD... 600 At approximately 2-6 hours, bacterial cells were collected and transferred to BMMY medium supplemented with 0.5 mM CuSO4 and 0.2% biotin. The cells were then cultured at 18°C ​​with shaking at 200 rpm, and expression was induced daily with 0.5% methanol. Bp Lac samples were taken to measure laccase activity. When the enzyme activity reached its maximum, the bacteria were harvested, and the supernatant was retained. The supernatant was concentrated using an ultrafiltration system with a molecular cutoff of 10 kDa for the membrane.

[0073] Laccase purification: Ni column was equilibrated at pH 8.0 with 50 mM phosphate buffer, then impurities were eluted with pH 8.0 phosphate buffer containing 0.5 M NaCl and 20 mM imidazole, and the target protein was eluted with pH 8.0 phosphate buffer containing 300 mM imidazole and 0.5 M NaCl.

[0074] After purification, the eluent was ultrafiltered using a 10 kDa ultrafiltration tube. For example... Figure 4 As shown, the laccase obtained after purification and ultrafiltration Bp The SDS-PAGE gel electrophoresis image of Lac showed that its enzyme activity was 20.35 U / mL and its protein concentration was 0.37 mg / mL.

[0075] Laccase Bp Lac enzyme activity assay: Lac enzyme activity was determined using ABTS as a substrate in a sodium acetate buffer system (0.2 M, pH 4.0) at room temperature. The final ABTS concentration was 1 mM. The reaction mixture consisted of 940 μL of 0.2 M sodium acetate buffer (pH 4.0) and 10 μL of the laccase to be tested. The oxidation of ABTS was measured at λ = 420 nm (ε = 36000 M). -1 cm -1 One enzyme activity unit is the amount of 1 μmol ABTS oxidized per minute.

[0076] Example 4 Bp Preparation of Lac@IPM

[0077] By adsorption method Bp Lac was immobilized on the inorganic porous material IPM. The specific procedure was as follows: 2.5 mg of the inorganic porous material IPM (obtained in the first group of Example 1) was weighed and mixed with 1 ml of... Bp After mixing with Lac (obtained in Example 3), the mixture was shaken in a shaker at 25°C and 150 rpm for 24 h, and the immobilized laccase was collected by centrifugation. Bp Lac@IPM, then wash three times with phosphate buffer (pH 7.0, 50 mM) to remove unbound molecules. Bp Lac, after freeze-drying, yields immobilized laccase. Bp Lac@IPM.

[0078] Example 5 Bp Characterization of particle shape and surface properties of Lac@IPM

[0079] Combination Figure 5 As shown, immobilized laccase Bp The FESEM image of Lac@IPM shows that after fixation... Compared to the original IPM, Lac@IPM did not undergo any significant changes in morphology.

[0080] The crystal structure and phase changes before and after IPM immobilization were analyzed by X-ray diffraction (XRD). Bp As shown, the comparison reveals that IPM material has... Figure 6 Both before and after Lac loading, high-intensity diffraction peaks were observed at similar positions, indicating that... The introduction of Lac did not cause any destructive changes to the properties of the support material itself. The strongest peak position (intensity) of the IPM support material before loading was 26.62◦ (504.70), and the position of the maximum diffraction peak remained unchanged after loading.

[0081] IPM and Bp The FTIR spectrum of Lac@IPM is as follows: Bp As shown, the FTIR spectra of the material before and after adsorption exhibit high similarity. At 447 cm⁻¹ -1 809 cm -1 and 1079 cm -1 Characteristic infrared absorption bands of Si-O were observed at all locations. Furthermore, Bp Lac@IPM at 610 cm -1 The skeletal vibration band remains intact. (Explanation) Figure 7 The combination of Lac and IPM does not affect the skeletal structure of IPM, which is similar to the XRD findings.

[0082] BET pore size distribution before and after IPM immobilization is as follows: Bp As shown. Combined with Bp It can be seen Figure 8 The Lac load occupies part of the mesopores and macropores of the IPM, resulting in a reduction in surface area during measurement. Referring to Table 6, and... Figure 8 After Lac bonding, the pore size increased from 8.71 nm to 21.16 nm. This is because... Bp Lac molecules are larger than some pore sizes in the IPM, causing localized expansion of the IPM. This phenomenon further proves... Bp The successful integration of Lac with IPM.

[0083] Table 6 Physical properties of IPM

[0084]

[0085] Example 6 Bp Lac@IPM and Bp Enzymatic properties of Lac

[0086] 6.1 Bp Lac and Bp Substrate binding capacity and catalytic rate of Lac@IPM

[0087] Referring to Table 7, Bp Lac and Bp The maximum reaction rates of Lac@IPM were 0.62 and 0.73 μM min, respectively. -1 ,show Bp Lac@IPM reacts faster with ABTS. m The values ​​were 2287.90 and 2008.74 μM, respectively, indicating that... Bp The affinity ratio between Lac@ IPM and ABTS Bp Lac is larger. Specificity constant (K) cat / K m The concentrations were 0.018 and 0.024 μM, respectively. -1 s -1 These findings suggest that, under specific conditions, Bp Lac@IPM catalyzes ABTS substrates more efficiently, and also constructs... Bp Lac@IPM does not cause Bp Lac activity is reduced.

[0088] Table 7 Bp Lac and Bp Dynamic parameters of Lac@IPM

[0089]

[0090] 6.2 Bp Lac and Bp Optimal pH and temperature for Lac@IPM

[0091] The reaction temperature was 25℃, and the results were measured in buffer solutions with pH values ​​ranging from 3 to 10. Lac and Bp The enzyme activity of Lac@IPM, and the effect of different pH values ​​on enzyme activity, are as follows: Bp As shown. The results indicate... Bp Lac activity and Bp Lac@IPM is sensitive to pH changes, with an optimal pH of pH 4. Furthermore, under pH 3 conditions, Figure 9 Lac and Bp Lac@IPM showed activities of 48.84%±0.52% and 77.26%±0.21%, respectively, indicating that... Bp Lac@IPM exhibits greater resistance to acidic conditions. Studies using ABTS as a substrate... Bp Lac and Bp The catalytic ability of Lac@IPM at different temperatures, such as Bp As shown. It was found that with changes in temperature, Bp Lac and Bp The catalytic activity of Lac@IPM shows a similar trend, with the optimal temperature for both being 60℃.

[0092] 6.3 Figure 10 Lac and Bp Stability of Lac@IPM

[0093] High stability is a crucial guarantee for the practical application of biocatalysts; therefore, using ABTS as a reaction substrate is appropriate. Bp Lac and Bp A comparative analysis was conducted on the pH and room temperature storage stability of Lac@IPM. Details are as follows: Bp and Bp As shown, it was found that under acidic (pH 4) and neutral (pH 7) conditions, [the following is a description of the effect of pH 4 on the relationship between pH 4 and pH 7]. Bp Compared to Lac, Figure 11 Lac@IPM exhibits better stability after 10 days of storage at room temperature (25°C). Figure 12 Lac retains 3.27% ± 0.43% of its relative activity, while Bp Lac@IPM exhibits a relative activity of 75.44% ± 2.84%. These results indicate that IPM is... Bp Lac provides a protective layer, mitigating conformational changes or denaturation caused by temperature and pH. Bp The good pH stability and room temperature storage stability of Lac@IPM indicate its application potential.

[0094] 6.3 IPM and Bp Lac binding stability

[0095] Will Bp After centrifuging Lac@IPM at 8000 mpm for 4 minutes, the mixture was collected for enzyme activity determination. Bp As shown, after 5 cycles, Bp Lac@IPM still retains 99.47% activity, indicating that the IPM material and... Bp Lac bonding exhibits good bonding stability. IPM materials contain abundant mesopores and macropores, forming groove-shaped angles. Figure 13 Lac enters the IPM surface and is completely adsorbed onto the rough surface of the nanosheets, especially due to the groove-shaped angle between them, making desorption difficult. Furthermore, the hydroxyl groups on the IPM surface form hydrogen bonds with the NH2 groups of the laccase molecules, further promoting adsorption. Bp The combination of Lac and IPM is more stable.

[0096] 6.4 Bp Lac@IPM's reusability

[0097] Reusability is Bp Lac is a commonly used performance evaluation metric in industrial applications. Bp Unlike Lac, immobilized enzymes can be reused and are easily separated from the reaction system. Bp It can be seen that as the number of reuses increases, Bp The activity of Lac@IPM gradually decreased, and after 5 cycles, 87.84 ± 2.07% of the enzyme activity remained. This phenomenon indicates that immobilization... Bp Lac has excellent reusability, which makes it possible to reduce its application costs.

[0098] 6.5 Figure 14 Calculation of Lac@IPM loading capacity, immobilization efficiency, and specific enzyme activity.

[0099] Immobilization System 1: Weigh 5 mg of inorganic porous material IPM (obtained from the first group of Example 1) and 1 ml of... Bp After mixing Lac (laccase obtained in Example 3), it was shaken in a shaker at 25°C and 150 rpm for 24 h.

[0100] Immobilization System 2: Weigh 5 mg of inorganic porous material IPM (obtained in Example 1) and 4 ml of... Bp After mixing Lac (laccase obtained in Example 3), it was shaken in a shaker at 25°C and 150 rpm for 24 h.

[0101] Determining the effect of IPM on two systems Bp The adsorption effect of Lac was calculated at 0h, 0.167h, 0.5h, 1h, 6h, 18h and 24h respectively. Bp The immobilization efficiency, loading capacity, and specific enzyme activity of Lac@IPM are shown in Tables 8 and 9, respectively.

[0102] Bp The specific steps for improving the immobilization efficiency of Lac@IPM are as follows:

[0103] The protein concentration of laccase at 0.0 h is recorded as the initial protein concentration A0, and the protein concentration of the supernatant in the mixed sample taken at time t is recorded as the residual protein concentration A0. t . Bp The Lac immobilization efficiency is shown in equation (1):

[0104] (1)

[0105] Among them, A0 and A t These represent the initial protein concentration (mg / mL) and the residual protein concentration (mg / mL), respectively.

[0106] Measurement Bp The method for loading Lac@IPM is as follows: the protein concentration of laccase at 0.0 h is recorded as the initial protein concentration C. a The protein concentration in the supernatant of the mixed sample taken at time t is denoted as the residual protein concentration C. b .

[0107] Bp Lac@IPM Bp The Lac load is shown in equation (2):

[0108] (2)

[0109] Where V is the volume of the immobilized system, m is the mass of the IPM, and C a and C b These represent the initial protein concentration and the residual protein concentration (mg / mL), respectively.

[0110] Measurement Bp The method for comparing the enzyme activity of Lac@IPM is as follows: ABTS is used as a substrate, and the reaction is carried out in a sodium acetate buffer system (0.2M, pH 4.0) at room temperature. The final concentration of ABTS is 1mM in the 0.2M sodium acetate buffer (pH 4.0). Immobilized samples from two different immobilization systems are taken separately. Bp Lac@IPM was used to measure the oxidation of ABTS at λ=420 nm, and the results were obtained. Bp The enzyme activity of Lac@IPM, oneBp Lac@IPM enzyme activity is measured in units of the amount of ABTS oxidized per minute (1 μmol). a Enzyme activity per gram of carrier (U / g); B o For formula (2) Bp Lac load capacity;

[0111] Bp The specific enzyme activity of Lac@IPM is shown in formula (3):

[0112] (3)

[0113] Among them, E a It is the enzyme activity per unit carrier (U / g), B o unit carrier Bp The Lac protein concentration (mg / g) is also obtained from the above formula (2). Bp Lac load capacity.

[0114] Two systems Bp The experimental results of Lac@IPM loading capacity, immobilization efficiency, and specific enzyme activity are shown in Tables 8 and 9.

[0115] Table 8. Immobilization System 1 Bp Immobilization efficiency and catalytic performance of Lac@IPM

[0116]

[0117] Table 9. Immobilization System 2 Bp Immobilization efficiency and catalytic performance of Lac@IPM

[0118]

[0119] As can be seen from Table 8, Bp The immobilization efficiency of Lac@IPM can reach 100%, and the specific enzyme activity of free laccase in immobilization system 1 was measured to be 54.61±5.84 U / mg. This was achieved after an immobilization process of 18-24 hours. Bp The specific enzyme activity of Lac@IPM was 52.23±2.82~55.77±2.89 U / mg, indicating that the immobilized enzyme activity... Bp The laccase activity of Lac@IPM was not affected.

[0120] As can be seen from Table 9, when Bp Lac@IPM Bp Excessive loading of Lac will decrease the specific activity of immobilized laccase, therefore it needs to be controlled. Bp Lac@IPM Bp Lac's load capacity, in order to improveBp The specific enzyme activity of Lac@IPM was optimized by immobilization parameters, and orthogonal experiments were conducted. The results are shown in Table 10.

[0121] Table 10 Optimization Table of Immobilization Parameters

[0122]

[0123] As can be seen from Table 10 above, when the IPM in the immobilized system is 5 mg, Bp Increasing the Lac concentration to 3 ml decreased the immobilization efficiency and enzyme activity; therefore, control was necessary. Bp The amount of Lac is 0.5~2ml, and the specific enzyme activity of the immobilized laccase is basically the same as that of the free laccase.

[0124] Example 7: Removal effect of quinolone antibiotics

[0125] The catalytic reaction experiment was conducted at 25°C in a 50 mM phosphate buffer solution at pH 7.0. The buffer solution contained 30 mg / L of contaminants (ciprofloxacin CIP, norfloxacin NOR, enrofloxacin ENR, and moxifloxacin MOX), 1 mM of syringaldehyde (SA), and 300 U / L of [unspecified substance]. Bp Lac@IPM or Bp Lac. investigated the effects of different systems on the removal efficiency of quinolone organic pollutants. Specifically, the removal efficiency of five systems on quinolone organic pollutants was studied, namely... Bp Lac system, SA system Bp Lac+SA system Bp Lac@IPM system Bp Lac@IPM+SA system.

[0126] The specific components of each system are as follows:

[0127] Bp Lac system: 30 mg / L pollutant, 300 U / L Bp Lac;

[0128] SA system: 30 mg / L pollutant, 1 mM SA;

[0129] Bp Lac+SA system: 30 mg / L pollutant, 300 U / L Bp Lac and 1mM SA;

[0130] Bp Lac@IPM system: 30 mg / L pollutant, 300 U / L Bp Lac@IPM;

[0131] Bp Lac@IPM+SA system: 30 mg / L pollutant, 300 U / L Bp Lac@IPM, 1mM SA.

[0132] The above Bp The Lac and SA systems showed no significant effect in removing quinolone antibiotics (CIP, NOR, ENR, and MOX), as detailed in Table 11. Table 11 shows that... Bp The Lac and SA systems showed no significant degradation effects on quinolone antibiotics (CIP, NOR, ENR, and MOX). Therefore, the data will not be presented in the following charts. Bp The corresponding data for the Lac and SA systems are presented below. Specific research follows. Bp Lac+SA system Bp Lac@ IPM system and Bp The removal effect of the Lac@ IPM+SA system on quinolone antibiotics.

[0133] Table 11 Degradation of Quinolone Pollutants in Each System

[0134]

[0135] 7.1 Removal efficacy of ciprofloxacin (CIP):

[0136] The three systems studied are:

[0137] Bp Lac+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L CIP and 300 U / L Bp Lac, 1mM SA;

[0138] Bp Lac@IPM system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L CIP and 300 U / L CIP. Bp Lac@IPM;

[0139] Bp Lac@IPM+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L CIP and 300 U / L CIP. Bp Lac@IPM, 1mM SA.

[0140] Results combined Bp As shown, with Bp Compared to the Lac+SA system (86.15±2.27%), BpThe Lac@ IPM+SA system exhibits better removal performance, with a removal rate of 94.70±1.08% after 24 hours. Furthermore, comparisons revealed that it is superior to... Figure 15 Compared to Lac, Bp Lac@IPM+SA achieves a faster removal rate for CIP. For example, in the first 6 hours... Bp Lac+SA and Bp The removal rates of CIP by Lac@IPM+SA were 9.20±0.92 μg / h and 14.76±0.84 μg / h, respectively.

[0141] 7.2 Removal efficacy of norfloxacin (NOR):

[0142] The three systems studied are:

[0143] Bp Lac+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L NOR and 300 U / L Bp Lac, 1mM SA;

[0144] Bp Lac@IPM system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L NOR and 300 U / L Bp Lac@IPM;

[0145] Lac@IPM+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L NOR and 300 U / L Bp Lac@IPM, 1mM SA;

[0146] Norfloxacin (NOR) has a similar removal effect to CIP, such as... Bp As shown, Bp The removal rate of Lac@IPM+SA was 92.11±2.55% after 24 h.

[0147] 7.3 ENR removal effect:

[0148] The three systems studied are:

[0149] Bp Lac+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L ENR and 300 U / L Bp Lac, 1mM SA;

[0150] Figure 16Lac@IPM system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L ENR and 300 U / L Bp Lac@IPM;

[0151] Bp Lac@IPM+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L ENR and 300 U / L Bp Lac@IPM, 1mM SA;

[0152] Combination Bp As shown, Bp The ENR removal rate of the Lac+SA system was only 15.85% ± 3.77%, while Bp Lac@IPM system and Bp The removal rates of the Lac@IPM+SA system were as high as 85.15±3.99% and 77.08±1.97%.

[0153] 7.4 MOX Removal Effect:

[0154] The three systems studied are:

[0155] Figure 17 Lac+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L MOX and 300 U / L... Bp Lac, 1mM SA;

[0156] Bp Lac@IPM system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L MOX and 300 U / L... Bp Lac@IPM;

[0157] Bp Lac@IPM+SA system: 50mM, pH 7.0 phosphate buffer containing 30 mg / L MOX and 300 U / L... [[ID= Lac@IPM, 1mM SA;

[0158] Combination ​ As shown, ​ The removal rate of MOX in the Lac+SA system was only 10.37% ± 4.30%, while ​ Lac@IPM system and ​ The removal rates of the Lac@IPM+SA system were as high as 75.68±3.11% and 81.77±5.69%.

[0159] As can be seen from this embodiment 7, ​Compared to the Lac@IPM+SA system ​ The Lac+SA system exhibits better removal efficiency for quinolone pollutants. In particular, ​ Lac+SA achieved removal rates of only 15.85%±3.77% for ENR and 10.37%±4.30% for MOX. Meanwhile... ​ Lac@IPM+SA has a broader range of quinolone pollutant removal capabilities, and it also shows good removal effects on CIP, NOR, ENR and MOX.

[0160] Example 8 ​ Effects of Lac@IPM on the biotoxicity of quinolone antibiotics

[0161] Combination ​ As shown, processing with unremoved CIP. ​ OD of DH5α 12 h 600 For 0.20, use ​ Lac@IPM, ​ Lac@ IPM +SA and ​ OD after removing CIP in the Lac+SA system 600 The values ​​reached 0.19, 0.72, and 1.11 respectively, while the blank control was 3.0. It can be seen that... ​ After Lac@IPM processing, CIP... ​ The biotoxicity of DH5α is reduced, among which ​ The Lac@ IPM +SA system showed the most significant reduction in biotoxicity.

[0162] Example 9 ​ Lac@IPM removal mechanism of quinolone antibiotics

[0163] Combination ​ The contributions of adsorption and enzymatic catalysis to the removal of quinolone antibiotics were evaluated, from... ​ It can be seen that the adsorption and enzymatic catalytic efficiency of quinolone antibiotics gradually and slowly increases with the extension of reaction time. FTIR analysis was performed... ​ The binding of Lac@IPM with ciprofloxacin, as detailed below. ​ As shown, observations revealed that after binding with quinolone antibiotics... ​ The main characteristic peaks of Lac@IPM did not change significantly, indicating that ​ Lac@IPM is stable. 1717 cm⁻¹ -1The spectral bands represent the carbon-oxygen double bonds in the carbonyl group. When these bands bind to quinolone antibiotics (CIP, NOR, ENR, MOX), their absorption peak intensities increase. CIP, NOR, ENR, and MOX contain abundant carbonyl groups, directly demonstrating the successful binding of quinolone antibiotics. It is known that CIP, NOR, ENR, and MOX have an absorption peak intensity at 1396 cm⁻¹. -1 -1558 cm -1 There are many absorption peaks at this point. ​ The absorption peak in this region significantly increased after Lac@IPM bound to quinolone antibiotics, further demonstrating the binding of quinolone antibiotics with... ​ The combination of Lac@IPM.

[0164] Combination ​ Further explanation ​ The removal mechanism of quinolone antibiotics by Lac@IPM ​ Lac@IPM effectively removes QNs through a combination of adsorption and biocatalysis. Taking the removal mechanism of CIP as an example, IPM carries a strong negative charge and has a large number of hydroxyl groups on its surface, thus exhibiting strong hydrophilicity. The median isoelectric point of laccase is 3.9, and in a neutral environment, CIP carries a positive charge. In addition, CIP also contains carboxyl groups as hydrogen bond acceptors, giving it hydrophilicity. ​ The hydroxyl groups on the Lac@IPM surface also facilitate hydrogen bonding interactions with CIP. ​ It can be seen that CIP and ​ The interactions of Lac@IPM are achieved through a synergistic mechanism of electrostatic attraction, hydrogen bonding, and hydrophilic interactions, which together lead to ​ A significant increase in CIP concentration was observed on the Lac@IPM surface. Therefore, ​ The biocatalytic rate of Lac@IPM is greatly enhanced, thereby accelerating the degradation of CIP.

Claims

1. An immobilized laccase based on inorganic porous materials, characterized in that: The immobilized laccase comprises an inorganic porous material and laccase adsorbed and immobilized on the surface of the inorganic porous material. The inorganic porous material is prepared by a dynamic hydrothermal synthesis method using piperidine as a template agent, aluminum isopropoxide as an aluminum source, boric acid as a boron source, and fumed silica as a silicon source. The inorganic porous material is in the form of microspheres, with a large number of irregularly arranged nanosheets distributed on its surface. Adjacent nanosheets enclose pores to accommodate laccase molecules. The inner walls of these pores are rough and have a groove-shaped angled structure. The roughness and groove-shaped angled structure of the inner walls of the pores form a gripping force to immobilize the laccase. The surface of the inorganic porous material also has hydroxyl groups, which form hydrogen bonds with the NH2 of the laccase molecules. The laccase molecules are immobilized on the surface of the inorganic porous material by the hydrogen bonds and the gripping force of the inner walls of the pores. The specific process for preparing the inorganic porous material is as follows: First, prepare the raw materials: deionized water, piperidine, aluminum isopropoxide, boric acid, and fumed silica in a molar ratio of 20~25:1~2:0.01~0.02:1~2:1~2; then add aluminum isopropoxide to the deionized water, and after complete hydrolysis, add piperidine, stir magnetically until uniform, then add boric acid, and stir again until uniform to obtain a first mixed solution. Add fumed silica to the first mixed solution and stir until uniform to obtain a second mixed solution. Place the second mixed solution in a homogeneous reactor for aging and crystallization. After crystallization, cool, clean, and dry in sequence. Finally, calcine in a muffle furnace to remove piperidine, and finally grind into powder to obtain the inorganic porous material. The laccase was obtained through the recombinant strain X33 / pPICZαA- BpLac The recombinant strain X33 / pPICZαA- was obtained through exogenous expression. BpLac The recombinant plasmid pPICZαA- was obtained by inserting the laccase gene, as shown in SEQ ID NO: 1, into the pPICZαA plasmid. BpLac, Then the recombinant plasmid pPICZαA‐ BpLac It is formed by electroporation into Pichia pastoris X33.

2. The immobilized laccase based on inorganic porous materials according to claim 1, characterized in that, The pores on the surface of the inorganic porous material are multi-level pores, including micropores, mesopores and macropores, with an average pore diameter of 8~9 nm.

3. The immobilized laccase based on inorganic porous materials according to claim 2, characterized in that, The laccase molecules are immobilized within the mesopores and macropores of the inorganic porous material.

4. A method for preparing immobilized laccase based on inorganic porous materials, characterized in that, Including the following steps: (1) Preparation of inorganic porous materials: Inorganic porous materials were prepared by dynamic hydrothermal synthesis using piperidine as a template agent, aluminum isopropoxide as an aluminum source, boric acid as a boron source, and fumed silica as a silicon source. The surface of the inorganic porous materials is covered with a large number of irregularly arranged nanosheets. Adjacent nanosheets enclose pores to accommodate laccase molecules. The inner walls of these pores are rough and have a groove-shaped angle structure. The roughness and groove-shaped angle structure of the inner walls of the pores form a gripping force to fix the laccase. The surface of the inorganic porous materials also has hydroxyl groups that can form hydrogen bonds with NH2 laccase molecules. (2) Adsorption and fixation Inorganic porous materials are mixed with laccase to obtain a mixture. In the mixture, laccase molecules enter the pores of the inorganic porous materials under the adsorption of the inorganic porous materials. Furthermore, under the hydrogen bonding force of the inorganic porous materials and the gripping force of the inner wall of the pores, the laccase molecules are fixed on the surface of the inorganic porous materials. (3) Collect immobilized laccase: The mixture was centrifuged to collect the solids, and then the solids were washed to remove unbound laccase molecules, resulting in immobilized laccase. The specific process for preparing the inorganic porous material is as follows: First, prepare the raw materials: deionized water, piperidine, aluminum isopropoxide, boric acid, and fumed silica in a molar ratio of 20~25:1~2:0.01~0.02:1~2:1~2; then add aluminum isopropoxide to the deionized water, and after complete hydrolysis, add piperidine, stir magnetically until uniform, then add boric acid, and stir again until uniform to obtain a first mixed solution. Add fumed silica to the first mixed solution and stir until uniform to obtain a second mixed solution. Place the second mixed solution in a homogeneous reactor for aging and crystallization. After crystallization, cool, clean, and dry in sequence. Finally, calcine in a muffle furnace to remove piperidine, and finally grind into powder to obtain the inorganic porous material.

5. The method for preparing immobilized laccase based on inorganic porous materials according to claim 4, characterized in that, The aging process is carried out at a speed of 50-60 rpm, a temperature of 120-130℃, and a time of 8-12 hours. The crystallization process is carried out at a temperature of 160-170℃ for 2-3 days. The muffle furnace calcination process is carried out at a temperature of 500-600℃ for 10-12 hours.

6. The method for preparing immobilized laccase based on inorganic porous materials according to claim 5, characterized in that, In step (2), the adsorption and fixation parameters are: 5 mg of inorganic porous material, 0.5-2 ml of laccase, temperature of 20-30℃, rotation speed of 100-200 rpm, and time of 18-24 hours. The enzyme activity of laccase is 20.35 U / mL, and the concentration of laccase protein is 0.37 mg / mL.

7. The application of the immobilized laccase as described in any one of claims 1 to 3 in the removal of quinolone antibiotics from wastewater, characterized in that, The catalytic system for removing quinolone antibiotics from wastewater includes: quinolone antibiotics, immobilized laccase, and syringaldehyde.

8. The application according to claim 7, characterized in that, The quinolone antibiotics include ciprofloxacin, enrofloxacin, norfloxacin, and moxifloxacin. The catalytic system comprises: 30 mg / L quinolone antibiotics, 300 U / L immobilized laccase, and 1 mM syringaldehyde.

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