Polymer brush coated SiO2 microsphere immobilized penicillin G acylase as well as preparation and application thereof

Abstract

The invention discloses a polymer brush coated SiO2 microsphere immobilized penicillin G acylase as well as preparation and application thereof. The preparation method comprises the following steps: grafting poly (tert-butyl acrylate) / polyethylene glycol phenyl ether methacrylate on the surface of a silicon dioxide microsphere through an electron activation regeneration atom transfer radical polymerization reaction, and carrying out acidolysis to obtain a polyacrylic acid / polyethylene glycol phenyl ether methacrylate polymer brushed SiO2 microsphere; and immobilizing penicillin G acylase on the polymer brush through EDC / NHS reaction. The method is simple in reaction condition, high in enzyme loading capacity and enzyme activity recovery rate and good in biocompatibility, and has good application potential in catalytic synthesis of beta-lactam antibiotics and antibiotics.

Description

Technical Field

[0001] The present invention belongs to the field of immobilized enzymes, and particularly relates to an immobilized penicillin G acylase using polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microspheres as a carrier, and a preparation method and application thereof. Background Art

[0002] Penicillin G acylase (PGA) has the advantages of green catalysis, high selectivity, and mild reaction conditions, and has important application value in the field of β-lactam antibiotic synthesis. However, due to its nature as a protein, it is greatly affected by the environment, is easily inactivated under high temperature and relatively acidic or alkaline conditions, and has a small volume, making it difficult to recover and reuse from the reaction system, which limits its industrial application. In practical applications, PGA is usually immobilized on an insoluble carrier to improve the enzyme recovery ability, enhance the enzyme stability, and increase the tolerance to temperature and solvent composition changes.

[0003] Enzyme immobilization technology refers to a process of restricting free enzymes in a specific space through physical or chemical means to achieve their reuse and operational stability. Currently, the mainstream methods can be divided into four categories: adsorption method, entrapment method, covalent binding method, and crosslinking method, each with its own characteristics and applicable ranges. The adsorption method immobilizes the enzyme on the surface of the carrier based on physical adsorption or ionic binding, and the interaction between the enzyme and the carrier is weak, which is prone to enzyme leakage; the entrapment method is divided into grid type (such as polymer gel) and microcapsule type (encapsulated by a semipermeable membrane), which protects the enzyme active center through spatial confinement effect. This method has a high enzyme loading capacity but the enzyme is prone to leakage; the covalent binding method realizes immobilization through the covalent bond binding between the enzyme molecule and the functionalized carrier (such as epoxy silica gel, amino resin), endowing the product with excellent stability and reusability. It is worth noting that the surface immobilization strategy is often limited by the low loading rate caused by the specific surface area of the carrier, while the internal entrapment method increases the substrate mass transfer resistance due to the spatial hindrance effect of the three-dimensional network structure.

[0004] Polymer brush is an organic macromolecule grafted on the surface of a high-density substrate. Due to the steric hindrance effect between polymer chains, a brush-like structure is extended, which has a high specific surface area and multiple binding sites, and can load enzymes at a high density. Spherical polymer brush is to graft polymer chains on the surface of colloidal particles or metal particles to construct a three-dimensional structure. Compared with planar polymer brush, it shows the advantage of significantly increased specific surface area, and can exhibit good dispersion in solution. The fully extended polymer chains can bind to the enzyme, exposing the enzyme active sites directly to the reaction system. It can not only load enzymes at a high density, effectively avoid the problem of low enzyme loading in surface immobilization, but also avoid the common problem of large mass transfer resistance in traditional entrapment methods, and at the same time contribute to the high loading rate and recovery and reuse of immobilized enzymes.

[0005] Polyacrylic acid (PAA) brushes, due to their excellent hydrophilicity and carboxyl functional groups densely distributed on the surface, can provide abundant enzyme binding sites through electrostatic interaction, hydrogen bonding or covalent coupling, thus significantly increasing the enzyme loading amount and being widely used in the immobilized bio-enzyme system. Y. Zhao et al. (Talanta 155 (2016) 265 - 271) used PAA brush-nano spherical silica polymer brushes prepared by surface-initiated reversible addition-fragmentation chain transfer for the immobilization of glucose oxidase and horseradish peroxidase cascade catalysis for ultrasensitive monitoring of glucose, amplifying the detection signal change by 120 times. Z. Qu et al. (Journal of Colloid and Interface Science 398 (2013) 82 - 87) used the prepared PAA brush-nano spherical silica polymer brushes for the immobilization of streptavidin and peroxidase, and the binding ability of proteins to the brushes was significantly improved. Due to the high carboxyl content and brush-like structure of PAA brushes, polyacrylic acid brushes have good immobilization effects on enzymes. However, due to the abundant fixed sites on the polymer chains, it will cause the dense accumulation of enzymes, resulting in the mutual extrusion of enzymes, thus reducing the apparent enzyme activity; at the same time, irreversible inactivation may be caused by the direct chemical modification of the enzyme active center during the covalent immobilization process. To break through this bottleneck, introducing a second monomer to construct a binary copolymerization system has become a key strategy: by flexibly combining the hydrophilic, hydrophobic or dynamic responsive units of different monomers, functional synergy and precise microenvironment regulation can be achieved at the molecular level. In addition, based on the monomer synchronous grafting strategy of controlled polymerization technology (such as ATRP, RAFT), the brush layer thickness and surface charge distribution can be precisely regulated, providing a customizable carrier platform with high loading, efficient mass transfer and long-term stability for the construction of complex catalytic systems. Summary of the Invention

[0006] To solve the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a preparation method for immobilizing penicillin G acylase based on polyacrylic acid / ethylene glycol phenyl ether methacrylate brush@SiO2 microspheres.

[0007] Through surface-initiated electron-activated regenerative atom transfer radical polymerization, a second monomer, ethylene glycol phenyl ether methacrylate (EGPMA), is introduced on the basis of PAA, and a copolymer chain of polyacrylic acid and poly(ethylene glycol phenyl ether methacrylate) is grafted on the surface of silica microspheres, and penicillin acylase is chemically immobilized through EDC / NHS (EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, NHS is N-hydroxysuccinimide) (the preparation principle of the immobilized enzyme is as Figure 1)。The EGPMA segment weakens the non-specific aggregation between enzyme molecules through steric hindrance effect, reducing the risk of conformational distortion and inactivation of the enzyme caused by physical collision; its phenyl ether hydrophobic unit and the hydrophilic carboxyl group of PAA cooperate to form a hydrophilic-hydrophobic microphase separation interface, further optimizing the directional anchoring of enzyme molecules on the surface of the carrier, and enhancing its stability in different pH and temperature environments.

[0008] Another object of the present invention is to provide an immobilized penicillin G acylase with a high enzyme loading, catalytic activity and small mass transfer resistance prepared by the above preparation method.

[0009] Another object of the present invention is to provide the application of the above polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase.

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] A preparation method of a binary copolymerized polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase, comprising the following steps:

[0012] (1) Perform an ammoniation modification reaction on silica microspheres, ammonia water and a silane coupling agent to obtain ammoniated modified silica microspheres;

[0013] (2) React the ammoniated modified silica microspheres with an ATRP initiator under the action of an acid-binding agent to obtain silica microspheres SiO2-Br with ATRP initiation sites immobilized on the surface;

[0014] (3) Perform a polymerization reaction on SiO2-Br, tert-butyl acrylate (tBA), and ethylene glycol phenyl ether methacrylate (EGPMA) under the action of a transition metal catalyst, a ligand and a reducing agent to obtain SiO2-PtBA-EGPMA; then remove the tert-butyl group in SiO2-PtBA-EGPMA to obtain SiO2-PAA-EGPMA;

[0015] (4) Activate the carboxyl group of SiO2-PAA-EGPMA, and then perform a covalent immobilization reaction with PGA enzyme solution to obtain an immobilized enzyme PGA@SiO2-PAA-EGPMA.

[0016] Preferably, the particle size of the silica microspheres in step (1) is 40-1000 nm, more preferably 800 nm.

[0017] Preferably, the ratio of the silica microspheres to ammonia water in step (1) is 2-8 g: 4-6 mL; more preferably 1 g: 1 mL; the mass concentration of the ammonia water is 28%.

[0018] Preferably, the silane coupling agent in step (1) is at least one of 3-aminopropyltrimethoxysilane (APTES) and 3-aminopropyltriethoxysilane.

[0019] Preferably, the mass ratio of the silica microspheres to the silane coupling agent in step (1) is 1:(0.6 - 0.9), more preferably 1:0.7.

[0020] Preferably, the temperature of the ammoniation modification reaction in step (1) is 30 - 80 °C, and the reaction time is 6 - 15 h; further preferably, the temperature of the ammoniation modification reaction is 60 °C, and the reaction time is 12 h.

[0021] Preferably, the ATRP initiator in step (2) is 2-bromoisobutyryl bromide (BiBB).

[0022] Preferably, the mass ratio of the ammoniated silica microspheres to the ATRP initiator in step (2) is 1:(1 - 3), more preferably 1:1.5.

[0023] Preferably, the acid-binding agent in step (2) is triethylamine; the mass ratio of the ATRP initiator to the acid-binding agent is 1:(1 - 3), more preferably 1:2.

[0024] Preferably, in step (2), the reaction is first carried out in an ice bath for 10 - 50 min, and then at 20 - 40 °C for 16 - 24 h. More preferably, the reaction is first carried out in an ice bath for 30 min, and then at 30 °C for 20 h.

[0025] Preferably, the tert-butyl acrylate (tBA) in step (3) accounts for 92 - 98% of the total molar amount of tert-butyl acrylate (tBA) and ethylene glycol phenyl ether methacrylate (EGPMA), more preferably 97%.

[0026] Preferably, the total mass ratio of SiO2-Br to tert-butyl acrylate (tBA) and ethylene glycol phenyl ether methacrylate (EGPMA) in step (3) is 1:(6.1 - 10.0), more preferably 1:8.0.

[0027] Preferably, the transition metal catalyst in step (3) is at least one of CuCl2, CuBr2, and FeCl3, more preferably CuCl2; the ligand is pentamethyldiethylenetriamine (PMDETA).

[0028] Preferably, the molar ratio of the total molar amount of tert-butyl acrylate (tBA) and ethylene glycol phenyl ether methacrylate (EGPMA) to the transition metal catalyst and the ligand in step (3) is 100:(20 - 26):(20 - 26); more preferably 100:24:24.

[0029] Preferably, the reducing agent in step (3) is at least one of ascorbic acid and stannous octoate, and more preferably ascorbic acid.

[0030] Preferably, the ratio of SiO2-Br to the reducing agent in step (3) is 1 g: 1.6 - 4.8 mmol.

[0031] Preferably, nitrogen is passed through to remove oxygen before the reaction in step (3), and the time is 20 - 50 min, more preferably 30 min.

[0032] Optimally, the polymerization reaction temperature in step (3) is 30 - 60 °C, and the reaction time is 2 - 6 h; further preferably, the polymerization reaction temperature is 40 °C and the reaction time is 4 h.

[0033] Optimally, the method for removing the tert-butyl group from SiO2-PtBA-EGPMA in step (3) is: dispersing SiO2-PtBA-EGPMA in a solvent, adding trifluoroacetic acid for the tert-butyl group removal reaction to obtain SiO2-PAA-EGPMA.

[0034] More preferably, the tert-butyl group removal reaction temperature is room temperature, the reaction time is 46 - 50 h, more preferably 48 h; the mass ratio of SiO2-Br to trifluoroacetic acid in SiO2-PtBA-EGPMA is 1: (4 - 6), more preferably 1:5.

[0035] More preferably, the ratio of SiO2-Br to the solvent in SiO2-PtBA-EGPMA is 0.025 g: 1 mL; the solvent is dichloromethane.

[0036] Preferably, the activator for carboxyl activation in step (4) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) with a molar ratio of 2:1; the ratio of SiO2-PAA-EGPMA to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 g: 4 - 12 mmol.

[0037] Preferably, the temperature for carboxyl activation in step (4) is 30 - 60 °C, more preferably 30 °C, and the time is 30 - 60 min, more preferably 45 min.

[0038] Preferably, the addition amount of the PGA enzyme solution in step (4) is 2 - 7 mL / g SiO2-PAA-EGPMA, more preferably 5 mL / g SiO2-PAA-EGPMA, and the concentration of the PGA enzyme solution is 20 mg / mL.

[0039] Preferably, the temperature of the immobilization reaction in step (4) is 20-40 °C, and the time is 3-8 h; more preferably, the temperature of the immobilization reaction is 30 °C and the time is 5 h.

[0040] Preferably, the pH of the buffer solution for the immobilization reaction in step (4) is 5-10, and more preferably the pH is 8.

[0041] The preparation method of the above-mentioned polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase by binary copolymerization includes the following steps:

[0042] (1) Add silica microspheres, ammonia water and silane coupling agent into a solvent, heat for ammoniation modification reaction, after the reaction is completed, centrifuge and separate, and dry to obtain ammoniated modified silica microspheres;

[0043] (2) Dissolve the ammoniated modified silica microspheres and acid-binding agent in a solvent, dropwise add an ATRP initiator under an ice bath, and then heat for reaction. After the reaction is completed, centrifuge, wash, and dry to obtain silica microspheres SiO2-Br with ATRP initiation sites immobilized on the surface;

[0044] (3) Dissolve SiO2-Br, tert-butyl acrylate (tBA), ethylene glycol phenyl ether methacrylate (EGPMA), transition metal catalyst and ligand in a solvent. Under an inert gas atmosphere, add a reducing agent solution for polymerization reaction. After the reaction is completed, centrifuge and wash to obtain SiO2-PtBA-EGPMA;

[0045] (4) Disperse SiO2-PtBA-EGPMA in a solvent, add trifluoroacetic acid for de-tert-butylation reaction, remove the solvent, and dry to obtain SiO2-PAA-EGPMA;

[0046] (5) Disperse SiO2-PAA-EGPMA in PBS buffer solution, add a carboxylating reagent for carboxyl activation reaction, and then add PGA enzyme solution for covalent immobilization reaction. After the reaction is completed, centrifuge and wash to obtain immobilized enzyme PGA@SiO2-PAA-EGPMA.

[0047] Preferably, the solvent in step (1) is ethanol, and the concentration of the silica microspheres in the solvent is 0.02-0.08 g / mL, more preferably 0.05 g / mL.

[0048] Preferably, the centrifugation speed in step (1) is 8000-15000 rpm.

[0049] Preferably, the concentration of the ammoniated modified silica microspheres in the solvent in step (2) is 0.02-0.05 g / mL, more preferably 0.04 g / mL; the solvent is tetrahydrofuran.

[0050] Preferably, the solvent in step (3) is N,N-dimethylformamide (DMF); the concentration of SiO2-Br in the solvent is 0.005 - 0.02 g / mL, more preferably 0.01 g / mL.

[0051] Preferably, the concentration of the reducing agent solution in step (3) is 0.2 - 0.6 mol / L, more preferably 0.4 mol / L.

[0052] Preferably, the solvent in step (4) is dichloromethane; the concentration of SiO2-PtBA-EGPMA in the solvent is 0.02 - 0.03 g / mL; more preferably 0.025 g / mL.

[0053] Preferably, the concentration of SiO2-PAA-EGPMA in the PBS buffer solution in step (5) is 0.005 mg / mL; the pH of the PBS buffer solution is 5 - 10; more preferably pH is 8.

[0054] The polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase prepared by the above preparation method.

[0055] The application of the polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase.

[0056] Preferably, the application is in the synthesis of β-lactam antibiotics and the key intermediates for synthesizing antibiotics.

[0057] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0058] 1. The polymer brush microspheres adopted in the present invention have a large specific surface area and abundant active sites. The carboxyl groups of PAA bind to penicillin G acylase, thus achieving a high enzyme loading amount, being able to immobilize a large amount of enzyme on the carrier per unit mass, and improving the catalytic efficiency. At the same time, since the enzyme and the carrier are bonded by chemical bonds relatively firmly, and the carrier has stable structure and properties, the immobilized enzyme is easy to be separated from the reaction system after completing one catalytic reaction, with a high reuse rate, reducing the production cost and improving the resource utilization rate.

[0059] 2. The polymer brush microspheres of the present invention significantly improve the carrier performance by introducing the synergistic effect of PAA and EGPMA. PAA enhances the interaction between the carrier and the enzyme with its flexibility and hydrophilicity, effectively inhibiting the shedding of the enzyme during the catalytic reaction; the polyethylene glycol segment in EGPMA solves the problem of enzyme aggregation caused by the dense structure of a single PAA brush through steric hindrance effect, reduces enzyme molecule aggregation and denaturation, and improves the stability of the enzyme under different environmental conditions.

[0060] 3. In the immobilized enzyme prepared in the present invention, PGA is fixed on the polymer chain and directly contacts the substrate solution, with almost no mass transfer resistance, which is conducive to the rapid approach of the substrate to the active center of the enzyme. At the same time, the product can also diffuse out from the enzyme active site in a timely manner, reducing the mass transfer resistance of the substrate and the product, and improving the efficiency and rate of the enzyme-catalyzed reaction.

[0061] 4. The microspheres of the present invention have excellent mechanical strength and can withstand operating stresses such as stirring and centrifugation, ensuring their structural integrity and long-term use reliability in complex reaction systems, and providing a stable carrier support for the construction of an efficient biocatalytic system. The introduction of EGPMA enhances the mechanical strength of the microsphere polymer segments and the operating stress of repeated centrifugation through hydrophobic interaction and covalent crosslinking network, and also ensures the structural integrity of the carrier in continuous flow reactions or large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 It is the preparation flow chart of the present invention.

[0063] Figure 2 It is the Fourier transform infrared spectrum of SiO2-PAA-EGPMA prepared in Example 1 and Example 2 and SiO2-PAA prepared in Comparative Example 1.

[0064] Figure 3 It is the thermogravimetric curve of SiO2-Br, SiO2-PAA-EGPMA prepared in Example 1 and SiO2-PAA prepared in Comparative Example 1.

[0065] Figure 4 It is the transmission electron microscope image of SiO2 and SiO2-PAA-EGPMA prepared in Example 1 (left figure is SiO2, right figure is SiO2-PAA-EGPMA).

[0066] Figure 5 It is the laser confocal image of the immobilized penicillin G acylase of SiO2-PAA-EGPMA prepared in Example 1, where a is the dark field image, b is the bright field image, and c is the superposition image of the bright and dark fields.

[0067] Figure 6 It is the enzyme activity, enzyme loading and enzyme activity recovery rate of the immobilized penicillin G acylase of Comparative Examples 1-3 and Examples 1-6.

[0068] Figure 7 For the recyclability of the immobilized penicillin G acylase in Example 1 and Comparative Example 1. Detailed implementation manners

[0069] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto.

[0070] In the examples of the present invention, those without specific conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. The raw materials, reagents, etc. without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0071] Example 1

[0072] (1) 1 g of silica microspheres (800 nm) was evenly dispersed in 20 mL of an ammonia-ethanol mixed solution (where 28 wt% ammonia accounted for 5 v% of the mixed solution), 0.7 g of APTES was added, and the mixture was continuously stirred in a constant-temperature reaction system at 60 °C for 12 h. After that, the solid product was centrifuged at 10000 rpm - ethanol washed, and vacuum dried for 24 h to obtain amino-functionalized silica microspheres (SiO2-NH2).

[0073] (2) 1 g of SiO2-NH2 and 3 g of triethylamine were dispersed in 25 mL of tetrahydrofuran (THF). Under ice bath conditions, 1.5 g of BiBB was added dropwise to the above solution. After reacting in the ice bath for 30 min, the reaction system was raised to 30 °C and continuously stirred for 20 h. After the reaction, it was centrifuged at 10000 rpm - anhydrous ethanol washed 3 times to remove unreacted reagents and by-products. The washed solid product was vacuum dried at 60 °C for 12 h to obtain functionalized silica microspheres SiO2-Br with surface-fixed ATRP initiation sites.

[0074] (3) 1 g of SiO2-Br was dispersed in 100 mL of DMF, and 7.76 g of tBA, 0.39 g of EGPMA, 25 mg of CuCl2, and 15 mg of PMDETA were added in sequence. After the system was purged with nitrogen for 30 min to remove air, under continuous nitrogen protection, 20 mL of a newly prepared 0.2 mol / L aqueous solution of ascorbic acid was slowly injected through a syringe, and the reaction was carried out at 40 °C for 6 h. After the reaction, it was centrifuged at 10000 rpm - anhydrous ethanol washed three times and then vacuum dried to obtain composite microspheres SiO2-PtBA-EGPMA.

[0075] To prepare the carboxylic acid-functionalized product, 1.0 g of the above product was weighed and dispersed in 20 mL of dichloromethane, and 5.0 g of trifluoroacetic acid was added for reaction. After the system was magnetically stirred at 25 °C for 48 h, the solvent and low-boiling by-products were removed by a rotary evaporator (30 °C, 0.06 MPa). The solid product was vacuum-dried for 24 h, and finally, the SiO2-PAA-EGPMA carrier material with surface-grafted polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer was obtained.

[0076] (4) 1 g of SiO2-PAA-EGPMA was dispersed in 200 mL of PBS (pH = 8, 0.02 mol / L) buffer solution containing EDC (40 mmol / L) / NHS (20 mmol / L), and the activation reaction was carried out for 45 min under the condition of constant temperature oscillation at 30 °C. 5 mL of PGA enzyme solution (20 mg / mL) was added to the system, and the reaction was carried out for 5 hours under the condition of constant temperature oscillation at 30 °C. After the reaction was completed, the unfixed free enzyme was removed by centrifugal washing, and finally, the immobilized enzyme was obtained.

[0077] Example 2

[0078] (1) 1 g of silica (800 nm) was uniformly dispersed in 50 mL of an ammonia-ethanol mixed solution (where 28 wt% ammonia accounted for 4 v% of the mixed solution), 0.6 g of 3-aminopropyltriethoxysilane was added, and the reaction system was continuously stirred at 40 °C for 9 h. After centrifugation at 10000 rpm, the solid product was washed with ethanol and vacuum-dried for 24 h to obtain amino-functionalized silica microspheres SiO2-NH2.

[0079] (2) 1 g of SiO2-NH2 and 1 g of triethylamine were dispersed in 50 mL of tetrahydrofuran (THF). Under ice-bath conditions, 1 g of BiBB was added dropwise to the above solution. After reacting in the ice bath for 10 min, the reaction system was raised to 20 °C and continuously stirred for 16 h. After centrifugation at 10000 rpm and washing with anhydrous ethanol three times, the unreacted reagents and by-products were removed. The washed solid product was vacuum-dried for 12 h to obtain functionalized silica microspheres (SiO2-Br) with surface-fixed ATRP initiation sites.

[0080] (3) 1 g of SiO2-Br was dispersed in 200 mL of DMF, and 6.65 g of tBA, 0.56 g of EGPMA, 43.6 mg of CuBr2, and 33.8 mg of PMDETA were added in sequence. After the system was purged with nitrogen for 10 min, under continuous nitrogen protection, 8 mL of a newly prepared 0.2 mol / L aqueous solution of ascorbic acid was slowly injected through a syringe, and the reaction was carried out at 60 °C for 2 h. After centrifugation at 10000 rpm and washing with anhydrous ethanol three times, it was vacuum-dried to obtain composite microspheres SiO2-PtBA-EGPMA.

[0081] To prepare the carboxylic acid-functionalized product, 1.0 g of the above product was weighed and dispersed in 20 mL of dichloromethane, and 5.0 g of trifluoroacetic acid was added for the tert-butoxycarbonyl deprotection reaction. After the system was magnetically stirred at 25 °C for 48 h, the solvent and low-boiling by-products were removed by a rotary evaporator (30 °C, 0.06 MPa). The residue was vacuum dried for 24 h to finally obtain the SiO2-PAA-EGPMA composite material with polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer grafted on the surface.

[0082] (4) 1 g of SiO2-PAA-EGPMA was dispersed in 200 mL of PBS (pH = 5, 0.02 mol / L) buffer solution containing EDC (20 mmol / L) / NHS (10 mmol / L), and the reaction was activated under constant temperature oscillation at 30 °C for 30 min. 2 mL of PGA enzyme solution (20 mg / mL) was added to the system, and the reaction was carried out under constant temperature oscillation at 30 °C for 3 h. After the reaction was completed, the unimmobilized free enzyme was removed by centrifugal washing to finally obtain the immobilized enzyme complex.

[0083] Example 3

[0084] (1) 1 g of silica microspheres (1000 nm) was uniformly dispersed in 25 mL of an ammonia-ethanol mixed solution (where 28 wt% ammonia accounted for 5 v% of the mixed solution), 0.6 g of APTES was added, and the reaction system was continuously stirred at 50 °C for 12 h. After centrifugation at 10000 rpm, the solid product was washed with ethanol and vacuum dried for 24 h to obtain amino-functionalized silica microspheres (SiO2-NH2).

[0085] (2) 1 g of SiO2-NH2 and 4 g of triethylamine were dispersed in 33.3 mL of tetrahydrofuran (THF). Under ice bath conditions, 2 g of BiBB was added dropwise to the above solution. After reacting in the ice bath for 20 min, the reaction system was raised to 40 °C and continuously stirred for 18 h. Centrifugal separation was carried out at 10000 rpm, and the solid product was washed 3 times with absolute ethanol to remove the unreacted reagents and by-products. The washed solid product was vacuum dried at 60 °C for 12 h to obtain functionalized silica microspheres with ATRP initiation sites immobilized on the surface (SiO2-Br).

[0086] (3) Disperse 1 g of SiO2-Br in 100 mL of DMF, and successively add 5.82 g of tBA, 0.29 g of EGPMA, 43.6 mg of CuCl2, and 33.8 mg of PMDETA. After the system is purged with nitrogen bubbling for 40 min, under continuous nitrogen protection, slowly inject 8 mL of freshly prepared 0.6 mol / L ascorbic acid aqueous solution through a syringe, and react at 50 °C for 4 h. After centrifuging at 10000 rpm and washing three times with absolute ethanol, vacuum dry to obtain composite microspheres SiO2-PtBA-EGPMA.

[0087] To prepare the carboxylic acid-functionalized product, weigh 1.0 g of the above product and disperse it in 20 mL of dichloromethane, and add 5.0 g of trifluoroacetic acid for tert-butoxycarbonyl deprotection reaction. After the system is magnetically stirred at 25 °C for 48 h, remove the solvent and low-boiling by-products through a rotary evaporator (30 °C, 0.06 MPa). The residue is vacuum dried for 24 h to finally obtain the SiO2-PAA-EGPMA composite material with polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer grafted on the surface.

[0088] (4) Disperse 1 g of SiO2-PAA-EGPMA in 200 mL of PBS (pH = 6, 0.02 mol / L) buffer solution containing EDC (20 mmol / L) / NHS (10 mmol / L), activate the reaction under constant temperature oscillation conditions at 37 °C for 30 min, add 3 mL of PGA enzyme solution (20 mg / mL) to the system, and react under constant temperature oscillation conditions at 37 °C for 4 hours. After the reaction, remove the unfixed free enzyme by centrifugal washing to finally obtain the immobilized enzyme complex.

[0089] Example 4

[0090] (1) Uniformly disperse 1 g of silica microspheres (500 nm) in 16 mL of ammonia ethanol mixed solution (where 28 wt% ammonia accounts for 4 v% of the mixed solution), add 0.6 g of 3-aminopropyltriethoxysilane, continuously stir in a constant temperature reaction system at 50 °C for 12 h, then centrifuge at 10000 rpm, wash the solid product with ethanol, and vacuum dry for 24 h to obtain amino-functionalized silica microspheres SiO2-NH2.

[0091] (2) Disperse 1 g of SiO2-NH2 and 5 g of triethylamine in 20 mL of tetrahydrofuran. Under ice bath conditions, add 2.5 g of BiBB dropwise to the above solution. After reacting in the ice bath for 40 min, raise the reaction system to 30 °C and continuously stir and react for 20 h. Centrifuge and separate at 10000 rpm, wash three times with absolute ethanol to remove unreacted reagents and by-products. The washed solid product is vacuum dried at 60 °C for 12 h to obtain functionalized silica microspheres (SiO2-Br) with ATRP initiation sites immobilized on the surface.

[0092] (3) Disperse 1 g of SiO2-Br in 100 mL of DMF, and successively add 8.55 g of tBA, 0.72 g of EGPMA, 65.3 mg of CuCl2, and 50.7 mg of PMDETA. After the system is purged with nitrogen bubbling for 50 min, 8 mL of freshly prepared 0.4 mol / L stannous octoate aqueous solution is slowly injected through a syringe under continuous nitrogen protection, and the reaction is carried out at 40 °C for 4 h. After centrifugation at 10000 rpm and washing three times with absolute ethanol, it is dried in vacuum to obtain composite microspheres SiO2-PtBA-EGPMA.

[0093] To prepare the carboxylic acid-functionalized product, weigh 1.0 g of the above product and disperse it in 20 mL of dichloromethane, and add 5.0 g of trifluoroacetic acid for the tert-butoxycarbonyl deprotection reaction. After the system is magnetically stirred at 25 °C for 48 h, the solvent and low-boiling by-products are removed by a rotary evaporator (30 °C, 0.06 MPa). The residue is dried in vacuum for 24 h to finally obtain the SiO2-PAA-EGPMA composite material with polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer grafted on the surface.

[0094] (4) Disperse 1 g of SiO2-PAA-EGPMA in 200 mL of PBS (pH = 7, 0.02 mol / L) buffer solution containing EDC (50 mmol / L) / NHS (25 mmol / L), activate the reaction under constant temperature oscillation at 30 °C for 60 min, add 4 mL of PGA enzyme solution (20 mg / mL) to the system, and react under constant temperature oscillation at 30 °C for 6 hours. After the reaction is completed, the unimmobilized free enzyme is removed by centrifugal washing to finally obtain the immobilized enzyme complex.

[0095] Example 5

[0096] (1) Uniformly disperse 1 g of silica microspheres (100 nm) in 12.5 mL of ammonia-ethanol mixed solution (where 28 wt% ammonia accounts for 6 v% of the mixed solution), add 0.8 g of APTES, continuously stir in a constant temperature reaction system at 70 °C for 9 h, then centrifuge at 10000 rpm, wash the solid-phase product with ethanol, and dry it in vacuum for 24 h to obtain amino-functionalized silica microspheres SiO2-NH2).

[0097] (2) Disperse 1 g of SiO2-NH2 and 3 g of ethylamine in 20 mL of tetrahydrofuran (THF). Under ice bath conditions, add 3 g of BiBB dropwise to the above solution. After reacting in the ice bath for 50 min, raise the reaction system to 30 °C and continuously stir and react for 24 h. Centrifuge at 10000 rpm, wash three times with absolute ethanol to remove unreacted reagents and by-products. The washed solid product is vacuum dried at 60 °C for 12 h to obtain functionalized silica microspheres (SiO2-Br) with ATRP initiation sites immobilized on the surface.

[0098] (3) Disperse 1 g of SiO2-Br in 50 mL of DMF, and successively add 9.8 g of tBA, 0.32 g of EGPMA, 52.7 mg of CuCl2, and 56.3 mg of PMDETA. After bubbling nitrogen through the system to remove air for 30 min, under continuous nitrogen protection, slowly inject 8 mL of freshly prepared 0.2 mol / L stannous octoate aqueous solution through a syringe, and react at 30 °C for 6 h. After centrifuging at 10000 rpm - washing three times with absolute ethanol and then vacuum drying, obtain composite microspheres SiO2-PtBA-EGPMA.

[0099] To prepare the carboxylic acid-functionalized product, weigh 1.0 g of the above product and disperse it in 20 mL of dichloromethane, and add 5.0 g of trifluoroacetic acid for tert-butoxycarbonyl deprotection reaction. After the system is magnetically stirred at 25 °C for 48 h, remove the solvent and low-boiling by-products through a rotary evaporator (30 °C, 0.06 MPa). The residue is vacuum dried for 24 h to finally obtain the SiO2-PAA-EGPMA composite material with polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer grafted on the surface.

[0100] (4) Disperse 1 g of SiO2-PAA-EGPMA in 200 mL of PBS (pH = 9, 0.02 mol / L) buffer solution containing EDC (60 mmol / L) / NHS (30 mmol / L), activate and react under constant temperature oscillation conditions at 50 °C for 30 min, add 6 mL of PGA enzyme solution (20 mg / mL) to the system, and react under constant temperature oscillation conditions at 30 °C for 7 hours. After the reaction is completed, remove the unfixed free enzyme by centrifugal washing to finally obtain the immobilized enzyme complex.

[0101] Example 6

[0102] (1) Uniformly disperse 1 g of silica microspheres (40 nm) in 20 mL of ammonia-ethanol mixed solution (where 28 wt% ammonia accounts for 5 v% of the mixed solution), add 0.9 g of APTES, continuously stir in the constant temperature reaction system at 80 °C for 15 h, then centrifuge at 10000 rpm, wash the solid phase product with ethanol, and vacuum dry for 24 h to obtain amino-functionalized silica microspheres (SiO2-NH2).

[0103] (2) 1 g of SiO2-NH2 and 0.9 g of triethylamine were dispersed in 25 mL of tetrahydrofuran. Under an ice bath condition, 1.5 g of BiBB was added dropwise to the above solution. After reacting for 30 min under the ice bath, the reaction system was raised to 30 °C and continuously stirred for 20 h. Centrifugation was carried out at 10000 rpm and washed three times with absolute ethanol to remove unreacted reagents and by-products. The washed solid product was dried in vacuo at 60 °C for 12 h to obtain functionalized silica microspheres (SiO2-Br) with ATRP initiation sites immobilized on the surface.

[0104] (3) 1 g of SiO2-Br was dispersed in 50 mL of DMF, and 5.76 g of tBA, 0.39 g of EGPMA, 31.6 mg of CuCl2, and 33.8 mg of PMDETA were added in sequence. After the system was bubbled with nitrogen to remove air for 50 min, under continuous nitrogen protection, 8 mL of freshly prepared 0.6 mol / L stannous octanoate aqueous solution was slowly injected through a syringe, and the reaction was carried out at 40 °C for 3 h. After centrifugation at 10000 rpm - washing three times with absolute ethanol and then drying in vacuo, composite microspheres SiO2-PtBA-EGPMA were obtained.

[0105] To prepare the carboxylic acid-functionalized product, 1.0 g of the above product was weighed and dispersed in 20 mL of dichloromethane, and 5.0 g of trifluoroacetic acid (TFA) was added for tert-butoxycarbonyl deprotection reaction. After the system was magnetically stirred at 25 °C for 48 h, the solvent and low-boiling by-products were removed by a rotary evaporator (30 °C, 0.06 MPa). The residue was dried in vacuo for 24 h, and finally, SiO2-PAA-EGPMA composite materials grafted with polyacrylic acid-polyethylene glycol phenyl methyl ether copolymer on the surface were obtained.

[0106] (4) 1 g of SiO2-PAA-EGPMA was dispersed in 200 mL of PBS (pH = 10, 0.02 mol / L) buffer solution containing EDC (40 mmol / L) / NHS (20 mmol / L). The activation reaction was carried out under constant temperature oscillation at 60 °C for 45 min. 7 mL of PGA enzyme solution (20 mg / mL) was added to the system, and the reaction was carried out under constant temperature oscillation at 30 °C for 8 hours. After the reaction, the unimmobilized free enzyme was removed by centrifugation and washing, and finally, an immobilized enzyme complex was obtained.

[0107] Comparative Example 1

[0108] To confirm the effect of EGPMA on the enzyme activity of the immobilized enzyme, penicillin G acylase immobilized based on pure PAA microspheres was prepared with reference to Example 1, with the difference that: the monomer added in step (3) of Example 1 was changed to an equimolar amount of tBA (8 g), and other reaction conditions remained unchanged, and SiO2-PAA was prepared.

[0109] Comparative Example 2

[0110] To confirm the effect of the addition amount of EGPMA on the activity of the immobilized enzyme, penicillin G acylase immobilized based on pure PAA microspheres was prepared with reference to Example 1, with the difference that: the monomers added in step (3) of Example 1 were changed to equimolar tBA (7.92 g) and EGPMA (0.13 g). At this time, EGPMA accounted for 1% of the total monomers, and other reaction conditions remained unchanged, and SiO2-PAA-EGPMA was prepared.

[0111] Comparative Example 3

[0112] To confirm the effect of the addition amount of EGPMA on the activity of the immobilized enzyme, penicillin G acylase immobilized based on pure PAA microspheres was prepared with reference to Example 1, with the difference that: the monomers added in step (3) of Example 1 were changed to equimolar tBA (7.2 g) and EGPMA (1.29 g). At this time, EGPMA accounted for 10% of the total monomers, and other reaction conditions remained unchanged, and SiO2-PAA-EGPMA was prepared.

[0113] Test conditions:

[0114] I. Determination of penicillin G acylase activity

[0115] Currently, the alkali titration method can be used to determine the activity of penicillin G acylase. Since the free penicillin G acylase used in the present invention is an enzyme solution, the determination of the activity of the immobilized enzyme and the free enzyme is slightly different. Put 50 mL of phosphate buffer solution with pH = 8 into a 100 mL beaker, add 0.4 g of potassium penicillin G, then adjust the pH to 8 with 0.1 mol / L sodium hydroxide solution, add the immobilized enzyme to be tested (or free penicillin G acylase enzyme solution), and then adjust the pH to 8 with 0.1 mol / L sodium hydroxide solution again. Start timing when the solution pH drops to 8. During the reaction, use 0.1 mol / L standard sodium hydroxide solution calibrated with potassium hydrogen phthalate to maintain the pH of the substrate solution at about pH = 8, keep the reaction time for 5 min, and record the volume of sodium hydroxide solution consumed.

[0116]

[0117] In the formula, EA represents the enzyme activity U / g of the immobilized penicillin G acylase; C NaOH represents the concentration of the titrated sodium hydroxide mol / L, V NaOH represents the volume of the titrated NaOH mL, T represents the titration time min, and W represents the mass of the immobilized enzyme g.

[0118]

[0119] Wherein, E represents the enzyme activity of free penicillin G acylase, U / mL; C NaOH represents the concentration of titrated sodium hydroxide, mol / L, V NaOH represents the volume of titrated NaOH, mL, T represents the titration time, min, and V represents the volume of added free penicillin G acylase solution, mL.

[0120] II. Enzyme loading

[0121] The Bradford method was used to measure the enzyme content. A standard curve was made with bovine serum albumin as the standard. The supernatant after static culture of the immobilized enzyme and Coomassie Brilliant Blue G250 were mixed in amounts of 5 μL and 200 μL respectively, shaken well and left to stand at room temperature for 5 min. The absorbance was measured at 595 nm with an enzyme-labeling instrument, and the enzyme concentration was converted according to the standard curve. The calculation of enzyme loading and enzyme activity recovery rate is specifically based on the following formula:

[0122]

[0123] Wherein, EAL represents the enzyme loading of the immobilized enzyme, mg / g, C0 (mg / mL) and V0 (mL) respectively represent the enzyme concentration of the original enzyme solution and the added amount of the original enzyme solution in the immobilization process; C1 (mg / mL) and V1 (mL) respectively represent the enzyme concentration in the immobilized enzyme and the volume of the supernatant after centrifugation of the whole immobilized enzyme solution; W represents the mass of the immobilized enzyme, g.

[0124] III. Enzyme activity recovery rate

[0125] The enzyme activity recovery rate refers to the percentage of the catalytic activity retained by the immobilized enzyme after the immobilization process in the total activity of the original free enzyme. This parameter is used to evaluate the effect of the immobilization process on enzyme activity and reflects the biocompatibility and technical feasibility of the carrier material and the immobilization method. According to the enzyme activity and enzyme loading, the following formula can be deduced:

[0126]

[0127] Wherein, EA represents the enzyme activity of the immobilized penicillin G acylase, U / g, C0 (mg / mL) represents the enzyme concentration of the original enzyme solution, E represents the enzyme activity of free penicillin G acylase, U / mL, and EAL represents the enzyme loading of the immobilized enzyme, mg / g.

[0128] Figure 1 is the preparation schematic diagram of the present invention, which details the reaction mechanism of the invention and the composition of the prepared product.

[0129] Figure 2 are the infrared spectra of Example 1, Example 2 and Comparative Example 1. The characteristic peak of the out-of-plane bending vibration of C-H in the monosubstituted benzene ring of EGPMA (750 cm -1) and the characteristic peak of the stretching vibration of the conjugated aromatic ring C═C skeleton (1500 cm -1 ), which appears in the infrared spectra of Example 1 and Example 2, indicates the successful polymerization of EGPMA. The characteristic peak of the C═O stretching vibration of the carboxylic acid group in the single polymer system of Comparative Example 1 is located at 1705 cm -1 , while in the copolymer polymer systems of Example 1 and Example 2, this characteristic peak is located at 1720 cm -1 with a significant blue shift, indicating that the incorporation of EGPMA affects the peak position of polyacrylic acid.

[0130] Figure 3 The thermogravimetric curves show the thermogravimetric curves of SiO2-Br, SiO2-PAA-EGPMA in Example 1 and SiO2-PAA in Comparative Example 1. The first stage (30 - 250 °C) corresponds to the removal of physically adsorbed water, and the second stage (250 - 600 °C) is attributed to the thermal decomposition of the grafted polymer chains. Silicon dioxide has excellent thermal stability and does not decompose above 600 °C. Based on the weight loss percentage, it can be calculated that the polymer grafted on the surface of SiO2-PAA contains 66.2%, and the polymer grafted on the surface of SiO2-PAA-EGPMA contains 65.7%. The similar grafting rates of the two indicate that the introduction of the EGPMA monomer does not significantly affect the polymerization efficiency of the PAA main chain, verifying the synthesis controllability of adding the two monomers.

[0131] Figure 4 are the transmission electron microscope images of SiO2 and SiO2-PAA-EGPMA in Example 1. It can be seen from the left figure that the unmodified SiO2 presents a monodisperse morphology, with a smooth surface and an average particle size of 700 nm, while the right figure shows that the surface of SiO2-PAA-EGPMA is coated with a layer of film, confirming the surface polymerization of the polymer. Due to the requirement of the TEM test environment for the sample to be dry, the polymer brush is transformed into a collapsed coating layer in the solution stretching state.

[0132] Penicillin G acylase is labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and can exhibit green fluorescence under the excitation of a 488 nm laser, while SiO2-PAA-EGPMA itself has no fluorescence, Figure 5 The laser confocal microscope images of

[0133] Figure 6Table 1 shows the enzyme activity performance, enzyme loading, and enzyme activity recovery of the immobilized penicillin G acylase obtained in Examples 1-6 and Comparative Examples 1-3. Examples 1 and Comparative Examples 1-3 used the same mass ratio of SiO2 and monomer. Among them, Comparative Example 1 was a single polyacrylic acid brush, and the polyacrylic acid / ethylene glycol phenyl ether methacrylate ratios of Comparative Example 2, Example 1, and Comparative Example 3 decreased in sequence. It can be seen from the figure that the immobilized enzyme in Comparative Example 1 had the lowest enzyme activity. The enzyme activity of the copolymer brush-immobilized penicillin G acylase was significantly improved compared to that of the single polyacrylic acid brush, and the binary copolymer EGPMA could effectively enhance the enzyme activity. However, although Comparative Example 3 had the highest proportion of EGPMA compared to Comparative Example 2 and Example 1, the enzyme activity decreased. This may be because too much EGPMA would lead to a decrease in the chemical binding efficiency between the enzyme and the loading, resulting in a decrease in enzyme activity.

[0134] By adjusting the mass ratio of SiO2 and monomer, as well as the polyacrylic acid / ethylene glycol phenyl ether methacrylate ratio, Examples 1-6 can obtain immobilized enzymes with excellent performance. From the data in Table 1, it can be seen that their enzyme activity is 1238-1284 U / g, the enzyme loading is 92 mg / g-95 mg / g, and the enzyme activity recovery is 75.8-78.8%, all of which are better than the immobilized enzyme of the single polyacrylic acid brush in Comparative Example 1. It is proved that the penicillin G acylase immobilized by the polyacrylic acid / ethylene glycol phenyl ether methacrylate brush of the present invention has more excellent enzyme activity performance and can further improve the enzyme activity recovery performance of the immobilized enzyme.

[0135] Table 1 Enzyme activity, enzyme loading, and enzyme activity recovery of the immobilized penicillin G acylase in Examples 1-6 and Comparative Examples 1-3

[0136]

[0137] As Figure 7 shown, PGA@SiO2-PAA-EGPMA of Example 1 still retained 81.4% of the initial enzyme activity after 10 cycles, which was significantly higher than that of PGA@SiO2-PAA in Comparative Example 1 (60%). This indicates that EGPMA improves the stability of the immobilized enzyme through its molecular structure synergistic effect. The EGPMA segment inhibits enzyme aggregation through steric hindrance and forms a dynamic hydration layer to buffer the shear stress. The phenyl ether hydrophobic unit and the hydrophilic carboxyl group of PAA construct a microphase separation interface, which synergistically enhances enzyme anchoring and substrate mass transfer, thereby reducing the shedding of the enzyme during repeated use.

[0138] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Preparation method of polyacrylic acid / polyethylene glycol phenyl ether methacrylate brush@SiO2 microsphere immobilized penicillin G acylase, characterized in that, It includes the following steps: (1) Carry out an ammoniation modification reaction on silica microspheres, ammonia water and a silane coupling agent to obtain ammoniated modified silica microspheres; (2) React the ammoniated modified silica microspheres with an ATRP initiator under the action of an acid-binding agent to obtain silica microspheres SiO2-Br with ATRP initiation sites immobilized on the surface; (3) Carry out a polymerization reaction on SiO2-Br, tert-butyl acrylate, and ethylene glycol phenyl ether methacrylate under the action of a transition metal catalyst, a ligand, and a reducing agent to obtain SiO2-PtBA-EGPMA; then remove the tert-butyl group in SiO2-PtBA-EGPMA to obtain SiO2-PAA-EGPMA; (4) Activate the carboxyl group of SiO2-PAA-EGPMA, and then carry out a covalent immobilization reaction with PGA enzyme solution to obtain immobilized enzyme PGA@SiO2-PAA-EGPMA.

2. The preparation method according to claim 1, characterized in that, The tert-butyl acrylate described in step (3) accounts for 92-98% of the total molar amount of tert-butyl acrylate and ethylene glycol phenyl ether methacrylate; And / or, the mass ratio of SiO2-Br to the total mass of the comonomers tert-butyl acrylate and ethylene glycol phenyl ether methacrylate in step (3) is 1:(6.1-10.0).

3. The preparation method according to claim 1, characterized in that, The transition metal catalyst described in step (3) is at least one of CuCl2, CuBr2, and FeCl3; And / or, the ligand described in step (3) is pentamethyldiethylenetriamine; And / or, the molar ratio of the total molar amount of tert-butyl acrylate and ethylene glycol phenyl ether methacrylate to the transition metal catalyst and the ligand in step (3) is 100:(20-26):(20-26); And / or, the reducing agent described in step (3) is at least one of ascorbic acid and stannous octoate, and more preferably ascorbic acid; And / or, the ratio of SiO2-Br to the reducing agent in step (3) is 1 g:1.6-4.8 mmol; And / or, the polymerization reaction in step (3) is carried out under an inert gas atmosphere; the inert gas is at least one of nitrogen, helium, and argon; And / or, the polymerization reaction temperature in step (3) is 30-60 °C, and the reaction time is 2-6 h; And / or, the method for removing the tert-butyl group in SiO2-PtBA-EGPMA in step (3) is: disperse SiO2-PtBA-EGPMA in a solvent, add trifluoroacetic acid to carry out a tert-butyl group removal reaction to obtain SiO2-PAA-EGPMA; And / or, the tert-butyl group removal reaction temperature is room temperature, and the reaction time is 46-50 h; the mass ratio of SiO2-Br to trifluoroacetic acid in SiO2-PtBA-EGPMA is 1:(4-6).

4. The preparation method according to claim 1, characterized in that, The addition amount of the PGA enzyme solution described in step (4) is 2-7 mL / g SiO2-PAA-EGPMA; And / or, the concentration of the PGA enzyme solution is 20 mg / mL; And / or, the temperature of the immobilization reaction in step (4) is 20-40 °C, and the time is 3-8 h; And / or, the activator for carboxyl activation in step (4) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide with a molar ratio of 2:1; And / or, the mass ratio of SiO2-PAA-EGPMA to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 g:4 - 12 mmol; And / or, the temperature for carboxyl activation in step (4) is 30 - 60 °C, and the time is 30 - 60 min.

5. The preparation method according to claim 1, characterized in that, The ATRP initiator in step (2) is 2-bromoisobutyryl bromide; And / or, the mass ratio of the aminated modified silica microspheres to the ATRP initiator in step (2) is 1:(1 - 3); And / or, the acid-binding agent in step (2) is triethylamine; And / or, the mass ratio of the ATRP initiator to the acid-binding agent in step (2) is 1:(1 - 3); And / or, the reaction in step (2) is to first react in an ice bath for 10 - 50 min, and then react at 20 - 40 °C for 16 - 24 h.

6. The preparation method according to claim 1, wherein The particle size of the silica microspheres in step (1) is 40 - 1000 nm; And / or, the mass ratio of the silica microspheres to ammonia water in step (1) is 2 - 8 g:4 - 6 mL; And / or, the silane coupling agent in step (1) is at least one of 3-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane; And / or, the mass ratio of the silica microspheres to the silane coupling agent in step (1) is 1:(0.6 - 0.9); And / or, the temperature of the amination modification reaction in step (1) is 30 - 80 °C, and the reaction time is 6 - 15 h.

7. According to the preparation method described in any one of claims 1 to 6, it is characterized in that Comprising the following steps: (1) Add silica microspheres, ammonia water and silane coupling agent to a solvent, heat for amination modification reaction, after the reaction is completed, centrifuge and separate, dry, to obtain aminated modified silica microspheres; (2) Dissolve the aminated modified silica microspheres and the acid-binding agent in a solvent, dropwise add the ATRP initiator under ice bath, then heat for reaction, after the reaction is completed, centrifuge, wash, dry, to obtain silica microspheres SiO2-Br with ATRP initiator sites immobilized on the surface; (3) Dissolve SiO2-Br, tert-butyl acrylate, ethylene glycol phenyl ether methacrylate, transition metal catalyst and ligand in a solvent, under an inert gas atmosphere, add a reducing agent solution for polymerization reaction, after the reaction is completed, centrifuge, wash, to obtain SiO2-PtBA-EGPMA; (4) Disperse SiO2-PtBA-EGPMA in a solvent, add trifluoroacetic acid for de-tert-butylation reaction, remove the solvent, dry, to obtain SiO2-PAA-EGPMA; (5) Disperse SiO2-PAA-EGPMA in PBS buffer solution, add a carboxylation reagent for carboxyl activation reaction, then add PGA enzyme solution for covalent immobilization reaction, after the reaction is completed, centrifuge and wash, to obtain immobilized enzyme PGA@SiO2-PAA-EGPMA.

8. The preparation method according to claim 7, characterized in that, The solvent in step (1) is ethanol, and the concentration of the silica microspheres in the solvent is 0.02 - 0.08 g / mL; And / or, the concentration of the ammoniated modified silica microspheres described in step (2) in the solvent is 0.02 - 0.05 g / mL; the solvent is tetrahydrofuran; And / or, the solvent described in step (3) is N,N-dimethylformamide; the concentration of SiO2-Br in the solvent is 0.005 - 0.02 g / mL; And / or, the concentration of the reducing agent solution described in step (3) is 0.2 - 0.6 mol / L, and the solvent is dichloromethane; And / or, the solvent described in step (4) is dichloromethane; the concentration of SiO2-PtBA-EGPMA in the solvent is 0.02 - 0.03 g / mL; And / or, the concentration of SiO2-PAA-EGPMA in the PBS buffer solution described in step (5) is 0.005 mg / mL; the pH of the PBS buffer solution is 5 - 10.

9. Polyacrylic acid / poly(ethylene glycol phenyl ether methacrylate) brush@SiO2 microsphere-immobilized penicillin G acylase obtained by the preparation method according to any one of claims 1 to 8.

10. Application of the polyacrylic acid / poly(ethylene glycol phenyl ether methacrylate) brush@SiO2 microsphere-immobilized penicillin G acylase according to claim 9.

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