A method for preparing highly catalytically selective immobilized penicillin G acylase hydrogel microspheres

Abstract

This invention discloses a method for preparing highly catalytically selective immobilized penicillin G acylase hydrogel microspheres. The method comprises three steps: First, sodium alginate, gelatin, and zwitterionic monomers are mixed to form a sodium alginate stock solution; second, an initiator and a crosslinking agent are added to the sodium alginate stock solution, and polymerization is carried out under ultraviolet light to obtain a polymerized stock solution; third, a penicillin G acylase solution is mixed with the polymerized stock solution to form a hydrogel stock solution, which is then injected into an inorganic salt solution to obtain immobilized penicillin G acylase hydrogel microspheres. This invention, by adding gelatin and zwitterionic monomers, provides a method that can improve the activity, stability, and catalytic selectivity of immobilized enzymes in a sodium alginate system, and is considered a green, controllable, and highly promising method for immobilizing penicillin G acylase.

Description

Technical Field

[0001] This invention relates to a method for preparing a sodium alginate stock solution containing gelatin and zwitterions, wherein the sodium alginate stock solution mixed with an enzyme is crosslinked with inorganic salt ions to construct immobilized penicillin G acylase hydrogel microspheres for biomedical use. Background Technology

[0002] In industrial applications, enzyme-catalyzed reactions are often reversible processes. The selectivity of the reaction direction often depends on the thermodynamic equilibrium and kinetic control of the reaction system. In the biopharmaceutical field, penicillin G acylase is frequently used to synthesize important semi-synthetic β-lactam antibiotics. However, due to the accumulation of antibiotic drug during the reaction, it promotes the reverse hydrolysis of the antibiotic to generate the antibiotic nucleus, thereby inhibiting the forward synthesis reaction and reducing industrial yield. Precise regulation of enzyme reaction equilibrium directly determines the product synthesis efficiency and feedstock conversion rate. Suppressing undesirable side reactions can prevent a decrease in product purity and reduce separation costs. This process intensification strategy can comprehensively improve the space-time yield and specificity of the process, promoting the greening and sustainability of biomanufacturing. Therefore, precise regulation of enzyme reaction equilibrium is not only a core scientific issue in basic enzyme catalysis research but also a key technological challenge in promoting the cross-application of enzyme catalysis and continuous flow.

[0003] From a thermodynamic perspective, the direction of an enzyme-catalyzed reaction depends on the Gibbs free energy of the system, and modifying reaction conditions or the properties of the enzyme itself is an important approach to controlling reaction equilibrium. Using non-aqueous solvents or adjusting reaction conditions such as pH and temperature often leads to unavoidable enzyme inactivation. Modifying or altering enzyme molecules can fundamentally change the catalytic equilibrium. For example, adjusting the electrostatic environment of the enzyme's catalytic site through site-directed mutagenesis or directed evolution can make hydrolases more inclined towards synthetic reactions. Furthermore, enzyme immobilization technology endows enzymes with scalable functions by altering the microenvironment surrounding the enzyme; for example, by changing the mass transfer process between substrate and product around the enzyme, it can improve the catalytic performance and regioselectivity of various enzymes. Immobilized enzymes, by regulating the microenvironment, not only achieve a trade-off between activity and stability but also advance the research of continuous catalysis.

[0004] Sodium alginate (ALG / SA) is widely used in immobilized enzymes, producing biomaterials that are low-cost, recyclable, and biocompatible. By cross-linking sodium alginate with inorganic salts and controlling the process, hydrogels of various shapes can be obtained. Its excellent physicochemical properties have broad applications in biomedicine. However, the traditional application of sodium alginate is hampered by limited enzyme aggregation and diffusion, resulting in a significant decrease in relative catalytic activity, which is a major challenge. To meet the demands of industrial applications, it is necessary to increase the cross-linking density to improve the mechanical strength of the hydrogel. However, this characteristic hinders the mass transfer of substrates and products, as well as the diffusion of water molecules, further leading to a loss of enzyme activity and decreased stability. Therefore, it is urgent to explore a hydrogel that achieves an optimal balance between mechanical strength and mass transfer efficiency. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a method for preparing highly catalytically selective immobilized penicillin G acylase hydrogel microspheres. First, a sodium alginate stock solution containing zwitterions and gelatin is prepared. Then, the sodium alginate stock solution mixed with penicillin G acylase is cross-linked with inorganic salt ions to form immobilized enzyme microspheres.

[0006] A method for preparing highly catalytically selective immobilized penicillin G acylase hydrogel microspheres, the method comprising the following steps: Step 1: Dissolve sodium alginate (SA), gelatin, and zwitterionic monomers in a solvent in a certain proportion to obtain sodium alginate stock solution.

[0007] Step 2: Add a certain amount of initiator and crosslinking agent to the sodium alginate stock solution, mix well, and after the liquid becomes turbid, irradiate it under ultraviolet light for a period of time to allow the added zwitterionic monomers to polymerize, and obtain the polymerized stock solution.

[0008] Step 3: Mix the penicillin G acylase (PGA) solution with the polymerized stock solution to obtain a mixture. As The hydrogel stock solution was then injected into an inorganic salt solution using an injection molding machine to solidify and form microspheres. The microspheres were then cross-linked with sodium alginate using inorganic salt ions and washed with a solvent to obtain immobilized penicillin G acylase hydrogel microspheres.

[0009] In the preferred embodiment of the above-described technical solution, the molecular weight of the sodium alginate material is 50-100 kDa; the molecular weight of the gelatin is 20-50 kDa, and the gel strength is 100-250 bloom.

[0010] In the preferred embodiment of the above-described technical solution, the zwitterionic monomer is one or more of lysine (Lys), glutamic acid (Glu), aspartic acid (Asp), sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), cocamidopropyl betaine (CAB), phosphatidylcholine (PC), carboxybetaine acrylamide (CBAA), and N,N-dimethyl-N-(3-acrylamido)propanediamine oxide (TMAO), preferably one or two of sulfobetaine methacrylate, carboxybetaine methacrylate, cocamidopropyl betaine, and carboxybetaine acrylamide.

[0011] In the preferred embodiment of the above-described technical solution, the solvent of the sodium alginate stock solution includes one or more of the following: water (ultrapure water), phosphate buffer, methanol, ethanol, acetonitrile, acetone, diethyl ether, N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane, preferably one or two of the following: water (ultrapure water) and phosphate buffer.

[0012] In the preferred embodiment of the above-described technical solution, the ratio of sodium alginate, gelatin, and solvent in the sodium alginate stock solution is 0.1~100 g: 0.1~20 g: 100 mL, more preferably 1~10 g: 5~20 g: 100 mL. The concentration of the zwitterionic monomer in the sodium alginate stock solution is 50~1000 mM, preferably 150-1000 mM, and more preferably 500 mM.

[0013] In the preferred embodiment of the above-described technical solution, sodium alginate, gelatin, zwitterionic compound, and solvent are mixed in a certain proportion and then heated at 30-100 ℃ for 1-6 hours (preferably at 60 ℃ for 2 hours) to dissolve them and obtain sodium alginate stock solution.

[0014] In the preferred embodiment of the above-described technical solution, the initiator is one or more of the following: benzoyl peroxide, dicumyl peroxide, azobisisobutyronitrile, 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and potassium persulfate. Preferably, it is one or two of the following: 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone. The ratio of zwitterionic monomer to initiator is 0.1~50 g:0.1~100 μL, preferably 1~10 g:1~10 μL. For example, the amount of initiator used is 0.1~100 μL, preferably 1~10 μL.

[0015] In the preferred embodiment of the above-described technical solution, the crosslinking agent is one or more of dicumyl peroxide, ethylene glycol dimethacrylate, diphenylmethane diisocyanate, triethylene glycol dimethacrylate, diethylenetriamine, and methyltrimethoxysilane, preferably one or two of ethylene glycol dimethacrylate and triethylene glycol dimethacrylate. The ratio of zwitterionic monomer to crosslinking agent is 0.1~50 g:0.1~100 μL, preferably 1~10 g:1~10 μL, for example, the amount of crosslinking agent used is 0.1~100 μL, preferably 1~10 μL. When the crosslinking agent is ethylene glycol dimethacrylate and triethylene glycol dimethacrylate, the volume ratio of ethylene glycol dimethacrylate to triethylene glycol dimethacrylate is 1:1~2.

[0016] In the above-described technical solution, in a preferred embodiment, the penicillin G acylase is derived from one or more of the strains Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens, and Providenciarettgeri, preferably from one or both of Escherichia coli and Kluyveracryocrescens.

[0017] In the above-described technical solution, preferably, the concentration of penicillin G acylase in the PBS buffer solution is 0.1~10 mg / mL, preferably 1~6 mg / mL; wherein the pH of the PBS buffer solution is 5~9, preferably 6~8, and more preferably 7.3.

[0018] In the preferred embodiment of the above-described technical solution, the second step involves ultrasonic oscillation to achieve uniform mixing, with the oscillation time being 30-60 seconds.

[0019] In the preferred embodiment of the above-described technical solution, the reaction conditions in the second step are as follows: the wavelength of the ultraviolet lamp used to initiate the polymerization of zwitterionic monomers is 100-300 nm, preferably 150-250 nm; the polymerization temperature is 10-60℃, preferably 20-40℃; and the polymerization time is 10-120 min, preferably 10-30 min.

[0020] In the preferred embodiment of the above-described technical solution, the inorganic salt comprises one or more of calcium chloride, calcium carbonate, magnesium chloride, strontium chloride, magnesium sulfate, barium chloride, and barium sulfate, preferably one or two of calcium chloride, strontium chloride, and barium chloride. The mass fraction of the inorganic salt solution used is 0.1%-10%, preferably 1%-3%. The volume of the inorganic salt solution used is 100-500 mL, preferably 500 mL.

[0021] In the preferred embodiment of the above-described technical solution, in the third step, the volume ratio of the penicillin G acylase (PGA) solution to the polymerized stock solution is 1-5:5-10, preferably 1-2:10. For example, the volume of the penicillin G acylase (PGA) solution is 1-5 mL, and the volume of the polymerized stock solution is 5-10 mL. The injection speed of the injection machine is 0.1-2.0 mL / s, preferably 0.5-1.0 mL / s.

[0022] The penicillin G acylase hydrogel microspheres prepared by the above method have good mechanical and catalytic properties, and the diameter of the microspheres is 0.5 ~ 5.0 mm.

[0023] The above-mentioned immobilized penicillin G acylase hydrogel microspheres are used as catalysts in the reversible synthesis of drugs, such as β-lactam antibiotics.

[0024] In the preferred embodiment of the above-described technical solution, the reactants for synthesizing β-lactam antibiotics are 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-phenylglycine amide (D-PGA), with a molar ratio of 7-aminodeacetoxycephalosporanic acid (7-ADCA) to D-phenylglycine amide (D-PGA) of 1:1 to 5, preferably 1:1 to 2. The mass ratio of penicillin G acylase hydrogel microspheres to 7-aminodeacetoxycephalosporanic acid (7-ADCA) is 1 to 5:0.1 to 1.0, preferably 1 to 2:0.2 g to 0.5. The reaction is carried out in PBS buffer. The concentration of 7-aminodeacetoxycephalosporanic acid (7-ADCA) in the PBS buffer is 1 to 200 mM, preferably 100 to 150 mM.

[0025] In the above-described technical solution, preferably, the reaction pH of the catalytic reaction of the immobilized penicillin G acylase hydrogel microspheres is 3 to 11, preferably 6 to 8, and the reaction temperature is 10 to 80°C, preferably 20 to 40°C.

[0026] This invention improves the mechanical properties of sodium alginate by adding gelatin and mixing in zwitterionic monomers, and provides a method to improve the activity, stability and catalytic selectivity of immobilized enzymes in sodium alginate systems. It is considered a green, controllable and promising method for immobilizing enzymes.

[0027] The method for preparing highly catalytically selective penicillin G acylase hydrogel microspheres described in this invention has the following advantages over other methods: (1) The immobilized enzyme microspheres have stable physicochemical properties and can be applied in a variety of scenarios; (2) Immobilized enzyme microspheres have micropores, which can increase the enzyme loading capacity.

[0028] (3) Immobilized enzyme microspheres have good mechanical properties; (4) Immobilized enzyme microspheres improve the activity and stability of enzyme catalysis; (5) Immobilized enzyme microspheres improve the catalytic selectivity of penicillin G acylase in reversible drug synthesis; (6) Good biocompatibility. No toxic chemicals are used in the whole process, which ensures the biodegradability and biocompatibility of the microspheres.

[0029] In summary, this method is a simple, environmentally friendly method for immobilizing penicillin G acylase, exhibiting good mechanical properties, excellent catalytic activity, stability, and catalytic selectivity. Attached Figure Description

[0030] Figure 1 : The prepared sodium alginate stock solution containing gelatin and zwitterionic monomers.

[0031] Figure 2 Immobilized enzyme microspheres obtained by cross-linking with inorganic salt ions.

[0032] Figure 3 Infrared spectral characterization of immobilized enzyme microspheres.

[0033] Figure 4 Scanning electron microscopy was used to characterize the lyophilized immobilized enzyme microspheres.

[0034] Figure 5 : PGA loading of immobilized enzyme microspheres.

[0035] Figure 6 Determination of tensile and compressive moduli of immobilized enzymes.

[0036] Figure 7 The effect of concentration and type of zwitterions in immobilized enzyme microspheres on catalytic efficiency.

[0037] Figure 8 Determination of synthetic selectivity of immobilized enzymes in reversible drug synthesis.

[0038] Figure 9 pH, temperature stability, reusability, and storage stability of immobilized enzyme microspheres. Detailed Implementation

[0039] This invention provides a method for preparing highly catalytically selective penicillin G acylase hydrogel microspheres. The method involves dissolving sodium alginate (molecular weight 50-100 kDa), gelatin (molecular weight 20-50 kDa, gel strength 100-250 bloom), and zwitterionic monomers in a solvent to form a sodium alginate stock solution. An initiator and crosslinking agent are added, and after thorough mixing until the liquid becomes turbid, it is irradiated under ultraviolet light for a period of time to polymerize the added zwitterionic monomers, yielding a polymerized stock solution. A penicillin G acylase solution is mixed with the polymerized stock solution, and then crosslinked with sodium alginate using an inorganic salt. Microspheres are then prepared using an injection molding machine. After washing with a solvent, immobilized enzyme hydrogel microspheres are obtained. The diameter of the penicillin G acylase hydrogel microspheres prepared by the above method is 0.5-5.0 mm.

[0040] The following specific embodiments are for further explanation of the content of the present invention and should not be construed as limiting the present invention in any way.

[0041] In the examples below, the penicillin G acylase was obtained from the Kluyvera cryocrescens strain (Cheng, T., Chen, M., Zheng, H., Wang, J., Yang, S., & Jiang, W. (2006). Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis / hydrolysis ratios for cephalexin. Protein Expression and Purification, 46(1), 107-113.).

[0042] Example 1: Preparation of sodium alginate stock solution Experiment 1: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa and 1.6 g of gelatin with a molecular weight of 20-50 kDa and a gel strength of 100-250 bloom were mixed in 8 mL of PBS buffer (pH 7.3). Different concentrations (150, 300, 500, 1000 mM) of the zwitterionic monomer SBMA were added, and the mixture was heated at 60 °C for 2 h to dissolve, yielding the sodium alginate stock solution (denoted as SA-SBMA). Figure 1As shown, the 500mM SBMA sodium alginate stock solution appears as a slightly turbid light yellow.

[0043] Experiment 2: 0.2g of sodium alginate with a molecular weight of 50-100 kDa and 1.6g of gelatin with a molecular weight of 20-50 kDa and a gel strength of 100-250 bloom were mixed in 8 mL of PBS buffer (pH 7.3). Different concentrations (150, 300, 500, 1000 mM) of zwitterionic monomer CBMA were added respectively. After heating at 60 °C for 2 h, the mixture was dissolved to obtain sodium alginate stock solution (denoted as SA-CBMA).

[0044] Experiment 3: 0.2g of sodium alginate with a molecular weight of 50-100 kDa and 1.6g of gelatin with a molecular weight of 20-50 kDa and a gel strength of 100-250 bloom were mixed in 8 mL of PBS buffer (pH 7.3). Different concentrations (150, 300, 500, 1000 mM) of zwitterionic monomer CBAA were added respectively. After heating at 60 °C for 2 h, the mixture was dissolved to obtain sodium alginate stock solution (denoted as SA-CBAA).

[0045] Experiment 4: 0.2g of sodium alginate with a molecular weight of 50-100 kDa and 1.6g of gelatin with a molecular weight of 20-50 kDa and a gel strength of 100-250 bloom were mixed in 8 mL of PBS buffer (pH 7.3). Different concentrations (150, 300, 500, 1000 mM) of zwitterionic monomer TMAO were added respectively. After dissolving by heating at 60 °C for 2 h, sodium alginate stock solution (denoted as SA-TMAO) was obtained.

[0046] Example 2: Preparation of immobilized enzyme microspheres Experiment 1: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa was added to 8 mL of PBS buffer (pH 7.3) and heated at 60 °C for 2 h to dissolve, yielding the sodium alginate stock solution. This stock solution was then mixed with 2 mL of PBS buffer (pH 7.3) containing 3 mg / mL penicillin G acylase. The mixture was then injected into 500 mL of 2% calcium chloride solution using a syringe (1 mL) at an injection rate of 0.8 mL / s to solidify and form microspheres. After washing with PBS, the immobilized enzyme microspheres were obtained and named SA@PGA. The resulting microspheres are shown in the figure. Figure 2 As shown, the diameter of the microspheres is approximately 2 mm.

[0047] Experiment 2: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa and 1.6 g of gelatin with a glue strength of 100-250 bloom were added to 8 mL of PBS buffer (pH 7.3) and heated at 60 °C for 2 h to dissolve, yielding the sodium alginate stock solution. This stock solution was then mixed with 2 mL of a PBS solution containing 3 mg / mL penicillin G acylase to obtain a mixture. The mixture was then injected into 500 mL of a 2% (w / w) calcium chloride solution using a syringe (1 mL) at an injection rate of 0.8 mL / s to solidify and form microspheres. After washing with PBS, the immobilized enzyme microspheres were obtained and named SA-gel@PGA. The obtained microspheres are shown below. Figure 2 As shown, the diameter of the microspheres is approximately 4 mm.

[0048] Experiment 3: 1 μL of 2-hydroxy-2-methylphenylacetone, 1 μL of ethylene glycol dimethacrylate, and 1 μL of triethylene glycol dimethacrylate were added to the sodium alginate stock solution prepared in Example 1. The solution was sonicated and shaken for 30 seconds in an ultrasonic vibrator until turbid. Then, it was irradiated under a 220 nm UV lamp for 20 min to allow the added zwitterionic monomers SBMA, CBMA, CBAA, and TMAO to polymerize, yielding the polymerized stock solution. 2 mL of PBS buffer (pH 7.3) containing 3 mg / mL penicillin G acylase from Kluyveracryocrescens was mixed with the polymerized stock solution to obtain a mixture. This mixture was then injected (1 mL) into 500 mL of a 2% (w / w) calcium chloride solution to solidify and form microspheres. After washing with PBS, immobilized enzyme microspheres were obtained and named SA-gel-pSBMA@PGA (polymerized using the zwitterionic monomer SBMA), SA-gel-pCBAA@PGA (polymerized using the zwitterionic monomer CBAA), SA-gel-pCBMA@PGA (polymerized using the zwitterionic monomer CBMA), and SA-gel-pTMAO@PGA (polymerized using the zwitterionic monomer TMAO). Microspheres prepared at 500 mM with different types of zwitterionic monomers are shown below. Figure 2 As shown, the diameter of the microspheres is approximately 4 mm.

[0049] Example 3: Infrared spectroscopy characterization of the above microspheres The microspheres prepared in Example 2 (prepared from 500mM of different types of zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) were freeze-dried to remove the internal solvent. Fourier transform infrared spectroscopy was used to test the microspheres, and the test results are as follows: Figure 3 As shown, the results indicate that the range is 3100-3500 cm. 1 The peak at 1600 cm⁻¹ is attributed to the tensile vibrations of the hydroxyl groups in sodium alginate and the amino groups in gelatin. After the addition of the zwitterionic polymer, the peak at 1600 cm⁻¹... -1 Tensile vibrations of the carbonyl group in amides and esters were discovered, with a value of 1316 cm⁻¹. 1 The peak value at this point is caused by the stretching vibration of the sulfone group. After PGA immobilization and backfilling, peak values ​​of 980-1060 cm⁻¹ were observed. 1 The peak of the ether group at the location confirms the successful preparation of the material.

[0050] Example 4: Scanning electron microscopy characterization of lyophilized immobilized enzyme microspheres The microspheres prepared in Example 2 (prepared from 500mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) were freeze-dried to remove the internal solvent. The cross-sections of the microspheres were measured using a field emission scanning electron microscope (SEM5000). The test results are as follows: Figure 4 As shown, the sodium alginate group and the sodium alginate-gelatin group exhibit a relatively smooth cross-section, while the cross-section becomes rough and some small pore structures appear after the addition of the zwitterionic polymer.

[0051] Example 5: PGA loading of immobilized enzyme microspheres The residual enzyme content in the supernatant (calcium chloride solution) after the microspheres (prepared with 500 mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) prepared in Example 2 was measured using a micro-BCA protein concentration kit. The PGA loading of the immobilized enzyme microspheres was obtained by subtracting the residual enzyme content from the total enzyme content. Figure 5As shown, the loading was 3.17~5.62 mg / g, and the loading after adding zwitterionic polymer was about 1.77 times higher than that without it.

[0052] Example 6: Determination of the mechanical properties of immobilized enzymes Experiment 1: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa was mixed in 8 mL of PBS buffer (pH 7.3) and heated at 60 °C for 2 h to dissolve, yielding a sodium alginate stock solution. This sodium alginate stock solution was then mixed with 2 mL of PBS buffer (pH 7.3) containing 3 mg / mL penicillin G acylase to obtain a mixture. The mixture was poured into mechanical testing molds. The compression and tension molds were cylindrical (with a cross-sectional area of ​​28.26 mm²). 2 The molds were prepared in two shapes: a height of 5.2 mm and a dumbbell shape (length: 40 mm, width: 6 mm, thickness: 4 mm). The molds were then immersed in 500 mL of a 2% (w / w) calcium chloride solution for 10 min. After drying at room temperature, the mechanical testing model was obtained and named SA@PGA.

[0053] Experiment 2: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa and 1.6 g of gelatin with a glue strength of 100-250 bloom were added to 8 mL of PBS buffer (pH 7.3). The mixture was heated at 60 °C for 2 h to dissolve the alginate stock solution. This sodium alginate stock solution was then mixed with 2 mL of PBS buffer (pH 7.3) containing 3 mg / mL penicillin G acylase. The mixture was poured into mechanical testing molds. The compression and tension molds were cylindrical (cross-sectional area 28.26 mm²). 2 The molds were prepared in two shapes: a height of 5.2 mm and a dumbbell shape (length: 40 mm, width: 6 mm, thickness: 4 mm). The molds were then immersed in 500 mL of a 2% calcium chloride solution for 10 min. After drying at room temperature, the mechanical testing model was obtained and named SA-gel@PGA.

[0054] Experiment 3: 0.2 g of sodium alginate with a molecular weight of 50-100 kDa and 1.6 g of gelatin with a molecular weight of 20-50 kDa and a gel strength of 100-250 bloom were mixed in 8 mL of PBS buffer (pH 7.3). 500 mM of zwitterionic monomers SBMA, CBMA, CBAA, and TMAO were added separately. After dissolving by heating at 60 ℃ for 2 h, a sodium alginate stock solution was obtained. 1 μL of 2-hydroxy-2-methylphenylacetone, 1 μL of ethylene glycol dimethacrylate, and 1 μL of triethylene glycol dimethacrylate were added to the prepared sodium alginate stock solution. The solution was ultrasonicated and shaken for 30 s to make it turbid. Then, it was irradiated under a 220 nm UV lamp for 20 min to allow the added zwitterionic monomers SBMA, CBMA, CBAA, and TMAO to polymerize, resulting in a polymerized stock solution. The polymerized stock solution was then mixed with 2 mL of PBS buffer (pH 7.3) containing 3 mg / mL penicillin G acylase to obtain a mixture. The mixture was then poured into mechanical testing molds. The compression and tension molds were cylindrical (with a cross-sectional area of ​​28.26 mm²). 2 Gel specimens were prepared in two shapes: a height of 5.2 mm and a dumbbell shape (length: 40 mm, width: 6 mm, thickness: 4 mm). The molds were then immersed in 500 mL of a 2% (w / w) calcium chloride solution for 10 min. After drying at room temperature, mechanical testing models were obtained and named SA-gel-pSBMA@PGA (polymerized using the zwitterionic monomer SBMA), SA-gel-pCBAA@PGA (polymerized using the zwitterionic monomer CBAA), SA-gel-pCBMA@PGA (polymerized using the zwitterionic monomer CBMA), and SA-gel-pTMAO@PGA (polymerized using the zwitterionic monomer TMAO).

[0055] The compression and tensile behavior of the gel was evaluated using a universal testing machine (E43, MTS Instruments, USA) equipped with a 50N load cell. All mechanical tests were performed under conditions exceeding 60% humidity to prevent moisture evaporation from the gel. Compression and tensile tests were conducted at 0.5 mm... 1 The loading rate was determined. To prevent slippage between the specimen surface and the geometrically shaped clamps, both ends of the clamps were covered with sandpaper to increase roughness and secure the dumbbell-shaped specimen. The compressive and tensile moduli were calculated based on the average slope of the initial portion (0%-10% strain) of the stress-strain curve, such as... Figure 6 As shown, the polyzwitterionic group showed an increase of approximately 1.18 to 1.53 times compared to the sodium alginate and sodium alginate-gelatin groups.

[0056] Example 7: Effect of zwitterion concentration in immobilized enzyme on enzyme activity Weigh 1.5 g of each of the immobilized enzyme microspheres prepared with different concentrations and types of zwitterionic monomers as described in Example 2 (SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA, respectively). Catalyze the reaction in 2 mL of 1 mM PBS solution of 2-nitro-5-phenylacetaminobenzoic acid (pH 7.3) at room temperature for 1 min. Dilute 100 μL of the catalytic product with 100 μL of ethanol. Analyze the absorbance at 410 nm. Repeat the reaction three times and take the average value. Figure 7 The results showed that the activity of the immobilized enzymes first increased and then decreased with increasing concentration of the four zwitterions, reaching a maximum at 500 mM, which was 1.18 to 1.37 times that of the 150 mM group. Furthermore, the addition of zwitterions significantly improved the immobilized enzyme activity compared to the sodium alginate group and the sodium alginate-gelatin group, by approximately 1.02 to 1.13 times.

[0057] Example 8: Determination of synthetic selectivity of immobilized enzymes in reversible drug synthesis 1.0 g of the microspheres prepared in Example 2 (prepared with 500 mM of different zwitterionic monomers, namely SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA as experimental groups, and free PGA and SA@PGA of equivalent immobilized enzyme amount as control groups) were added. The mixture was reacted at room temperature for 60 min in 2 mL of PBS solution (pH 7.3) containing 133 mM 7-aminodeacetoxycephalosporanic acid (7-ADCA) and 267 mM D-phenylglycine amide (D-PGA). The supernatant was then diluted 20-fold with ethanol. HPLC analysis was performed. The reaction was repeated three times, and the average value was taken. A reaction selectivity parameter was defined: the S / H ratio, i.e., the synthesis-hydrolysis ratio, is the molar ratio of the synthesized product cephalexin to the hydrolyzed product D-phenylglycine when an equal amount of 7-ADCA is consumed. Figure 8 The results showed that the S / H ratio of the immobilized enzyme was significantly higher than that of the free enzyme, increasing by approximately 1.59 times. The addition of zwitterions increased the ratio by approximately 1.70 times compared to the free enzyme.

[0058] Example 9: pH, temperature stability, reusability, and storage stability of immobilized enzymes Experiment 1: To evaluate the heat resistance of immobilized PGA, free PGA (2 mg PGA) or immobilized enzyme microspheres prepared in Example 2 (prepared with 500 mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, SA-gel-pTMAO@PGA, and 2 mg PGA) were incubated in PBS solution (pH 7.3) for 20 min at 10, 20, 30, 40, 50, and 60 °C. Then, they were catalyzed in 2 mL of 1 mM PBS solution of 2-nitro-5-phenylacetaminobenzoic acid (pH 7.3) at room temperature for 1 min. 100 μL of the catalytic product was diluted with 100 μL of ethanol. The absorbance was measured at 410 nm for analysis. The reaction was repeated three times as parallel experiments, and the average value was taken to evaluate the enzyme activity.

[0059] Experiment 2: To evaluate the pH tolerance of immobilized PGA, free PGA (2 mg PGA) or immobilized enzyme microspheres prepared in Example 2 (prepared with 500 mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) were incubated at room temperature for 20 min at pH = 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Catalysis was then performed for 1 min at room temperature in 2 mL of 1 mM PBS solution of 2-nitro-5-phenylacetaminobenzoic acid (pH 7.3). 100 μL of the catalytic product was diluted 1 / 2 with 100 μL of ethanol. The absorbance was measured at 410 nm for analysis. The reaction was repeated three times as parallel experiments, and the average value was used to evaluate the enzyme activity.

[0060] Experiment 3: To evaluate the storage stability of immobilized PGA, 30 mL of 1 mg / mL free enzyme or immobilized enzyme microspheres prepared in Example 2 (prepared with 500 mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) were stored in PBS solution (pH 7.3) at 37°C for 30 days. During the storage period, every 5 days, 2 mg (free or immobilized enzyme microspheres) was taken out and catalyzed in 2 mL of 1 mM PBS solution of 2-nitro-5-phenylacetaminobenzoic acid (pH 7.3) at room temperature for 1 min. 100 μL of the catalytic product was diluted with 100 μL of ethanol. The absorbance was measured at 410 nm for analysis. The reaction was repeated 3 times as a parallel experiment, and the average value was used to evaluate the enzyme activity.

[0061] All three assessments above used free PGA as a control.

[0062] Experiment 4: To evaluate the repetitive catalytic stability of immobilized PGA, 1.5 g of the immobilized enzyme microspheres prepared in Example 2 (prepared with 500 mM of different zwitterionic monomers, namely SA@PGA, SA-gel@PGA, SA-gel-pSBMA@PGA, SA-gel-pCBAA@PGA, SA-gel-pCBMA@PGA, and SA-gel-pTMAO@PGA) were catalyzed in 2 mL of 1 mM PBS solution of 2-nitro-5-phenylacetaminobenzoic acid (pH 7.3) at room temperature for 1 min. 100 μL of the catalytic product was diluted 1 / 2 with 100 μL of ethanol. The absorbance was measured at 410 nm for analysis. Repeated catalytic reactions were performed. After each reaction, the immobilized enzyme was washed three times with PBS solution (pH 7.3) before the next reaction, repeated 6 times. The enzyme activity of each reaction was evaluated. Three replicates were considered parallel experiments, and the average value was taken.

[0063] like Figure 9 As shown, the microspheres with the addition of zwitterionic polymer exhibit improved temperature, pH stability, storage stability, and repeated catalytic stability compared to those without zwitterionic polymer, demonstrating the "buffering" effect of zwitterionic polymer.

Claims

1. A method for preparing highly catalytically selective immobilized penicillin G acylase hydrogel microspheres, characterized in that, The method includes the following steps: Step 1: Dissolve sodium alginate, gelatin, and zwitterionic monomers in a solvent to obtain sodium alginate stock solution; Step 2: Add initiator and crosslinking agent to sodium alginate stock solution, mix well, and polymerize under ultraviolet light to obtain polymerized stock solution; Step 3: Mix the penicillin G acylase solution with the polymerized stock solution to obtain a mixture. Inject the mixture into an inorganic salt solution to solidify and form microspheres. After cleaning with a solvent, immobilized penicillin G acylase hydrogel microspheres are obtained.

2. The preparation method according to claim 1, characterized in that, The sodium alginate has a molecular weight of 10-200 kDa; the gelatin has a molecular weight of 10-100 kDa and a gel strength of 100-500 bloom.

3. The preparation method according to claim 1, characterized in that, The zwitterionic monomers are one or more of the following: lysine, glutamic acid, aspartic acid, sulfobetaine methacrylate, carboxybetaine methacrylate, cocamidopropyl betaine, phosphatidylcholine, carboxybetaine acrylamide, and N,N-dimethyl-N-(3-acrylamido)propanediamine oxide. The solvents used in the sodium alginate stock solution include one or more of the following: ultrapure water, phosphate buffer, methanol, ethanol, acetonitrile, acetone, diethyl ether, N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane.

4. The preparation method according to claim 1, characterized in that, In the first step, the ratio of gelatin, sodium alginate and solvent in the sodium alginate stock solution is 0.1~100 g: 0.1~20 g: 100 mL; the concentration of zwitterionic monomer in the sodium alginate stock solution is 50~1000 mM.

5. The preparation method according to claim 1, characterized in that, The initiator is a solution of one or more of the following: benzoyl peroxide, dicumyl peroxide, azobisisobutyronitrile, 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and potassium persulfate; the ratio of zwitterionic monomer to initiator is 0.1~50 g: 0.1~100 μL. The crosslinking agent is one or more of dicumyl peroxide, ethylene glycol dimethacrylate, diphenylmethane diisocyanate, triethylene glycol dimethacrylate, diethylenetriamine, and methyltrimethoxysilane; the ratio of zwitterionic monomer to crosslinking agent is 0.1~50 g: 0.1~100 μL; When the crosslinking agent is ethylene glycol dimethacrylate and triethylene glycol dimethacrylate, the volume ratio of ethylene glycol dimethacrylate to triethylene glycol dimethacrylate is 1:1~2.

6. The preparation method according to claim 1, characterized in that, In the second step, the method used to achieve uniform mixing is ultrasonic vibration, and the ultrasonic vibration time is 30~60s. The reaction conditions are: ultraviolet light wavelength of 100~300 nm, polymerization temperature of 10~60℃, and polymerization time of 10~120 min.

7. The preparation method according to claim 1, characterized in that, The penicillin G acylase is derived from one or more of the strains Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens, and Providenciarettgeri; the concentration of penicillin G acylase in the penicillin G acylase solution is 1 to 10 mg / mL. The inorganic salts include one or more of calcium chloride, calcium carbonate, magnesium chloride, strontium chloride, magnesium sulfate, barium chloride, and barium sulfate; the mass fraction of the inorganic salt solution is 0.1% to 10%.

8. The preparation method according to claim 1, characterized in that, In the third step, the volume ratio of penicillin G acylase (PGA) solution to the polymerized stock solution is 1~5:5~10; The injection speed of the injection machine is 0.1~2.0mL / s.

9. Immobilized penicillin G acylase hydrogel microspheres prepared by the preparation method according to any one of claims 1-8.

10. The application of the immobilized penicillin G acylase hydrogel microspheres according to claim 9 as a catalyst in reversible drug synthesis.

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