An immobilized carrier with molecular chaperone-like function and its application

Abstract

This invention discloses a flexible intelligent immobilization carrier with molecular chaperone-like function and its applications. The flexible polymer immobilization carrier obtained by this invention using a high-throughput screening method exhibits high affinity and good selectivity for enzyme proteins, and can replace traditional immobilization carriers. This immobilization carrier is a flexible polymer prepared by chemical methods, possessing high stability, long service life, and strong resistance to harsh environments, overcoming the shortcomings of traditional immobilized enzyme carriers such as poor selectivity for enzyme proteins, high preparation costs, and reduced enzyme catalytic activity. The immobilization carrier prepared by this invention is a flexible polymer, which is beneficial for adapting to the flexible conformation of enzymes, enabling it to perform molecular chaperone functions, bind to denatured and inactivated enzymes, and utilize the specific molecularly imprinted holes of the flexible polymer to restore the structure and catalytic performance of enzymes without the need for external conditions and complex experimental equipment, thus achieving the refolding of denatured enzymes.

Description

Technical Field

[0001] This invention belongs to the field of enzyme immobilization technology, and specifically relates to a flexible intelligent immobilization carrier with molecular chaperone-like function and its application. Background Technology

[0002] Enzymes are essential to life, playing a crucial role in catalyzing almost all metabolic processes. Enzymes are the "chips" of green biotechnology, highly valuable in industrial biosynthesis due to their high selectivity, specificity, and mildness. High activity and stability are universal requirements for enzyme catalysis. However, a major drawback of enzymes in engineered applications is their poor stability and susceptibility to inactivation under complex environments. For example, changes in temperature and pH, organic solvents, small molecule inhibitors, and even the presence of other proteins can affect enzyme activity and stability. Current solutions to improve enzyme performance include molecular modification and immobilization, modifying the enzyme molecule itself and its environment, respectively. However, a trade-off effect exists between enzyme activity and stability; simultaneously improving both stability and activity under complex application environments remains a research challenge.

[0003] Conventional immobilized enzyme carriers are mostly rigid, providing short relaxation times for enzymes. The interaction between the microenvironment and enzyme molecules is non-specific. While this can improve enzyme stability, it generally results in a loss of at least 30-50% of the enzyme's apparent activity, significantly limiting its practical applications. As disclosed in patent (publication number CN110343693A) by Chen Chao, Tang Wen, et al., a method for preparing a magnetically immobilized enzyme carrier, this covalent immobilization method can maximize the stability of the immobilized enzyme; however, only 69.2% of the lipase activity is retained after immobilizing the enzyme on the carrier.

[0004] Enzymes often undergo changes in their secondary structure during application. When the secondary structure of an enzyme changes, its activity is severely damaged. In order to restore the secondary structure of an enzyme, people have designed some methods to restore enzyme activity. However, these methods are often complex, require harsh conditions, and have significant drawbacks.

[0005] Dong Xiaoyan, Liu Hu, and others disclosed a metal chelate nanomedia and its preparation method, as well as a method for enhancing the refolding and integrated purification of inclusion body proteins, in patent (publication number CN105111390A). The specific steps are as follows: A method for enhancing the refolding and integrated purification of inclusion body proteins is prepared by grafting polymers onto the surface of silica nanoparticles with an average particle size of 20-30 nm. The charge density of the metal chelate nanomedia obtained by this method reaches 2520-5680 μmol / g, which is far higher than the charge density of other media used in current studies on the refolding of proteins with the same charge. The extremely high charge density enhances the electrostatic repulsion between the media and protein molecules with the same charge, thereby more effectively inhibiting protein aggregation. The extremely high specific surface area of ​​the nanomedia allows for more effective spatial orientation of protein molecules with the same charge, thus being more conducive to the refolding of proteins with the same charge. However, the composite carrier synthesized by this method is only effective for enzymes with the same charge, resulting in poor universality and limited application.

[0006] Xiang Kaijun disclosed a universal, electric field-driven solid-phase protein refolding method in patent (publication number CN111777659A). The specific steps are as follows: First, two closely contacting solid-phase gels are prepared. The first layer (bottom or pre-layer) is a very thin, non-denaturing, low-concentration polyacrylamide gel (or other gels with similar properties), serving as the site for protein refolding. The second layer (top or post-layer) is a solid-phase denaturing agarose gel (or other gels with similar properties) containing the denaturing agent urea and denatured proteins. After mixing the inclusion body protein denaturing lysis solution with the liquid agarose gel (or other similar gels) containing the denaturing agent urea, it is added onto the non-denaturing thin-layer solid-phase polyacrylamide gel (or other similar gels) and solidified at a low temperature (preferably but not limited to 4℃~7℃) to form a urea-denaturing agarose gel containing denatured proteins (top or post-layer). Then, the two closely packed gel layers are placed in a direct current electric field. Electrophoresis causes the denatured proteins in the denaturing agarose gel to migrate into the non-denaturing polyacrylamide gel for refolding. The refolded proteins continue to migrate in the electric field, passing through the non-denaturing polyacrylamide gel and entering a dialysis bag or electrophoresis tank for recovery. This method provides a convenient way to refold enzymes, but it requires complex experimental setups and external conditions, limiting its usability. Summary of the Invention

[0007] This invention discloses an immobilized carrier with molecular chaperone-like function and its applications. For enzyme proteins with different properties, flexible polymer immobilized carriers that promote enzyme performance are designed and screened simply and rapidly using high-throughput screening methods. The properties of the immobilized carrier are utilized to regulate the enzyme's microenvironment, significantly improving the enzyme's catalytic activity and efficiency under various reaction conditions. Simultaneously, molecular imprinting technology is used to enhance the specific binding affinity of the immobilized carrier to the enzyme, overcoming the shortcomings of previous immobilized carriers, such as poor enzyme loading specificity, weak binding, and easy enzyme detachment. The carrier's intelligent responsiveness enables its regeneration and reuse. The finally synthesized immobilized carrier can specifically bind to denatured and inactivated enzymes through the molecularly imprinted cavity, exerting a molecular chaperone-like function. Because the enzyme molecule and the flexible carrier form a complementary host-guest pair during molecular imprinting, this method can customize a confined space for the enzyme molecule, restoring the structure and performance of denatured enzymes without external additives, eliminating the need for complex experimental equipment. This immobilized carrier has low preparation cost, high specificity and selectivity, and can be used for enzyme immobilization, leveraging the carrier's charge and hydrophobicity to promote enzyme activity.

[0008] The method for preparing the immobilized carrier with molecular chaperone-like function is as follows:

[0009] (1) Dissolve N-tert-butylacrylamide (TBAm) or N-phenylacrylamide (PAM) in ethanol, and dissolve N-isopropylacrylamide (NIPAm), functional monomers, crosslinking agents and initiators in water. Mix the above reactants in different proportions and add them to the detection cell. After removing the oxygen in the mixture by ultrasonic nitrogen gas for 5-10 minutes, polymerize in situ at the bottom of the detection cell to obtain a polymer thin layer. After the reaction, wash the polymer thin layer with water 3-5 times to remove unreacted monomers.

[0010] (2) Add enzyme solution to the detection cell for incubation. The enzyme is immobilized on the polymer thin layer at the bottom of the detection cell. Remove the supernatant and measure the catalytic activity of the enzyme immobilized on the polymer thin layer. Screen out polymer synthesis formulas with high affinity for enzyme molecules and great activity enhancement.

[0011] (3) Prepare raw materials according to the formula selected in step (2), then add template agent and surfactant, remove oxygen from the mixture by ultrasonic nitrogen gas for 5-10 minutes, stir and polymerize to obtain polymer nanoparticles, wash off template agent and dialysis to purify, and obtain immobilized carrier with molecular chaperone-like function.

[0012] The functional monomer is selected from one or more of acrylic acid (AAc), itaconic acid (IA), methacrylic acid (MAA), N-(3-aminopropyl)methacrylamide (APM), (3-acrylamidopropyl)trimethylammonium chloride (ATC), 1-vinylimidazolium (IM), N-[(3-(dimethylamino)propyl]methacrylamide (DMAPAA), 4-vinylphenylboronic acid (VPBA), acrylamide (AAm), hydroxyethyl methacrylate (HEMA), and N-(2-aminoethyl)acrylamide (AEM), 2-methacryloyloxyethyl phosphocholine (MPC).

[0013] The crosslinking agent is one or more of N,N'-methylenebisacrylamide, N,N'-vinylbisacrylamide, and ethylene glycol dimethacrylate.

[0014] The initiator is ammonium persulfate, azobisisobutyronitrile, or tetramethylethylenediamine. The amount of the initiator used is 0.3-2 mg / mL.

[0015] The molar ratio of the N-tert-butylacrylamide or N-phenylacrylamide to N-isopropylacrylamide, functional monomer, and crosslinking agent is 8-98:38-80:5-50:2-10.

[0016] The total concentration of the N-tert-butylacrylamide or N-phenylacrylamide, N-isopropylacrylamide, and functional monomers is 10-1000 mM.

[0017] The polymerization reaction in step (1) is carried out in a sealed or nitrogen atmosphere, the polymerization temperature is 25℃-80℃, and the polymerization time is 2-72h.

[0018] The method for determining the catalytic activity of the enzyme immobilized on the polymer thin layer in step (2) is as follows: add a colorimetric reagent to the detection cell, react for 2 minutes, and then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value and calculate the effect of the polymer on the enzyme activity.

[0019] The surfactant is sodium dodecyl sulfate or hexadecyltrimethylammonium bromide, and the amount of surfactant used is 0.01-0.2 mg / mL.

[0020] The template agent is an enzyme molecule, an epitope polypeptide of an enzyme molecule, or silica microspheres, magnetite spheres, or glass microspheres modified with the surface of an enzyme molecule or its epitope polypeptide. The amount of the template agent used is 0.01-0.4 mg / mL, based on the content of the enzyme molecule or its epitope polypeptide.

[0021] The polymerization reaction in step (3) is carried out in a sealed or nitrogen atmosphere, with a polymerization temperature of 25℃-65℃ and a polymerization time of 2-72h.

[0022] The stirring speed in step (3) is 300-1000 rpm.

[0023] After the polymerization reaction in step (3) is completed, the template agent is eluted by raising the temperature to 50-90℃, lowering the temperature to 0-10℃, or adding a NaCl aqueous solution with a concentration of 0.5M-5M and stirring for 2-24 hours.

[0024] One method for enzyme immobilization involves dispersing an immobilized carrier with chaperone-like function in water, adding free enzyme, incubating at 0-10°C for 1-48 hours, and then dialysis to purify the enzyme to obtain a solid-phase enzyme preparation. The concentration of the immobilized carrier is 0.5-100 mg / mL, and the enzyme concentration is 0.1-50 mg / mL.

[0025] One method for regenerating an inactivated enzyme is as follows: first, incubate the inactivated enzyme with a 0.5-100 mg / mL immobilized carrier having a molecular chaperone-like function at 35-40℃ for 0.5-2 h, and then incubate at 3-5℃ for 20-30 h.

[0026] The immobilized support prepared in this invention is a flexible polymer, which is beneficial for adapting to the flexible conformation of enzymes and can perform molecular chaperone functions. It binds to denatured and inactivated enzymes, utilizing the specific molecularly imprinted holes of the flexible polymer, without the need for external conditions or complex experimental equipment, to restore the enzyme's structure and catalytic performance, achieving the refolding of denatured enzymes. The flexible polymer immobilized support obtained by this invention based on a high-throughput screening method exhibits high affinity and good selectivity for enzyme proteins and can replace traditional immobilized supports. This immobilized support is a flexible polymer prepared by chemical methods, possessing high stability, long service life, and strong resistance to harsh environments, overcoming the shortcomings of traditional immobilized enzyme supports such as poor selectivity for enzyme proteins, high preparation costs, and reduced enzyme catalytic activity. The flexible immobilization carriers prepared by this method are flexibly designed for the enzymes requiring immobilization, significantly reducing costs. They enable rapid identification of immobilization carriers with optimal charge, hydrophobicity, and affinity, greatly improving synthesis efficiency. This method is applicable to the screening of immobilization carriers for various enzymes and proteins. Furthermore, molecular imprinting technology significantly enhances the affinity and selectivity of the immobilization carriers for enzymes. The customized confinement space for enzyme molecules using molecular imprinting mediates correct enzyme folding, achieving simultaneous improvement in enzyme activity and catalytic efficiency, and restoration of inactivated enzyme activity. Simultaneously, the intelligent response properties of the carriers to temperature and pH enable carrier regeneration and reuse. Attached Figure Description

[0027] Figure 1These are scanning electron microscope (SEM) images of the flexible, intelligent immobilized enzyme carrier with chaperone-like function synthesized in Example 2. The synthesis formulation in Figure (a) is 10% AAc, 35% TBAm, 2% Bis, and 53% NIPAm; the synthesis formulation in Figure (b) is 10% ATC, 60% TBAm, 2% Bis, and 28% NIPAm; and the synthesis formulation in Figure (c) is 10% ATC, 40% TBAm, 2% Bis, and 48% NIPAm.

[0028] Figure 2 This is Example 2, which describes the adsorption capacity of lipase on the non-imprinted and imprinted carriers synthesized based on the optimal monomer formulation. The functional monomer is (3-acrylamidopropyl)trimethylammonium chloride (ATC).

[0029] Figure 3 This refers to the adsorption capacity of laccase on the non-imprinted and imprinted carriers synthesized based on the optimal monomer formulation in Example 2. The functional monomer is acrylic acid (AAc).

[0030] Figure 4 Example 3 shows the relative enzyme activities of non-imprinted and imprinted laccases synthesized based on different functional monomer formulations (functional monomers: acrylic acid AAc, acrylamide AAm, (3-acrylamidopropyl)trimethylammonium chloride ATC).

[0031] Figure 5 This refers to the relative enzyme activity of laccase immobilized on an imprinted carrier synthesized using itaconic acid as a monomer, as described in Example 3.

[0032] Figure 6 The results are circular dichroism chromatograms of the non-blotted carrier and the blotted carrier synthesized in Example 5, which promote the recovery of the secondary structure of the inactivated enzyme.

[0033] Figure 7 This is the result of the non-imprinted carrier and imprinted carrier synthesized based on the optimal monomer formulation in Example 5 promoting the restoration of enzyme activity of inactivated enzymes.

[0034] Figure 8 The results are FTIR values ​​from Example 5, showing the effects of non-imprinted and imprinted carriers synthesized based on the optimal monomer formulation on the recovery of inactivated lipase structure.

[0035] Figure 9 The results are circular dichroism chromatograms of the non-imprinted carrier and the imprinted carrier synthesized in Example 5, which promote the structural recovery of inactivated lipase.

[0036] Figure 10 The results are circular dichroism chromatograms of the non-imprinted carrier and the imprinted carrier synthesized in Example 5, which promote the structural recovery of inactivated laccase.

[0037] Figure 11The results are from negative staining transmission electron microscopy of proteins synthesized in Example 5, specifically the non-blotting carrier and the blotted carrier that promote the structural recovery of inactivated lipases.

[0038] Figure 12 The results are from negative staining transmission electron microscopy of proteins synthesized in Example 5 using non-blotting and blotted vectors to promote the structural recovery of inactivated laccase. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, a flexible intelligent immobilization carrier with molecular chaperone-like function and its application are described in detail below with reference to embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0040] The polymer design strategy utilizes biomimetic molecular design methods to study enzyme-flexible polymer interactions by mimicking enzyme-molecular chaperone interactions. Enzymes and molecular chaperones achieve specific recognition of their binding domains through various interactions such as hydrogen bonding, electrostatic forces, and hydrophobicity. Therefore, monomer combinatorial chemistry libraries are constructed by selecting positively charged, negatively charged, hydrophobic, and easily hydrogen-bonding amino acid-like functional monomers. High-throughput screening methods are used to obtain monomer formulations, and polymerization reactions are conducted to prepare immobilized supports with chaperone-like functions and enhanced enzyme activity.

[0041] Example 1: Synthesis of immobilized carriers with high affinity for enzyme molecules and the most significant improvement in enzyme activity based on high-throughput screening.

[0042] (1) Prepare a 200mM stock solution for each monomer. Add the stock solutions of each monomer and crosslinking agent according to the molar ratio in Table 1 to obtain a total volume of 200μL. Add 50μL of ammonium persulfate initiator of the concentration in Table 1 to the mixture. Then place each mixture into the well of a 96-well plate. After removing the oxygen in the mixture by purging nitrogen gas for 5-10 minutes, seal the 96-well plate with a sealing sticker. Then place it in an oven at 80℃ for 3 hours to react. A polymer thin layer is obtained at the bottom of each well of the 96-well plate. After the reaction, wash the polymer thin layer with water 3 times to remove unreacted monomers.

[0043] (2) Add 100 μL of laccase (50 μg / mL), lipase (2.5 mg / mL), cytochrome C (100 μg / mL), or D-amino acid oxidase (50 μg / mL) to each well of a 96-well plate containing the bottom polymer film obtained in step (1). Incubate on a shaker for 3 h, and the enzymes will be immobilized on the polymer at the bottom of the 96-well plate. Remove the solution. Add chromogenic reagents to each well (100 μL of 10 mM ABTS for laccase; 20 μL of 1.665 mg / mL p-nitrophenyl laurate for lipase; 100 μL of 5 mM ABTS for cytochrome C; and 30 μL of 100 μM fluorescent red for DAAO). After reacting for 2 min, measure the absorbance value using a microplate reader, calculate the effect of the polymer on enzyme activity, and screen out polymer synthesis formulations with high affinity for enzyme molecules and significant enzyme activity enhancement.

[0044] Table 1. Synthesis formulation of immobilized enzyme carriers

[0045]

[0046]

[0047] Taking laccase, lipase, cytochrome C (Cyt C), and D-amino acid oxidase (DAAO) as examples, based on high-throughput screening, the optimal formulations were finally determined as follows: Laccase (Formula 1): IA 10%, TBAm 40%, Bis 2%, NIPAm 48%; Lipase (Formula 2): ATC 10%, TBAm 60%, Bis 2%, NIPAm 28%; Cytochrome C (Formula 3): AAc 40%, TBAm 35%, Bis 2%, NIPAm 23%; D-amino acid oxidase (DAAO, Formulation 4): ATC 10%, TBAm 35%, Bis 2%, NIPAm 53%. The polymers synthesized using the above formulations showed the most significant improvement in enzyme activity, thus the optimal immobilization carrier for enzyme performance was determined through high-throughput screening.

[0048] Example 2: Improving the affinity and selectivity of immobilized carriers for enzymes using molecular imprinting technology

[0049] Following Example 1, after obtaining the formulation that promotes enzyme performance through high-throughput screening, lipase and laccase were selected as template molecules. Then, 50 mL of monomer solutions were prepared using the optimal formulation obtained in Example 1, with 500 μL of initiator and 50 mg of monomer added, resulting in a total monomer concentration of 20 mM. Next, 0.02 mg / mL sodium dodecyl sulfate and 5 mg of lipase or laccase were added as template molecules. The reaction solution was then added to a reaction vessel, and the mixture was stirred magnetically or mechanically at 40°C for 24 h. Polymer nanoparticles were synthesized through precipitation polymerization or reverse emulsion polymerization. After the reaction, 1 M NaCl aqueous solution was added to the nanoparticles and stirred for 2 h to remove the enzyme template. The nanoparticles were then dialyzed with pure water to obtain the molecularly imprinted immobilized carrier. Alternatively, the enzyme template molecules can be imprinted onto a material. First, a solid support carrier is prepared, such as silica microspheres, magnetic iron oxide microspheres, or glass beads. Amino, carboxyl, and other chemical bonds are modified on the surface of the microspheres, and then the enzyme molecules are immobilized on the surface of the solid support. Taking the immobilization of enzyme template molecules on iron oxide microspheres as an example, the specific operation process is as follows: (1) Synthesis of magnetic iron oxide microspheres MagNP: Weigh 1.3g FeCl3·6H2O, 0.62g hexadecyltrimethylammonium bromide, and 2.6g anhydrous sodium acetate into a round-bottom flask, add 40mL ethylene glycol, stir at 80℃ for one hour until completely dissolved, and transfer to a reaction vessel. React at 200℃ for 10h, wash with water and ethanol, and dry at 60℃ for later use. (2) Synthesis of SiO2 thin layer (MagNP@SiO2) on the surface of magnetic iron oxide microspheres: Disperse 100mg MagNP uniformly in 87.1mL 80% (v / v) ethanol solution, add 1.4mL 25% NH3·H2O, sonicate for 1min; then add 11.5mL tetraethyl orthosilicate (TEOS), and react in a 30℃ water bath at 600-700rpm for 6h. After the reaction, wash with water until the pH is neutral, then wash with ethanol, and dry at 60℃ for later use. (3) Modification of MagNP@SiO2 surface with amino groups: Disperse 300mg MagNPs@SiO2 in 180mL ethanol solution (ethanol:water = 3 / 1, v / v), sonicate at room temperature for 30 minutes. Purge with nitrogen for 30 minutes, place in a 40℃ water bath, inject 4mL of 3-(aminopropyl)trimethoxysilane (APTMS) into the flask, stir mechanically overnight to obtain MagNP@SiO2-NH2 with amino groups on the surface, wash with water and ethanol, and dry at 60℃ for later use. (4) Immobilization of template molecules: Disperse 50mg MagNP@SiO2-NH2 in 30mL solution containing 3mL glutaraldehyde (5% v / v) at room temperature, stir mechanically at 600-700rpm for 3h to bind aldehyde groups to the surface of MagNP@SiO2-NH2. Wash with PBS (0.1M, pH 7.2-7.4) to remove unreacted glutaraldehyde.MagNP@SiO2-NH2 was then dispersed in PBS (0.1M, pH 7.2-7.4) containing 10 mL of 1 mg / mL template molecules and incubated for 2 h. The particles were collected and separated by magnetic means and washed with PBS (0.1M, pH 7.2-7.4) to obtain a solid template. (5) Synthesis of solid-phase imprinted immobilized carrier, taking laccase as an example: 100 mg of laccase solid template was mixed evenly with 20 mM 50 mL monomer solution of Formula 1, nitrogen gas was purged for 30 min, and the reaction was carried out at 40 °C for 24 h. The mixture was eluted by raising the temperature to 65 °C or lowering the temperature to 4 °C to obtain a flexible intelligent immobilized enzyme carrier with molecular chaperone-like function. The scanning electron microscopy results of the flexible intelligent immobilized enzyme carrier with molecular chaperone-like function are shown below. Figure 1 .

[0050] The molecularly imprinted carriers prepared in Example 2 were used to immobilize lipase and laccase, respectively. The specific steps are as follows: 5 mg of the molecularly imprinted immobilized carrier was dispersed in 5 mL of pure water, and 0.25 mg of free enzyme was added to the solution. After the enzyme was completely dissolved, the solution was stored at low temperature for 24 h. The incubation system was then purified by dialysis, and the immobilization rate of the molecularly imprinted carrier on the target protein was measured.

[0051] like Figure 2 As shown, the non-imprinted immobilized enzyme carrier synthesized with the optimal formulation obtained in Example 1 had an adsorption capacity of 23.5 mg / g for lipase, while the molecularly imprinted immobilized enzyme carrier obtained in Example 2 based on the optimal formulation obtained in Example 1 and molecular imprinting technology had an adsorption capacity of 146.7 mg / g for lipase and an imprinting factor of 6.24. This indicates that molecular imprinting technology further improved the affinity and selectivity of the immobilized carrier for the enzyme.

[0052] Similarly, such as Figure 3 As shown, the non-imprinted flexible immobilized enzyme carrier synthesized with the optimal formulation obtained in Example 1 had an adsorption capacity of 53.9 mg / g for laccase, while the molecularly imprinted flexible immobilized enzyme carrier obtained in Example 2 based on the optimal formulation obtained in Example 1 and molecular imprinting technology had an adsorption capacity of 235.6 mg / g for laccase and an imprinting factor of 4.37. This indicates that molecular imprinting technology further improved the affinity and selectivity of the immobilized carrier for the enzyme.

[0053] Application Example 3: Molecularly imprinted immobilized carriers enhance enzyme activity

[0054] Lipase (147 μg / mL) and laccase (235 μg / mL) were immobilized with the immobilized enzyme carriers (1 mg / mL) synthesized in Formula 2 and Formula 1, respectively, at 37 °C and 200 rpm for 2 h. The catalytic activity of the immobilized enzymes was measured and compared with that of the free enzymes. Lipase activity assay: 50 μL of 0.33 mg / mL 4-nitrobenzene laurate was dissolved in isopropanol to prepare a solution. 200 μL of phosphate buffer (pH=8) and 20 μL of 1.25 mg / mL molecularly imprinted lipase, non-imprinted lipase, or free lipase were added. The absorbance change of the product at 410 nm within 6 min was measured using a microplate reader. Laccase activity assay: Take 100 μL of laccase immobilized on a molecularly imprinted carrier or immobilized on a non-imprinted carrier or free laccase at a concentration of 0.05 mg / mL, add 100 μL of 10 mM 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and measure the change in absorbance at 420 nm over 2 min using a microplate reader.

[0055] Experimental results are as follows Figure 4 and Figure 5 The results showed that the molecularly imprinted immobilized carrier significantly improved the catalytic activity of lipase and laccase, with the enzyme activities of lipase and laccase being 2.06 and 8.44 times higher than the corresponding free enzyme levels, respectively.

[0056] Application Example 4: Molecularly imprinted immobilized supports improve enzyme catalytic efficiency

[0057] Lipase kinetic assay: 50 μL of 0.5-5 mM 4-nitrobenzene laurate was dissolved in isopropanol to prepare a solution. 200 μL of phosphate buffer (pH=8) and 20 μL of 1.25 mg / mL molecularly imprinted lipase, non-imprinted lipase, or free lipase were added. The absorbance change of the product at 410 nm within 6 min was measured using a microplate reader.

[0058] Lacase kinetic assay: Take 100 μL of laccase immobilized on a molecularly imprinted carrier or immobilized on a non-imprinted carrier or free laccase at a concentration of 0.05 mg / mL, add 100 μL of ABTS at a concentration of 0.2-10 mM, and measure the change in absorbance at 420 nm over 2 min using a microplate reader.

[0059] The experimental results are shown in Tables 2 and 3. It can be seen that the catalytic efficiency (kJ) of the enzyme immobilized on the molecularly imprinted carrier is... cat / K mThe catalytic efficiency of laccase immobilized on molecularly imprinted carriers was significantly superior to that of free laccase. Specifically, the catalytic efficiency of laccase immobilized on molecularly imprinted carriers was 2.8 times that of free laccase, and the catalytic efficiency of lipase immobilized on molecularly imprinted carriers was 3 times that of free lipase. Furthermore, the catalytic efficiency of enzymes immobilized on molecularly imprinted carriers was significantly better than that of enzymes immobilized on non-imprinted carriers, further confirming the superiority of the constructed molecularly imprinted recognition sites in improving enzyme catalytic performance.

[0060] Table 2 Comparison of catalytic reaction kinetic parameters between immobilized laccase and free laccase

[0061]

[0062] Table 3 Comparison of catalytic reaction kinetic parameters between immobilized lipase and free lipase

[0063]

[0064] Application Example 5: Refolding of inactivated enzymes on molecularly imprinted immobilized supports

[0065] First, lipase and laccase were inactivated by treatment with 6M guanidine hydrochloride at 37°C and 200 rpm for 3 hours. Then, guanidine hydrochloride molecules were removed by dialysis for 48 hours. After inactivation, the denatured enzyme proteins were subjected to circular dichroism spectroscopy to characterize the differences in their secondary structure from the native enzymes, demonstrating that the secondary conformation of the enzyme proteins was disrupted after denaturation. The experimental results are as follows: Figure 6 As shown, the denatured enzyme protein exhibits a significant decrease in characteristic peak intensity compared to the native enzyme, indicating that the secondary structure at this wavelength has been disrupted. Then, 5 mg / mL of the molecularly imprinted immobilization carrier (Formula 1) was incubated with either laccase (0.5 mg / mL) or lipase (1.25 mg / mL) of the denatured enzyme at 37°C and 200 rpm for 1 h, followed by further incubation at 4°C for 24 h. After incubation, circular dichroism spectroscopy was performed on the molecularly imprinted immobilization carrier combined with the denatured enzyme solution to characterize the restoration effect of the immobilization carrier on the denatured enzyme structure. The differences in its secondary structure compared to the denatured and native enzymes were observed, demonstrating that the immobilization carrier can effectively restore the disrupted secondary conformation of the enzyme protein. After 24 h of incubation with the molecularly imprinted immobilization carrier, the secondary conformation of the denatured enzyme protein was restored to a level close to that of the native enzyme, and its characteristic peak intensity was significantly enhanced compared to the denatured enzyme, indicating that the disrupted secondary structure at this wavelength was restored.

[0066] After refolding denatured enzymes onto molecularly imprinted immobilized carriers, the catalytic activity of the refolded enzymes was measured. The specific experimental procedure is as follows: Laccase (50 μg / mL), cytochrome C (100 μg / mL), and D-amino acid oxidase (50 μg / mL) were incubated with their corresponding 1 mg / mL molecularly imprinted immobilized carriers, or lipase (2.5 mg / mL) was incubated with its corresponding 10 mg / mL molecularly imprinted immobilized carriers in a shaker at 200 rpm and 37 °C for 2 h, and then incubated at 4 °C for 24 h. After the enzymes were removed and allowed to return to room temperature, their activity was measured.

[0067] Experimental results are as follows Figure 7 As shown in the figure, the catalytic activity of the enzyme after denaturation and inactivation is less than 10% of that of the native enzyme, indicating that the destruction of the enzyme protein structure affects its catalytic activity. When the molecularly imprinted immobilized carrier is co-incubated with the denatured enzyme, the enzyme activity recovers to or even exceeds the level of the native enzyme. Further characterization of the molecular chaperone function of the molecularly imprinted immobilized carrier for the denatured enzyme was performed using Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and negative staining TEM, with results shown in the figure. Figure 8-12 As shown, this demonstrates that molecularly imprinted immobilization carriers can mediate the correct folding of denatured enzymes, restoring them to their native conformation and thus restoring the activity of inactivated enzymes.

Claims

1. A method for producing an immobilized carrier having a chaperone-like function, characterized by, The specific steps of the preparation method are as follows: (1) N -tert-butylacrylamide or N -Phenylacetamide is dissolved in ethanol, N -Isopropylacrylamide, functional monomers, crosslinking agents and initiators are dissolved in water. The above reactants are mixed in different proportions and added to the detection cell. After removing the oxygen in the mixture by ultrasonic nitrogen purging for 5-10 minutes, the polymer thin layer is obtained by in-situ polymerization at the bottom of the detection cell. After the reaction, the polymer thin layer is washed with water 3-5 times to remove unreacted monomers. (2) Add enzyme solution to the detection cell for incubation. The enzyme is immobilized on the polymer thin layer at the bottom of the detection cell. Remove the supernatant and measure the catalytic activity of the enzyme immobilized on the polymer thin layer. Screen out polymer synthesis formulas with high affinity for enzyme molecules and great activity enhancement. (3) Prepare raw materials according to the formula selected in step (2), then add template agent and surfactant, remove oxygen from the mixture by ultrasonic nitrogen gas for 5-10 min, stir and polymerize to obtain polymer nanoparticles, wash off template agent and dialysis to purify, and obtain immobilized carrier with molecular chaperone-like function. The template agent is an enzyme molecule; The polymerization reaction in step (1) is carried out in a sealed or nitrogen atmosphere, the polymerization temperature is 25℃-80℃, and the polymerization time is 2-72 h. The polymerization reaction in step (3) is carried out in a sealed or nitrogen atmosphere, with a polymerization temperature of 25℃-65℃ and a polymerization time of 2-72 h.

2. The preparation method according to claim 1, characterized in that, The functional monomers are selected from acrylic acid, itaconic acid, methacrylic acid, etc. N -(3-aminopropyl)methacrylamide, (3-acrylamidopropyl)trimethylammonium chloride, 1-vinylimidazolium, N-[(3-(dimethylamino)propyl]methacrylamide, 4-vinylphenylboronic acid, acrylamide, hydroxyethyl methacrylate, N One or more of (2-aminoethyl)acrylamide and 2-methacryloyloxyethyl phosphocholine; the crosslinking agent is... N,N' -methylenebisacrylamide, N,N One or more of '-vinylbisacrylamide and ethylene glycol dimethacrylate; wherein the initiator is ammonium persulfate, azobisisobutyronitrile, or tetramethylethylenediamine.

3. The preparation method according to claim 1, characterized in that, The method for determining the catalytic activity of the enzyme immobilized on the polymer thin layer in step (2) is as follows: add a colorimetric reagent to the detection cell, react for 2 min, and then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value and calculate the effect of the polymer on the enzyme activity.

4. The preparation method according to claim 1, characterized in that, The surfactant is sodium dodecyl sulfate, and the amount of surfactant used is 0.01-0.2 mg / mL.

5. The preparation method according to claim 1, characterized in that, After the polymerization reaction in step (3) is completed, the template agent is eluted by raising the temperature to 50-90℃, lowering the temperature to 0-10℃, or adding a NaCl aqueous solution with a concentration of 0.5 M-5 M and stirring for 2-24 h.

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