A method for targeted immobilization of nuclease P1 and a computationally-aided design-based method for targeted affinity immobilization of nuclease P1.

Abstract

This invention relates to the fields of computational chemistry and enzyme engineering, specifically to a method for the targeted immobilization of nuclease P1 and a computationally-aided design-based method for the targeted affinity immobilization of nuclease P1. This invention, for the first time, combines computational and experimental methods, such as molecular dynamics simulations, to achieve precise affinity immobilization of nuclease P1 through targeted design. Based on the charge distribution and hydrophilic / hydrophobic regions of nuclease P1, combined with the spatial distribution of its key residues, dopamine and charged polymers are used as modifiers, co-deposited on the surface of a mesoporous resin support, exhibiting excellent adsorption capacity and immobilization effect. Furthermore, through dual optimization of glutaraldehyde crosslinking and concanavalin A modification, the binding efficiency and catalytic activity stability of nuclease P1 are significantly improved. This invention not only solves the problem of poor stability of nuclease P1 in the free state but also achieves efficient targeted affinity immobilization through novel molecular design and interface optimization techniques, providing a feasible technical path for industrial-scale applications.

Description

Technical Field

[0001] This invention relates to the fields of computational chemistry and enzyme engineering, specifically to a method for the directional immobilization of nuclease P1 and a method for the directional affinity immobilization of nuclease P1 based on computation-aided design. Background Technology

[0002] In the fields of enzyme engineering and biocatalysis, immobilization technology, as an important means to effectively improve the stability, activity retention, and reusability of enzymes, has been widely used in the immobilization of various enzyme preparations in industrial production processes. Immobilized enzymes can avoid the inactivation and degradation problems that easily occur in the free state of enzymes, while also helping to improve reaction efficiency and reduce production costs. However, traditional enzyme immobilization technologies, such as physical adsorption, covalent binding, and encapsulation, usually suffer from problems such as complex immobilization processes, significant loss of enzyme activity, and limited carrier selection, which restrict their large-scale application.

[0003] Nuclease P1, an important nucleic acid degrading enzyme, was first discovered in the fermentation of *Penicillium citrinum*. It possesses dual activities as both a phosphodiesterase and a phosphomonomerase, and is widely used in DNA and RNA degradation, nucleotide extraction, and analytical detection, demonstrating significant industrial application value. Nuclease P1 consists of 270 amino acids with a molecular weight of approximately 44 kDa. Its active site contains three Zn groups coordinated to phosphorus. 2+ These enzymes, located at the bottom of the binding cleft, are crucial to their catalytic function. However, nuclease P1 exhibits poor stability in its free state and is easily affected by environmental factors (such as temperature, pH, and organic solvents), resulting in low reusability and complex operation in practical applications, thus failing to fully realize its potential industrial value. In recent years, molecular simulation technology and computational-aided design (CAD) have gained increasing attention in enzyme engineering. Through molecular dynamics simulations and electrostatic analysis, the structural characteristics, active sites, and interactions with the support of enzyme molecules can be effectively predicted, thereby providing guidance for the targeted immobilization of enzymes. Therefore, achieving efficient and stable immobilization of nuclease P1 is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the existing nuclease P1 has significant industrial application potential due to its high efficiency in DNA and RNA degradation and nucleotide extraction. However, its poor stability and low reusability in the free state greatly limit its promotion in large-scale industrial applications. The present invention provides a computationally assisted design-based method for the directional affinity immobilization of nuclease P1.

[0005] The technical problem that this invention also aims to solve is to provide a directionally immobilized nuclease P1 based on a modifier-modified directionally immobilized nuclease P1.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0007] A computationally-aided design-based method for the directional affinity immobilization of nuclease P1 includes the following steps:

[0008] (1) Obtain and analyze the crystal structure of nuclease P1, and identify the electrostatic potential distribution and hydrophilic and hydrophobic regions on its surface;

[0009] (2) Based on the electrostatic potential distribution and hydrophilic / hydrophobic region results of nuclease P1 surface in step (1), dopamine molecule DA and charged polymer molecule were selected as modifiers, and corresponding molecular models were constructed respectively.

[0010] (3) Optimize the structure and energy of the molecular model constructed in step (2);

[0011] (4) Using the molecular model optimized in step (3), a simulated system containing water, nuclease P1 and modifiers was constructed, and the net charge of the system was neutralized by adding sodium ions;

[0012] (5) Perform molecular dynamics simulation on the simulation system of step (4) to analyze the dynamic behavior of nuclease P1 in the modifying agent environment;

[0013] (6) Based on the analysis results of the dynamic behavior of nuclease P1, a directionally immobilized nuclease P1 modified by a modifier was constructed.

[0014] In step (1), before identifying the electrostatic potential distribution and hydrophilic / hydrophobic regions on the surface of nuclease P1, the crystal structure of nuclease P1 needs to be converted into a molecular simulation topology file.

[0015] Specifically, in step (1), the crystal structure of nuclease P1 is obtained from the PDB library, and after removing the water of crystallization, removing the ligands, and protonating, it is processed by the Gromacs program to obtain the topology file.

[0016] In step (1), molecular visualization software (such as Pymol, Chimera, VMD, etc.) is used to analyze the electrostatic potential distribution and hydrophilic / hydrophobic regions on the surface of nuclease P1.

[0017] Specifically, the surface electrostatic potential region distribution of nuclease P1 in step (2) was calculated by the Pymol program using the APBS tool, and the surface hydrophilic and hydrophobic region distribution was obtained by Chimera analysis of residue distribution.

[0018] Specifically, the surface electrostatic potential region distribution of the nuclease P1 is of great significance for analyzing the enzyme's catalytic mechanism and its binding mode with substrates or other molecules. Mapping the surface hydrophilic and hydrophobic region distribution helps to assess its stability in water-soluble environments and possible interaction interfaces.

[0019] In step (2), the dopamine molecules and charged polymer molecules need to be protonated when constructing the corresponding molecular models.

[0020] In step (2), the charged polymer molecule is any one or a combination of several of the following: polyethyleneimine (PEI), polyethylene glycol (PEG), and polylysine (EPL). Preferably, it is polyethyleneimine (PEI).

[0021] In step (3), after the molecular model constructed in step (2) is structurally and energy optimized, it needs to be converted into a molecular model topology file for calculation.

[0022] In step (4), in the simulation system containing water, nuclease P1 and the modifier, water, nuclease P1 and the modifier are used to construct a cubic box with boundary periodicity conditions.

[0023] Specifically, the size of the cubic box is 8.9nm × 8.9nm × 8.9nm.

[0024] Specifically, the nuclease P1 is located at a distance of >1.0 nm from the edge of the cubic box; the modifier is a dopamine molecule and a charged polymer molecule, the positions of the dopamine molecule and the charged polymer molecule in the cubic box are random, and the molar ratio of the dopamine molecule and the charged polymer molecule is 1 to 3:1.

[0025] Preferably, the molar ratio of dopamine molecules to charged polymer molecules is 3:1. In some embodiments of the invention, 300 dopamine molecules and 100 charged polymer molecules are added to the cubic box.

[0026] In step (5), before formally conducting molecular dynamics simulation, the steepest descent method is used to simulate 1000 to 10000 steps to minimize the energy of the simulation system. Then, under constant conditions of reaction temperature of 298.15 K and reaction pressure of 1 bar, an ensemble simulation of 2 to 10 ns is conducted to ensure that the solute and solvent in the simulation system are fully mixed while allowing the cubic box to be compressed to a reasonable density.

[0027] Preferably, the steepest descent method is used to simulate 5000 steps to minimize the energy of the simulated system and eliminate unreasonable contacts caused by the system construction.

[0028] Specifically, the ensemble simulation includes NVT and NPT ensemble simulations, with a preferred duration of 5 ns.

[0029] Specifically, the reaction temperature of 298.15K is constant using the V-rescale method with a coupling time constant of 0.2 ps; the reaction pressure of 1 bar is constant using the Parrinello-Rahman method with a coupling time constant of 0.5 ps.

[0030] In step (6), the molecular dynamics simulation is set to a duration of 10–100 ns and a time step of 1–2 fs.

[0031] Preferably, in step (6), the molecular dynamics simulation is set to a duration of 100 ns and a time step of 2 fs.

[0032] Specifically, the molecular dynamics simulations employed the Leap-frog algorithm for integration. The cut-off algorithm was used to calculate van der Waals interactions, with a cutoff radius set to 1.1 nm. The Particle Mesh Ewald (PME) algorithm was used to calculate long-range electrostatic interactions, with a cutoff distance set to 1.1 nm.

[0033] In step (5), the dynamic behavior includes the interaction energy between nuclease P1 and the modifier, root mean square deviation, root mean square fluctuation, solvent accessible area, etc.

[0034] A directionally immobilized nuclease P1 based on a modifier is also within the scope of protection of this invention. The directionally immobilized nuclease P1 is constructed based on the directionally affinity immobilization method of the nuclease P1.

[0035] The modifier is dopamine (DA) and a charged polymer; the charged polymer molecule is any one or a combination of several of polyethyleneimine (PEI), polyethylene glycol (PEG), and polylysine (EPL); the preferred charged polymer is polyethyleneimine (PEI).

[0036] Specifically, the directional immobilized nuclease P1 is obtained by modifying mesoporous resin with DA and PEI to obtain a modified carrier, and then dispersing the modified carrier in a nuclease P1 solution for incubation.

[0037] or,

[0038] The directional immobilized nuclease P1 is obtained by modifying mesoporous resin with DA and PEI to obtain a modified carrier, then modifying the modified carrier with glutaraldehyde, and then adding the modified carrier obtained from the second modification to a concanavalin A (ConA) solution to obtain a specifically modified carrier. Finally, the specifically modified carrier is dispersed in a nuclease P1 solution and incubated to obtain the directional immobilized nuclease P1.

[0039] The mesoporous resin is any one of epoxy resin (LX-1000EPB, LX-103B), amino resin (HA, LXEP120), and quaternized resin (LX-1000EA, LX-1000EPN, LX-1000NH). Preferably, the mesoporous resin is LX-1000EPB, LX-103B, LXEP120, LX-1000NH, or HA. More preferably, the mesoporous resin is HA (i.e., amino-functionalized polystyrene-divinylbenzene resin).

[0040] The modified support was obtained by modifying mesoporous resin with DA and PEI as follows: The mesoporous resin was dispersed in 50 mM Tris buffer (pH 7.0–9.0). Then, 3 g / L of DA and PEI were added, and the mixture was stirred at 30°C for 30 h to allow it to co-deposit on the surface of the mesoporous resin. The resulting support was filtered with pure water, washed twice, and stored at 4°C to obtain the modified support.

[0041] Specifically, the mass-to-volume ratio of the mesoporous resin to 50–100 mM Tris buffer solution with pH 7.0–9.0 is 1:10.

[0042] Specifically, the molar ratio of DA to PEI is 1 to 3:1, preferably 3:1.

[0043] The nuclease P1 solution is in the form of 0.05–0.20 M PBS buffer with a pH of 6.0–8.0.

[0044] The process of modifying the carrier by using glutaraldehyde is as follows: the modified carrier is mixed with 50-100mM, pH 6.0-8.0 PBS buffer, and then 1-5% v / v glutaraldehyde is added.

[0045] Specifically, the modified carrier is mixed with 50-100 mM PBS buffer at pH 7.0-9.0 at a mass-to-volume ratio of 1:40.

[0046] The canavalia brevicornuate A (ConA) solution is prepared in the form of a PBS buffer solution with a pH of 6.0 to 8.0 containing 0.05 to 0.20 M of metal ions; specifically, the metal ions are any one of manganese ions, calcium ions, and potassium ions.

[0047] The incubation is carried out under the following conditions: 25°C and 180 rpm / min for 2 to 5 hours.

[0048] The aforementioned dopamine and polyethyleneimine-modified directional immobilized nuclease P1 was experimentally verified, showing that the simulation results using the computational-aided design method of this invention closely matched the experimental results, validating the accuracy of the prediction simulation. Experiments show that the directional immobilized enzyme HA-PDA / PEI-GA-ConA-NP1 prepared in this invention exhibits optimal thermostability, maintaining over 91% relative activity below 60℃; it retains over 88% enzyme activity even at pH 8; after 10 batches of repeated reactions, the enzyme activity retention rate of the DA / PEI-modified immobilized nuclease P1 (i.e., HA-PDA / PEI-NP1) was 26%, while the immobilized nuclease P1 co-modified with PDA / PEI and ConA (i.e., HA-PDA / PEI-GA-ConA-NP1) still maintained over 70% relative activity. Compared to single covalently linked, physically adsorbed, or affinity-adsorbed immobilized enzymes, the performance of the immobilized enzyme in this invention is significantly improved.

[0049] Beneficial effects:

[0050] (1) Innovative Application of Computation-Aided Design: This invention combines molecular dynamics simulation and molecular visualization analysis to conduct the first systematic study on the immobilization design of nuclease P1. By accurately calculating the charged region and hydrophilic / hydrophobic distribution of nuclease P1, a scientific basis is provided for selecting suitable modification materials and immobilization strategies, breaking through the traditional experience-based design methods and significantly improving the efficiency of carrier development. It not only solves the problem of poor stability of nuclease P1 in the free state but also achieves efficient directional affinity immobilization through novel molecular design and interface optimization technologies, providing a feasible technical path for industrial-scale applications.

[0051] (2) This invention optimizes the selection of modification materials by analyzing the charge distribution and hydrophilicity of the nuclease P1 surface. Dopamine (DA) and charged polymers are used as modifiers and co-deposited on the surface of a mesoporous resin support, exhibiting excellent adsorption capacity and immobilization effect. This modifier co-deposition strategy improves the immobilization effect and helps to achieve customized design for different application needs. Furthermore, through dual optimization of glutaraldehyde crosslinking and concanavalin A modification, the binding efficiency and catalytic activity stability of nuclease P1 are significantly improved.

[0052] (3) This invention combines dynamic simulation with experimental verification, using GROMACS for energy optimization and molecular dynamics simulation. The obtained dynamic behavior data (such as root mean square deviation, root mean square fluctuation, solvent access area, etc.) are highly consistent with the experimental results, enhancing the understanding of the interaction mechanism between nuclease P1 and the modifier. This method provides quantifiable theoretical support for optimizing the immobilization process, effectively reducing experimental costs and time.

[0053] (4) By comprehensively considering the surface characteristics and molecular dynamics parameters of nuclease P1, this invention has obtained an innovative immobilization method, which greatly improves the activity and reusability of the immobilized enzyme. This method is not limited to the immobilization of nuclease P1, but can also be extended to the directional immobilization design of other enzymes. It has strong versatility and scalability, and is applicable to a wider range of biocatalysis and related application fields, with broad application prospects. Attached Figure Description

[0054] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0055] Figure 1 The diagram shows the surface features of nuclease P1. A represents the structural features of nuclease P1, where nuclease P1 is cyan, zinc ions are orange, and asparagine residues are purple; B represents the surface electrostatic potential distribution of nuclease P1, where blue areas represent positively charged regions, red areas represent negatively charged regions, and white areas represent neutral regions; C represents the distribution of hydrophilic and hydrophobic regions of nuclease P1, where blue areas represent hydrophilic regions, orange-red areas represent hydrophobic regions, and white areas represent neutral regions.

[0056] Figure 2 The initial and final conformations of the water-nuclease P1-DA / PEI system with polyethyleneimine (PEI) as the charged polymer are shown within 100 ns.

[0057] Figure 3 The root mean square deviation (RMSD) of nuclease P1 in pure water, DA, and DA / PEI environments.

[0058] Figure 4 The root mean square fluctuation (RMSF) of nuclease P1 is shown in pure water, DA, and DA / PEI environments. The inset shows the B-factor conformation of nuclease P1 in the three environments: pure water, DA, and DA / PEI. Red indicates rigidity and blue indicates flexibility.

[0059] Figure 5 Solvent accessible surface area (SASA) of nuclease P1 in pure water, DA, and DA / PEI environments.

[0060] Figure 6 This represents the change in the interaction energy between nuclease P1 and DA within 100 ns.

[0061] Figure 7 This represents the change in the interaction energy between nuclease P1 and DA / PEI within 100 ns.

[0062] Figure 8 This is a diagram showing the distribution of residues on the front and back sides of nuclease P1 that adsorb the most DA within 100 ns in the DA environment.

[0063] Figure 9 This is a graph showing the amount of DA adsorbed on the top ten nuclease P1 residues within 100 ns in the DA environment.

[0064] Figure 10 The graph shows the enzyme activity and protein loading of seven different resins used for immobilizing nucleases.

[0065] Figure 11 The image shows the enzyme activity and specific enzyme activity of immobilized nuclease P1.

[0066] Figure 12 This is a graph showing the thermal stability of immobilized nuclease P1.

[0067] Figure 13 This is a pH stability diagram of immobilized nuclease P1.

[0068] Figure 14 This is a batch stability graph of immobilized nuclease P1. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0070] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0071] In the following examples, the LX-1000EPB, LX-103B, LX-1000EA, LX-1000EPN, LXEP120, LX-1000NH, and HA resins were purchased from Xi'an Lanxiao Technology New Materials Co., Ltd., the dopamine, polyethyleneimine, glutaraldehyde, and concanavalin A were purchased from Aladdin Reagent (Shanghai), and the nuclease P1 and substrate RNA solution were purchased from Nanjing Tongkai Zhaoye Co., Ltd.

[0072] Example 1: Computational-aided design of dopamine (DA) and dopamine / charged polymer (DA / charged polymer) adsorption simulation

[0073] 1. Three-dimensional structure and distribution of characteristic regions of nuclease P1

[0074] The PDB file (PDB id: 1ak0) of the nuclease P1 (NP1) crystal structure was downloaded from the Protein Data Bank on the RCSB website as the research object. The downloaded P1 crystal structure was then optimized. Specifically, Pymol was used to remove excess water of crystallization molecules and subunits. The obtained PDB file was then converted into a GRO file and a confinement potential ITP file for computation using the pdb2gmx command in Gromacs, and a TOP file was obtained.

[0075] The three-dimensional structure and feature regions of nuclease P1 were drawn using the VMD program. First, the .gro file of nuclease P1 was loaded. Using the Representations tool of the Graphics module, the secondary structure was displayed using the New-cartoon model, the asparagine residues and the trizinc structure were displayed using the VDW model, and the glycosyl group was displayed using the CPK model. All textures were selected as Glossy.

[0076] Using the APBS plugin of the Pymol program, the electric field and potential distribution were simulated according to the Poisson-Boltzmann equation, with a dielectric constant of 80, an ionic strength of 0.15 M, and a solvent radius of... At a temperature of 300K, the electrostatic potential distribution region on the surface of nuclease P1 was calculated and plotted. The distribution of hydrophilic and hydrophobic regions on the surface was plotted using the interactive 3 command in the preset module of the Chimera program based on the distribution of hydrophobic / hydrophilic residues.

[0077] Figure 1 The results show the surface characteristics of nuclease P1. Figure 1 The A in the figure represents the three-dimensional structural features of nuclease P1. It can be seen that the asparagine residues (Asn92, Asn138, Asn184, Asn197) in nuclease P1 are glycosylated by glucose side chains, and the glycosylation sites are all located on the back side of the active pocket region of nuclease P1, which makes concanavalin A (ConA) an ideal bridge for directional affinity adsorption of NP1. Figure 1 B in the figure represents the electrostatic potential distribution on the surface of nuclease P1. As can be seen from the figure, its charge property shows a significant positive potential in the region where the active site is located. Figure 1 In the figure, C represents the distribution of hydrophilic and hydrophobic regions on the surface of nuclease P1. The blue area represents the hydrophilic region, and it can be seen that a large number of hydrophilic regions are distributed on the surface of nuclease P1.

[0078] 2. Construction of dopamine (DA) and dopamine / charged polymer (DA / charged polymer) mimic molecules

[0079] Dopamine (DA) molecular models and dopamine / charged polymer (DA / charged polymer) molecular models were constructed using Avogadro software (version 1.2.0). All DA molecules and charged polymer molecules were constructed as protonated DA molecules and charged polymer molecules, respectively. The charged polymers were polyethyleneimine (PEI), polyethylene glycol (PEG), or polylysine (EPL). The Gaussian 09W program was used to perform structural optimization and vibrational frequency calculations on the DA and DA / charged polymer molecules at the B3LYP-D3(BJ) / 6-311G* level, and their single-point energies in gas and liquid phases were calculated. Finally, with the help of Multiwfn 3.8, the final RESP2 charges of the two simulated molecules were obtained, generating mol2 and chg files, which were then converted into GAFF force field-based molecular topology files (itp and top files) using the sobtop program.

[0080] 3. All-atom molecular dynamics simulation

[0081] All-atom molecular dynamics simulations were performed on two simulated molecules using the GROMACS2018.4 software package. The simulation parameters, including atom type, charge, bonding, and non-bonding terms, were provided by the amber99sb-ildn force field. The water molecule model used was TIP3P, with periodic boundary conditions set in the x, y, and z directions. Specifically, nuclease P1 was placed at the center of a cubic box with a side length of 8.9 nm, ensuring that nuclease P1 was more than 1.0 nm from the edge of the box. Then, 400 DA molecules and 300 DA molecules + 100 charged polymer molecules were randomly added to the box, respectively. Water molecules were then added to construct the water-nuclease P1-DA system (NP1-DA) and the water-nuclease P1-DA / PEI system (NP1-DA / charged polymer), respectively. Ten sodium ions were added to neutralize the net charge of each system, resulting in two simulated systems.

[0082] Before formally performing the phase generation simulation, a 5000-step energy minimization process was performed using the steepest descent method to stabilize and optimize the simulation system, eliminating unreasonable contact caused by the construction of the two systems. Subsequently, the Velocity-rescale and Parrinello-Rahman methods were used to maintain the system at constant temperature and pressure (298.15 K and 1 bar), respectively, allowing for micro-fluctuations. The coupling time constants for temperature control and pressure control were 0.2 ps and 2.5 ps, respectively, to ensure that the average system density of each box reached a reasonable state. Afterwards, the system was fully relaxed and balanced, i.e., positional constraints were imposed on all atoms in nuclease P1. At 298.15 K, a 5 ns NVT ensemble simulation was performed to bring the system to the set temperature. A second 5 ns NPT ensemble simulation was then performed to maintain the system pressure at 1 bar, ensuring that the solute and solvent in the water-nuclease P1-DA system (NP1-DA) and the water-nuclease P1-DA / charged polymer system (NP1-DA / charged polymer) were fully mixed while allowing the boxes to compress to a reasonable density. During this process, the positions of all bonds containing hydrogen atoms are constrained by the LINCS algorithm.

[0083] Formal molecular dynamics simulations were performed with a duration of 100 ns and a time step of 2 fs, using the Leap-frog algorithm for integration. Van der Waals interactions were calculated using the Cut-off algorithm, with a cutoff radius set to 1.1 nm. Long-range electrostatic interactions were calculated using the Particle MeshEwald (PME) algorithm, with a cutoff distance set to 1.1 nm. To ensure the accuracy and reliability of the calculation results, three parallel simulations were performed for each system with randomly generated initial velocities, and the final results were averaged.

[0084] The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible area (SASA), and interaction energy of nuclease P1 were obtained by the built-in calculation program of GROMACS; the dynamic evolution of the active pocket region of nuclease P1, the distribution of the temperature factor B-factor, and the distribution regions of DA and PEI molecules in the water-nuclease P1-DA / PEI system (NP1-DA / PEI) were obtained by the visualization program VMD.

[0085] Figure 2 The initial and final conformations of the water-nuclease P1-DA / PEI system, using polyethyleneimine (PEI) as the charged polymer, are shown during the 100 ns timeframe of the formal kinetic simulation. As can be seen from the figure, after 100 ns of kinetic simulation, most DA and PEI molecules are adsorbed onto the surface of nuclease P1, while its active site region remains unimpeded.

[0086] The overall conformational stability of nuclease P1 in different environments was measured using root mean square deviation (RMSD). Results are as follows: Figure 3 As shown, in pure water (i.e., NP1 in the figure), nuclease P1 requires approximately 50 ns to reach conformational equilibrium, with an RMSD value of 0.3 nm. After adding DA molecules (i.e., NP1-DA in the figure), the equilibrium time is shortened to less than 10 ns, with an RMSD value of 0.25 nm. This indicates that DA molecules help to rapidly enhance the conformational change of NP1, but the stability is slightly reduced. After adding DA and PEI molecules (i.e., NP1-DA-PEI in the figure), the equilibrium time is shortened to less than 10 ns. In the presence of DA and PEI, the final RMSD value of NP1 decreases to 0.27 nm, indicating smaller and more stable conformational shifts, suggesting that the DA / PEI environment enhances the stability of NP1.

[0087] Figure 4 The root mean square fluctuation (RMSF) results of nuclease P1 in pure water (NP1 in the figure), DA (NP1-DA in the figure), and DA / PEI (NP1-DA-PEI in the figure) environments were presented. By comparing the RMSF under different environments, the regions where DA and DA / PEI have stabilizing / unstable effects on nuclease P1 can be determined. As shown in the figure, both the DA and DA / PEI environments reduced the RMSF values ​​of residues 45–89 and 208–252 of nuclease P1, and increased the irregular coils and rigidity of the C-terminal α-helix in the secondary structure of nuclease P1. The DA / PEI environment showed a slightly more significant advantage than the DA environment.

[0088] Figure 5 The figure shows the solvent accessible surface area (SASA) results for nuclease P1 in pure water, DA, and DA / PEI environments. As can be seen from the figure, the SASA of nuclease P1 after DA and PEI adsorption is almost identical to that of nuclease P1 without DA and PEI adsorption, and no significant hydrophobic surface exposure is observed that would lead to nuclease P1 inactivation. The slight decrease in hydrophobic accessible area due to DA and the slight increase in hydrophobic accessible area due to DA / PEI may indicate further opening of the active site.

[0089] Figure 6 and Figure 7 The interaction energies of nuclease P1 with DA and DA / PEI over 100 ns were shown. Electrostatic interactions were found to play a dominant role in both the DA and DA / PEI environments. When both DA and PEI molecules were present, the interaction energy stabilized more quickly, reaching stable adsorption within 35 ns; when only DA molecules were present, stable adsorption was reached within 60 ns. The energy values ​​indicate that DA and nuclease P1 can bind stably, but the DA / PEI combination environment showed a more pronounced interaction, further enhancing stable binding.

[0090] Further identification of specific residues involved in DA binding in the DA environment, Figure 8 The distribution of the residues with the highest DA adsorption within 100 ns in the DA environment is shown. It can be seen that most of these residues are located on the back and sides of NP1, far from the active site. This ensures that the active site of NP1 is not obstructed, thus not hindering its catalytic activity. Figure 9 It can be seen that basic amino acids such as R207, K174, and H223 play the main adsorption role. Given that basic amino acids account for only 8.89% of NP1 residues, the concentrated interaction of DA on the surface of nuclease P1 helps to determine the fixed orientation of NP1 without producing excessively strong interactions that could disrupt its function.

[0091] Example 2: Experimental Validation of Dopamine / Polyethyleneimine (DA / PEI) Adsorption Model

[0092] 1. Carrier screening

[0093] Seven mesoporous resins with different pore sizes, particle sizes, and surface modifications (LX-1000EPB, LX-103B, LX-1000EA, LX-1000EPN, LXEP120, LX-1000NH, and HA) were used as carriers for immobilized nuclease experiments. 0.6 g of nuclease P1 powder was dissolved in 100 mL of PBS buffer (0.1 M, pH 6.0) to obtain nuclease P1 solution. 1 g of each of the LX-1000EPB, LX-103B, LX-1000EA, LX-1000EPN, LXEP120, LX-1000NH, and HA carriers were dispersed in 30 mL of nuclease P1 solution, and the mixtures were shaken at 25℃ and 180 rpm for 2 h to obtain the corresponding immobilized nuclease P1.

[0094] from Figure 10 As can be seen, the enzyme activity of immobilized enzymes on LX-1000EA and LX-1000EPN carriers was almost zero. This is partly due to the poor hydrophilicity of these two carriers and their pore size of 50-60 nm, which makes them prone to floating on the liquid surface during enzyme reactions, leading to incomplete catalysis. Furthermore, for nuclease molecules with a particle size of around 10 nm, the enzyme molecules are easily detached during repeated washing of the carrier. Except for HA resin, the other carriers all exhibited low protein loading. Considering both enzyme activity and protein loading, HA resin performed best, achieving an enzyme activity of 1009 U and a protein loading of 0.27 mg / g. Therefore, HA resin was chosen for subsequent modification and enzyme immobilization experiments.

[0095] One unit of enzyme activity is defined as the amount of enzyme that produces a nucleotide difference of 1.0 at 260 nm per minute. Enzyme activity is calculated using the following formula, where α is the dilution factor of the supernatant.

[0096]

[0097] 2. Carrier modification and immobilization

[0098] (1) Synthesize HA-PDA / PEI vector and immobilized nuclease P1.

[0099] 5 g of HA resin was dispersed in 50 mL of Tris buffer (50 mM, pH 7.0–9.0). 3 g / L of DA / PEI (DA to PEI molar ratio 3:1) was added, and the mixture was stirred at 30 °C for 30 h to allow it to co-deposit on the HA surface, yielding the HA-PDA / PEI carrier. The obtained carrier was filtered with pure water, washed twice, and stored at 4 °C.

[0100] 0.6 g of nuclease P1 powder was dissolved in 100 mL of PBS buffer (0.1 M, pH 6.0) to obtain nuclease P1 solution. 1 g of HA-PDA / PEI carrier was dispersed in 30 mL of nuclease P1 solution and shaken at 25 °C and 180 rpm for 2 h to obtain nuclease P1 immobilized using HA-PDA / PEI carrier, named HA-PDA / PEI-NP1.

[0101] (2) Synthesize HA-PDA / PEI-GA-ConA vector and immobilized nuclease P1.

[0102] Take 5g of HA-PDA / PEI vector from (1) and mix it with 200mL of PBS buffer (0.1M, pH 8.0), then add 2% v / v glutaraldehyde. Stir the mixture at 180rpm for 2h at room temperature to obtain the HA-PDA / PEI-GA vector. Filter the obtained vector with pure water, wash twice, and then store it at 4℃.

[0103] ConA was activated by suspending it in PBS buffer (0.1M, pH 7.0) containing 1mM MnCl2, 1mM CaCl2, or 1mM KCl. 1g of the HA-PDA / PEI-GA vector was added to 25mL of ConA solution (pH 7.0) with a concentration of 0.10–5.00g / L, and the mixture was incubated at 25℃ and 180rpm for 12h to obtain the HA-PDA / PEI-GA-ConA vector.

[0104] 0.6 g of nuclease P1 powder was dissolved in 100 mL of PBS buffer (0.1 M, pH 6.0). 1 g of HA-PDA / PEI-GA-ConA carrier was dispersed in 30 mL of nuclease P1 solution and shaken at 25 °C and 180 rpm for 2 h to obtain nuclease P1 immobilized using HA-PDA / PEI-GA-ConA carrier, named HA-PDA / PEI-GA-ConA-NP1.

[0105] (3) Synthesis of HA-GA and HA-GA-ConA vectors and immobilization of nuclease P1 using HA, HA-GA and HA-GA-ConA vectors

[0106] Mix 5g of HA with 200mL of PBS buffer (0.1M, pH 8.0), and then add 2% v / v glutaraldehyde. Stir the mixture at room temperature for 2h to complete the crosslinking reaction between HA and glutaraldehyde. Wash the carrier with pure water to remove excess glutaraldehyde, and then store it at 4℃ to obtain the HA-GA carrier.

[0107] Dissolve 0.6 g of nuclease P1 powder in 100 mL of PBS buffer (0.1 M, pH 6.0) to obtain an enzyme solution. Suspend ConA in PBS buffer (0.1 M, pH 7.0) containing 1 mM MnCl2, 1 mM CaCl2, or 1 mM KCl. Add 1 g of HA-GA vector to 25 mL of ConA solution with a concentration of 0.10–5.00 g / L, and incubate at 25 °C and 180 rpm for 12 h to obtain the HA-GA-ConA vector.

[0108] 1 g of HA, HA-GA, and HA-GA-ConA carriers were dispersed in 30 mL of nuclease P1 solution for reaction. The reaction was carried out at 25 °C and 180 rpm for 2 h, which immobilized nuclease P1 on the surface of the HA carrier, and nuclease P1 immobilized using HA, HA-GA, and HA-GA-ConA carriers were obtained, and named HA-NP1, HA-GA-NP1, and HA-GA-ConA-NP1, respectively.

[0109] (2) Enzyme activity assay

[0110] Dissolve 0.6 g of nuclease P1 powder in 100 mL of PBS buffer (0.1 M, pH 6.0) to obtain a free nuclease P1 solution. Incubate 1.9 mL aliquots of 5% substrate RNA solution in a 70°C water bath for 5 min, then add 0.1 mL of the free nuclease P1 solution (or 0.1 g of nuclease P1 immobilized on different carriers) and react for 15 min. Then add 2 mL of nucleic acid precipitant to terminate the reaction. After incubating on ice for 10 min, centrifuge the reaction mixture at 4°C and 5000 rpm for 10 min. Subsequently, dilute the supernatant with distilled water as needed and measure the absorbance at 260 nm. For the blank control, add nucleic acid precipitant before the enzyme solution. One unit of enzyme activity is defined as the amount of enzyme that produces an absorbance difference of 1.0 at 260 nm per minute. The specific calculation formula is as follows:

[0111]

[0112] Where α is the dilution of the supernatant; X1 is the initial enzyme activity; X0 is the enzyme activity after treatment; and C1 is the protein concentration.

[0113] from Figure 11 As can be seen, DA / PEI co-deposition (i.e., the HA-PDA / PEI-NP1 carrier) enhanced enzyme activity, with an enzyme activity of 1316.0 U, a 28% increase compared to HA-NP1. Simultaneously, the specific enzyme activity reached 2905.1 U / mg, indicating that the DA / PEI co-deposition surface facilitates the directional adsorption of NP1, ensuring unobstructed active sites and thus maintaining enzyme function. Further investigation revealed that the DA / PEI and ConA co-modified carrier used to immobilize nuclease P1 (i.e., HA-PDA / PEI-GA-ConA-NP1) achieved an enzyme activity of 1475.18 U, a 43% increase compared to the unmodified immobilized enzyme HA-NP1, and a specific enzyme activity of 3590 U / mg, a 27% increase compared to the unmodified immobilized enzyme.

[0114] 3. Stability of immobilized enzymes

[0115] (3) Temperature stability test

[0116] The free or immobilized enzyme was cultured in a water bath at 4–85°C for 30 min, and the retention rate of enzyme activity was measured. Figure 12 It can be seen that after incubation at all temperatures, HA-PDA / PEI-GA-ConA-NP1 exhibits the best thermal stability, maintaining more than 91% of its relative activity below 60°C, and its activity loss is lower than that of other NP1s as the temperature increases.

[0117] (4) pH stability test

[0118] Free or immobilized enzymes were cultured in phosphate buffer (pH 4.5–8.0) for 1 hour, and the retention rate of enzyme activity was measured. Figure 13 It can be seen that HA-PDA / PEI-GA-ConA-NP1 can still maintain more than 88% of its enzyme activity after the pH value reaches 8, while the free enzyme loses more than 30% of its enzyme activity.

[0119] (5) Batch stability test

[0120] After an enzyme reaction at 70°C for 5 minutes, the immobilized enzyme pump was filtered and washed twice to remove residual substrate RNA. Fresh substrate RNA was then added for the next batch of reaction. This process was repeated for 10 batches. The enzyme activity of the first batch was taken as 100%, and the enzyme activity retention rate after each batch was calculated. Figure 14 It can be seen that after 10 batches of reaction, the immobilized enzyme without modification had an activity of 19%, the immobilized nuclease P1 (i.e., HA-PDA / PEI-NP1) modified with DA / PEI had an activity retention rate of 26%, and the immobilized nuclease P1 (i.e., HA-PDA / PEI-GA-ConA-NP1) modified with DA / PEI and ConA still maintained a relative activity of more than 70%, which is more than 250% higher than that of the unmodified immobilized enzyme (i.e., HA-NP1).

[0121] This invention provides a method and approach for the targeted immobilization of nucleases and a computationally assisted design-based method for the targeted affinity immobilization of nuclease P1. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A directionally immobilized nuclease P1 based on dopamine and charged polymer modification, characterized in that, The directional immobilized nuclease P1 is obtained by modifying a mesoporous resin with dopamine molecules and charged polymer molecules to obtain a modified carrier, and then dispersing the modified carrier in a nuclease P1 solution for incubation. Wherein, the charged polymer molecule is polyethyleneimine molecule PEI, and the mesoporous resin is polystyrene-divinylbenzene resin HA; The molar ratio of dopamine molecules to charged polymer molecules is 1-3:1, and the solvent of the nuclease P1 solution is 0.05-0.20 M PBS buffer with pH 6.0-8.

0.

2. A directionally immobilized nuclease P1 based on dopamine and charged polymer modification, characterized in that, The directional immobilized nuclease P1 is obtained by modifying a mesoporous resin with dopamine molecules and charged polymer molecules to obtain a modified carrier, then modifying the modified carrier with glutaraldehyde, and then adding the modified carrier obtained from the second modification to a concanavalin A solution to obtain a specifically modified carrier. Finally, the specifically modified carrier is dispersed in a nuclease P1 solution and incubated to obtain the directional immobilized nuclease P1. Wherein, the charged polymer molecule is polyethyleneimine molecule PEI, and the mesoporous resin is polystyrene-divinylbenzene resin HA; The molar ratio of dopamine molecules to charged polymer molecules is 1-3:1, and the solvent of the nuclease P1 solution is 0.05-0.20 M PBS buffer with pH 6.0-8.

0.

3. The directed immobilized nuclease P1 according to claim 2, characterized in that, The canavon bean lectin A solution is prepared in the form of 0.05-0.20 M PBS buffer with pH 6.0-8.0 containing 0.05-0.20 M metal ions.

4. The directed immobilized nuclease P1 according to claim 3, characterized in that, The metal ion is any one of manganese ion, calcium ion, and potassium ion.

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