Directional immobilized nuclease P1 and nuclease P1 directional affinity immobilization method based on computational aided design

Abstract

The invention relates to the field of computational chemistry and enzyme engineering, in particular to directional immobilization nuclease P1 and a nuclease P1 directional affinity immobilization method based on computational aided design. According to the method, calculation and experiment means such as molecular dynamics simulation are combined for the first time, and accurate affinity immobilization is carried out on nuclease P1 through directional design. According to charge distribution and hydrophilic and hydrophobic areas of nuclease P1, spatial distribution of key residues of nuclease P1 is combined, dopamine and an electrified polymer are adopted as modifiers and co-deposited on the surface of the mesoporous resin carrier, and excellent adsorption capacity and immobilization effect are shown. Furthermore, through double optimization of glutaraldehyde crosslinking and concanavalin A modification, the binding efficiency and the catalytic activity stability of the nuclease P1 are remarkably improved. According to the method, the problem that nuclease P1 is poor in stability in a free state is solved, efficient directional affinity immobilization is achieved through a brand new molecular design and interface optimization technology, and a feasible technical path is provided for industrial-scale application.

Description

Technical Field

[0001] The present invention relates to the fields of computational chemistry and enzyme engineering, and in particular to a directed immobilized nuclease P1 and a method for directed affinity immobilization of nuclease P1 based on computational-aided design. Background Art

[0002] In the field of enzyme engineering and biocatalysis, immobilization technology has been widely used to immobilize various enzyme preparations in industrial production processes as an important means to effectively improve the stability, activity retention and reuse rate of enzymes. Immobilized enzymes can avoid the inactivation and degradation problems that are prone to occur in the free state of enzymes, and at the same time help to improve reaction efficiency and reduce production costs. However, traditional enzyme immobilization technologies, such as physical adsorption, covalent bonding and embedding, usually have problems such as complex immobilization process, large enzyme activity loss, and limited carrier selection, which restricts their large-scale application.

[0003] Nuclease P1 is an important nucleic acid degrading enzyme. It was first discovered from the fermentation of Penicillium citrinum. It has the dual activity of phosphodiesterase and phosphomonoesterase. It is widely used in the fields of DNA and RNA degradation, nucleotide extraction, analysis and detection, and has significant industrial application value. Nuclease P1 is composed of 270 amino acids with a molecular weight of about 44kDa. Its active site contains three Zn 2+ , they are all located at the bottom of the binding cleft and are crucial to its catalytic function. However, nuclease P1 has poor stability in the free state and is easily affected by environmental factors (such as temperature, pH value, organic solvents, etc.), resulting in low reuse rate and complex operation in practical applications, and unable to fully exert its potential industrial value. In recent years, the application of molecular simulation technology and computer-aided design (CAD) in enzyme engineering has received increasing attention. Through molecular dynamics simulation and electrostatic analysis, the structural characteristics, active sites and interactions of enzyme molecules with carriers can be effectively predicted, thereby providing guidance for the directional immobilization of enzymes. Therefore, how to achieve efficient and stable immobilization of nuclease P1 is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the nuclease P1 in the prior art has significant industrial application potential due to its efficient catalytic performance in DNA and RNA degradation and nucleotide extraction. However, its current poor stability in the free state and low reuse rate have greatly limited its promotion in large-scale industrial applications. A method for directional affinity immobilization of nuclease P1 based on computer-aided design is provided.

[0005] Another technical problem to be solved by the present invention is to provide a directional immobilized nuclease P1 based on the directional immobilized nuclease P1 modified by a modifier.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

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

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

[0009] (2) According to the electrostatic potential distribution and hydrophilic and hydrophobic region results of the nuclease P1 surface in step (1), dopamine molecules DA and charged polymer molecules are selected as modifiers, and corresponding molecular models are constructed respectively;

[0010] (3) performing structural optimization and energy optimization on the molecular model constructed in step (2);

[0011] (4) using the molecular model optimized in step (3), constructing a simulation system including water, nuclease P1 and a modifier, and neutralizing the net charge of the system by adding sodium ions;

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

[0013] (6) Based on the analysis results of the dynamic behavior of nuclease P1, a directional immobilized nuclease P1 modified with a modifying agent was constructed.

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

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

[0016] Wherein, in step (1), the electrostatic potential distribution and hydrophilic and hydrophobic regions on the surface of nuclease P1 are analyzed using molecular visualization software (such as Pymol, Chimera, VMD, etc.).

[0017] Specifically, the surface electrostatic potential area distribution of the nuclease P1 described in step (2) is calculated by the Pymol program using the APBS tool, and the surface hydrophilicity and hydrophobicity area distribution is obtained by analyzing the residue distribution using Chimera.

[0018] Specifically, the surface electrostatic potential area distribution of the nuclease P1 is of great significance for analyzing the catalytic mechanism of the enzyme and its binding mode with substrates or other molecules, and the mapping of the surface hydrophilic and hydrophobic area distribution helps to evaluate its stability in a water-soluble environment and possible interaction interfaces.

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

[0020] Wherein, in step (2), the charged polymer molecule is any one or a combination of polyethyleneimine molecule PEI, polyethylene glycol molecule PEG, polylysine molecule EPL, preferably polyethyleneimine molecule PEI.

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

[0022] Wherein, in step (4), in the simulation system comprising water, nuclease P1 and modifier, water, nuclease P1 and modifier are constructed into a cubic box with boundary periodicity conditions.

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

[0024] Specifically, the nuclease P1 is greater than 1.0 nm away from the edge of the cubic box; the modifiers are dopamine molecules and charged polymer molecules, the positions of the dopamine molecules and the charged polymer molecules in the cubic box are random, and the molar ratio of the dopamine molecules to the charged polymer molecules is 1 to 3:1.

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

[0026] Among them, in step (5), before formally carrying out molecular dynamics simulation, the steepest descent method is used to simulate 1000 to 10000 steps to minimize the energy of the simulation system, and then an ensemble simulation is carried out for 2 to 10 ns under constant conditions of a reaction temperature of 298.15 K and a reaction pressure of 1 bar, so 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 first used to simulate 5000 steps to minimize the energy of the simulation system to eliminate unreasonable contacts generated by the system construction.

[0028] Specifically, the ensemble simulation includes NVT and NPT ensemble simulations, and the preferred durations are 5 ns respectively.

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

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

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

[0032] Specifically, the molecular dynamics simulation was integrated using the Leap-frog algorithm. The van der Waals interactions were calculated using the Cut-off algorithm, and the cutoff radius was set to 1.1 nm. The long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) algorithm, and the cutoff distance was set to 1.1 nm.

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

[0034] A directional immobilized nuclease P1 modified by a modifier is also within the scope of protection of the present invention, wherein the directional immobilized nuclease P1 is constructed under the guidance of the directional affinity immobilization method of the nuclease P1.

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

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

[0037] or,

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

[0039] Wherein, the mesoporous resin is any one of epoxy resin (LX-1000EPB, LX-103B), aminated resin (HA, LXEP120), quaternary ammonium resin (LX-1000EA, LX-1000EPN, LX-1000NH), preferably, the mesoporous resin is LX-1000EPB, LX-103B, LXEP120, LX-1000NH, HA; more preferably, the mesoporous resin is HA (i.e., amino-functionalized polystyrene-divinylbenzene resin).

[0040] The modified carrier is obtained by modifying the mesoporous resin with DA and PEI, and the process is as follows: the mesoporous resin is dispersed in 50mM, pH 7.0-9.0 Tris buffer. Then 3g / L DA and PEI are added, and the mixture is stirred at 30°C for 30h to be co-deposited on the surface of the mesoporous resin. The obtained carrier is filtered with pure water, washed twice, and stored at 4°C to obtain the modified carrier.

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

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

[0043] Wherein, the solvent of the nuclease P1 solution is 0.05-0.20M PBS buffer with a pH of 6.0-8.0.

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

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

[0046] The solvent of the ConA solution is a PBS buffer solution with a pH of 6.0 to 8.0 and containing 0.05 to 0.20 M metal ions; specifically, the metal ions are any one of manganese ions, calcium ions, and potassium ions.

[0047] Wherein, the incubation conditions are 25° C. and 180 rpm / min for 2 to 5 hours.

[0048] The above-mentioned directional immobilized nuclease P1 modified with dopamine and polyethyleneimine was experimentally verified and found that the simulation results of the computationally assisted design method of the present invention were highly compatible with the experimental results, verifying the accuracy of the predictive simulation. The experiment shows that the directional immobilized enzyme HA-PDA / PEI-GA-ConA-NP1 prepared by the present invention shows the best thermal stability, and still maintains more than 91% relative activity below 60°C; it can still maintain more than 88% enzyme activity after the pH value reaches 8; after repeating 10 batches of reactions, the enzyme activity retention rate of the immobilized nuclease P1 (ie, HA-PDA / PEI-NP1) modified by DA / PEI is 26%, and the immobilized nuclease P1 (ie, HA-PDA / PEI-GA-ConA-NP1) co-modified with PDA / PEI and ConA still maintains more than 70% relative activity. Compared with single covalently linked, physically adsorbed, and affinity adsorbed immobilized enzymes, the performance of the immobilized enzyme in the present invention is greatly improved.

[0049] Beneficial effects:

[0050] (1) Innovative application of computer-aided design: This invention combines molecular dynamics simulation with molecular visualization analysis to conduct a systematic study on the immobilization design of nuclease P1 for the first time. By accurately calculating the charged region and hydrophilic and hydrophobic distribution of nuclease P1, it provides a scientific basis for selecting suitable modification materials and immobilization strategies, breaking through the traditional design method that relies on experience and greatly improving the efficiency of carrier research and development. It not only solves the problem of poor stability of nuclease P1 in the free state, but also realizes efficient directional affinity immobilization through new molecular design and interface optimization technology, providing a feasible technical path for industrial-scale application.

[0051] (2) The present invention optimizes the selection of modification materials by analyzing the charge distribution and hydrophilicity of the surface of nuclease P1, and uses dopamine (DA) and charged polymers as modifiers, which are co-deposited on the surface of the mesoporous resin carrier, showing excellent adsorption capacity and immobilization effect. This modifier co-deposition strategy improves the immobilization effect and helps to achieve customized design for different application requirements. Furthermore, through the dual optimization of glutaraldehyde cross-linking and concanavalin A modification, the binding efficiency and catalytic activity stability of nuclease P1 were significantly improved.

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

[0053] (4) The present invention obtains an innovative immobilization method by comprehensively considering the surface characteristics and molecular dynamics parameters of nuclease P1, and the activity and reusability of the immobilized enzyme are greatly improved. 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, is suitable for a wider range of biocatalysis and related application fields, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 1 Figure 1 is a surface feature map of nuclease P1. A is the structural feature of nuclease P1, where nuclease P1 is cyan, zinc ions are orange, and asparagine residues are purple; B is the surface electrostatic potential distribution map of nuclease P1, where the blue area is the part with positive potential, the red area is the part with negative potential, and the white area is the neutral potential; C is the hydrophilic and hydrophobic region distribution map of nuclease P1, where the blue area is the hydrophilic area, the orange-red area is the hydrophobic area, and the white area is the neutral area.

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

[0057] Figure 3 is 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 in pure water, DA and DA / PEI environments. The inset shows the B-factor conformation of nuclease P1 in pure water, DA and DA / PEI environments, with red representing rigidity and blue representing flexibility.

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

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

[0061] Figure 7 The interaction energy change between nuclease P1 and DA / PEI within 100 ns.

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

[0063] Fig. 9 This is a graph showing the number of DA adsorbed by the first ten nuclease P1 residues within 100 ns in a DA environment.

[0064] Fig.10 The figure shows the enzymatic activity and protein loading capacity of seven resins used to immobilize nucleases.

[0065] Fig.11 The enzymatic activity and relative enzyme activity of immobilized nuclease P1 are shown in FIG.

[0066] Fig.12 The thermal stability diagram of immobilized nuclease P1.

[0067] Fig.13 The pH stability diagram of immobilized nuclease P1.

[0068] Fig.14 Batch stability graph of immobilized nuclease P1. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

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

[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), dopamine / charged polymer (DA / charged polymer) adsorption simulation

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

[0074] The pdb file (PDB id: 1ak0) of the crystal structure of nuclease P1 (NP1) was downloaded from the Protein Data Bank on the RCSB website as the research object, and the downloaded nuclease P1 crystal structure was optimized. Specifically, the Pymol program was used to remove excess crystal water molecules and excess subunits, and the obtained pdb file was converted into a gro file and a restriction potential itp file that can be used for calculations through the pdb2gmx command of Gromacs, and the top file was obtained.

[0075] The three-dimensional structure and characteristic regions of nuclease P1 were drawn by VMD program. First, load the gro file of nuclease P1, use the Representations tool of Graphics module, use New-cartoon model for secondary structure, VDW model for asparagine residues and tri-zinc structure, CPK model for sugar group, and select Glossy for material.

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

[0077] Figure 1 Surface characterization results of nuclease P1. Figure 1 The A in the figure is the three-dimensional structural feature of nuclease P1. It can be seen that the asparagine residues (Asn92, Asn138, Asn184, Asn197) in nuclease P1 are glycosylated with glucose side chains, and the glycosylation sites are all present on the back away from the active pocket area of ​​nuclease P1, making ConA an ideal bridge for directional affinity adsorption of NP1. Figure 1 B in the figure is the surface electrostatic potential distribution result of nuclease P1. It can be seen from the figure that its charge properties show a significant positive potential in the area where the active site is located. Figure 1 The C in the figure is the distribution result of hydrophilic and hydrophobic regions on the surface of nuclease P1. The blue area is the hydrophilic area. It can be found that there are a large number of hydrophilic areas distributed on the surface of nuclease P1.

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

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

[0080] 3. All-atom molecular dynamics simulation

[0081] The GROMACS2018.4 software package was used to perform all-atom molecular dynamics simulations on the two simulated molecules. The simulation parameters such as atomic type, charge, bonding, and non-bonding terms required for the simulation system were provided by the amber99sb-ildn force field. The water molecule model used TIP3P, and periodic boundary conditions were set in the x, y, and z directions. Specifically, the nuclease P1 was placed in the center of the box to construct a cubic box with a side length of 8.9 nm, and ensure that the nuclease P1 was more than 1.0 nm away from the edge of the box. Then, 400 DA molecules, 300 DA molecules + 100 charged polymer molecules were added to random positions in the box, and water molecules were added to construct the water-nuclease P1-DA system (NP1-DA) and the water-nuclease P1-DA / PEI system (NP1-DA / charged polymer). At the same time, 10 sodium ions were added to neutralize the net charge of the system, and finally two simulation systems were obtained.

[0082] Before the formal phase simulation, the steepest descent method was used to perform 5000 steps of energy minimization to stabilize and optimize the simulation system and eliminate the unreasonable contact caused by the construction of the two systems. Subsequently, the Velocity-rescale and Parrinello-Rahman methods were used to keep the system at constant temperature and pressure (298.15K and 1bar), respectively, and microscopic fluctuations were allowed. The coupling time constants of temperature control and pressure control were 0.2ps and 2.5ps, respectively, to ensure that the average density of the system in each box reached a reasonable state. After that, the system was fully relaxed and balanced, that is, position constraints were imposed on all atoms in the nuclease P1. At a temperature of 298.15K, a 5ns NVT ensemble simulation was first performed to allow the system to reach the set temperature. Another 5ns NPT ensemble simulation was set to maintain the system pressure at 1bar, 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 box to be compressed to a reasonable density. During this process, the positions of all bonds containing hydrogen atoms were constrained by the LINCS algorithm.

[0083] The duration of the formal molecular dynamics simulation was set to 100 ns, the time step was 2 fs, and the Leap-frog algorithm was used for integration. The Cut-off algorithm was used to calculate the van der Waals interaction, and the cut-off radius was set to 1.1 nm. The Particle Mesh Ewald (PME) algorithm was used to calculate the long-range electrostatic interaction, and the cut-off distance was set to 1.1 nm. To ensure the accuracy and reliability of the calculation results, three parallel simulations were performed on each system with different initial velocities randomly generated by the system, 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 results of nuclease P1 were obtained by the GROMACS built-in calculation program; the dynamic evolution process of the active pocket area of ​​nuclease P1, the temperature factor B-factor distribution, and the distribution area 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 with polyethyleneimine PEI as the charged polymer in the 100ns duration of the formal kinetic simulation are shown. As can be seen from the figure, after 100ns of kinetic simulation, most of the DA molecules and PEI molecules are adsorbed to the surface of nuclease P1, while its active site area is not hindered.

[0086] The overall conformational stability of nuclease P1 in different environments was measured by the root mean square deviation (RMSD). Figure 3 As shown in the figure, in pure water (i.e., NP1 in the figure), nuclease P1 takes about 50ns to reach conformational equilibrium, and the RMSD value is 0.3nm. After adding DA molecules (i.e., NP1-DA in the figure), the equilibrium time is shortened to less than 10ns, and the RMSD value is 0.25nm, which indicates that DA molecules help to quickly enhance the conformational changes of NP1, but the stability is slightly poor. After adding DA molecules and PEI molecules (i.e., NP1-DA-PEI in the figure), the equilibrium time is shortened to less than 10ns, and the final RMSD value of NP1 in the presence of DA and PEI is reduced to 0.27nm, indicating that the conformational movement is smaller and more stable, indicating 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 (i.e. NP1 in the figure), DA (i.e. NP1-DA in the figure) and DA / PEI (i.e. NP1-DA-PEI in the figure) environments. By comparing the RMSF in different environments, the regions where DA and DA / PEI have a stabilizing / destabilizing effect on nuclease P1 can be determined. Combined with the figure, it can be seen that both DA and DA / PEI environments reduce the RMSF values ​​of residues 45-89 and 208-252 of nuclease P1, increase the rigidity of the irregular coil and C-terminal α-helix in the secondary structure of nuclease P1, and the DA / PEI environment has a slightly more obvious advantage than the DA environment.

[0088] Figure 5 The results of the solvent accessible surface area (SASA) of nuclease P1 in pure water, DA and DA / PEI environments. As can be seen from the figure, the SASA shows that the SASA of nuclease P1 after DA and PEI adsorption is almost the same as that of nuclease P1 without DA and PEI adsorption, and does not show a large amount of hydrophobic surface exposure that causes nuclease P1 inactivation. The slight decrease in the hydrophobic accessible area caused by DA and the slight increase in the hydrophobic accessible area caused by DA / PEI may indicate further opening of the active site.

[0089] Figure 6 and Figure 7 The changes in the interaction energy between nuclease P1 and DA and DA / PEI within 100ns were shown respectively. It was found that electrostatic interaction played a dominant role in both the DA environment and the DA / PEI environment. When DA molecules and PEI molecules existed at the same time, the interaction energy stabilized faster and reached stable adsorption within 35ns; when only DA molecules existed, stable adsorption was achieved within 60ns. The energy value showed that DA and nuclease P1 could be stably bound, but the DA / PEI combination environment showed a more obvious interaction, and the stable binding was further enhanced.

[0090] Further identification of specific residues involved in DA binding in the DA context, Figure 8 The distribution of the residues that adsorbed the most DA 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, away from the active site. This ensures that the active site of NP1 is not obstructed, thereby not hindering its catalytic activity. Fig. 9 It can be seen that basic amino acids such as R207, K174, and H223 play a major role in adsorption. Given that basic amino acids account for only 8.89% of the residues in NP1, the concentrated interaction of DA on the surface of nuclease P1 helps to determine the fixed orientation of NP1 without generating overly strong interactions that may disrupt its function.

[0091] Example 2: Experimental verification of dopamine / polyethyleneimine (DA / PEI) adsorption model

[0092] 1. Vector screening

[0093] Seven mesoporous resins with different pore sizes, particle sizes and surface modifications, including 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 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 corresponding immobilized nuclease P1 was obtained by shaking at 25 ° C and 180 rpm for 2 h.

[0094] from Fig.10 It can be seen that the immobilized enzyme activity of LX-1000EA and LX-1000EPN carriers is almost 0. On the one hand, the hydrophilicity of these two carriers is poor, and the pore size is between 50 and 60 nm. During the enzyme reaction, the carrier easily floats on the liquid surface, resulting in insufficient catalytic reaction. In addition, for nuclease molecules with a particle size of about 10 nm, the enzyme molecules are easy to fall off during repeated washing of the carrier. Except for HA resin, the other carriers all show low protein loading. Considering the enzyme activity and protein loading, HA resin performed best, with an enzyme activity of 1009U and a protein loading of 0.27 mg / g. Therefore, HA resin was selected for subsequent modification and immobilized enzyme experiments.

[0095] One unit of enzyme activity is defined as the amount of enzyme that produces 1.0 absorbance difference at 260 nm per minute for the amount of nucleotides generated. Enzyme activity is calculated according to the following formula, where α is the dilution factor of the supernatant.

[0096]

[0097] 2. Carrier modification and immobilization

[0098] (1) Synthesis of HA-PDA / PEI carrier and immobilized nuclease P1.

[0099] Disperse 5g of HA resin in 50mL of Tris buffer (50mM, pH 7.0-9.0). Add 3g / L of DA / PEI (the molar ratio of DA to PEI is 3:1), and stir the mixture at 30°C for 30h to allow it to co-deposit on the HA surface to obtain a HA-PDA / PEI carrier. Filter the obtained carrier with pure water, wash twice, and store 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 a 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 on HA-PDA / PEI carrier, named HA-PDA / PEI-NP1.

[0101] (2) Synthesis of HA-PDA / PEI-GA-ConA carrier and immobilized nuclease P1.

[0102] 5 g of HA-PDA / PEI carrier in (1) was mixed with 200 mL of PBS buffer (0.1 M, pH 8.0), and then 2% v / v glutaraldehyde was added. The mixture was stirred at 180 rpm for 2 h at room temperature to obtain HA-PDA / PEI-GA carrier. The obtained carrier was filtered with pure water, washed twice, and then stored at 4°C.

[0103] ConA was suspended in 1 mM MnCl 2 , 1 mM CaCl 2 Or 1mM KCl PBS buffer (0.1M, pH 7.0) for activation. 1g HA-PDA / PEI-GA carrier was added to 25mL ConA solution (pH 7.0) with a concentration of 0.10-5.00g / L, and cultured at 25°C and 180rpm for 12h to obtain HA-PDA / PEI-GA-ConA carrier.

[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 on HA-PDA / PEI-GA-ConA carrier, named HA-PDA / PEI-GA-ConA-NP1.

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

[0106] 5 g of HA was mixed with 200 mL of PBS buffer (0.1 M, pH 8.0), and then 2% v / v glutaraldehyde was added. The mixture was stirred at room temperature for 2 h to complete the cross-linking reaction between HA and glutaraldehyde, and the carrier was washed with pure water to remove excess glutaraldehyde, and then stored at 4°C to obtain 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 a PBS solution containing 1 mM MnCl 2 , 1 mM CaCl 2 Or 1mM KCl PBS buffer (0.1M, pH 7.0). 1g HA-GA carrier was added to 25mL ConA solution with a concentration of 0.10-5.00g / L, and cultured at 25°C and 180rpm for 12h to obtain HA-GA-ConA carrier.

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

[0109] (2) Enzyme activity assay

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

[0111]

[0112] Where α is the dilution of the supernatant; X 1 is the initial enzyme activity; X 0 is the enzyme activity after treatment; C 1 is the protein concentration.

[0113] from Fig.11 It can be seen that DA / PEI co-deposition (i.e., HA-PDA / PEI-NP1 carrier) enhances the enzyme activity, with an enzyme activity of 1316.0U, which is 28% higher than that of HA-NP1, and the specific enzyme activity also reaches 2905.1U / mg, indicating that the DA / PEI co-deposition surface is conducive to the directional adsorption of NP1, ensuring that the active sites are not hindered, thereby maintaining the function of the enzyme. It was further found that the DA / PEI and ConA co-modified carrier was used to immobilize the nuclease P1 (i.e., HA-PDA / PEI-GA-ConA-NP1) with an enzyme activity of 1475.18U, which is 43% higher than that of the unmodified immobilized enzyme HA-NP1, and the specific enzyme activity reached 3590U / mg, which is 27% higher than that of the unmodified immobilized enzyme.

[0114] 3. Stability of immobilized enzyme

[0115] (3) Temperature stability test

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

[0117] (4) pH stability test

[0118] The free enzyme or immobilized enzyme was cultured in a phosphate buffer with a pH value of 4.5 to 8.0 for 1 hour, and the retention rate of the enzyme activity was determined. Fig.13 It can be seen that HA-PDA / PEI-GA-ConA-NP1 can still maintain more than 88% of the enzyme activity after the pH value reaches 8, while the free enzyme loses more than 30% of the enzyme activity.

[0119] (5) Batch stability test

[0120] After 5 min of enzyme reaction at 70°C, the immobilized enzyme was filtered and washed twice to wash away the residual substrate RNA on the surface, and fresh substrate RNA was added for the next batch of reaction. This was repeated for 10 batches of reactions. The enzyme activity of the first batch of reactions was taken as 100%, and the retention rate of enzyme activity after each batch of reactions was calculated. Fig.14 It can be seen that after 10 batches of reactions, the unmodified immobilized enzyme had an activity retention rate 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) co-modified with DA / PEI and ConA still maintained a relative activity of more than 70%, which was more than 250% higher than that of the unmodified immobilized enzyme (i.e., HA-NP1).

[0121] The present invention provides a directional immobilized nuclease, and a method and idea for a directional affinity immobilization method of nuclease P1 based on computer-aided design. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for directional affinity immobilization of nuclease P1 based on computer-aided design, characterized in that: The steps include: (1) Obtain and analyze the crystal structure of nuclease P1 to identify the electrostatic potential distribution and hydrophilic and hydrophobic regions on its surface; (2) According to the electrostatic potential distribution and hydrophilic and hydrophobic region results of the nuclease P1 surface in step (1), dopamine molecules DA and charged polymer molecules are selected as modifiers, and corresponding molecular models are constructed respectively; (3) performing structural optimization and energy optimization on the molecular model constructed in step (2); (4) using the molecular model optimized in step (3), constructing a simulation system including water, nuclease P1 and a modifier, and neutralizing the net charge of the system by adding sodium ions; (5) performing molecular dynamics simulation on the simulation system of step (4) to analyze the dynamic behavior of nuclease P1 in the modifier environment; (6) Based on the analysis results of the dynamic behavior of nuclease P1, a directional immobilized nuclease P1 modified with a modifying agent was constructed.

2. The method for directional affinity immobilization of nuclease P1 according to claim 1, characterized in that: In step (2), the dopamine molecules and the charged polymer molecules need to be protonated when constructing the corresponding molecular models; the charged polymer molecules are any one or a combination of polyethyleneimine molecules PEI, polyethylene glycol molecules PEG, and polylysine molecules EPL.

3. The method for directional affinity immobilization of nuclease P1 according to claim 1, characterized in that: In step (4), in the simulation system comprising water, nuclease P1 and a modifier, water, nuclease P1 and a modifier are constructed into a cubic box with boundary periodicity conditions; The distance between the nuclease P1 and the edge of the cubic box is greater than 1.0 nm; the modifiers are dopamine molecules and charged polymer molecules, the positions of the dopamine molecules and the charged polymer molecules in the cubic box are random, and the molar ratio of the dopamine molecules to the charged polymer molecules is 1 to 3:

1.

4. The method for directional affinity immobilization of nuclease P1 according to claim 1, characterized in that: 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, and then an ensemble simulation is performed for 2 to 10 ns under constant conditions of 298.15 K and 1 bar to allow the solute and solvent in the simulation system to be fully mixed while allowing the cubic box to be compressed to a reasonable density.

5. The method for directional affinity immobilization of nuclease P1 according to claim 1, characterized in that: In step (6), the duration of the molecular dynamics simulation is set to 10 to 100 ns, and the time step is 1 to 2 fs.

6. A directional immobilized nuclease P1 modified with dopamine and charged polymer, characterized in that: The directed immobilized nuclease P1 is constructed under the guidance of the directed affinity immobilization method for nuclease P1 according to any one of claims 1 to 5.

7. The directional immobilized nuclease P1 according to claim 6, 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 to obtain the directional immobilized nuclease P1.

8. The directional immobilized nuclease P1 according to claim 6, characterized in that: The directional immobilized nuclease P1 is a modified carrier obtained by modifying a mesoporous resin with dopamine molecules and charged polymer molecules, and the modified carrier is secondarily modified with glutaraldehyde. The modified carrier obtained by the second modification is then added to a concanavalin A solution to obtain a specifically modified modified carrier. Finally, the specifically modified modified carrier is dispersed in a nuclease P1 solution for incubation to obtain the directional immobilized nuclease P1.

9. The directional immobilized nuclease P1 according to claim 7 or 8, characterized in that: The charged polymer molecule is any one of polyethyleneimine molecule PEI, polyethylene glycol molecule PEG, and polylysine molecule EPL, or a combination of several of them; the molar ratio of the dopamine molecule to the charged polymer molecule is 1 to 3:1; the mesoporous resin is any one of hydroxylated resin, aminated resin, and quaternized resin; and the solvent of the nuclease P1 solution is 0.05 to 0.20 M, pH 6.0 to 8.0 PBS buffer.

10. The directional immobilized nuclease P1 according to claim 8, characterized in that: The solvent of the Concanavalin A solution is a 0.05-0.20M PBS buffer solution containing 0.05-0.20M metal ions and a pH of 6.0-8.0; wherein the metal ions are any one of manganese ions, calcium ions, and potassium ions.

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