Molecular modification design method for improving hydrophobicity of catalytic center of bacillus subtilis protease

Abstract

The invention belongs to the field of bioengineering. The invention provides a molecular modification design method for improving the hydrophobicity of a bacillus subtilis protease catalytic center, which comprises the following steps: searching an analyzed bacillus subtilis protease crystal structure in a protein database PDB, docking with a hexapeptide substrate through a Glide mode of # imgabs0 # software to generate an enzyme-substrate compound, and carrying out molecular modification on the enzyme-substrate compound to obtain the bacillus subtilis protease catalytic center hydrophobicity. The method comprises the following steps: calculating polar charges of polar atoms N and O and combined H of each amino acid residue in a 10 angstrom range of a catalytic central amino acid of the subtilisin by using a PPC force field, calculating the sum of absolute values of the polar charges, selecting a potential mutation site of the subtilisin, and carrying out virtual single-point mutation on the potential mutation site to obtain a large-steric-hindrance non-polar amino acid, the method comprises the following steps: calculating the sum of absolute values of polar charges of polar atoms N, O and H in a 10 angstrom range of a catalytic center amino acid of a single-point mutant by using a PPC force field, selecting a virtual subtilisin single-point mutant, expressing the mutant, carrying out iterative mutation, and carrying out re-expression to obtain a modified subtilisin mutant, and carrying out hydrophobicity verification. The molecular modification design method can quickly and effectively reduce water molecules in the catalytic center of the subtilisin and improve the hydrophobicity of the subtilisin, and the enzyme modification method is proved to be effective and reliable.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a rational design method for the binding ability of water molecules in a charge characterization region. Background Art

[0002] The term "hydrophobic" was proposed in 1915, specifically referring to the phenomenon that non-polar compounds have very low solubility in water or tend to aggregate in water. The hydrophobic and hydrophilic effects are gradually recognized as one of the dominant driving forces of biochemical processes, playing an important role in many key areas such as protein folding, nucleic acid stability, molecular recognition and binding. These effects are crucial foundations for the stability of molecular structure, functional realization, and the understanding and research of disease mechanisms.

[0003] Hydrophobicity can be characterized by measuring the contact angle of a droplet at the macroscopic scale, but it cannot be directly observed or accurately described at the molecular and atomic scales. Whether it is the characterization method of the hydration index between water and amino groups proposed by Millard-Stafford, the protein hydrophobicity proposed by Tang et al. that should depend on the chemical properties of the side chain and its position in the protein, the clustering method based on the water density and single chain of the residues used by Gao et al., or the dynamic hydrogen bond network of the protein and solvent interface described by Rienzo et al. using molecular dynamics simulation at the atomic level, all studies on protein hydrophobicity are based on the amino acid molecular level. More accurate analysis and design of protein hydrophobic regions at the atomic level remains a huge challenge.

[0004] This is because the local hydrophobic region of the protein is not uniformly non-polar, but under the influence of the surrounding amino acids under the action of geometric conformation, the polarity, that is, the charge changes, are displayed at the nanoscale. Therefore, it is necessary to comprehensively consider the impact of the environment of this local area and the interaction between several residues on the charge. However, the molecular force field parameters of traditional protein calculations currently solidify the charge of each amino acid side chain atom, and cannot consider the impact of environmental changes, making it difficult to achieve accurate calculation and design of local hydrophobic regions.

[0005] The polarizable force field method proposed by Shi et al. in 2015 is expected to solve this problem better. This is a variable molecular force field that can simulate local environmental changes and truly reflect the changes in the dipole moment of water molecules in the system, the electrostatic polarization effect, and the local structural rearrangement of the protein. Therefore, the polarizable molecular force field can more accurately describe the charge state of each atom of the protein in the solvent environment than the traditional fixed charge force field, thereby truly characterizing the overall polarity state (i.e., hydrophobicity) of a specific region. In addition, there is a high-intensity and universal polarization-induced effect inside the protease. Since the fixation of water molecules is mainly achieved through H-bonding with surrounding polar atoms, the stronger the polarity of the atom, the stronger the H-bond, and thus the stronger the ability of the protease to bind and transfer water molecules. For N and O atoms, the more negative the polar charge, the higher the electron cloud density, the higher the degree of polarization, and the stronger its ability to form H bonds with H atoms of water molecules; on the contrary, for H atoms on N and O atoms, the more positive their polar charge, the easier it is to form H bonds with other polar atoms, thus showing high polarization intensity. In summary, the selection of a precise polarizable molecular force field "protein-specific polarization charge" (PPC) and the calculation of the polar charges of N, O and H atoms of proteases on this basis, thereby characterizing the ability to bind water molecules, are of great significance for drug design, material science, and biomedical engineering. A series of protein engineering fields designed for hydrophobic modification of proteins, especially enzymes, have attracted the attention of scientists. Computer rational design methods are widely used because of their high efficiency, small experimental screening workload, and the ability to quickly obtain better mutants.

[0006] Therefore, in view of the problem that the currently commonly used hydrophobic calculation design mainly starts from the fixed hydrophobic value of amino acid residues or fixed charge force field parameters, and rarely starts from the polarizable molecular force field method, it is impossible to directly observe and it is difficult to accurately describe the charged state of each atom of the protein in the solvent environment and its true characterization of the local hydrophobic area, the present invention uses a series of computer rational calculations to carry out targeted transformation to improve the hydrophobicity of the catalytic center of Bacillus subtilis protease, thereby realizing the expulsion of water molecules from the catalytic center. Summary of the invention

[0007] In order to solve the above-mentioned prior art problems, the present invention provides a molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilisin. The molecular modification design method of the present invention can quickly and effectively reduce the water molecules in the catalytic center of subtilisin, improve the hydrophobicity, confirm the water content of the catalytic center through the crystal structure of subtilisin and the substrate, and further confirm the ability to exclude water molecules through binding free energy calculation. The verification results show that the design method can quickly and effectively improve the hydrophobicity of the catalytic center of subtilisin, and the enzyme modification method of the present invention is reliable.

[0008] The object of the present invention is to provide a molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilisin, comprising the following steps:

[0009] (1) Search the protein database PDB for the solved crystal structure of Bacillus subtilisin (Subtilisin BPN′) The Glide mode of dinger software was docked with the hexapeptide substrate to generate the subtilisin-substrate complex, and the docked structure was simulated by 500ns MD. The subtilisin-substrate complex structure conformation with the lowest potential energy in the simulation process was selected as the analysis conformation. The amino acid sequence of the hexapeptide substrate was Suc-AAPFAF-amide.

[0010] (2) Based on the analytical conformation obtained in step (1), the polar charges of the polar atoms N, O and H bound to each amino acid residue within 10 angstroms of the catalytic center amino acid His64 of the subtilisin are calculated by polarizing the protein-specific charge PPC force field, the absolute values ​​of the polar charges are summed, and the absolute sums are sorted from high to low. After excluding the amino acid residues that constitute the catalytic triad of the subtilisin, at least the top 10 amino acid residues with the highest absolute sums are selected as potential mutation sites of the subtilisin;

[0011] (3) Virtually single-point mutation of the potential mutation site of subtilisin obtained in step (2) to a large steric hindrance non-polar amino acid to obtain a virtual subtilisin single-point mutant, and using the PPC force field to calculate the polar charges of the polar atoms N, O and H bound to them within 10 angstroms of the catalytic center amino acid His64 of the virtual subtilisin single-point mutant, summing the absolute values ​​of the polar charges, and sorting the absolute values ​​from low to high, and selecting at least the top 10 virtual subtilisin single-point mutants with low absolute value sums, and waiting for further expression, wherein the large steric hindrance non-polar amino acids are Val, Leu, Ile, Phe, Trp and Met;

[0012] (4) Expressing the virtual subtilisin single-point mutant selected in step (3), iteratively mutating the successfully expressed mutant, expressing it again, obtaining the subtilisin mutant and verifying its hydrophobicity.

[0013] Specifically, the catalytic triad of subtilisin in the above step (2) consists of serine at position 221, histidine at position 64, and aspartic acid at position 32 of subtilisin. The key amino acids of the catalytic center of subtilisin are serine at position 221, histidine at position 64, and aspartic acid at position 32 that constitute its catalytic triad, and the mutation point excludes the key amino acids of the catalytic center.

[0014] The hydrophobicity verification step in the above step (4) is specifically to confirm whether the obtained subtilisin mutant has a crystalline water molecule within 4.5 angstroms of the catalytic center through crystal structure, and calculate the binding free energy of the mutant with the substrate Suc-AAPFAF-amide by MM / GBSA to verify the hydrophobicity of the subtilisin mutant.

[0015] The modified subtilisin mutant finally obtained by the molecular modification design method of the present invention has a crystal structure confirmation that the catalytic center of the subtilisin mutant There are no crystal water molecules in the In addition, the binding free energy of the subtilisin mutant and the original subtilisin with the substrate was calculated by MM / GBSA. The binding free energy of the subtilisin mutant with the substrate was significantly lower than that of the original protease, and the binding between the substrate and the subtilisin mutant was more "tight", excluding water molecules.

[0016] During the docking process of step (1) above, subtilisin was prepared in the Protein Preparation Wizard, the hydrogen bond network was optimized in the OPLS2005 force field, the substrate was prepared using the LigPrep panel, the catalytic triad of subtilisin was defined as the center of the grid box, and docking was performed using the precision SP mode with default parameters in the receptor rigid docking RRD protocol.

[0017] The MD simulation in the above step (1) includes the following steps:

[0018] (1) The simulation parameters are provided by the ff14SB force field of AMBER18. The truncated periodic octahedral box of TTIP3P water molecules is used as the solvent environment, and the buffer distance is Sodium ions are added as counterions;

[0019] (2) First, the solvent was optimized using 5000 steps of the steepest descent method and 5000 steps of the conjugate gradient method. The force constant constraint; secondly, the whole system was optimized using 25000 steps of the steepest descent method and 25000 steps of the conjugate gradient method without any constraints;

[0020] (3) The system is heated to 333K for 300ps and the solute is confined in The weak force constants were calculated. Three MD simulations of 500 ns were performed for each system under the NPV ensemble. Berendsen barostat and Langevin dynamics were used to control pressure and temperature, respectively. The SHAKE algorithm was used to constrain the bond lengths of all hydrogen atoms involved. The cutoff distance for long-range interactions was set to

[0021] In the above step (2), the polar charges of the polar atoms N, O and H of subtilisin are calculated using the polarized protein specific charge PPC force field, which includes the following steps:

[0022] (1) The subtilisin molecule was cleaved into amino acid fragments at the peptide bond by molecular differentiation and conjugated cap MFCC method. A pair of conjugated caps was added to each cleaved peptide bond position to complete the electronic structure. The electron density distribution of each amino acid fragment was calculated using Gaussian 16 at the B3LYP / 6-31G* level to obtain the electrostatic potential on the surface of each amino acid fragment.

[0023] (2) The restrained electrostatic potential RESP program was used to fit the atomic charges of the subtilisin molecule. The electrostatic solubilization energy and induced surface charge were obtained by solving the Poisson-Boltzmann (PB) equation. The induced surface charge and the atomic charges of other molecular fragments were used as background charges to repeatedly calculate the new charge density distribution. The solute and solvent were polarized with each other until the solubilization energy and the charge of the subtilisin were consistent.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention characterizes the binding ability of water molecules in the charge characterization region, calculates the charges of polar atoms N, O and their bound H atoms according to methods such as molecular docking and all-atom quantization calculation, characterizes the polarization ability of amino acid residues within 10 angstroms of the catalytic center, that is, the ability to bind water molecules, and virtually screens out mutant sites with potential increased hydrophobicity. After using virtual mutation to convert single-point mutations into large sterically hindered non-polar amino acids (Val, Leu, Ile, Phe, Trp, Met), mutants with weak ability to bind water molecules within 10 angstroms of the catalytic center amino acids are evaluated and screened, and the successfully expressed single-point mutants of subtilisin are selected, and then the subtilisin mutants are obtained through iterative mutation and expression;

[0026] (2) The hydrophobicity of the mutant protease was verified by the catalytic center of There are no crystal water molecules in the In addition, the binding free energy of the subtilisin mutant, the original subtilisin and the substrate was calculated by MM / GBSA. The binding free energy of the subtilisin mutant and the substrate was significantly lower than that of the original protease, and the binding between the substrate and the subtilisin mutant was more "tight", excluding water molecules.

[0027] Therefore, the subtilisin mutant obtained by this design method has high hydrophobicity, achieving the expulsion of water molecules from the catalytic center, which will be of extremely important value in drug design, materials science, biomedical engineering, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the molecular docking diagram of subtilisin and Suc-Ala-Ala-Pro-Phe-Ala-Phe-amide;

[0029] Figure 2 is the RMSD value of subtilisin during MD simulation;

[0030] Figure 3 This is a graph of the ability of amino acid residues within 10 angstroms of the catalytic center amino acid His64 of subtilisin to bind to water molecules;

[0031] Figure 4 A map of potential mutation sites within 10 angstroms of the amino acid His64 in the catalytic center of subtilisin;

[0032] Figure 5 It is the SDS-PAGE protein gel image after the purification of subtilisin and mutants;

[0033] Figure 6 A comparison of the crystal structures of local water molecules of subtilisin and its mutants. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The subtilisin of the present invention is selected from the subtilisin derived from Bacillus amyloliquefaciens.

[0036] Example 1 Molecular docking and MD simulation of Bacillus amyloliquefaciens subtilisin

[0037] 1. Search the solved crystal structure of Bacillus subtilisin (Subtilisin BPN') in the Protein Data Bank (PDB) (PDB ID: 1TO1).

[0038] 2. Use Dinger's Glide mode was docked with the hexapeptide substrate Suc-AAPFAF-amide to generate the enzyme-substrate complex structure, such as Figure 1As shown. During the docking process, proteins were prepared in Protein Preparation Wizard, hydrogen bond networks were optimized in OPLS2005 force field to alleviate potential steric clashes, hexapeptide substrates were prepared by LigPrep panel, and low-energy 3D structures were generated by tautomers, stereoisomers, and ring conformations. To predict the binding mode, the catalytic triad in the protein (serine at position 221, histidine at position 64, and aspartic acid at position 32) was defined as the center of the grid box, and docking was performed using the precision (SP) mode with default parameters in the RRD (receptor rigid docking) protocol.

[0039] 3. Further 500ns MD simulation was performed on the docked structure to obtain stable results. The structure with the lowest potential energy during the simulation was selected as the stable structure of subtilisin. The RMSD value of subtilisin during the simulation is shown in Figure 2 shown.

[0040] The steps of MD simulation are as follows:

[0041] The simulation parameters were provided by the ff14S3B force field of AMBER18. A truncated periodic octahedral box of TTIP3P water molecules was used as the solvent environment with a buffer distance of Sodium ions were added as counterions to maintain the electroneutrality of the whole system. Then, the whole system was optimized in two steps to avoid poor contact between solute and solvent molecules. First, the solvent was optimized using 5000 steps of the steepest descent method and 5000 steps of the conjugate gradient method. The whole system was then optimized using 25,000 steps of the steepest descent method and 25,000 steps of the conjugate gradient method without any constraints. Next, the system was heated to 333 K for 300 ps while the solute was confined in The weak force constants were used. Finally, the system was simulated three times for 500 ns under the NPV ensemble. Berendsen barostat and Langevin dynamics were used to control pressure and temperature, respectively. At the same time, the SHAKE algorithm was used to constrain the bond lengths of all hydrogen atoms involved, and the cutoff distance for long-range interactions was set to

[0042] Example 2 Screening of potential mutation sites within 10 angstroms of the amino acid in the catalytic center of subtilisin

[0043] Based on the stable enzyme-substrate complex structure obtained in Example 1, the charges of the polar atoms N, O and the H atoms (protons H) bound to the residues within 10 angstroms around the amino acid His64 in the catalytic center of subtilisin were calculated by the polarized protein-specific charge (PPC) force field calculated by all-atom quantum mechanics, and the binding capacity of the amino acid residues to water molecules (i.e., the sum of the absolute values ​​of the polar charges of N, O and the H atoms bound to them in the amino acid residues) was calculated using the following formula:

[0044]

[0045] Among them, C is the ability of amino acids to bind to water molecules. The higher the C, the higher the polarization degree of the amino acid, and the stronger the ability to bind to water molecules; ppc,i represents the charge of the ith atom calculated by the PPC force field; n is the total number of polar atoms (N, O, H protons (H)) in the amino acid. The calculation results are as follows Figure 3 shown.

[0046] The amino acid residues within 10 angstroms of the catalytic center amino acid His64 of subtilisin were sorted from high to low according to the absolute sum of their polarity charges, and after excluding the amino acid residues that constitute the catalytic triad of subtilisin, at least the first 10 amino acid residues with high absolute sum were selected as potential mutation sites of subtilisin, and potential mutation sites within 10 angstroms of the catalytic center amino acid His64 were virtually screened, such as Figure 4 shown.

[0047] The steps of the above PPC force field calculation are as follows:

[0048] The protein was cleaved into amino acid fragments at the peptide bonds by the molecular differentiation and conjugated cap (MFCC) method to achieve all-atom quantum mechanical calculations. A pair of conjugated caps was added at each cleaved peptide bond position to complete the electronic structure. The electron density distribution of each molecular fragment was then calculated using Gaussian 16 at the B3LYP / 6-31G* level. At the same time, the electrostatic potential on the surface of each molecular fragment was obtained. Next, the restrained electrostatic potential (RESP) program was used to fit the atomic charges of the entire protein. The electrostatic solvation energy and induced surface charge were obtained by solving the Poisson-Boltzmann (PB) equation. The induced surface charge and the atomic charges of other molecular fragments were used as background charges to repeatedly calculate the new charge density distribution. Finally, the solute and solvent were polarized to each other until the solvation energy and protein charge converged.

[0049] Example 3 Virtual screening of subtilisin single-point mutants

[0050] Based on the stable enzyme-substrate complex structure obtained in Example 1, the potential mutation sites with higher absolute values ​​screened out in Example 2 were subjected to virtual mutation, and each site was half-saturated and mutated to a large sterically hindered non-polar amino acid (Val, Leu, Ile, Phe, Trp, Met), and a virtual subtilisin single-point mutant was obtained, and a virtual single-point mutant with weaker ability to bind water molecules was selected. The virtual screening method is to use the PPC force field to calculate the water binding ability of the virtual single-point mutant within 10 angstroms of the His64 amino acid residue in the catalytic center, that is, the absolute value of the polar charge of N, O and its bound H atoms within 10 angstroms of the His64 amino acid residue in the catalytic center is added, and the virtual subtilisin single-point mutant is sorted from low to high according to the absolute value of the polar charge within 10 angstroms of the His64 amino acid residue in the catalytic center, and the virtual subtilisin single-point mutant with the lowest absolute value of the polar charge is selected as a virtual screened mutant, waiting for further expression. The ability to bind water molecules within the 10 angstroms region of the His64 amino acid residue in the catalytic center is calculated using the following formula:

[0051]

[0052] Among them, C t The ability of the His64 amino acid residue in the catalytic center to bind water molecules within the 10 angstrom region. The higher the C, the higher the degree of polarization of the region, and the stronger the ability to bind water molecules. i represents the binding ability of the ith amino acid residue in the region with water molecules, and the calculation method is the same as that in Example 2; n is the total number of amino acid residues in the region.

[0053] Example 4 Expression, purification and iterative mutagenesis of subtilisin and mutants

[0054] 1. Use Bacillus subtilis WB600 expression strain, culture in 50 mL, 2xYT medium (containing 100 μg / ml, Kanamycin, 5 mM CaCl2) (37°C, 200 rpm, 20-24 h), and collect the supernatant by centrifugation (4000g, 15 min, 4°C).

[0055] 2. Slowly add 3 times the volume of pre-cooled 95% ethanol to the supernatant, stir at 4°C for 1-2h, centrifuge (18000g, 20min, 4°C) and dry the precipitate at room temperature. Resuspend the precipitate with 10m nickel column purification balance solution (20mM PBS, 500mM NaCl, 30mM imidazole, pH=7.4), centrifuge (18000g, 20min, 4°C), retain the supernatant, use HisTrap HP1mL, affinity chromatography column for protein purification, use nickel column elution buffer (20mM PBS, 500mM NaCl, 500mM imidazole) AKTA linear gradient elution to obtain the purified protein, use 10K concentrating centrifuge tube, use buffer 20mM PBS, 500mM NaCl, pH8.0 to concentrate and centrifuge three times to remove the imidazole of the purified protein, and obtain high-concentration protein.

[0056] 3. Select a successfully expressed single-point mutant of subtilisin, perform directed mutagenesis on its recombinant plasmid to obtain an iterative mutant plasmid, and the mutation site is the other successfully expressed single-point mutation site. The iterative mutant plasmid was transferred into Bacillus subtilis WB600, expressed and purified again, and the purity was detected by SDS-PAGE. The results of subtilisin and mutant detection are as follows: Figure 5 shown.

[0057] Example 5 Comparison of crystal structures of subtilisin and mutants

[0058] Comparison of the crystal structures of water molecules in local areas of Subtilisin and mutants, such as Figure 6 As shown in the figure, two real crystal structures are shown, which can intuitively show the number of water molecules in the catalytic center during the catalytic process. The number of red spheres is the number of water molecules in the catalytic center of the aqualigase mutant during the catalytic process; the number of blue spheres is the number of water molecules in the catalytic center of the wild type during the catalytic process. Figure 6 It can be clearly observed that at a distance from the catalytic center In the range, the aqualigase mutant (red) has no water molecules present, while the wild type (blue) has three water molecules present.

[0059] The crystal structure confirmed that the mutant had a change in His64 and Ser221 at the catalytic center. There are no crystal water molecules in the In addition, this is different from the catalytic triad in wild-type subtilisin (Subtilisin BPN') The distribution of three crystal water molecules is obviously different, indicating that the mutant obtained by the design method of the present invention has higher hydrophobicity.

[0060] Example 6 Determination of binding free energy of subtilisin and mutants

[0061] The binding free energy of mutant and wild-type Subtilisin with the substrate Suc-AAPFAF-amide was calculated using the MM / GBSA method.

[0062] The MM / GBSA method can be expressed by the following formula:

[0063] △G bind =△G gas +△G solv (1)

[0064] Where △G gas and △G solv Represent gas phase energy and dissolution free energy respectively. gas It consists of electrostatic interaction and van der Waals (vdW) interaction, as follows:

[0065] △G gas =△E ele +△E vdw (2)

[0066] △G solv It contains two parts: polar and non-polar dissolution free energy, as follows:

[0067] △G solv =△G gb +△G np (3)

[0068] Polar dissolution free energy △G gb Calculated by GB module, nonpolar dissolution free energy △G np Calculated by:

[0069] △G np =γ×SASA+β(4)

[0070] SASA stands for solvent accessible surface area, which is calculated by the MSMS program. γ and β are set to and 0.00 kcal·mol -1 .

[0071] The measurement results are shown in the following table:

[0072]

[0073] The measurement results showed that the binding free energy of the mutant and wild-type Subtilisin were -33.9 kcal / mol and -8.05 kcal / mol, respectively. The binding free energy was significantly reduced by 25.8492 kcal / mol compared with the wild-type, that is, the binding of the substrate and the enzyme was more "tight", excluding water molecules. These data indicate that the mutant has relatively high hydrophobicity.

[0074] The present invention takes subtilisin (Bacillus amyloliquefaciens) as an example, based on the rational design of the binding ability of water molecules in the charge characterization region, the charges of polar atoms N, O and H atoms are calculated according to methods such as molecular docking and all-atom quantitative calculation, and the polarization ability of amino acid residues within 10 angstroms of the catalytic center amino acid His64, that is, the ability to bind water molecules, is characterized, and a mutant site that has the potential to improve hydrophobicity is obtained by virtual screening. After mutating it to a large sterically hindered non-polar amino acid (Val, Leu, Ile, Phe, Trp, Met) by virtual mutation, a virtual single-point mutant with weak ability to bind water molecules within 10 angstroms of the catalytic center amino acid His64 is evaluated and screened. The single-point mutant is expressed, mutated and expressed iteratively, and finally a mutant with high hydrophobicity in the catalytic center is obtained. The crystal structure confirms that His64 and Ser221 in the catalytic center There are no crystal water molecules in the In addition, the catalytic triad of wild-type subtilisin (Subtilisin BPN') The distribution of three crystal water molecules is obviously different, and the binding free energy of the mutant and wild-type Subtilisin with the substrate Suc-AAPFAF-amide is calculated by MM / GBSA. The binding free energy of the enzyme mutant with the substrate is significantly lower than that of the wild-type, that is, the binding of the substrate and the enzyme mutant is more "tight", excluding the water molecules, realizing the expulsion of water molecules in the catalytic center, and finally obtaining a mutant with high hydrophobicity in the catalytic center, verifying that the molecular modification design method of the present invention is reliable and effective.

[0075] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. A molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilisin, It is characterized in that The following steps are included: (1) Based on the analyzed conformation obtained in step (1), the resolved crystal structure of Bacillus subtilisin in the protein database PDB is searched by using the polarized protein-specific charge PPC force field meter. The Glide mode of dinger software was docked with the hexapeptide substrate to generate a subtilisin-substrate complex, and its docking structure was simulated by 500ns MD, and the subtilisin-substrate complex structure conformation with the lowest potential energy in the simulation process was selected as its analysis conformation, wherein the amino acid sequence of the hexapeptide substrate was Suc-AAPFAF-amide; (2) Based on the analytical conformation obtained in step (1), the polar charges of the polar atoms N, O and H bound to each amino acid residue within 10 angstroms of the catalytic center amino acid His64 of the subtilisin are calculated by polarizing the protein-specific charge PPC force field, the absolute values ​​of the polar charges are summed, and the absolute sums are sorted from high to low. After excluding the amino acid residues that constitute the catalytic triad of the subtilisin, at least the top 10 amino acid residues with the highest absolute sums are selected as potential mutation sites of the subtilisin; (3) Virtually single-point mutating the potential mutation site of subtilisin obtained in step (2) to a large steric hindrance non-polar amino acid to obtain a virtual subtilisin single-point mutant, using the PPC force field to calculate the polar charges of the polar atoms N, O and H bound to them within 10 angstroms of the catalytic center amino acid His64 of the virtual subtilisin single-point mutant, summing the absolute values ​​of the polar charges, sorting the absolute values ​​from low to high, and selecting at least the top 10 virtual subtilisin single-point mutants with the lowest absolute value sum, waiting for further expression, wherein the large steric hindrance non-polar amino acids are Val, Leu, Ile, Phe, Trp and Met; (4) Expressing the virtual subtilisin single-point mutant selected in step (3), iteratively mutating the successfully expressed mutant, expressing it again, obtaining the subtilisin mutant and verifying its hydrophobicity.

2. A molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilisin according to claim 1, It is characterized in that The catalytic triad of the subtilisin in step (2) is composed of serine at position 221, histidine at position 64, and aspartic acid at position 32 of the subtilisin.

3. A molecular modification design method for improving the hydrophobicity of the catalytic center of Bacillus subtilisin according to claim 1 or 2, It is characterized in that The hydrophobicity verification step in step (4) is specifically to confirm whether the obtained subtilisin mutant has a crystalline water molecule within 4.5 angstroms of the catalytic center through crystal structure, and calculate the binding free energy of the mutant with the substrate Suc-AAPFAF-amide by MM / GBSA to verify the hydrophobicity of the subtilisin mutant.

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