Molecular dynamics calculation-based assembled cyclic peptide immobilized protease variant

Abstract

The invention belongs to the technical field of protein engineering modification, and particularly relates to an assembled cyclopeptide immobilized protease variant based on molecular dynamics calculation. In order to improve the structure and performance of the protease, cyclopeptide covalent assembly calculation simulation is carried out on the protease structure with the distance between an N-terminal alpha amino group and a C-terminal alpha carboxyl group being between # imgabs0 #, and through molecular dynamics calculation, by taking reduction of the turning radius of the protease and the accessible area of a solvent as a benchmark, the content of the protease in the protease is reduced. And finding the position and chain length structure of the covalent binding fusion connection short polypeptide with the greatest contribution to the protease molecular stability to obtain the stably assembled cyclopeptide alkaline protease variant of # imgabs2 # with the turning radius reduced than that of the original parent protease # imgabs1 # and the accessible area reduced than that of the original parent protease # imgabs2 #.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering modification, and particularly relates to an assembled cyclic peptide-immobilized protease variant based on molecular dynamics calculation and its application in the field of washing or cleaning. Background Art

[0002] Enzymes have wide applications in the fields of biocatalysis, food, medicine, etc. However, free proteases are easily affected by environmental factors during use, resulting in poor stability and easy inactivation. Therefore, the enzyme immobilization technology emerged, combining enzymes with carriers to improve their stability and enzyme activity in complex environments. Along with the progress of genetic engineering technology, the modification technology of enzyme molecules has also attracted much attention from researchers. The emergence of new methods for immobilizing enzymes can make the functions of enzymes play better and make the production process more efficient. In traditional enzyme immobilization methods, enzymes are linked to carriers at random sites, and the common linking sites are generally lysine. Since enzymes usually contain multiple lysines, enzymes will react with carriers at many sites. However, in this immobilization method, the reaction at some sites will hinder the substrate from entering the active site of the enzyme.

[0003] In recent years, the rapid development of computational chemistry has improved the understanding of the relationship between the three-dimensional structure and function of proteins, thus having the ability to better evaluate the improvement of specific properties of proteins by changing a certain region of proteins.

[0004] Therefore, the present invention provides an assembled cyclic peptide-immobilized protease variant based on molecular dynamics calculation. By using the protein conjugation method, the enzyme and the carrier short peptide are connected at both ends of the enzyme, so that the enzyme is linked by covalent bonds formed between the carrier short peptides to achieve the purpose of immobilization. By using the protein conjugation method, the special side chains of amino acid residues are used to control the immobilization direction. The activity of the directionally immobilized enzyme is much higher than that of the randomly immobilized enzyme and plays a role in stabilizing the peptide chain in the molecule. The greater the stability of the protein molecule against the influence of external factors. Summary of the Invention

[0005] In order to improve the structure and performance of proteases, the present invention provides an assembled cyclic peptide-immobilized protease variant based on molecular dynamics calculation, by embedding two peptide chains at the N-terminus and C-terminus of the protease respectively, mediating covalent cyclization to generate a cyclic topological structure.

[0006] The present invention uses the molecular dynamics calculation method to study and evaluate the influence of introducing short peptide protein conjugation on the structural characteristics and enzyme efficacy of proteases, and can screen the best assembled cyclic peptide-immobilized protease gene sequence by means of analyzing the hydration radius, radius of gyration, and minimizing the ability of secondary structure composition of enzyme molecules through Gromacs software. Through molecular dynamics calculation, it is found that the protease used for the cyclic peptide variant has a distance between the N-terminus and the C-terminus less than The protease between them is most suitable for cyclization.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an assembled cyclic peptide-immobilized protease variant based on molecular dynamics calculation. The parental protease is selected as a protease with a distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in the natural conformation of The N-terminal domain and the C-terminal domain of the parental protease are respectively fused with a short peptide module containing a reactive group through an amino acid flexible linker, and the short peptide module forms a cyclic assembly structure through specific covalent crosslinking to achieve protease immobilization.

[0009] A short polypeptide is respectively embedded and fused in the N-terminal and C-terminal regions of the alkaline protease, and protease immobilization is achieved through specific covalent bond binding and assembly. Using the molecular dynamics calculation method to test the introduction of the alkaline protease's radius of gyration and solvent accessible surface as a reference, after immobilization, the radius of gyration of the alkaline protease is reduced compared with the original protease by The solvent accessible surface is reduced by The best covalent cyclization produces a cyclic topological structure polypeptide alkaline protease variant. Based on this, a suitable optimal cyclic peptide structure is screened from the covalently cyclized circular topological structure polypeptide alkaline protease variants for protein engineering transformation, so as to obtain a protease variant suitable for the detergent industry. Such cyclic peptide protease variants exhibit excellent thermal tolerance and inhibitor tolerance under various conditions, have good compatibility with other chemical components in detergents, and have good application prospects in the industrial field.

[0010] Furthermore, 0 to 28 amino acid positions are truncated in the N-terminal or N-terminal Loop region of the parental protease, and 0 to 28 amino acid positions are truncated in the C-terminal or C-terminal Loop region, and each is respectively fused with a polypeptide through an amino acid flexible chain, so as to achieve both protease immobilization and enzyme catalytic function;

[0011] The amino acid flexible chain is composed of glycine residues (Gly) and serine residues (Ser). Using the amino acid sequence GGGGS or GSG as the basic unit of the flexible chain, 1 to 6 flexible chain basic units are selected to combine the amino acid flexible chain for immobilization between the target protease and the polypeptide;

[0012] The amino acid chain length of the short polypeptide is 8 to 138. The short polypeptide is an amino acid polypeptide containing aspartic acid or asparagine, and is a hydrophilic short polypeptide with a hydrophilic value of the peptide chain amino acids between -0.10 and 0.60. The short polypeptide may also be an amino acid polypeptide containing lysine and glutamic acid or methionine, and is a hydrophobic short polypeptide with a hydrophilic value of the peptide chain amino acids between -0.15 and 0.36. Specific covalent bonds are formed by dehydration condensation of aspartic acid or asparagine and lysine to immobilize between two short polypeptides.

[0013] Furthermore, the parental protease is PB92 protease of Bacillus subtilis, 2709 protease of Bacillus licheniformis, subtilisin BPN' alkaline protease, Nocardiopsis sp. NRRL18262 protease or proteinase K of Candida albicans.

[0014] Still further, the radius of gyration of the protease variant is reduced compared to the original parental protease The solvent accessible surface area is reduced

[0015] The present invention also provides a preparation method of an assembled cyclic peptide protease variant, which performs computational simulation of cyclic peptide covalent assembly on a protease structure with a distance between the N-terminal α-amino group and the C-terminal α-carboxyl group within By molecular dynamics calculation, taking the reduction of the radius of gyration and the solvent accessible surface area of the protease as the benchmark, the position and chain length structure of the covalently bound fusion-linked short polypeptide that contribute the most to the molecular stability of the protease are found, and a stable assembled cyclic peptide alkaline protease variant with a reduced radius of gyration compared to the original parental protease The solvent accessible surface area is reduced is obtained.

[0016] The present invention also provides an application of the assembled cyclic peptide protease variant, which is used in the field of washing or cleaning.

[0017] The present invention also provides a plasmid, which contains a nucleic acid sequence encoding the assembled cyclic peptide protease variant.

[0018] The present invention also provides a host cell comprising the plasmid described above. It is a recombinant host cell constructed by transferring the plasmid containing the nucleic acid sequence encoding the assembled cyclic peptide immobilized protease variant into the host cell. The present invention also relates to a recombinant host cell comprising a polynucleotide encoding the assembled cyclic peptide immobilized protease variant of the present invention operably linked to one or more regulatory sequences that direct the production of the polypeptide of the present invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. The term "host cell" includes any progeny of a parental cell that differs from the parental cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source. The host cell can be any cell useful in the recombinant production of the polypeptide of the present invention, for example, a prokaryotic or eukaryotic cell. The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Marinobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The bacterial host cell can be any Bacillus cell, including, but not limited to, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0019] The method of DNA introduction into the target bacterial strain varies depending on the cell type. For example, for Bacillus cells, protoplast transformation, electroporation, or conjugation methods are commonly used; for Escherichia coli, chemical transformation (using competent cells) or electroporation is typically employed; for Streptomyces cells, protoplast transformation, electroporation, conjugation, or transduction can be used; for Pseudomonas cells, electroporation and conjugation are relatively commonly used techniques.

[0020] The classification of host cells should clearly distinguish different microbial groups, and the host cells include Gram-positive bacteria, Gram-negative bacteria, and yeast. Among them, the Gram-positive strains can be selected from Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus lentus; the Gram-negative strains mainly include Escherichia coli; and the yeast is preferably Pichia pastoris.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The stability and chemical component tolerance of the assembled cyclic peptide alkaline protease variant designed based on the molecular dynamics algorithm of the present invention are improved to varying degrees compared with the unassembled alkaline protease. Among them, without adding any protectants and stabilizers, the residual enzyme activity of the standard liquid detergent added with the assembled cyclic peptide immobilized alkaline protease variant of the present invention is higher and the stability is better, and it can be used in detergents or cleaners. Detailed implementation mode

[0023] In order to further elaborate the technical solution of the present invention, the present invention will be further described below through examples.

[0024] Example 1

[0025] Screening of assembled cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant based on molecular dynamics simulation calculation

[0026] In this example, the molecular dynamics simulation technology is used to optimize the structure of Bacillus subtilis PB92 alkaline protease (SEQ ID NO: 1) to screen out an assembled cyclic peptide immobilized variant with higher stability. In the molecular structure (PDB ID: 1AH2) of PB92 alkaline protease, the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group is First, homologous modeling is performed by calling the Modeller program through the Chimera software, and molecular dynamics simulation is performed using the GROMACS 2022.6 version. The simulation system is solvated using the TIP3P water model, and NaCl is added to simulate the physiological environment. After the steepest descent energy minimization below 1000 kJ / (mol·nm), equilibrium simulations of 500 ps are performed under the NVT and NPT ensembles respectively, and the system temperature (298.15 K) and pressure (1 bar) are gradually relaxed to the target values. The formal simulation is carried out for 300 ns, using the AMBER ff19SB force field, the V-rescale method to control the temperature, the Parrinello-Rahman method to control the pressure, and the LINCS algorithm is enabled to support a 2 fs time step. The van der Waals interaction adopts the truncation radius, and the long-range electrostatic interaction is processed by the particle mesh Ewald (PME) method, and the Fourier grid spacing is The direct space truncation radius is By analyzing classical parameters such as the backbone RMSD, residue RMSF, hydrogen bond network and free energy change, combined with conformational clustering analysis, the conformational stability of the protein is evaluated, and finally highly stable mutants are screened out, and their thermal stability is verified by experiments.

[0027] The structure of PB92 alkaline protease after being immobilized by assembled cyclic peptides derived from Bacillus subtilis was used to reduce the gyration radius of the alkaline protease. Based on the solvent-accessible area, the optimal gene sequence of the assembled cyclic peptide-immobilized alkaline protease variant of PB92 alkaline protease derived from Bacillus subtilis was calculated. The calculation results showed the C-terminal and N-terminal in the molecular structure of the truncated PB92 alkaline protease. (Here, the truncated PB92 alkaline protease refers to the PB92 alkaline protease after removing the first 12 amino acids at the N-terminal of its original amino acid sequence, and its amino acid sequence is included in the amino acid sequence from position 13 to 269 of SEQ ID NO:1). The assembled cyclic peptide-immobilized PB92 alkaline protease molecular variant, which uses the amino acid flexible short-chain linker (SEQ ID NO:2) to connect the short polypeptide (SEQ ID NO:3) to the N-terminal of the truncated PB92 alkaline protease molecule and the long polypeptide (SEQ ID NO:4) to the C-terminal of the same PB92 alkaline protease molecule, significantly improved the stability while maintaining the enzyme activity.

[0028] An assembled cyclic peptide-immobilized PB92 alkaline protease molecular variant was obtained, and its amino acid sequence is as shown in SEQ ID NO:5. The short polypeptide and the long polypeptide fused at both ends of the truncated PB92 alkaline protease molecule were assembled through the dehydration condensation of aspartic acid or asparagine and lysine to form a specific covalent bond for protease cyclization immobilization.

[0029] The gyration radius of the immobilized PB92 alkaline protease was reduced compared to the amino acids of the parental sequence of the original Bacillus subtilis PB92 alkaline protease The solvent-accessible area was reduced PB92 alkaline protease was immobilized by specific covalent bond assembly. Based on this result, the assembled cyclic peptide-immobilized Bacillus subtilis PB92 alkaline protease variant with the optimal stability was selected.

[0030] Preparation of the assembled cyclic peptide-immobilized Bacillus subtilis PB92 alkaline protease variant:

[0031] Design the upstream and downstream primers according to the FastMutagenesis System of Beijing Quanshi Jin Biotechnology Co., Ltd. Construct the recombinant plasmid by using the homologous recombination method through the restriction enzyme sites BamHI and XhoI. Take the target alkaline protease backbone protein as the design template, and design the functional region and assembly region of the backbone protein respectively. Design of the functional region: including constructing the target protease backbone protein with truncation treatment at the N-terminus for protein expression. Design of the assembly region: including constructing heterologous short polypeptides and long polypeptides, where the short polypeptide and long polypeptide are obtained by fusion PCR with an amino acid flexible chain linker to obtain a complete assembled cyclic peptide for immobilizing the 2709 alkaline protease variant gene. Select two common restriction endonucleases, BamHI and XhoI, to obtain the recombinant plasmid through homologous recombination. Send the successfully constructed plasmid to Sangon Biotech Co., Ltd. for sequencing and sequence identification. Use the recombinant plasmid pET-28a(+) of the assembled cyclic peptide immobilized alkaline mutant constructed as the template for PCR amplification. The pET-28a(+) plasmid contains the T7 / lac promoter and is generally used as a protein expression plasmid. Transfer the plasmid with successful sequencing results into B. subtilis WB600 and culture it overnight for protein expression. After induction expression and purification, finally obtain an assembled cyclic peptide immobilized alkaline protease variant with improved stability compared to the original Bacillus subtilis PB92 alkaline protease, and the amino acid sequence is as shown in SEQ ID NO:5.

[0032] Ferment and express the target recombinant strain, and collect the expressed assembled cyclic peptide immobilized alkaline protease variant, whose size is consistent with the expected result.

[0033] Determine the enzyme activities of the purified wild-type Bacillus subtilis PB92 alkaline protease and the assembled cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant respectively. The protein concentrations of Bacillus subtilis PB92 alkaline protease and the assembled cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant are 0.2 mg / mL, and the protein buffer is 50 mM Tris-HCl, 100 mM NaCl, pH = 8.0. The method for enzyme activity determination refers to the Folin method in Appendix B of GB / T23527-2009. By evaluating the influence of the assembled cyclic peptide Bacillus subtilis PB92 alkaline protease variant on the stability in aqueous solution and surfactant, it is found that the assembled cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant has excellent thermal tolerance and inhibitor tolerance stability, and the results are shown in Table 1.

[0034] Example 2

[0035] Screening of the assembled cyclic peptide immobilized Bacillus licheniformis alkaline protease 2709 variant based on molecular dynamics simulation calculation:

[0036] For the alkaline protease 2709 derived from Bacillus licheniformis, the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group is The amino acid sequence is as shown in SEQ ID NO:6. The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation are the same as those in Example 1. The gene sequence of the assembled cyclic peptide-immobilized alkaline protease variant of Bacillus licheniformis 2709 is screened. The amino acid sequence of the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant with the optimal stability is as shown in SEQ ID NO:7. The C-terminal and N-terminal in the molecular structure of the truncated Bacillus licheniformis alkaline protease 2709 (here, the truncated Bacillus licheniformis alkaline protease 2709 refers to the Bacillus licheniformis alkaline protease 2709 after removing the first 5 amino acids at the N-terminal of its original amino acid sequence, and its amino acid sequence is included from position 6 to 274 of SEQ ID NO:6) are used. The short polypeptide (SEQ ID NO:3) is linked to the N-terminal of the truncated Bacillus licheniformis alkaline protease 2709 molecule using the amino acid flexible short-chain linker (SEQ ID NO:8), and the long polypeptide (SEQ ID NO:9) is linked to the C-terminal of the same Bacillus licheniformis alkaline protease 2709 molecule. The radius of gyration of the immobilized Bacillus licheniformis 2709 alkaline protease variant is reduced compared to the amino acids of the original Bacillus licheniformis 2709 alkaline protease parental sequence The solvent-accessible area is reduced

[0037] Preparation of the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant: The same as in Example 1, the assembled cyclic peptide-immobilized 2709 alkaline protease variant was finally obtained.

[0038] The expressed assembled cyclic peptide-immobilized alkaline protease variant was collected, with a size of approximately 37 kDa, which was consistent with the expected result. Multiple protein bands appeared at 74 kDa and 63 - 110 kDa in SDS-PAGE (4B). This is due to the influence of the cyclic structure on the protein migration rate. In addition, the assembled cyclic peptide-immobilization technology will cause the cyclization of one or more Bacillus licheniformis 2709 alkaline protease variant proteins, which also leads to the appearance of multiple target bands in the gel pattern.

[0039] The enzyme activity assays of the purified wild-type alkaline protease 2709 and the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant were the same as those in Example 1. It was found that the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant has excellent thermal tolerance and inhibitor tolerance stability performance, and the results are shown in Table 1.

[0040] Example 3

[0041] Screening of Immobilized Subtilisin BPN' Alkaline Protease Variants by Cyclic Peptide Assembly Based on Molecular Dynamics Simulation Calculation

[0042] The amino acid sequence of subtilisin-derived BPN' alkaline protease is shown in SEQ ID NO: 10. The distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its molecular structure (PDBID: 1SBT) The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation are the same as in Example 1. A series of calculations were performed to find the gene sequence of the cyclic peptide-assembled immobilized alkaline protease variant of subtilisin BPN' protease molecule. The calculation results showed that the radius of gyration of the amino acid variant of immobilized subtilisin BPN' alkaline protease was reduced compared to the amino acid parent sequence of the original subtilisin BPN' alkaline protease The solvent-accessible area was reduced Based on this result, the amino acid variant of the cyclic peptide-assembled immobilized subtilisin BPN' alkaline protease with the optimal stability truncation treatment was selected, and its amino acid sequence is shown in SEQ ID NO: 11. The C-terminal and N-terminal of the truncated BPN' alkaline protease molecule structure (here, the truncated BPN' alkaline protease refers to the BPN' alkaline protease after removing the first 5 amino acids at the N-terminal of its original amino acid sequence. The truncated BPN' alkaline protease is included in the amino acid sequence of SEQ ID NO: 10 from 6 to 274). The short polypeptide (SEQ ID NO: 3) was linked to the N-terminal of the truncated BPN' alkaline protease molecule using the amino acid flexible short-chain linker (SEQ ID NO: 12), and the long polypeptide (SEQ ID NO: 13) was linked to the C-terminal of the same BPN' alkaline protease molecule

[0043] Specific covalent bond binding assembly was achieved between the short polypeptide and the long polypeptide fused at both ends of the truncated BPN' alkaline protease molecule through dehydration condensation of aspartic acid or asparagine and lysine to immobilize the protease

[0044] Preparation of cyclic peptide-assembled immobilized BPN' alkaline protease variant: The same as in Example 1, and finally the cyclic peptide-assembled immobilized BPN' alkaline protease variant was obtained. The target recombinant strain was fermented and expressed, and the expressed cyclic peptide-assembled immobilized alkaline protease variant was collected, with a size of about 41 kDa, which was consistent with the expected result

[0045] The method for evaluating the stability and measuring the enzyme activity of the mutants was the same as in Example 1. It was found that the cyclic peptide-assembled immobilized subtilisin BPN' protease variant had excellent thermal tolerance and inhibitor tolerance stability, and the results are shown in Table 1

[0046] Example 4

[0047] Molecular dynamics simulation selects the best assembled cyclic peptide-immobilized Nocardiopsis sp. NRRL18262 protease variant:

[0048] The amino acid sequence of the protease from Nocardiopsis sp. NRRL18262 is SEQ ID NO:14. The distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its molecular structure (PDBID: 2OUA) is The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation are the same as in Example 1. The gene sequence of the assembled cyclic peptide-immobilized alkaline protease variant of Nocardiopsis sp. NRRL18262 protease molecule is screened. The optimal stable structure of the truncated Nocardiopsis sp. NRRL18262 protease molecule variant gene sequence is selected. Here, the Nocardiopsis sp. NRRL18262 protease molecule (the truncated Nocardiopsis sp. NRRL18262 protease, which refers to the Nocardiopsis sp. NRRL18262 protease with the first 7 amino acids removed from the N-terminal of its original amino acid sequence, and its amino acid sequence is included in the 8th to 188th of the SEQ ID NO:14 amino acid sequence). The short polypeptide (SEQ ID NO:16) is linked to the N-terminal of the truncated Nocardiopsis sp. NRRL18262 protease molecule using the amino acid flexible short-chain linker (SEQ ID NO:15), and the long polypeptide (SEQ ID NO:17) is linked to the C-terminal of the same Nocardiopsis sp. NRRL18262 protease molecule.

[0049] The short polypeptide and the long polypeptide at both ends of the truncated Nocardiopsis sp. NRRL18262 protease molecule are covalently bonded and assembled specifically to achieve protease immobilization. The amino acid sequence of the best self-assembled cyclic peptide-immobilized Nocardiopsis sp. NRRL18262 protease molecule variant is SEQ ID NO:18.

[0050] Preparation of the assembled cyclic peptide-immobilized Nocardiopsis sp. NRRL18262 protease variant: The same as in Example 1, and finally the assembled cyclic peptide-immobilized Nocardiopsis sp. NRRL18262 protease variant is obtained.

[0051] The target recombinant strain is fermented and expressed, and the expressed assembled cyclic peptide-immobilized alkaline protease variant is collected, with a size of about 34 kDa, which is consistent with the expected result.

[0052] The method for evaluating the stability and measuring the enzyme activity of the mutant is the same as in Example 1. It is found that the assembled cyclic peptide-immobilized Nocardiopsis sp. NRRL18262 protease variant has excellent thermal tolerance and inhibitor tolerance stability. The results are shown in Table 1.

[0053] Example 5

[0054] Selection of the best assembled cyclic peptide-immobilized variant of proteinase K from Tritirachium album limber by molecular dynamics simulation

[0055] The amino acid sequence of proteinase K from Tritirachium album limber is shown in SEQ ID NO:19. The distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its molecular structure (PDB ID: 2ID8) The software, parameters and screening criteria for homology modeling and molecular dynamics simulation are the same as those in Example 1. The best gene sequence of the assembled cyclic peptide-immobilized proteinase K variant derived from proteinase K of Tritirachium album limber was calculated. The calculation results showed the molecular structure of the truncated proteinase K (here, the truncated proteinase K of Tritirachium album limber refers to the proteinase K of Tritirachium album limber with the first 6 amino acids removed from the N-terminal of its original amino acid sequence, and its amino acid sequence is included in the amino acid sequence of SEQ ID NO:19 from positions 7 to 279). The short polypeptide (SEQ ID NO:20) was linked to the N-terminal of the truncated proteinase K molecule using the amino acid flexible short-chain linker (SEQ ID NO:15), and the long polypeptide (SEQ ID NO:21) was linked to the C-terminal of the same proteinase K molecule.

[0056] The short polypeptide and the long polypeptide at both ends of the truncated proteinase K molecule of Tritirachium album limber were assembled by specific covalent bond binding to achieve protease immobilization. An assembled cyclic peptide-immobilized variant of proteinase K molecule of Tritirachium album limber was obtained, and its amino acid sequence is shown in SEQ ID NO:22.

[0057] Preparation of the assembled cyclic peptide-immobilized variant of proteinase K from Tritirachium album limber: Same as in Example 1, and finally the assembled cyclic peptide-immobilized variant of proteinase K from Tritirachium album limber was obtained. The target recombinant strain was fermented and expressed, and then SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the molecular weight of the purified assembled cyclic peptide-immobilized protease. The size was about 42 kDa, which was consistent with the expected result, and a protein band appeared at 84 kDa in SDS-PAGE (4B).

[0058] The purified proteinase K from Tritirachium album limber and the assembled cyclic peptide-immobilized variant of proteinase K from Tritirachium album limber were respectively assayed for enzyme activity in the same manner as in Example 1. It was found that the assembled cyclic peptide-immobilized variant of proteinase K from Tritirachium album limber had excellent thermal tolerance and inhibitor tolerance stability. The results are shown in Table 1.

[0059] Example 6

[0060] Screening of assembled cyclic peptide-immobilized variant of Bacillus subtilis PB92 alkaline protease based on molecular dynamics simulation calculation

[0061] The amino acid sequence of the PB92 alkaline protease derived from Bacillus subtilis is shown in SEQ ID NO:1. The distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its amino acid molecular structure (PDBID: 1AH2) is The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation are the same as those in Example 1. The best gene sequence for the cyclic peptide immobilized alkaline protease variant of PB92 alkaline protease derived from Bacillus subtilis was calculated. The calculation results show that the C-terminal and N-terminal in the molecular structure of the truncated PB92 alkaline protease (here, the truncated PB92 alkaline protease refers to the PB92 alkaline protease after removing the last 22 amino acids at the C-terminal of its original amino acid sequence, and its amino acid sequence is included in the amino acid sequence of SEQ ID NO:1 from position 1 to 247). A short polypeptide (SEQ ID NO:3) was connected to the N-terminal of the truncated PB92 alkaline protease molecule using an amino acid flexible short-chain linker (SEQ ID NO:2), and a long polypeptide (SEQ ID NO:4) was connected to the C-terminal of the same PB92 alkaline protease molecule. The cyclic peptide immobilized PB92 alkaline protease molecular variant significantly improved stability while maintaining enzyme activity.

[0062] The cyclic peptide immobilized PB92 alkaline protease molecular variant was obtained. The amino acid sequence is shown in SEQ ID NO:23. The short polypeptide and the long polypeptide at both ends of the truncated PB92 alkaline protease molecule were assembled by specific covalent bond binding to achieve protease immobilization.

[0063] The obtained immobilized PB92 alkaline protease had a reduced radius of gyration compared to the amino acids of the parental sequence of the PB92 alkaline protease of the original Bacillus subtilis The solvent accessible area decreased PB92 alkaline protease was immobilized by specific covalent bond binding and assembly. Based on this result, the cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant with the best stability was selected.

[0064] Preparation of the cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant: The same as in Example 1, and finally the cyclic peptide immobilized Bacillus subtilis PB92 alkaline protease variant was obtained.

[0065] The target recombinant strain was fermented and expressed, and the molecular weight of the purified cyclic peptide immobilized protease was detected, and the size was consistent with the expected result.

[0066] The purified Bacillus subtilis PB92 alkaline protease and the assembled cyclic peptide-immobilized Bacillus subtilis PB92 alkaline protease variant were respectively subjected to the same enzyme activity assay as in Example 1, and it was found that the assembled cyclic peptide-immobilized Bacillus subtilis PB92 alkaline protease variant had excellent thermal tolerance and inhibitor tolerance stability performance, and the results are shown in Table 1.

[0067] Example 7

[0068] Screening of assembled cyclic peptide-immobilized Bacillus licheniformis alkaline protease 2709 variant based on molecular dynamics simulation

[0069] Derived from Bacillus licheniformis alkaline protease 2709, in which the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group is The amino acid sequence is as shown in SEQ ID NO:6, and computational simulation was performed. The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation were the same as in Example 1, and the amino acid sequence of the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant with the best stability was selected as shown in SEQ ID NO:24. The C-terminal and N-terminal of the truncated Bacillus licheniformis alkaline protease 2709 molecular structure (here, the truncated Bacillus licheniformis alkaline protease 2709 refers to Bacillus licheniformis alkaline protease 2709 after removing the last 22 amino acids at the C-terminal of its original amino acid sequence, and its amino acid sequence is included in positions 1 to 252 of SEQ ID NO:6), using the amino acid flexible short-chain linker (SEQ ID NO:8) to connect the short polypeptide (SEQ ID NO:3) to the N-terminal of the truncated Bacillus licheniformis alkaline protease 2709 molecule and the long polypeptide (SEQ ID NO:9) to the C-terminal of the same Bacillus licheniformis alkaline protease 2709 molecule. The radius of gyration of the amino acids of the immobilized Bacillus licheniformis 2709 alkaline protease variant was reduced compared to the original Bacillus licheniformis 2709 alkaline protease parental sequence The solvent accessible area was reduced

[0070] Preparation of assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant: The same as in Example 1, and finally the assembled cyclic peptide-immobilized Bacillus licheniformis 2709 alkaline protease variant was obtained.

[0071] The target recombinant strain was fermented and expressed, and the molecular weight of the purified assembled cyclic peptide-immobilized protease was detected, which was consistent with the expected result.

[0072] The purified Bacillus licheniformis 2709 alkaline protease and the assembled cyclic peptide immobilized Bacillus licheniformis 2709 alkaline protease variant were respectively subjected to the same enzyme activity assay as in Example 1, and it was found that the assembled cyclic peptide immobilized Bacillus licheniformis 2709 alkaline protease variant had excellent thermal tolerance and inhibitor tolerance stability performance, and the results are shown in Table 1.

[0073] Example 8

[0074] Screening of Assembled Cyclic Peptide Immobilized Subtilisin BPN' Alkaline Protease Variant Based on Molecular Dynamics Simulation Calculation

[0075] The amino acid sequence of BPN' alkaline protease derived from subtilisin is shown in SEQ ID NO: 10, and the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its molecular structure (PDBID: 1SBT) The software, parameters and screening criteria for homology modeling and molecular dynamics simulation are the same as in Example 1. The gene sequence of the assembled cyclic peptide immobilized alkaline protease variant of the BPN' alkaline protease molecule of subtilisin was searched for a series of calculations. The calculation results showed that the amino acid variant of the BPN' alkaline protease of subtilisin after immobilization had a reduced radius of gyration compared to the amino acid parent sequence of the original BPN' alkaline protease of subtilisin The solvent accessible area decreased Based on this result, the amino acid variant of the assembled cyclic peptide immobilized subtilisin BPN' alkaline protease with the optimal stability was selected. The C-terminal and N-terminal of the truncated BPN' alkaline protease molecule structure (here the truncated BPN' alkaline protease refers to the BPN' alkaline protease after removing the last 23 amino acids at the C-terminal of its original amino acid sequence, and the truncated BPN' alkaline protease is included in the amino acid sequence of SEQ ID NO: 10 from 1 to 252). The short polypeptide (SEQ ID NO: 3) was connected to the N-terminal of the truncated BPN' alkaline protease molecule using the amino acid flexible short-chain linker (SEQ ID NO: 12), and the long polypeptide (SEQ ID NO: 13) was connected to the C-terminal of the same BPN' alkaline protease molecule.

[0076] The protease was immobilized by specific covalent bond binding and assembly between the short polypeptide and the long polypeptide at both ends of the truncated BPN' alkaline protease molecule. The assembled cyclic peptide immobilized BPN' alkaline protease molecule variant, SEQ IDNO: 25, was obtained.

[0077] Preparation of assembled cyclic peptide immobilized BPN' alkaline protease variant: The same as in Example 1, and finally the assembled cyclic peptide immobilized BPN' alkaline protease variant was obtained.

[0078] The target recombinant strain was fermented and expressed, and the expressed assembled cyclic peptide immobilized alkaline protease variant was collected, and a protein band appeared at a size of about 39 kDa. The purified wild-type subtilisin BPN' alkaline protease and the assembled cyclic peptide immobilized subtilisin BPN' alkaline protease variant were respectively subjected to the same enzyme activity assay as in Example 1, and it was found that the assembled cyclic peptide immobilized subtilisin BPN' alkaline protease variant had excellent thermal tolerance and inhibitor tolerance stability, and the results are shown in Table 1.

[0079] Example 9

[0080] Molecular dynamics simulation selected the best assembled cyclic peptide to immobilize the protease variant of Nocardia spp. NRRL18262:

[0081] The amino acid sequence of the protease derived from Nocardia spp. NRRL18262 is SEQ ID NO: 14. In its molecular structure (PDBID: 2OUA), the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group is The software, parameters and screening criteria for homology modeling and molecular dynamics simulation are the same as those in Example 1, and the assembled cyclic peptide immobilized alkaline protease variant gene sequence of the Nocardia NRRL18262 protease molecule is screened. The optimal stability Nocardia NRRL18262 protease molecule variant gene sequence structure is selected, and the short polypeptide (SEQ ID NO: 16) is connected to the N-terminus of the Nocardia NRRL18262 protease molecule using an amino acid flexible short chain linker (SEQ ID NO: 15), and the long polypeptide (SEQ ID NO: 17) is connected to the C-terminus of the same Nocardia NRRL18262 protease molecule.

[0082] The short polypeptide and the long polypeptide at both ends of the Nocardiopsis NRRL18262 protease molecule are assembled by specific covalent bonds to achieve protease immobilization. The amino acid sequence of the best self-assembling cyclic peptide immobilized Nocardiopsis NRRL18262 protease molecule variant is SEQ ID NO: 26.

[0083] Preparation of assembled cyclic peptide-immobilized protease variant of Nocardiopsis NRRL18262: The assembled cyclic peptide-immobilized protease variant of Nocardiopsis NRRL18262 was finally obtained as in Example 1. The target recombinant strain was fermented and expressed, and the expressed assembled cyclic peptide-immobilized alkaline protease variant was collected, and a protein band appeared at a size of about 34 kDa.

[0084] The purified wild-type Nocardiopsis prasina NRRL18262 protease and the assembled cyclic peptide-immobilized Nocardiopsis prasina NRRL18262 protease variant were respectively subjected to the same enzyme activity assay as in Example 1, and it was found that the assembled cyclic peptide-immobilized Nocardiopsis prasina NRRL18262 protease variant had excellent thermal tolerance and inhibitor tolerance stability performance. (The results are shown in Table 1).

[0085] Example 10

[0086] Molecular dynamics simulation selects the best assembled cyclic peptide-immobilized Candida albicans protease K variant

[0087] The amino acid sequence of Candida albicans protease K is shown in SEQ ID NO: 19, and the distance between the N-terminal α-amino group and the C-terminal α-carboxyl group in its molecular structure (PDBID: 2ID8) The software, parameters, and screening criteria for homology modeling and molecular dynamics simulation are the same as in Example 1, and the best gene sequence calculation for the assembled cyclic peptide-immobilized protease K variant derived from Candida albicans protease K is searched. The calculation results show that the amino acid flexible short-chain linker (SEQ ID NO: 15) is used in the molecular structure of protease K to connect the short polypeptide (SEQ ID NO: 20) to the N-terminus of the protease K molecule and the long polypeptide (SEQ ID NO: 21) to the C-terminus of the same protease K molecule.

[0088] The short polypeptide and the long polypeptide at both ends of the Candida albicans protease K molecule are assembled by specific covalent bonds to achieve protease immobilization. An assembled cyclic peptide-immobilized Candida albicans protease K molecular variant is obtained, and the amino acid sequence is shown in SEQ ID NO: 27.

[0089] The protease variant sequence of Candida albicans protease K variant was used to prepare protease variants in the same way as in Example 1, and the target recombinant strain was obtained. The target recombinant strain was fermented and expressed, and the molecular weight of the purified assembled cyclic peptide-immobilized protease was detected. The size was about 42 kDa, which was consistent with the expected result, and a protein band appeared at 84 kDa in SDS-PAGE (4B).

[0090] The purified Candida albicans protease K and the assembled cyclic peptide-immobilized Candida albicans protease K variant were respectively subjected to the same enzyme activity assay method as in Example 1, and it was found that the assembled cyclic peptide-immobilized Candida albicans protease K variant had excellent thermal tolerance and inhibitor tolerance stability performance. (The results are shown in Table 1).

[0091] Table 1 Comparison of enzyme activity and stability before and after immobilization (50 °C / 30 minutes)

[0092]

[0093]

[0094] Description:

[0095] The "initial enzyme activity (%)" of each sample is based on the activity of the wild-type enzyme before treatment and is set to 100%. The "residual activity after treatment (%)" represents the percentage of enzyme activity measured after heat treatment or treatment with a detergent under specified temperature and time conditions.

Claims

1. A molecular dynamics simulation-based assembled cyclic peptide immobilized protease variant, characterized in that: The parent protease of the protease variant is selected from a protease having a distance between the N-terminal α amino group and the C-terminal α carboxyl group of The N-terminal domain and the C-terminal domain of the parent protease are respectively fused with a short peptide module containing a reactive group through an amino acid flexible connector, and the short peptide module forms a ring assembly structure through specific covalent cross-linking to achieve protease immobilization.

2. The assembled cyclic peptide immobilized protease variant based on molecular dynamics calculation according to claim 1, characterized in that: The N-terminus or the N-terminal Loop domain of the parent protease is truncated at 0 to 28 amino acid positions, and the C-terminus or the C-terminal Loop domain is truncated at 0 to 28 amino acid positions, and each is connected and fused to a short polypeptide through an amino acid flexible chain; The amino acid flexible chain is composed of glycine and serine, with the amino acid sequence GGGGS or GSG as the flexible chain basic unit, and 1 to 6 flexible chain basic units are selected to assemble the amino acid flexible chain; The amino acid chain length of the short polypeptide is 8 to 138, the short polypeptide is an amino acid polypeptide containing aspartic acid or asparagine, the peptide chain amino acid sum hydrophilicity value is a hydrophilic short polypeptide between -0.10 and 0.60, the short polypeptide is or an amino acid polypeptide containing lysine and glutamic acid or methionine, the peptide chain amino acid sum hydrophilicity value is a hydrophobic short polypeptide between -0.15 and -0.36, and the hydrophilicity value is calculated using the Kyte-Doolittle method. The specific covalent bond immobilized protease is formed by dehydration condensation of aspartic acid or asparagine and lysine between two short polypeptides.

3. The assembled cyclic peptide immobilized protease variant based on molecular dynamics calculation according to claim 1, characterized in that: The parent protease is PB92 protease of Bacillus subtilis, 2709 protease of Bacillus licheniformis, Bacillus subtilisin BPN' alkaline protease, Nocardia spp. NRRL18262 protease or Candida albicans protease K.

4. The assembled cyclic peptide immobilized protease variant based on molecular dynamics calculation according to any one of claims 1 to 3, characterized in that: The protease variant has a reduced radius of gyration compared to the original parent protease Reduced solvent accessible area 5. A method for preparing the assembled cyclic peptide protease variant according to any one of claims 1 to 4, characterized in that: The distance between the N-terminal α amino group and the C-terminal α carboxyl group is The protease structure between the two was simulated by cyclic peptide covalent assembly. Molecular dynamics calculations were used to find the position and chain length structure of the covalently bound fusion-linked short polypeptide that contributed most to the stability of the protease molecule, based on the reduction of the protease's gyration radius and solvent accessible area. The gyration radius of the original parent protease was reduced. Reduced solvent accessible area Stable assembly of cyclic peptide alkaline protease variants.

6. A use of the assembled cyclic peptide protease variant according to any one of claims 1 to 4, characterized in that: Used in washing or cleaning areas.

7. A plasmid, characterized in that The invention comprises a nucleic acid sequence encoding the assembled cyclic peptide immobilized protease variant according to any one of claims 1 to 4.

8. A host cell, characterized in that Comprising the plasmid as claimed in claim 7.

9. The host cell according to claim 8, characterized in that This includes Gram-positive or Gram-negative bacteria or yeast.

10. The host cell according to claim 9, characterized in that The Gram-positive bacteria are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus lentus; the Gram-negative bacteria are Escherichia coli; and the yeast is Pichia pastoris.

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