A method for improving the activity and thermal stability of lipase and mutant thereof

Abstract

The application provides a molecular modification design method for improving lipase activity and thermal stability and a mutant thereof. The method takes Bacillus subtilis lipase A (BSLA) as a modification object, identifies key region sites with enhanced flexibility and related to catalytic function under high-temperature conditions through molecular dynamics simulation and residue root mean square fluctuation analysis, screens candidate sites favorable to enhancing global skeleton rigidity through FireProt, FoldX folding free energy change, B-factor, ML pred. score, Conservation score and spatial structure position, then adopts NNK degenerate codon for site-directed saturation mutation and discontinuous multi-point simultaneous saturation mutation, constructs a combinatorial mutant library, and adopts a microplate screening method based on a spectrophotometric method to screen an excellent mutant with synchronous improvement of activity and thermal stability, wherein the thermal half-life and catalytic activity of the BSLA mutant of the application are improved by 94.52 times and 4.35 times, respectively, compared with those of the wild type, and it is proved that the application can realize a high-performance lipase mutant.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and protein engineering technology, specifically relating to a method for molecular modification and design of lipases based on molecular dynamics simulation, dynamic correlation analysis, sequence conservation analysis, thermal stability prediction and combinatorial mutation screening, and particularly relating to a method for simultaneously improving lipase activity and stability by maintaining flexibility in key regions and rigidifying the entire backbone. Background Technology

[0002] Lipases (EC3.1.1.3) belong to the serine hydrolases and typically possess a typical α / β hydrolase fold structure. They rely on a catalytic triad composed of Ser, Asp, and His residues to complete catalytic reactions. Lipases can catalyze a variety of reactions, including hydrolysis, esterification, and transesterification, and have been widely used in food processing, pharmaceutical intermediate synthesis, fine chemical preparation, and other industrial biocatalytic processes.

[0003] In practical industrial applications, increasing reaction temperature can enhance substrate solubility, reduce system viscosity, and improve mass transfer efficiency. However, most natural enzymes are prone to conformational perturbations, structural unfolding, or inactivation at higher temperatures or in organic solvent environments. Although protein engineering techniques such as rational design, semi-rational design, or directed evolution can improve enzyme stability, excessively enhancing the overall rigidity of the protein often limits conformational changes in catalytically relevant regions, leading to decreased enzyme activity and forming an "activity-stability tradeoff."

[0004] Previous studies have explored ways to improve the thermal stability of lipases by modifying flexible regions. Ahmad et al. (Protein Science, 2012, 21(3), 433-446) performed saturation mutations on 86 flexible sites of BSLA, and after screening approximately 16,000 clones, only 17 stable mutants were obtained, with a positive rate of approximately 0.11%, and their melting temperature increased by only 1-6℃. Jones et al. (Biochemistry, 2017, 56(50), 6521-6532) found that, using the α / β hydrolase SABP2 as a model enzyme, only 4 of the 13 mutations predicted by Rosetta and FoldX showed improved stability, and the catalytic efficiency of some of the more stable mutants decreased by about 80%, indicating that a single computational parameter is difficult to balance stability and catalytic activity.

[0005] Guan et al. (CN106047838B) designed five groups of disulfide bond mutations in the Yersinia lipase Lip2. Three of these groups exhibited problems such as failure to form disulfide bonds, decreased thermal stability, or impaired catalytic activity, while only two groups balanced stability and activity. Their half-life at 50℃ increased from 1.65 min to 13.61 min and 12.57 min, respectively, but the catalytic efficiency only improved by 4.0% and 12.8%. Therefore, existing methods still suffer from low screening efficiency, limited predictive accuracy, and excessive rigidity that damages catalytic dynamics. A modification method to simultaneously improve lipase activity and stability is needed.

[0006] The purpose of this invention is to provide a molecular modification design method that can simultaneously improve the thermal stability and catalytic activity of lipases. This method identifies key flexible regions related to catalysis through molecular dynamics simulations, and screens for mutation sites that can maintain the flexibility of these key regions through dynamic correlation analysis and conservation analysis. Simultaneously, it utilizes FireProt, FoldX, B-factor, and conservation analysis to screen for candidate mutation sites that are beneficial to the rigidification of the entire scaffold. Finally, it obtains lipase mutants with both high activity and high thermal stability through saturation mutations and combinatorial mutation screening. Summary of the Invention

[0007] This invention provides a molecular modification design method and its mutants for improving lipase activity and thermal stability, comprising the following steps:

[0008] 1. Structure acquisition and preprocessing: Bacillus subtilis lipase A was selected as the target for modification. Its crystal structure was obtained from a protein database, with PDB ID 1I6W being preferred. Unnecessary ligands and water molecules were removed from the crystal structure to obtain the initial structure for subsequent simulation and computational analysis.

[0009] 2. Molecular Dynamics Simulation and Identification of Key Flexible Regions: Molecular dynamics simulations were performed on the pretreated BSLA structure at different temperatures for hundreds of nanoseconds. RMSF analysis was used to analyze residue fluctuations under high-temperature conditions. Combined with the spatial positions of the catalytic triplet Ser77, Asp133, and His156, key flexible regions related to catalytic function were identified.

[0010] 3. Screening of flexible maintenance sites in key regions: Dynamic correlation matrix analysis was performed on the key flexible regions identified in step 2 to screen residues that have strong dynamic coupling with the key flexible regions; highly conserved residues were eliminated based on the ConSurf analysis results; catalytic triples Ser77, Asp133 and His156 were excluded to obtain candidate sites for flexible maintenance of key regions.

[0011] 4. Screening of sites for global skeleton rigidity: FireProt online server was used to predict whole-enzyme-scale stabilization mutations in BSLA; further, combined with FoldX-predicted folding free energy change ΔΔG, conservation score and spatial structural location of mutation sites, candidate single-point mutations that can enhance global skeleton rigidity without directly interfering with the catalytic center were screened.

[0012] 5. Single-strategy validation and template mutant screening: NNK site-directed saturation mutagenesis or site-directed mutagenesis were performed on candidate sites for flexible maintenance of key regions and candidate sites for rigidification of the whole-domain skeleton, respectively, to construct a single mutant library and perform expression screening.

[0013] 6. Dual-strategy synergistic combination mutation: Using the optimal single-site mutant as a template, potential sites are introduced and saturated with mutations to obtain double mutants. Then, based on the optimal double mutant, discontinuous multi-site simultaneous saturation mutations are performed on the remaining potential sites to construct a combination mutant library containing triple, quadruple, and quintuple mutants.

[0014] 7. Mutant expression and performance screening: The mutant plasmid was transformed into the expression host, and the enzyme solution was obtained after induction of expression. The relative enzyme activity and residual enzyme activity after heat treatment were determined by the p-nitrophenyl palmitate (pNPP) colorimetric method, and the thermal half-life was further determined. Finally, the preferred mutants with simultaneous improvement in activity and stability were screened.

[0015] In a preferred embodiment of the present invention, the preferred mutant obtained by the synergistic combination mutation is M4, which contains A15K / F17E / A132D / R142Q mutations.

[0016] In a preferred embodiment of the present invention, the residual activity of the M4 mutant after heat treatment is 21.37 times higher than that of the wild type; the catalytic activity is 4.35 times higher than that of the wild type; and the half-life is 94.52 times higher than that of the wild type.

[0017] Beneficial effects

[0018] (1) This invention combines maintaining the flexibility of key regions with rigidifying the entire framework, thus avoiding the problem of reduced catalytic activity caused by simply enhancing the rigidity of enzyme molecules.

[0019] (2) This invention uses multi-dimensional information such as RMSF, DCCM, ConSurf, FireProt, FoldX, B-factor and structural position analysis to screen mutation sites, thereby improving the rationality and success rate of candidate mutation site screening.

[0020] (3) The present invention uses discontinuous multi-point simultaneous saturation mutations to construct a combined mutant library, which can expand the sequence search space and avoid missing advantageous combinations due to simple superposition of single-point mutations.

[0021] (4) The preferred mutant M4 obtained by this invention has improved thermal stability, catalytic activity and organic solvent tolerance compared with the wild type, which can provide a feasible solution for the development of high-performance lipases.

[0022] (5) The molecular modification process established in this invention has strong versatility and can provide a reference for the synergistic improvement of stability and activity of other lipases or industrial enzymes with well-defined structures. Attached Figure Description

[0023] Figure 1 A flowchart of a molecular modification design to improve lipase activity and thermal stability;

[0024] Figure 2 : RMSF analysis and key flexible region identification results of BSLA under different temperature conditions;

[0025] Figure 3 Results of mutant screening based on the flexible maintenance strategy in key regions;

[0026] Figure 4 Comparison of relative enzyme activity and residual enzyme activity of WT and mutants M1-M4;

[0027] Figure 5 Comparison of relative enzyme activity and residual enzyme activity of WT and mutant M4 in 50mL culture system. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Experimental methods not specified in the embodiments can be performed according to conventional methods in the art or reagent instructions.

[0029] Example 1: BSLA Structure Acquisition and Molecular Dynamics Simulation

[0030] BSLA was selected as the target for modification. The BSLA encoding gene with a 6×His tag at the C-terminus was used as the original enzyme gene and fused into the recombinant expression vector pET-28a(+). The amino acid sequence of wild-type BSLA is shown in SEQ ID NO.1, and the encoding nucleotide sequence is shown in SEQ ID NO.2.

[0031] The BSLA crystal structure was obtained from the RCSB Protein Data Bank database. The PDB ID is 1I6W, and the resolution is [missing information]. After removing inherent ligands and unnecessary water molecules from the crystal structure, molecular dynamics simulations were performed using GROMACS software. The preferred protein force field was AMBER14SB_parmbsc1, and the preferred water model was TIP3P.

[0032] After energy minimization, NPT pre-equilibration, and production simulation, the simulation system yielded trajectory files for subsequent analysis. Simulations were performed for hundreds of nanoseconds at different temperatures. RMSF analysis was conducted on the equilibrated trajectory, and key flexible regions were identified based on RMSF variations.

[0033] Example 2: Screening of Flexible Maintenance Sites in Key Regions

[0034] Based on the RMSF results obtained in Example 1, key flexible regions in BSLA that are enhanced in flexibility under high temperature conditions and are spatially related to the catalytic center were identified.

[0035] Further analysis of the dynamic coupling relationship between residues was conducted using a dynamic correlation matrix to screen residues that showed strong positive or negative correlations with key flexible regions. The sequence conservation of candidate residues was analyzed using the ConSurf online server, and amino acid sites with low conservation were retained. At the same time, the catalytic triplet Ser77, Asp133 and His156 were excluded.

[0036] Based on the above screening criteria, candidate sites for flexible maintenance in key regions were obtained. Figure 2 For candidate sites, saturation mutant primers were designed using NNK degenerate codons (Table 4), and a site-directed saturation mutant library was constructed. The results are shown in Table 1 and [other tables]. Figure 3 As shown.

[0037] Table 1: Saturation mutagenesis results of candidate sites identified by the key region flexibility maintenance strategy

[0038]

[0039] Example 3: Screening of sites for global skeleton rigidification

[0040] The BSLA crystal structure was uploaded to the FireProt online server for whole-enzyme-scale stabilization mutation prediction; then, FoldX was used to calculate the folding free energy change ΔΔG of candidate mutations. Candidate mutations with ΔΔG < 0, high B-factor, high MLpred. score, and moderate conservation score were preferentially selected.

[0041] Candidate mutation sites also need to be manually verified in conjunction with their spatial structural location to exclude mutations that are directly located near the catalytic triplet Ser77, Asp133, and His156 and may interfere with substrate binding or the catalytic process. A total of 11 candidate mutations were obtained based on the above criteria (Table 2).

[0042] Table 2: Candidate stable mutation sites for BSLA selected based on the global skeleton rigidification strategy

[0043]

[0044] Candidate mutations were expressed and their thermal stability was verified. The results are shown in Table 3. The single-point mutant with the best thermal stability was selected and used as the template for subsequent dual-strategy synergistic combination mutations.

[0045] Table 3: Activity and thermal stability of BSLA single-point mutants identified based on global scaffold stiffening strategy

[0046]

[0047] Example 4: Construction and Screening of Combinatorial Mutant Library

[0048] Using the single-site mutant with the best thermal stability as a template, saturation mutations were performed on potential sites identified through a flexible maintenance strategy in key regions to construct a dual mutant library. After screening for activity and thermal stability, dual mutants with superior performance were obtained (Table 7).

[0049] Based on the optimal double mutant, other potential sites were further selected for discontinuous multi-site simultaneous saturation mutations to construct combined mutant libraries containing triple, quadruple, and quintuple mutants. After sequencing validation, the mutants were subjected to recombinant expression and initial screening.

[0050] Table 8 and Figure 4 Screening results showed that the preferred mutant M4 contained the A15K / F17E / A132D / R142Q mutation. Under scale-up verification conditions, after heat treatment, the thermal stability of M4 was increased by 21.37 times compared to the wild type, and the catalytic activity was increased by 4.35 times compared to the wild type. Figure 5 ).

[0051] Table 4: Primer Design Used for Mutant Construction

[0052]

[0053]

[0054] Table 5: Mutant amplification system (50 μL)

[0055]

[0056] Table 6: Thermal cycling conditions for PCR amplification

[0057]

[0058] Table 7: Screening results of BSLA double mutants

[0059]

[0060] Table 8: Screening results of BSLA combinatorial mutants

[0061]

[0062] Example 5: Mutant Expression and Enzymatic Properties Determination

[0063] The correctly sequenced wild-type and mutant plasmids were transformed into E. coli expression hosts, and positive single clones were selected for liquid culture. Cultured to OD... 600 IPTG was added to a final concentration of 0.1 mM when the bacterial culture temperature was between 0.6 and 0.8, and the expression was induced at 18°C ​​for 24 hours. After induction, the bacterial cells were collected, resuspended in buffer, sonicated, and centrifuged to obtain the enzyme solution.

[0064] Lipase activity was determined using a p-nitrophenol substrate colorimetric method. Enzyme activity units were defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute under the assay conditions. Thermal stability screening included determination of relative enzyme activity, residual enzyme activity after heat treatment, and thermal deactivation kinetic parameters.

[0065] The results are shown in Tables 9 and 10. The thermal half-life of the M4 mutant was extended from 3.78 min to 357.30 min, an increase of 94.52 times; K m The value decreased from 1.285 mM to 0.301 mM; k cat Increased by 1.51 times.

[0066] Table 9: Thermal half-life parameters of wild-type and its mutants at 52℃

[0067]

[0068] Table 10: Enzyme catalytic kinetic parameters of wild type and its mutant M4

[0069]

[0070] SEQ ID NO.1: Wild-type Bacillus subtilis lipase A amino acid sequence:

[0071]

[0072] SEQ ID NO.2: Nucleotide sequence encoding wild-type Bacillus subtilis lipase A:

[0073]

[0074] SEQ ID NO.3: M4 mutant amino acid sequence:

[0075] SEQ ID NO.4: The nucleotide sequence encoded by the M4 mutant:

[0076]

[0077] The above-mentioned experiments demonstrate that this invention, based on a rational design strategy of co-evolution of key region flexibility maintenance and global framework rigidity, molecularly modifies BSLA, ultimately obtaining the mutant M4 (A15K / F17E / A132D / R142Q). Compared with wild-type BSLA, mutant M4 exhibits a 4.35-fold increase in catalytic activity and a 94.52-fold increase in half-life, proving that this method can effectively overcome the enzyme's "activity-stability trade-off" and has good industrial application potential, applicable to food processing, pharmaceutical synthesis, and fine chemicals.

[0078] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. A molecular modification design method for improving lipase activity and thermal stability, and its mutants, characterized in that, Includes the following steps: (1) Bacillus subtilis lipase A was selected as the target for modification. The crystal structure and amino acid sequence of the lipase to be modified were obtained from the protein database. (2) Perform molecular dynamics simulation on the crystal structure obtained in step (1), identify the key regions for enhanced flexibility under high temperature conditions based on the root mean square fluctuations of residues in the simulation trajectory, and determine the key flexible regions related to catalytic function by combining the spatial position of the catalytic triplet. (3) For the key flexible regions identified in step (2), screen the key region flexibility maintenance sites by combining dynamic correlation matrix and ConSurf conservation analysis. The sites satisfy the following conditions: they are dynamically coupled with the key flexible regions, have low or moderate conservation, and do not contain catalytic triple residues. (4) Based on the overall structure obtained in step (1), FireProt, FoldX, B-factor, ML pred. score, Conservation score and spatial structural location of mutation sites are used to screen candidate mutation sites that are conducive to the rigidification of the whole domain skeleton. (5) For the candidate sites determined in steps (3) and (4), site-directed saturation mutation or discontinuous multi-site simultaneous saturation mutation is performed using NNK degenerate codons to construct a mutant library. (6) The mutants obtained in step (5) were recombinantly expressed, and their relative enzyme activity and residual enzyme activity after heat treatment were determined by the p-nitrophenyl palmitate (pNPP) colorimetric method. Lipase mutants with simultaneous improvement in activity and thermal stability were screened.

2. The molecular modification design method according to claim 1, characterized in that, The crystal structure of Bacillus subtilis lipase A in step (1) is preferably PDB ID: 1I6W, with a resolution of [missing information]. The amino acid sequence of the wild-type Bacillus subtilis lipase A is shown in SEQ ID NO.1, and the encoding nucleotide sequence is shown in SEQ ID NO.

2.

3. The molecular modification design method according to claim 1, characterized in that, The molecular dynamics simulation described in step (2) is preferably performed under at least two different temperature conditions, with a simulation time of several hundred nanoseconds.

4. The molecular modification design method according to claim 1, characterized in that, The screening criteria for the key region flexibility maintenance sites in step (3) include: 1) the candidate residue is located in the key flexible region or its adjacent structural unit; 2) the candidate residue is dynamically related to the key flexible region; 3) the candidate residue has low ConSurf conservation; 4) the candidate residue is not Ser77, Asp133 or His156.

5. The molecular modification design method according to claim 1, characterized in that, The screening criteria for candidate sites for global framework rigidification in step (4) include: 1) FoldX predicted change in folding free energy ΔΔG < 0; 2) B-factor at a high level; 3) ML pred. score at a high level; 4) Conservation score at low or moderate conservation; 5) Mutation site far from the catalytic triad composed of Ser77, Asp133 and His156.

6. The molecular modification design method according to claim 1, characterized in that, In step (5), the optimal mutant obtained by the whole-domain skeleton rigidification strategy is used as a template, and potential sites obtained by the key region flexible maintenance strategy are introduced for saturation mutation to obtain double mutants. Using the optimal double mutant as a template, discontinuous multi-point simultaneous saturation mutation is performed on the remaining potential sites to construct a combined mutant library containing triple, quadruple and quintuple mutants.

7. A Bacillus subtilis lipase A mutant obtained by the method according to any one of claims 1-6, characterized in that, The mutant contains at least F17E and R142Q mutations relative to wild-type Bacillus subtilis lipase A; the mutant is M4, containing A15K / F17E / A132D / R142Q mutations.

8. The Bacillus subtilis lipase A mutant according to claim 7, characterized in that, The amino acid sequence of the M4 mutant is shown in SEQ ID NO.3, and the encoding nucleotide sequence is shown in SEQ ID NO.

4.

9. The Bacillus subtilis lipase A mutant according to claim 7 or 8, characterized in that, The M4 mutant exhibited 21.37 times higher residual enzyme activity, 94.52 times higher half-life, and 4.35 times higher catalytic activity after heat treatment compared to the wild type.

10. The application of the molecular modification design method according to any one of claims 1-6 or the Bacillus subtilis lipase A mutant according to any one of claims 7-9 in the preparation of highly active, highly thermostable lipases.

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