A method for improving the thermal stability of lipase by molecular modification design

Abstract

The application provides a rational design method for improving thermal stability of lipase, and belongs to the field of enzyme engineering. A flexible region of the lipase to be reformed is determined through molecular dynamics simulation, a reforming region is determined through structure analysis, a reforming site is determined through comprehensive measurement of multiple calculation tools, a mutant library is established through site-directed saturation mutation using NNK degenerate codon, and an excellent mutant with improved thermal stability is screened through a microplate screening method based on spectrophotometry. The half-life of the Candida antarctica lipase B mutant of the application at 50 DEG C is 9 times that of the wild type, and the specific activity is also 6.32% higher than that of the wild type. The application breaks the trade-off between stability and activity, and realizes the improvement of the thermal stability of the lipase, which is conducive to the important role of the lipase mutant in the industrial application in the fields of food, medicine or biodiesel.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to a method for modifying and designing lipases based on structural analysis, thereby improving the thermal stability of lipases. Background Technology

[0002] Lipase (EC3.1.1.3) is a serine hydrolase with a classic α / β hydrolase domain, alternating α-helices and β-chains, and a catalytic triad composed of Ser, His, and Asp residues. Lipases can be used in a variety of reactions, including hydrolysis, esterification, and transesterification, and have achieved success in catalyzing various industrially valuable processes. Furthermore, the demand for the production of novel lipases is increasing. Lipase B (CALB) from *Candida antarcticis* is one of the most important enzymes in industry, serving as a highly efficient catalyst for the preparation of single-chiral isomer drugs and the synthesis of biodegradable polyesters. Developing more functionally improved CALBs and elucidating their mechanisms will bring significant benefits to scientific and industrial applications. Previous studies have found that CALBs gradually deactivate above 60°C, leading to a decrease in reaction rate, and many substrates (such as glycerol and dimethyl carbonate) require higher temperatures to completely dissolve. Therefore, further improving the thermal stability of CALBs is crucial for their industrial applications.

[0003] Thermal stability is considered an important parameter for evaluating the feasibility of enzymes in industrial applications. Generally, higher thermal stability makes enzymes more competitive and popular in industry. This is because operating at higher temperatures is highly advantageous because (i) the catalytic reaction rate approximately doubles every 10°C, (ii) the medium viscosity decreases and substrate diffusion is enhanced with increasing temperature, and (iii) the risk of potential contamination is lower. Considering that thermostable enzymes can adapt to a wider range of industrial application conditions, modifying existing mesophilic enzymes has significant industrial application implications. Protein engineering is an ideal method for improving enzyme properties. In recent years, semi-rational design based on structure, such as introducing non-covalent interactions and covalent bonds, adding proline or reducing glycine, and truncating loop rings, has been widely used to improve the thermal stability of proteins. For example, the optimal temperature of GH10 endoxylanase increased by 10°C after the introduction of a salt bridge; and the half-life of α-l-rhamnosase at 65°C (t) was increased after the introduction of a strong hydrophobic interaction through K573V substitution. 1 / 2 The time was increased by 11 minutes. However, the enzyme activity of both mutants decreased by about 40% compared to the wild type. The negative correlation between enzyme activity and stability has long been defined as the activity-stability trade-off. Therefore, a series of studies on thermophilic proteins are needed to elucidate the trade-off between enzyme activity and stability, thereby improving the thermostability of enzymes without sacrificing catalytic activity. Summary of the Invention

[0004] The purpose of this invention is to provide a rational design method for rapidly modifying and improving the catalytic efficiency of lipases to address the aforementioned problems. This method primarily involves two aspects: rapid target site screening using computer-aided calculations, followed by experimental verification of lipase thermostability. Lipase B from *Candida antarcticis* was selected as the research model to screen for mutants with potentially improved thermostability. Heterologous expression was performed using an *E. coli* expression system, and the purified lipase was used to determine enzymatic parameters and enzyme-catalyzed reaction kinetics. This invention synergistically combines irrational and rational design to improve the thermostability of CALB and deepen the understanding of the structure-function relationship of CALB. This invention develops a rational design method for molecular modification of lipases based on structural analysis. Based on the lipase structure, methods such as molecular docking, molecular dynamics simulation, FireProt, and FoldX are used for virtual screening to obtain mutants with potentially improved thermostability. The residual enzyme activity and half-life of the obtained mutants are experimentally verified, ultimately yielding mutants with higher thermostability. The method described yielded an Antarctic Candida lipase B mutant. The amino acid sequence of the Antarctic Candida lipase B mutant M1 is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4. After heat treatment, the Antarctic Candida lipase B mutant M1 still retained 79.525% of its residual activity, the relative enzyme activity of mutant M1 was 106.98%, and the half-life of mutant M1 was 81.355 minutes.

[0005] To solve the technical problem of this invention, the proposed technical solution is as follows: This invention provides a molecular modification design method for improving the thermal stability of lipases, comprising the following steps:

[0006] 1) Lipase B from Candida antarctica 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 (PDB).

[0007] 2) The AMBER18 software was used to further perform MD simulation on the above structure. The root mean square fluctuation value obtained by the MD simulation was used to determine the flexible region. Combined with the structure of the lipase to be modified, the target region for modification was determined.

[0008] 3) For the target region identified in step 2), based on the thermal stability prediction results of FireProt Web and FoldX, the binding energy ΔG is preliminarily determined. fold <0 potential target sites; combining the ConSurf conservation analysis results and B-factor values, sites with a conservation score <5 and a high B-factor (top 50%) were selected as the final target sites.

[0009] 4) For the potential target sites identified in step 3), site-directed saturation mutagenesis was performed using NNK degenerate codons, followed by recombination expression. The thermostability of the modified lipase was determined using the p-nitrophenol method, and single mutants with improved thermostability were screened.

[0010] 5) For the single mutants screened in step 4), use FoldX to perform virtual screening and recombination to select more promising virtual multiple mutants;

[0011] 6) The virtual multiple mutants screened in step 5) were recombinantly expressed and purified to obtain the modified lipase. The thermostability of the modified lipase was determined by the p-nitrophenol method, and the single mutant with the highest thermostability was screened.

[0012] The molecular modification design method for improving the thermostability of lipase, step 5) of which involves virtual screening, includes the following steps:

[0013] All single mutants were recombined, and the binding free energy of all multiple mutants was calculated using FoldX. The binding free energies were sorted from low to high, and at least the top 20 mutants with the lowest binding free energies were selected for further experimental verification.

[0014] The molecular modification design method for improving the thermal stability of lipase, in step 6), includes the determination of relative enzyme activity, residual enzyme activity, and thermal stability parameters.

[0015] This invention selects lipases from *Candida antarcticis* and develops a novel computer-aided design method based on structural analysis and virtual screening. Through a series of theoretical calculations, including molecular dynamics simulations and FoldX calculations of binding energy, mutants with potential improved thermal stability were virtually screened. Enzyme activity and various parameters were measured. Validation results show that this design method can rapidly and effectively improve the thermal stability of lipases.

[0016] The method yielded an Antarctic Candida lipase B mutant. The amino acid sequence of the Antarctic Candida lipase B mutant M1 is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0017] The Antarctic Candida lipase B mutant, after heat treatment, still had 79.525% residual activity, a relative enzyme activity of 106.98%, and a half-life of 81.355 minutes.

[0018] Beneficial effects

[0019] This invention provides a rational design method for improving the thermostability of lipases, belonging to the field of enzyme engineering. Molecular dynamics simulations are used to initially determine the flexible region of the lipase to be modified. Structural analysis is then used to determine the modification region, and multiple computational tools are used to comprehensively evaluate and determine the modification site. Site-directed saturation mutagenesis using NNK degenerate codons is employed to build a mutant library. Finally, a spectrophotometric microplate screening method is used to select superior mutants with improved thermostability. The *Candida antarcticis* lipase B mutant of this invention exhibits a 9-fold increase in half-life at 50°C compared to the wild type, and its specific activity is also increased by 6.32%. This invention overcomes the "stability-activity trade-off" and achieves improved thermostability of lipases, which is beneficial for the industrial applications of lipase mutants in food, pharmaceuticals, and biodiesel. Attached Figure Description

[0020] Figure 1 Crystal structure of Candida antarcticis lipase B;

[0021] Figure 2 The root mean square oscillation of lipase during MD simulation;

[0022] Figure 3 ConSurf predicted conservation of lipase sequences;

[0023] Figure 4 The point binding energy estimated by FoldX;

[0024] Figure 5 Target sites selected through comprehensive screening;

[0025] Figure 6 ; PCR identification diagram of Candida antarcticis lipase B mutant; M: Marker, 1-3, PCR product;

[0026] Figure 7 SDS-PAGE gel image of purified Candida antarcticis lipase B expression; M: Marker, 1. Supernatant, 2. Cell lysis buffer, 3. Transmission buffer, 4-6. 10mM imidazole elution buffer, 7-10. 30mM imidazole elution buffer, 11. 100mM imidazole elution buffer; The target protein is highlighted in the box.

[0027] Figure 8 SDS-PAGE gel images of purified wild-type and mutant Candida antarcticis lipase B; M: Marker, 1. Wild-type, 2. Mutant SPPDD, 3. Mutant SPPED, 4. Mutant GPPSE;

[0028] Figure 9 Comparison of residual enzyme activity and residual enzyme activity between wild-type and single-point mutant of Candida antarcticis lipase B;

[0029] Figure 10 Comparison of relative and residual enzyme activities of wild-type and quintuplet mutant of Candida antarcticis lipase B;

[0030] Figure 11 Half-life of wild-type and mutant Candida antarcticis lipase B. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but is not limited thereto. Unless otherwise specified, the reagent raw materials described in the following embodiments are all commercially available common raw materials, and the reagents are prepared using conventional methods. Methods not detailed in the embodiments are all conventional operations in the art.

[0032] Example 1: Method for Modifying the Thermal Stability of Candida Antarctica Lipase B

[0033] 1. Lipase B from *Candida antarcticis* was selected as the target for modification. Its crystal structure is shown below. Figure 1 As shown, the amino acid sequence of this lipase is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0034] 2. Molecular dynamics simulations of the above structures were performed using AMBER18 software for 50 ns. The structure with the lowest potential energy during the simulation was taken as the stable lipase structure. The RMSF changes of the lipase structure during the dynamic simulation are shown below. Figure 2 As shown, the flexible regions were initially identified as α5 and α10. Based on the structure of the lipase to be modified, the target region for modification was determined.

[0035] 3. Upload the crystal structure or amino acid sequence of the lipase to be modified to FireProt Web (an automated design platform for thermostable proteins based on evolution and energy). Select user-defined specific location analysis, choose the target region, and perform virtual saturation mutagenesis on its amino acids. Based on the output results, screen for sites with high B-factor values ​​(top 50%). Combine the results with the conservation analysis output from the ConSurf online tool. The results are as follows: Figure 3 As shown, sites with a conservation score <5 were selected to initially identify potential target sites; FoldX was used to further estimate the binding energy of the initial screening targets, and the results are as follows. Figure 4 As shown, ΔΔG was further filtered out. fold ≤0 kcal·mol -1 The site was determined to be the final target site. The results of the comprehensive site selection strategy are as follows: Figure 5 As shown.

[0036] The amino acid sequence of Candida antarcticis lipase B (SEQ ID NO.1):

[0037] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP

[0038] Nucleotide sequence of Candida antarctica lipase B (SEQ ID NO.2):

[0039] CTGCCTAGTGGTAGCGATCCGGCCTTTAGCCAGCCGAAAAGCGTGCTGGATGCAGGTCTGACCTGCCAGGGCGCCAGTCCGAGCAGCGTGAGCAAACCGATTCTGCTGGTGCCGGGTACAGGCACCACCGGTCCGCAGAGCTTTGATAGCAATTGGATTCCGCTGAGCACCCAGCTGGGTTATACCCCGTGTTGGATT AGCCCGCCGCCGTTTATGCTGAATGATACCCAGGTTAATACCGAATATATGGTGAATGCAATTACCGCACTGTATGCCGGCAGTGGCAATAATAAGCTGCCGGTTCTGACCTGGAGCCAGGGCGGTCTGGTTGCCCAGTGGGGTCTGACCTTTTTCCCGAGCATTCGCAGTAAAGTTGATCGCCTGATGGCCTTTGCACCGGATTATAAAGGTACAGTTCTGGCCGGCCCGCTGGATGCCCTGGCAGTTAGCGCACCGAGCGTGTGGCAGCAGACCACCGGTAGTGCCCTGACCACCGCCCTGCGTAATGCAGGTGGTCTGACCCAGATTGTTCCGACCACCAATCTGTATAGTGCAACCGATGAAATTGTGCAGCCGCAGGTGAGTAATAGTCCGCTGGATAGCAGCTATCTGTTTAATGGTAAAAATGTTCAGGCCCAGGCCGTTTGCGGCCCGCTGTTTGTGATTGATCATGCAGGTAGTCTGACCAGTCAGTTTAGTTATGTTGTTGGCCGCAGCGCCCTGCGCAGCACCACAGGTCAGGCACGCAGTGCAGATTATGGTATTACCGATTGCAATCCGCTGCCGGCAAATGATCTGACCCCGGAACAGAAAGTGGCAGCAGCAGCCCTGCTGGCCCCGGCAGCAGCAGCGATTGTTGCAGGCCCGAAACAGAATTGCGAACCGGATCTGATGCCGTATGCCCGTCCGTTTGCAGTTGGTAAACGTACCTGCAGTGGCATTGTTACCCCG Example 2: Expression of Candida antarctica lipase B

[0040] 1. Using the lipase B gene from Candida antarctica as a template, the enzyme was named CALB, and its amino acid sequence is shown in SEQ ID NO:1. It was inserted into the pET25b(+) vector with EcoRI-NcoI as the cloning site, and the wild-type recombinant expression plasmid pET25b-CALB was synthesized.

[0041] 2. The plasmid was transformed into BL21(DE3) competent cells by heat shock. 500 μL of LB medium without any antibiotics was added, and the cells were incubated at 37°C for 60 min. After a brief centrifugation at 3000 rpm, the remaining 100 μL of supernatant was mixed with the cells. An appropriate amount was spread on LB agar plates containing 100 mg / L ampicillin and cultured overnight at 37°C until clear single colonies appeared.

[0042] 3. Pick a single colony from the overnight plate and transfer it to 5 mL of LB liquid medium containing 100 mg / L ampicillin resistance. Incubate at 37°C in a shaker for 12 h to preserve the bacteria and then sequence them.

[0043] 4. Select successfully validated single clones and culture them in LB liquid medium containing 100 mg / L ampicillin resistance at 37°C and 220 rpm for 12-16 h. Then, transfer 1-2% of the clones to 50 mL of TB medium containing 100 mg / L ampicillin resistance and culture at 37°C and 220 rpm for 2-4 h until OD (Organic Closing Rate) is reached. 600 Add 0.6-0.8% IPTG to a final concentration of 0.1 mM and induce for 24 h at a temperature of 18 °C and a stirring speed of 220 rpm.

[0044] 5. Centrifuge the fermentation broth at 4℃ and 6000rpm for 20min to collect the bacterial cells. Resuspend the bacterial cells in 10mL of PBS buffer per 1g of bacterial cells. Sonicate the cells at 30% power for 3s followed by 5s pause, 20min per 10mL. Centrifuge the disrupted bacterial solution at 4℃ and 6000rpm for 20min to remove cell debris and collect the supernatant.

[0045] 6. The disrupted supernatant was passed through a 0.22 μm inorganic water membrane and purified by nickel column chromatography. The imidazole elution gradient was 10-30-100 mM. The 100 mM elution buffer was collected and subjected to 10% SDS-PAGE electrophoresis to identify the expression and purification status of the target protein. The expression and purification process of *Candida antarcticis* lipase B is as follows: Figure 7 As shown.

[0046] 7. Determination of lipase activity: Prepare solution A: 50 mM p-nitrophenol butyrate pNPB (0.15% Triton X-100, w / v); solution B: 50 mM Tris-HCl buffer solution, pH 8. Mix solutions A and B at a ratio of 1:9 to prepare the substrate solution. Add 0.1 mL of the obtained wild-type lipase crude enzyme solution to 4.5 mL of the substrate solution, and add 0.1 mL of blank culture medium as a control to 4.5 mL of the substrate solution. Incubate at 40℃ for 5 min, and add 1 mL of acetone to terminate the reaction. Lipase hydrolyzes pNPB to release p-nitrophenol (pNP). The p-nitrophenol solution is yellow-green under alkaline conditions. Enzyme activity is defined as: 1 μmol of p-nitrophenol released by 1 mL of lipase hydrolyzing pNPB for 1 min is 1 enzyme activity unit (U), μmol / mL. -1 ·min -1 .

[0047] Example 3: Establishment and screening of a Candida antarcticis lipase B mutant library

[0048] 1. Mutant construction: Specifically, this includes using pET25b-CALB as the amplification template, for... Figure 5 Primers were designed for the final target sites (L147, T244, S250, T256, N292) as shown in Table 1. A saturated mutant library was constructed by whole plasmid PCR, and the specific amplification parameters are shown in Tables 2-3. The amplified products were digested and then gel-recovered. The recovered products were transformed into BL21(DE3) competent cells and replicated and expressed in them. The cells were plated, cultured, and single clones were selected for sequencing. After successful sequencing verification, the saturated mutants of the target sites were obtained.

[0049] Table 1: Primer design for saturated lipase mutants of Candida antarctica

[0050]

[0051]

[0052] Table 2: Mutant amplification system (50 μL)

[0053] Components Dosage (μL) pET25b-CALB (template DNA) 1 Upstream primer (10 μM) 2 Downstream primer (10 μM) 2 dNTP mix (10mM) 1 2×Max Buffer 25 Phanta Max Super-Fidelity DNA Polymerase 1 <![CDATA[ddH2O]]> Up to 50

[0054] Table 3: Mutant Amplification Reaction Procedure

[0055]

[0056] 2. Mutant Expression: Mutants were cultured and expressed in 96-well plates (2.1 mL volume). Specifically, single clones were picked and placed in 96-well plates, each well containing 500 μL of LB liquid medium containing 100 mg / L ampicillin resistance. Five wild-type single clones were included in each well as a negative control. The plates were incubated overnight at 37°C and 300 rpm. 20 μL of the mutant was then transferred to 500 μL of TB liquid medium containing 100 mg / L ampicillin resistance and incubated at 37°C and 220 rpm for 2-4 hours until OD (Organic Growth Rate) was reached. 600 Add 20 μL of IPTG to a final IPTG concentration of 0.1 mM, and induce for 24 h at 18 °C and 300 rpm.

[0057] 3. Mutant screening:

[0058] 1) Before screening, the expressed bacterial culture needs to be lysed and centrifuged. Lysozyme is added to the cultured bacterial culture to a final concentration of 0.4 mg / mL. The culture is then incubated on ice for 30 min, heat-treated at 37°C for 5 min, and incubated on ice for another 30 min. Finally, the culture is centrifuged at 4000 rpm and 4°C for 30 min, and the supernatant is collected.

[0059] 2) The enzyme solution was incubated in a 60°C water bath for 15 min, and the thermostability of the lipase was determined. Specifically, the enzyme solution obtained by centrifugation was placed in another clean 96-well plate and incubated at 60°C. Samples were taken at different treatments, with the activity at 0 min incubation considered 100%. The remaining lipase activity was calculated using the pNPB method described in Example 2. The results are as follows: Figure 9 As shown, the residual enzyme activity of wild-type lipase is 11.250%, while single-point mutants such as L147S and N292D still have 20-30% residual activity. The top 2-5 single-point mutants with the highest residual enzyme activity at each site were screened, as shown in Table 4.

[0060] Table 4: Screening results of single-point mutants of Candida antarcticis lipase B

[0061] site Mutation situation 147 L147S, L147G, L147E, L147D, L147P 244 T244E, T244D, T244P, T244S, T244V 250 S250E, S250D, S250P 256 T256E, T256D, T256P, T256S 292 N292D, N292E

[0062] Example 4: Recombination and screening of Candida antarcticis lipase B mutant

[0063] 1. The top 20% of single-point mutants with the highest residual enzyme activity in Example 3 were arranged and combined to form duplex / triple / quadruple / quintuple mutants. FoldX was used to estimate the ΔΔG of all multiple mutants. fold Filter out ΔΔG fold The 20 lowest-ranking mutants were experimentally verified, as shown in Table 5.

[0064] 2. Construction and expression of multiple mutants: Using pET25b-CALB as the amplification template, primers were designed according to the mutants shown in Table 5, as shown in Table 6, to construct multiple mutants; the amplification products were digested and then gel recovered, and the recovered products were transformed into BL21(DE3) competent cells for replication and expression. The cells were plated, cultured, and single clones were selected for sequencing. After successful sequencing verification, the target multiple mutants were obtained.

[0065] 3. Screening of multiple mutants: The mutants were cultured and treated using the method described in Example 3, and the residual enzyme activity of the obtained multiple mutants was determined using the pNPB method. The relative enzyme activity results without heat treatment are shown in Table 7. Figure 10 As shown, the residual enzyme activity of wild-type lipase after heat treatment was 11.250%, while the best-performing mutant M1 (L147S / T244P / S250P / T256D / N292D, abbreviated as SPPDD) still retained 79.525% of its residual activity. Meanwhile, the relative enzyme activity of mutant M1 was 106.98% (wild-type was 100%), indicating that mutant M1 did not lose its original enzyme activity due to increased thermostability. Furthermore, the half-life of wild-type and mutant M1 at 50℃ was determined, as shown in Table 8. Figure 11 As shown in the figure, the half-life of mutant M1 is 81.355 minutes (compared to only 8.665 minutes for the wild type), approximately 9.4 times that of the wild type, further demonstrating the significantly improved thermostability of mutant M1. The amino acid sequence of mutant M1 is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0066] Table 5: Results of virtual recombination screening of Candida antarctic lipase B mutants (the letters in the table from left to right correspond to the abbreviated amino acids after mutations at sites 147, 244, 250, 256, and 292).

[0067] SPPPD SPPED SPPDD SPPSD SDPPD SPPPE SPPEE SPPDE SPPSE SDPPE GPPPD GPPED GPPDD GPPSD GDPPD GPPPE GPPEE GPPDE GPPSE GDPPE

[0068] Table 6: Primer design for multiple mutants of Candida antarcticis lipase B

[0069]

[0070]

[0071] Table 7: Enzyme activity (%) of wild-type and some pentapex mutants of Candida antarctica lipase B

[0072] name Residual enzyme activity (%) Relative enzyme activity (%) WT 11.250 100.000 SDPPD 60.300 114.100 SPPED 66.487 105.417 SPPDD 79.525 106.981 SPPPD 46.138 108.380 SPPSD 40.200 100.200

[0073] Table 8: Half-life of wild-type and mutant SPPDD M1 of Candida antarcticis lipase B at 50℃

[0074] <![CDATA[k d ]]> <![CDATA[t 1 / 2 ]]> WT 0.07999 8.665 SPPDD 0.00852 81.355 SPPED 0.01743 39.767 GPPSE 0.05831 11.887

[0075] Lipase mutant SPPDD SEQ ID NO.3:

[0076] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDASAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSPTGQARPADYGIDDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQDCEPDLMPYARPFAVGKRTCSGIVTP

[0077] Lipase mutant SPPDD SEQ ID NO.4:

[0078] CTGCCTAGTGGTAGCGATCCGGCCTTTAGCCAGCCGAAAAGCGTGCTGGATGCAGGTCTGACCTGCCAGGGCGCCAGTCCGAGCAGCGTGAGCAAACCGATTCTGCTGGTGCCGGGTACAGGCACCACCGGTCCGCAGAGCTTTGATAGCAATTGGATTCCGCTGAGCACCCAGCTGGGTTATACCCCGTGTTGGATTAGCCCGCCGCCGTTTATGCTGAATGATACCCAGGTTAATACCGAATATATGGTGAATGCAATTACCGCACTGTATGCCGGCAGTGGCAATAATAAGCTGCCGGTTCTGACCTGGAGCCAGGGCGGTCTGGTTGCCCAGTGGGGTCTGACCTTTTTCCCGAGCATTCGCAGTAAAGTTGATCGCCTGATGGCCTTTGCACCGGATTATAAAGGTACAGTTCTGGCCGGCCCGCTGGATGCCTCGGCAGTTAGCGCACCGAGCGTGTGGCAGCAGACCACCGGTAGTGCCCTGACCACCGCCCTGCGTAATGCAGGTGGTCTGACCCAGATTGTTCCGACCACCAATCTGTATAGTGCAACCGATGAAATTGTGCAGCCGCAGGTGAGTAATAGTCCGCTGGATAGCAGCTATCTGTTTAATGGTAAAAATGTTCAGGCCCAGGCCGTTTGCGGCCCGCTGTTTGTGATTGATCATGCAGGTAGTCTGACCAGTCAGTTTAGTTATGTTGTTGGCCGCAGCGCCCTGCGCAGCCCCACAGGTCAGGCACGCCCCGCAGATTATGGTATTGATGATTGCAATCCGCTGCCGGCAAATGATCTGACCCCGGAACAGAAAGTGGCAGCAGCAGCCCTGCTGGCCCCGGCAGCAGCAGCGATTGTTGCAGGCCCGAAACAGGATTGCGAACCGGATCTGATGCCGTATGCCCGTCCGTTTGCAGTTGGTAAACGTACCTGCAGTGGCATTGTTACCCCG

[0079] The above-mentioned experiments show that the finally constructed lipase mutant M1 has superior thermal stability and stable enzyme activity, avoiding the previous situation where enzyme activity decreased due to improved thermal stability. It can be widely used in food processing, pharmaceutical synthesis, leather production or papermaking industry and has high commercial value.

[0080] The present invention describes a design method for modifying *Candida antarcticis* lipase B to enhance its thermostability. This method primarily involves virtual screening using multiple techniques, such as FireProt and FoldX, to identify mutants with potential thermostability enhancements. The enzyme was expressed using an *E. coli* expression system, purified by nickel column chromatography, and its activity and thermostability were determined using the pNPB assay. The results demonstrate that the molecular modification design method of this invention can rapidly and effectively improve the thermostability of lipases without sacrificing enzyme activity, indicating the reliability of the enzyme modification method.

[0081] 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 the thermal stability of lipases, characterized in that, Includes the following steps: (1) Lipase B from Candida antarctica 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 PDB. (2) The above structure was further simulated using AMBER18 software. The region with the largest fluctuation was identified as the flexible region by the root mean square fluctuation value obtained by MD simulation, and the target region for modification was determined by combining the structure of the lipase to be modified. (3) For the target region determined in step (2), the binding energy ∆∆G is initially determined by combining the thermal stability prediction results of FireProt Web and FoldX. fold <0 potential target sites; Based on the combined results of ConSurf conservation analysis and B-factor values, sites with a conservation score of <5 and a high B-factor in the top 50% were selected as the final target sites. (4) For the potential target sites identified in step (3), primers were designed using NNK degenerate codons to perform site-directed saturation mutagenesis and recombination expression. The thermostability of the modified lipase was determined using the p-nitrophenol method, and single mutants with improved thermostability were screened. (5) For the single mutants screened in step (4), use FoldX to perform virtual screening and recombination to select more promising virtual multiple mutants; (6) The virtual multiple mutants screened in step (5) were recombinantly expressed and purified to obtain the modified lipase. The thermostability of the modified lipase was determined by the p-nitrophenol method, and the single mutant with the highest thermostability was screened. The amino acid sequence of the Candida antarcticis lipase B mutant M1 is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.

4.

2. The molecular modification design method for improving the thermal stability of lipase according to claim 1, characterized in that, The virtual screening described in step (5) includes the following steps: recombine all the selected single mutants, use FoldX to calculate the binding free energy of all multiple mutants, sort the binding free energy from low to high, and select at least the top 20 mutants with lower binding free energy for further experimental verification.

3. The molecular modification design method for improving the thermal stability of lipase according to claim 1, characterized in that, The thermal stability determination described in step (6) includes the determination of relative enzyme activity, residual enzyme activity and thermal stability parameters.

4. The molecular modification design method for improving the thermal stability of lipase according to claim 1, characterized in that: The Antarctic Candida lipase B mutant M1, after heat treatment, still has 79.525% residual activity, the relative enzyme activity of mutant M1 is 106.98%, and the half-life of mutant M1 is 81.355 minutes.

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