High-stability pectin methylesterase mutant, ancestor sequence reconstruction method based on GRASP algorithm, preparation method and application of high-stability pectin methylesterase mutant

Abstract

The invention discloses a high-stability pectin methylesterase mutant as well as an ancestor sequence reconstruction method, a preparation method and application thereof based on a GRASP algorithm, evolution information of homologous sequences of 456 bacteria is integrated, a maximum posterior probability ancestor sequence is constructed by adopting the GRASP algorithm optimized by a graph theory, and 32 key stability degeneration sites are identified for ancestor residue recovery. The Tm value of the mutant reaches 76.8 DEG C and is increased by 11.2 DEG C compared with that of a wild type. The mutant keeps high activity in a pH range of 2.5-9.0, and is successfully applied to a high-temperature juice processing production line, so that the consumption of an enzyme preparation is reduced by 60%. The invention provides a new methodological framework for thermal stability modification of industrial enzyme.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of computational biology and enzyme engineering, and specifically relates to a highly stable pectin methylesterase mutant and a method for reconstructing its ancestral sequence based on the GRASP algorithm, a preparation method and an application thereof. Background Art

[0002] Pectin methylesterase (PME) catalyzes the demethylesterification of pectin. Its thermal stability requires repeated addition of enzyme preparations during high-temperature food processing, such as citrus juice pasteurization. Traditional site-directed mutagenesis methods struggle to achieve multi-site coordinated optimization, while existing ASR techniques suffer from the following limitations: 1) ignoring three-dimensional structural constraints; 2) low sensitivity to evolutionary events in long lineages; and 3) inability to effectively process large-scale sequence data (>400 sequences). Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention proposes a highly stable pectin methylesterase mutant and its ancestral sequence reconstruction method based on the GRASP algorithm, as well as its preparation and application. The present invention combines graph theory network analysis with Bayesian inference in conjunction with the GRASP algorithm. By constructing a Markov random field constrained by a residue contact network, introducing an adaptive temperature annealing strategy to optimize sampling efficiency, and integrating coevolutionary coupling information, the accuracy of ancestral sequence reconstruction is significantly improved. Ultimately, this method is applied to the thermostability modification of pectin methylesterase.

[0004] The technical solution adopted by the present invention is as follows: a highly stable pectin methylesterase mutant, the amino acid sequence of the stable pectin methylesterase mutant PME-ASR02 is shown in SEQ ID NO: 1.

[0005] The highly stable pectin methylesterase mutant maintains >85% activity within the pH range of 2.5-9.0, has an optimum reaction temperature of 65°C, and a Tm value of 76.8°C.

[0006] A method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant based on the GRASP algorithm comprises the following steps:

[0007] 1) Integrate the evolutionary information of bacterial homologous sequences and use the graph-theoretic optimized GRASP algorithm to construct the maximum a posteriori probability ancestral sequence;

[0008] 1.1) Integrate 456 homologous sequences of pectin methylesterases from γ-Proteobacteria;

[0009] 1.2) Perform trajectory analysis on the homologous sequences in 1.1):

[0010] 1.2.1) Construct a Markov random field model with structural constraints and integrate sequence coevolution information: 456 homologous sequences were aligned using MAFFT, and sites with a confidence score > 0.9 were retained after filtering;

[0011] 1.2.2) Ancestral state probabilistic inference using a modified Metropolis-Hastings sampler: The GRASP algorithm was used to construct the maximum a posteriori probability ancestral sequence, with a Markov chain length of 5 million generations and a convergence criterion of PSRF < 1.05.

[0012] 2) Identification of 32 key stability degradation sites for ancestral residue restoration;

[0013] Rosetta ddG scanning was used to identify 32 evolutionarily degenerate sites that contributed >1.5 kcal / mol to ΔΔG. Golden Gate assembly technology was used to construct a combinatorial library containing four mutation modules, and differential scanning fluorimetry (DSF) was used to screen for preferred mutants with a Tm increase of 11.2°C.

[0014] The 32 key stability degradation sites include: G15S, V28T, S42A, K59Q, D80G, N93S, T106P, R118H, G132E, A149V, L160M, S172C, K183E, V196I, P209S, R221Q, N232D, A244V, T257S, K269N, V282L, S295A, E307D, L319M, N331S, K343R, V355I, Q12R, H24D, F36Y, M48L, and W60G.

[0015] The preparation method of the highly stable pectin methylesterase mutant:

[0016] 1) Design overlapping fragments containing BsaI restriction sites, with a length of 80-120 bp, and construct the full gene by Golden Gate assembly;

[0017] 2) Clone the mutant gene into the Pichia pastoris expression vector pPIC9K and achieve soluble inducible expression;

[0018] 3) Use Prometheus NT.48 to perform DSF scanning and screen clones with ΔTm>10°C.

[0019] The highly stable pectin methylesterase mutant is used in high-temperature debittering of citrus juice, biological degumming of coffee bean mucilage, and pretreatment of lignocellulose.

[0020] Application of high-stability pectin methylesterase mutant in tobacco leaf degradation technology.

[0021] The present invention provides a highly stable pectin methylesterase mutant, utilizing a GRASP-based ancestral sequence reconstruction method. This mutant maintains high activity within a pH range of 2.5-9.0 and has been successfully applied to high-temperature juice processing lines, reducing enzyme consumption by 60%. This invention provides a new methodological framework for thermostability modification of industrial enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the GRASP evolutionary tree diagram;

[0023] Figure 2 for the soluble expression of wild-type PME-1QJV and PME-ASR02;

[0024] Figure 3 for wild-type PME-1QJV and PME-ASR02;

[0025] Figure 4 To identify the thermal stability of PME-ASR02. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below based on the accompanying drawings and preferred embodiments. The specific embodiments described herein are only used to explain the present invention but are not intended to limit the scope of protection of the present invention.

[0027] A highly stable pectin methylesterase mutant, the amino acid sequence of the stable pectin methylesterase mutant PME-ASR02 is shown in SEQ ID NO: 1.

[0028] The highly stable pectin methylesterase mutant maintains >85% activity within the pH range of 2.5-9.0, has an optimum reaction temperature of 65°C, and a Tm value of 76.8°C.

[0029] Thermodynamic analysis of highly stable pectin methylesterase mutants:

[0030] 1) Use Alphafold to predict protein structure and compare structural differences, such as Figure 3 As shown;

[0031] 2) Circular dichroism spectroscopy showed that the α-helix content of the mutant increased by 12.3% and the β-sheet decreased by 8.7%;

[0032] 3) Molecular dynamics simulation (100 ns) showed that the back mutation reduced the RMSF of the protein core region by 64%;

[0033] 4) Hydrogen bond network analysis found 8 new stable hydrogen bonds (R15 and D93 formed a salt bridge). Its thermal stability is as follows Figure 4 shown.

[0034] A method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant based on the GRASP algorithm comprises the following steps:

[0035] 1) Integrate the evolutionary information of bacterial homologous sequences and use the graph-theoretic optimized GRASP algorithm to construct the maximum a posteriori probability ancestral sequence;

[0036] 1.1) Integrate 456 homologous sequences of pectin methylesterases from γ-Proteobacteria;

[0037] 1.2) Perform trajectory analysis on the homologous sequences in 1.1):

[0038] 1.2.1) Construct a Markov random field model with structural constraints and integrate sequence coevolution information: 456 homologous sequences were aligned using MAFFT, and sites with a confidence score > 0.9 were retained after filtering;

[0039] 1.2.2) Ancestral state probabilistic inference using a modified Metropolis-Hastings sampler: The GRASP algorithm was used to construct the maximum a posteriori probability ancestral sequence, with a Markov chain length of 5 million generations and a convergence criterion of PSRF < 1.05.

[0040] 2) Identification of 32 key stability degradation sites for ancestral residue restoration;

[0041] Rosetta ddG scanning was used to identify 32 evolutionarily degenerate sites that contributed >1.5 kcal / mol to ΔΔG. Golden Gate assembly technology was used to construct a combinatorial library containing four mutation modules, and differential scanning fluorimetry (DSF) was used to screen for preferred mutants with a Tm increase of 11.2°C.

[0042] The 32 key stability degradation sites include: G15S, V28T, S42A, K59Q, D80G, N93S, T106P, R118H, G132E, A149V, L160M, S172C, K183E, V196I, P209S, R221Q, N232D, A244V, T257S, K269N, V282L, S295A, E307D, L319M, N331S, K343R, V355I, Q12R, H24D, F36Y, M48L, and W60G.

[0043] The preparation method of the highly stable pectin methylesterase mutant:

[0044] 1) Design overlapping fragments containing BsaI restriction sites, with a length of 80-120 bp, and construct the full gene by Golden Gate assembly;

[0045] 2) Clone the mutant gene into the Pichia pastoris expression vector pPIC9K and achieve soluble inducible expression;

[0046] 3) Use Prometheus NT.48 to perform DSF scanning and screen clones with ΔTm>10°C.

[0047] The highly stable pectin methylesterase mutant is used in high-temperature debittering of citrus juice, biological degumming of coffee bean mucilage, and pretreatment of lignocellulose.

[0048] Application of high-stability pectin methylesterase mutant in tobacco leaf degradation technology.

[0049] Example 1

[0050] Method for ancestral sequence reconstruction based on GRASP algorithm:

[0051] 1) 456 γ-Proteobacterial PME homologous sequences were obtained from the UniRef90 database and de-redundanted using CD-HIT with a threshold of 70%;

[0052] 2) Multiple sequence alignment was performed using the MAFFT-L-INS-i algorithm, and confident sites were screened using Guidance2;

[0053] 3) GRASP v2.1.0 was used to reconstruct the ancestral sequence. The parameters were as follows: Markov chain length: 5 × 10^6 generations; temperature annealing coefficient: 0.95; structure constraint weight: γ = 0.7; coevolution coupling threshold: MI > 0.3.

[0054] 4) Obtain the ancestral sequence of node N5 (95 Myr ago) through posterior decoding Figure 1 ,like Figure 1 As shown, the confidence probability is >0.85.

[0055] Example 2

[0056] 1) Gene cloning and vector construction

[0057] First, based on the wild-type pectin methylesterase (PME) gene sequence, a series of overlapping DNA fragments containing the BsaI restriction endonuclease recognition site were designed. Each fragment was controlled to be between 80 and 120 bp in length, and adjacent fragments were designed to have a 20-25 bp overlap region to ensure specific ligation. The specific design method is as follows:

[0058] The target gene sequence was divided into several fragments of about 90 bp each, and a BsaI restriction site (5'-GGTCTC-3') and a corresponding 4 bp sticky end were added to the 5' and 3' ends of each fragment, respectively. The sequence added to the 5' end is 5'-GGTCTCN-NNNN-3', and the sequence added to the 3' end is 5'-NNNN-NGAGACC-3', where NNNN is a specific sticky end, and complementary sequences are designed based on adjacent fragments. A total of 12 fragments were designed for the whole gene, numbered F1-F12. The codons were optimized using DNAStar software, and the OligoAnalyzer tool was used to check the GC content of the fragments (controlled at 40%-60%) and the possibility of secondary structure formation. All DNA fragments were synthesized and purified by Jinweizhi Biotechnology (Shanghai) Co., Ltd. to at least the PAGE purity level. The GoldenGate assembly reaction system (20 μL) contained 2 μL of 10× T4 DNA ligase buffer, 1 μL of each DNA fragment (10 ng / μL), 0.5 μL of BsaI-HF v2 (20 U / μL, NEB), 0.5 μL of T4 DNA ligase (400 U / μL, NEB), and ddH2O to 20 μL. The reaction cycle was as follows: (37°C, 5 min, digestion) → (16°C, 10 min, ligation) repeated 30 times → 55°C, 10 min (termination) → 80°C, 20 min (enzyme inactivation) → storage at 4°C. The reaction product was examined by 1.5% agarose gel electrophoresis, and the target band (approximately 1.2 kb) was recovered and purified using a DNA purification kit (Qiagen).

[0059] The purified target gene fragment was ligated with the pPIC9K vector, which had been double-digested with EcoRI and NotI. The ligation reaction mixture (20 μL) contained: 2 μL of 10× T4 DNA ligase buffer, 1 μL of vector DNA (50 ng / μL), 5 μL of insert DNA (30 ng / μL) (approximately a 1:3 molar ratio), and 1 μL of T4 DNA ligase (5 U / μL, Thermo Scientific). Sterile ultrapure water was added to bring the mixture to 20 μL. The reaction mixture was incubated at 16°C overnight (16-18 hours). The ligation product was transformed into E. coli DH5α competent cells. 5 μL of the ligation product was mixed with 50 μL of competent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 60 seconds, and immediately incubated on ice for 2 minutes. 950 μL of SOC medium preheated to 37°C was added and the cells were cultured at 37°C with shaking (220 rpm) for 1 hour. Transformed cells were plated on LB plates containing 50 μg / mL kanamycin and incubated at 37°C for 16 hours. Ten single colonies were randomly selected for colony PCR identification, and positive clones were sent to BGI for sequencing verification.

[0060] 2) Enzyme expression, purification and structure prediction

[0061] The correctly sequenced recombinant plasmid pPIC9K-PME_mutant was linearized using SacI digestion. The digestion system (100 μL) consisted of: 10 μL of 10× FastDigest buffer, 5 μg of plasmid DNA, 20 U of SacI, and sterile ultrapure water to 100 μL. After digestion at 37°C for 3 hours, the linearized product was purified by agarose gel electrophoresis.

[0062] The linearized plasmid was transformed into Pichia pastoris GS115 competent cells using electroporation. Mix 5 μg of linearized DNA with 80 μL of competent cells and transfer to a pre-chilled 0.2 cm electroporation cuvette. The electroporation parameters were set to 1.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, 1 mL of pre-warmed 1 M sorbitol solution was added. The cells were transferred to a sterile culture tube and allowed to recover at 30°C for 1 hour. The transformed cells were plated onto MD plates (1.34% YNB, 4 × 10 -5 % biotin, 2% glucose, 2% agar), and cultured at 30°C for 3-4 days.

[0063] A single colony was picked from the MD plate and inoculated into 5 mL YPD medium (1% yeast extract, 2% peptone, 2% glucose), and cultured at 30°C with shaking for 16 hours until the OD600 reached 6-8. The colony was transferred to 50 mL BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34% YNB, 4×10 -5 % biotin, 1% glycerol) in a 250 mL conical flask and cultured with shaking at 30°C (250 rpm) until OD 600 Up to 2-6 (about 16-20 hours).

[0064] The cells were collected (1,500 × g, 5 min, room temperature) and resuspended in 50 mL of BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34% YNB, 4 × 10 -5 % biotin, 0.5% methanol) in a 250 mL conical flask and shake cultured at 30°C (250 rpm). Methanol was added every 24 hours to a final concentration of 0.5% and expression was induced for 96 hours. Figure 2 As shown, SDS-PAGE analysis showed that the target protein (about 45 kDa) was efficiently expressed in the soluble fraction.

[0065] Alphafold3 was used to predict the three-dimensional structure of the mutant. The amino acid sequence of the mutant was input, and the high-precision prediction mode was used. The MSA method was set to MMseqs2, the template search method was set to HHsearch, and the prediction confidence score was greater than 90%. The obtained structure was visualized and analyzed using PyMOL software, as shown in the following example: Figure 3 As shown, the mutated enzyme molecule maintains the original structural skeleton, while the surface hydrophobicity and density are improved.

[0066] 3) High-throughput screening of stability:

[0067] The expression product was initially purified by ammonium sulfate precipitation. The culture supernatant was collected (10,000×g, 15 minutes, 4°C), ammonium sulfate was added to 70% saturation, and stirred at 4°C for 2 hours to fully precipitate the protein. The precipitate was collected (12,000×g, 30 minutes, 4°C), dissolved in 50mM sodium phosphate buffer (pH 6.0), and dialyzed overnight to remove ammonium sulfate. After the dialysate was filtered through a 0.22μm filter membrane, the target protein (His tag fusion protein) was purified using Ni-NTA affinity chromatography. Elution and collection were performed according to standard operating procedures to obtain an enzyme protein with a purity greater than 95%, and the protein concentration was determined using the BCA method.

[0068] High-throughput differential scanning fluorimetry (DSF) thermostability analysis was performed using a Prometheus NT.48 instrument (NanoTemper Technologies). Sample preparation was as follows: purified mutant enzyme samples were diluted to 1 mg / mL, 10× SYPRO Orange fluorescent dye was added (final concentration 5×), and 10 μL / tube was loaded using standard capillaries (NanoTemper Technologies' proprietary consumables). Instrument parameters included a temperature gradient from 25°C to 95°C at a rate of 1°C / min, an excitation wavelength of 470 nm, and fluorescence detection wavelengths of 570 nm and 350 nm. Each sample was measured in triplicate.

[0069] A total of 128 single clones were screened from E. coli and Pichia pastoris expression systems for DSF analysis. One-dimensional heat map analysis was used to compare the changes in fluorescence intensity of different clones under temperature gradients. Using the wild-type enzyme as a control (Tm value of 65.6°C), 36 mutants with ΔTm>5°C were screened, including 8 highly stable clones with ΔTm>10°C. The optimal mutant showed a ΔTm of 11.2°C and a Tm value of 76.8°C ( Figure 4 Data were analyzed using ProteinInn software, and the Tm and ΔTm of each sample were determined based on the first-order derivative fluorescence curve.

[0070] Example 3

[0071] Enzyme solution preparation: Dissolve the purified high-stability pectin methylesterase mutant in 50 mM citric acid-phosphate buffer (pH 3.8) and adjust the enzyme activity to 500 U / mL. Preheat at 4°C for 2 hours to allow the enzyme molecular conformation to fully adapt to the pH environment.

[0072] Tobacco leaf sample pretreatment: Select high-quality tobacco leaves (K326), remove the midrib, and cut into 2 cm × 2 cm pieces. Weigh 100 g of treated tobacco leaves and place them in a 2 L stainless steel reactor. Add 900 mL of 50 mM citric acid-phosphate buffer (pH 3.8) preheated to 60°C and soak for 15 minutes to allow the leaves to fully absorb moisture and soften.

[0073] Enzyme hydrolysis: Add 100 mL of preheated enzyme solution (final enzyme activity 50 U / g tobacco leaf) to the reactor. Adjust the reaction temperature to 75 ± 1°C and maintain the pH within 3.8 ± 0.1 using an automated pH adjustment system. Start the stirring system (80 rpm) to ensure uniform mixing without damaging the tobacco leaf structure. Allow the reaction to proceed for 60 minutes. A control group was treated with the wild-type enzyme under the same conditions for 120 minutes.

[0074] Sampling and monitoring: Every 15 minutes, 10 mL of the reaction mixture was sampled and rapidly cooled to room temperature. 1 mL of the sample was centrifuged (12,000 × g, 5 minutes). The supernatant was used to determine the degree of methylation and reducing sugar content.

[0075] Termination and Collection: After the reaction is complete, cool the system to 40°C and add 2 L of cold water to quench the reaction and dilute the enzyme solution. Filter the mixture to separate the tobacco leaves from the liquid. Dry the tobacco leaf sample in a 45°C oven to a constant weight for subsequent physical and chemical analysis.

Claims

1. A highly stable pectin methylesterase mutant, characterized in that: The amino acid sequence of the highly stable pectin methylesterase mutant PME-ASR02 is shown in SEQ ID NO:

1.

2. A highly stable pectin methylesterase mutant according to claim 1, characterized in that: The highly stable pectin methylesterase mutant maintains >85% activity within the pH range of 2.5-9.0, has an optimum reaction temperature of 65°C, and a Tm value of 76.8°C.

3. A method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant according to claim 1 based on the GRASP algorithm, characterized in that: The steps are: 1) Integrate the evolutionary information of bacterial homologous sequences and use the graph-theoretic optimized GRASP algorithm to construct the maximum a posteriori probability ancestral sequence; 2) 32 key stability degradation sites were identified and ancestral residue restoration was performed.

4. The method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant based on the GRASP algorithm according to claim 3, characterized in that: In the above 1), the specific method is: 1.1) Integrate 456 homologous sequences of pectin methylesterases from γ-Proteobacteria; 1.2) Perform trajectory analysis on the homologous sequences in 1.1): 1.2.1) Construct a Markov random field model with structural constraints and integrate sequence coevolution information: 456 homologous sequences were aligned using MAFFT, and sites with a confidence score > 0.9 were retained after filtering; 1.2.2) Ancestral state probabilistic inference using a modified Metropolis-Hastings sampler: The GRASP algorithm was used to construct the maximum a posteriori probability ancestral sequence, with a Markov chain length of 5 million generations and a convergence criterion of PSRF < 1.

05.

5. The method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant based on the GRASP algorithm according to claim 3, characterized in that: In the above 2), the specific method is: 32 evolutionarily degenerate sites contributing >1.5 kcal / mol to ΔΔG are identified by Rosetta ddG scanning; a combinatorial library containing four mutation modules is constructed using Golden Gate assembly technology, and preferred mutants with a Tm increase of 11.2°C are screened using differential scanning fluorimetry (DSF).

6. The method for reconstructing the ancestral sequence of the highly stable pectin methylesterase mutant based on the GRASP algorithm according to claim 3, characterized in that: The 32 key stability degradation sites include: G15S,V28T,S42A,K59Q,D80G,N93S,T106P,R118H,G132E,A149V,L160M,S172C,K183E,V196I,P209S,R221Q, N232D, A244V, T257S, K269N, V282L, S295A, E307D, L319M, N331S, K343R, V355I, Q12R, H24D, F36Y, M48L, W60G.

7. A method for preparing the highly stable pectin methylesterase mutant according to claim 1, characterized in that: 1) Design overlapping fragments containing BsaI restriction sites, with a length of 80-120 bp, and construct the full gene by Golden Gate assembly; 2) Clone the mutant gene into the Pichia pastoris expression vector pPIC9K and achieve soluble inducible expression; 3) Use Prometheus NT.48 to perform DSF scanning and screen clones with ΔTm>10°C.

8. Use of the highly stable pectin methylesterase mutant according to claim 1 in high-temperature debittering of citrus juice, biodegumming of coffee bean mucilage, and pretreatment of lignocellulose.

9. Use of the highly stable pectin methylesterase mutant according to claim 1 in a tobacco leaf degradation process.

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