High-stability pectin lyase mutant as well as ancestor sequence reconstruction-based method, preparation method and application thereof

Abstract

The invention relates to a high-stability pectin lyase mutant and a method, a preparation method and application thereof based on ancestor sequence reconstruction, 28 key stability degeneration sites are screened out for ancestor residue recovery by analyzing evolution trajectories of homologous sequences of 382 bacteria and combining AlphaFold2 predicted contact diagram constraints. The half-life period of the mutant at 65 DEG C reaches 48 hours and is increased by 6 times compared with that of a wild type, and the Tm value is increased by 9.8 DEG C. When the mutant is applied to tobacco extract treatment, reducing sugar substances can be increased to 1.8 times, amino acid and other substances are increased by 35%, and a novel biological catalysis tool is provided for tobacco deep processing.

Description

Technical Field

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

[0002] In tobacco processing, pectate lyase is generally used to treat tobacco leaves. However, there are two major bottlenecks in the use of traditional pectate lyase in tobacco processing:

[0003] 1) High temperature treatment (>55°C) leads to rapid inactivation (half-life <8h);

[0004] 2) The nicotine binding-induced conformational collapse site-directed mutagenesis used in patent number CN202510500.X only achieved an improvement of ΔTm = 5.2°C, and has a small scope of application; while the diffusion-based method used in patent number CN202510001.8 improved activity but lacked thermal stability. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention proposes a highly stable pectin lyase mutant and a method, preparation method and application thereof based on ancestral sequence reconstruction, which can solve the technical problems of the existing technology such as small scope of application, poor stability and rapid inactivation of pectin lyase.

[0006] The technical solution adopted by the present invention is as follows: a highly stable pectin lyase mutant, the amino acid sequence of the highly stable pectin lyase mutant PEC-ASR03 is shown in SEQ ID NO: 1.

[0007] The highly stable pectin lyase mutant maintains >90% activity within the pH range of 3.0-8.5, has an optimum reaction temperature of 60°C, and a Tm value of 68.5±0.3°C.

[0008] A method for reconstructing the highly stable pectate lyase mutant based on the ancestral sequence comprises the following steps:

[0009] 1) Analyze the evolutionary trajectory of bacterial homologous sequences and use AlphaFold2 to predict contact graph constraints:

[0010] A multiple sequence alignment of γ-Proteobacteria pectate lyase homologs was performed using MAFFT, and conserved sites with a retention rate greater than 80% were screened using Gblocks. A Bayesian inference model incorporating tertiary structure constraints was constructed, and GRASPv3.0 was used to construct the maximum a posteriori probability ancestral sequence. The Markov chain length was set to 8 million generations, and the convergence criterion was a Gelman-Rubin R value of less than 1.02. The ancestral state probability calculation was optimized by incorporating a network weight of residue contact γ = 0.75.

[0011] 2) Identify key stability degradation periods and screen out 28 degradation sites that affect α-helix stability, that is, restore ancestral residues for key stability degradation sites.

[0012] The 28 degenerate sites that affect α-helix stability include:

[0013] D84A,K107R,V29T,S45A,Q61E,G78S,N92D,T114P,R127H,G141E,A158V,L169M,S183C,K194E,V20 7I, P220S, R232Q, N243D, A255V, T268S, K280N, V293L, S306A, E318D, L330M, N342S, K354R, V366I.

[0014] A method for preparing the highly stable pectin lyase mutant:

[0015] 1) Design a mutagenesis primer set covering 28 sites;

[0016] 2) Using pET-28a-wtPEC as a template, multiple rounds of overlapping PCR were used to construct mutant genes;

[0017] 3) Clone the mutant gene into the Pichia pastoris expression vector pPICZαA;

[0018] 4) Electroporation of the GS115 host and screening of positive clones using Zeocin resistance.

[0019] An application of the highly stable pectin lyase mutant in tobacco extract processing.

[0020] Application of the highly stable pectin methylesterase mutant in tobacco extract processing:

[0021] 1) mixing tobacco leaves with a 200 U / g concentration of a high-stability pectin methylesterase mutant solution in proportion;

[0022] 2) Using three-stage enzymatic hydrolysis process:

[0023] Stage I (50°C, pH 4.5, 2h): rapid degradation of pectin;

[0024] Stage II (60°C, pH 5.0, 3h): release of aroma precursors;

[0025] Stage III (55°C, pH 4.8, 1h): synergistic action of polyphenol oxidase;

[0026] 3) Complete the treatment to obtain the final tobacco extract.

[0027] The tobacco leaves and the 200U / g concentration of the high-stability pectin methylesterase mutant solution are mixed in a ratio of 1000:1.

[0028] The present invention offers the following beneficial effects: It provides a method for ancestral sequence reconstruction, resulting in a novel, highly stable pectate lyase mutant that can be used in the preparation of tobacco extracts. This mutant has a half-life of 48 hours at 65°C, a six-fold increase compared to the wild-type, and a Tm value increased by 9.8°C. Application of this mutant to tobacco extracts can increase reducing sugars by 1.8-fold and amino acids by 35%, providing a novel biocatalytic tool for tobacco deep processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Reconstruct schematic diagrams for phylogenetic trees and ancestral nodes;

[0030] Figure 2 This is a structural comparison diagram of wild-type PEL-3VMW and PEL-ASR03;

[0031] Figure 3 This is a stability comparison chart of wild-type PEL-3VMW and PEL-ASR03;

[0032] Figure 4 This is a graph showing the effect of different enzymatic hydrolysis times on the results of enzyme treatment of tobacco extract;

[0033] Figure 5 This is a diagram showing the effects of different material-liquid ratios of tobacco extract on reducing sugars and amino acids. DETAILED DESCRIPTION

[0034] 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.

[0035] A highly stable pectin lyase mutant, the amino acid sequence of the highly stable pectin lyase mutant PEC-ASR03 is shown in SEQ ID NO: 1.

[0036] The highly stable pectin lyase mutant maintains >90% activity within the pH range of 3.0-8.5, has an optimum reaction temperature of 60°C, and a Tm value of 68.5±0.3°C.

[0037] The above high stability pectin lyase mutant was tested for thermal stability:

[0038] The purified protein was determined by DSC: the Tm value of the mutant was 68.5±0.3℃, and that of the wild type was 59.2±0.5℃( Figure 3 );

[0039] Incubation experiment at 60°C: the half-life of the mutant was 48 h (8 h for the wild type), and the thermal inactivation rate constant was reduced by 85%.

[0040] The method for reconstructing the highly stable pectate lyase mutant based on the ancestral sequence comprises the following steps:

[0041] 1) Analyze the evolutionary trajectory of bacterial homologous sequences and use AlphaFold2 to predict contact graph constraints:

[0042] A multiple sequence alignment of γ-Proteobacteria pectate lyase homologs was performed using MAFFT, and conserved sites with a retention rate greater than 80% were screened using Gblocks. A Bayesian inference model incorporating tertiary structure constraints was constructed, and GRASPv3.0 was used to construct the maximum a posteriori probability ancestral sequence. The Markov chain length was set to 8 million generations, and the convergence criterion was a Gelman-Rubin R value of less than 1.02. The ancestral state probability calculation was optimized by incorporating a network weight of residue contact γ = 0.75.

[0043] 2) Identify the key stability degradation period and screen out 28 degradation sites that affect the stability of the α-helix, that is, restore the ancestral residues of the key stability degradation sites. Figure 1 The figure shows a schematic diagram of the phylogenetic tree and ancestral node reconstruction, with the key evolutionary node N3 (PEC-ASR03) marked in red. The 28 degenerate sites that affect α-helical stability include: D84A, K107R, V29T, S45A, Q61E, G78S, N92D, T114P, R127H, G141E, A158V, L169M, S183C, K194E, V207I, P220S, R232Q, N243D, A255V, T268S, K280N, V293L, S306A, E318D, L330M, N342S, K354R, and V366I.

[0044] Example 1

[0045] Ancestral sequence reconstruction method:

[0046] 1) 382 homologous sequences of γ-proteobacterial PL (PDB: 3VMW) were obtained from the NCBI database and clustered by CD-HIT with a threshold of 60%;

[0047] 2) Use IQ-TREE2 to build Figure 1 The maximum likelihood phylogenetic tree shown was molecular clock-calibrated using BEAST2;

[0048] 3) GRASP v3.0 was used to reconstruct the ancestral sequence, with the following parameters: Markov chain length: 8 × 106 generations; structural constraint weight: γ = 0.75; temperature annealing gradient: 0.85-1.20;

[0049] The ancestral sequence of node N3 (118 Myr ago) was obtained with a posterior probability of > 0.88, and the protein structures of the two were compared and predicted. Figure 2 shown.

[0050] Example 2

[0051] 1) Design a mutation primer set containing 28 sites

[0052] Based on the 28 mutation targets, primers were designed with a length of 25-35 nucleotides, a Tm value controlled within the range of 58-65°C, and a GC content controlled between 40% and 60%. The mutation site was located in the center of the primer, with at least 10-15 bases of homologous sequence retained on each side. Primer Premier 5.0 software was used to check the secondary structure and dimer formation potential of the primers to ensure primer specificity. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by HPLC, dissolved in sterile double-distilled water to a final concentration of 10 μM, and stored at -20°C until use.

[0053] 2) Using pET-28a-wtPEC as a template, multiple rounds of overlapping PCR were used to construct mutant genes

[0054] The wild-type pectate lyase gene was cloned into the pET-28a vector to construct the pET-28a-wtPEC expression plasmid, which served as a PCR template. The PCR reaction system (50 μL) contained: 5 μL of 10× Pfu DNA polymerase buffer, 4 μL of 2.5 mM dNTP mix, 1 μL of 10 μM upstream and downstream primers, 1 μL of template DNA (10 ng / μL), 1 μL of Pfu DNA polymerase (2.5 U / μL), and sterile double-distilled water to 50 μL. The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C (1 kb / min) for 90 seconds; and a final extension at 72°C for 10 minutes.

[0055] For the 28 mutation sites, a multi-round overlapping PCR strategy was employed. The first round of PCR generated single-point mutations at each mutation site. The second round of PCR, using the first-round PCR product as a template, linked adjacent single-point mutations. The third round of PCR constructed gene fragments encompassing multiple mutation sites. The final round of PCR linked all fragments into the complete mutant gene. PCR products were examined by 1% agarose gel electrophoresis, and the desired fragments were recovered from the gel and purified using a PCR product purification kit (TIANGEN, DP204).

[0056] 3) Clone the mutant gene into the Pichia pastoris expression vector pPICZαA

[0057] The purified final mutant gene PCR product and the pPICZαA vector were digested with EcoRI and XbaI restriction endonucleases, respectively, at 37°C for 4 hours. The digestion system (50 μL) consisted of: 5 μL of 10× buffer, 2 μg of DNA, 10 U each of EcoRI and XbaI, and sterile double-distilled water to 50 μL. The digestion products were separated by 1% agarose gel electrophoresis, and the desired bands were recovered using a gel extraction kit (TIANGEN, DP209).

[0058] The ligation reaction was carried out at 16°C overnight (12-16 hours). The ligation system (20 μL) contained: 2 μL of 10× T4 DNA ligase buffer, 50 ng of vector DNA, 150 ng of insert DNA (molar ratio approximately 1:3), 1 μL of T4 DNA ligase (5 U / μL), and sterile double-distilled water to 20 μL.

[0059] Transform the ligation product into E. coli DH5α competent cells. Add 5 μL of the ligation product to 100 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and immediately incubate on ice for 2 minutes. Add 900 μL of resistance-free LB medium and culture at 37°C with shaking (220 rpm) for 1 hour. Spread the plate onto an LB plate containing 25 μg / mL Zeocin and incubate at 37°C overnight. Pick a single colony for colony PCR identification and send for sequencing to verify the correct recombinant plasmid.

[0060] 4) Electroporation of GS115 host and screening of positive clones using Zeocin resistance

[0061] The correctly sequenced pPICZαA-mutPEC recombinant plasmid was extracted and linearized with the SacI restriction endonuclease. The digestion system (100 μL) contained: 10 μL of 10× buffer, 10 μg of plasmid DNA, 20 U of SacI, and sterile double-distilled water to 100 μL. Digestion was performed at 37°C overnight. The linearized plasmid DNA was purified by phenol-chloroform extraction and ethanol precipitation and dissolved in 10 μL of sterile double-distilled water.

[0062] Follow EasySelect TM Prepare Pichia pastoris GS115 competent cells according to the instructions of Pichia Expression Kit (Invitrogen). Mix 80 μL of GS115 competent cells and 5-10 μg of linearized plasmid DNA and transfer to a pre-cooled 0.2 cm electroporation cuvette. TM The parameters of the electroporation system (Bio-Rad) were set as follows: voltage 1500 V, capacitance 25 μF, and resistance 200 Ω. Immediately after electroporation, 1 mL of 1 M sorbitol solution pre-warmed to 30°C was added, transferred to a sterile test tube, and allowed to recover at 30°C for 1 hour.

[0063] Recovered cells were plated onto YPDS plates (1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, 2% agar containing 100 μg / mL Zeocin) and cultured at 30°C for 3-5 days. Eight to 12 single colonies were randomly selected and plated onto YPD plates containing a gradient of Zeocin concentrations (100, 500, and 1000 μg / mL). The plates were cultured at 30°C for 2-3 days to screen for multicopy integration transformants. Highly resistant clones were verified by PCR to confirm integration of the target gene into the Pichia pastoris genome.

[0064] Example 3

[0065] 1) Mix tobacco leaves with a 200 U / g concentration of a high-stability pectin methylesterase mutant solution in a ratio of 1000:1.

[0066] Select mature high-quality tobacco leaves (such as Yunnan K326 and other tobacco varieties), remove the stems and impurities, and chop them into 2-5cm 2 Weigh 100g of treated tobacco leaves and place them in a 2L glass reactor. Prepare the enzyme solution: Dilute the purified high-stability pectin methylesterase mutant with 0.1M phosphate buffer (pH 4.5) to an activity of 20,000U / L. Add 100mL of enzyme solution (equivalent to 200U enzyme activity per gram of tobacco leaves) to the reactor and mix with the tobacco leaves, maintaining a solid-liquid ratio of 1000:1 (w / v). Use a thermostatic stirrer at 60rpm to gently stir the mixture, ensuring full contact between the enzyme solution and the tobacco leaves without damaging the tobacco tissue structure.

[0067] 2) Using three-stage enzymatic hydrolysis process

[0068] Stage I (50°C, pH 4.5, 2h): Rapid degradation of pectin

[0069] The reactor temperature was set at 50 ± 1°C. The pH of the reaction system was monitored in real time using a pH meter and adjusted to 4.5 ± 0.1 using 1 M citric acid or 1 M NaOH solution. Enzymatic hydrolysis was performed under these conditions for 2 hours. During this stage, the highly stable pectin lyase mutant primarily rapidly degraded pectin in the tobacco leaves, increasing cell wall permeability. Samples of 100 μL were taken every 30 minutes, and the reducing sugar content was determined using the DNS method to monitor degradation progress. Constant stirring at 60 rpm was maintained throughout the reaction to ensure uniform reaction progress.

[0070] Phase II (60°C, pH 5.0, 3h): Release of aroma precursors

[0071] After the first stage, the reaction system temperature was raised to 60 ± 1°C and the pH was adjusted to 5.0 ± 0.1. Enzymatic hydrolysis was continued under these conditions for 3 hours. This stage facilitated the release of aroma precursor compounds and the degradation of complex polysaccharides in the tobacco. 100 μL of the sample was sampled every 60 minutes and analyzed by GC-MS for changes in volatile compound composition. During the reaction, the stirring speed was increased to 80 rpm to promote release and dissolution.

[0072] Phase III (55°C, pH 4.8, 1h): Synergistic action of polyphenol oxidase

[0073] After the second stage, the reaction system temperature was lowered to 55 ± 1°C and the pH was adjusted to 4.8 ± 0.1. These conditions favored the activity of endogenous polyphenol oxidase in the tobacco leaves, which synergized with the exogenously added highly stable pectate lyase mutant to promote the oxidation of polyphenols and the formation of aroma compounds. The reaction was continued for 1 hour, with the stirring speed reduced to 50 rpm to avoid excessive oxidation.

[0074] 3) Complete the treatment to obtain the final tobacco extract

[0075] After the enzymatic hydrolysis treatment is completed, the reactant is filtered through a 100-mesh sieve to separate the solid residue and the liquid extract. The liquid extract is centrifuged (8,000 rpm, 15 minutes, 4°C) to remove insoluble matter. The supernatant is concentrated under reduced pressure (50°C, -0.08MPa) to 1 / 5 of the original volume. After the concentrate is cooled to room temperature, an equal volume of 95% food-grade ethanol is added and allowed to stand overnight at 4°C to precipitate polysaccharides and proteins. The supernatant is collected by centrifugation again (10,000 rpm, 20 minutes, 4°C), and the ethanol is evaporated under reduced pressure at 45°C to obtain the final tobacco extract.

[0076] The reducing sugar and amino acid contents in the extract were analyzed by HPLC. Figure 4As shown in the results, the reducing sugar content in tobacco extract after enzyme treatment increased significantly from 110 mg / g to 180 mg / g, an increase of 63.6%, and the amino acid content increased from 15 mg / g to 28 mg / g, an increase of 86.7%. The increase in these compounds is beneficial to the improvement of the flavor of tobacco products.

[0077] In order to determine the optimal process parameters, the effects of material-liquid ratio and reaction time on the quality of the extract were further investigated. Figure 4 As shown in Figure 2, when the material-liquid ratio varies from 800:1 to 1200:1, the material-liquid ratio of 1000:1 can obtain the highest reducing sugar and amino acid content. Figure 5 As shown, the total reaction time is extended from 4 hours to 8 hours, and the product content gradually increases, but the increase slows down after more than 6 hours. Considering the production efficiency and product quality comprehensively, the optimal reaction time is determined to be 6 hours.

Claims

1. A highly stable pectin lyase mutant, characterized in that: The amino acid sequence of the highly stable pectate lyase mutant PEC-ASR03 is shown in SEQ ID NO:

1.

2. A highly stable pectin lyase mutant according to claim 1, characterized in that: The highly stable pectin lyase mutant maintains >90% activity within the pH range of 3.0-8.5, has an optimum reaction temperature of 60°C, and a Tm value of 68.5±0.3°C.

3. A method for reconstructing the highly stable pectate lyase mutant according to claim 1 based on the ancestral sequence, characterized in that: The steps are: 1) Analyze the evolutionary trajectory of bacterial homologous sequences and use AlphaFold2 to predict contact graph constraints: Multiple sequence alignments of γ-proteobacterial pectate lyase homologs were performed using MAFFT, and conserved sites with a retention rate greater than 80% were screened using Gblocks. A Bayesian inference model incorporating tertiary structure constraints was constructed, and GRASP v3.0 was used to construct the maximum a posteriori probability ancestral sequence. The Markov chain length was set to 8 million generations, and the convergence criterion was a Gelman-Rubin R value of less than 1.

02. Ancestral state probabilities were optimized by incorporating a network weight of residue contacts γ = 0.

75. 2) Identify key stability degradation periods and screen out 28 degradation sites that affect α-helix stability, that is, restore ancestral residues for key stability degradation sites.

4. The method for reconstructing a highly stable pectate lyase mutant based on an ancestral sequence according to claim 3, characterized in that: The 28 degenerate sites that affect α-helix stability include: D84A,K107R,V29T,S45A,Q61E,G78S,N92D,T114P,R127H,G141E,A158V,L169M,S183C,K194E,V20 7I, P220S, R232Q, N243D, A255V, T268S, K280N, V293L, S306A, E318D, L330M, N342S, K354R, V366I.

5. A method for preparing the highly stable pectin lyase mutant according to claim 1, characterized in that: 1) Design a mutagenesis primer set covering 28 sites; 2) Using pET-28a-wtPEC as a template, multiple rounds of overlapping PCR were used to construct mutant genes; 3) Clone the mutant gene into the Pichia pastoris expression vector pPICZαA; 4) Electroporation of GS115 host cells and screening of positive clones using Zeocin resistance.

6. Use of the highly stable pectate lyase mutant according to claim 1 in the treatment of tobacco extract.

7. Use of the highly stable pectin methylesterase mutant according to claim 1 in tobacco extract processing, characterized in that: 1) mixing tobacco leaves with a 200 U / g concentration of a high-stability pectin methylesterase mutant solution in proportion; 2) Using three-stage enzymatic hydrolysis process: Stage I (50°C, pH 4.5, 2h): rapid degradation of pectin; Stage II (60°C, pH 5.0, 3h): release of aroma precursors; Stage III (55°C, pH 4.8, 1h): synergistic action of polyphenol oxidase; 3) Complete the treatment to obtain the final tobacco extract.

8. Use of the highly stable pectin methylesterase mutant according to claim 7 in the treatment of tobacco extract, characterized in that: The mixing ratio of the tobacco leaves and the 200U / g concentration of the high-stability pectin methylesterase mutant solution is 1000:1.

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