Xanthan lyase ancestral enzyme based on ancestral sequence reconstruction and mutants and applications thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a xanthan gum cleavage ancestral enzyme AncXLY196 based on ancestral sequence reconstruction, an amino acid sequence of which is shown as SEQ ID NO. 2. Based on the xanthan gum cleavage ancestral enzyme, a mutant AncXLY196-X7 is further obtained, an amino acid sequence of which is shown as SEQ ID NO. 4, xanthan gum degradation activity of which reaches 2.5 times of that of the xanthan gum cleavage ancestral enzyme, and the optimal catalytic temperature of the xanthan gum cleavage ancestral enzyme and the mutant thereof is increased to 45 DEG C, and the enzyme activity of the mutant of the xanthan gum cleavage ancestral enzyme is reserved by 50-80% after being treated at 45-55 DEG C for 12-24 h. The application provides a new enzyme resource for efficient enzymatic degradation of xanthan gum and preparation of functional oligosaccharides.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a xanthan gum cleavage ancestor enzyme based on ancestor sequence reconstruction, its mutants, and their applications. Background Technology

[0002] Xanthan gum is an extracellular polysaccharide produced by the Gram-negative bacterium *Xanthomonas brassicae*. It has a main chain of β-1,4-D-glucopyranoside, with side chains consisting of mannose, glucuronic acid, and terminal mannose residues linked to the main chain via α-1,3-glycosidic bonds to alternating glucose residues. Xanthan gum exhibits unique pseudoplastic rheological properties and strong stability in salt, acid, alkali, and enzymatic environments, making it widely used in the food, pharmaceutical, cosmetic, and petroleum industries.

[0003] Xanthan gum oligosaccharides are polysaccharide polymers that are structurally similar to natural xanthan gum but with a significantly lower molecular weight. Compared to their higher molecular weight forms, they exhibit enhanced solubility, higher antioxidant activity, and better antibacterial properties, thus demonstrating broader application potential in various fields such as the food, pharmaceutical, and petroleum industries.

[0004] Currently, xanthan gum can be degraded using three methods: physical, chemical, and enzymatic. Physical degradation is often used as an auxiliary method in combination with other degradation methods because it easily triggers side reactions such as molecular branching and cross-linking. Chemical degradation is difficult to control precisely and consumes large amounts of chemical reagents, potentially leading to safety hazards in the final product. Enzymatic degradation, with its high reaction specificity and minimal environmental impact, is gaining increasing attention. Currently reported xanthan gum-degrading enzymes typically have an optimal temperature range of 30-40℃ and exhibit poor thermal stability. In tertiary oil recovery chemical flooding, xanthan gum and other polymers need to be injected. After the process, degrading xanthan gum is crucial to prevent its prolonged and excessive retention in the oil reservoir, which could lead to decreased permeability or reservoir blockage. Therefore, degrading enzymes with good temperature resistance are needed to meet industrial applicability.

[0005] Ancestor sequence reconstruction is an emerging and highly promising computational strategy that uses phylogenetic analysis to obtain the inferred ancestral sequences of existing enzymes. The designed ancestral enzymes often exhibit higher thermostability and / or a wider substrate range. Gu et al. successfully discovered an ancestral nitrile hydrolase with significantly enhanced thermostability through ancestral sequence reconstruction. Furthermore, Zhu et al. revealed the evolutionary mechanism of the stereoselectivity of imine reductases through ancestral sequence reconstruction. Therefore, obtaining ancestral enzymes through ancestral sequence reconstruction technology to improve the thermostability and activity of xanthan gum lyase has significant research value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a xanthan gum cleavage ancestor enzyme AncXLY196, which addresses the shortcomings of the prior art.

[0007] Another technical problem to be solved by the present invention is to provide a mutant of the xanthan gum cleavage ancestor enzyme AncXLY196, AncXLY196-X7.

[0008] Another technical problem to be solved by the present invention is to provide a nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7.

[0009] Another technical problem to be solved by the present invention is to provide an expression cassette or recombinant expression vector containing a nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7.

[0010] Another technical problem to be solved by the present invention is to provide recombinant bacteria containing the aforementioned expression cassette or recombinant expression vector.

[0011] The final technical problem to be solved by this invention is to provide the application of the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 in the preparation of xanthan gum oligosaccharides.

[0012] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0013] The first aspect of the present invention provides a xanthan gum cleavage ancestor enzyme AncXLY196, the amino acid sequence of which is shown in SEQ ID NO.2.

[0014] From Bacillus ( BacillusThe amino acid sequence of xanthan lyase (XLY) from *Bacillus sp. GL1*, as shown in SEQ ID NO.1, was entered into UniProt (https: / / www.uniprot.org) and NCBI (https: / / www.ncbi.nlm.nih.gov). Homologous sequences were screened, and an amino acid substitution model was determined using IQ-TREE software to construct a phylogenetic tree. Ancestor sequences were reconstructed using the pamlX software package, and the protein sequence corresponding to node 196 in the phylogenetic tree was cloned and expressed. This node represents the most recent common ancestor of the target xanthan lyase group. Enzyme expression and enzymatic characterization confirmed that the xanthan lyase ancestor enzyme AncXLY196 is a thermostable xanthan lyase with a full-length nucleotide sequence of 2715 bases, encoding 905 amino acids, as shown in SEQ ID NO.3, and the encoded amino acid sequence as shown in SEQ ID NO.2. The xanthan lyase ancestor enzyme AncXLY196 and the xanthan lyase from *Bacillus sp. GL1* (…) are related to… Bacillus The xanthan lyase sp. GL1 has 69.25% sequence similarity.

[0015] The second aspect of the present invention provides a xanthan gum cleavage ancestor enzyme mutant AncXLY196-X7, whose amino acid sequence has at least 99% sequence identity with SEQ ID NO.2.

[0016] Preferably, the xanthan gum cleavage ancestor enzyme mutant AncXLY196-X7 has an amino acid sequence that is 99.2% identical to that of SEQ ID NO.2.

[0017] Most preferably, the xanthan gum cleavage ancestor enzyme mutant AncXLY196-X7 is obtained by mutating glutamic acid at position 73 of SEQ ID NO.2 to arginine, serine at position 120 to leucine, aspartic acid at position 135 to lysine, tyrosine at position 230 to glutamine, glutamine at position 236 to aspartic acid, aspartic acid at position 399 to proline, and valine at position 589 to alanine; the amino acid sequence of the xanthan gum cleavage ancestor enzyme mutant AncXLY196-X7 is shown in SEQ ID NO.4.

[0018] Based on the xanthan gum cleavage ancestor enzyme AncXLY196, a high-performance combinatorial mutant, AncXLY196-X7, was obtained by rationally modifying key sites using computational design methods such as molecular docking, molecular dynamics simulation, and energy residue decomposition. The mutation sites are E73R, S120L, D135K, Y230Q, Q236D, N399P, and V589A. The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.5.

[0019] The optimal catalytic temperature of the xanthan gum cleavage progenitor enzyme and its mutants was increased from 40℃ for the wild-type xanthan gum cleavage enzyme to 45℃. The xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 has better thermal stability than the xanthan gum cleavage progenitor enzyme AncXLY196, retaining 50-80% of its enzyme activity after treatment at 45-55℃ for 12-24 h, while the xanthan gum cleavage progenitor enzyme AncXLY196 retains 0% of its enzyme activity under the same conditions.

[0020] A third aspect of the present invention provides a nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7.

[0021] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme AncXLY196 is shown in SEQ ID NO.3; the nucleotide sequence of the nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 is shown in SEQ ID NO.5.

[0022] The fourth aspect of the present invention provides an expression cassette or recombinant expression vector containing a nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7.

[0023] In some embodiments, the starting vector of the recombinant expression vector is any one of the pET series vectors, pHT series vectors, pBE series vectors, and pPIC series vectors, preferably pET-22b.

[0024] The fifth aspect of the present invention provides recombinant bacteria containing the aforementioned expression cassette or recombinant expression vector.

[0025] In some embodiments, the originating strain of the recombinant bacteria is any one of Escherichia coli, Bacillus, and Pichia pastoris, preferably Escherichia coli. E. coli BL21(DE3).

[0026] The sixth aspect of the present invention provides the use of the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 in the preparation of xanthan gum oligosaccharides and / or the degradation of xanthan gum.

[0027] In some embodiments, the method of applying the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 to prepare xanthan gum oligosaccharides and / or degrade xanthan gum includes the following steps: using xanthan gum as a substrate, the xanthan gum is degraded by catalyzing the xanthan gum using the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7, thereby generating the xanthan gum oligosaccharides.

[0028] In some embodiments, when the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 is used to catalyze the degradation of xanthan gum, the catalytic reaction conditions are: temperature of 30-50°C and pH of 4-9.

[0029] In some embodiments, when the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 is used to catalyze the degradation of xanthan gum, the catalytic reaction conditions are: a temperature of 40-50°C and a pH of 5-8.

[0030] In some embodiments, a method for producing the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 includes the following steps:

[0031] (1) The encoding gene of the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 is ligated with plasmid pET-22b to obtain a recombinant expression vector, which is then introduced into Escherichia coli BL21(DE3) to obtain a recombinant strain.

[0032] (2) Cultivate the recombinant strain described in step (1) to induce the expression of the encoding gene of the xanthan gum cleavage ancestor enzyme AncXLY196 or the xanthan gum cleavage ancestor enzyme mutant AncXLY196-X7.

[0033] (3) Collect the bacterial cells obtained after the culture in step (2), sonicate them, separate the solid and liquid, collect the supernatant, and obtain the crude enzyme solution of xanthan gum cleavage progenitor enzyme AncXLY196 or xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7.

[0034] In some embodiments, the method for producing the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 further includes a separation and purification step, which includes the following steps:

[0035] (I) The crude enzyme solution of the xanthan gum cleavage progenitor enzyme AncXLY196 or the xanthan gum cleavage progenitor enzyme mutant AncXLY196-X7 is loaded onto a nickel ion affinity chromatography column equilibrated with binding buffer at a flow rate of 0.5 ~ 5 mL / min, and the permeate is collected and the loading is repeated once.

[0036] (II) Wash 5 to 15 column volumes each with washing buffers containing 50 mM and 100 mM imidazole to remove contaminating proteins.

[0037] (III) Elute with elution buffer containing 150-500 mM imidazole and collect the target protein peak. Finally, desalt and concentrate the eluent using an ultrafiltration centrifuge tube to obtain purified xanthan gum lyase or its mutant.

[0038] Beneficial effects:

[0039] This invention employs ancestral sequence reconstruction technology and, based on evolutionary relationships inferred from phylogenetic analysis, computationally designed an ancestral xanthan gum lysin. Compared to the wild-type xanthan gum lysin, its enzyme activity and thermostability are significantly improved. Specifically, the optimal reaction temperature of the ancestral enzyme AncXLY196 is 5°C higher than that of the wild-type enzyme, reaching 45°C. After incubation at 45°C for 2 hours, the enzyme activity showed no significant decrease, while the wild-type enzyme was almost completely inactivated under the same conditions. Building upon this, this invention rationally designed and obtained a mutant of the xanthan gum lysin, AncXLY196-X7. Under the same conditions, its xanthan gum degradation activity reached 2.5 times that of the ancestral enzyme, and its thermostability was further improved: after treatment at 45-55°C for 12-24 hours, the enzyme activity retained 50-80%, while the ancestral enzyme retained 0% of its activity under the same conditions. Attached Figure Description

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0041] Figure 1 The phylogenetic tree is reconstructed from the ancestral sequence in Example 1.

[0042] Figure 2The image shows SDS-PAGE electrophoresis images of wild-type xanthan gum lyase, xanthan gum lysin ancestor enzyme, and crude and purified ancestor enzyme mutants in Example 2. Lane M represents the DL 180 kDa protein molecule standard, lane 1 represents the crude wild-type xanthan gum lyase, lane 2 represents the purified wild-type xanthan gum lyase, lane 3 represents the crude xanthan gum lysin ancestor enzyme, lane 4 represents the purified xanthan gum lysin ancestor enzyme, lane 5 represents the crude ancestor enzyme mutant, and lane 6 represents the purified ancestor enzyme mutant.

[0043] Figure 3 The relative hydrolytic activities of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant on xanthan gum substrates in Example 5 are shown.

[0044] Figure 4 The optimal temperature and temperature stability analysis of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant in Example 6 are shown in Figure a. Figure a shows the relative enzyme activity of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant at different catalytic temperatures, and Figure b shows the relative enzyme activity of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant after incubation at 45°C for different durations.

[0045] Figure 5 The optimal pH and acid-base tolerance of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant in Example 7 are analyzed. Figure a shows the relative enzyme activities of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant at different catalytic pH, and Figure b shows the relative enzyme activities of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant after incubation at different pH. Detailed Implementation

[0046] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0047] Example 1: Reconstruction of the ancestral sequence of xanthan gum lyase

[0048] Derived from Bacillus ( BacillusUsing the amino acid sequence (UniProt ID: Q9AQS0) of xanthan lyase (XLY) sp. GL1 as the starting template, a BLASTP homology search was performed in the UniProt and NCBI databases, yielding 136 homologous protein sequences. A phylogenetic tree was constructed using the IQ-TREE software based on the LG amino acid substitution model. Based on the constructed phylogenetic tree, ancestral sequences were reconstructed using the maximum likelihood method with the PAML software package (CodeML module). The ancestral sequence corresponding to node 196, which is closely related to the evolution of the target modern enzyme, was selected for further study and named AncXLY196. The reconstructed AncXLY196 sequence was used with SignalP 6.0 software to predict and remove its N-terminal signal peptide, obtaining the mature catalytic domain sequence. The amino acid sequence of this xanthan lyase ancestor enzyme AncXLY196 is shown in SEQ ID NO.2, and its encoding gene sequence is shown in SEQ ID NO.3.

[0049] Example 2 Construction of a xanthan gum cleavage ancestor enzyme mutant

[0050] The differences in the binding ligand PyrMan between the wild-type enzyme XLY and the ancestral enzyme AncXLY196 were analyzed using molecular docking, molecular dynamics simulations (GROMACS), and binding free energy calculations (gmxMMPBSA). Based on the energy decomposition results, key residues of the ancestral enzyme were rationally designed, and corresponding sites were reverted to the amino acids of the wild-type enzyme XLY. The mutation sites were E73R, S120L, D135K, Y230Q, Q236D, N399P, and V589A, thus obtaining the combinatorial mutant AncXLY196-X7. The amino acid sequence of this xanthan gum cleavage ancestral enzyme AncXLY196 is shown in SEQ ID NO.4, and its encoding gene sequence is shown in SEQ ID NO.5.

[0051] Example 3: Heterologous expression and preparation of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant

[0052] The Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the whole genomes of the ancestral sequence obtained in Example 1, the mutant AncXLY196-X7 obtained in Example 2, and the wild-type xanthan gum lyase XLY. The genes were then cloned into the expression vector pET-22b to construct the recombinant expression vectors pET-22b-AncXLY196, pET-22b-AncXLY196-X7, and pET-22b-XLY.

[0053] The recombinant expression vectors pET-22b-AncXLY196, pET-22b-AncXLY196-X7, and pET-22b-XLY were transformed into *E. coli* BL21(DE3) competent cells using the heat shock method, and positive clones were screened. Single colonies were picked and inoculated into LB broth containing ampicillin and cultured at 37°C with shaking until OD500. 600 The concentration was 0.4–0.8. IPTG was added to a final concentration of 0.2–1.0 mM, and expression was induced at 16–28°C for 20–24 h. The induced bacterial cells were collected (centrifuged at 8000 rpm for 10 min), washed three times with sterile physiological saline, and resuspended in PBS buffer (pH 7.0–7.5). The bacterial suspension was sonicated and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected to obtain the crude enzyme solution containing the target protein.

[0054] The above three crude enzyme solutions were subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown, the molecular weight of wild-type xanthan gum lyase is 97.6 kDa. Both the xanthan gum lyase ancestor and its mutant showed a distinct band at 97.6 kDa, indicating that the three target proteins were successfully induced to express.

[0055] Example 4: Isolation and purification of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant

[0056] Recombinant wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant all have a six-histidine (His) tag fused to their C-terminus; therefore, nickel affinity chromatography was used for purification. The cell lysis supernatant (crude enzyme solution) obtained in Example 3 was used as the purification sample. Using an AKTA protein purification system equipped with a 5 mL nickel column, the column was first equilibrated with binding buffer. The sample was then loaded at a flow rate of 0.5–5 mL / min, and the permeate was collected and the loading was repeated once to ensure sufficient adsorption of the target protein. Washing buffers containing 50 and 100 mM imidazole were used sequentially, eluting approximately 5–15 column volumes each, to remove non-specific contaminants that did not bind well to the nickel column. Finally, elution buffers containing 150–500 mM imidazole were used to elute the specifically bound His-tagged protein, and the elution peak was collected. For further removal of salt and imidazole and protein concentration, Amicon was used. ® The eluent was desalted and concentrated using Ultra-15 ultrafiltration centrifuge tubes. The purity of the final purified protein was determined by SDS-PAGE electrophoresis.

[0057] SDS-PAGE electrophoresis results are as follows: Figure 2As shown, the molecular weight of wild-type xanthan gum lyase is 97.6 kDa. Both the xanthan gum lyase ancestor and its mutants show a distinct band at 97.6 kDa, and almost no bands of other molecular weights are observed after purification.

[0058] Example 5 Enzyme activity assay of wild-type xanthan gum lyase, ancestral enzyme and ancestral enzyme mutant

[0059] The DNS method was used to determine the enzyme activities of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant. The specific procedures were as follows: 100 μL of purified enzyme solutions of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant were mixed with 900 μL of 10 g / L xanthan gum solution (solvent: pH 5.5, 50 mM sodium acetate buffer) to make a total reaction volume of 1 mL. The mixture was incubated at 45℃ for 1 h, then heated in a boiling water bath for 10 min to terminate the reaction. After the reaction solution cooled to room temperature, 200 μL was taken, and 300 μL of DNS reagent was added. The mixture was incubated in a boiling water bath for 5 min, and then rapidly cooled. The resulting solution was then added to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader. The reducing sugar content was calculated based on a standard curve prepared using glucose as a standard, and the enzyme activity was then calculated accordingly. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the degradation of xanthan gum to produce 1 μmol of reducing sugar per minute at 45°C and pH 5.5.

[0060] The calculated enzyme activities of wild-type xanthan gum lyase, the ancestral enzyme, and the ancestral enzyme mutant were 3.72 U / mL, 2.91 U / mL, and 7.27 U / mL, respectively. Using the wild-type xanthan gum lyase activity as 100%, the relative enzyme activities of the ancestral enzyme and the ancestral enzyme mutant relative to wild-type xanthan gum lyase were calculated, and the results are as follows: Figure 3 As shown, the relative enzyme activity of the ancestral enzyme mutant is 2.5 times higher than that of the ancestral enzyme.

[0061] Example 6 Determination of Optimal Reaction Temperature and Thermal Stability

[0062] (1) Determination of optimal temperature

[0063] The purified enzyme solutions of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant obtained in Example 4 were diluted to a certain concentration, and 100 μL of each solution was added to a test tube containing 900 μL of xanthan gum substrate at a concentration of 10 g / L (solvent: pH 5.5, 50 mM sodium acetate buffer). The reactions were carried out at 30, 40, 45, 50, 55, 60, and 65 °C for 1 h, respectively. The enzyme activities of the three enzymes at various temperatures were calculated according to the method in Example 5. The highest enzyme activity of each enzyme was taken as 100%, and the relative enzyme activities of the three enzymes at different temperatures were calculated. Curves of enzyme activity versus temperature were plotted.

[0064] The results are as follows Figure 4 As shown in Figure a, the optimal reaction temperature for wild xanthan gum lyase is 40℃, while the optimal temperature for the ancestral enzyme and the ancestral enzyme mutant is 45℃. When the temperature is higher than 55℃, the enzyme activities of xanthan gum lyase, ancestral enzyme, and mutant enzyme all decrease significantly.

[0065] (2) Thermal stability test

[0066] The purified enzyme solutions of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant obtained in Example 4 were incubated at 45°C for 2 to 24 hours. Following the method described in Example (1), an enzyme catalytic reaction system was prepared, and the enzymes were subjected to their respective optimal temperatures for 1 hour. Enzyme activity was then determined using the method described in Example 5. The relative enzyme activity was calculated using the pure enzyme solutions of the three enzymes that had not undergone 45°C incubation as controls.

[0067] The results are as follows Figure 4 As shown in b, the wild-type enzyme exhibited the worst thermostability, almost completely losing its activity after incubation at 45°C for 2 hours. In contrast, the ancestral enzyme AncXLY196 showed significantly enhanced thermostability, with no significant decrease in activity observed after incubation at 45°C for 2 hours. Further examination of long-term thermostability revealed that after incubation at 45°C for 24 hours, the ancestral enzyme AncXLY196 completely lost its activity, while the mutant AncXLY196-X7 still maintained more than 60% of its residual enzyme activity.

[0068] Example 7 Determination of optimal reaction pH and acid-base tolerance

[0069] Prepare buffer solutions with different pH values: 50 mM sodium acetate buffer with pH values ​​of 3.0 to 6.0, 50 mM HEPES buffer with pH values ​​of 6.0 to 8.0, and 50 mM glycine-sodium hydroxide buffer with pH values ​​of 9.0 to 11.0. Use the above buffer solutions as solvents to prepare xanthan gum solutions with different pH values ​​and a concentration of 10 g / L.

[0070] (1) Determination of the optimal reaction pH

[0071] The purified enzyme solutions of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant obtained in Example 4 were diluted to a certain concentration, and 100 μL was added to test tubes containing 900 μL of xanthan gum substrate at different pH values ​​with a concentration of 10 g / L. The reaction was carried out at 45°C for 1 h. The enzyme activities of the three enzymes at different pH values ​​were calculated according to the method in Example 5. The highest enzyme activity of each of the three enzymes was taken as 100%, and the relative enzyme activities of the three enzymes at different pH values ​​were calculated. Curves of enzyme activity versus substrate pH were plotted.

[0072] The results are as follows Figure 5 As shown in a, the optimal reaction pH for wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant is 5.5. Under the condition of pH 5.5, they have the highest hydrolytic activity. As the pH increases or decreases, the activity of all three enzymes decreases.

[0073] (2) Acid-base tolerance test

[0074] The purified enzyme solutions of wild-type xanthan gum lyase, ancestral enzyme, and ancestral enzyme mutant obtained in Example 4 were diluted with buffers of different pH values ​​and incubated at 40°C for 1 h. Immediately after incubation, 100 μL of the enzyme solution was added to 900 μL of a 10 g / L xanthan gum substrate solution at pH 5.5, and the reaction was carried out at 45°C for 1 h. The remaining enzyme activity was then determined using the method described in Example 5. Using the enzyme activity of the three enzymes pre-incubated at pH 5.5 as 100%, the relative enzyme activities of the three enzymes after incubation at various pH conditions were calculated, and pH stability curves were plotted.

[0075] The results are as follows Figure 5 As shown in b, the stability of the ancestral enzyme mutant was significantly better than that of the wild-type xanthan gum lyase and the ancestral enzyme in the pH range of 6.0 to 11.0. After incubation for 1 h under various pH conditions, it could retain more than 80% of the enzyme activity. However, under the condition of pH 3.0, the stability of the wild-type xanthan gum lyase, the ancestral enzyme and the ancestral enzyme mutant all decreased significantly.

[0076] In summary, this invention not only verifies the effectiveness of ancestral sequence reconstruction technology in industrial enzymatic modification, but also specifically obtains a class of xanthan gum lyase variants with superior catalytic performance and significantly enhanced thermal stability, providing a competitive new enzyme resource for the efficient biodegradation and industrial application of xanthan gum.

[0077] This invention provides a method and approach for the reconstruction of xanthan gum cleavage ancestor enzymes based on ancestral sequences, their mutants, and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A xanthan gum cleavage ancestral enzyme, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A xanthan gum cleavage ancestral enzyme mutant, characterized in that, Its amino acid sequence has at least 99% sequence identity with SEQ ID NO.

2.

3. The xanthan gum cleavage ancestor enzyme mutant according to claim 2, characterized in that, The xanthan gum cleavage ancestor enzyme mutant was obtained by mutating glutamic acid at position 73 of SEQ ID NO.2 to arginine, serine at position 120 to leucine, aspartic acid at position 135 to lysine, tyrosine at position 230 to glutamine, glutamine at position 236 to aspartic acid, asparagine at position 399 to proline, and valine at position 589 to alanine.

4. A nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme of claim 1 or a mutant of the xanthan gum cleavage progenitor enzyme of claim 2 or 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme is shown in SEQ ID NO.3; the nucleotide sequence of the nucleic acid molecule encoding the xanthan gum cleavage progenitor enzyme mutant is shown in SEQ ID NO.

5.

6. An expression cassette or recombinant expression vector containing the nucleic acid molecule of claim 4.

7. The expression cassette or recombinant expression vector according to claim 6, characterized in that, The starting vector of the recombinant expression vector can be any one of the pET series vectors, pHT series vectors, pBE series vectors, and pPIC series vectors, preferably pET-22b.

8. Recombinant bacteria containing the expression cassette or recombinant expression vector as described in claim 6.

9. The recombinant bacteria according to claim 8, characterized in that, The recombinant bacteria originating from any one of Escherichia coli, Bacillus, and Pichia pastoris, preferably Escherichia coli. E. coli BL21(DE3).

10. The application of the xanthan gum cleavage progenitor enzyme of claim 1 or the xanthan gum cleavage progenitor enzyme mutant of claim 2 or 3 in the preparation of xanthan gum oligosaccharides and / or degradation of xanthan gum; preferably, the method for preparing xanthan gum oligosaccharides and / or degrading xanthan gum using the xanthan gum cleavage progenitor enzyme of claim 1 or the xanthan gum cleavage progenitor enzyme mutant of claim 2 or 3 comprises the following steps: using xanthan gum as a substrate, using the xanthan gum cleavage progenitor enzyme or the xanthan gum cleavage progenitor enzyme mutant to catalyze the degradation of xanthan gum, thereby generating the xanthan gum oligosaccharides.

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