Alkali-resistant xanthan side chain lyase mutants and methods for making same

Abstract

This invention discloses an alkali-resistant xanthan gum side-chain lyase mutant and its preparation method, which is effective against alkali-resistant xanthan gum from Bacillus subtilis (Bacillus spp.). Paenibacillus nanensis Site-directed mutagenesis was performed on wild-type xanthan gum side-chain lyase to obtain alkali-resistant xanthan gum side-chain lyase mutants. The site-directed mutagenesis included at least one of Y260V, T387A, and I422S. The xanthan gum side-chain lyase mutants of this invention showed significantly increased enzyme activity compared to the original enzyme after treatment with an alkaline buffer system at 40°C, with the three mutants Y260V / T387A / I422S showing the greatest increase. These mutants are suitable for large-scale degradation of xanthan gum side chains under alkaline conditions, possessing significant social and economic benefits and broad application prospects.

Description

Technical Field

[0001] This invention relates to a xanthan gum lyase mutant and its preparation method, and more particularly to an alkali-resistant xanthan gum side chain lyase mutant and its preparation method. Background Technology

[0002] Xanthan lyase is a polysaccharide degrading enzyme that specifically degrades xanthan gum. Xanthan gum has a complex macromolecular structure, with its main chain consisting of alternating β-(1→4)-D-glucose and β-(1→4)-D-glucuronic acid. The side chains are regularly linked to the main chain and consist of trisaccharide residues composed of mannose, glucuronic acid, and mannose. The terminal mannose often carries modifying groups such as acetyl or propionyl groups.

[0003] Xanthan gum lyases can be classified into main-chain lyases and side-chain lyases based on their sites of action. Main-chain lyases directly degrade the polysaccharide backbone by breaking the glycosidic bonds in the main chain, showing significant effects in altering molecular weight; however, the substrate recognition of these enzymes is limited by the complex spatial configuration of the main and side chains, making their screening and expression challenging. In contrast, side-chain lyases specifically act on the glycosidic bonds between mannose and glucuronic acid in the xanthan gum side chain, rapidly reducing the viscosity of xanthan gum and altering its solution rheology through hydrolysis. They are widely used in the regulation of high-viscosity systems, demonstrating good substrate adaptability and promising practical applications.

[0004] Naturally derived xanthan gum side-chain lyases generally suffer from low catalytic activity and instability in alkaline environments, which particularly limits their application in industrial fields such as papermaking and textile wastewater treatment using alkaline reaction systems. Although some literature exists on xanthan gum derived from... Paenibacillus nanensis The wild-type xanthan gum side chain lyase was modified, but the mutant only showed maximum enzyme activity at pH=6 and did not show excellent tolerance under alkaline conditions. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a xanthan gum side chain lyase mutant with high catalytic activity and excellent alkali resistance, and to provide the relevant nucleotide sequence, recombinant vector, and recombinant cells. Another purpose of this invention is to provide a method for preparing the enzyme mutant.

[0006] Technical solution: The alkali-resistant xanthan gum side chain lyase mutant of the present invention is obtained by site-directed mutagenesis of xanthan gum lyase with the sequence shown in SEQ ID NO.1, wherein the mutation site of the site-directed mutagenesis includes at least one of Y260V, T387A, and I422S.

[0007] Furthermore, the alkali-resistant xanthan gum lyase mutants are Y260V, T387A, I422S, Y260V / I422S, and Y260V / T387A / I422S.

[0008] This invention uses Bacillus subtilis (B.) Paenibacillus nanensis The xanthan gum side-chain lyase is the original enzyme (wild type), whose amino acid sequence is SEQ ID NO.1, and the gene sequence encoding the enzyme is SEQ ID NO.2. This invention obtained a xanthan gum side-chain lyase mutant with high catalytic activity and excellent alkali resistance by performing site-directed mutagenesis on this enzyme.

[0009] This invention uses standard single-letter codes and standard substitution notation for amino acids. For example, Y260V means that tyrosine (Y) at position 260 is mutated to valine (V); Y260V / T387A means that tyrosine (Y) at position 260 is mutated to valine (V), and threonine (T) at position 387 is mutated to alanine (A); Y260V / T387A / I422S means that tyrosine (Y) at position 260 is mutated to valine (V), threonine (T) at position 387 is mutated to alanine (A), and isoleucine (I) at position 422 is mutated to serine (S).

[0010] The nucleotide sequence of the present invention encodes the alkali-resistant xanthan gum side chain lyase mutant, and the nucleotide sequence can be obtained by base mutation of the sequence shown in SEQ ID NO.2.

[0011] The recombinant vector of the present invention comprises the nucleotide sequence described herein. The recombinant vector includes a cloning vector or an expression vector, optionally a plasmid or a virus, which can maintain replication ability in a host cell and amplify or express the nucleotide sequence.

[0012] The recombinant cells of the present invention comprise the recombinant vector, wherein the recombinant cells are preferably... E. coli DH5α or E. coli BL21.

[0013] The method for preparing the alkali-resistant xanthan gum side-chain lyase mutant of the present invention includes the following steps:

[0014] (1) Using the wild-type xanthan gum side chain lyase gene as a template, a PCR reaction was performed using point mutation primers to obtain the xanthan gum side chain lyase mutant gene;

[0015] (2) Insert the xanthan gum side chain lyase mutant gene into the expression vector, and then transfer it into the host cell to obtain recombinant cells, and induce expression of xanthan gum side chain lyase mutant.

[0016] Preferably, in step (1), the point mutation primers are Y260V-F, Y260V-R, T387A-F, T387A-R, I422S-F, and I422S-R, with sequences from SEQ ID NO.3 to SEQ ID NO.8. Using computer simulation, the key mutation sites are determined by predicting the three-dimensional structure and functional sites of the original xanthan gum side chain lyase, and specific primers required for site-directed mutagenesis are designed.

[0017] Preferably, in step (1), the PCR reaction system is as shown in Table 1:

[0018] Table 1 PCR reaction system

[0019] .

[0020] Preferably, in step (1), the PCR reaction conditions are as shown in Table 2:

[0021] Table 2 PCR reaction conditions

[0022] .

[0023] Preferably, in step (2), the xanthan gum side chain lyase mutant gene is inserted into the expression vector by DNA homologous recombination.

[0024] Preferably, in step (2), the host cell is *Escherichia coli*, including... E. coli DH5α and E. coli BL21.

[0025] Preferably, in step (2), after expression is completed, the expressed cells are collected; or the cells are broken, crude enzyme solution is extracted, and the xanthan gum side chain lyase mutant is collected and purified.

[0026] The product of this invention comprises the alkali-resistant xanthan gum side-chain lyase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell.

[0027] The present invention also provides the application of the product in the degradation of xanthan gum side chains, wherein the pH of the application is 8-12.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides an alkali-resistant xanthan gum side chain lyase mutant. At 40°C, the enzyme activity of the mutants treated with an alkaline buffer system is significantly improved compared with the original enzyme, and the triple mutant Y260V / T387A / I422S shows the greatest improvement. It is suitable for the large-scale degradation of xanthan gum side chains under alkaline conditions, has important social and economic benefits, and has broad application prospects. Attached Figure Description

[0029] Figure 1 A schematic diagram of the mutation sites Y260V, T387A, and I422S on the original xanthan gum side chain lyase;

[0030] Figure 2 A standard curve for colorimetric determination of enzyme activity;

[0031] Figure 3 This is a schematic diagram showing the relative enzyme activities of the original xanthan gum side chain lyase and the mutant after alkaline treatment.

[0032] Figure 4 The relative enzyme activity changes of the original xanthan gum side chain lyase and the mutant under different pH conditions;

[0033] Figure 5 The remaining relative enzyme activity of the original xanthan gum side chain lyase and the mutant after pretreatment at different pH for 1 h is given. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] The acquisition methods of the materials used in the embodiments:

[0036] 1. Strains and plasmids: Synthesis of original strains Paenibacillus nanensis The pET-22b(+) expression vector of the xanthan gum side-chain lyase gene, the synthesis of mutant primers and plasmid construction primers, and the sequencing verification of the target gene were all completed by Genewiz (Suzhou) Co., Ltd. The expression plasmid of the xanthan gum side-chain lyase mutant was constructed by the inventors. The strain used for plasmid amplification... E. coli DH5α and strains used for protein expression E. coli BL21(DE3) was purchased from Shenzhen Kangti Life Technology Co., Ltd.

[0037] 2. Experimental reagents and culture media: The enzymes and buffers used in PCR were all from the MutUFO rapid site-directed mutagenesis kit provided by Nanjing Jujiang Biotechnology Co., Ltd., the plasmid extraction kit and DNA marker were from Shanghai Sangon Biotech Co., Ltd., the DpnI endonuclease used to remove template DNA was from Adamas, the DNS reagent used to determine enzyme activity was purchased from Phygene, and the remaining experimental reagents were all domestically produced analytical grade reagents.

[0038] The LB medium formula is: yeast extract 5g / L, tryptone 10g / L, NaCl 5g / L. If a solid medium is to be prepared, add 15g / L of agar powder.

[0039] The composition of TB liquid culture medium includes: yeast extract 12 g / L, tryptone 12 g / L, glycerol 4 mL / L, dipotassium hydrogen phosphate 12.5 g / L and potassium dihydrogen phosphate 2.3 g / L.

[0040] Example 1: Alkali-resistant xanthan gum side-chain lyase mutant Y260V

[0041] Bacillus subtilis ( Paenibacillus nanensis Using xanthan gum side-chain lyase from xanthan gum as a template, the tyrosine (Tyr) at position 260 of its amino acid sequence was mutated to valine (Val), constructing the single-point mutant Y260V. The mutation site is as follows: Figure 1 As shown. The preparation method is as follows: (1) Design and synthesize point mutation primers, perform PCR reaction to obtain the mutant gene; (2) Construct expression vector and transform into host bacteria for induced expression. The details are as follows:

[0042] (1) Design and synthesize point mutation primers, perform PCR reaction to obtain the mutant gene Y260V, and the primers for the mutation site are shown in Table 3. PCR reaction was performed using pET-22b(+) plasmid containing the original xanthan gum side chain lyase gene as a template. The PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5.

[0043] Table 3. Primers for point mutation and primers for plasmid construction

[0044]

[0045] Note: The underlined part in the primers indicates the mutation site. "F" represents the upstream primer and "R" represents the downstream primer.

[0046] Table 4 PCR reaction system

[0047]

[0048] Table 5 PCR reaction conditions

[0049]

[0050] After verification by nucleic acid electrophoresis, the PCR products were digested with the restriction enzyme DpnI. The template digestion system shown in Table 6 was prepared and digested at 37℃ for 1-2 hours.

[0051] Table 6 Template Digestion System

[0052]

[0053] After template digestion, the product was subjected to DNA homologous recombination. The homologous recombination system shown in Table 7 was prepared, and one-step cloning was performed at 37℃ for 30 min to construct the expression plasmid of the mutant enzyme.

[0054] Table 7 Homologous Recombination System

[0055]

[0056] The obtained plasmid was transformed by engineered bacteria E. coli After amplification of DH5α, recombinant plasmids were extracted and sequenced for verification.

[0057] (2) Constructing the expression vector and inducing expression after transformation into the host bacteria: The recombinant plasmid containing the Y260V mutant gene was constructed and transformed into the expression host. E. coli In BL21(DE3), the culture was spread on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C for positive screening. A single colony was picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured with shaking at 37°C and 140 rpm for 12 h. 2.5 mL of the seed culture was transferred to 50 mL of TB medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 140 rpm for 6 h until the bacterial culture reached the logarithmic growth phase. Filtered and sterilized IPTG solution to a final concentration of 0.4 mM was added to the fermentation broth, and expression was induced for another 22 h with shaking at 22°C and 140 rpm. After incubation, the bacterial cells were collected by centrifugation.

[0058] Example 2: Alkali-resistant xanthan gum side-chain lyase mutant T387A

[0059] This embodiment uses the same original enzyme as in Example 1, but mutates threonine (Thr) at position 387 of its amino acid sequence to alanine (Ala) to construct a single-point mutant T387A. The mutation site is as follows: Figure 1 As shown in Table 8. The preparation method is basically the same as in Example 1, except for the mutation primers.

[0060] Table 8. Primers for point mutation and primers for plasmid construction

[0061]

[0062] Example 3: Alkali-resistant xanthan gum side-chain lyase mutant I422S

[0063] This embodiment uses the same original enzyme as in Example 1, but mutates isoleucine (Ile) at position 422 of its amino acid sequence to serine (Ser), constructing a single-point mutant I422S. The mutation site is as follows: Figure 1 As shown in Table 9. The preparation method is basically the same as in Example 1, except for the mutation primers.

[0064] Table 9. Primers for point mutation and primers for plasmid construction

[0065]

[0066] Example 4: Alkali-resistant xanthan gum side-chain lyase double mutant Y260V / I422S

[0067] This embodiment uses the same original enzyme as in Example 1, but introduces two mutation sites: tyrosine (Tyr) at position 260 of its amino acid sequence is mutated to valine (Val), and isoleucine (Ile) at position 422 of its amino acid sequence is mutated to serine (Ser), constructing the double mutant Y260V / I422S. The preparation method is basically the same as in Example 1, the difference being in the mutation primers, as shown in Table 10.

[0068] Table 10. Primers for point mutation and primers for plasmid construction

[0069]

[0070] Example 5: Alkali-resistant xanthan gum side-chain lyase triple mutant Y260V / T387A / I422S

[0071] This embodiment uses the same original enzyme as in Example 1, but introduces three mutation sites: tyrosine (Tyr) at position 260 of its amino acid sequence is mutated to valine (Val), threonine (Thr) at position 387 is mutated to alanine (Ala), and isoleucine (Ile) at position 422 is mutated to serine (Ser), constructing the triple mutant Y260V / T387A / I422S. The preparation method is basically the same as in Example 1, the difference being in the mutation primers, as shown in Table 11.

[0072] Table 11 Point mutation primers and plasmid construction primers

[0073]

[0074] Performance testing:

[0075] 1. Enzyme activity assay of xanthan gum side chain lyase and its mutants at pH 7.5:

[0076] DNS method for determination Paenibacillus nanensis The catalytic activity of wild-type xanthan gum side chain lyase (WT) and mutants obtained in Examples 1-5 (Y260V, T387A, I422S, Y260V / I422S, Y260V / T387A / I422S) at pH=7.5 was compared to evaluate the enzymatic effects of different mutants.

[0077] Crude enzyme solution was prepared using wild-type xanthan gum side-chain lyase and recombinant mutant cells: The fermentation broth of the recombinant bacteria was centrifuged at 11,000 rpm and the cells were collected. The cells were resuspended in a 20 mL buffer system. The OD of the system was adjusted. 600 The concentration was 40. Cells were lysed by sonication for 10 minutes and centrifuged at 12,000 rpm. The collected supernatant was the crude enzyme solution.

[0078] The buffer system is a KPi buffer (potassium phosphate buffer), prepared by mixing KH2PO4 and K2HPO4 in a certain ratio, adjusting the final concentration to 100mM, and setting the pH to 7.5. The specific ratio is: 0.680g KH2PO4, 0.870g K2HPO4, with ddH2O added to bring the volume to 100mL. The solution is then sterilized using a 0.22μm filter membrane before use.

[0079] Add 30 μL of crude enzyme solution to 120 μL of 0.5% (w / v) xanthan gum solution, mix well, and incubate in a 40°C water bath for 5 min. After the reaction, add 150 μL of DNS reagent, heat in a boiling water bath for 10 min, and immediately cool to room temperature. Measure the absorbance at 540 nm using a UV spectrophotometer. For the negative control, replace the crude enzyme solution with the same volume of buffer solution and follow the same procedure.

[0080] To accurately calculate the glucose concentration released during the enzymatic reaction, glucose standard solutions of 0.7500, 0.5000, 0.3750, 0.2500, 0.1875, and 0.1250 mg / mL were prepared. 150 μL of each glucose standard solution was mixed with 150 μL of DNS reagent, heated in a boiling water bath for 10 min, and then rapidly cooled to room temperature after the reaction. The absorbance was measured at 540 nm using a UV spectrophotometer. The standard curve was plotted using the same volume of buffer solution instead of the crude enzyme solution, following the same procedure. Figure 2 The standard curve shown is the glucose standard curve at 540 nm:

[0081] y = 5.4781x - 0.0857, R 2 = 0.9989.

[0082] Where y is the absorbance at 540 nm and x is the glucose concentration (mg / mL). A standard curve was prepared for each batch of samples and detected simultaneously to ensure the accuracy of the colorimetric analysis and the repeatability of the experimental results.

[0083] Enzyme activity unit is defined as: under the given reaction conditions, the amount of enzyme required to catalyze the hydrolysis of xanthan gum and release 1 nmol of glucose per minute is 1 U. Specific enzyme activity represents the catalytic capacity (U / mL) of a unit volume of crude enzyme solution, which is calculated by converting the released sugar concentration from the standard curve.

[0084] Test results are as follows Figure 3 As shown in Table 12, from Figure 3 It was found that, after treatment with a buffer system at pH 7.5 at 40℃, the mutants all showed higher enzyme activity compared to the original enzyme. Among them, the triple mutant (Y260V / T387A / I422S) had the best enzyme activity, with its specific enzyme activity being 3.4 times higher than that of the wild-type enzyme.

[0085] Table 12 Enzyme activities of xanthan gum side chain lyase and different mutants at pH 7.5

[0086]

[0087] 2. Enzyme activity assay of xanthan gum side chain lyase and its mutants under different pH conditions:

[0088] Systematic evaluation was conducted using different buffer systems with pH values ​​ranging from 4.0 to 12.0 at 40°C. Paenibacillus nanensis The catalytic activity changes of wild-type xanthan gum side chain lyase (WT) and its mutants (Y260V, Y260V / I422S, Y260V / T387A / I422S) under acidic, neutral and alkaline conditions were investigated to determine the optimal pH and the adaptability of the mutants over a wide pH range.

[0089] (1) Preparation of crude enzyme solution: The preparation method of crude enzyme solution is as described above. Take the fermentation broth of WT and each mutant recombinant bacteria, collect the bacterial cells by centrifugation at 11000 rpm, resuspend in 100 mM KPi buffer, and adjust the OD of the system. 600 Adjust to 40. After ultrasonic disruption, centrifuge at 12,000 rpm and collect the supernatant as crude enzyme solution for later use.

[0090] The required buffer solutions of different pH values ​​are all prepared using the KH2PO4 / K2HPO4 or Gly-NaOH system, with pH ranges covering 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0.

[0091] (2) Enzyme activity assay method: The enzyme activity assay method is as described above, except that the pH of the reaction system is changed. The specific operation is as follows: Take 30 μL of crude enzyme solution and add it to 120 μL of 0.5% (w / v) xanthan gum substrate solution prepared with the corresponding pH buffer. Mix well and place in a 40℃ water bath for 5 min. After the reaction is complete, add 150 μL of DNS reagent, heat in a boiling water bath for 10 min, and immediately cool to room temperature after the reaction is complete. Measure the absorbance at 540 nm using a UV spectrophotometer. The standard curve is prepared by replacing the crude enzyme solution with the same volume of buffer solution, and the rest of the operation is exactly the same.

[0092] The quantitative method for reducing sugars is as described above, with the amount produced converted from the glucose standard curve. The calculation methods for enzyme activity units and specific enzyme activity are as described above. To facilitate comparison of the catalytic abilities of different enzymes, the enzyme activity of WT at pH=7.0 was set to 100%, and all other test conditions were converted to relative enzyme activity (%).

[0093] Experimental results are as follows Figure 4 As shown, the relative enzyme activity of the single mutant Y260V was increased compared to WT across the entire pH range, reaching approximately 2.13–2.45 times at pH 7–9. However, it decreased to 1.33 times under strongly alkaline conditions (pH 12), indicating a limited increase. The relative enzyme activity of the double mutant Y260V / I422S was further increased compared to the single mutant Y260V, reaching 2.62–2.83 times at pH 7–9, with the highest value at pH 8. Even under strongly alkaline conditions at pH 12, it maintained a relative enzyme activity of 2.01 times, demonstrating good alkaline activity retention. The optimal triple mutant Y260V / T387A / I422S exhibited the best performance across the entire pH range. At pH 7–8, its relative enzyme activity reached 3.23–3.60 times, the highest among all samples. Even at higher pH levels (pH 10–11), it maintained a relative enzyme activity of 2.49–2.63 times, and at pH 12, it maintained a relative enzyme activity of 2.16 times, significantly superior to other enzymes. These results indicate that enzyme activity gradually increases with the number of mutants. The triple mutant, in particular, exhibits significantly enhanced catalytic efficiency under both neutral and alkaline conditions and is the key mutant in this invention.

[0094] 3. Determination of pH stability of xanthan gum side chain lyase and its mutants under different pH conditions:

[0095] To further evaluate the structural stability of each enzyme under acidic and alkaline conditions, the tolerance of WT and its mutants (Y260V, Y260V / I422S, Y260V / T387A / I422S) in the pH range of 4.0–12.0 was tested. Enzyme solutions were added to different buffer solutions ranging from pH 4.0 to 12.0 and incubated at 40°C for 1 hour. After incubation, the system was immediately adjusted to standard reaction conditions (pH 7.0), and the enzyme catalytic reaction was carried out. The remaining activity was then determined using the DNS method described above. The enzyme activity of WT at pH 7.0 was set as 100%, and the remaining relative enzyme activity (%) of each sample was calculated.

[0096] Experimental results are as follows Figure 5As shown, for the wild-type enzyme WT, its residual relative enzyme activity decreased significantly in both acidic and alkaline environments, especially at pH ≥ 10, where the residual relative enzyme activity dropped to 0.46–0.77 times. For the single mutant Y260V, it showed some improvement under weakly alkaline conditions at pH 9–11, but the overall retention rate was still insufficient. For the double mutant Y260V / I422S, it maintained high stability in the pH range of 7–11, with a residual relative enzyme activity of 1.35–1.44 times, showing a significant improvement. For the triple mutant Y260V / T387A / I422S, its stability was the most outstanding, with a residual relative enzyme activity of 2.23–2.61 times in the pH range of 7–11, reaching its highest value at pH 8, and even under the extreme alkaline environment of pH 12, it could still maintain a residual relative enzyme activity of 1.33 times.

[0097] The above results indicate that the three mutants Y260V / T387A / I422S exhibit excellent stability over a wide pH range, especially in alkaline environments where they significantly outperform other mutants, thus meeting the alkalinity requirements of xanthan gum side chain lyase in industrial applications.

Claims

1. An alkali-resistant xanthan gum side-chain lyase mutant, characterized in that, The xanthan gum lyase of the sequence shown in SEQ ID NO.1 was obtained by site-directed mutagenesis, wherein the mutation site of the site-directed mutagenesis is one of Y260V, Y260V / I422S, or Y260V / T387A / I422S.

2. A nucleotide sequence, characterized in that, Encodes the alkali-resistant xanthan gum side-chain lyase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the nucleotide sequence of claim 2, wherein the recombinant vector comprises a cloning vector or an expression vector.

4. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 3.

5. A method for preparing an alkali-resistant xanthan gum side-chain lyase mutant as described in claim 1, characterized in that, Includes the following steps: (1) Using the wild-type xanthan gum side chain lyase gene shown in SEQ ID NO.1 as a template, a PCR reaction was performed using point mutation primers to obtain the xanthan gum side chain lyase mutant gene; (2) Insert the xanthan gum side chain lyase mutant gene into the expression vector, and then transfer it into the host cell to obtain recombinant cells, and induce expression of xanthan gum side chain lyase mutant.

6. The preparation method according to claim 5, characterized in that, In step (1), the point mutation primers are Y260V-F, Y260V-R, T387A-F, T387A-R, I422S-F, and I422S-R, and their sequences are SEQ ID NO.3 to SEQ ID NO.8, respectively.

7. The preparation method according to claim 5, characterized in that, In step (2), the host cell is Escherichia coli.

8. A product characterized in that, The invention comprises the alkali-resistant xanthan gum side-chain lyase mutant of claim 1, or the nucleotide sequence of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4.

9. The use of the product of claim 8 in the degradation of xanthan gum side chains, wherein the pH of the application is 8-12.

Images