Highly active xanthan backbone-cleaving enzyme mutants and methods for making same

Abstract

The application discloses a high-enzyme-activity xanthan backbone cleavage enzyme mutant and a preparation method thereof. Microbacterium sp. ​ The application discloses a high-enzyme-activity xanthan backbone cleavage enzyme mutant and a preparation method thereof, wherein the wild-type xanthan backbone cleavage enzyme of XT11 is subjected to site-directed mutation, and the mutation point of the site-directed mutation comprises at least one of W753V, H755G and L810S. The xanthan backbone cleavage enzyme mutant of the application can directly degrade the xanthan backbone without side chain pretreatment, thereby simplifying an enzymolysis system, and the enzyme activity of the mutant after being treated by using an alkaline buffer system at 55 DEG C is obviously improved compared with the original enzyme, wherein the three-mutant W753V / H755G / L810S has the largest improvement range, and the three-mutant W753V / H755G / L810S overcomes the limitation that a traditional xanthan cleavage system depends on side chain pre-deconstruction.

Description

Technical Field

[0001] This invention relates to a xanthan gum lyase mutant and its preparation method, and more particularly to a xanthan gum backbone lyase mutant with high enzyme activity and its preparation method. Background Technology

[0002] Xanthan gum is an extracellular polysaccharide secreted by Xanthomonas campestris, with wide applications in food, oil extraction, medicine, and papermaking. The polysaccharide backbone consists of β-(1→4)-D-glucose residues, while the side chains are composed of trisaccharide units consisting of mannose, glucuronic acid, and mannose. Some mannose residues are also modified with acetyl or propionyl. Due to its highly branched and stable structure, xanthan gum exhibits good heat resistance, acid and alkali resistance, and high viscosity, and is not easily degraded during use.

[0003] Currently, enzymatic degradation of xanthan gum is the primary method. However, commonly used xanthan gum-degrading enzymes mostly act on the side chains, leaving the main chain intact after degradation, making it difficult to further disrupt its stable structure. Although some studies have attempted to degrade the xanthan gum main chain using glycosidases such as endo-β-1,4-glucanase, these methods require pretreatment of the xanthan gum side chains, resulting in a complex process. In recent years, some literature has reported on... Microbacterium sp. A novel xanthan gum backbone lyase (NCBI protein number: ALX66163.1) was isolated from XT11. This enzyme can directly act on the backbone structure, effectively degrading xanthan gum and simplifying the process. However, the natural enzyme activity is relatively low, making it difficult to meet the high-efficiency catalytic requirements of actual production. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a xanthan gum backbone lyase mutant with high catalytic activity that can act directly on the xanthan gum backbone without side chain pretreatment, and to provide related nucleotide sequences, recombinant vectors, and recombinant cells. Another purpose of this invention is to provide a method for preparing the enzyme mutant.

[0005] Technical solution: The high-enzyme-activity xanthan gum backbone 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 W753V, H755G, and L810S.

[0006] Furthermore, the high-enzyme-activity xanthan gum lyase mutants are W753V, H755G, L810S, W753V / H755G, and W753V / H755G / L810S.

[0007] This invention uses xanthan sugar to degrade microorganisms Microbacterium spThe xanthan gum backbone lyase derived from XT11 is the original enzyme (wild type), with its amino acid sequence SEQ ID NO.1 (corresponding to NCBI number ALX66163.1), and the gene sequence encoding this enzyme is SEQ ID NO.2. This invention obtained a xanthan gum backbone lyase mutant with high enzyme activity through site-directed mutagenesis of this enzyme.

[0008] This invention uses standard single-letter codes for amino acids and standard substitution notation. For example, W753V means that tryptophan (W) at position 753 is mutated to valine (V); W753V / H755G means that tryptophan (W) at position 753 is mutated to valine (V), and histidine (H) at position 755 is mutated to glycine (G); W753V / H755G / L810S means that tryptophan (W) at position 753 is mutated to valine (V), histidine (H) at position 755 is mutated to glycine (G), and leucine (L) at position 810 is mutated to serine (S).

[0009] The nucleotide sequence of the present invention encodes the high-enzyme-activity xanthan gum backbone lyase mutant, and the nucleotide sequence can be obtained by base mutation of the sequence shown in SEQ ID NO.2.

[0010] 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.

[0011] The recombinant cells of this invention comprise the recombinant vector, and the recombinant cells are preferably *Escherichia coli*. E. coli BL21(DE3).

[0012] This invention also provides a method for preparing the high-enzyme-activity xanthan gum backbone lyase mutant, comprising the following steps:

[0013] (1) Using the wild-type xanthan gum backbone lyase gene as a template, a PCR reaction was performed using point mutation primers to obtain the xanthan gum backbone lyase mutant gene;

[0014] (2) Insert the xanthan gum backbone 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 backbone lyase mutant.

[0015] Further, in step (1), the point mutation primers are W753V-F, W753V-R, H755G-F, H755G-R, L810S-F, and L810S-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 backbone lyase, and specific primers required for site-directed mutagenesis are designed.

[0016] Preferably, in step (1), the PCR reaction system is as shown in Table 1:

[0017] Table 1 PCR reaction system

[0018] .

[0019] Preferably, in step (1), the PCR reaction conditions are as shown in Table 2:

[0020] Table 2 PCR reaction conditions

[0021] .

[0022] Preferably, in step (2), the xanthan gum backbone lyase mutant gene is inserted into the expression vector by DNA homologous recombination.

[0023] Preferably, in step (2), the host cell is *Escherichia coli*, including... E. coli DH5α and E. coli BL21.

[0024] 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 backbone lyase mutant is collected and purified.

[0025] The product of this invention comprises the high-enzyme-activity xanthan gum backbone lyase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell.

[0026] The present invention also provides the application of the product in the degradation of xanthan gum backbone, wherein the pH of the application is greater than or equal to 7.5, and the xanthan gum is unbranched.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides a xanthan gum lyase mutant with high enzyme activity, which can directly degrade the xanthan gum backbone without side chain pretreatment, simplifying the enzymatic hydrolysis system; and at 55°C, the enzyme activity of the mutants treated with a buffer system with a pH range of 4 to 12 is significantly improved compared with the original enzyme, among which the triple mutant W753V / H755G / L810S has the largest increase, overcoming the limitation of the traditional xanthan gum lysis system that depends on the pre-deconstruction of side chains. Attached Figure Description

[0028] Figure 1 A schematic diagram of the mutation sites W753V, H755G, and L810S on the original xanthan gum side chain lyase;

[0029] Figure 2 A standard curve for colorimetric determination of enzyme activity;

[0030] Figure 3 This is a schematic diagram showing the relative enzyme activities of the original xanthan gum backbone lyase and the mutant after alkaline treatment.

[0031] Figure 4 The relative enzyme activities of the original xanthan gum backbone lyase and the mutant under different pH conditions were shown.

[0032] Figure 5 The remaining relative enzyme activity of the original xanthan gum backbone lyase and the mutant after pretreatment at different pH for 1 h is given. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] The acquisition methods of the materials used in the embodiments:

[0035] 1. Strains and plasmids: Synthesis of original strains Microbacterium sp. The pET-22b(+) expression vector of the XT11 xanthan gum backbone 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 backbone lyase mutant was constructed by the inventors themselves. 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1: High-activity xanthan gum backbone lyase mutant W753V

[0040] Microorganisms that degrade xanthanose Microbacterium sp. Using xanthan gum backbone lyase derived from XT11 as a template, tryptophan (W) at position 753 of its amino acid sequence was mutated to valine (V), constructing a single-point mutant W753V. 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:

[0041] (1) Design and synthesize point mutation primers, and perform PCR reaction to obtain the mutant gene W753V. The primers for the mutation site are shown in Table 3. The pET-22b(+) plasmid containing the original xanthan gum backbone lyase gene is used as a template for PCR reaction. The PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5.

[0042] Table 3. Primers for point mutation and primers for plasmid construction

[0043]

[0044] Note: The underlined part in the primer indicates the mutation site. "F" represents the upstream primer and "R" represents the downstream primer.

[0045] The PCR amplification system consisted of 20 μL, including the Proofreading high-fidelity enzyme system, target primers, and template expression plasmid (the wild-type gene had been cloned into pET-22b(+)).

[0046] Table 4 PCR reaction system

[0047]

[0048] Table 5 PCR reaction conditions

[0049]

[0050] The amplified product was digested with the restriction enzyme DpnI to remove template DNA. The template digestion system was prepared as shown in Table 6, and digestion was carried out at 37°C 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 homologous recombination product was transformed into competent E. coli. E. coli In DH5α, after screening positive clones, plasmids were extracted and sent for sequencing to confirm that the W753V mutation was correctly inserted into the target sequence.

[0057] (2) After constructing the expression vector and transforming it into the host bacteria, expression was induced: The W753V mutant expression plasmid, which was verified by sequencing, was transformed into the expression host. E. coli In BL21(DE3), positive clones were selected and inoculated into LB liquid medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 140 rpm in a shaker. The seed culture was then inoculated into TB medium at a 1:20 ratio and cultured until the cells reached the logarithmic growth phase. When the system OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced for another 22 h at 22°C. After the culture was completed, the bacterial cells were collected by centrifugation.

[0058] Example 2: High-activity xanthan gum backbone lyase mutant H755G

[0059] In this embodiment, the same original enzyme as in Example 1 was used, but the histidine (H) at position 755 of its amino acid sequence was mutated to glycine (G) to construct a single-point mutant H755G. 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: High-activity xanthan gum backbone lyase mutant L810S

[0063] This embodiment uses the same original enzyme as in Example 1, but mutates leucine (L) at position 810 of its amino acid sequence to serine (S), constructing a single-point mutant L810S. 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: High-activity xanthan gum backbone lyase double mutant W753V / H755G

[0067] In this embodiment, the same original enzyme as in Example 1 was used, but the tryptophan (W) at position 753 of its amino acid sequence was mutated to valine (V), and the histidine (H) at position 755 of its amino acid sequence was mutated to glycine (G), constructing a double mutant W753V / H755G. The preparation method was basically the same as in Example 1, except for the mutation primers. A one-step site-directed mutagenesis method was used, introducing two mutation sites in a single PCR reaction using the same pair of primers, as shown in Table 10.

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

[0069]

[0070] Example 5: High-activity xanthan gum backbone lyase triple mutant W753V / H755G / L810S

[0071] In this embodiment, the W753V / H755G double mutant plasmid obtained in Example 4 was used as a PCR template. The leucine (L) at position 810 of its amino acid sequence was mutated to serine (S) to construct the triple mutant W753V / H755G / L810S. The preparation method is basically the same as in Example 1, except for 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 backbone lyase and its mutants at pH 7.5:

[0076] DNS method for determination Microbacterium sp. The catalytic activity of wild-type xanthan gum backbone lyase (WT) derived from XT11 and mutants obtained in Examples 1-5 (W753V, H755G, L810S, W753V / H755G, W753V / H755G / L810S) 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 backbone lyase and recombinant cells with mutants: The fermentation broth of the recombinant bacteria was centrifuged at 11,000 rpm for 10 min 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. The cells were sonicated for 10 minutes to break them up. The cells were then centrifuged at 12,000 rpm for 10 minutes. 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 at 55°C for 5 min. After the reaction, add 150 μL of DNS reagent, incubate at boiling water 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 calculate the concentration of reducing sugars released during the 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, and the mixture was reacted in a boiling water bath for 10 min. After the reaction, the mixture was immediately cooled to room temperature. 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 = 5.4699x - 0.1095, R 2 = 0.9979

[0082] Where y is the absorbance at 540 nm and x is the glucose concentration (mg / mL).

[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, by Figure 3 It can be seen that, compared with wild type (WT), the mutants showed varying degrees of enhanced enzyme activity. Among them, the triple mutant W753V / H755G / L810S showed the best performance, with enzyme activity increased to 1.94 times that of WT. The other mutants also showed varying degrees of improvement.

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

[0086]

[0087] 2. Enzyme activity assay of xanthan gum backbone lyase and its mutants at different pH conditions:

[0088] At 55℃, different buffer systems with pH ranging from 4.0 to 12.0 were used to investigate... Microbacterium sp The catalytic activity changes of wild-type xanthan gum backbone lyase (WT) derived from XT11 and its mutants (W753V, W753V / H755G, W753V / H755G / L810S) 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 ​​were all prepared with KH2PO4 / K2HPO4 to a final concentration of 100 mM. The pH range was adjusted by hydrochloric acid or sodium hydroxide to cover 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. Specifically, 30 μL of crude enzyme solution is added to 120 μL of 0.5% (w / v) xanthan gum substrate solution prepared with the corresponding pH buffer, mixed well, and then placed in a 55℃ water bath for 5 min. After the reaction is complete, 150 μL of DNS reagent is added, and the mixture is heated in a boiling water bath for 10 min. After the reaction is complete, the mixture is immediately cooled to room temperature, and the absorbance is measured 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 all other operations are exactly the same.

[0092] The reducing sugar concentration was converted using the aforementioned glucose standard curve, and the calculation methods for enzyme activity units and specific enzyme activity were 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 wild-type enzyme WT exhibits the highest activity at pH 7.0, but its activity decreases with both increasing and decreasing pH. It retains only 67% of its activity at pH 4.0 and 64% at pH 12.0, indicating a significant pH-dependent effect. The single mutant W753V shows higher activity than WT at all pH conditions, reaching its maximum activity at pH 7.0 with a relative enzyme activity of 144%. It maintains 116% and 127% of its activity at pH 6.0 and pH 8.0, respectively, demonstrating good neutrality adaptability. The main chain cleavage ability of the double mutant W753V / H755G is further enhanced. At pH 7.0, its activity is about 1.54 times that of the wild type. At pH 6.0, pH 8.0 and pH 9.0, it reaches about 125%, 167% and 144% respectively. At pH 10.0 and pH 11.0, it also maintains 137% and 125% respectively, showing a wide range of applicability. The triple mutant W753V / H755G / L810S exhibited the best overall performance, achieving a relative enzyme activity of 198% at pH 7.0, 1.98 times that of WT, the highest among all mutants. It reached 163%, 188%, and 158% at pH 6.0, 8.0, and 9.0, respectively. Furthermore, it maintained a relative enzyme activity of approximately 130%–150% within the pH range of 10.0–12.0, indicating that this mutant maintained high main-chain cleavage capacity over a wide pH range. These results demonstrate that all mutants improved the pH range and catalytic efficiency of the main-chain lyase to varying degrees, with the triple mutant showing the most outstanding performance.

[0094] 3. Determination of pH stability of xanthan gum backbone 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 (W753V, W753V / H755G, W753V / H755G / L810S) 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 55°C for 1 h. 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 5 As shown, the single mutant W753V exhibited slightly higher residual relative enzyme activity than WT under neutral and weakly alkaline conditions, with an increase of approximately 1.05–1.20 times. The double mutant W753V / H755G showed a more significant increase in residual relative enzyme activity within the pH range of 6.0–10.0, reaching 1.10–1.31 times that of WT. The triple mutant W753V / H755G / L810S maintained the highest stability across all test ranges, with a residual relative enzyme activity reaching 1.74 times that of WT at pH 8.0; even after treatment under strongly alkaline conditions at pH 12.0, it remained at 1.08 times that of WT. These experimental results demonstrate that the triple mutant W753V / H755G / L810S not only exhibited the highest activity under optimal reaction conditions but also demonstrated stronger structural tolerance under a wide pH range, especially with a significantly higher residual relative enzyme activity after alkaline treatment compared to other mutants, further proving its advantages in industrial applications.

Claims

1. A xanthan gum backbone lyase mutant with high enzyme activity, 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 W753V, W753V / H755G, or W753V / H755G / L810S.

2. A nucleotide sequence, characterized in that, The xanthan gum backbone lyase mutant with high enzyme activity as described in claim 1 is encoded.

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 the xanthan gum backbone lyase mutant with high enzyme activity as described in claim 1, characterized in that, Includes the following steps: (1) Using the wild-type xanthan gum backbone 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 backbone lyase mutant gene; (2) Insert the xanthan gum backbone 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 backbone lyase mutant.

6. The preparation method according to claim 5, characterized in that, In step (1), the point mutation primers are W753V-F, W753V-R, H755G-F, H755G-R, L810S-F, and L810S-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 xanthan gum backbone lyase mutant with high enzyme activity as described in claim 1, or the nucleotide sequence as described in claim 2, or the recombinant vector as described in claim 3, or the recombinant cell as described in claim 4.

9. The application of the product of claim 8 in the degradation of xanthan gum backbone, wherein the pH of the application is 7.5-12.

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