Dittomycin lyase and mutant thereof and application thereof

Abstract

The application discloses a diutan lyase and a mutant and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. Based on computer-aided technology, a diutan lyase DpsR coding sequence is mined from a genome database of a sphingomonas ATCC 53159, and is subjected to heterologous expression to obtain the diutan lyase DpsR. After the diutan lyase DpsR coding sequence is subjected to molecular modification, truncated expression is carried out to obtain diutan lyase mutants DpsR1, DpsR2 and DpsR3 with better enzyme activity, and the enzyme activity of the diutan lyase mutants is 1.5-4.5 times that of the original diutan lyase DpsR. The diutan lyase and the mutant provided by the application are bases for preparation, research and identification of diutan oligosaccharide.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a Deuterium lyase, its mutants, and their applications. Background Technology

[0002] Sphingosine gum (gellan gum, velan gum, rhamnose gum, Dettol gum, S88, S198, S7, etc.) is a type of gum produced by Sphingospora (… Sphingomonas Extracellular polysaccharides synthesized by *Sphingomonas ATCC* have the same main chain structure, consisting of tetrasaccharide repeating units composed of [→4)-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-β-D-GlcpA-(1→4)-β-D-Glcp-(1→]. They exhibit diverse side chain structures and unique physical and rheological properties, making them suitable as emulsifiers, suspending agents, and thickeners in food, pharmaceuticals, and oil extraction. With an annual output value of up to $30 billion, they are among the most commercially valuable microbial polysaccharides. Diutan gum (or S-657) is produced by *Sphingomonas ATCC*. The microbial polysaccharide synthesized by 53159 has a branched chain formed by α-L-Rha(1→4)-α-L-Rha(1→) substitution at the O3 position of the (1→4)-β-D-Glcp unit. In addition to possessing the properties of sphingosine gum, its superior temperature and salt stability is more important, making it one of the novel polysaccharides with potential application value in recent years.

[0003] Studies have found that oligosaccharides obtained from the degradation of natural polysaccharides possess excellent biological activities. For example, xanthan gum oligosaccharides have antioxidant properties, fucoidan oligosaccharides have anti-tumor effects, and chondroitin sulfate oligosaccharides have anti-inflammatory and cartilage repair-promoting functions. However, research on dextrose oligosaccharides is relatively limited. Currently, methods for preparing dextrose oligosaccharides include physical, chemical, and enzymatic methods. Compared to traditional physical and chemical methods, enzymatic methods offer high specificity, accurately identifying and cleaving specific chemical bonds to obtain specific oligosaccharides with well-defined structures and narrow molecular weight distributions. Furthermore, enzymatic methods offer advantages such as mild reaction conditions, rapid reaction rates, high extraction rates, and low pollution. This allows for the rapid, environmentally friendly, and specific preparation of dextrose oligosaccharides. While gellan gum and vilan gum, which belong to the same sphingosine gum family as dextrose, have reported corresponding lysins, research on dextrose lysins has not yet been reported. Therefore, obtaining a dextrose lysin with both high enzyme activity and high degradation efficiency is of great significance for achieving the preparation of high-purity, specific-structure, small-molecule dextrose oligosaccharides. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Detergent gel lysin to address the shortcomings of the prior art.

[0005] Another technical problem to be solved by the present invention is to provide a Detergum lysin mutant.

[0006] Another technical problem to be solved by the present invention is to provide a nucleic acid molecule encoding the Deuterium lyase or a Deuterium lyase mutant.

[0007] Another technical problem to be solved by the present invention is to provide biological materials comprising nucleic acid molecules encoding the Deuterium lyase or a Deuterium lyase mutant.

[0008] Another technical problem to be solved by the present invention is to provide a recombinant strain containing a nucleic acid molecule encoding the Deuterium lyase or a Deuterium lyase mutant.

[0009] The final technical problem to be solved by the present invention is to provide the application of the Deuterium lyase or the Deuterium lyase mutant in the preparation of Deuterium oligosaccharides.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0011] The first aspect of the present invention provides a Deuterium lysin DpsR, the amino acid sequence of which is shown in SEQ ID NO:1.

[0012] A second aspect of the present invention provides a Deutz-Gum lysin mutant, the amino acid sequence of which is shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 correspond to the amino acid sequences of the Deutz-Gum lysin mutants DpsR1, DpsR2, and DpsR3, respectively.

[0013] A third aspect of the present invention provides a nucleic acid molecule encoding the Deuterium lyase or a Deuterium lyase mutant.

[0014] The nucleotide sequence encoding the DpsR1 lyase is shown in SEQ ID NO:5; the nucleotide sequence encoding the DpsR2 mutant is shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. The nucleotide sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 correspond to the nucleotide sequences encoding the genes of the DpsR1, DpsR2, and DpsR3 mutants, respectively.

[0015] A fourth aspect of the present invention provides a biomaterial containing a nucleic acid molecule encoding the Deutergum lyase or a Deutergum lyase mutant, wherein the biomaterial is recombinant DNA, an expression cassette, a transposon, a plasmid vector, or a viral vector.

[0016] In some embodiments, the plasmid vector is pET-28a.

[0017] A fifth aspect of the present invention provides a recombinant strain comprising a nucleic acid molecule encoding the Dieter gum lyase or a mutant of the Dieter gum lyase.

[0018] In some embodiments, the recombinant strain originates from Escherichia coli BL21(DE3).

[0019] The sixth aspect of the present invention provides the use of the Deuterium lyase or the Deuterium lyase mutant in the degradation of Deuterium or the preparation of Deuterium oligosaccharides.

[0020] The method for degrading Dieter gum or preparing Dieter gum oligosaccharides using the Dieter gum lyase or the Dieter gum lyase mutant includes the following steps: using Dieter gum as a substrate, the Dieter gum lyase or the Dieter gum lyase mutant catalyzes the depolymerization of the Dieter gum to generate the Dieter gum oligosaccharides.

[0021] The depolymerization reaction is carried out at a temperature of 25-50°C, a pH of 5.0-8.0, and a reaction time of 10-120 min.

[0022] Beneficial effects:

[0023] This invention, based on computer-aided technology, mined the coding sequence of the Dietragole lyase DpsR from the genome database of *Sphingomonas ATCC 53159*, and obtained the Dietragole lyase DpsR through heterologous expression. Furthermore, molecular modification and truncation of the Dietragole lyase DpsR coding sequence yielded mutant Dietragole lyases DpsR1, DpsR2, and DpsR3 with improved enzyme activity, ranging from 1.5 to 4.5 times that of the original Dietragole lyase DpsR. The Dietragole lyases and their mutants provided by this invention form the basis for the preparation, research, and identification of Dietragole oligosaccharides. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is an amino acid sequence alignment diagram of DpsR, WelR, and GelR, the gellan gum lysins used in Example 1.

[0026] Figure 2 This is an SDS-PAGE electrophoresis image of the pure enzyme solution of DpsR, the lysin DpR prepared in Example 1.

[0027] Figure 3 The following is a statistical chart showing the evaluation results of the optimal enzymatic hydrolysis temperature and temperature stability of DpsR, a lyase for Detergent gum, in Example 2; where, Figure a shows the relative enzyme activity of DpsR at different temperatures during the enzymatic hydrolysis reaction; and Figure b shows the relative enzyme activity of DpsR after incubation at different temperatures for 1 h during the enzymatic hydrolysis reaction.

[0028] Figure 4 The following is a statistical chart showing the evaluation results of the optimal pH value and pH stability of DpsR, the gelatin lysin, in Example 2; where, Figure a shows the relative enzyme activity of DpsR at different pH values ​​during the enzymatic hydrolysis reaction; and Figure b shows the relative enzyme activity of DpsR after incubation at different pH values ​​for 1 h during the enzymatic hydrolysis reaction.

[0029] Figure 5 The relative enzyme activities of DpsR, the lyase for Dieter gum lysin in Example 2, in the presence of different metal ions during the enzymatic hydrolysis reaction are shown.

[0030] Figure 6 The relative enzyme activity of DpsR, the lyase for Dieter gum lysin in Example 2, when it hydrolyzes different substrates is shown.

[0031] Figure 7 The image shows the MALDI-TOF-MS mass spectrum of the hydrolysate of Dietrich glycoside after hydrolysis with Dietrich glycoside lyase DpsR in Example 3.

[0032] Figure 8 This is a statistical chart showing the relative enzyme activity of DpsR and its mutants DpsR1, DpsR2, and DpsR3 in Example 4.

[0033] Figure 9 The image shows the three-dimensional protein structure of DpsR and its mutant DpsR2 from Example 4. Detailed Implementation

[0034] 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.

[0035] Example 1: Heterologous expression and purification of DpsR, a dilatational lyase.

[0036] (1) By comparing the amino acid sequences of the Dieter gum synthesis gene cluster (GenBank: EU026118.1), Welan gum lyase WelR and Gellen gum lyase GelR, a putative Dieter gum lyase DpsR sequence with 40-70% homology was obtained. Its nucleotide sequence is shown in SEQ ID NO:5. The pET-28a-DpsR plasmid containing the putative Dieter gum lyase DpsR gene was synthesized from the whole gene.

[0037] (2) Add 50 μg of plasmid pET-28a-DpsR to BL21(DE3) competent cells under sterile conditions, mix by pipetting and aspiration, and incubate on ice for about 30 min. Then quickly place in a 42℃ water bath for 90 s heat shock, immediately remove and cool on ice for 2-4 min, add 900 μL of LB medium and mix well, and incubate at 37℃ with shaking for 50 min. After centrifugation, remove an appropriate amount of supernatant and mix again. Spread the culture medium on LB solid medium plates containing a final concentration of 50 μg / mL kanamycin and incubate at 37℃ for 12 h to obtain positive single colonies, namely Escherichia coli BL21(DE3) containing pET-28a-DpsR plasmid.

[0038] (3) Inoculate the Escherichia coli BL21(DE3) containing the pET-28a-DpsR plasmid from step (2) into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and incubate at 37°C until OD. 600 When the concentration reaches 0.6 ~ 1.0, IPTG with a final concentration of 0.1 ~ 1 mM is added for induction, and the induction is carried out at 20℃ for 20 h.

[0039] (4) Centrifuge the bacterial culture after induction of expression in step (3) at 8000 rpm for 10 min to collect the bacterial cells. Wash the bacterial cells 2 to 3 times with PBS buffer at pH 7 to 7.5 and resuspend them in PBS buffer. Sonicate the bacterial cells under ice-water bath conditions, centrifuge at 12000 rpm for 10 min at low temperature, and take the supernatant to obtain the crude enzyme solution of DpsR lyase.

[0040] (5) Purification was performed using Ni column affinity chromatography. After passing the crude enzyme solution through a nickel column, the target protein was eluted using elution buffer (50 mM Tris-HCl, 300 mM NaCl) containing different concentration gradients (0, 50, 100, 150, and 500 mM) of imidazole at pH=8. The elution products were collected. The collected elution products were desalted and concentrated using a 30 kDa ultrafiltration tube to remove imidazole and salt, thus obtaining the pure enzyme solution of Dieter gum lyase DpsR. The pure DpsR enzyme solution was subjected to SDS-PAGE electrophoresis, and the results are shown below. Figure 2 As shown, this demonstrates that a relatively single target protein band can be obtained after elution with gradient concentrations of imidazole.

[0041] Example 2 Enzymatic properties of DpsR, a gelatin lyase

[0042] (1) Detection of DpsR enzyme activity of Dieter gum lysin

[0043] Enzyme activity unit (U) definition: 1 U is the amount of enzyme required to produce 1 μmol of reducing sugar per minute in an enzyme reaction system under conditions of 50℃ and pH=6.

[0044] The enzyme activity of Dieter gum lyase DpsR was detected by determining the amount of reducing sugar released using the 3,5-dinitrosalicylic acid (DNS) method. The specific procedure was as follows: 100 μL of the purified DpsR enzyme solution prepared in Example 1 was added to 900 μL of Dieter gum solution containing 5 g / L (the solvent was 0.1 mol / L phosphate buffer at pH 6). The mixture was thoroughly mixed and reacted at 50°C for 30 min. The enzyme was then inactivated by boiling in a water bath for 5 min. After centrifugation, 200 μL of the supernatant was mixed with 300 μL of DNS reagent and boiled in a water bath for 5 min. After cooling to room temperature, 200 μL was taken and the enzyme activity was detected using a microplate reader. 540 The value was then used to calculate the amount of reducing sugar produced using a glucose standard curve. Finally, the specific enzyme activity of the DpsR pure enzyme solution prepared in Example 1 was determined to be 1 U / mL.

[0045] (2) Optimal enzyme activity temperature and temperature stability of DpsR, a gelatin lyase

[0046] The enzyme activity of DpsR, a lyase for Detergent gum, was determined at temperatures of 25, 30, 35, 40, 45, 50, 55, 60, and 65°C (except for the temperature, other reaction parameters remained unchanged) according to the method in Example (1) of this embodiment, to determine the optimal reaction temperature for DpsR to degrade Detergent gum. The relative enzyme activity at each temperature was calculated using the highest enzyme activity as a reference. All experiments were conducted in triplicate, and the average value was taken. The results are as follows: Figure 3 As shown in a, the optimal reaction temperature of DpsR, a gelatin lyase, is 50 °C, and it exhibits a maximum activity of over 50% in the temperature range of 40 ~ 55 °C.

[0047] To evaluate the temperature stability of DpsR, the DpsR was first incubated at nine different temperatures (25, 30, 35, 40, 45, 50, 55, 60, and 65°C) for 1 hour each. Then, the enzyme activity of DpsR after incubation at each temperature was measured according to the method described in Example (1). The enzyme activity without incubation was considered 100%. The relative enzyme activity under each temperature condition was calculated. All experiments were conducted in triplicate, and the average value was taken. The results are as follows: Figure 3 As shown in b, the Dieter gum lyase DpsR retained over 90% of its activity after incubation at 25–40 °C for 1 h. After treatment at 45 °C or higher for 1 h, the relative enzyme activity gradually decreased. These results indicate that the Dieter gum lyase DpsR exhibits good temperature stability below 40 °C.

[0048] (3) Optimal pH and pH stability of DpsR, a gelatin lyase

[0049] Prepare buffer solutions with different pH values: 0.1 mol / L disodium hydrogen phosphate-citric acid buffer for pH 3, 4, and 5; 0.1 mol / L phosphate buffer for pH 6, 6.5, and 7; 0.1 mol / L Tris-HCl buffer for pH 8 and 9; and 0.1 mol / L glycine-sodium hydroxide buffer for pH 10 and 11. Use these buffer solutions as solvents to prepare 5 g / L Dieter gum solutions at different pH values.

[0050] The enzyme activity of DpsR, a diterbinafine lysin, was determined at pH values ​​of 3, 4, 5, 6, 6.5, 7, 8, 9, 10, and 11 according to the method in Example (1) of this embodiment (except for pH value, other reaction parameters remained unchanged) to determine the optimal pH value for DpsR in the degradation of diterbinafine. The relative enzyme activity under each pH condition was calculated using the highest enzyme activity as a reference. All experiments were performed in triplicate, and the average value was taken. The results are as follows: Figure 4 As shown in figure a, the optimal reaction pH for the Detergent gel lyase DpsR is 6.0.

[0051] To test pH stability, the pure enzyme was first incubated at 30°C for 1 h at pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, and 11. Then, the enzyme activity of DpsR (a diterpenoid lyase) after incubation at each pH value was determined according to the method described in Example (1). The relative enzyme activity at each pH condition was calculated with the untreated enzyme activity as 100%. All experiments were performed in triplicate, and the average value was taken. The results are as follows: Figure 4 As shown in b, the stability of the Detergum lyase DpsR remains above 50% of its initial activity in the pH range of 5 to 9, indicating that the Detergum lyase DpsR has good pH stability.

[0052] (4) Effect of metal ions on DpsR, a gelatin lyase

[0053] Different metal ions and EDTA·2Na were added to the purified DpsR enzyme solution of Dietrich lyase prepared in Example 1 at final concentrations of 1 mmol / L or 10 mmol / L. The metal ions included K+. + Na + Zn 2+ Ca 2+ Mg 2+ Mn 2+ Ni 2+ Cu 2+ Co 2+ Fe2+ and Fe 3+ The effects of metal ions and EDTA·2Na on the activity of DpsR, a dilatation enzyme, were then investigated according to the method described in Example (1). The relative enzyme activity was calculated with the enzyme activity without added metal ions as 100%. All experiments were performed in triplicate, and the average value was taken. The results are as follows: Figure 5 As shown, 1 mmol / L Ca 2+ 10 mmol / L Fe 2+ It can promote the activity of DpsR, an enzyme that lyses DpR, with 10 mmol / L EDTA·2Na and Cu. 2+ and Fe 3+ It will significantly inhibit the activity of DpsR, a gelatin lyase.

[0054] (5) Substrate specificity of DpsR, a gelatin lyase

[0055] To determine the substrate specificity of the sphingosine gum lyase DpsR for sphingosine gums (Detara gum, Wilan gum, and rhamn gum) with the same main chain structure but different branched chain structures.

[0056] The enzyme catalytic activities of DpsR, a lyase for Dettol gum, on substrates Dettol gum, Vilan gum, and rhamnosus gum were detected according to the method described in Example (1) (all reaction parameters remained unchanged except for the substrates). The relative enzyme activities of different substrates were calculated using the highest enzyme activity as a reference. All experiments were performed in triplicate, and the average value was taken. The results are as follows: Figure 6 As shown, the enzyme exhibits the best degradation effect on Dieter gum, good degradation activity on Wilan gum, and weak degradation activity on Rhamn gum. This indicates that the enzyme can recognize sphingosine gum substrates with the same main chain structure, while the degradation efficiency for different substrates is affected by the side chain structure.

[0057] Example 3: Product analysis of Degradation of Degradation Gum by Degradation Enzyme DpsR

[0058] 100 μL of the purified DpsR enzyme solution prepared in Example 1 was added to 900 μL of PBS buffer (5 mM, pH=6) containing 5 g / L DpsR and mixed well. The mixture was reacted at 50 °C for 1 h, and then the enzyme was inactivated by boiling in a water bath for 5 min. After centrifugation, the supernatant was filtered through a 0.22 μm aqueous filter. The results were analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0059] Depend on Figure 7Mass spectrometry analysis showed that the Dieter gum lyase DpsR acts on the β-1,4 glycosidic bonds of the Dieter gum backbone, mainly degrading the Dieter gum into pentasaccharide units with the structure α-L-Rhap-(1→4)-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-α-L-Rhap-(1→3)-β-D-Glcp, with the (1→3)-β-D-Glcp containing one or two acetyl groups; and a small amount of α- The trisaccharide unit of L-Rhap-(1→3)-β-D-Glcp-(1→4)-β-D-GlcAp is a diploid polysaccharide with a tetrasaccharide repeating unit of the structure [α-L-Rhap-(1→4)-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-β-D-GlcAp]2, carrying four acetyl groups.

[0060] Example 4 Expression and purification of the DpsR mutant of Dieter gum lyase

[0061] The three-dimensional structure of the DpsR lyase, predicted by AlphaFold2, was simulated. It can be mainly divided into two domains. Molecular dynamics and Rosetta were used to select appropriate truncation sites, and the C-terminal domain was truncated to construct mutants DpsR1, DpsR2, and DpsR3.

[0062] The specific implementation method is as follows:

[0063] Using the plasmid pET-28a-DpsR from Example 1 as a template, and using DpsR-F / DpsR1-R, DpsR-F / DpsR2-R, and DpsR-F / DpsR3-R as primers, respectively, the coding gene fragments of the truncated enzymes DpsR1, DpsR2, and DpsR3 were amplified by PCR reaction.

[0064] The PCR reaction system consisted of: 25 μL Gloria Nova HS 2X Master Mix V2 (purchased from Wuhan Aiboteke Biotechnology Co., Ltd.), 1 μL upstream primer, 1 μL downstream primer, 1 μL recombinant plasmid pET-28a-DpsR, and up to 50 μL ddH2O. The nucleotide sequences of the primers are shown in Table 1.

[0065] The PCR amplification program is as follows: pre-denaturation 95 ℃, 3 min; denaturation 95 ℃, 15 sec, annealing 62 ℃, 30 sec, extension 72 ℃, 10 s / kb, 30 cycles; complete extension at 72 ℃, 5 min.

[0066] Table 1. Sequences of primers required for constructing the Dieter gum lysin mutant.

[0067]

[0068] After the PCR amplification was completed, the reaction product was recovered by agarose gel extraction to obtain the target gene fragment.

[0069] The plasmid pET-28a-DpsR was double-digested with restriction endonucleases NdeI and XhoI at 37°C for 30 min. The digestion products were recovered by gel extraction to obtain the linearized vector pET-28a. The linearized vector pET-28a was ligated with the coding gene fragments of DpsR1, DpsR2, and DpsR3, respectively, and then transformed into Escherichia coli BL21(DE3). After selecting positive clones, the mutant strains pET-28a-DpsR1, pET-28a-DpsR2, and pET-28a-DpsR3 were expressed and purified according to steps (3) to (5) of Example 1. The enzyme activities of the purified enzymes DpsR, DpsR1, DpsR2, and DpsR3 were detected according to the method in (1) of Example 2. The relative enzyme activities of DpsR1, DpsR2, and DpsR3 were calculated with the enzyme activity of DpsR as a reference. The results are as follows. Figure 8 As shown, the enzyme activity of the mutants obtained after truncation increased by 1.5 to 4.5 times, with the mutant DpsR2 showing the highest enzyme activity. Figure 9 Three-dimensional protein structures of DpsR and its mutant DpsR2.

[0070] This invention provides a method and approach for developing a Deuterium lyase, its 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 Deuterium lysin, characterized in that, The amino acid sequence of the Detergum lysin is shown in SEQ ID NO:

1.

2. A Deuterium lyase mutant, characterized in that, The amino acid sequence of the Detergum lysin mutant is shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. A nucleic acid molecule encoding the Dieter gum lyase of claim 1 or a mutant of the Dieter gum lyase of claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the Deuterium lysin is shown in SEQ ID NO:5; the nucleotide sequence of the nucleic acid molecule encoding the Deuterium lysin mutant is shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

5. A biomaterial containing the nucleic acid molecule of claim 3 or 4, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, or viral vector.

6. A recombinant bacterial strain, characterized in that, It includes the nucleic acid molecule described in claim 3 or 4.

7. The use of the Dieter gum lyase of claim 1 or the Dieter gum lyase mutant of claim 2 in the degradation of Dieter gum or the preparation of Dieter gum oligosaccharides.

8. The application according to claim 7, characterized in that, The method for degrading Diet gum or preparing Diet gum oligosaccharides using the Diet gum lyase or Diet gum lyase mutant includes the following steps: using Diet gum as a substrate, the Diet gum undergoes a depolymerization reaction catalyzed by the Diet gum lyase or Diet gum lyase mutant to generate the Diet gum oligosaccharides.

9. The application according to claim 8, characterized in that, The depolymerization reaction is carried out at a temperature of 25-50°C, a pH of 5.0-8.0, and a reaction time of 10-120 min.

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