Method for preparing ultra-low molecular weight dendrobium oligosaccharides by enzyme and application thereof

Abstract

This invention discloses a method for preparing ultra-low molecular weight Dendrobium oligosaccharides using an enzymatic process and its applications, belonging to the field of bioengineering technology. This invention utilizes a glycosidase derived from the thermophilic bacterium *Thermobifida halotolerans*, recombinantly expressed in an *E. coli* expression system, achieving high-activity expression. The recombinant ThDPS enzyme can hydrolyze Dendrobium polysaccharides, producing oligosaccharide products mainly including ultra-low molecular weight (molecular weight <1000 Daltons) Dendrobium disaccharides, Dendrobium trisaccharides, Dendrobium tetrasaccharides, and Dendrobium pentasaccharides, and low molecular weight (molecular weight <10000 Daltons) Dendrobium hexasaccharides, Dendrobium heptasaccharides, Dendrobium octasaccharides, and higher Dendrobium oligosaccharide products. This invention lays the technical foundation for the biological preparation of ultra-low molecular weight Dendrobium oligosaccharides and is suitable for industrial production and application.

Description

Technical Field

[0001] This invention relates to a method for preparing ultra-low molecular weight Dendrobium oligosaccharides using an enzymatic process and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] Dendrobium officinale (Kimura et Migo, D. officinale) is a rare traditional Chinese medicine belonging to the genus Dendrobium in the Orchidaceae family, encompassing over 1500 species, and is currently listed in the catalogue of medicinal and edible plants. With the improvement of people's living standards, the demand for traditional Chinese medicinal materials is becoming increasingly widespread. Among these, the chemical composition and pharmacological activity of Dendrobium officinale are currently a hot topic and focus in the field of Dendrobium research. Numerous pharmacological activity studies have shown that Dendrobium officinale possesses immunomodulatory, antitumor, antimutagenic, blood pressure-lowering, blood lipid-lowering, blood sugar-lowering, antiviral, antioxidant, anti-radiation, anti-ulcer, and anti-aging effects. Besides its medicinal uses, Dendrobium has been used as a health food for thousands of years. Therefore, the development of these active ingredients into finished traditional Chinese medicine preparations and health products for improving immunity, anti-aging, and anti-fatigue is highly promising. In recent years, nearly 100 active compounds have been isolated from more than 40 kinds of Dendrobium species with pharmacological activity, including Dendrobium polysaccharides, Dendrobium alkaloids, bibenzylphenol, amino acids, flavonoids, terpenoids, and trace elements.

[0003] Among the most widely studied active components of Dendrobium officinale polysaccharides are polysaccharides, with Dendrobium officinale polysaccharide (DOP) being primarily glucomannan formed from mannose and glucose via glycosidic bonds. Studies have found that DOP possesses various activities, including protecting the gastric mucosa, regulating blood sugar, protecting the liver, enhancing immunity, and lowering blood lipids. Recent research has revealed that low molecular weight polysaccharides exhibit stronger biological activity. For example, low molecular weight mannooligosaccharides can stimulate the liver to secrete mannobinding protein, activating the complement system to exert opsonization and natural anti-infective immune functions, and demonstrating excellent recognition, adhesion, and elimination of intestinal pathogens. However, the high molecular weight and viscosity of natural Dendrobium officinale polysaccharides limit their use in pharmaceutical and food processing. The glycosidic bonds of Dendrobium officinale polysaccharides are easily broken in acidic solutions, forming hydrolysates with varying degrees of polymerization. Therefore, acid-catalyzed hydrolysis is a commonly used method for degrading plant polysaccharides. Hydrolyzing Dendrobium officinale polysaccharides using sulfuric acid, trifluoroacetic acid, or hydrochloric acid catalytic-assisted boiling water bath heating or direct high-temperature heating equipment is time-consuming, involves high acid concentrations, and is prone to equipment damage, resulting in high costs. Trifluoroacetic acid and hydrochloric acid are highly volatile, irritate the respiratory system, and are toxic; their concentrations are difficult to control during operation, making them unsuitable for large-scale hydrolysis of polysaccharides to prepare mannose. While a recently reported technique for preparing low-molecular-weight Dendrobium officinale polysaccharides through mold fermentation has been developed, its fermentation time is long, the reaction products are complex and difficult to separate, and the prepared molecular weight is above 10,000 Da, making industrial-scale production difficult. These factors contribute to the persistently high production costs of pharmaceutical-grade and food-grade oligosaccharides. Summary of the Invention

[0004] To address the numerous shortcomings in the preparation of small-molecule active oligosaccharides from Dendrobium polysaccharides, this invention employs a bio-enzymatic method to catalytically degrade Dendrobium polysaccharides. Because the bio-enzymatic method offers mild reaction conditions, high specificity, and high product purity, it is easily scalable for industrial production.

[0005] This invention provides the application of glycosidases derived from thermophilic bacteria (Thermobifida halotolerans) or recombinant microorganisms expressing said glycosidases in the hydrolysis of Dendrobium polysaccharides.

[0006] In one embodiment, the glycosidase derived from the thermophilic bacterium *Thermobifida halotolerans* has the amino acid sequence shown in SEQ ID NO. 3.

[0007] In one embodiment, the gene encoding the glycosidase has the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0009] The present invention also provides a method for preparing ultra-low molecular weight Dendrobium oligosaccharides, wherein the method involves using the glycosidase or a cell culture containing the glycosidase for the degradation of Dendrobium polysaccharides.

[0010] In one embodiment, the ultra-low molecular weight Dendrobium oligosaccharide has a molecular weight ≤1000 Da, including but not limited to Dendrobium disaccharide, Dendrobium triaccharide, and Dendrobium tetraaccharide.

[0011] In one embodiment, the method involves adding the glycosidase or a cell culture containing the glycosidase to a high molecular weight Dendrobium polysaccharide solution.

[0012] In one embodiment, the molecular weight of the polysaccharide in the high molecular weight Dendrobium polysaccharide solution is ≥1×10⁻⁶. 4 Da.

[0013] In one embodiment, the molecular weight of the polysaccharide in the high molecular weight Dendrobium polysaccharide solution is 1×10⁻⁶. 4 ~1×10 7 Da.

[0014] In one embodiment, the Dendrobium polysaccharide is prepared into a solution with a concentration of 1-100 g / L using a 50 mM, pH 7.0 phosphate buffer.

[0015] In one embodiment, the concentration of Dendrobium polysaccharide in the solution is 10 g / L.

[0016] In one embodiment, the amount of the glycosidase added is ≥1 U / g. 石斛多糖 Specifically, it can be 1-1000 U / g 石斛多糖 or 500U / g 石斛多糖 .

[0017] In one embodiment, the glycosidase involved in the degradation of Dendrobium polysaccharides is a pure enzyme solution without other salts, and the concentration of the enzyme in the reaction system is 10-10000 U / L, specifically 5000 U / L.

[0018] In one embodiment, the pH value of the reaction system solution is controlled between 5.0 and 10.0, specifically pH 7.0.

[0019] In one embodiment, the temperature of the reaction system is between 10°C and 75°C, specifically 60°C.

[0020] In one embodiment, the reaction time of the reaction system is 0.5-16 hours, specifically 2 hours.

[0021] The present invention also provides the application of the glycosidase or the method in the production of products containing Dendrobium oligosaccharides.

[0022] In one embodiment, the high molecular weight Dendrobium polysaccharide can be hydrolyzed into ultra-low molecular weight Dendrobium oligosaccharides with a molecular weight of ≤1000 Daltons, including but not limited to Dendrobium disaccharide, Dendrobium triaccharide, Dendrobium tetraaccharide, Dendrobium pentasaccharide, or hydrolyzed into Dendrobium hexasaccharide, Dendrobium heptasaccharide, Dendrobium octasaccharide and above oligosaccharides with a molecular weight of 1000 to 10000 Daltons.

[0023] Beneficial effects:

[0024] (1) This invention provides the application of glycosidases derived from thermophilic bacterium Thermobifida halotolerans in the hydrolysis of Dendrobium polysaccharides.

[0025] (2) In this invention, a glycosidase derived from thermophilic bacterium Thermobifida halotolerans is heterologously expressed in recombinant Escherichia coli. By optimizing the coding gene of the glycosidase, a ThDPS expression product with significantly improved expression activity is obtained, thus achieving efficient expression of ThDPS enzyme. Since Escherichia coli has a strong protein expression ability, it is easy to prepare ThDPS enzyme by large-scale fermentation. The thermophilic glycosidase ThDPS expressed by this invention can be directly used for efficient hydrolysis of Dendrobium polysaccharide, realizing the large-scale enzymatic catalytic preparation of active oligosaccharides.

[0026] (3) This invention utilizes enzymes produced by fermentation using genetically engineered strains to directly degrade Dendrobium polysaccharides to prepare small oligosaccharides. The reaction conditions are extremely simple, requiring no specific equipment and can be carried out at room temperature and pressure. The enzymatic hydrolysis process does not require the addition of any organic reagents and generates no pollutants or waste. The conversion yield of small oligosaccharides reaches over 95%, and the product is relatively simple in aqueous solution, making it easy to purify and recover. Based on application analysis, the method of this invention has very broad industrial value for preparing single-function oligosaccharides and their derivatives. Attached Figure Description

[0027] Figure 1 The results show the activity of various glycoside hydrolases in hydrolyzing Dendrobium polysaccharides as detected by the DNS method.

[0028] Figure 2 The relative enzyme activities before and after glycosidase sequence optimization are shown.

[0029] Figure 3 Electrophoresis diagrams showing the expression and purification of ThDPS recombinant protein.

[0030] Figure 4 The specific enzyme activity of ThDPS enzyme catalyzing the hydrolysis of Dendrobium polysaccharides under different substrate concentrations was determined.

[0031] Figure 5Effect of different reaction temperatures on the hydrolysis of Dendrobium polysaccharides by ThDPS enzyme: relative enzyme activity refers to the percentage value relative to the maximum reaction enzyme activity.

[0032] Figure 6 Effect of different reaction pH on the hydrolysis of Dendrobium polysaccharides by ThDPS enzyme: relative enzyme activity refers to the percentage value relative to the maximum reaction enzyme activity.

[0033] Figure 7 To detect the oligosaccharide products of Dendrobium nobile catalyzed by ThDPS enzyme hydrolysis by fluorescent electrophoresis. Detailed Implementation

[0034] The expression host and culture medium for ThDPS glycosidase are as follows:

[0035] The pET21a plasmid and E. coli BL21(DE3) used in the following examples were purchased from Stratagene, La Jolla, CA, USA.

[0036] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, ampicillin concentration 100 μg / mL.

[0037] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, ampicillin concentration 100 μg / mL, agar 15 g / L.

[0038] ThDPS glycosidase activity assay:

[0039] Enzyme activity is defined as the amount of enzyme that can convert 1 micromolar of substrate per minute at 37°C, which is defined as one unit of enzyme activity (U).

[0040] Enzyme activity assay: ThDPS glycosidase can decompose Dendrobium polysaccharides into mannooligosaccharides. Mannooligosaccharides have reducing properties and can undergo a certain chemical reaction with DNS reagent under alkaline conditions to generate 3-amin-5-nitrosalicylic acid, which is brownish-red under boiling conditions. The amount of reducing sugar generated can be measured with a spectrophotometer, and the intensity of its color is related to the reducing sugar content.

[0041] The specific steps for enzyme activity assay are as follows: A total reaction system of 2 mL contains 1 mL of 4 g / L Dendrobium polysaccharide solution, 200 μL of 200 mM phosphate buffer, 10 μL of enzyme solution, and 10 μL of distilled water for the control group. The total volume of all reaction solutions is then adjusted to 2 mL. The mixed reaction solution is incubated at 37°C for 30 min. After incubation, the enzyme is inactivated by boiling in a water bath for 1 min. 2 mL of DNS solution is added to each tube, and the tube is incubated by boiling in a water bath for 3 min, then rapidly cooled. 1 mL of distilled water is added to bring the volume to 5 mL. The absorbance is measured at 540 nm to determine enzyme activity.

[0042] Hydrolyzed product Dendrobium oligosaccharide gel fluorescence electrophoresis

[0043] Reagent preparation: Prepare DMSO (acetic acid:water:DMSO = 3:17:20), 0.2M ANTS (dissolved in water:acetic acid = 17:3), and 1M NaCNBH3 (dissolved in DMSO solution);

[0044] Sample preparation: 80 μL of oligosaccharide hydrolysate and 80 μL of sucrose aqueous solution were placed in EP tubes. 10 μL of DMSO, 5 μL of ANTS, and 5 μL of NaCNBH3 were accurately pipetted into each EP tube. The EP tubes were placed in a 40°C water bath for 16 h. The oven was then set to 45°C, and the EP tubes were placed in the oven with the caps off for 6 h. When the sample volume was reduced to half of its original volume, 100 μL of 3M urea was added to the sample, and 10 μL of 6M urea was added to the standard sample. The mixture was shaken and placed in a -20°C refrigerator overnight.

[0045] Separating gel preparation: Mix 5.33 mL of 30% acrylamide, 1 mL of 1.5 M Tirs-HCl (pH 8.8), 1.67 mL of distilled water, 30 μL of 10% ammonium persulfate, and 10 μL of TEMEO to form a gel.

[0046] After electrophoresis, place the extracted gel under a UV lamp to observe the bands.

[0047] Example 1: Heterologous expression and preparation of recombinant Escherichia coli with multiple glycosidases

[0048] 1) Recombinant construction of glycosidase genes

[0049] To screen for glycosidases that can efficiently hydrolyze Dendrobium polysaccharides, bioinformatics comparisons were used to screen glycosidase encoding genes from different species and different glycosidase families. After phylogenetic tree and conserved site comparison analysis, four representative glycosidases were selected for heterologous recombination expression to prepare glycosidase proteins.

[0050] Using THGH (SEQ ID NO. 1) from *Thermobifida halotolerans*, TFGH (Genbank accession number: AID 15578.1) from *Thermobifida fusca*, KPGH (Genbank accession number: CP035202.1(146142-148334)) from *Klebsiella pneumoniae*, and TAGH (Genbank accession number: BBA57841.1) from *Thermobifida alba* as templates, the encoding genes were amplified by PCR. pET21a was recombined using seamless cloning. The recombinant plasmids THGH-pET21a and T... FGH-pET21a, KPGH-pET21a, THGH-pET21a, and TAGH-pET21a were transformed into *E. coli* BL21(DE3) to obtain recombinant strains THGH-pET21a / BL21(DE3), TFGH-pET21a / BL21(DE3), KPGH-pET21a / BL21(DE3), and TAGH-pET21a / BL21(DE3), respectively. A suitable amount of transformation solution was plated on LB agar plates and incubated overnight at 37°C. After successful colony PCR, single colonies were picked and cultured in shake flasks for plasmid extraction. Further sequencing confirmed the correctness of the results. Strains containing the recombinant plasmids were then selected as recombinant bacteria and proceeded to the next expression stage.

[0051] 2) Induction and activity detection of recombinant bacterial proteins

[0052] The recombinant bacteria constructed in step 1 were inoculated into test tubes containing 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured in shake flasks at 37°C and 200 rpm for 16 h as seed culture. This seed culture was then transferred at a 1% inoculation rate to 100 mL of LB liquid medium (containing 100 μg / mL ampicillin) and cultured in shake flasks at 37°C and 200 rpm until the OD600 reached 0.6. 0.1 mM IPTG was added to induce expression, and the culture was continued at 37°C and 200 rpm for 24 h with shaking. The obtained fermentation broth was centrifuged at 8000 × g at 4°C for 10 min, the supernatant was removed, and the bacterial cells were collected. The bacterial cells were resuspended in 8 mL of 50 mM pH 7.0 PBS buffer, sonicated, and centrifuged at 4°C and 12000 × g for 10 min to remove the precipitate. The supernatant was collected and used for hydrolysis of Dendrobium polysaccharides, and the hydrolysis products were detected using the DNS method. The results are as follows: Figure 1 As shown, only the THGH expression product exhibited significant hydrolytic activity against Dendrobium polysaccharides and produced a large amount of reducing oligosaccharides, with an enzyme activity reaching 68 U / OD. 菌体 The other glycosidases, however, failed to exhibit significant hydrolytic activity.

[0053] Example 2: Optimized expression of ThDPS Dendrobium polysaccharide hydrolase

[0054] Meanwhile, to further improve the expression level and enzyme activity of THGH protein, using the THGH gene sequence in Thermobifidahalotolerans as a template, the codons were optimized according to the degeneracy of E. coli host codons to synthesize a 1326bp ThDPS gene (nucleotide sequence shown in SEQ ID NO.2). Homologous recombination primers ThDPS-21AF (5`-AAGAAGGAGATATACATATGCGTAAACGCCTGACCGTG-3`) and ThDPS-21AR (5`-CAGTGGTGGTGGTGGTGATCGGTGGTGCAGGTCAGGGTC-3`) were designed, retaining the downstream 6x His-tag of the recombinant protein. The prepared gene fragment product was seamlessly cloned with the pET21a empty plasmid (double digested with NdeI and XhoI). The ligation product was transformed into E. coli BL21(DE3), and after sequencing verification, expression was induced according to the method in Example 1. Enzyme activity analysis of hydrolyzed Dendrobium polysaccharides was performed on the expression product after codon optimization. The results are as follows: Figure 2 As shown, compared with the THGH expression enzyme activity level without codon optimization (68 U / OD cells), the optimized sequence ThDPS expression enzyme activity level was significantly increased by 2.75 times, reaching 187 U / OD cells. This lays the technical foundation for the subsequent high-level preparation of Dendrobium polysaccharide hydrolase.

[0055] Example 3: Purification and Enzyme Activity Characterization of ThDPS Dendrobium Polysaccharide Hydrolase

[0056] The enzyme solution prepared in Example 2 was collected and purified using a nickel column to produce ThDPS enzyme, which was then stored at -20°C for later use. SDS-PAGE protein electrophoresis was performed to detect the enzyme. Figure 3 As shown, the purified protein bands are approximately 50-52 kDa in size, which is consistent with the theoretical size.

[0057] The enzymatic properties of ThDPS were characterized using Dendrobium polysaccharide as a substrate.

[0058] 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, and 3 g of Dendrobium officinale polysaccharide (Mw > 100,000 Da) were weighed and dissolved in 1 L of distilled water to prepare Dendrobium officinale polysaccharide solutions. Then, 20 μL of enzyme solution was added, and the reaction was carried out at 37°C for 30 min. The enzyme solution was the ThDPS enzyme solution prepared in Example 1, diluted with deionized water to a concentration of 100 μg / mL. Figure 4As shown, the catalytic activity of ThDPS increased rapidly as the initial concentration of Dendrobium polysaccharide increased from 0.2 g / L to 3 g / L. These results indicate that there was no significant substrate inhibition in the hydrolysis of Dendrobium polysaccharide, and the specific enzyme activity reached 5100 U / mg at a substrate concentration of 2.4 g / L.

[0059] 2. Determination of Optimal Temperature: A 10 g / L Dendrobium polysaccharide was prepared as the reaction substrate. 0.4 mL of Dendrobium polysaccharide (final concentration 2 g / L) was added to the reaction system, along with 200 μL of 500 mM Tris-HCl buffer and 20 μL of enzyme solution (providing 10 U of catalytic activity). 20 μL of distilled water was added to the control group. The total volume of all reaction solutions was adjusted to 2 mL with water. After thorough mixing, the solutions were incubated in water baths at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 30 min each. The enzyme solution was the ThDPS enzyme solution prepared in Example 1, diluted with deionized water to a concentration of 100 μg / mL. The amount of newly generated reducing sugar in each reaction system was determined using the DNS-reducing sugar method.

[0060] The results are as follows Figure 5 As shown, ThDPS exhibits an optimal reaction temperature of 60 °C and retains over 85% activity at temperatures between 40 °C and 60 °C. This indicates that the enzyme is highly thermostable. The thermostability of this Dendrobium polysaccharide hydrolase is significantly higher than that of glycosidases from other sources. These results suggest that ThDPS can serve as an excellent high-temperature biocatalytic enzyme for large-scale hydrolysis of Dendrobium polysaccharides to prepare low molecular weight oligosaccharides.

[0061] 3. Determination of optimal pH:

[0062] Prepare 500mM buffer solutions with different pH values: citrate buffer (pH 2, 3, 4, 5, 6), phosphate buffer (pH 6, 7, 8), and Tris-HCl buffer (pH 8, 9, 10, 11).

[0063] A 10 g / L Dendrobium polysaccharide was prepared as the reaction substrate. 0.4 mL of Dendrobium polysaccharide sample was added to the reaction system to a final concentration of 2 g / L. Different pH buffers were then added to bring the total to 2 mL. Three tubes were prepared for each group; two tubes contained 20 μL (50 μg / mL) of enzyme solution, and the remaining tube contained 20 μL of distilled water. The substrate autodecomposition at different pH values ​​was measured. The mixture was incubated at 37°C for 30 min. After incubation, the enzyme was inactivated by boiling in a water bath for 1 min. 2 mL of DNS solution was added to each tube, and the mixture was incubated by boiling in a water bath for 3 min, followed by rapid cooling. The enzyme solution was the ThDPS enzyme solution prepared in Example 1 diluted with deionized water to a concentration of 100 μg / mL.

[0064] Zero the instrument with deionized water and measure the absorbance at 540 nm. Refer to the standard curve to calculate enzyme activity, plot the relationship between reaction conditions at different pH values ​​and enzyme activity, and determine the optimal pH. The reaction temperature corresponding to the maximum absorbance value is the optimal temperature for the recombinant enzyme. Relative enzyme activity (RA) is defined as the percentage of each absorbance value relative to the maximum absorbance value.

[0065] The results are as follows Figure 6 As shown, ThDPS exhibited maximum activity in 50 mM phosphate buffer at pH 7.0, and showed over 90% activity in the pH range of 6.0 to 10.0. Acid-base stability tests indicated that ThDPS is also highly stable over a wide pH range, which is beneficial for its large-scale catalytic hydrolysis preparation of Dendrobium oligosaccharides.

[0066] Example 4: High-efficiency preparation of Dendrobium oligosaccharide products by hydrolyzing Dendrobium polysaccharides with ThDPS enzyme solution.

[0067] To achieve maximum hydrolysis efficiency, based on the optimized hydrolysis reaction conditions described above, the concentration of Dendrobium polysaccharide was increased to 10 g / L (resulting in viscosity, essentially reaching the maximum concentration) for the hydrolysis reaction. 0.1 g of Dendrobium polysaccharide powder was weighed and dissolved in 10 mL of pH 7.0 phosphate buffer to prepare a Dendrobium polysaccharide solution. Then, 1 mL of ThDPS enzyme solution (enzyme protein concentration of 100 μg / mL) was added, and the reaction was carried out at 60°C for 2 h. The enzyme solution was the ThDPS enzyme solution prepared in Example 1 diluted with deionized water to a concentration of 100 μg / mL.

[0068] After derivatization, the samples were subjected to gel fluorescence electrophoresis. The results were observed under ultraviolet light as follows: Figure 7 As shown: Using unhydrolyzed Dendrobium polysaccharide sample solution as a control, after adding ThDPS enzyme solution to fully hydrolyze Dendrobium polysaccharide, fluorescence glucose electrophoresis analysis showed that the Dendrobium polysaccharide control group did not contain low molecular weight oligosaccharide products under the same conditions; while after using ThDPS enzyme to hydrolyze Dendrobium polysaccharide, a large number of ultra-low molecular weight Dendrobium disaccharide, Dendrobium triaccharide, Dendrobium tetraaccharide, Dendrobium pentasaccharide, and low molecular weight Dendrobium hexasaccharide, Dendrobium heptasaccharide, Dendrobium octasaccharide and above Dendrobium oligosaccharide products were produced. That is, the recombinantly expressed thermophilic mannosidase ThDPS achieved efficient hydrolysis of Dendrobium polysaccharide substrate, and ultra-low molecular weight Dendrobium oligosaccharide products can be directly prepared in one step.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of thermophilic bacteria-derived glycosidases or recombinant microorganisms expressing said glycosidases in the hydrolysis of Dendrobium polysaccharides, characterized in that, The amino acid sequence of the glycosidase is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The gene encoding the glycosidase has the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The recombinant microorganisms include Escherichia coli.

4. The application according to claim 1 or 2, characterized in that, The recombinant microorganism uses Escherichia coli BL21(DE3) as the host and pET21a as the expression vector to express the glycosidase.

5. A method for preparing ultra-low molecular weight Dendrobium oligosaccharides, characterized in that, The glycosidase with the amino acid sequence shown in SEQ ID NO.3 or cell cultures containing the glycosidase were used for the degradation of Dendrobium polysaccharides.

6. The method according to claim 5, characterized in that, The ultra-low molecular weight Dendrobium oligosaccharides have a molecular weight ≤1000 Da and include Dendrobium disaccharide, Dendrobium triaccharide or Dendrobium tetraaccharide.

7. The method according to claim 5 or 6, characterized in that, The glycosidase or cell culture containing the glycosidase is added to a reaction system containing Dendrobium polysaccharide; the amount of the glycosidase added is ≥ 1 U / g. 石斛多糖 .

8. The method according to claim 7, characterized in that, The pH value of the reaction system solution is controlled between 5.0 and 10.0, and the reaction temperature is between 20℃ and 70℃.

9. A recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET21a as the expression vector, the glycosidase gene with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 was expressed.

10. A glycosidase with an amino acid sequence as shown in SEQ ID NO.3, or the method described in any one of claims 5 to 7, or the use of the recombinant Escherichia coli as described in claim 9 in the production of products containing Dendrobium oligosaccharides.

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