Thermophilic beta-1, 4-glucosidase and application thereof
By providing the amino acid sequence of thermophilic β-1,4-glucosidase, the problem of catalyzing the hydrolysis of β-glycosidic bonds at high temperatures was solved, and the enzyme activity and structural stability were maintained at high temperatures, making it suitable for biomass degradation, biofuels and food processing.
Patent Information
- Application Number
- CN202511083301.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-04
- Publication Date
- 2025-10-10
AI Technical Summary
There is currently no commercial thermophilic β-glucosidase, which is unable to efficiently catalyze the hydrolysis of β-glycosidic bonds at high temperatures and maintain structural stability and catalytic activity under high temperature conditions, limiting its application in biomass degradation, biofuels, food processing and other fields.
Provided is an amino acid sequence of a thermophilic β-1,4-glucosidase, which is derived from a thermophilic bacterium from the Eryuan hot springs in Yunnan Province. After full gene synthesis and heterologous expression in Escherichia coli, the enzyme has an optimal reaction temperature at 95°C and good temperature stability, and is capable of hydrolyzing substrates such as p-nitrophenyl-β-D-pyranoglucoside.
Thermophilic β-glucosidase maintains enzyme activity at 95°C, has efficient catalytic performance and good temperature stability, and is suitable for biomass degradation, biofuel production and food processing under high temperature conditions, reducing the risk of microbial contamination.
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Figure CN120758485A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a thermophilic beta-1,4-glucosidase and an application thereof. Background Art
[0002] β-Glucosidase (GH, EC 3.2.1.21) is a class of glycoside hydrolases that catalyze the hydrolysis of β-glycosidic bonds. They act on the β-D-glucosidic bonds at the non-reducing ends of glycosides or oligosaccharides, releasing glucose or other products. They are widely involved in carbohydrate metabolism and natural product transformation. They play a variety of roles in archaea, bacteria, and eukaryotes, including biomass conversion in microorganisms, breakdown of glycolipids and exogenous glucosides in animals, lignification in plants, degradation of cell wall oligosaccharides, defense, activation of plant hormone conjugates and odor release, and plant-microbe and plant-insect interactions.
[0003] β-glucosidase has a wide range of applications in the food, biofuel, and pharmaceutical industries. In the food industry, β-glucosidase-mediated hydrolysis can be used to modify flavor precursors to improve the quality of food and beverages; in engineering, it can be used to improve catalytic properties to enhance flavor, improve stability, and improve nutrition. In biofuel production, β-glucosidase, as part of the cellulase complex, hydrolyzes cellulosic sugars and oligosaccharides to produce glucose, which can be fermented by yeast into the biofuel ethanol. In the pharmaceutical field, β-glucosidase plays an important role in the synthesis of drug precursors and the conversion of naturally active monomers. For example, in the soy food industry, the use of additional β-glucosidase can hydrolyze inactive soy isoflavones into aglycones to activate them. β-glucosidase can also hydrolyze ginsenosides into more active rare ginsenosides.
[0004] Thermophilic β-glucosidases are a class of glycoside hydrolases derived from thermophilic microorganisms. They can efficiently catalyze the hydrolysis of β-glycosidic bonds at temperatures of 60-100°C or even higher, while maintaining structural stability and catalytic activity. Research on thermophilic β-glucosidases is currently attracting considerable attention. Compared to normothermic and mesophilic enzymes, thermostable β-glucosidases maintain thermal stability and catalytic efficiency in biomass degradation, biofuels, food processing, and other applications requiring high temperatures. They also significantly enhance substrate solubility and molecular thermal motion at high temperatures, improving production efficiency and reducing the risk of microbial contamination, making them crucial for industrial production. Currently, there are no commercially available thermophilic β-glucosidases. Summary of the Invention
[0005] The present invention aims to provide a thermophilic β-1,4-glucosidase; the second purpose is to provide the application of the thermophilic β-1,4-glucosidase.
[0006] The first object of the present application is achieved by the amino acid sequence of the thermophilic β-1, 4-glucosidase shown as SEQ ID NO: 1, in particular by: MRLERALFLAHGAEGPEGLPEEGWREVRLPHQWSLEGLEAEVGWYRLALPEGGPRRFLRSLADYYQEAWVEGVHLGRHEGYFFPWLVELPPGRELLLRVSAPKEPLGQWPRFKRQIKGVFGQHDCRPGGTTERGQERGTGGLWGGVEVWEREEVALLGLTHRLYPRPGGWRLLVRLLLDAPRPFREGVRLSLWPENFPGEAMEKEAVLEGEGGRAWREVVWDLPEMPLWEVWERGFPHLYRLEAHLLGATLATPVGFRTVALDPEGWLLLNGRRLFLRGTNIIPTQWLAGYTEAQAERDVALLKEANLNAVRVHAHITHPAFYRACDREGVLVWQDFPLQWGYAPDEAFAQEAVRQARAMVETLGAHPALYLWCAQNEPTHGRHTLGPLLAGEIRTQDPTRPVKEASDFREHPYPGWYWGHLRDFLALPGAPLPSEFGAQALPRAELLKRVLGEAAWPPRWEAYAYHNFQPHETFRVAGVEVGESLEAFVENSQAYQARLLEFAVHAYRRAKGKVVGYFQFMFVEPWEGITWAVLDVERVPKKGFYALKEASSPVLLSLVPYREVLEVGGVPLQEAWLVSDVERPLRLKVRLWLEGPTYLPLYEEEVALAPQEVRRVLSLGELWESPLEAQARFLPIQEALKGLPPGRYLLVGEAWEEGKLWSRQVLGLTYLEPLLPLGVAW The amino acid sequence of the thermophilic β-1, 4-glucosidase is shown as SEQ ID NO: 2, in particular by: YRLEAHLLGATLATPVGFRTVALDPEGWLLLNGRRLFLRGTNIIPTQWLAGYTEAQAER The amino acid sequence of the thermophilic β-1, 4-glucosidase is shown as SEQ ID NO: 3, in particular by: TEAQAERDVALLKEANLNAVRVHAHITHPAFYR The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 4, specifically: ITHPAFYRACDREGVLVWQDFPLQWGYAPDEAFAQEAVRQARAMVETLGAHPALYLWCAQNEPTHGRHTLGPLLAGEIRTQ The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 5, specifically: VRQARAMVETLGAHPALYLWCAQNEPTHGRHTLGPLLAGEIRTQ The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 6, specifically: RWEAYAYHNFQPHETFRVAGVEVGESLEAFVENSQAYQARLLEFAVHAYRRAKGK The second object of the present invention is achieved by using the thermophilic β-1,4-glucosidase in the hydrolysis of p-nitrophenyl-β-D glucopyranoside (pNPGlu), p-nitrophenyl-β-D galactopyranoside (pNPGal), p-nitrophenyl-β-D xylopyranoside (pNPXyl), p-nitrophenyl-α-D glucopyranoside (pNPαGlu), p-nitrophenyl-N-acetylglucosamine, geniposide, polydatin, salicin and arbutin.
[0007] In the present invention, the term "amino acid sequence identity" refers to the Per.Ident parameter of the BlastP search results in NCBI.
[0008] The thermophilic β-glucosidase described in the present invention is a thermophilic β-glucosidase gene mined from the genome of thermophilic bacteria derived from Eryuan hot springs in Yunnan Province. The thermophilic β-glucosidase is obtained by whole gene synthesis and heterologous expression in Escherichia coli.
[0009] The thermophilic β-glucosidase of the present invention has an amino acid sequence of SEQ ID No. 1, which has a full length of 682 amino acid residues.
[0010] The thermophilic β-glucosidase described in the present invention has an optimal reaction temperature of 95°C and no loss of enzyme activity after incubation at 70°C for 48 hours. Compared with currently reported β-glucosidases, it has better temperature stability. Its optimal pH is 8. Under optimal conditions, the specific activity of the pure enzyme is 7.38 U / mg.
[0011] The amino acid sequence of the thermophilic β-glucosidase of the present invention, SEQ ID No. 1, was searched by BlastP in NCBI (nr). The sequence with the highest amino acid sequence identity was derived from Thermus igniterraeThe amino acid sequence identity of the β-glucosidase (WP_018110934.1) is 87.54%. Therefore, the thermophilic β-glucosidase with an amino acid sequence identity greater than 87.00% with the thermophilic β-glucosidase described in the present invention is within the scope of protection of this patent.
[0012] The amino acid sequence SEQ ID No. 2 of the thermophilic β-1,4-glucosidase of the present invention is retrieved by BlastP in NCBI (nr). The sequence with the highest amino acid sequence identity comes from Thermus igniterrae The amino acid sequence identity of WP_018110934.1 is 91.53%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 92.00% with the SEQ ID No.2 described in the present invention is within the scope of protection of this patent.
[0013] The amino acid sequence SEQ ID No. 3 of the thermophilic β-1,4-glucosidase of the present invention is retrieved by BlastP in NCBI (nr). The sequence with the highest amino acid sequence identity is derived from Thermoflexus sp. HXF70058.1, the amino acid sequence identity is 92.31%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 92.50% with the SEQ ID No. 3 described in the present invention is within the scope of protection of this patent.
[0014] The amino acid sequence SEQ ID No. 4 of the thermophilic β-1,4-glucosidase of the present invention is retrieved by BlastP in NCBI (nr). The sequence with the highest amino acid sequence identity comes from Thermus igniterrae The amino acid sequence identity of WP_018110934.1 is 92.59%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 93.00% with the SEQ ID No.4 described in the present invention is within the scope of protection of this patent.
[0015] The amino acid sequence of the thermophilic β-1,4-glucosidase of the present invention is SEQ ID No. 5, and the sequence with the highest amino acid sequence identity is retrieved from BlastP in NCBI (nr). Thermus igniterrae The amino acid sequence identity of WP_018110934.1 is 90.91%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 91.00% with the SEQ ID No.5 described in the present invention is within the scope of protection of this patent.
[0016] The amino acid sequence of the thermophilic β-1,4-glucosidase of the present invention is SEQ ID No. 6, which is retrieved by BlastP in NCBI (nr). The sequence with the highest amino acid sequence identity comes from Thermus igniterrae The amino acid sequence identity of WP_018110934.1 is 90.91%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 91.00% with the SEQ ID No.5 described in the present invention is within the scope of protection of this patent.
[0017] The catalytic mechanism of the thermophilic β-glucosidase described in the present invention was studied by molecular docking using pNPGlu as a ligand. The results showed that the catalytic mechanism of β-1,4-glucosidase is as follows: Glu377 acts as a nucleophilic residue and jointly completes acid-base catalysis with Glu435. Its catalytic mechanism is consistent with that of GH2 family glycoside hydrolases. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Schematic diagram of the optimal reaction temperature of thermophilic β-glucosidase Bgl68203 in Example 3; Figure 2 This is a schematic diagram of the temperature stability of the thermophilic β-glucosidase Bgl68203 in Example 3; Figure 3 Schematic diagram of the effect of pH on the activity of thermophilic β-glucosidase Bgl68203 in Example 4; Figure 4 Schematic diagram of pH stability of thermophilic β-glucosidase Bgl68203 in Example 4; Figure 5 Schematic diagram of the "one-pot method" for preparing blue pigment using thermophilic β-glucosidase Bgl68203 in Example 6. DETAILED DESCRIPTION
[0019] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.
[0020] The amino acid sequence of the thermophilic β-1,4-glucosidase of the present invention is shown in SEQ ID NO: 1.
[0021] The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 2.
[0022] The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 3.
[0023] The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 4.
[0024] The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 5.
[0025] The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 6.
[0026] DNA sequence encoding thermophilic β-1,4-glucosidase.
[0027] The recombinant vector of the DNA sequence.
[0028] The host cell is transformed, transduced or transfected by the recombinant vector.
[0029] The application of the thermophilic β-1,4-glucosidase of the present invention is the application of the thermophilic β-1,4-glucosidase in the hydrolysis of p-nitrophenyl-β-D glucopyranoside (pNPGlu), p-nitrophenyl-β-D galactopyranoside (pNPGal), p-nitrophenyl-β-D xylopyranoside (pNPXyl), p-nitrophenyl-α-D glucopyranoside (pNPαGlu), p-nitrophenyl-N-acetylglucosamine, geniposide, polydatin, salicin and arbutin.
[0030] The reaction temperature of the hydrolysis is 80-100°C.
[0031] The reaction temperature of the hydrolysis is 90°C.
[0032] The present invention will be further described below with reference to specific implementation cases: Example 1
[0033] Expression of thermophilic β-glucosidase Synthesis of the Thermophilic β-glucosidase Gene: A thermophilic β-glucosidase gene with 682 amino acid residues was predicted from the metagenome. The gene was synthesized by Sangon Biotech Co., Ltd. using Escherichia coli as the host, after codon optimization, and the recombinant plasmid pET-28(a)-Bgl68203 was constructed.
[0034] Plasmid extraction: Use SanPrep column-based plasmid DNA small-scale extraction kit according to the instructions.
[0035] Transformation of E. coli competent cells: Using the heat shock transformation method, take the recombinant plasmid dry powder (about 4µg) pET-28(a)-Bgl68203 after gene synthesis and place it in a centrifuge at 12000r / min for 2 minutes. Add 40µL ddH2O to a final concentration of 200ng / µL of recombinant plasmid. Transform into the prepared E. coli competent cells. When the E. coli competent cells are in the ice-water mixture, immediately take 20µL of the recombinant plasmid and add it to the E. coli Gently pipette to mix thoroughly, then place on ice for 30 minutes. Place the mixture in a 42°C water bath for 90 seconds (to open the cell membrane and allow the plasmid to enter the cells). Immediately remove the mixture and place it back on ice for 5 minutes (to close the cell membrane). Add 500µL of antibiotic-free LB medium to the centrifuge tube and incubate the medium in a shaker at 37°C, 220 rpm, for 30 minutes. Use a sterile spreader to spread 200µL of the culture onto solid LB medium containing kanamycin sulfate (final concentration 50µg / mL). Place the inoculated medium upright in a 37°C incubator for 30 minutes, then incubate it upside down for 12-16 hours.
[0036] Inducible expression of thermophilic β-glucosidase: A. Single clone small volume (5 mL) culture: Pick recombinant Escherichia coli E. coli BL21(DE3) monoclonal cells were cultured in 10 mL centrifuge tubes with 5 mL of LB resistance liquid medium (Kan, 50 µg / mL) and incubated at 37°C, 220 rpm, in a constant temperature shaker for 12-16 h.
[0037] B. Expansion culture (300 mL): Take 3 mL of bacterial solution (1% inoculum size) and inoculate it into 300 mL of fresh LB liquid medium (Kan, 50 μg / mL). Incubate in a constant temperature shaker at 37°C, 220 rpm for 4-6 hours until the OD 600 =0.4-0.6, add 714µL IPTG (final concentration 0.5mM), culture in a constant temperature shaker at 16℃, 220rpm for 20h, centrifuge at 8000rpm / min for 5min, discard the supernatant and collect the cells, ensuring that 4.0g of cells are collected in each centrifuge tube, and store at -20℃.
[0038] Example 2
[0039] Determination of thermophilic β-glucosidase activity Thermophilic β-glucosidase activity was determined using a p-nitrophenol colorimetric method. Specifically, 50 μL of 10 mmol / L pNPGlu and 50 μL of enzyme solution were reacted at the optimal pH and temperature for 20 minutes. After a 5-minute ice bath, 50 μL of 1 M Na₂CO₃ solution was added to terminate the enzymatic reaction. 100 μL of the mixture was aliquoted and measured for absorbance at 405 nm on a microplate reader. The enzyme activity unit (U) was defined as the amount of enzyme required to release 1 μmol of pNP per minute. A standard curve was generated using p-nitrophenol (pNP) to determine enzyme activity. The expression level of thermophilic β-glucosidase was 11.02 U / mL.
[0040] Example 3
[0041] Study on the Optimum Temperature and Thermostability of Thermophilic β-glucosidase To determine the optimal temperature for the thermophilic β-glucosidase Bgl68203, pNPGlu was used as a substrate at a final concentration of 10 mmol / L. A diluted enzyme solution was added and incubated at a series of temperatures (30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 95°C, 100°C, and 110°C) for 20 minutes. Enzyme activity was then measured. Two blank controls were included: an enzyme blank (containing enzyme solution and buffer) and a substrate blank (containing substrate and buffer). Five replicates were performed for each experimental group to determine the optimal reaction temperature. The optimal temperature curve was plotted, with the temperature at which enzyme activity reached 100% activity.
[0042] To investigate the temperature stability of thermophilic β-glucosidase Bgl68203, the enzyme was diluted to an appropriate concentration and incubated at 70°C, 80°C, and 90°C for 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours, respectively. The enzyme activity was measured after 20 minutes at the optimal pH and temperature. Two blank controls were included: an enzyme blank (containing enzyme solution and buffer) and a substrate blank (containing substrate and buffer). Five replicates were performed for each experimental group, and the enzyme activity without temperature tolerance treatment was taken as 100%. The temperature stability curve was then plotted based on this activity.
[0043] The experimental results show that the optimal reaction temperature of Bgl68203 is 90℃ ( Figure 1 ); The temperature stability of Bgl68203 is as follows Figure 2 As shown in the figure, after treatment at 70℃ for 48h, the residual enzyme activity was greater than 95%, indicating excellent thermal stability at 70℃. After treatment at 80℃ for 36h, nearly 50% of the residual enzyme activity was still retained. After treatment at 90℃ for 2h, the residual enzyme activity was 48.2%.
[0044] Example 4
[0045] Study on the Optimal pH and pH Stability of Thermophilic β-Glucosidase To determine the optimal pH for thermophilic β-glucosidase Bgl68203, pNPGlu was used as a substrate at a final concentration of 10 mmol / L. A diluted enzyme solution was added and incubated at a range of pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) for 20 minutes. Enzyme activity was then measured. Two blank controls were included: an enzyme blank (containing enzyme solution and buffer) and a substrate blank (containing substrate and buffer). Five replicates were performed for each experimental group to determine the optimal reaction pH. The optimal pH curve was plotted, with the pH at which enzyme activity reached its peak being defined as 100%.
[0046] To investigate the pH stability of thermophilic β-glucosidase Bgl68203, the enzyme was diluted to an appropriate concentration and incubated at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 for 24 hours. The enzyme activity was then measured after 20 minutes at the optimal pH and temperature. Two blank controls were included: an enzyme blank (containing enzyme solution and buffer) and a substrate blank (containing substrate and buffer). Five replicates were performed for each experimental group, and the enzyme activity without pH tolerance treatment was taken as 100%. pH stability curves were then constructed based on these results.
[0047] like Figure 3 As shown, the optimum pH of thermophilic β-glucosidase Bgl68203 is 8.0; Figure 4 As shown in the figure, the relative enzyme activity was greater than 50% when the pH was between 7.5 and 8.5. In the pH range of 7.5-9.0, the thermophilic β-glucosidase Bgl68203 showed good pH stability, with relative enzyme activity greater than 80%.
[0048] Example 5
[0049] Study on the substrate spectrum of thermophilic β-glucosidase β-glucosidases from different sources vary in their ability to hydrolyze β-glycosidic bonds. Therefore, the substrate specificity of Bgl68203 was investigated using a series of synthetic pNP substrates and natural substrates. The pNP standard substrates included p-nitrophenyl-β-D-glucopyranoside (pNPGlu), p-nitrophenyl-β-D-galactopyranoside (pNPGal), p-nitrophenyl-β-D-xylopyranoside (pNPXyl), p-nitrophenyl-α-D-glucopyranoside (pNPαGlu), and p-nitrophenyl-N-acetylglucosamine, all at a final concentration of 10 mmol / L. Under the same conditions, the reaction was carried out at 90°C for 20 min. Enzyme activity was determined using the p-nitrophenol colorimetric method, with absorbance measured at 405 nm. Natural substrates included geniposide, polydatin, salicin, arbutin, and sodium carboxymethylcellulose, all at a final concentration of 10 mmol / L. After reacting at 90°C for 20 minutes, the enzyme activity was determined by measuring the absorbance at a wavelength of 540 nm using the DNS method. The above analysis analyzed the hydrolysis ability of thermophilic β-glucosidase on different substrates.
[0050] As shown in Table 1, the enzyme activity of p-nitrophenyl-β-D-pyranoside was 100%, the relative enzyme activity of hydrolyzing geniposide was 100%, and sodium carboxymethylcellulose had no enzyme activity.
[0051] Table 1 Study on substrate specificity of thermophilic β-glucosidase Note: “ND” means that the data set was not detected.
[0052] Example 6
[0053] Study on the one-pot preparation of blue pigment using thermophilic β-glucosidase The optimal pH for producing gardenia blue pigment by the "one-pot method" of thermophilic β-glucosidase Bgl68203, gardeniain and methionine was 6.0, the optimal temperature was 90℃, the optimal ratio of gardeniain to methionine was 1:8, the optimal concentration of gardeniain was 25mmol / L when the raw material was 98% gardeniain, the optimal concentration of gardeniain was 10mmol / L when the raw material was 30% gardeniain, and the optimal reaction time was 12h ( Figure 5 High-quality gardenia blue pigment can be prepared by a one-pot method using thermophilic β-glucosidase Bgl68203, 98% geniposide, and methionine, indicating that thermophilic β-glucosidase Bgl68203 has the potential for industrial application in the production of gardenia blue pigment.
Claims
1. A thermophilic β-1,4-glucosidase, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
1.
2. The thermophilic β-1,4-glucosidase according to claim 1, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
2.
3. The thermophilic β-1,4-glucosidase according to claim 1, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
3.
4. The thermophilic β-1,4-glucosidase according to claim 1, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
4.
5. The thermophilic β-1,4-glucosidase according to claim 1, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
5.
6. The thermophilic β-1,4-glucosidase according to claim 1, characterized in that The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO:
6.
7. A DNA sequence encoding any one of claims 1 to 6.
8. A recombinant vector of the DNA sequence according to claim 7.
9. A host cell transformed, transduced or transfected with the recombinant vector according to claim 8.
10. Use of the thermophilic β-1,4-glucosidase according to any one of claims 1 to 6, characterized in that: The thermophilic β-1,4-glucosidase is used in the hydrolysis of p-nitrophenyl-β-D glucopyranoside (pNPGlu), p-nitrophenyl-β-D galactopyranoside (pNPGal), p-nitrophenyl-β-D xylopyranoside (pNPXyl), p-nitrophenyl-α-D glucopyranoside (pNPαGlu), p-nitrophenyl-N-acetylglucosamine, geniposide, polydatin, salicin and arbutin.