Thermophilic beta-1, 4-glucosidase and application thereof

Abstract

The invention discloses thermophilic beta-1, 4-glucosidase and an application of the thermophilic beta-1, 4-glucosidase. The base sequence of the thermophilic beta-1, 4-glucosidase is as shown in SEQ ID NO: 1. The thermophilic beta-glucosidase provided by the invention can be used for hydrolyzing p-nitrophenyl-beta-D glucopyranoside (pNPGlu), p-nitrophenyl-beta-D galactopyranoside (pNPGal), p-nitrophenyl-beta-D xylopyranoside (pNPXyl), p-nitrophenyl-alpha-D glucopyranoside (pNP alpha Glu), p-nitrophenyl-N-acetylglucosamine, geniposide, polydatin, glucosyltransferase, glucosyltransferase and the like. And salicin and arbutin. The optimum reaction temperature of the enzyme is 95 DEG C, and after the enzyme is treated at 70 DEG C for 24 hours, the residual enzyme activity is greater than 40%. In an n-hexyl alcohol-water phase (or n-caprylic alcohol-water phase), geniposide can be hydrolyzed into genipin, and industrial production of genipin is facilitated.

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.

[0005] Genipin is an iridoid ether terpenoid compound with diverse biological activities, found widely in plants such as Gardenia jasminoides. Its unique cross-linking properties and pharmacological effects, including anti-inflammatory, anti-tumor, and antioxidant properties, have attracted significant attention in the biopharmaceutical, food processing, and biomaterials sectors. Gardeniaside, a precursor compound of genipin, is found in high concentrations in plants and can be obtained through the specific hydrolysis of its glycosidic bond by β-glucosidase. Summary of the Invention

[0006] 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.

[0007] The first object of the present invention is achieved as follows: the amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 1; specifically: MGEFKDLVFPSGFLWGCAVAAHQVEGNNTNNTWWKWEQEGHTLDPSGIACDHYNRYEEDLDIAKKLSLNTFRTSVEWSRIEPKEGVWSDKEIKHYRRVLEAMRERGLT PMITLHHFTDPLWFAEKGGWERPESTDIFARFVRKVVEELGDLIPFYNTINEPMVYVVLGYAFGVFPPGERDLLKALTVARHLLLAHAKAYRVIHEVCKEKGYPKPKVG IVHNMIVFEPLDPNDERHVNEANTSDAIYNRWFLESIHAGAVQPPAGQSEEVEELKGAWDFIGLNYYTRNICIPSPDPARRFNVVPMDAELTDMHYEVYPEGLYKLLVSLKKYGKPVYITENGIATSSDRQRCRFILRHLVEAHRAMREGVDLKGYIYWSLIDNFEWNEGFSKRFGIVEVDYKTLKRTPRESAYMYAEIARKNKVTSDLMQKYLGRK The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 2, specifically: VFPPGERDLLKALTVARHLLLAHAKAYRVIHEVCKEKGYPKPKVGIVHNMI The amino acid sequence of the thermophilic β-1,4-glucosidase is shown in SEQ ID NO: 3, specifically: SEEVEELKGAWDFIGLNYYTRNICIPSPDPARRFNVV The amino acid sequence of the thermophilic beta-1,4-glucosidase is shown in SEQ ID NO: 4, specifically: LLKALTVARHLLLAHAKAYRVIHEVCKEKGYPKPKVGIVHNMIVFEPLDPNDERHVNEANTSDAIYNRWFLESIHAGAVQPPAGQSEEVEELKGAWDFIGLNYYTRNICIPSPDPARRFNVV The amino acid sequence of the thermophilic beta-1,4-glucosidase is shown in SEQ ID NO: 5, specifically: SEEVEELKGAWDFIGLNYYTRNICIPSPDPARRFNVVPMDAELTDMHYEVYPEGLYKLLVSLKKYGKPVYITENGIATSSDRQRCRFILRHLV The second object of the application is achieved in that the thermophilic beta-1,4-glucosidase is applied in hydrolysis of p-nitrophenyl-beta-D glucopyranoside (pNPGlu), p-nitrophenyl-beta-D galactopyranoside (pNPGal), p-nitrophenyl-beta-D xylopyranoside (pNPXyl), p-nitrophenyl-alpha-D glucopyranoside (pNP alpha Glu), p-nitrophenyl-N-acetylglucosamine, geniposide, polydatin, salicin and arbutin.

[0008] In the application, the term "amino acid sequence identity" refers to the Per.Ident parameter of the BlastP search result in NCBI.

[0009] The thermophilic beta-glucosidase described in the application is a thermophilic beta-glucosidase gene mined from the genome of a thermophilic bacterium from the Eryuan hot spring in Yunnan Province, and the thermophilic beta-glucosidase is obtained by whole gene synthesis and heterologous expression in Escherichia coli.

[0010] The thermophilic beta-glucosidase described in the application has an amino acid sequence of SEQ ID No. 1, the sequence has a total length of 434 amino acid residues, and the theoretical molecular weight is 51.8 kDa.

[0011] The thermophilic beta-glucosidase described in the application has an optimal reaction temperature of 95℃, and 50% of the residual enzyme activity is still present after incubation at 80℃ for 8h, and has better temperature stability; the optimal pH is 6.0. Under the optimal conditions, the specific activity of the pure enzyme is 5.44 U / mg.

[0012] The amino acid sequence of the thermophilic beta-glucosidase described in the application is SEQ ID No. 1, and the sequence with the highest amino acid sequence identity is the sequence derived from Jordarchaeia archaeonThe MBS7247257.1 sequence has an amino acid sequence identity of 95.62%. Therefore, the thermophilic β-glucosidase with an amino acid sequence identity greater than 96.00% with the thermophilic β-glucosidase described in the present invention is within the scope of protection of this patent.

[0013] 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 is derived from Jordarchaeia archaeon The MBS7247257.1 sequence has an amino acid sequence identity of 90.20%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 91.00% with the SEQ ID No.2 described in the present invention is within the scope of protection of this patent.

[0014] 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 Jordarchaeia archaeon The amino acid sequence identity of the MEM3467137.1 sequence is 91.89%, so the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 92.00% with the SEQ ID No.3 described in the present invention is within the scope of protection of this patent.

[0015] 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 is derived from Jordarchaeia archaeon The MBS7247257.1 sequence has an amino acid sequence identity of 92.62%. Therefore, the β-1,4-glucosidase with an amino acid sequence identity of greater than or equal to 93% with the SEQ ID No.4 described in the present invention is within the scope of protection of this patent.

[0016] The amino acid sequence SEQ ID No. 5 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 Jordarchaeia archaeon The MBS7247257.1 sequence has an amino acid sequence identity of 93.55%. Therefore, the β-1,4-glucosidase with an amino acid sequence identity greater than or equal to 94.00% 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: Glu166 acts as a nucleophilic residue and jointly completes acid-base catalysis with Glu338, which is consistent with the catalytic mechanism of the glycoside hydrolase GH1 family. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the optimal reaction temperature of thermophilic β-glucosidase in Example 3; Figure 2 This is a schematic diagram of the temperature stability of thermophilic β-glucosidase in Example 3; Figure 3 Schematic diagram of the effect of pH on thermophilic β-glucosidase activity in Example 4; Figure 4 Schematic diagram of pH stability of thermophilic β-glucosidase in Example 4; Figure 5 Schematic diagram of the standard curve of genipin in Example 6; Figure 6 Schematic diagram of the reaction curve of geniposide and genipin in n-hexanol-water phase in Example 6; Figure 7 Schematic diagram of the reaction curve of geniposide and genipin in n-octanol-water phase 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 base 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] A DNA sequence encoding the thermophilic β-1,4-glucosidase.

[0026] The recombinant vector of the DNA sequence.

[0027] The host cell is transformed, transduced or transfected by the recombinant vector.

[0028] The thermophilic β-1,4-glucosidase described herein can be used to hydrolyze 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. The enzyme has an optimal reaction temperature of 95°C, and after treatment at 70°C for 24 hours, the residual enzyme activity is greater than 40%. In an n-hexanol-water phase (or n-octanol-water phase), geniposide can be hydrolyzed to genipin, facilitating the industrial production of genipin.

[0029] The present invention will be further described below with reference to specific implementation cases: Example 1

[0030] Expression of thermophilic β-glucosidase Synthesis of the Thermophilic β-glucosidase Gene: A thermophilic β-glucosidase gene with 434 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)-Bgl68 was constructed.

[0031] Plasmid extraction: Use SanPrep column-based plasmid DNA small-scale extraction kit according to the instructions.

[0032] Transformation of E. coli competent cells: Using the heat shock transformation method, take the recombinant plasmid dry powder (about 4µg) pET-28(a)-Bgl68 after gene synthesis and place it in a centrifuge at 12000r / min. Centrifuge for 2 minutes and add 40µL ddH2O. The final concentration of the recombinant plasmid is 200ng / µL. 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. coliGently pipette to mix thoroughly, then place on ice for 30 minutes. Place the competent colon cell 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 cell 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.

[0033] 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.

[0034] 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℃.

[0035] Example 2

[0036] 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.

[0037] Example 3

[0038] Study on the Optimum Temperature and Thermostability of Thermophilic β-glucosidase To determine the optimum temperature of the thermophilic β-glucosidase, the enzyme solution was diluted to a certain multiple, and the final concentration of pNPGlu was 10 mmol / L. The enzyme solution was placed in a series of temperature environments (30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 95°C, 100°C, 110°C) for 20 min, and the enzyme activity was determined. Double blank controls were set: enzyme blank group (containing enzyme solution and buffer) and substrate blank group (containing substrate and buffer). Each experimental group was set with 5 repeats to determine the optimum reaction temperature. The highest enzyme activity was taken as 100% enzyme activity, and the optimum temperature curve was plotted.

[0039] To explore the temperature stability of the thermophilic β-glucosidase, the enzyme was diluted to an appropriate concentration and incubated at different temperatures of 70°C, 80°C and 90°C for 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h and 48h, respectively. The enzyme activity was determined under the optimum pH and temperature for 20 min. Double blank controls were set: enzyme blank group (containing enzyme solution and buffer) and substrate blank group (containing substrate and buffer). Each experimental group was set with 5 repeats, and the enzyme activity without temperature tolerance treatment was taken as 100%. The temperature stability curve was plotted accordingly.

[0040] As shown in Figure 1 , the optimum temperature of the thermophilic β-glucosidase was 95°C. As shown in Figure 2 , after 24h of treatment at 70°C, the remaining enzyme activity was nearly 40%, indicating that it had good temperature stability.

[0041] Example 4

[0042] Optimum pH and pH stability of thermophilic β-glucosidase To determine the optimum pH of the thermophilic β-glucosidase, the enzyme solution was diluted to a certain multiple, and the final concentration of pNPGlu was 10 mmol / L. The enzyme solution was placed in a series of pH environments (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) for 20 min, and the enzyme activity was determined. Double blank controls were set: enzyme blank group (containing enzyme solution and buffer) and substrate blank group (containing substrate and buffer). Each experimental group was set with 5 repeats to determine the optimum reaction pH. The highest enzyme activity was taken as 100% enzyme activity, and the optimum pH curve was plotted.

[0043] To investigate the pH stability of thermophilic β-glucosidase, 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.

[0044] like Figure 3 As shown in Figure 2, the optimum pH of thermophilic β-glucosidase is 6.0. Figure 4 As shown in the figure, when the pH is between 5.0-9.0, the relative enzyme activity of thermophilic β-glucosidase is greater than 60%, showing good pH stability.

[0045] Example 5

[0046] 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 thermophilic β-glucosidases 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, with 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, with 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.

[0047] 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.

[0048] Table 1 Study on substrate specificity of thermophilic β-glucosidase Note: “ND” means that the data set was not detected.

[0049] Example 6

[0050] Study on the Preparation of Genipin by Thermophilic β-Glucosidase β-glucosidase specifically hydrolyzes gardeniaside to produce genipin. However, β-glucosidase at room temperature has low catalytic efficiency and poor stability. Furthermore, the substrate solubility is limited in a single aqueous phase, resulting in low yields that fail to meet industrial demand. In this study, thermophilic β-glucosidase was used to catalyze the conversion of gardeniaside to genipin in an organic-aqueous biphasic system.

[0051] (1) Drawing of the genipin standard curve Accurately weigh 10 mg of genipin standard, dissolve it fully in methanol, and dilute to a 100 mL volumetric flask. Then, draw different volumes of genipin and dilute to the mark with methanol to prepare a standard gradient solution of 20, 40, 60, 80, and 100 μg / mL. Use HPLC to detect genipin, and draw a standard curve with genipin concentration as the horizontal axis and the corresponding peak area as the vertical axis. Figure 5 As shown, the linear equation is y=32.506x-23.82, R 2 =0.9991.

[0052] (2) Study on the distribution coefficient of two-phase system To select suitable organic solvents for preparing the biphasic system, the partition coefficients of geniposide and genipin were determined in biphasic systems formed by isoamyl alcohol, n-butanol, ethyl acetate, n-hexanol, and n-octanol with water. As shown in Table 2, the partition coefficients of geniposide and genipin in isoamyl alcohol and ethyl acetate were similar, indicating that separation of geniposide and genipin was difficult in these systems. In the n-butanol-water system, although the partition coefficient of geniposide in the organic phase was higher, this conflicted with the research objective of producing large quantities of genipin. The partition coefficients of n-hexanol and n-octanol differed significantly, exceeding 30-fold and 90-fold, respectively. In the biphasic systems formed by n-hexanol and n-octanol with water, genipin primarily partitioned into the organic phase, while geniposide partitioned into the aqueous phase. This facilitated the enzyme reaction in the aqueous phase, while the product genipin was directly extracted into the organic phase, facilitating product separation and purification. Therefore, n-hexanol and n-octanol were selected for further studies.

[0053] Table 2 Partition coefficients of geniposide and genipin in different organic-aqueous systems (3) Thermophilic β-glucosidase converts gardeniaside to genipin in a biphasic system Depend on Figure 6 and Figure 7As shown, in both the n-hexanol and n-octanol-water systems, the conversion of geniposide increased with reaction time between 0 and 3.5 h. In the n-hexanol-water system, the yield of genipin increased with reaction time, exceeding 80.0% after 5 h. In the n-octanol-water system, the yield of genipin reached a maximum of 81.5% after 3.5 h, then gradually decreased after 3.5 h. At the same reaction time, the reaction rate in the n-octanol-water system was faster than that in the n-hexanol-water system, but the yield of genipin decreased with increasing reaction time. This may be due to the instability of genipin in the n-octanol-water system for extended periods (>3.5 h).

[0054] The reaction is carried out in a two-phase system, and the hydrophobic product genipin is preferentially distributed into the organic phase (n-hexanol or n-octanol) during the preparation process through the two-phase distribution effect of the organic solvent and water. By efficiently extracting genipin in situ, the product concentration in the aqueous phase is reduced, the product inhibition effect is reduced, and the yield is improved.

Claims

1. A thermophilic β-1,4-glucosidase, characterized in that The base 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. A DNA sequence encoding any one of claims 1 to 5.

7. A recombinant vector of the DNA sequence according to claim 6.

8. A host cell transformed, transduced or transfected with the recombinant vector according to claim 7.

9. Use of the thermophilic β-1,4-glucosidase according to any one of claims 1 to 5, 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.

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