Beta-glycosidase derived from ophiorrhiza pumila and application of beta-glycosidase

Abstract

The invention provides a beta-glycosidase OpBGlu1 (beta-glycosidase OpBGlu1) derived from Ophiorrhziza pumila and an application of the beta-glycosidase OpBGlu1 in catalytic synthesis of aglycone. The amino acid sequence of the beta-glucosidase is as shown in SEQ ID No.1, and the nucleotide sequence of the beta-glucosidase is as shown in SEQ ID No.2. The OpBGlu1 has a catalytic effect on a plurality of substrates, and especially can be used for efficiently producing loganin and strictosamide aglycone. The beta-glucosidase disclosed by the invention has a catalytic hydrolysis effect on a plurality of monoterpenoid indole alkaloids in the hairy root extract of the ophiorrhiza pumila. The beta-glucosidase and the method for producing various aglycones by using the beta-glucosidase can provide a new thought for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and relates to β-glucosidase, specifically to a β-glucosidase derived from *Hedyotis diffusa* (…). Ophiorrhiza pumila β-glycosidase OpBGlu1 and its application in catalytic synthesis of aglycones. Background Technology

[0002] β-Glucosidase (EC3.2.1.21) is an important component of the cellulolytic enzyme system, capable of hydrolyzing terminal non-reducing β-D-glucose bonds, releasing β-D-glucose and the corresponding aglycone. This enzyme has been widely used in food processing (e.g., flavor enhancement of fruit juices, flavor improvement of fruit wines), production of pharmaceutical active ingredients (e.g., preparation of soybean isoflavone aglycones), and cellulose degradation. However, β-glucosidases with broad substrate profiles and glycosylation activity remain relatively scarce, making the development of novel β-glucosidases with both industrial applicability crucial.

[0003] Aglycones, characterized by poor water solubility and high separation difficulty (such as loganin), face significant limitations in their pharmacological research and application development due to constraints in their production processes. Traditional acid / alkali hydrolysis methods are inefficient for preparing these aglycones (e.g., the ester structure of loganin is easily degraded under extreme conditions), while β-glycosidase-catalyzed methods offer advantages such as mild reaction conditions, single product, and ease of purification. For example, loganin can be used in the treatment of multidrug-resistant Gram-negative bacterial infections, liver injury, and anti-tumor applications, but its efficient production depends on the discovery of novel β-glycosidases.

[0004] Reported β-glycosidases using terpenoid alkaloids as substrates all exhibit high substrate specificity. For example, those derived from periwinkle (Catharanthus roseus) Catharanthus roseus CrSGD from snake root ( Rauvolfia serpentina RsSGD from [a specific plant] can efficiently catalyze only isoprogin, while RsRGD from [another plant] can efficiently catalyze only rocasine. Currently, there are no reports on the rapid enzymatic preparation of loganin.

[0005] Given the substrate specificity of most β-glucosidases, it is of great significance to discover new β-glucosidases to meet the diversity of aglycone production processes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel β-glycosidase derived from *Hedyotis diffusa*, and to provide its application in the conversion process of various glycoside compounds into corresponding aglycones.

[0007] In a first aspect, the present invention provides a β-glycosidase of *Hedyotis diffusa*, the β-glycosidase comprising the amino acid sequence shown in SEQ ID No. 1.

[0008] Secondly, the present invention provides a gene encoding the above-mentioned *Hedyotis diffusa* β-glycosidase, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0009] Thirdly, the present invention provides a recombinant vector carrying the aforementioned genes.

[0010] Fourthly, the present invention provides a recombinant cell containing the above-mentioned recombinant vector or containing the above-mentioned gene.

[0011] Fifthly, the present invention provides an engineered strain carrying the above-mentioned recombinant vector, wherein the engineered strain is a recombinant Escherichia coli Rosetta (DE3) strain.

[0012] In a sixth aspect, the present invention provides a method for producing the above-mentioned *Hedyotis diffusa* β-glycosidase, wherein the above-mentioned engineered strain is used for fermentation to produce the enzyme.

[0013] In a seventh aspect, the present invention provides the use of the above-mentioned *Hedyotis diffusa* β-glucosidase for catalytic hydrolysis of the non-reducing β-D-glucosidic bond at the end of glycoside compounds to generate the corresponding aglycone.

[0014] Furthermore, the glycoside compound is any one of strychnine, loganic acid, strychnine, loganic acid, iso-strychnine, isovincinolactam, eugenol, and deoxyeugenol.

[0015] Eighthly, the present invention provides an application of the above-mentioned *Hedyotis diffusa* β-glycosidase for catalytic hydrolysis of monoterpenoid indole alkaloids in the hairy root extract of *Hedyotis diffusa*.

[0016] Furthermore, the glycosaminoglycan indole alkaloid is one or more of isothioglycan, isovincinolactam, eugenol, and deoxyeugenol.

[0017] In a ninth aspect, the present invention provides a method for hydrolyzing glycoside compounds, the method comprising the step of contacting the glycoside compound with the *Hedyotis diffusa* β-glucosidase, the method being carried out under conditions that enable the enzymatic catalytic action of the *Hedyotis diffusa* β-glucosidase to hydrolyze the β-D-glucosidic bond at the non-reducing end of the compound.

[0018] Furthermore, the reaction was carried out under conditions of pH 5-6 and temperature 30-50℃.

[0019] Preferably, the enzyme maintains high activity at pH 6.0.

[0020] One embodiment provides a protein comprising, or substantially comprising, the amino acid sequence shown in SEQ ID NO. 1. It should be understood that those skilled in the art can, based on the amino acid sequence disclosed in this invention, substitute, delete, and / or add one or more amino acids to obtain mutant sequences of the protein with the same activity without affecting its activity. Therefore, the β-glycosidase of this invention also includes proteins derived from the protein shown in SEQ ID No. 1 by substituting, deleting, or adding one or more amino acids from the amino acid sequence shown in SEQ ID No. 1 and having equivalent activity. For example, by adding a tag sequence, such as a His-tag, to the end of the protein. For example, the protein or the enzyme may contain an amino acid sequence having 80% or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or higher homology to the amino acid sequence of SEQ ID NO. 1 while retaining the activity of converting strychnine to loganin.

[0021] The polynucleotide encoding this protein may comprise, or consist essentially of, the nucleotide sequence shown in SEQ ID NO. 2 or a nucleotide sequence substantially identical to SEQ ID NO. 2. As used herein, the term "substantially identical" means, as analyzed by an alignment procedure, that the polynucleotide comprises a nucleic acid sequence having at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99%, for example, 100% identity with a reference sequence, and that the protein encoded by this polynucleotide sequence retains the activity of converting strychnine, loganic acid, strychnine glycoside, strychnine acid, isopigenin, isovincinolactam, eugenol, and deoxyeugenol into their corresponding aglycones. Those skilled in the art will understand that, as a result of genetic code degeneracy, many different polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can perform nucleotide substitutions using conventional techniques, and such substitutions will not affect the polypeptide sequence encoded by the polynucleotides used in this invention. In addition, polynucleotides can be modified using methods known in the art to enhance the activity or survival time of the polynucleotides of this invention in vivo.

[0022] As long as the host cell possesses the ability to express a protein (including overexpression), it can be transformed into a host cell for the recombinant vector. For example, Pichia pastoris. Examples of Pichia pastoris include, but are not limited to: GS115, X-33, KM71, KM71H, SMD1168, and SMD1168H. Furthermore, the host cell can also be *Escherichia coli*, such as BL21 and JM109.

[0023] Beneficial effects: The β-glycosidase in this invention has catalytic activity on a variety of substrates, providing diversity and possibilities for the production process of aglycones; The β-glycosidase in this invention can efficiently produce loganin and isocinolone lactam. In industrial applications, the β-glucosidase of this invention is cost-effective, has mild and simple reaction conditions, short reaction time, and the product is easy to purify. The β-glycosidase in this invention catalyzes the hydrolysis of various monoterpenoid indole alkaloids in the hairy root extract of *Hedyotis diffusa*, allowing for direct enzymatic processing of the crude extract without the need for separate separation of various glycosides, releasing aglycones in one step and simplifying the process. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 According to Embodiment 1 of the present invention pCold-TF-OpBGlu1 SDS of enzyme proteins obtained from shake-flask fermentation - PAGE electrophoresis image; Figure 2 The effect of reaction temperature on the activity of recombinant β-glucosidase; Figure 3 The effect of buffer pH on the activity of recombinant β-glucosidase; Figure 4A This is the HPLC chromatogram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 5; Figure 4B This is the extraction ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 5; Figure 4C This is an ion fragment diagram of the reaction product loganin in Example 5; Figure 5A This is the HPLC chromatogram of the OpBGlu1-catalyzed deglycosylation reaction of loganic acid in Example 6; Figure 5B This is the extraction ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of loganic acid in Example 6; Figure 5C This is an ion fragment diagram of the reaction product loganic acid aglycone in Example 6; Figure 6A This is the HPLC chromatogram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 7; Figure 6B This is the extraction ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 7; Figure 6CThis is an ion fragment diagram of the strychnos nux-ointense aglycone, the reaction product from Example 7. Figure 7A This is the HPLC chromatogram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 8; Figure 7B This is the extracted ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of strychnine in Example 8; Figure 7C This is an ion fragment diagram of the reaction product strychnoside in Example 8; Figure 8A This is the HPLC chromatogram of the OpBGlu1-catalyzed deglycosylation reaction of isopigenin in Example 9; Figure 8B This is the extraction ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of isopigenin in Example 9; Figure 8C This is an ion fragment diagram of the reaction product isopigenin in Example 9; Figure 9A This is the HPLC chromatogram of the deglycosylation reaction of isocinolone lactam catalyzed by OpBGlu1 in Example 10; Figure 9B This is the extracted ion diagram of the OpBGlu1-catalyzed deglycosylation reaction of isocinolone lactam in Example 10; Figure 9C This is an ion fragment diagram of the reaction product isovinctin lactam aglycone in Example 10; Figure 10 These are the Michaelis-Menten curves of OpBGlu1 catalyzing various substrates; Figure 11 This is the UPLC-QTOF / MS total ion chromatogram of the OpBGlu1-catalyzed extract of *Hedyotis diffusa* in Example 11. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by the claims of this application. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0026] Example 1: Obtaining the nucleotide and amino acid sequences of β-glycosidase and its protein expression and purification. The roots of *Hedyotis diffusa* were used as tissue samples, and total RNA was extracted from them. The obtained total RNA was isolated and enriched as mRNA, then reverse transcribed into cDNA, and a cDNA library was constructed. Using this cDNA library as a template, PCR amplification was performed using forward and reverse primers (see Table 1) to obtain β-glycosidase (…). OpBGlu1 The gene sequence of ) is shown in SEQ ID NO. 2.

[0027] The specific PCR amplification system and conditions were as follows: A 50 μL PCR amplification system included 1 μL cDNA, 25 μL 2×PrimeSTAR Max DNA Polymerase, 1 μL primer (10 μmol), and 22 μL sterile ultrapure water. The PCR reaction conditions were: 98℃ pre-denaturation for 5 minutes; 98℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for 35 cycles; and a final extension at 72℃ for 10 minutes.

[0028] The obtained gene sequence was translated from the DNA sequence to obtain the amino acid sequence of β-glycosidase, as shown in SEQ ID NO.1.

[0029] The PCR products obtained above were then reacted with the vector. pCold-TF The ligation site, the double restriction site is ( Bam HI and Hin(dIII). The ligation product was directly transformed into *E. coli* Top10 for plasmid amplification. DNA sequencing of the plasmid confirmed the correct plasmid construction. The recombinant plasmid was transformed into *E. coli* Rosetta (DE3) competent cells. Transformant clones were picked and inoculated into an appropriate amount of LB medium (with ampicillin added to a final concentration of 50 μg / mL), and cultured at 37°C until the absorbance at 600 nm reached 0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.02%, and induction was performed at 16°C for 18 h. After centrifugation at 5,000 rpm for 10 min, the cells were collected and resuspended in buffer: 20 mM PBS, pH 6.0, at a cell:buffer ratio of 1 (g): 10 (mL). The cells were then lysed using sonication: 60% power, 3 s on, 5 s off, for a total of 15 min. The resuspended bacterial cells were placed on a tray wrapped with ice. After disruption, the cells were centrifuged at 12,000 rpm for 40 min to remove precipitate and other particulate impurities. The supernatant was conjugated with Ni-NTA affinity medium, and then washed with a buffer containing 500 mM sodium chloride, 100 mM phosphate, and 20 mM imidazole to remove contaminating proteins. Finally, the target protein was eluted from the affinity medium with an elution buffer containing 500 mM sodium chloride, 100 mM phosphate, and 250 mM imidazole. The eluted proteins were collected and analyzed by SDS-PAGE electrophoresis. The SDS-PAGE results are shown below. Figure 1 As shown. Maker used Takara SDS-polyacrylamide gel electrophoresis (SDS). - The protein is a molecular weight standard from the PAGE (part of a photogrammetry analysis). The protein is recombinant β-glycosidase OpBGlu1 purified by nickel column chromatography.

[0030] Table 1 shows the synthesis of the following primers based on the β-glycosidase sequences in the transcriptome of *Hedyotis diffusa*:

[0031] Example 2: Determination of β-glycosidase hydrolysis activity Enzyme activity was determined using p-nitrophenol-D-glucopyranoside as a substrate. First, p-nitrophenol solutions of different concentrations were prepared. Then, 500 µL of the p-nitrophenol solution was added to 500 µL of 1M Na₂CO₃ solution. Using a blank as a reference, the absorbance (A) was measured at a wavelength of 405 nm. 405nmTo obtain the standard curve for p-nitrophenol, the specific method is as follows: Accurately weigh 0.0139 g of p-nitrophenol and dilute to 100 mL with distilled water to prepare a 1 mM solution. Dilute with distilled water to prepare 1 mL of p-nitrophenol solutions of 0, 20, 80, 100, 240, 360, 700, and 800 µmol / L, respectively. Take 500 µL of each of the above p-nitrophenol solutions into a 1.5 mL centrifuge tube, and then add 500 µL of 1 M Na2CO3 solution to each. Using a blank as a reference, measure the absorbance value A at an absorption wavelength of 405 nm. 405nm Value, p-nitrophenol µmol number - A 405nm Find the standard curve and fit a linear equation. The resulting standard curve is: y = 0.0034x + 0.0367, R². 2 =0.9992 (Linear range: 0) <x<800 µmol)。

[0032] Enzyme activity assay: In a 1.5 mL centrifuge tube, add 10 µL of 50 mM p-nitrophenol-D-glucoside solution, then add 440 µL of 50 mM PBS buffer (pH 6.0). Incubate at 37°C for 15 min. After the temperature of the solution in the centrifuge tube stabilizes at 37°C, add 50 µL of enzyme solution pre-incubated at 37°C. Shake at 200 rpm for 5 min in a metal bath, then immediately add 500 µL of 1 M Na₂CO₃ solution to terminate the reaction. Using a blank as a control, measure the absorbance (A) at a wavelength of 405 nm. 405nm Value. If the measured A 405nm If the value exceeds the linear range of the standard curve, it should be diluted and measured again. Glycosidase activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of p-nitrophenol within 1 min under the above reaction conditions, defined as one unit of enzyme activity (U).

[0033] Example 3: Effect of reaction temperature on β-glucosidase hydrolytic activity The hydrolytic activity of β-glucosidase was determined using p-nitrophenol-D-glucoside as a substrate. The hydrolysis reaction temperatures were set at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. The reaction system consisted of 10 µL of 50 mM p-nitrophenol-D-glucoside solution, supplemented with 440 µL of 50 mM PBS buffer (pH 6.0). The reaction time was 5 min, and the reaction was terminated by adding 500 µL of 1 M Na₂CO₃ solution. The reaction solution was then placed in a spectrophotometer, and the absorbance was measured at 405 nm. Under the above reaction conditions, the amount of enzyme that catalyzes the conversion of 1 µmol of p-nitrophenol within 1 min was defined as one unit of enzyme activity (U). The group with the highest activity in the reaction was set as 100% relative enzyme activity.

[0034] Test results: Relative enzyme activity of β-glucosidase at different reaction temperatures, such as Figure 2 As shown, the highest β-glucosidase activity was 89.34 U / mg at a reaction temperature of 45℃. Between 30℃ and 50℃, the relative activity of OpBGlu1 remained above 80%.

[0035] Example 4: Effect of reaction buffer pH on β-glucosidase hydrolytic activity The hydrolytic activity of β-glucosidase was determined using p-nitrophenol-D-glucoside as a substrate. The pH of the hydrolysis reaction buffer was set to 3, 4, 5, 6, 7, 8, 9, 10, and 11. The reaction system consisted of 10 µL of 50 mM p-nitrophenol-D-glucoside solution, supplemented with 440 µL of 50 mM buffer solution at different pH values. The reaction time was 5 min, the reaction temperature was 40 °C, and the reaction was terminated by adding 500 µL of 1 M Na₂CO₃ solution. The reaction solution was then placed in a spectrophotometer, and the absorbance was measured at 405 nm. Under the above reaction conditions, the amount of enzyme that catalyzes the conversion of 1 µmol of p-nitrophenol within 1 min was defined as one unit of enzyme activity (U). The group with the highest activity in the reaction was set as 100% relative enzyme activity.

[0036] Test results: Relative enzyme activity of β-glucosidase in different reaction buffer pH values, such as Figure 3 As shown, the highest β-glucosidase activity was 104.625 U / mg at a reaction buffer pH of 6. Furthermore, the relative enzyme activity remained above 85% at a reaction buffer pH of 5.

[0037] Example 5: Application of the preparation of loganin by hydrolyzing strychnine with β-glycosidase as described above. The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of loganin at final concentrations of 0.2, 0.5, 1, 2, 4, and 5 mM, bringing the total volume to 100 µL. After reacting at 37°C for 10 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0038] The test results are shown in Figures 4A-4C. HPLC detection of enzyme activity revealed that OpBGlu1 can react with strychnine to generate loganin. Enzyme kinetics analysis determined the β-glycosidase's reaction with strychnine. k cat / K m is 15.984 mM -1 ·s -1 (See Figure 10 ).

[0039] Example 6: Application of β-glycosidase hydrolysis of loganic acid obtained above The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of loganic acid at final concentrations of 0.05, 0.1, 0.2, 0.25, and 1 mM, bringing the total volume to 100 µL. After reacting at 37°C for 10 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0040] Test results are as follows Figures 5A-5C As shown, HPLC detection of enzyme activity revealed that OpBGlu1 can hydrolyze the glucose residues of loganic acid. Enzyme kinetics analysis determined the activity of β-glucosidase on loganic acid. k cat / K m is 2.269 mM -1 ·s -1 (See Figure 10 ).

[0041] Example 7: Application of the above-mentioned β-glycosidase hydrolysis of strychnine The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of strychnos nuciferine at final concentrations of 0.8, 1, 2, 4, 5, and 10 mM, bringing the total volume to 100 µL. After reacting at 37°C for 10 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0042] Test results are as follows Figures 6A-6C As shown, HPLC detection of enzyme activity revealed that OpBGlu1 can hydrolyze the glucose residues of strychnine. Enzyme kinetic analysis confirmed the effect of β-glycosidase on the strychnine cleavage. k cat / K m is 14.049 mM -1 ·s -1 (See Figure 10 ).

[0043] Example 8: Application of the above-obtained β-glycosidase hydrolysis of strychnine The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of strychnine at final concentrations of 0.05, 0.1, 0.2, 0.25, 0.5, 0.75, and 1 mM, bringing the total volume to 100 µL. The reaction was carried out at 37°C for 10 minutes, then the reaction was terminated by adding an equal volume of methanol, and the product was analyzed by HPLC.

[0044] Test results are as follows Figures 7A-7C As shown, HPLC detection of enzyme activity revealed that OpBGlu1 can hydrolyze the glucose residues of strychnine. Enzyme kinetic analysis determined the β-glycosidase's ability to hydrolyze strychnine.k cat / K m is 0.0704 mM -1 ·s -1 (See Figure 10 ).

[0045] Example 9: Application of β-glycosidase hydrolyzing isopigenin obtained above The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of isopigenin at final concentrations of 0.5, 1, 2, 5, and 8 mM, bringing the total volume to 100 µL. After reacting at 40°C for 10 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0046] Test results are as follows Figures 8A-8C As shown, HPLC detection of enzyme activity revealed that OpBGlu1 can hydrolyze the glucose residues of isopigenin. Enzyme kinetic analysis determined the β-glycosidase activity of isopigenin. k cat / K m is 0.296 mM -1 ·s -1 (See Figure 10 ).

[0047] Example 10: Application of the β-glycosidase hydrolyzing isoclavicularin lactam obtained above The reaction system consisted of 95 µL of purified OpBGlu1 protein dissolved in PBS buffer, followed by the addition of isocinolone lactone at final concentrations of 0.2, 0.25, 0.5, 1, and 2 mM, bringing the total volume to 100 µL. After reacting at 40 °C for 10 minutes, an equal volume of methanol was added to terminate the reaction, and the product was analyzed by HPLC.

[0048] Test results are as follows Figures 9A-9C As shown, HPLC detection of enzyme activity revealed that OpBGlu1 can hydrolyze the glucose residues of isoclavillarin lactone. Enzyme kinetics analysis determined the β-glycosidase activity of isoclavillarin lactone. k cat / K m is 1.889mM -1 ·s -1 (See Figure 10 ).

[0049] Example 11: Application of the above-mentioned method to obtain multiple monoterpenoid indole alkaloids from the extract of hairy roots of *Hedyotis diffusa* hydrolyzed by β-glycosidase. The reaction system consisted of 95 µL of purified β-glucosidase protein dissolved in PBS buffer, followed by the addition of methanol extract of *Hedyotis diffusa* hairy root, bringing the total volume to 100 µL. After reacting at 40 °C for 60 minutes, an equal volume of methanol was added to terminate the reaction. The product was analyzed by UPLC-QTOF-MS.

[0050] Test results are as follows Figure 11 The results showed that β-glucosidase could react with isopigenin, isovendin, pumiloside, and deoxypumiloside in the methanol extract of *Hedyotis diffusa* hairy root. UPLC-QTOF-MS analysis revealed that β-glucosidase could hydrolyze the glucosyl groups of isopigenin, isovendin, pumiloside, and deoxypumiloside in the *Hedyotis diffusa* extract.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A β-glycosidase from *Hedyotis diffusa*, characterized in that, The amino acid sequence of the β-glycosidase is shown in SEQ ID No.

1.

2. A gene encoding the β-glycosidase of *Hedyotis diffusa* as described in claim 1, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.2, or the gene is a gene encoding the β-glycosidase of *Hedyotis diffusa* as described in claim 1, formed by replacing one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, It carries the gene described in claim 2.

4. A recombinant cell, characterized in that, It contains the recombinant vector of claim 3 or the gene of claim 2.

5. An engineered bacterial strain, characterized in that, The engineered strain carries the recombinant vector of claim 3, wherein the engineered strain is a recombinant Escherichia coli Rosetta (DE3) strain.

6. A method for producing the *Hedyotis diffusa* β-glycosidase according to claim 1, characterized in that, The engineered strain described in claim 5 was used for fermentation to produce enzymes.

7. The use of the *Hedyotis diffusa* β-glucosidase described in claim 1 for catalytic hydrolysis of the non-reducing β-D-glucosidic bond at the end of glycoside compounds to generate the corresponding aglycone.

8. The use according to claim 7, characterized in that, The glycoside compound is any one of strychnine, loganic acid, strychnine, loganic acid, iso-strychnine, isovincinolactam, eugenol, and deoxyeugenol.

9. The application of the *Hedyotis diffusa* β-glucosidase described in claim 1 for catalytic hydrolysis of monoterpenoid indole alkaloids in the hairy root extract of *Hedyotis diffusa*, wherein the monoterpenoid indole alkaloids are one or more of isothiostigin, isovincinolactam, hornwortin, and deoxyhornwortin.

10. A method for hydrolyzing glycoside compounds, characterized in that, The method includes the step of reacting a glycoside compound with the *Hedyotis diffusa* β-glucosidase of claim 1, under conditions that allow the non-reducing β-D-glucosidic bond of the compound to be hydrolyzed by the enzymatic catalysis of the *Hedyotis diffusa* β-glucosidase.

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