Application of beta-glucosidase BgLE or its mutant in synthesis of ginsenoside Rg3, and a synthesis method of ginsenoside Rg3

Abstract

This invention provides the application of β-glucosidase BglE or its mutants in the synthesis of ginsenoside Rg3; the β-glucosidase BglE is derived from *Thermoproteota* archaeon, and its amino acid sequence is shown in SEQ ID NO:2. This invention also provides a β-glucosidase BglE mutant, characterized in that it uses β-glucosidase BglE as the parent, with the following mutations: tyrosine (Y) at position 210 (F) mutated to phenylalanine (F), serine (S) at position 307 (A) mutated to alanine (A) / glycine (G), valine (V) at position 481 (A) mutated to alanine (A) or / and threonine (T) at position 517 (S) mutated to serine (S). The synthesis method of this invention allows for whole-cell feeding under single-enzyme catalysis, eliminating the need for cell disruption and additional hydrogen donors. It achieves a conversion rate exceeding 90% even at high substrate concentrations (40 g / L), enabling efficient one-pot preparation of the target product. The process is simple, offering better cost-effectiveness and promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the application of β-glucosidase BglE or its mutant in the synthesis of ginsenoside Rg3, and a method for synthesizing ginsenoside Rg3. Background Technology

[0002] Ginsenoside Rg3 (English name: 20(R)-GinsenosideRg3), CAS No. 38243-03-7, molecular formula C 42 H 72 O 13 It appears as a white to light yellow powder or crystals, extracted from the dried root of ginseng, a plant in the Araliaceae family. Common specifications range from 20mg to 1g, with a purity of not less than 98%. It is mainly used for drug content determination and has anti-tumor and cancer cell metastasis-inhibiting activities. This substance belongs to the tetracyclic triterpenoid ginsenoside type, and studies have shown that it has angiogenic inhibitory activity, anti-fatigue effects, and immune-enhancing effects.

[0003] Currently, recombinant lactic acid bacteria biotransformation is the method for targeted synthesis of Rg3, as illustrated in the literature: Su Min, et al., Isolation and Identification of β-glucosidase-producing yeasts and their application in the conversion of ginsenoside Rg3, Food Science, 2018, Vol.39, No.14, screening of β-glucosidase-producing microorganisms and their application in the conversion of ginsenoside Rg3. Using *Kluyveromyces martensii* fermentation to produce β-glucosidase catalyzes the conversion of Rb1 and Rd to Rg3, fermentation requires 3 days, and the conversion rate is 248%. However, in this method, the conversion rate is calculated as (ash mass of unfermented sample - ash mass of fermented sample) / ash mass of unfermented sample * 100.

[0004] Application No. 202510634060.7, Invention Title: A β-glucosidase and its preparation method, and its application in the production of ginsenoside Rg3, relates to the field of ginsenoside technology. In view of the problem of low conversion rate in the existing microbial transformation method for preparing ginsenoside Rg3, this invention specifically discloses a β-glucosidase and its preparation method, and its application in the production of ginsenoside Rg3. The conversion rate is 98% after reaction at 85℃ for 6 hours, but the calculation method of this conversion rate is not mentioned in the text.

[0005] CN201610219176.5, Invention Title: A Method for Enzymatic Preparation of Rare Ginsenoside 20(S)-Rg3, utilizing β-glucosidase from Thermotogapetrophila and arabinofuranase from Thermotogathermarum DSM5069 to degrade ginsenosides Rb1, Rb2, and Rc to produce 20(S)-Rg3. However, the enzyme preparation in this method is a complex enzyme preparation, requiring the simultaneous addition of β-glucosidase and arabinofuranase for catalytic conversion.

[0006] Application No.: CN202311376189.X, Invention Title: A β-glucosidase and its application in the preparation of ginsenosides. This invention discloses a β-glucosidase and its application in the preparation of ginsenosides. The β-glucosidase of this invention, or recombinant microorganisms expressing this enzyme, can generate rare ginsenosides Rg3 and / or Rh1 through whole-cell catalysis, thus proposing the application of β-glucosidase in the preparation of ginsenosides. The amino acid sequence of the β-glucosidase is numbered RLF05189.1 on NCBI. However, this method requires the addition of a hydrogen donor for catalytic conversion, increasing costs and hindering subsequent separation and purification.

[0007] In summary, the reported biotransformation methods generally suffer from problems such as cumbersome operation and processing steps, high production costs, and low conversion and utilization rates of reaction substrates (i.e., raw materials), which limit the feasibility of industrial-scale production. Summary of the Invention

[0008] This invention provides the application of β-glucosidase BglE or its mutant in the synthesis of ginsenoside Rg3, and a method for synthesizing ginsenoside Rg3.

[0009] This invention provides the application of β-glucosidase BglE or its mutant in the synthesis of ginsenoside Rg3; the β-glucosidase BglE is derived from Thermoproteota archaeon, and its amino acid sequence is shown in SEQ ID NO:2.

[0010] The synthetic substrates are ginsenoside Rb1 and / or Rd.

[0011] The ginsenoside Rg3 mentioned above is 20(S)-Rg3.

[0012] This invention provides a β-glucosidase BglE mutant, which is based on β-glucosidase BglE as the parent, by mutating tyrosine (Y) at position 210 (F) to phenylalanine (F), serine (S) at position 307 (G) to alanine (A) / glycine (G), valine (V) at position 481 (A) to alanine (A) and / or threonine (T) at position 517 (G) to serine (S).

[0013] The mutant is one in which the 307th serine (S) is mutated to alanine (A) and the 517th threonine (T) is mutated to serine (S).

[0014] The present invention provides a polynucleotide that encodes a β-glucosidase mutant as described above.

[0015] The present invention provides an expression vector comprising the aforementioned polynucleotide.

[0016] The present invention also provides a recombinant cell comprising the polynucleotide or the expression vector.

[0017] This invention also provides a method for synthesizing ginsenoside Rg3, which uses ginsenoside Rb1 or / and Rd as substrates and prepares ginsenoside Rg3 by catalysis of β-glucosidase BglE or a mutant of claim 4 or 5, wherein the β-glucosidase BglE is derived from Thermoproteota archaeon and its amino acid sequence is shown in SEQ ID NO:2.

[0018] The beneficial effects of this invention are:

[0019] The synthesis method of this invention can be carried out by whole-cell feeding under the catalysis of a single enzyme, without cell disruption or additional hydrogen donors. It also has a conversion rate of over 90% for high-concentration (40 g / L) substrates. It can efficiently prepare the target product in one pot, with a simple process and better cost-effectiveness and industrial application prospects. Attached Figure Description

[0020] Figure 1 The reaction pathway for converting Rb1 and Rd to produce Rg3 using total ginsenosides as substrates via β-glucosidase catalysis is described.

[0021] Figure 2 SDS-polyacrylamide gel electrophoresis image after β-glucosidase BglE expression; lane M is protein marker (purchased from Sangon Biotech Co., Ltd.), lane 1 is supernatant after cell lysis, and lane 2 is precipitate after cell lysis.

[0022] Figure 3This is a liquid chromatogram of the conversion of total ginsenosides into product Rg3 catalyzed by β-glucosidase BglE. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention.

[0024] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. All materials and reagents used in the embodiments are commonly used in this field and can be obtained through conventional commercial channels. The main materials and reagents are shown in Table 1.

[0025] Table 1 List of Main Materials and Reagents

[0026] name supplier Product Number Escherichia coli BL21(DE3) Beijing Qingke Biotechnology Co., Ltd. DZC201-96B100 Common restriction enzyme BamHI Baori Biotechnology (Beijing) Co., Ltd. 1010S Conventional restriction enzyme Xho I Baori Biotechnology (Beijing) Co., Ltd. 1094A T4 DNA Ligase Baori Biotechnology (Beijing) Co., Ltd. 2011A yeast powder OXOID LP0021 trypsin OXOID LP0042B Sodium chloride Sinopharm Chemical Reagent Co., Ltd. 10019318 Agar powder Sinopharm Chemical Reagent Co., Ltd. 10000561 Protein Marker YEASEN 20313ES76 Acetonitrile TEDIA 25016150 methanol TEDIA 210359

[0027] 1. Culture medium preparation

[0028] In the implementation of this invention, Escherichia coli is cultured using LB medium (Luria-Bertani medium), with the following formula: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. For solid culture media, an additional 1.5% agar powder is required. Appropriate antibiotics are added based on the resistance carried by the plasmid.

[0029] 2. Strain recovery and activation

[0030] Take a small amount of bacteria from the -80℃ glycerol-preserved or freeze-dried bacteria and streak it onto an LB agar plate using an inoculation loop in a clean bench. Incubate at 37℃ for 12-16 hours until single colonies grow. Select a single colony with regular morphology and no contamination and inoculate it into 5 mL of liquid LB medium. Incubate at 37℃ and 200 rpm for 8-10 hours to complete the activation of the strain.

[0031] 3. Expand training

[0032] Inoculate the activated bacterial culture into fresh liquid LB medium at a ratio of 1:100 (fill the conical flask with no more than 1 / 3 of the volume, ensuring aeration), and incubate at 37°C and 200-250 rpm on a shaker until the logarithmic growth phase (OD200). 600 (≈0.6-0.8) can be used for subsequent experiments such as plasmid extraction and protein expression.

[0033] The detection method using liquid chromatography-mass spectrometry (LC-MS) is as follows:

[0034] The configuration and concentration of product Rg3 were determined by liquid chromatography (HPLC). The chromatographic column was a Supersil ODS 25µm C18 reverse-phase column, and the mobile phase was acetonitrile:water. The detection wavelength was 203nm, the column temperature was 30℃, and the flow rate was 1ml / min. According to the HPLC results, the retention time of standard 20(S)-Rg3 was 21.45 min, and the retention time of standard 20(R)-Rg3 was 23.7 min. Comparison of the retention times of the sample with those of standards 20(S)-Rg3 and 20(R)-Rg3 confirmed that the target product of this technical solution is 20(S)-Rg3.

[0035] Standard solutions of different concentrations were prepared by dissolving 20(S)-Rg3 and 20(R)-Rg3 standards in methanol, and concentration-peak area curves were plotted. The concentration of Rg3 in the test sample was calculated based on the standard curves. The product configuration was determined based on the difference in retention time between the 20(S)-Rg3 and 20(R)-Rg3 standards.

[0036] Table 2 shows the HPLC detection conditions as follows:

[0037] Time (min) A (pure water)% B (acetonitrile)% 0-5 70 30 5-10 70-55 30-45 10-20 55-30 45-70 20-25 30-0 70-100 25-30 70 30

[0038] The total ginsenosides contain 50% Rb1 and 7% Rd. The molecular weight of Rb1 is 1109, the molecular weight of Rd is 947, and the molecular weight of Rg3 is 785.

[0039] After the catalytic reaction is complete, the concentration of Rg3 in the reaction solution is calculated using the method described above. The conversion rate can then be calculated using the following formula:

[0040] Conversion rate = (Number of moles of product Rg3) ÷ (Number of moles of reactants Rb1 + Rd) × 100%

[0041] To examine the catalytic activity and conversion ability of the relevant enzymes for the main substrate Rb1, the following formula can be used for calculation:

[0042] Conversion rate of Rb1 = (Number of moles of product Rg3 ÷ Number of moles of reactant Rb1) × 100%

[0043] Using total ginsenosides (of which the effective substrate Rb1 accounts for 50% and Rd accounts for 7%) as substrates, Rb1 and Rd can be sequentially hydrolyzed to generate the target product Rg3 under the catalysis of β-glucosidase. The specific reaction pathway is as follows: Figure 1 As shown.

[0044] In this transformation process, the catalytic performance of the enzyme plays a decisive role. It is important to note that proteins annotated as "isoenzymes" in databases do not necessarily possess the same catalytic activity. Even with consistent functional annotations, β-glucosidases from different sources may still differ in their amino acid sequences, thus affecting their spatial conformation and active site structure, ultimately leading to significant differences in actual biological activity. Therefore, the functional characteristics of isoenzymes must be verified experimentally in conjunction with specific reaction systems.

[0045] To address the shortcomings of existing technologies, the inventors obtained a batch of high-performance β-glucosidases through targeted mining and screening. These enzymes not only significantly improve the conversion efficiency of total ginsenosides and efficiently prepare Rg3, but also allow for direct whole-cell feeding, greatly simplifying post-processing. Furthermore, they maintain stable and efficient catalytic activity even at high temperatures, making them well-suited to the harsh environments of industrial production and demonstrating significant application advantages.

[0046] Example 1: Construction and Expression of β-glucosidase

[0047] 1. Enzyme library mining and construction

[0048] Three target enzyme gene sequences from different species (bacteria, fungi, etc.) were screened from the NCBI database and can be used for the transformation of rare ginsenoside Rg3. The relevant enzyme numbers, microbial sources and sequences are shown in Table 3.

[0049] Table 3 Enzyme IDs, Microbial Sources, and Sequences

[0050] Enzyme number Microbial source amino acid sequence BglC CandidatusBathyarchaeiaarchaeon SEQIDNO:1 BglE Thermoproteotaarchaeon SEQIDNO:2 BglF Thermotogacaldifontis SEQIDNO:3

[0051] Homology comparison was performed using the NCBIBLAST tool to exclude redundant genes with sequence similarity >95%; amino acid sequences were analyzed using ExPASyProtParam to predict molecular weight, isoelectric point, and conservation of active sites, ensuring the structural rationality of candidate enzymes.

[0052] Based on the screened gene sequences, specific primers containing the restriction enzyme sites BamHI and XhoI were designed. Using DNA from the BglC, BglE, and BglF genes as templates, and with gene sequences synthesized by Qingke Biotechnology Co., Ltd., the target fragments were amplified by PCR. The reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58-62℃ annealing for 30 s, and 72℃ extension (adjusting the time according to the gene length). After 30 cycles, the residue was filled at 72℃ for 10 min.

[0053] The PCR product and expression vector pET-28a(+) were digested with BamHI and XhoI restriction endonucleases at 37℃ for 4 h, respectively. The target fragment and vector backbone were recovered by agarose gel electrophoresis. The recombinant plasmid was constructed by ligation with T4 DNA ligase overnight at 16℃.

[0054] The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin, and incubated at 37°C for 12 h. Single colonies were picked for colony PCR identification, and positive clones were sequenced to verify the accuracy of the gene sequence.

[0055] The correctly sequenced recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on antibiotic-resistant plates, and positive engineered bacteria were screened. Single colonies were picked and inoculated into 5 ml LB broth (containing kanamycin) and incubated at 37°C and 200 rpm until OD500. 600 ≈0.6-0.8; Add IPTG to a final concentration of 0.5 mM, and induce expression at 28℃ and 180 rpm for 8-12 h. Collect the induced bacterial culture, centrifuge at 4℃ and 8000 rpm for 10 min, resuspend the bacterial cells in PBS buffer and sonicate; take the supernatant (soluble protein) and precipitate (inclusion bodies) separately for SDS-PAGE electrophoresis (e.g., ≈0.6-0.8); Figure 2 As shown in the figure, the expression and solubility of the target enzyme were verified.

[0056] Example 2: Screening of β-glucosidase was conducted in a 100 mL reaction system with a total ginsenoside feed amount of 40 g / L.

[0057] Three β-glucosidases derived from different microorganisms were preliminarily screened to evaluate their ability to catalyze the conversion of ginsenosides Rb1 and Rd to Rg3. Whole-cell β-glucosidase enzymes in the form of microbial sludge were prepared according to the method described in Example 1. Enzyme screening was performed based on the conversion rate of total ginsenosides to Rg3. The total volume of the reaction system was 100 ml, containing 40 g / L total ginsenosides (approximately 50% Rb1 and 7% Rd), 20 mM phosphate buffer (pH 5.5), and whole-cell enzyme resuspension (OD=110). The reaction was carried out in an 80°C water bath for 6 hours. After the reaction, enzyme activity was immediately terminated by boiling in a water bath for 5 minutes.

[0058] An equal volume of methanol was added to the reaction solution, and the mixture was vortexed. The supernatant was then filtered through a 0.22 μm filter membrane for subsequent analysis. The contents of Rg3, Rb1, and Rd were determined by high-performance liquid chromatography (HPLC) under the following conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), acetonitrile-water gradient elution mobile phase, flow rate 1.0 mL / min, and detection wavelength 203 nm. The conversion rate was calculated based on the molar amounts of reactants and products before and after the reaction.

[0059] The results showed that, in a 100 ml reaction system with a total ginsenoside feed concentration of 40 g / L, the catalytic efficiencies of each β-glucosidase were as shown in Table 4. Among them, BglE catalyzed a conversion rate of over 70% for the substrate to Rg3, and exhibited high utilization of substrate Rb1, indicating good specificity.

[0060] Table 4 shows the catalytic efficiency of β-glucosidase in a 100 mL reaction system under the condition that the total ginsenoside feed amount is 40 g / L.

[0061] Enzyme number Rb1 concentration (g / L) Rd concentration (g / L) Rg3 concentration (g / L) Conversion rate % Conversion rate % for Rb1 BglC 20 2.8 1.02 6.19 7.20 BglE 20 2.8 11.59 70.33 81.86 BglF 20 2.8 1.51 9.16 10.67

[0062] Example 3

[0063] The reactivity of β-glucosidase BglE was verified in a 1L reaction system with a total ginsenoside feed amount of 40g / L.

[0064] The reactivity of BglE was verified under the following conditions: pH 5.5, temperature 80℃, and reaction time 6h.

[0065] Prepare a 1L enzyme reaction conversion system. Add 40g / L total ginsenosides (with 50% effective substrate Rb1 and 7% Rd) to this system, with a final whole-cell loading concentration of OD=50, and bring the volume to 1L with pH 5.5, 20mM PBS buffer. Place the enzyme reaction solution in an 80℃ water bath and stir. Terminate the reaction after 6 hours of conversion.

[0066] Take 100 μL of the converted reaction solution and add 900 μL of methanol. After shaking and mixing, centrifuge and collect the supernatant. Analyze the Rg3 concentration in the converted solution using HPLC. The converted solution is then analyzed by HPLC (e.g., ...). Figure 3 As shown in the figure, the conversion rate is calculated based on the number of moles of reactants and products before and after the reaction.

[0067] Table 5 shows the catalytic efficiency of β-glucosidase BglE in a 1L reaction system with a total ginsenoside feed amount of 40 g / L.

[0068] Enzyme number Rb1(g / L) Rd(g / L) Rg3 concentration (g / L) Conversion rate (%) Conversion rate (%) for Rb1 BglE 20 2.8 13.27 80.51 93.71

[0069] Finally, in a 1L reaction system with a total ginsenoside feed amount of 40g / L, the catalytic efficiencies of each β-glucosidase were shown in Table 5, all exhibiting significant improvements. Among them, BglE catalyzed a substrate conversion rate exceeding 80% to Rg3, fully utilizing the raw materials and demonstrating promising industrial application prospects.

[0070] Example 4

[0071] Construction and expression of the β-glucosidase BglE mutant (single-point mutation)

[0072] This embodiment provides a method for rationally designing mutations targeting a protein (amino acid sequence as shown in SEQ ID NO:2) to optimize its stability, solubility, or regulate its functional activity. To achieve functional optimization of the aforementioned protein, bioinformatics tools combined with a protein structure-function prediction model are used to analyze the target protein sequence:

[0073] 1) Predict conservative domains using NCBIConservedDomainDatabase(CD);

[0074] 2) Homology modeling was performed using SWISS-MODEL to predict the three-dimensional structure of proteins;

[0075] 3) Analyze the physicochemical properties of amino acids using ExPASyProtParam; predict secondary structures (α-helix, β-sheet, random coil) using PSIPRED; and locate active sites and key interaction regions by combining functional annotations of homologous proteins in the Uniprot database, and screen suitable sites for mutation.

[0076] Based on the above analysis, the following suitable mutation sites were screened from the target protein sequence. Finally, the following mutations were selected: alanine (A) at position 148 of BglE was mutated to serine (S); tyrosine (Y) at position 210 was mutated to phenylalanine (F) / histidine (H); serine (S) at position 307 was mutated to alanine (A) / glycine (G); histidine (H) at position 374 was mutated to glutamine (Q) / asparagine (N); valine (V) at position 481 was mutated to alanine (A) / leucine (L); and threonine (T) at position 517 was mutated to serine (S).

[0077] Based on the protein sequence of wild-type glucosidase BglE (i.e., the enzyme that has not undergone the mutation treatment described in this example, defined as wild-type) as described in Example 1, the corresponding coding gene fragment was synthesized by Qingke Biotechnology Co., Ltd. Subsequently, the synthesized gene fragment and the expression vector pET-28a were double-digested using restriction endonucleases BamHI and XhoI, respectively. The gene fragment was then directionally cloned into the pET-28a vector via a ligation reaction to construct the recombinant plasmid.

[0078] Using the recombinant plasmid as a template, specific forward and reverse primers targeting the BglE mutation site were designed and synthesized (see Table 6). Site-directed mutagenesis PCR (as described in Example 1) was used to amplify the full-length plasmid fragment containing a single-point mutation. The PCR product was digested with DpnI restriction endonuclease at 37°C for 2 hours to degrade the methylated parental plasmid template while retaining the newly synthesized mutant strand. After digestion, the product was directly transformed into *E. coli* BL21(DE3) competent cells. The transformed bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down overnight at 37°C.

[0079] Table 6 Primers for BglE gene mutation

[0080] Primer name Primer sequence A148S-F GAAATGGCAGCAAGCTTCGTAAAAGGCG A148S-R CGCCTTTTACGAAGCTTGCTGCCATTTC Y210F-F CATGAGCGCGTTCAACAAGCTGAATGG Y210F-R CCATTCAGCTTGTTGAACGCGCTCATG Y210H-F CATGAGCGCGCACAACAAGCTGAATGG Y210H-R CCATTCAGCTTGTTGTGCGCGCTCATG S307A-F CTGGTGAATGCTCCGTCTTTCAAAGG S307A-R CCTTTGAAAGACGGAGCATTCACCAG S307G-F CTGGTGAATGGTCCGTCTTTCAAAGG S307G-R CCTTTGAAAGACGGACCATTCACCAG H374Q-F CTGGCGACACCCAGCCGCGTTACGTC H374Q-R GACGTAACGCGGCTGGGTGTCGCCAG H374N-F CTGGCGACACCAACCCGCGTTACGTC H374N-R GACGTAACGCGGGTTGGTGTCGCCAG V481A-F CTGGAACGCGCGTCCAAAGCGTTCC V481A-R GGAACGCTTTGGACGCGCGTTCCAG V481L-F CTGCTGGAACGCCTGTCCAAAGCGTTC V481L-R GAACGCTTTGGACAGGCGTTCCAGCAG T517S-F CATCCTGCTGTCCTGGCAGGCAGGC T517S-R GCCTGCCTGCCAGGACAGCAGGATG

[0081] Single colonies were selected, expanded, and plasmids were extracted. Sanger sequencing was used to verify the sequence of the target region. After confirmation that the expected mutation was successfully introduced at the target site without other non-specific mutations, the correct glucosidase single-point mutant was obtained (see Table 7). This mutant plasmid can be used for subsequent protein expression and functional analysis.

[0082] Table 7 BglE Single-Point Mutants

[0083] enzyme name mutation site BglE WT BglE1 A148S BglE2 Y210F BglE3 Y210H BglE4 S307A BglE5 S307G BglE6 H374Q BglE7 H374N BglE8 V481A BglE9 V481L BglE10 T517S

[0084] Example 5

[0085] The reactivity of the β-glucosidase BglE mutant was detected in a 1 mL reaction system with a total ginsenoside dosage of 40 g / L.

[0086] The single-point mutant successfully constructed in Example 4 was used to prepare mutant whole-cell enzymes according to the method described in Example 1, and the catalytic activity of BglE wild-type and its mutants was verified. Specifically, a 1 mL enzyme reaction system was constructed for activity screening: the reaction system contained 40 g / L total ginsenosides (of which the effective substrate Rb1 accounted for 50% and Rd accounted for 7%), the whole cell feed amount was calculated as OD600=50, and the volume was adjusted to 1 mL using pH 5.5, 20 mM PBS buffer. The prepared reaction solution was placed in an 80°C water bath and stirred continuously for 6 hours before termination. 100 μL of the transformed reaction solution was taken, 900 μL of methanol was added, and the mixture was thoroughly shaken and mixed. The supernatant was then collected by centrifugation, and the concentration of product Rg3 was determined by high performance liquid chromatography (HPLC). The reactivity of wild-type BglE was defined as 1 (wild-type reactivity was calculated by conversion rate, and the conversion rate calculation formula is as described above), and the relative activity of each mutant was calculated (see Table 8) to evaluate the changes in their catalytic performance. Relative activity fold = mutant transformation rate / wild-type transformation rate * 100%.

[0087] Table 8. Activity verification of BglE single-point mutants

[0088] enzymes Relative activity factor WT-BglE 1 BglE-A148S 0.88 BglE-Y210F 1.22 BglE-Y210H 0.76 BglE-S307A 1.27 BglE-S307G 1.07 BglE-H374Q 0.69 BglE-H374N 0.98 BglE-V481A 1.18 BglE-V481L 0.92 BglE-T517S 1.30

[0089] The final results showed that, under the conditions of pH 5.5, temperature 80℃ and reaction time of 6h, the single-point mutants T517S and S307A of BglE had relatively better catalytic activity than wild-type BglE, which were 1.30 and 1.27 times that of wild-type, respectively.

[0090] Example 6

[0091] Construction and expression of β-glucosidase BglE double mutant, and detection of its reactivity.

[0092] Based on the single-point mutation activity screening results in Example 5, in order to further synergistically optimize the physicochemical properties or biological activities of proteins, this invention rationally combines the single-point mutations with excellent performance to construct double-point mutants.

[0093] During the rational modification of the BglE protein, both single-point mutants T517S and S307A exhibited superior enzymatic performance compared to the wild type, including higher catalytic efficiency, enhanced substrate specificity (especially for ginsenoside substrates), and improved thermostability. Given that these two mutation sites are located in different functional regions of the substrate-binding pocket (T517 is closer to the catalytic center, and S307 is located in the substrate recognition loop region), and each independently improves key enzyme performance parameters, this invention further constructs a double-point mutant BglE-T517S-S307A based on a synergistic optimization strategy, combining T517S and S307A. T517 is typically located near the catalytic center or a conserved domain; its mutation to serine (T→S) may optimize transition state stability by fine-tuning the hydrogen bond network, water molecule arrangement, or local flexibility, thereby improving catalytic efficiency. S307 is often located at the edge of the substrate recognition loop or binding pocket. Its mutation to alanine (S→A) removes the polar hydroxyl group, reduces non-specific interactions, enhances selectivity for target substrates (such as ginsenosides Rb1 and Rc), or reduces product inhibition. Combining these two to construct BglE-T517S-S307A, with the amino acid sequence shown in SEQ ID NO:4, can synergistically reshape the microenvironment of the substrate binding cavity, achieving highly efficient hydrolysis of specific substrates (such as rare ginsenoside Rg3 precursors), which is particularly important in the biotransformation of natural products.

[0094] Using the single-point mutant plasmid constructed in Example 4 as a template, and with forward and reverse primers for another site, site-directed mutagenesis PCR was performed (as described in Example 1) to amplify full-length plasmid fragments containing two-point and three-point mutations. The PCR product was digested with DpnI restriction endonuclease at 37°C for 2 hours to degrade the methylated parental plasmid template while retaining the newly synthesized mutant strand. After digestion, the product was directly transformed into *E. coli* BL21(DE3) competent cells. The transformed bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down overnight at 37°C.

[0095] Single colonies were selected, expanded, and plasmids were extracted. Sanger sequencing was used to verify the sequence of the target region. After confirmation that the target site successfully introduced the expected mutation without other non-specific mutations, the correct two- and three-point mutants of glucosidase were obtained (see Table 9). These mutant plasmids can be used for subsequent protein expression and functional analysis.

[0096] The obtained two-point mutants were used to prepare mutant whole-cell enzymes according to the method shown in Example 1, and the catalytic activity of BglE wild-type and its two-point mutants was verified. Specifically, a 1 mL enzyme reaction system was constructed for activity screening: the reaction system contained 40 g / L total ginsenosides (of which the effective substrate Rb1 accounted for 50% and Rd accounted for 7%), the whole cell feed amount was calculated as OD600=50, and the volume was adjusted to 1 mL with pH 5.5, 20 mM PBS buffer. The prepared reaction solution was placed in an 80°C water bath and stirred continuously for 6 hours before termination. 100 μL of the transformed reaction solution was taken, 900 μL of methanol was added, and the mixture was thoroughly shaken and mixed. The supernatant was then collected by centrifugation, and the concentration of product Rg3 was determined by high performance liquid chromatography (HPLC). With the activity of wild-type BglE defined as 1, the relative activity of each mutant was calculated (see Table 9) to evaluate the change in its catalytic performance. Relative activity fold = mutant conversion rate / wild-type conversion rate * 100%.

[0097] Table 9. Activity verification of BglE double mutants

[0098] enzymes Relative activity factor WT-BglE 1 BglE-T517S-S307A 1.42

[0099] The final results showed that, under the conditions of pH 5.5, temperature 80℃, and reaction time of 6 h, the BglE double mutant BglE-T517S-S307A exhibited relatively better catalytic activity than the wild-type BglE, with an activity 1.42 times that of the wild-type. The amino acid sequence of the BglE double mutant BglE-T517S-S307A is shown in SEQ ID NO:4.

[0100] Example 7

[0101] The reactivity of the β-glucosidase BglE double mutant BglE-T517S-S307A was verified in a 1L reaction system with a total ginsenoside feed amount of 40 g / L.

[0102] Prepare a 1L enzyme reaction conversion system. Add 40g / L total ginsenosides (with 50% effective substrate Rb1 and 7% Rd) to this system, with a final whole-cell loading concentration of OD=50, and bring the volume to 1L with pH 5.5, 20 mM PBS buffer. Place the enzyme reaction solution in an 80℃ water bath and stir. Terminate the reaction after 6 hours of conversion.

[0103] Take 100 μL of the converted reaction solution and add 900 μL of methanol. After shaking and mixing, centrifuge and collect the supernatant. Then, determine the concentration of Rg3 in the converted solution by HPLC. After the converted solution is detected by HPLC, calculate the conversion rate based on the molar number of reactants and products before and after the reaction.

[0104] Table 10 Transformation rate of BglE double mutant BglE-T517S-S307A

[0105] enzymes Conversion rate (%) Conversion rate (%) for Rb1 WT-BglE 80.51 98% BglE double mutant BglE-T517S-S307A 95.7 ＞99%

[0106] Finally, in a 1L reaction system, with a substrate concentration of 40 g / L total ginsenosides, the catalytic efficiency of the BglE double mutant BglE-T517S-S307A is shown in Table 10. All of them are significantly improved compared with wild-type BglE, and the conversion rate of Rb1 exceeds 99%.

[0107] amino acid sequence listing

[0108] SEQIDNO:1 MPLKYVDEIISKMTLEEKAKIVVGVGLPGLFGNPPSRVPGAAGETHPIERLKIPSAVFADGPAGLRISPRREGDELTYHATAFPVETMLASTWNRNVLEEVGKAVGEEVREYGVDILLAPAINIHRNPLCGRNFEYYSEDPFLTGEMAAAFVRGVQSQGVGACLKHFAANEQETNRMFIDTLISERALREIYLKGFEIAIKKSKPWAVMSAYNKLNGEYCSQNKWLLTKVLREEWGFEGFVMTDWFAGDDPVKQMEAGNDMIMPGKAYQINPKRRDEVEEILSAVKEGRLSEDVLNRNVRNILKVLVNSPSFKGYKYSNRPDLDAHARVAYEAGAEGVILLKNNGALPIGVDKRIALFGTGQIETIKGGTGSGDTHPRYVISILDGVRERGLKIDENLAGIYAKYVGDMRGIEEYRVRRGVFGMPIMPKIPEDFLSEEDIEGFAENNDVAFIVISRISGEGYDRRPEKGDFYLSDDERNLVEKVSKIFHRHGRKVVAVLNIGSPIEVASWRELVDAILLIWQAGQETGRIFADVITGRINPSGKLPTTFPRDYWDVPSWSFPGEPKDNPQMVSYDEGIYVGYRYYDTFGVESAYEFGFGLSYTTFRYGNLKVEVSDNIIKISFEITNTGNYPGKEVAQVYIRAPKGVIDKPFQELKGFHKTRLLNPGETERVEIEVDVDSLAVYNGEKWIIERGRYEVRVGSSSRDIRLTGSFII SEQIDNO:2 MEEIPEIVSKMTLEEKARIVVGVGMPGILGNPQPRVPGAAGETHPVERLGIPSAVLADPAGLRINPKRENDDKTYHATAFPVETMLASTWNRELLEAVGRCFGEEVREYGVDILLAPAMNIHRNPLCGRNFEYYSEDPVLTGEMAAAFVKGVQSQGVGACLKHFAANNQETNRHILDTLVSERALREIYLKGFEIAVRKSKPWTVMSAYNKLNGQHCSQNEWLLTRVLRGDWGFEGFVMTDWFAKDDPVEMMRAGNDMIPGKAHQIDPKRKDEVEAIINAVKEGRLDEEILIRNVKNILRVLVNSPSFKGYKYSNRPDLDAHARVAYEAGVEGVVVLLKNNGALPVGRDSRIALFG TGQMETIKGGTGSGDTHPRYVISILDGMKEKHLRVDEELVEAHVKYLAEIRSKDEYRIEKTIFGEIFKPVPQDFLSEEIARFAERNDVAFIVISRISGEGYDRRPEKGDFYLSDDERRLLERVSKAFHERGKRVVAILNIGSPIEVASWRSMVDSILLTWQAGQETGRIIADIVSGERNPSGKLPTTFPDYSDVPSWSFPGEPRDNPQRVVYDEGIYVGYRYYDTFGVEPAYEFGFGLSYTSFEYRDLKVEMINNKVIVSFEVVNTGNLPGKEVAQIYIRAPKGRIDKPFQELKGFHKTRLLNPGEKEKITVELDFDSLASYDGEKWVVEKGGYEVRVGASSRDIRLLGGFTISQ SEQ ID NO:3 MDPNEILSQLTLEEKVKLVVGVGLPGLFGNPHSKVVGVAGETHTISRLNIPSIRLADGPAGLRINPVRENDPHTYHATAFPIASMLASTWNREIVEEVGRAMGEEVREYGVDILLAPAMNIHRNPLCGRNFEYYSEDPVLSGEMAAAFVRGVQSQGVGACLKHFAANNQETNRFTVDTIVSERALREIYLKGFEIAVKKSRPWTVMSAYNKLNGKYCSQNEWLLKKVLKKEWKFEGFVMTDWYAGDDPVAQLKSGNDMIMPGKVYQVNPQRKDEMEEILQGIREGKLSEEELDECVRNILNVLVKCPSFKNYSYSNRPNLEAHAKIAYEAGAEGLVLLKNVRALPLNENDRIAIFGTGQIETVKGGTGSGDTHPRYTVSILEGFRERTLRIDEELAKTYEDYVNTMRQKEEYKPRLDQWGTQIKPKLPEDFLSEEEIEKLAERNDVAIVVISRISGEGYDRKPVKGDYYLSDDELRLVRLVSKIFHRRGKRVIALLNVGGPIEVASWRDEVDAMMLVWQAGQETGRIVADAIAGRINPSGKLPTTFPMDYSDVPSWSFPGEPRQDPKRVIYEEDIYVGYRYYDTFGVEPAYEFGFGLSYTRFDYSDLSVSVENDTLKVSFTVKNIGDRAGKEIAQIYVKAPKGKIDKPFQELKAFHKTKNLSPNEEERIVLEIPVRDLASFDGERWIVENGEYELRVGASSRDIRLKGQLFLKERIYEL SEQIDNO:4 MEEIPEIVSKMTLEEKARIVVGVGMPGILGNPQPRVPGAAGETHPVERLGIPSAVLADPAGLRINPKRENDDKTYHATAFPVETMLASTWNRELLEAVGRCFGEEVREYGVDILLAPAMNIHRNPLCGRNFEYYSEDPVLTGEMAAAFVKGVQSQGVGACLKHFAANNQETNRHILDTLVSERALREIYLKGFEIAVRKSKPWTVMSAYNKLNGQHCSQNEWLLTRVLRGDWGFEGFVMTDWFAKDDPVEMMRAGNDMIPGKAHQIDPKRKDEVEAIINAVKEGRLDEEILIRNVKNILRVLVNAPSFKGYKYSNRPDLDAHARVAYEAGVEGVVLLKNNGALPVGRDSRIALFG TGQMETIKGGTGSGDTHPRYVISILDGMKEKHLRVDEELVEAHVKYLAEIRSKDEYRIEKTIFGEIFKPVPQDFLSEEIARFAERNDVAFIVISRISGEGYDRRPEKGDFYLSDDERRLLERVSKAFHERGKRVVAILNIGSPIEVASWRSMVDSILLSWQAGQETGRIIADIVSGERNPSGKLPTTFPDYSDVPSWSFPGEPRDNPQRVVYDEGIYVGYRYYDTFGVEPAYEFGFGLSYTSFEYRDLKVEMINNKVIVSFEVVNTGNLPGKEVAQIYIRAPKGRIDKPFQELKGFHKTRLLNPGEKEKITVELDFDSLASYDGEKWVVEKGGYEVRVGASSRDIRLLGGFTISQ

Claims

1. Application of β-glucosidase BglE or its mutant in the synthesis of ginsenoside Rg3; wherein the β-glucosidase BglE is derived from Thermoproteota archaeon, and the amino acid sequence is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that: The synthetic substrates are ginsenosides Rb1 and / or Rd.

3. The application according to claim 1 or 2, characterized in that: The ginsenoside Rg3 mentioned is 20(S)-Rg3.

4. A β-glucosidase BglE mutant, characterized in that: It uses β-glucosidase BglE as the parent, and mutates tyrosine (Y) at position 210 of BglE to phenylalanine (F), serine (S) at position 307 to alanine (A) / glycine (G), valine (V) at position 481 to alanine (A) or / and threonine (T) at position 517 to serine (S).

5. The β-glucosidase BglE mutant according to claim 4, characterized in that: The mutant is one in which the 307th serine (S) is mutated to alanine (A) and the 517th threonine (T) is mutated to serine (S).

6. A polynucleotide, characterized in that: The polynucleotide encodes the β-glucosidase mutant as described in claim 4 or 5.

7. An expression vector, characterized in that: The expression vector comprises the polynucleotide as described in claim 6.

8. A recombinant cell, characterized in that: The recombinant cells comprise the polynucleotide as described in claim 6 or the expression vector as described in claim 7.

9. A method for synthesizing ginsenoside Rg3, characterized in that: It is prepared as ginsenoside Rg3 by using ginsenoside Rb1 or / and Rd as substrates and catalyzing a reaction with β-glucosidase BglE or a mutant of claim 4 or 5. The β-glucosidase BglE is derived from Thermoproteota archaeon and its amino acid sequence is shown in SEQ ID NO:2.

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