β-glucosidase SS-BGL mutant for preparing rare ginsenosides and its application

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a β-glucosidase SS-BGL mutant for preparing rare ginsenosides and its application. The β-glucosidase SS-BGL mutant is a single-site mutation or a double-site mutation of the β-glucosidase SS-BGL with the amino acid sequence shown in SEQ ID NO.1. Specifically: a single-site mutation involves mutating phenylalanine at position 229 of SEQ ID NO.1 to proline; a double-site mutation involves mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 301 to proline; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and leucine at position 337 to valine; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 338 to proline. Based on the natural β-glucosidase SS-BGL, this invention utilizes site-directed mutagenesis to modify its molecular structure, resulting in four β-glucosidase SS-BGL mutants with enhanced saponin conversion activity.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a β-glycosidase SS-BGL mutant for the preparation of rare ginsenosides and its application. Background Technology

[0002] In recent years, saponins, one of the main active components of ginseng, have attracted attention due to their broad anti-cancer activity. Studies have found that secondary metabolites of ginsenosides, namely rare ginsenosides, exhibit stronger antitumor activity and can be obtained through in vitro degradation of total saponins. Rare ginsenosides include ginsenoside compounds K, Rh2, Rg2, and Rg3; among them, ginsenoside compound K, abbreviated as CK, or 20-o-diglucosyl-20(S)-protophenyl glycol, is the main deglycosylated metabolite of ginsenosides. It possesses various biological activities, including anti-cancer, anti-diabetic, anti-inflammatory, anti-allergic, anti-angiogenic, anti-aging, neuroprotective, and hepatoprotective effects. However, the difficulty in its preparation greatly limits the further application of CK. To increase the yield of CK, various methods have been developed for its production.

[0003] The structural modification of ginsenosides mainly involves the hydrolysis of glycosyl groups at specific sites. Glycosidases are among the most common industrial enzymes, responsible for the hydrolysis and cleavage of glycosyl bonds, and are used as catalysts for lactose hydrolysis in dairy products in the food industry. SS-BGL, a β-glycosidase derived from the thermophilic archaea (Sulfolobus solfataricus) grown at 87°C and pH 3.0, has been proven to be a highly efficient CK-producing enzyme using glycosylated PPD-type ginsenosides as substrates due to its broad hydrolytic activity, including β-D-glucosidase, β-D-galactosidase, β-D-hydroxyglucosidase, and α-glucosidase activities. Common glycosylated PPD-type ginsenosides include Rb1, Rb2, Rb3, and Rd. The hydrolytic pathway of SS-BGL in preparing rare ginsenoside CK using ginsenoside Rb1 as a substrate is: Rb1→Rd→F2→CK. High temperatures imply high reaction rates and low contamination, giving thermophilic glycosidases significant advantages in industrial applications. Studies show that the optimal temperature for wild-type thermophilic β-glucosidase SS-BGL is 80℃, and the optimal pH is 6. However, the efficiency of wild-type thermophilic β-glucosidase SS-BGL in converting rare ginsenosides is not high, which greatly limits its further application. Therefore, a new strategy for the efficient conversion of rare ginsenosides is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a β-glycosidase SS-BGL mutant for the preparation of rare ginsenosides, which solves the defect of low efficiency in the conversion of rare ginsenosides by wild-type thermophilic enzyme SS-BGL in the prior art.

[0005] The technical solution adopted in this invention is:

[0006] The first aspect of this invention provides a β-glucosidase SS-BGL mutant for preparing rare ginsenosides, wherein the β-glucosidase SS-BGL mutant is a single-point mutation or a two-site mutation of the β-glucosidase SS-BGL with the amino acid sequence as shown in SEQ ID NO.1, wherein:

[0007] The single point mutation is: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline;

[0008] The two-site mutations are: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 301 to proline; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and leucine at position 337 to valine; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 338 to proline.

[0009] Preferably, the rare ginsenosides include at least one of 20-o-diglucosyl-20(S)-orthophenylenediol, ginsenoside Rh2, ginsenoside Rg2, and ginsenoside Rg3.

[0010] A second aspect of the present invention provides a recombinant vector comprising the nucleotide sequence of the β-glycosidase SS-BGL mutant.

[0011] A third aspect of the present invention provides a method for preparing the recombinant vector, comprising the following steps:

[0012] The nucleotide sequence of the β-glycosidase SS-BGL mutant was ligated into an expression vector and site-directed mutagenesis was performed to obtain the recombinant vector.

[0013] Preferably, the expression vector is a pET series vector.

[0014] Preferably, the pET series carrier includes any one of pET-28a(+), pET-24a(+), and pET-23a(+).

[0015] A fourth aspect of the present invention provides a genetically engineered bacterium comprising the recombinant vector, wherein the genetically engineered bacterium is obtained by transforming the recombinant vector into Escherichia coli.

[0016] The fifth aspect of the present invention provides an application of the β-glucosidase SS-BGL mutant, the recombinant vector, or the genetically engineered bacteria, wherein the β-glucosidase SS-BGL mutant, the recombinant vector, or the genetically engineered bacteria is used to prepare rare ginsenosides.

[0017] Preferably, the substrate for preparing the rare ginsenosides is a glycosylated protopanaxadiol-type ginsenoside.

[0018] Preferably, the glycosylated protopanaxadiol-type ginsenosides include at least one of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rd.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a β-glucosidase SS-BGL mutant for the preparation of rare ginsenosides. The β-glucosidase SS-BGL mutant is a single-point or double-site mutation of the β-glucosidase SS-BGL with the amino acid sequence shown in SEQ ID NO.1, wherein:

[0021] The single point mutation is: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline;

[0022] The two-site mutation is as follows: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 301 to proline; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and leucine at position 337 to valine; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 338 to proline. Based on the natural β-glucosidase SS-BGL, this invention modifies the molecular structure of natural β-glucosidase SS-BGL through design and site-directed mutagenesis biotechnology. The effects of the mutated residues on the enzymatic properties were analyzed, and finally, one single-point mutation F229P and three double-point mutations F229P / G301P, F229P / L337V and F229P / G338P with improved saponin conversion activity were obtained. The ginsenoside conversion rate at 80℃ was increased to 1.85 times, 1.50 times, 1.79 times and 1.91 times that of the original, respectively.

[0023] While significantly improving transformation efficiency, the aforementioned single-point or double-point mutants maintained their excellent thermostability as thermophilic enzymes, with their thermal stability remaining essentially unchanged. In the residual enzyme activity assay after heat treatment at 95℃, compared to the wild-type with 85% residual enzyme activity, all four mutants maintained above 80%, with the double-point mutant F229P / L337V reaching 89%, slightly higher than the wild-type. After heat treatment at 80℃ for 24 hours, both the wild-type and the four mutants maintained more than 30% activity, with half-lives exceeding 16 hours, showing no significant difference compared to the wild-type. Attached Figure Description

[0024] Figure 1 The bands of the purified enzyme solutions WT, F229P, F229P / G301P, F229P / L337V, and F229P / G338P are displayed sequentially after analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0025] Figure 2 The results of transforming rare ginsenoside CK with wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P at 80℃ are presented.

[0026] Figure 3 The results show the residual enzyme activity of wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P after heat treatment at 95℃ for 20 min.

[0027] Figure 4 The results show the residual enzyme activity of wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P after heat treatment at 80℃ for 24 h. Detailed Implementation

[0028] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0029] The inventive concept of this invention is as follows:

[0030] The structural modification of ginsenosides mainly involves the hydrolysis of glycosyl groups at specific sites. Glycosidases are among the most common industrial enzymes, responsible for the hydrolysis and cleavage of glycosyl bonds, and are used as catalysts for lactose hydrolysis in dairy products in the food industry. β-glycosidase SS-BGL has been proven to be a highly efficient CK-producing enzyme using glycosylated PPD-type ginsenosides as substrates. The hydrolytic pathway of SS-BGL in preparing rare ginsenoside CK from ginsenoside Rb1 is: Rb1→Rd→F2→CK. However, the conversion efficiency of wild-type thermophilic β-glycosidase SS-BGL to rare ginsenosides is not high, which greatly limits its further application.

[0031] Based on this, the present invention provides a β-glucosidase SS-BGL mutant for preparing rare ginsenosides, wherein the β-glucosidase SS-BGL mutant is a single-point mutation or a two-site mutation of the β-glucosidase SS-BGL with the amino acid sequence as shown in SEQ ID NO.1, wherein:

[0032] The single point mutation is: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline;

[0033] The two-site mutation is as follows: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 301 to proline; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and leucine at position 337 to valine; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 338 to proline.

[0034] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0035] The culture media and formulations involved in the following examples are as follows:

[0036] Each liter of LB liquid medium contains 10g peptone, 5g yeast extract, and 10g NaCl, with the remainder made up with water.

[0037] LB solid medium: Add 2% agar to each liter of LB liquid medium.

[0038] The detection methods involved in the following embodiments are as follows:

[0039] (1) Method for determining the activity of β-glucosidase SS-BGL.

[0040] Enzyme activity was tested by detecting the release of p-nitrophenol from pNPG. 100 μL of 1.0 mg / mL enzyme solution was added to 500 μL of 4 mM pNPG solution, and the reaction was carried out at 80 °C for 3 min. The reaction was then terminated by adding 500 μL of 0.6 mM sodium carbonate solution. The release of p-nitrophenol was obtained by measuring the absorbance at 405 nm. Enzyme activity was defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute. All experiments were performed in triplicate.

[0041] (2) Method for determining saponin conversion rate.

[0042] Using 5 mg / mL Rb1 as a substrate, 1 mL of enzyme solution containing 0.5 mg / mL of enzyme dissolved in 50 mM citrate / phosphate buffer was added. The reaction was carried out at 80 °C for 30 min, and then an equal volume of methanol was added to terminate the reaction. The mixture was sonicated for 30 min and filtered through a 0.45 μm sterile filter. The formation of CK was monitored by high-performance liquid chromatography (HPLC). A standard curve was established using standard saponin CK, and the actual yield was calculated from the peak area of ​​the HPLC results to obtain the corresponding saponin conversion rate.

[0043] The list of abbreviations for this invention is shown in Table 1.

[0044] Table 1 List of Abbreviations

[0045]

[0046]

[0047] Example 1

[0048] A β-glycosidase SS-BGL mutant for the preparation of rare ginsenosides is described in detail below:

[0049] Step 1: Construct a recombinant vector containing the β-glucosidase SS-BGL mutant.

[0050] (1) Construction of the SS-BGL vector containing wild-type β-glucosidase.

[0051] The nucleotide sequence of wild-type β-glucosidase SS-BGL, as shown in SEQ ID NO.2, was chemically synthesized. This nucleotide sequence was then linked to the pET-28a(+) vector to prepare the vector pET-28a(+)-SS-BGL. The amino acid sequence corresponding to the nucleotide sequence of wild-type β-glucosidase SS-BGL is shown in SEQ ID NO.1.

[0052] (2) Obtaining recombinant vectors containing mutants.

[0053] Using whole plasmid PCR technology, site-directed mutagenesis was performed on pET-28a(+)-SS-BGL as a template to obtain the recombinant vector pET-28a(+)-SS-BGL-F229P containing the mutated gene.

[0054] The PCR amplification program used to amplify the whole plasmid was set as follows: First, pre-denaturation at 95℃ for 5 min; then 20 cycles of denaturation at 95℃ for 1 min, annealing at 68℃ for 30 s, extension at 72℃ for 7 min; and finally incubation at 4℃.

[0055] After the PCR reaction, 1 μL Dpn I was added directly to the PCR reaction system, mixed well, and incubated at 37°C for 5 min. After the Dpn I completely digested the template plasmid in the PCR product, the PCR product was verified by electrophoresis using a 1% agarose gel. After confirming the electrophoretic bands of the PCR product, the PCR product was recovered, transformed into E. coli DH5α competent cells, and plasmids were extracted and sequenced for verification.

[0056] Step 2: Construction of recombinant E. coli containing the β-glucosidase SS-BGL mutant, and expression, isolation, and purification of the β-glucosidase SS-BGL mutant. The specific steps are as follows:

[0057] (1) The recombinant vector pET-28a(+)-SS-BGL-F229P obtained in step 1 was transformed into E.coli BL21(DE3) competent cells to prepare the genetically engineered bacteria E.coli / pET-28a(+)-SS-BGL-F229P.

[0058] (2) The genetically engineered bacteria prepared above were inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm to prepare seed culture. The prepared seed culture was transferred to 100 mL of LB liquid medium containing 50 μg / mL kanamycin at a volume fraction of 0.5% and cultured at 37°C and 220 rpm until the OD600 was 0.6. IPTG was added to a final concentration of 1 mM and cultured for another 16 h at 16°C to obtain fermentation broth. The prepared fermentation broth was centrifuged at 10000 rpm and 4°C for 5 min to collect the bacterial cells and resuspend the bacterial cells in 50 mM citrate / phosphate buffer. The resuspended cells were treated with an ultrasonic disruptor under ice bath conditions for 10 min, then centrifuged at 10000 rpm and 4℃ for 10 min. The supernatant was collected to obtain the crude enzyme solution, which is the β-glucosidase SS-BGL mutant F229P. The nucleotide sequence of F229P is shown in SEQ ID NO.3.

[0059] (3) After heating the crude enzyme solution at 80°C for 30 min, filter it through a 0.45 μm sterile filter membrane. The filtrate is the pure enzyme solution. The protein concentration is determined using the BCA protein concentration kit.

[0060] Examples 2 to 4

[0061] A method for preparing a β-glycosidase SS-BGL mutant is as follows:

[0062] In Examples 2 to 4, site-directed mutagenesis was performed using whole-plasmid PCR with pET-28a(+)-SS-BGL as a template to sequentially obtain recombinant vectors containing the mutant gene: pET-28a(+)-SS-BGL-F229P / G301P, pET-28a(+)-SS-BGL-F229P / L337V, and pET-28a(+)-SS-BGL-F229P / G338P. The genetically engineered bacteria obtained sequentially using these recombinant vectors containing the mutant gene were E. coli / pET-28a(+)-SS-BGL-F229P / G301P, E. coli / pET-28a(+)-SS-BGL-F229P / L337V, and E. coli / pET-28a(+)-SS-BGL-F229P / G338P. Then, pure enzyme solutions of the β-glucosidase SS-BGL mutants F229P / G301P, F229P / L337V, and F229P were obtained by expression in genetically engineered bacteria. The experimental methods described above are exactly the same as in Example 1.

[0063] The nucleotide sequences of F229P / G301P, F229P / L337V and F229P / G338P are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

[0064] F229P is a single-point mutation, while F229P / G301P, F229P / L337V, and F229P / G338P are double-point mutations.

[0065] The primer sequences used for site-directed mutagenesis in the above embodiments are shown in Table 2.

[0066] Table 2 Primer sequences

[0067] sequence Primer name Sequence information SEQ ID NO.7 F229P_F TACTTGAGCCCGGAACTTTCTCGTCGTGCAATGTAC SEQ ID NO.8 F229P_R AGAAAGTTCCGGGCTCAAGTAACCCGGCGGAAAGCC SEQ ID NO.9 G301P_F AAATCACTCGTCCGAACGAGAAGATCGTTCGTGACG SEQ ID NO.10 G301P_R ATCTTCTCGTTCGGACGAGTGATTTCGCCACGGATG SEQ ID NO.11 L337V_F GTGTCTGTGGGTGGTTACGGTCACGGTTG SEQ ID NO.12 L337V_R CACCCACAGACACGTAGCCTTTCTCGGTA SEQ ID NO.13 G338P_F TACGTGTCTCTGCCGGGTTACGGTCACGGTTGCGAAC SEQ ID NO.14 G338P_R GTGACCGTAACCCGGCAGAGACACGTAGCCTTTCTCG

[0068] Note: The underlined bases in Table 2 indicate the bases that need to be replaced by site-directed mutations.

[0069] The pure enzyme solutions obtained in Examples 1 to 4 were analyzed by SDS-PAGE, and the results are as follows: Figure 1As shown. From left to right, lanes 1 to 4 contain purified enzyme solutions of F229P, F229P / G301P, F229P / L337V, and F229P / G338P, respectively. The results show a clear band at 60 kDa, confirming the expression of β-glucosidase SS-BGL.

[0070] The purified enzymes obtained in Examples 1 to 4 were subjected to pNPG enzyme activity tests, with the enzyme activity of the wild-type enzyme WT as 100%, and the relative enzyme activity of each mutant was calculated. The test results are shown in Table 3.

[0071] Table 3. Relative enzyme activities of each mutant

[0072]

[0073]

[0074] As shown in Table 3, single-point and double-point mutations have a significant impact on pNPG enzyme activity. Compared with the wild type, the relative enzyme activity of F229P and F229P / G301P decreased, with F229P / G301P showing only 46.82%, while the two mutants F229P / L337V and F229P / G338P showed varying degrees of improvement, with the highest value reaching 119.91%.

[0075] The pure enzyme solutions obtained in Examples 1 to 4 were appropriately diluted and heat-treated in a 95°C oven for 20 minutes. The residual enzyme activity of each mutant was calculated with the enzyme activity before treatment as 100%. The test results are shown in Table 4.

[0076] Table 4 shows the residual enzyme activity of each group after heat treatment at 95℃ for 20 min.

[0077] enzymes Residual enzyme activity, % WT 85.71 F229P 84.10 F229P / G301P 86.40 F229P / L337V 89.27 F229P / G338P 80.46

[0078] As shown in Table 4, single-point and double-point mutations had no significant effect on the thermostability of thermophilic enzymes at 95℃. In the residual enzyme activity assay after heat treatment at 95℃, all four mutants maintained above 80% activity compared to the wild type, with the double-point mutation F229P / L337V reaching 89%, slightly higher than the wild type.

[0079] Example 5

[0080] An application of the β-glycosidase SS-BGL mutant for the preparation of rare ginsenosides is as follows:

[0081] 1. Saponin conversion rate.

[0082] Take 1 mL of each of the pure enzyme solutions from Examples 1 to 4 (0.5 mg / mL), and weigh 5 mg / mL LRb1. React at 80°C for 30 min, then add an equal volume of methanol to terminate the reaction. Sonicate for 30 min, filter through a 0.45 μL filter membrane, and detect the formation of CK by HPLC. The results are shown in Table 5.

[0083] Table 5 Relative activity of saponin conversion

[0084] enzymes Relative enzyme activity, % WT 100.00 F229P 184.57 F229P / G301P 149.57 F229P / L337V 178.88 F229P / G338P 191.34

[0085] Table 5 shows that both single-point and double-point mutations significantly enhanced the enzyme-catalyzed preparation of rare ginsenosides, increasing the ginsenoside conversion rate at 80℃ to 1.85, 1.50, 1.79, and 1.91 times the original rate, respectively. Statistical results for the conversion of rare ginsenosides (CK) are shown in [Table 5]. Figure 2 .

[0086] Simultaneously, this invention also involves mixing 0.25 mL each of 0.5 mg / mL pure enzyme solutions of F229P, F229P / G301P, F229P / L337V, and F229P / G338P, and then reacting the mixture with Rb1 at 80°C for 30 min, detecting the formation of CK. The relative enzyme activity of the mixture of F229P, F229P / G301P, F229P / L337V, and F229P / G338P was determined to be 166.80%.

[0087] 2. Thermal stability.

[0088] Take 1 mL of 0.5 mg / mL pure enzyme solution from Examples 1 to 4 and heat-treat it in a 95°C oven for 20 min. Using the enzyme activity before treatment as 100%, calculate the residual enzyme activity of each mutant. See [link to relevant documentation]. Figure 3 The results showed that single-point and double-point mutations had no significant effect on the thermostability of the thermophilic enzyme at 95℃. In the residual enzyme activity assay after heat treatment at 95℃, all four mutants maintained above 80% activity compared to the wild type, with the double-point mutation F229P / L337V reaching 89%, slightly higher than the wild type.

[0089] 3. Half-life test at 80℃.

[0090] The residual enzyme activity of wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P after heat treatment at 80℃ for 24 h is shown in the figure. Figure 4The results showed that after heat treatment at 80℃ for 24 hours, both the wild type and the four mutant types could retain more than 30% of their activity and the half-life could reach more than 16 hours. There was no significant difference compared with the wild type. That is, the point mutation did not change the original excellent heat resistance of the enzyme and could still maintain its original stability.

[0091] The amino acid sequence of wild-type β-glucosidase SS-BGL, SEQ ID NO.1:

[0092] MYSFPNSFRFGWSQAGFQSEMGTPGSEDPNTDWYKWVHDPENMAAG LVSGDLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPQNFDESKQDVTEVEINENELKRLDEYANKDALNHYREIFKDLKSRGLYFILNMYHWPLPLWL HDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSRRAMYNIIQAHARAYDGIKSVSKKPVGIIYANSSFQ PLTDKDMEAVEMAENDNRWWFFDAIIRGEITRGNEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGCERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRY HLYMYVTENGIADDADYQRPYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFSMRFGLLKVDYNTKRLYWRPSALVYREIATNGAITDEIEHLNSVPPVKPLRH.

[0093] The nucleotide sequence of wild-type β-glucosidase SS-BGL, SEQ ID NO.2:

[0094]

[0095] The nucleotide sequence encoding F229P, SEQ ID NO.3:

[0096] ATGTACAGCTTTCCAAACTCTTTCCGTTTCGGTTGGAGCCAGGCTGGCTTCCAGTCCGAAATGGGCACTCCGGGCTCCGAAGATCCGAACACCGACTGGTACAAATGGGTTCATGATCCGGAGAACATGGCTGCAGGTCTGGTTTCTGGTGATTTGCCGGAGAACGGTCCAGGTTACTGGGGCAACTACAAGACCTTCCACGACAACGCTCAGAAGATGGGTCTGAAGATCGCGCGTCTGAACGTTGAATGGAGCCGTATCTTCCCAAACCCGCTGCCGCGTCCGCAGAACTTCGATGAATCCAAACAAGATGTGACCGAAGTTGAAATCAACGAGAACGAACTGAAACGTCTGGATGAATACGCGAACAAAGACGCTCTGAACCACTATCGTGAAATCTTCAAAGACCTGAAATCTCGTGGTCTGTACTTCATTCTGAACATGTACCATTGGCCGCTGCCGTTGTGGCTGCATGATCCGATCCGTGTTCGTCGCGGTGACTTCACCGGTCCGTCTGGTTGGCTGTCTACTCGTACCGTGTACGAGTTCGCTCGTTTCTCTGCGTACATCGCGTGGAAATTCGATGACCTGGTAGACGAATACTCCACCATGAACGAACCAAACGTGGTTGGTGGTCTGGGTTACGTTGGTGTTAAATCTGGCTTTCCGCCGGGTTACTTGAGCCCGGAACTTTCTCGTCGTGCAATGTACAACATCATCCAGGCACACGCTCGTGCTTATGATGGCATCAAATCTGTTTCCAAGAAACCGGTTGGTATCATCTACGCGAACTCTTCTTTCCAGCCGTTGACCGACAAAGACATGGAAGCTGTGGAAATGGCGGAGAACGACAACCGTTGGTGGTTCTTCGATGCAATCATCCGTGGCGAAATCACTCGTGGTAACGAGAAGATCGTTCGTGACGACCTGAAAGGTCGTCTGGATTGGATCGGCGTGAACTACTACACTCGTACTGTTGTTAAACGTACCGAGAAAGGCTACGTGTCTCTGGGTGGTTACGGTCACGGTTGCGAACGTAACTCCGTAAGCCTGGCAGGTCTGCCAACCTCTGACTTCGGCTGGGAGTTCTTTCCGGAAGGTTTGTACGACGTTCTGACCAAATACTGGAACCGTTACCACCTGTACATGTACGTTACCGAGAACGGTATCGCGGACGATGCTGACTACCAGCGTCCGTACTACCTGGTGTCTCACGTTTACCAGGTGCATCGTGCGATCAACTCTGGTGCAGACGTACGTGGTTACCTGCACTGGAGCCTGGCGGACAACTACGAATGGGCTTCCGGTTTCAGCATGCGCTTTGGCCTGCTGAAAGTTGACTACAACACCAAACGTCTGTATTGGCGTCCGTCTGCGCTGGTATACCGTGAAATCGCGACCAACGGTGCGATCACCGACGAAATCGAACACCTGAACAGCGTACCGCCGGTTAAACCACTGCGTCACTAA。

[0097] Nucleotide sequence encoding F229P / G301P, SEQ ID NO.4:

[0098]

[0099] The nucleotide sequence encoding F229P / L337V, SEQ ID NO.5:

[0100] ATGTACAGCTTTCCAAACTCTTTCCGTTTCGGTTGGAGCCAGGCTGGCTTCCAGTCCGAAATGGGCACTCCGGGCTCCGAAGATCCGAACACCGACTGGTACAAATGGGTTCATGATCCGGAGAACATGGCTGCAGGTCTGGTTTCTGGTGATTTGCCGGAGAACGGTCCAGGTTACTGGGGCAACTACAAGACCTTCCACGACAACGCTCAGAAGATGGGTCTGAAGATCGCGCGTCTGAACGTTGAATGGAGCCGTATCTTCCCAAACCCGCTGCCGCGTCCGCAGAACTTCGATGAATCCAAACAAGATGTGACCGAAGTTGAAATCAACGAGAACGAACTGAAACGTCTGGATGAATACGCGAACAAAGACGCTCTGAACCACTATCGTGAAATCTTCAAAGACCTGAAATCTCGTGGTCTGTACTTCATTCTGAACATGTACCATTGGCCGCTGCCGTTGTGGCTGCATGATCCGATCCGTGTTCGTCGCGGTGACTTCACCGGTCCGTCTGGTTGGCTGTCTACTCGTACCGTGTACGAGTTCGCTCGTTTCTCTGCGTACATCGCGTGGAAATTCGATGACCTGGTAGACGAATACTCCACCATGAACGAACCAAACGTGGTTGGTGGTCTGGGTTACGTTGGTGTTAAATCTGGCTTTCCGCCGGGTTACTTGAGCCCGGAACTTTCTCGTCGTGCAATGTACAACATCATCCAGGCACACGCTCGTGCTTATGATGGCATCAAATCTGTTTCCAAGAAACCGGTTGGTATCATCTACGCGAACTCTTCTTTCCAGCCGTTGACCGACAAAGACATGGAAGCTGTGGAAATGGCGGAGAACGACAACCGTTGGTGGTTCTTCGATGCAATCATCCGTGGCGAAATCACTCGTGGTAACGAGAAGATCGTTCGTGACGACCTGAAAGGTCGTCTGGATTGGATCGGCGTGAACTACTACACTCGTACTGTTGTTAAACGTACCGAGAAAGGCTACGTGTCTGTGGGTGGTTACGGTCACGGTTGCGAACGTAACTCCGTAAGCCTGGCAGGTCTGCCAACCTCTGACTTCGGCTGGGAGTTCTTTCCGGAAGGTTTGTACGACGTTCTGACCAAATACTGGAACCGTTACCACCTGTACATGTACGTTACCGAGAACGGTATCGCGGACGATGCTGACTACCAGCGTCCGTACTACCTGGTGTCTCACGTTTACCAGGTGCATCGTGCGATCAACTCTGGTGCAGACGTACGTGGTTACCTGCACTGGAGCCTGGCGGACAACTACGAATGGGCTTCCGGTTTCAGCATGCGCTTTGGCCTGCTGAAAGTTGACTACAACACCAAACGTCTGTATTGGCGTCCGTCTGCGCTGGTATACCGTGAAATCGCGACCAACGGTGCGATCACCGACGAAATCGAACACCTGAACAGCGTACCGCCGGTTAAACCACTGCGTCACTAA。

[0101] Nucleotide sequence encoding F229P / G338P, SEQ ID NO.6:

[0102] ATGTACAGCTTTCCAAACTCTTTCCGTTTCGGTTGGAGCCAGGCTGGCTTCCAGTCCGAAATGGGCACTCCGGGCTCCGAAGATCCGAACACCGACTGGTACAAATGGGTTCATGATCCGGAGAACATGGCTGCAGGTCTGGTTTCTGGTGATTTGCCGGAGAACGGTCCAGGTTACTGGGGCAACTACAAGACCTTCCACGACAACGCTCAGAAGATGGGTCTGAAGATCGCGCGTCTGAACGTTGAATGGAGCCGTATCTTCCCAAACCCGCTGCCGCGTCCGCAGAACTTCGATGAATCCAAACAAGATGTGACCGAAGTTGAAATCAACGAGAACGAACTGAAACGTCTGGATGAATACGCGAACAAAGACGCTCTGAACCACTATCGTGAAATCTTCAAAGACCTGAAATCTCGTGGTCTGTACTTCATTCTGAACATGTACCATTGGCCGCTGCCGTTGTGGCTGCATGATCCGATCCGTGTTCGTCGCGGTGACTTCACCGGTCCGTCTGGTTGGCTGTCTACTCGTACCGTGTACGAGTTCGCTCGTTTCTCTGCGTACATCGCGTGGAAATTCGATGACCTGGTAGACGAATACTCCACCATGAACGAACCAAACGTGGTTGGTGGTCTGGGTTACGTTGGTGTTAAATCTGGCTTTCCGCCGGGTTACTTGAGCCCGGAACTTTCTCGTCGTGCAATGTACAACATCATCCAGGCACACGCTCGTGCTTATGATGGCATCAAATCTGTTTCCAAGAAACCGGTTGGTATCATCTACGCGAACTCTTCTTTCCA.

[0103] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A β-glycosidase SS-BGL mutant for the preparation of rare ginsenosides, characterized in that, The β-glucosidase SS-BGL mutant is a single-point or double-site mutation of the β-glucosidase SS-BGL with the amino acid sequence shown in SEQ ID NO.1, wherein: The single point mutation is: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline; The two-site mutations are: mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 301 to proline; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and leucine at position 337 to valine; or mutating phenylalanine at position 229 of SEQ ID NO.1 to proline and glycine at position 338 to proline.

2. The β-glycosidase SS-BGL mutant as described in claim 1, characterized in that, The rare ginsenosides include at least one of 20-o-diglucosyl-20(S)-propenyl glycol, ginsenoside Rh2, ginsenoside Rg2, and ginsenoside Rg3.

3. A recombinant vector comprising the nucleotide sequence of the β-glycosidase SS-BGL mutant of claim 1.

4. The method for preparing the recombinant vector as described in claim 3, characterized in that, The steps are as follows: The nucleotide sequence of the β-glucosidase SS-BGL was ligated into an expression vector, and the nucleotide sequence of the β-glucosidase SS-BGL was subjected to site-directed mutagenesis to obtain the recombinant vector; the nucleotide sequence of the β-glucosidase SS-BGL is shown in SEQ ID NO.2; The nucleotide sequence of the site-directed mutated β-glucosidase SS-BGL is any one of SEQ ID NO.3 to SEQ ID NO.

6.

5. The preparation method according to claim 4, characterized in that, The expression vector is a pET series vector.

6. The preparation method according to claim 5, characterized in that, The pET series vectors include any one of pET-28a(+), pET-24a(+), and pET-23a(+).

7. A genetically engineered bacterium comprising the recombinant vector of claim 3, characterized in that, The genetically engineered bacteria are obtained by transforming the recombinant vector into Escherichia coli.

8. The application of the β-glucosidase SS-BGL mutant as described in claim 1, the recombinant vector as described in claim 3, or the genetically engineered bacteria as described in claim 7, characterized in that, The β-glucosidase SS-BGL mutant, the recombinant vector, or the genetically engineered bacteria are used to prepare rare ginsenosides. The rare ginsenoside is ginsenoside compound K; The substrate for preparing the rare ginsenosides is glycosylated protopanaxadiol-type ginsenosides; The glycosylated proginadindiol-type ginsenoside is ginsenoside Rb1.

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