Beta-glycosidase SS-BGL mutant for preparing rare ginsenoside and application of beta-glycosidase SS-BGL mutant

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a beta-glycosidase SS-BGL mutant for preparing rare ginsenosides and application of the beta-glycosidase SS-BGL mutant. The beta-glycosidase SS-BGL mutant is obtained by conducting single-site mutation or double-site mutation on beta-glycosidase SS-BGL with the amino acid sequence shown as SEQ ID NO.1. The beta-glycosidase SS-BGL mutant is obtained by conducting single-site mutation or double-site mutation on beta-glycosidase SS-BGL with the amino acid sequence shown as SEQ ID NO.2. Wherein the single site mutation is that phenylalanine at the 229 site of SEQ ID NO. 1 is mutated into proline; the double-site mutation is as follows: phenylalanine at the 229 site in SEQ ID NO. 1 is mutated into proline, and glycine at the 301 site is mutated into proline; or the phenylalanine at the 229 site in SEQ ID NO. 1 is mutated into proline, and the leucine at the 337 site is mutated into valine; or the phenylalanine at the 229 site in SEQ ID NO. 1 is mutated into the proline, and the glycine at the 338 site is mutated into the proline. On the basis of natural beta-glycosidase SS-BGL, the molecular structure of the beta-glycosidase SS-BGL is modified through site-directed mutagenesis, and four beta-glycosidase SS-BGL mutants with the saponin conversion activity improved are obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a beta-glycosidase SS-BGL mutant for preparing rare ginsenosides and an application thereof. Background Art

[0002] In recent years, saponins, one of the main medicinal components of ginseng, have garnered attention for their extensive anticancer activity. Studies have found that secondary metabolites of ginsenosides, namely rare ginsenosides, exhibit enhanced antitumor activity and can be obtained through in vitro degradation of total saponins. These rare ginsenosides include ginsenoside compounds K, Rh2, Rg2, and Rg3. Ginsenoside compound K, abbreviated as CK (20-o-diglucosyl-20(S)-protobenzenediol), is the primary deglycosylated metabolite of ginsenosides and exhibits diverse biological activities, including anticancer, antidiabetic, anti-inflammatory, anti-allergic, anti-angiogenic, anti-aging, neuroprotective, and hepatoprotective properties. However, the difficulty of preparing CK has significantly limited its further application. To increase its yield, various methods have been developed for its production.

[0003] The structural modification of ginsenosides primarily involves hydrolysis of glycosyl groups at specific sites. Glycosidases are among the most common industrial enzymes, responsible for the hydrolytic cleavage of glycosyl bonds. In the food industry, they are used as catalysts for the hydrolysis of lactose in dairy products. β-Glycosidase SS-BGL, derived from the thermophilic archaeon Sulfolobus solfataricus, which grows at 87°C and pH 3.0, has been demonstrated to be an efficient enzyme for the production of CK 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. Common glycosylated PPD-type ginsenosides include Rb1, Rb2, Rb3, and Rd. The hydrolysis pathway of β-Glycosidase SS-BGL for the production of rare ginsenoside CK using ginsenoside Rb1 as a substrate follows: Rb1 → Rd → F2 → CK. High temperatures mean high reaction rates and low pollution, giving thermophilic glycosidases excellent advantages in industrial applications. Studies have shown that the wild-type thermophilic enzyme β-glycosidase SS-BGL has an optimal temperature of 80°C and an optimal pH of 6. However, the efficiency of wild-type thermophilic β-glycosidase SS-BGL in converting rare ginsenosides is low, which greatly limits the further application of SS-BGL. Therefore, a new strategy for the efficient conversion of rare ginsenosides is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a β-glucosidase SS-BGL mutant for preparing rare ginsenosides, which solves the defect of low efficiency of wild-type thermophilic enzyme SS-BGL in converting rare ginsenosides in the prior art.

[0005] The technical solution adopted in the present invention is:

[0006] In a first aspect, the present invention provides a β-glucosidase SS-BGL mutant for preparing rare ginsenosides, wherein the β-glucosidase SS-BGL mutant is a β-glucosidase SS-BGL with an amino acid sequence as shown in SEQ ID NO. 1 subjected to a single-site mutation or a double-site mutation, wherein:

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

[0008] The double-site mutation is: mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the glycine at position 301 to proline; or mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the leucine at position 337 to valine; or mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the glycine at position 338 to proline.

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

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

[0011] The 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-glucosidase SS-BGL mutant is connected to an expression vector for site-directed mutagenesis to obtain the recombinant vector.

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

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

[0015] The 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-glycosidase SS-BGL mutant, the recombinant vector or the genetically engineered bacteria, wherein the β-glycosidase 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 ginsenoside is 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] The present invention provides a β-glycosidase SS-BGL mutant for preparing rare ginsenosides, wherein the β-glycosidase SS-BGL mutant is a β-glycosidase SS-BGL with an amino acid sequence as shown in SEQ ID NO.1 subjected to a single-site mutation or a double-site mutation, wherein:

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

[0022] The double-site mutation is: mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the glycine at position 301 to proline; or mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the leucine at position 337 to valine; or mutating the phenylalanine at position 229 of SEQ ID NO.1 to proline, and mutating the glycine at position 338 to proline. Based on the natural β-glycosidase SS-BGL, the present invention transforms the molecular structure of the natural β-glycosidase SS-BGL through design and combined with site-directed mutagenesis biotechnology, analyzes the influence of the mutated residues on the enzymatic properties, and finally obtains a single-point mutation F229P and three double-point mutations F229P / G301P, F229P / L337V and F229P / G338P with improved saponin conversion activity. The ginsenoside conversion rates at 80°C are increased to 1.85 times, 1.50 times, 1.79 times and 1.91 times the original ones, respectively.

[0023] While significantly improving conversion efficiency, these single- and double-point mutants maintain their excellent heat resistance as thermophilic enzymes, with their thermal stability remaining largely unchanged. In a 95°C heat treatment assay measuring residual enzyme activity, compared to the wild-type, which had a residual activity of 85%, all four mutants maintained activity above 80%. The double-point mutation F229P / L337V reached 89%, slightly higher than the wild-type. After heat treatment at 80°C for 24 hours, both the wild-type and four mutants retained activity above 30%, with half-lives exceeding 16 hours, showing no significant differences from the wild-type. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The bands of WT, F229P, F229P / G301P, F229P / L337V and F229P / G338P pure enzyme solutions after sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis are shown in sequence.

[0025] Figure 2 The results of conversion of rare ginsenoside CK by wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P at 80℃.

[0026] Figure 3 The residual enzyme activity results of wild-type β-glycosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P after heat treatment at 95°C for 20 minutes.

[0027] Figure 4 The residual enzyme activities of wild-type β-glycosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P after heat treatment at 80°C for 24 hours. DETAILED DESCRIPTION

[0028] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

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

[0030] The structural modification of ginsenosides primarily involves the hydrolysis of glycosyl groups at specific sites. Glycosidases are among the most common industrial enzymes, responsible for the hydrolytic cleavage of glycosyl bonds and used in the food industry as catalysts for the hydrolysis of lactose in dairy products. β-Glycosidase SS-BGL has been shown to be a highly efficient CK-producing enzyme using glycosylated PPD-type ginsenosides as substrates. The hydrolysis pathway for β-Glycosidase SS-BGL to produce rare ginsenoside CK using ginsenoside Rb1 as a substrate is: Rb1→Rd→F2→CK. However, the efficiency of wild-type thermophilic β-Glycosidase SS-BGL in converting rare ginsenosides is low, which greatly limits the further application of SS-BGL.

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

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

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

[0034] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0035] In the following examples, the culture medium and formulations involved are as follows:

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

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

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

[0039] (1) β-glucosidase SS-BGL enzyme activity determination method.

[0040] Enzyme activity was tested by measuring 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 incubated at 80°C for 3 minutes. The reaction was terminated by adding 500 μL of 0.6 mM sodium carbonate solution. The release of p-nitrophenol was determined by measuring 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) Determination of saponin conversion rate.

[0042] Using 5 mg / mL Rb1 as the substrate, 1 mL of 0.5 mg / mL enzyme solution dissolved in 50 mM citric acid / phosphate buffer was added. The reaction was incubated at 80°C for 30 minutes, then terminated with an equal volume of methanol. The reaction was sonicated for 30 minutes and filtered through a 0.45 μm sterile filter. CK formation was monitored by HPLC. A standard curve was established using saponin CK standards, and the actual yield was calculated from the peak area of ​​the HPLC results to provide the corresponding saponin conversion rate.

[0043] The abbreviations of the present invention are shown in Table 1.

[0044] Table 1 Abbreviations

[0045]

[0046]

[0047] Example 1

[0048] A β-glucosidase SS-BGL mutant for preparing rare ginsenosides, specifically described as follows:

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

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

[0051] The nucleotide sequence of wild-type β-glycosidase SS-BGL, as shown in SEQ ID NO. 2, was chemically synthesized and ligated with the pET-28a(+) vector to produce the vector pET-28a(+)-SS-BGL. The amino acid sequence corresponding to the nucleotide sequence of wild-type β-glycosidase SS-BGL is shown in SEQ ID NO. 1.

[0052] (2) Obtaining a recombinant vector containing a mutant.

[0053] The whole plasmid PCR technique was used to perform site-directed mutagenesis with pET-28a(+)-SS-BGL as a template to obtain the recombinant vector pET-28a(+)-SS-BGL-F229P containing the mutant gene.

[0054] The PCR amplification program for amplifying the whole plasmid was set as follows: first, pre-denaturation at 95°C for 5 min; then 20 cycles of denaturation at 95°C for 1 min, annealing at 68°C for 30 s, and extension at 72°C for 7 min; and finally, insulation at 4°C.

[0055] After the PCR reaction, add 1 μL of Dpn I directly to the PCR reaction mixture, mix thoroughly, and incubate at 37°C for 5 minutes. After Dpn I digests the template plasmid in the PCR product, verify the PCR product by electrophoresis on a 1% agarose gel. After confirming the electrophoretic band of the PCR product, recover the PCR product and transform it into E. coli DH5α competent cells. The plasmid is then extracted and sequenced.

[0056] Step 2: Construction of recombinant Escherichia 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 culture medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm to prepare a seed solution; the prepared seed solution was transferred to 100 mL of LB liquid culture medium containing 50 μg / mL kanamycin at an inoculum volume fraction of 0.5%, and cultured at 37°C and 220 rpm until OD600 reached 0.6, IPTG was added at a final concentration of 1 mM, and the culture was continued at 16°C for 16 h to obtain a fermentation broth; the prepared fermentation broth was centrifuged at 10,000 rpm and 4°C for 5 min to collect the cell bodies, and the cell bodies were resuspended in 50 mM citric acid / phosphate buffer. The resuspended cells were treated with an ultrasonic disruptor in an ice bath for 10 minutes, and then centrifuged at 10,000 rpm and 4°C for 10 minutes. The supernatant was collected to obtain a crude enzyme solution, namely, the β-glycosidase SS-BGL mutant F229P. The nucleotide sequence of F229P is shown in SEQ ID NO.3.

[0059] (3) The crude enzyme solution was heated at 80°C for 30 min and then filtered through a 0.45 μm sterile filter membrane. The filtrate was the pure enzyme solution, and the protein concentration was determined using a BCA protein concentration kit.

[0060] Example 2 to Example 4

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

[0062] In Examples 2 to 4, whole-plasmid PCR technology was used to perform site-directed mutagenesis using pET-28a(+)-SS-BGL as a template, and recombinant vectors containing the mutant genes, pET-28a(+)-SS-BGL-F229P / G301P, pET-28a(+)-SS-BGL-F229P / L337V, and pET-28a(+)-SS-BGL-F229P / G338P, were obtained, respectively. The genetically engineered bacteria obtained, respectively, using the recombinant vectors containing the mutant genes 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 / G338P were obtained by expression in genetically engineered bacteria.

[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] Among them, F229P is a single point mutation, and F229P / G301P, F229P / L337V and F229P / G338P are double point mutations.

[0065] The primer sequences used for site-directed mutagenesis in the above examples 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 base sequences in Table 2 indicate the bases that need to be replaced by site-directed mutagenesis.

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

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

[0071] Table 3 Relative enzyme activities of each mutant

[0072]

[0073]

[0074] As can be seen from Table 3, single-point and double-point mutations have a significant effect on the pNPG enzyme activity. Compared with the wild type, the relative enzyme activity of F229P and F229P / G301P decreased, among which F229P / G301P was only 46.82%, while the F229P / L337V and F229P / G338P mutants both increased to varying degrees, with the highest value reaching 119.91%.

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

[0076] Table 4 Residual enzyme activity of each group after heat treatment at 95℃ for 20min

[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- and double-point mutations had no significant effect on the thermostability of the thermophilic enzyme at 95°C. In the residual enzyme activity assay following 95°C heat treatment, all four mutants maintained activity above 80% 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 a β-glucosidase SS-BGL mutant for preparing rare ginsenosides, specifically as follows:

[0081] 1. Saponin conversion rate.

[0082] 1 mL of each 0.5 mg / mL pure enzyme solution from Examples 1 to 4 was taken, along with 5 mg / mL of Rb1. The reaction was incubated at 80°C for 30 min. An equal volume of methanol was added to terminate the reaction. The reaction was sonicated for 30 min, filtered through a 0.45 μL filter, and CK production was determined 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] As can be seen from Table 5, single-point and double-point mutations have a significant effect on the enzyme-catalyzed preparation of rare ginsenosides. The saponin conversion rate at 80°C is increased to 1.85 times, 1.50 times, 1.79 times and 1.91 times the original value, respectively. The statistical results of the conversion of rare ginsenoside CK are shown in Figure 2 .

[0086] The present invention also mixed 0.25 mL of 0.5 mg / mL pure enzyme solutions of F229P, F229P / G301P, F229P / L337V, and F229P / G338P, then reacted them with Rb1 at 80°C for 30 minutes to detect CK formation. Testing revealed that the relative enzyme activity of the F229P, F229P / G301P, F229P / L337V, and F229P / G338P mixtures was 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. The residual enzyme activity of each mutant is calculated with the enzyme activity before treatment as 100%. Figure 3 The results showed that single- and double-point mutations had no significant effect on the thermostability of the thermophilic enzyme at 95°C. In a 95°C heat treatment experiment, the residual enzyme activity of all four mutants remained above 80% 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 activities of wild-type β-glucosidase SS-BGL and its mutants F229P, F229P / G301P, F229P / L337V and F229P / G338P after heat treatment at 80℃ for 24h are shown in the table. Figure 4The results showed that after heat treatment at 80°C for 24 hours, the wild type and four mutant types could maintain more than 30% of their activity, with half-lives exceeding 16 hours. There was no significant difference compared with the wild type, indicating that the point mutations did not alter the enzyme's original excellent heat resistance and it still maintained its original stability.

[0091] 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-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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 above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

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

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

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

4. The method for preparing the recombinant vector according to claim 3, characterized in that: Here are the steps: The nucleotide sequence of the beta-glycosidase SS-BGL mutant is connected to an expression vector for site-directed mutagenesis to obtain the recombinant vector.

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 according to claim 3, characterized in that: The genetically engineered bacteria are obtained by transforming the recombinant vector into Escherichia coli.

8. Use of the β-glycosidase SS-BGL mutant according to claim 1, the recombinant vector according to claim 3 or the genetically engineered bacteria according to claim 7, characterized in that: The beta-glycosidase SS-BGL mutant, the recombinant vector or the genetically engineered bacteria are used for preparing rare ginsenosides.

9. The use according to claim 8, characterized in that The substrate for preparing the rare ginsenoside is glycosylated protopanaxadiol type ginsenoside.

10. The use according to claim 9, characterized in that The glycosylated protopanaxadiol-type ginsenosides include at least one of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3 and ginsenoside Rd.

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