A beta-glucosidase bglsk mutant and application thereof

Abstract

The application discloses a beta-glucosidase BglSK mutant and application thereof. The BglSK mutant is mutated based on the amino acid sequence shown in SEQ ID NO. 1. The beta-glucosidase BglSK mutant of the application significantly improves the thermal stability and / or catalytic activity of the beta-glucosidase by introducing one or more amino acid residue substitutions at specific sites. In addition, the beta-glucosidase BglSK mutant can efficiently catalyze ginsenoside Rd or an analogue thereof to direct generation of rare ginsenoside F2 and / or rare ginsenoside CK. The beta-glucosidase BglSK mutant of the application successfully realizes large-scale production of rare ginsenosides, and has great industrial application prospect and value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a β-glucosidase BglSK mutant and its applications. Background Technology

[0002] Ginseng (Panax ginseng), as one of the world's most representative traditional medicinal plants, has been used for disease prevention and health maintenance in Asia for thousands of years. Ginsenosides are the core material basis for ginseng's various biological and pharmacological activities, possessing a variety of pharmacological functions including immunomodulation, anti-tumor, anti-inflammatory, anti-diabetic, antihypertensive, and neuroprotective effects. To date, more than 150 natural saponins have been isolated and identified from ginseng, among which the six major saponins Rb1, Rb2, Rc, Rd, Re, and Rg1 constitute the main part of the total saponin content in Korean ginseng and American ginseng.

[0003] However, these major ginsenosides have extremely low absorption rates in the human gastrointestinal tract due to their large molecular weight, poor solubility, and limited transmembrane permeability, resulting in limited bioavailability. In contrast, rare saponins generated through deglycosylation (such as CK, CY, C-Mc, F2, and F1) exhibit higher membrane permeability and more significant pharmacological activity. Therefore, the efficient conversion of major saponins into low-polarity rare saponins has become a key technological direction for the deep processing of ginseng and modern pharmacological research.

[0004] Currently, researchers have isolated and cloned glycoside hydrolases (GHs) capable of converting ginsenosides from various microorganisms, including enzyme systems from the genera *Aspergillus spp.* and *Terrabacter ginsenosidimutans*, and classified them into types I–IV ginsenoside glycosides based on their catalytic pathways. In addition, there are reports of research using *Lactobacillus paracasei* PR strain, naringinase derived from *Penicillium sclerotium*, and recombinant glycosides to catalyze the production of rare ginsenoside F1.

[0005] However, for some rare saponins with outstanding pharmacological activity (such as CK), existing conversion systems generally suffer from problems such as insufficient substrate recognition specificity, low catalytic efficiency, and reduced yield due to side reactions, making it difficult to meet the requirements of industrial-scale production and thus limiting their pharmacological mechanism research and industrial development.

[0006] In summary, developing novel ginsenoside hydrolases with high catalytic activity and high substrate specificity, constructing efficient and controllable biotransformation systems, and realizing the large-scale preparation of rare saponins are the core technological bottlenecks that urgently need to be overcome in this field. Summary of the Invention

[0007] To overcome the limitations of existing technologies, this invention proposes a β-glucosidase BglSK mutant and its applications, particularly relating to the construction and use of the BglSK mutant. Specifically, this invention utilizes thermophilic microorganisms... Sanguibacter keddieii Based on the enzyme protein BglSK, its parent BglSK_AA (whose amino acid sequence is shown in SEQ ID NO: 1) was further directionally modified, and a new mutant with significantly improved thermal stability and its ability to prepare ginsenoside F2 and / or ginsenoside CK was successfully obtained.

[0008] Specifically, the present invention includes, but is not limited to, the following technical solutions:

[0009] The first aspect of the present invention provides a β-glucosidase BglSK mutant, based on the amino acid sequence shown in SEQ ID NO.1, wherein the BglSK mutant comprises one or more of the following: Q57H, G560A, T263P and Y309F.

[0010] MPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENAGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFAEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSI (SEQ ID NO.1)

[0011] The nucleotide sequence of the gene encoding SEQ ID NO: 1 is shown as SEQ ID NO: 2.

[0012]

[0013] The β-glucosidase BglSK mutant of the present invention is modified based on the parental BglSK_AA (SEQ ID NO.1), which is derived from wild-type BglSK (from the genus *Sanguisorba*). Sanguibacter keddieii It was obtained by modifying the original (SEQ ID NO.3).

[0014] In some embodiments, the BglSK mutant contains a substitution for Q57H. Compared to the parent SK_AA, the mutant Q57H exhibits better substrate tolerance at 40 and 50°C. p The relative activity of NPG was significantly improved, and the relative activity of catalyzing the conversion of the substrate ginsenoside Rd to ginsenoside CK was also significantly improved by 14.0%.

[0015] In some embodiments, the BglSK mutant contains a substitution for G560A. Compared to the parent SK_AA, the mutant G560A exhibits better substrate tolerance at 40 and 50°C. p The relative activity of NPG was significantly improved, and the relative activity of catalyzing the conversion of the substrate ginsenoside Rd to ginsenoside CK was also significantly improved by 6.4%.

[0016] In some embodiments, the BglSK mutant contains a substitution for T263P. Compared to the parent SK_AA, the mutant T263P exhibits better substrate tolerance at 40 and 50°C. p The relative activity of NPG was significantly improved, and the relative activity of catalyzing the conversion of the substrate ginsenoside Rd to ginsenoside CK was also significantly improved by 9.7%.

[0017] In some embodiments, the BglSK mutant contains a substitution for Y309F. Compared to the parent SK_AA, the mutant Y309F exhibits better substrate tolerance at 40 and 50°C. p The relative activity of NPG was significantly improved, and the relative activity of catalyzing the conversion of the substrate ginsenoside Rd to ginsenoside CK was also significantly improved by 21.7%.

[0018] In some embodiments, the BglSK mutant comprises any of the following:

[0019] 1) Q57H and Y309F;

[0020] 2) Q57H and G560A

[0021] 3) Q57H and T263P;

[0022] 4) G560A and T263P;

[0023] 5) G560A and Y309F;

[0024] 6) Q57H, G560A, and T263P;

[0025] 7) Q57H, G560A, and Y309F;

[0026] 8) Q57H, T263P, and Y309F;

[0027] 9) G560A, T263P, and Y309F;

[0028] 10) Q57H, G560A, T263P and Y309F.

[0029] Compared with the parent SK_AA, the relative activity of the mutants described in 1)-10) in catalyzing the conversion of the substrate ginsenoside Rd to ginsenoside CK was significantly increased by 39.4%, 12.5%, 20.4%, 14.7%, 12.3%, 28.5%, 47.8%, 34.5%, 30.0%, and 42.6%, respectively.

[0030] A second aspect of the present invention provides a polynucleotide encoding the BglSK mutant as described above.

[0031] A third aspect of the present invention provides a recombinant vector comprising the polynucleotides described above.

[0032] A fourth aspect of the present invention provides a host cell comprising the polynucleotides described above or the recombinant vectors described above.

[0033] In some embodiments, the host cell is a fungal cell, a bacterial cell, or a plant cell.

[0034] In some embodiments, the host cell is a bacterial cell. Preferably, the bacterial cell is an *Escherichia coli* cell. More preferably, the *E. coli* cell is *E. coli*. BL21(DE3) cell.

[0035] In some embodiments, the bacterial cells are Escherichia coli cells.

[0036] The fifth aspect of the invention provides the use of the BglSK mutant as described above, or the polynucleotide as described above, or the recombinant vector as described above, or the host cell as described above, in the preparation of ginsenoside F2 and / or ginsenoside CK.

[0037] Ginsenoside Compound K (CK) is a rare diol-type ginsenoside that does not exist in natural ginseng. It is the main degradation product of other diol-type ginsenosides in the human intestine and is the entity that is actually absorbed and exerts its effects. Rare ginsenoside CK possesses high safety and diverse biological functions, and may be a potential therapeutic agent for many diseases. For example, ginsenoside CK has been reported to have (adjunctive) therapeutic effects on various diseases such as cancer, tumors, inflammation, and diabetes. Ginsenoside CK is a metabolite of proto-ginsenosides (such as Rb1, Rb2, Rc, etc.) undergoing gradual deglycosylation under the action of intestinal microorganisms. Its typical metabolic pathway is: Rb1→Rd→F2→CK or Rb1→Gypenoside XVII→F2→CK.

[0038] Ginsenoside F2 (molecular formula C) 42 H 72 O 13 Ginsenoside CK (molecular weight 785.01) is an intermediate metabolite, which, after further removal of the glucose group at the C-20 position, generates ginsenoside CK (molecular formula C...). 36 H 62 O8, molecular weight 622.87).

[0039] A sixth aspect of the present invention provides a method for preparing ginsenoside F2 and / or ginsenoside CK, comprising the following steps:

[0040] Ginsenoside F2 and / or ginsenoside CK can be obtained by using the BglSK mutant, polynucleotide, recombinant vector, or host cell catalysis as described above to obtain ginsenoside Rd or similar substances.

[0041] In some embodiments, using ginsenoside Rd as a substrate, the amount of BglSK mutant added, by mass percentage, is 1 μg / mL to 1000 μg / mL.

[0042] In some embodiments, the CAS number of the ginsenoside Rd is 52705-93-8.

[0043] In some embodiments, the catalytic temperature is 16-45°C.

[0044] In some embodiments, the catalysis time is 1 h to 7 d.

[0045] In some implementations, the pH of the catalytic reaction system is 6.0-10.0.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1) The β-glucosidase BglSK mutant of the present invention is based on the mutant BglSK_AA and is molecularly modified. The resulting β-glucosidase BglSK mutant not only has improved enzyme activity, but also has significantly improved heat resistance.

[0048] 2) Compared with the parent BglSK_AA, the BglSK mutant of β-glucosidase in this invention showed a relative increase of 9.7%-42.6% in the catalysis of ginsenoside Rd. Among them, the mutant containing Q57H, G560A, T263P and Y309F showed the greatest relative activity increase, which was 42.6%; the mutant containing Y309F showed the least relative activity increase, which was 9.7%.

[0049] 3) Using the β-glucosidase BglSK mutant BglSK_AA_4M (BglSK_AA_Q57H / G560A / T263P / Y309F) obtained in this invention, with ginsenoside Rd as a substrate, the conversion rate of the mutant BglSK_AA_4M was increased from 85% to 98% after reacting at 30℃ for 2 h compared with the parent BglSK_AA. Therefore, the β-glucosidase BglSK mutant of this invention can realize the large-scale production of rare ginsenosides and has great industrial application prospects. Attached Figure Description

[0050] Figure 1 The diagram shows the alignment of the β-glucosidase BglSK mutant of the present invention with five homologous sequences. Sequences with >50% identity are considered conserved sequences. Based on the differences between BglSK and conserved sequences, the following 59 mutants were designed from the parental BglSK_AA: F28Y, S34P, T37E, N47T, Q57H, V96A, V113L, V125I, D151E, S152P, S177A, N201G, T206D, S229G, A230P, T236M, T263P, V276A, H280A, K281G, D294G, Y309F, V329C, V335L, I366L, H367D, C3 70V, D380E, L381V, E388S, E409R, D415N, L432F, S441R, V469E, L479Y, W500Y, S520P, A521E, V537T, L538V, I547V, L548 I, T549P, P550E, V552A, T556A, V559L, G560A, Y562F, T564A, V570L, A571D, A572V, T574F, V577A, Q586E, A602P, V609L.

[0051] Figure 2This invention demonstrates the results of preliminary screening of 59 mutants using pNPG. The activity of the initial parent BglSK_AA (abbreviated as SK-AA or SK_AA in the figure) was defined as 100%. The relative activities of the other 59 mutants were calculated. After preliminary screening, it was found that the BglSK mutants F28Y, S34P, Q57H, V96A, D151E, S177A, N201G, T206D, S229G, V113L, T263P, D294G, Y309F, L432F, S520P, V609L, G560A, and T564A had higher activities than the parent BglSK_AA.

[0052] Figure 3 The SDS-PAGE purification electrophoresis results of the parental BglSK_AA and the initially screened positive BglSK mutants L432F, S520P, V609L, G560A, and T564A show that high-purity proteins were obtained.

[0053] Figure 4 This paper presents a comparative chart showing the activity of the parental BglSK_AA and its BglSK mutants in generating rare ginsenoside CK from the hydrolysis of ginsenoside Rd. The amount of ginsenoside CK generated from the parental BglSK_AA was taken as 100%, and the relative activities of other mutants were calculated. The results show that, compared with the parental BglSK_AA, the relative activities of the BglSK mutants Q57H, G560A, T263P, and Y309F were increased by 14.0%, 6.4%, 9.7%, and 21.7%, respectively.

[0054] Figure 5 The activity of the combined mutants formed by 11 combinations of the BglSK mutants Q57H, G560A, T263P, and Y309F of the present invention was shown. Ultimately, compared with the parent BglSK_AA, the relative activities of the combined mutants Q57H / Y309F, Q57H / G560A / T263P, Q57H / G560A / Y309F, Q57H / T263P / Y309F, G560A / T263P / Y309F, and Q57H / G560A / T263P / Y309F were increased by 39.4%, 28.5%, 47.8%, 34.5%, 30.0%, and 42.6%, respectively.

[0055] Figure 6 This diagram illustrates the process by which the β-glucosidase BglSK mutant of the present invention catalyzes the production of ginsenoside Rd into ginsenoside F2 and / or CK.

[0056] Figure 7The HPLC chromatograms of the rare ginsenoside CK prepared by hydrolyzing ginsenoside Rd using the parental SK_AA and its mutant BglSKAA_4M (BglSKAA_Q57H / G560A / T263P / Y309F, SK_AA_4M) are shown. The retention time of the substrate ginsenoside Rd is 37.6 min, and the retention time of the product ginsenoside CK is 52.2 min. Detailed Implementation

[0057] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0058] Example 1: Construction and Screening of β-glucosidase BglSK Mutants

[0059] 1.1 Construction of the BglSK mutant

[0060] To enhance the catalytic activity of the BglSK_AA protein in the hydrolysis of ginsenoside Rd to generate the rare ginsenoside CK, a protein engineering strategy guided by a regression to conserved mutations was employed. This included the following:

[0061] Homologous sequence retrieval (BLAST) of the NCBI Nr database revealed five homologous sequences from thermophilic microorganisms; multiple sequence alignment analysis was performed using ClustalX2 software. Figure 1 The amino acid sites with a conservation rate of >50% were defined as conserved sites; then, 59 mutants were designed using a strategy of reverting to conservation (conserved-but-difference).

[0062] Including F28Y, S34P, T37E, N47T, Q57H, V96A, V113L, V125I, D151E, S152P, S177A, N201G, T206D, S229G, A230P, T236M, T263P, V276A, H280A, K281G, D294G, Y309F, V329C, V335L, I366L, H367D, C370V, D380E, L381V, E388S , E409R, D415N, L432F, S441R, V469E, L479Y, W500Y, S520P, A521E, V537T, L538V, I547V, L548I, T549P, P5 50E, V552A, T556A, V559L, G560A, Y562F, T564A, V570L, A571D, A572V, T574F, V577A, Q586E, A602P, V609L.

[0063] The circular plasmid containing the BglSK_AA gene (pET21a-BglSKAA) was synthesized by Qingke Biotechnology Co., Ltd., with NdeI and XhoI restriction sites. In our laboratory, it was transformed into Escherichia coli BL21(DE3). Here, the circular plasmid containing BglSK_AA was used as a template for PCR amplification, and 59 mutant plasmids were obtained.

[0064] Circular plasmid PCR system: 1 μL template; 1 μL each of forward and reverse single-point mutation primers (10 μM each), primer list is shown in Table 1; 4 μL dNTP (2.5 mM); 5 μL 10× high-fidelity enzyme buffer; 1 μL high-fidelity enzyme; add water to a reaction volume of 50 μL.

[0065] Table 1

[0066]

[0067] PCR thermal cycling conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, (Tm-5)℃ annealing for 20 s; 72℃ extension for 4 min 20 s; 4℃ incubation; PCR amplification was performed under these conditions for 30 cycles.

[0068] After digestion with Dpn I, 5 μL of the amplified product was added to 50 μL of BL21(DE3) competent cells for transformation. The specific transformation method was as follows:

[0069] Incubate on ice for 20 min; heat shock at 42℃ for 45 s; place on ice for 3 min; add 800 μL LB medium, activate at 37℃ and 220 rpm for 1 h, then spread on plates and incubate for 14 h-16 h until mature single colonies grow. Select bacteria for sequencing verification to complete the construction of the BglSK_AA mutant strain.

[0070] Plate cloning of mutants: single colonies were picked into 96-well plates (800 μL of LB ampicillin medium), (3 colonies of each mutant were picked into 3 wells) and incubated overnight at 37°C for 13-16 h to obtain seed culture.

[0071] Then, 20 μL of seed culture was inoculated into 1 mL of fresh LB medium (new 96-well plate, containing 0.1% ampicillin and 0.2 mM IPTG) and induced at 18°C ​​for 24 h. The cells were harvested by centrifugation at 4000 rpm for 10 min, added to 100 μL of 50 mM lysis buffer (pre-prepared, PBS: Hammer lysis buffer = 9:1, pH 7.0), mixed well, incubated for 5 min, and centrifuged to collect the supernatant as crude enzyme solution.

[0072] 1.2. Secondary screening and secondary screening

[0073] The substrate was 4-nitrophenyl-β-D-glucopyranoside (pNPG, Aladdin Company, China, stock solution concentration 0.1M). The p-nitrophenol released by β-glucosidase from the substrate showed the maximum absorption peak under alkaline conditions. Therefore, the absorbance of the solution at 405 nm after the reaction was detected by an enzyme-linked immunosorbent assay (ELISA) reader to obtain the enzyme activity.

[0074] Both the initial screening and the secondary screening were carried out at 25℃ in 0.1 M KH2PO4-NaOH buffer (pH 8.0), and the experiment was repeated 3 times.

[0075] 1.2.1 Initial screening

[0076] 200 μL reaction system: containing 188 μL 0.1 M KH2PO4-NaOH buffer, 2 μL 0.1 M pNPG, and 10 μL crude enzyme solution obtained in step 1.1.

[0077] Reaction conditions: Reaction at 40℃ for 10 minutes.

[0078] 1.2.2 Secondary screening

[0079] The reaction system is the same as the initial screening system.

[0080] The reaction conditions are the same as those for the initial screening, except that the reaction is carried out at a different temperature of 50℃ for 10 minutes.

[0081] Enzyme kinetic analysis was performed, and the point of highest reaction rate was taken as enzyme activity. The activity of the unmutated enzyme was defined as 100%. Three replicates were set up to compare the relative activities of the mutants. The results are shown in […]. Figure 2 .

[0082] from Figure 2 It can be seen that after incubation at 40℃ / 50℃ for 10 min, compared with the parent BglSK_AA, the mutants F28Y, S34P, Q57H, V96A, D151E, S177A, N201G, T206D, S229G, V113L, T263P, D294G, Y309F, L432F, S520P, A521E, G560A, T564A, and V609L showed better substrate response to the crude enzyme solution. p The relative activity of NPG was improved in all mutants, with the improvement ranging from 10% to 1200%. This may be due to the poor stability of BglSK_AA at 40 / 50℃, which led to a significant decrease in activity. These mutants enhanced the activity and stability of the enzyme.

[0083] Example 2: Expression and purification of parental BglSK_AA and its mutants

[0084] The strains successfully verified in step 1.2 of Example 1, and the positive mutants F28Y, S34P, Q57H, V96A, D151E, S177A, N201G, T206D, S229G, V113L, T263P, D294G, Y309F, L432F, S520P, A521E, G560A, T564A, V609L, and the recombinant strain of parent BglSK_AA, were inoculated into 5 mL of LB medium (15 mL centrifuge tube) containing 100 μg / mL (final concentration) of ampicillin and cultured for 13-16 h to obtain seed culture.

[0085] Then, inoculate 2 mL of seed culture into 100 mL of fresh LB medium (containing ampicillin, 100 μg / mL) in a 250-500 mL Erlenmeyer flask and incubate at 37°C and 220 rpm for approximately 1.5-2 h until the logarithmic growth phase (OD200). 600 When the concentration of 0.5 M IPTG reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM (20 μL of 0.5 M IPTG) for induction, and continue culturing at 18℃ and 220 rpm for 24 h.

[0086] After 24 hours of culture, the bacterial cells were harvested by centrifugation at 7000×g for 10 min. The cells were then added to 4.5 mL of Lysis Buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.5, 5% glycerol), vortexed, and 0.5 mL of Hammer Super Lysis Buffer was added. The mixture was then vortexed and placed at 4-25℃ (selected according to protein stability) for 1-10 minutes until the bacterial solution was clear and transparent.

[0087] Centrifuge the lysate at 8000×g for 10 min, collect the supernatant, filter it through a 0.22 μm filter, and then load it onto the sample.

[0088] Purification was performed using a Ni-NTA affinity chromatography column. After equilibration with 5 mL of lysis buffer, the supernatant (not exceeding 10 mL) was loaded. Impurities were eluted with 30 mL of Wash buffer (50 mM Tris-HCl, 1 M NaCl, 20 mM imidazole, pH 7.5, 5% glycerol). The target protein was then collected by elution with 3 mL of Elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, 5% glycerol) and analyzed by SDS-PAGE. The results are shown in the figure below. Figure 3 .

[0089] from Figure 3 It can be seen that the parental BglSK_AA and mutants F28Y, S34P, Q57H, V96A, V113L, N201G, S229G, T263P, D294G, Y309F, L432F, A521E, and G560A with high protein concentration and purity were finally obtained.

[0090] Take 40 μL of sample to verify whether the protein is expressed and the mode of expression: 1. lysis buffer, 2. lysis supernatant, 3. lysis precipitate (resuspended in an equal volume of lysis buffer), 4. flow-through buffer, 5. washing buffer, 6. elution buffer.

[0091] Add 10 μL of protein 5× loading buffer to the 40 μL sample, heat at 95℃ for 10 min, then take 10 μL for SDS-PAGE at 150V for approximately 40-60 min. Add rapid staining buffer and stain on a shaker at 12 rpm for 5-10 min.

[0092] Ni-NTA columns can be reused. Before use, discard the stock solution (20% ethanol), rinse with 10 mL of purified water, then add 5 mL of lysis buffer to equilibrate before loading the sample. After use (eluting proteins), rinse the column with 5 mL of Elution elution buffer, then add 20% ethanol solution and store at 4°C.

[0093] Add 3 mL of purified protein to a 10 kDa ultrafiltration tube, centrifuge at 4000×g to approximately 200 μL (20 min), add 3 mL of Tris-HCl storage buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5, 5% glycerol), centrifuge three times to concentrate, test the protein concentration, dilute to 1 mg / mL, aliquot into 100 μL and store at -80℃.

[0094] Example 3: Using BglSK_AA parent and its mutant to catalyze the production of ginsenoside Rd into ginsenoside CK

[0095] To further evaluate whether the activity of the positive mutants screened by pNPG in catalyzing the conversion of ginsenoside Rd (CAS No. 52705-93-8) to rare ginsenoside CK was truly enhanced, the purified BglSK_AA from Example 2 and mutants F28Y, S34P, Q57H, V96A, V113L, N201G, S229G, T263P, D294G, Y309F, L432F, A521E, and G560A (other positive mutants or proteins with insufficient purity, or whose expression levels were too low for this experiment) were subjected to the following catalytic experiments. A schematic diagram of the process by which the parental BglSK_AA and its mutants catalyze the conversion of ginsenoside Rd to rare ginsenoside CK is shown below. Figure 6 .

[0096] 3.1 Single-point mutation

[0097] The reaction system consisted of 1 mL of substrate ginsenoside Rd (purchased from Pusi Biotechnology) at a concentration of 5 mg / mL, and a reaction buffer of phosphate buffer (0.1 mol / L, pH 8.0). The purified parent BglSK_AA and its mutant were added separately to a final concentration of 0.1 mg / mL. After reacting at 30℃ for 2 h, the reaction was detected by HPLC under the same conditions as in Example 4. The peak area of ​​ginsenoside CK generated by the reaction was calculated, and the peak area of ​​ginsenoside CK generated by the parent BglSK_AA (SK_AA) was defined as 100%. The relative activities of each mutant were compared.

[0098] from Figure 4 Compared with the parent SK_AA, the relative activities of mutants Q57H, G560A, T263P, and Y309F were increased by 14.0%, 6.4%, 9.7%, and 21.7%, respectively. However, compared with the parent SK_AA, the relative activities of other mutants V96A, V113L, N201G, and S229G were decreased; while the mutants F28Y, S34P, L432F, D294G, and A521E showed no significant differences.

[0099] 3.2 Multi-point mutation

[0100] Subsequently, these four positive mutations were combined to form 11 combined mutants: Q57H / G560A, Q57H / T263P, Q57H / Y309F, G560A / T263P, G560A / Y309F, T263P / Y309F, Q57H / G560A / T263P, Q57H / G560A / Y309F, Q57H / T263P / Y309F, G560A / T263P / Y309F, and Q57H / G560A / T263P / Y309F. These 11 combined mutants were formed by sequentially adding other mutation sites to the original single-point mutation. The primers are shown in Table 1. The construction method is the same as in step 1.1. After construction, the activity of the mutant combinations in catalyzing the conversion of ginsenoside Rd to rare ginsenoside CK was tested.

[0101] The reaction system was the same as in step 3.1, and the reaction was carried out at 30℃ for 2 hours. The results are shown below. Figure 5 .

[0102] from Figure 5 It can be seen that, compared with the parent SK_AA, the relative activity increases of the combined mutants Q57H / G560A, Q57H / T263P, G560A / T263P and G560A / Y309F were 12.5%, 20.4%, 14.7% and 12.3%, respectively, while the activity of the mutant T263P / Y309F was almost not increased. Compared with the parental BglSK_AA, the relative activities of other combined mutants Q57H / Y309F, Q57H / G560A / T263P, Q57H / G560A / Y309F, Q57H / T263P / Y309F, G560A / T263P / Y309F, and Q57H / G560A / T263P / Y309F (BglSK_AA_4M) were increased by 39.4%, 28.5%, 47.8%, 34.5%, 30.0%, and 42.6%, respectively.

[0103] Example 4: Isolation and purification of rare ginsenoside CK

[0104] To separate and purify the rare ginsenoside CK obtained in Example 3, the following steps were performed:

[0105] After reacting for 24 hours, the reaction solution was placed at 4℃ and allowed to stand for 2 hours. Then, it was centrifuged at 5000 rpm for 15 minutes to obtain the precipitate, which was then washed twice with pure water. The precipitate was dissolved in 1 mL of anhydrous ethanol, and the supernatant was collected by centrifugation. The ethanol was evaporated at 55℃ and the solution was lyophilized overnight to obtain ginsenoside CK powder. 1 mg of the powder was dissolved in methanol and analyzed by HPLC. The detection method was as follows: a C18 column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The column temperature was 30℃; the flow rate was 1.0 mL / min; the detection wavelength was 203 nm; and the injection volume was 10 μL. The linear elution gradient was: 0–10 min: 18%–27% A; 10–30 min: 27%–33% A; 30–40 min: 33%–52% A; 40–70 min: 52%–68% A.

[0106] HPLC detection results are shown in Figure 7 By calculating the peak area, it can be found that the conversion rate of ginsenoside Rd to ginsenoside CK catalyzed by the parent SK_AA is 85%, while the conversion rate of the mutant SK_AA_4M (Q57H / G560A / T263P / Y309F) is 98%, which is much higher than the conversion efficiency of the parent SK_AA.

[0107] In summary, the β-glucosidase BglSK mutant of the present invention is more suitable for large-scale production of rare ginsenoside CK.

Claims

1. A β-glucosidase BglSK mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, mutations were performed at any of the following sites: 1) Q57H and Y309F; 2) Q57H and G560A 3) Q57H and T263P; 4) G560A and T263P; 5) G560A and Y309F; 6) Q57H, G560A, and T263P; 7) Q57H, G560A, and Y309F; 8) Q57H, T263P, and Y309F; 9) G560A, T263P, and Y309F; 10) Q57H, G560A, T263P and Y309F.

2. A polynucleotide, characterized in that, The code is for the BglSK mutant as described in claim 1.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the polynucleotide as described in claim 2 or the recombinant vector as described in claim 3.

5. The host cell as described in claim 4, characterized in that, The host cell is a fungal cell, a bacterial cell, or a plant cell.

6. The host cell as described in claim 5, characterized in that, The bacterial cells are Escherichia coli cells.

7. The use of the BglSK mutant as described in claim 1, the polynucleotide as described in claim 2, the recombinant vector as described in claim 3, or the host cell as described in any one of claims 4-6 in the preparation of ginsenoside F2 and / or ginsenoside CK.

8. A method for preparing ginsenoside F2 and / or ginsenoside CK, characterized in that, Includes the following steps: Ginsenoside F2 and / or ginsenoside CK are obtained by using the BglSK mutant as described in claim 1, the polynucleotide as described in claim 2, the recombinant vector as described in claim 3, or the host cell catalysis of ginsenoside Rd as described in any one of claims 4-6.

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