Preparation method of ginsenoside CK

Abstract

The application discloses a preparation method of ginsenoside CK, and steps are as follows: 1) integrating genes TpexyRKU-1 and GlacS into an expression type host cell to obtain an engineering bacterium 1; culturing; centrifuging to obtain a bacterium body 1; 2) adding the bacterium body 1 into a buffer solution with pH=3.5-6.5, resuspending to obtain a resuspension 1; 3) extracting ginseng root hairs, American ginseng root hairs or panax notoginseng with methanol to obtain an extraction liquid 1, drying to obtain a crude extract 1, dissolving the crude extract 1 with methanol to obtain a methanol solution of the crude extract 1; 4) mixing the resuspension 1 and the methanol solution of the crude extract 1, and performing a biological catalysis reaction to obtain the ginsenoside CK. The resuspension 1 or resuspension mixed solution of the application can catalyze and convert ginseng root hairs, American ginseng root hairs or panax notoginseng to generate the rare ginsenoside CK; the method of the application has low raw material cost and low production cost, and has high conversion rate of the rare ginsenoside CK and is green and efficient.

Description

Technical Field

[0001] This invention belongs to the field of ginsenoside preparation, and specifically relates to a method for preparing ginsenoside CK. Background Technology

[0002] Ginseng possesses high medicinal value. Ginsenosides are the main active components of ginseng, exhibiting anti-inflammatory, anti-cancer, immunomodulatory, and cardiovascular protective activities. Prototype ginsenosides directly isolated from ginseng, such as Rb, Rd, Rc, and Re, contain various glycosyl side chains, which to some extent hinder their pharmacological activity. Rare ginsenosides obtained by degrading the glycosyl side chains of prototype ginsenosides exhibit stronger pharmacological activity and higher medicinal value in terms of anti-tumor, immune-enhancing, anti-fatigue, and free radical scavenging activities. For example, rare ginsenoside Rg3 shows significantly better anti-tumor and anti-inflammatory effects than prototype ginsenosides, while rare ginsenoside CK has better hypoglycemic and anti-tumor effects. Rare ginsenoside CK oral tablets have entered the clinical research stage.

[0003] Although rare ginsenoside CK has a promising market prospect, its content in ginseng and other medicinal materials is extremely low, and the lack of sources makes it difficult to obtain in large quantities. Furthermore, using ginseng as the main body as a saponin raw material to obtain rare ginsenoside CK is too costly, making large-scale production of rare ginsenoside CK difficult. Therefore, how to obtain rare ginsenoside CK quickly, at low cost, and efficiently is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing ginsenoside CK.

[0005] The second objective of this invention is to provide another method for preparing ginsenoside CK.

[0006] The technical solution of this invention is summarized as follows:

[0007] The preparation method of ginsenoside CK includes the following steps:

[0008] 1) The β-glucosidase gene TpexyRKU-1 and the glycosidase gene GlacS were incorporated into an expression-type host cell to obtain engineered bacteria 1; engineered bacteria 1 was cultured; centrifuged to obtain bacterial cells 1;

[0009] 2) Add the bacterial cells 1 to a buffer solution with pH = 3.5-6.5 and resuspend them to obtain resuspension 1;

[0010] 3) Extract ginseng rootlets, American ginseng rootlets, or Panax notoginseng with methanol to obtain extract 1, dry to obtain crude extract 1; dissolve with methanol to obtain methanol solution 1 of crude extract;

[0011] 4) Mix the resuspended solution 1 with the methanol solution 1 of the crude extract and carry out a biocatalytic reaction to obtain ginsenoside CK.

[0012] The preferred host cells for expression are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Vibrionatriegens, or Vibrio natriegens.

[0013] The buffer solution is preferably 2-morpholinoethanesulfonic acid buffer, acetic acid-sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, or citrate-sodium citrate buffer.

[0014] The second method for preparing ginsenoside CK includes the following steps:

[0015] 1) The β-glucosidase gene TpexyRKU-1 was incorporated into the expression-type host cell to obtain engineered bacteria 2; engineered bacteria 2 was cultured; centrifuged to obtain bacterial cells 2; then the glycosidase gene GlacS was incorporated into the expression-type host cell to obtain engineered bacteria 3; engineered bacteria 3 was cultured; centrifuged to obtain bacterial cells 3;

[0016] 2) Add bacterial cells 2 to a buffer solution with pH = 3.5-6.5 and resuspend to obtain resuspension 2; add bacterial cells 3 to a buffer solution with pH = 3.5-6.5 and resuspend to obtain resuspension 3; mix resuspension 2 and resuspension 3 to obtain resuspension mixture 1;

[0017] 3) Extract ginseng rootlets, American ginseng rootlets, or Panax notoginseng with methanol to obtain extract 2;

[0018] 4) Add the resuspended mixture 1 to the extract 2 and carry out a biocatalytic reaction to obtain ginsenoside CK.

[0019] The preferred host cells for expression are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Vibrionatriegens, or Vibrio natriegens.

[0020] The buffer solution is preferably 2-morpholinoethanesulfonic acid buffer, acetic acid-sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, or citrate-sodium citrate buffer.

[0021] Advantages of this invention:

[0022] The resuspended solution 1 or resuspended mixture 1 of the present invention catalytically transforms ginseng rootlets, American ginseng rootlets, or Panax notoginseng to generate rare ginsenoside CK. The method of the present invention uses inexpensive raw materials, has low production costs, achieves a high conversion rate of rare ginsenoside CK, and is green and efficient, enabling the large-scale production of precious and rare ginsenoside CK. Attached Figure Description

[0023] Figure 1 This is a method for preparing ginsenoside CK (Example 1) to obtain the result of ginsenoside CK.

[0024] Figure 2 This is a method for preparing ginsenoside CK (Example 3) to obtain the result of ginsenoside CK. Detailed Implementation

[0025] The β-glucosidase gene TpexyRKU-1 (abbreviated as TpexyRKU-1) derived from the thermophyte *Thermotoga petrophila* RKU-1 involved in each embodiment. (GenBank: CP000702.1)

[0026] The glycosidase gene GlacS (abbreviated as GlacS) from the sulfur-bearing fungus *Saccharolobus solfataricus*. (GenBank: M34696.1)

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Equivalent substitutions or corresponding improvements made to the content of the present invention shall still fall within the scope of protection of the present invention.

[0028] The CAS number for ginsenoside CK is 39262-14-1.

[0029] Example 1

[0030] A method for preparing ginsenoside CK includes the following steps:

[0031] 1) The β-glucosidase gene TpexyRKU-1 and the glycosidase gene GlacS were incorporated into an expression-type host cell to obtain engineered bacteria 1; engineered bacteria 1 was cultured; centrifuged to obtain bacterial cells 1;

[0032] Specifically, the GlacS gene (synthesized by Genewiz Biotechnology Co., Ltd.) was used as a template, and GlaS-F (SEQ ID NO.1) and GlaS-R (SEQ ID NO.2) were used as upstream and downstream primers for PCR amplification to obtain the GlacS gene.

[0033] Using the TpexyRKU-1 gene (synthesized by Genewiz Biotechnology Co., Ltd.) as a template, PCR amplification was performed using TpexyRKU-F (SEQ ID NO.3) and TpexyRKU-R (SEQ ID NO.4) as upstream and downstream primers, and the TpexyRKU-1 gene was obtained as shown in Table 1;

[0034] PCR conditions are shown in Table 2.

[0035] Table 1. PCR primer information used for GlacS and TpexyRKU-1 genes.

[0036]

[0037] Table 2 PCR conditions

[0038]

[0039] The GlacS gene obtained by PCR and the E. coli plasmid pET-28a were digested with restriction endonucleases NheI / BamHI (purchased from NEB) at 37°C for 6 hours. After purification by agarose gel electrophoresis, the digested bands were extracted using a gel extraction kit purchased from TransGen Biotech. The correct fragment was recovered using the Quick GelExtraction Kit. Then, the digested GlacS gene fragment and the pET-28a plasmid fragment were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-GlacS.

[0040] The TpexyRKU-1 gene obtained by PCR and the recombinant plasmid pET-28a-GlacS were digested with restriction endonucleases BamHI / XhoI at 37°C for 6 h. After purification by agarose gel electrophoresis, the digested bands were extracted using a gel extraction kit purchased from TransGen Biotech. The correct fragment was recovered using the Quick Gel Extraction Kit. Then, the digested TpexyRKU-1 gene fragment and the pET-28a-GlacS recombinant plasmid fragment were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-GlacS-TpexyRKU-1.

[0041] The recombinant plasmid pET-28a-GlacS-TpexyRKU-1 was transformed into Escherichia coli competent cells BL21(DE3) (purchased from TransGen Biotech Ltd.) using the calcium chloride transformation method to obtain engineered bacteria, referred to as engineered bacteria 1-1.

[0042] A single colony of engineered bacteria 1-1 was inoculated into 50 mL of LB liquid medium containing kanamycin sulfate at a final concentration of 50 mg / L (LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) and cultured at 37°C and 220 rpm for 12 h as a seed culture. The seed culture was then transferred to 5 L of LB liquid medium containing kanamycin sulfate at a final concentration of 50 mg / L and cultured at 37°C and 220 rpm until the bacterial density reached the measured OD value. 600 The concentration ranged from 0.6 to 0.8. Then, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mM. The culture temperature was lowered to 20°C, the shaking speed was 150 rpm, and the culture was carried out for 20 h. Finally, the cells were centrifuged, and the resulting precipitate was cell 1-1 (i.e., *E. coli* cells containing both TpexyRKU-1 and GlacS glycosidases).

[0043] 2) The bacterial cells 1-1 were resuspended in a 50 mM potassium hydrogen phthalate-sodium hydroxide buffer solution with a pH of 3.5, and the bacterial cells were diluted to an OD value. 600 =20 resuspension 1-1;

[0044] 3) Ginseng rootlets were extracted by reflux with 6 times their weight of methanol for 30 minutes to obtain extract 1-1. The extract was dried to obtain crude extract 1-1. The crude extract was dissolved in methanol to obtain methanol solution 1-1 with a concentration of 100 mg / ml.

[0045] 4) Mix the resuspended solution 1-1 with the methanol solution 1-1 of the crude extract at a volume ratio of 99:1; place the reaction system in a 45℃ magnetically stirred water bath at 300 rpm for 24 h of biocatalytic reaction to obtain ginsenoside CK.

[0046] Take 0.1 mL of the reaction solution obtained in step 4) (experimental group 1-1) and 0.1 mL of the crude extract methanol solution 1-1 with a concentration of 100 mg / mL obtained in step 3) (the latter being the control group 1-1), respectively. Add 0.9 mL of methanol to each and vortex for 5 min. After centrifugation at 10000 rpm for 5 min, collect the supernatant, filter it through a 0.22 μM filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0047] The liquid chromatography conditions were as follows: Column: C18 column (2.7 μm, 4.6 × 100 mm, Waters, USA); Detection conditions: 0–5 min, 25% acetonitrile; 5–10 min, 25–30.5% acetonitrile; 10–40 min, 30.5% acetonitrile; 40–45 min, 30.5–95% acetonitrile; 45–47 min, 95% acetonitrile; 47–50 min, 95%–25% acetonitrile; 50–55 min, 25% acetonitrile. Detection wavelength: 270 nm; Flow rate: 0.5 mL / min.

[0048] After measurement, see Figure 1 The contents of several proto-ginsenosides in the control group 1-1 were as follows: Rb1 was 0.14 mg / mL, Rc was 0.12 mg / mL, Rd was 0.14 mg / mL, and the contents of other proto-ginsenosides were relatively low.

[0049] Based on the yield of CK obtained from the reaction conversion in experimental group 1-1, calculated according to the amounts of Rb1, Rc, and Rd, the CK yield was 96.5%.

[0050] Example 2

[0051] A method for preparing ginsenoside CK includes the following steps:

[0052] The construction of the recombinant plasmid pET-28a-GlacS-TpexyRKU-1 is described in Example 1;

[0053] The recombinant plasmid pET-28a-GlacS-TpexyRKU-1 and the Bacillus subtilis vector pHT01 (Fenghui Biotechnology) were digested using restriction endonucleases NheI / XhoI at 37℃ for 6 h. The digestion products were separated by agarose gel electrophoresis and recovered using a gel extraction kit. The Quick Gel Extraction Kit (TransGen Biotech Ltd.) was used to recover the GlacS-TpexyRKU-1 target band and the pHT01 band. The recovered GlacS-TpexyRKU-1 target band and pHT01 fragment were ligated using T4 DNA ligase to obtain the recombinant plasmid pHT01-GlacS-TpexyRKU-1.

[0054] The recombinant plasmid pHT01-GlacS-TpexyRKU-1 was introduced into Bacillus subtilis T7 (commercial) by electroporation to obtain Bacillus subtilis engineered strains co-expressing TpexyRKU-1 and GlacS (referred to as engineered strains 1-2).

[0055] Single clones of engineered bacteria (1-2) were inoculated into 50 mL of LB broth containing kanamycin sulfate at a final concentration of 50 mg / L and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. The seed culture was then transferred to 5 L of LB broth containing kanamycin sulfate at a final concentration of 50 mg / L and cultured at 37°C and 220 rpm until the bacterial density reached the measured OD value. 600 The concentration ranged from 0.6 to 0.8. Then, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM. The culture temperature was lowered to 20°C, the shaking speed was 150 rpm, and the culture was carried out for 20 hours. Finally, the cells were centrifuged, and the resulting precipitate was cell 1-2 (i.e., Bacillus subtilis cells containing both TpexyRKU-1 and GlacS glycosidases).

[0056] 2) Resuspend the bacterial cells 1-2 in 50mM acetate-sodium acetate buffer solution at pH 6.5 to obtain bacterial cells diluted to OD. 600 =20 resuspension 1-2;

[0057] 3) Extract the roots of American ginseng by reflux with 6 times their weight of methanol for 30 minutes to obtain extract 1-2. Dry the extract to obtain crude extract 1-2. Dissolve the crude extract in methanol to obtain methanol solution 1-2 with a concentration of 100 mg / ml.

[0058] 4) Mix the resuspended solution 1-2 with the crude extract methanol solution 1-2 at a volume ratio of 99:1; place the reaction system in an 80℃ magnetically stirred water bath and carry out the biocatalytic reaction at 300 rpm for 16 h to obtain ginsenoside CK.

[0059] Take 0.1 mL of the reaction solution obtained in step 4) (experimental group 1-2) and 0.1 mL of the crude extract methanol solution 1-2 with a concentration of 100 mg / mL obtained in step 3) (the latter being the control group 1-2), respectively. Add 0.9 mL of methanol to each and vortex for 5 min. After centrifugation at 10000 rpm for 5 min, collect the supernatant, filter it through a 0.22 μM filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0060] The liquid phase conditions are the same as in Example 1.

[0061] The contents of several proto-ginsenosides in control groups 1-2 were determined to be as follows: Rb1 0.14 mg / mL, Rc 0.16 mg / mL, Rb2 0.23 mg / mL, Rd 1.43 mg / mL, Rb3 1.07 mg / mL, and F2 0.89 mg / mL. The contents of other proto-ginsenosides were relatively low.

[0062] Based on the yield of CK obtained from the reaction transformation in experimental groups 1-2, and calculated according to the contents of several ginsenosides measured above, the yield of CK was 100.5%.

[0063] Example 3

[0064] A method for preparing ginsenoside CK includes the following steps:

[0065] The construction of the recombinant plasmid pET-28a-GlacS-TpexyRKU-1 is described in Example 1;

[0066] The recombinant plasmid pET-28a-GlacS-TpexyRKU-1 was directly transformed into sodium-dependent Vibrio (CGMCC1.5849) by electroporation to obtain engineered bacteria (referred to as engineered bacteria 1-3).

[0067] Single clones of engineered bacteria (1-3) were inoculated into 50 mL of modified LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride) and cultured at 37°C and 220 rpm for 8 h as seed culture. The seed culture was then transferred to 5 L of modified LB liquid medium and cultured at 37°C and 220 rpm until the bacterial density reached the measured OD value. 600 The concentration ranged from 0.6 to 0.8. Then, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM, and the cells were cultured for 20 h. Finally, the cells were centrifuged, and the resulting precipitate was cell 1-3.

[0068] 2) Resuspend the bacterial cells 1-3 in a 50 mM citrate-sodium citrate buffer solution at pH 4.5, and then dilute the bacterial cells to OD200. 600 =20 resuspension 1-3;

[0069] 3) Extract Panax notoginseng by reflux with 6 times its weight of methanol for 30 minutes to obtain extract 1-3. Dry the extract to obtain crude extract 1-3. Dissolve the crude extract in methanol to obtain methanol solution 1-3 with a concentration of 100 mg / ml.

[0070] 4) Mix the resuspended solution 1-3 with the methanol solution 1-3 of the crude extract at a volume ratio of 99:1; place the reaction system in an 80℃ magnetically stirred water bath and carry out the biocatalytic reaction at 300 rpm for 24 h to obtain ginsenoside CK.

[0071] Take 0.1 mL of the reaction solution obtained in step 4) (experimental group 1-3) and 0.1 mL of the crude extract methanol solution 1-3 with a concentration of 100 mg / mL obtained in step 3) (the latter being the control group 1-3), respectively. Add 0.9 mL of methanol to each and vortex for 5 min. After centrifugation at 10000 rpm for 5 min, collect the supernatant, filter it through a 0.22 μM filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0072] The liquid phase conditions are the same as in Example 1:

[0073] After measurement, see Figure 2 The contents of several prototype ginsenosides in the control groups 1-3 of the reaction system were as follows: Rb1 0.07 mg / mL, Rc 0.04 mg / mL, Rb2 0.14 mg / mL, and Rd 0.13 mg / mL. Standards for other prototype ginsenosides could not be obtained. Based on the yield of the CK obtained from the reaction conversion in experimental groups 1-3, and calculating according to the amounts of the aforementioned prototype ginsenosides, the CK yield was 146.5%.

[0074] Experiments have shown that replacing the Escherichia coli competent cells BL21(DE3) in Example 1 with Pichia pastoris GS115 (Fenghui Biotechnology) or Corynebacterium glutamicum competent cells (CGMCC1.15647) respectively, while following the same steps as in Example 1, yielded CK production rates similar to those in Example 1.

[0075] Experiments showed that replacing the pH 3.5 potassium hydrogen phthalate-sodium hydroxide buffer solution with a pH 3.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and a pH 3.5 2-morpholinoethanesulfonic acid buffer solution with a pH 3.5, respectively, with the other conditions remaining the same as in Example 1, yielded CK yields similar to those in Example 1.

[0076] Example 4

[0077] The second method for preparing ginsenoside CK includes the following steps:

[0078] For PCR amplification of the GlacS and TpexyRKU-1 genes, please refer to Example 1;

[0079] The GlacS gene, TpexyRKU-1 gene, and Pichia pastoris PPIC9 vector (Fenghui Biotechnology) obtained by PCR were digested with restriction endonucleases NheI / XhoI at 37℃ for 6 hours. After purification by agarose gel electrophoresis, the digested bands were recovered using a gel extraction kit. The PPIC9 band was recovered after digestion using the Quick Gel Extraction Kit (TransGen Biotech). Then, the PPIC9 and the GlacS and TpexyRKU-1 gene fragments obtained after PCR amplification were ligated using T4 DNA ligase, respectively, to obtain recombinant plasmids PPIC9-GlacS and PPIC9-TpexyRKU-1. The recombinant plasmids were linearized by digestion with restriction endonuclease Sal I. The linearized PPIC9-TpexyRKU-1 and PPIC9-GlacS plasmids were then introduced into Pichia pastoris GS115 (Fenghui Biotechnology) using electroporation. Screening was performed using histidine-deficient medium (1% YPD medium, purchased from Solarbio Science & Technology Co., Ltd.) to obtain engineered Pichia pastoris strains expressing TpexyRKU-1 (referred to as engineered strain 2-1) and GlacS expressing Pichia pastoris strains (referred to as engineered strain 3-1).

[0080] Single clones of engineered bacteria 2-1 and 3-1 were inoculated into 50 mL of YPD liquid medium and then cultured at 30 °C and 220 rpm for 18 h to obtain seed culture. The seed culture was then transferred to 500 mL of MGY (1.34% YNB, 1% glycerol, 4 × 10⁻⁶ ppm) medium. -5 In a 1% biotinylated liquid culture medium (known), the bacteria were incubated at 30°C and 220 rpm in a bacterial incubator until the bacterial density reached the measured OD value. 600 The range was 1.0. Afterwards, centrifuge at 10000 rpm for 5 min, discard the supernatant, and use fresh BMMY medium (100 mM potassium phosphate, 1.34% YNB, 4 × 10⁻⁶) to collect the bacterial pellet. -5 The cells were cultured continuously for 6 days with 0.1% biotin and 0.1% methanol. Finally, the cells were centrifuged, and the resulting precipitates were cell 2-1 (containing TpexyRKU-1 glycosidase) and cell 3-1 (containing GlacS glycosidase).

[0081] 2) Resuspend bacterial cells 2-1 and 3-1 separately in a 50 mM, pH 6.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain resuspension 2-1 and resuspension 3-1. Mix resuspension 2-1 and resuspension 3-1 in equal volumes to obtain resuspension mixture 1-1. Dilute the bacterial cells to OD200. 600 =30;

[0082] 3) Ginseng rootlets were extracted by reflux with 6 times their weight of methanol for 30 minutes to obtain extract 2-1;

[0083] 4) Mix the resuspended mixture 1-1 and the extract 2-1 at a volume ratio of 99:1. Place the reaction system in a 65℃ magnetically stirred water bath and carry out the biocatalytic reaction at 300 rpm for 24 hours to obtain ginsenoside CK.

[0084] Take 0.1 mL of the reaction solution from step 4) (experimental group 2-1) and 0.1 mL of the extract obtained from step 3) (the latter being the control group 2-1), respectively. Add 0.9 mL of methanol to each and vortex for 5 min. After centrifugation at 10000 rpm for 5 min, collect the supernatant, filter it through a 0.22 μM filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0085] The liquid phase conditions are the same as in Example 1:

[0086] The yield of CK obtained from the reaction conversion was calculated to be 99.5% according to the method in Example 1.

[0087] Example 5

[0088] The second method for preparing ginsenoside CK includes the following steps:

[0089] For PCR amplification of the GlacS and TpexyRKU-1 genes, please refer to Example 1;

[0090] The TpexyRKU-1 gene and GlacS gene obtained by PCR were integrated into the pXMJ19 vector suitable for Corynebacterium glutamicum using restriction endonucleases NheI / XhoI, respectively, to obtain two vectors: pXMJ19-TpexyRKU-1 and pXMJ19-GlacS.

[0091] The two vectors were transformed into *E. coli* JM109 competent cells (purchased from TransGen Biotech) using electroporation. The transformed competent cells were plated on solid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar) containing chloramphenicol at a final concentration of 30 mg / L for selection, and cultured overnight at 37°C. Single colonies with good growth were picked and transferred to 5 mL of LB liquid medium (containing chloramphenicol, final concentration 30 mg / L), cultured at 37°C and 220 rpm for 12 h, and then centrifuged at 10,000 rpm for 2 min. Subsequently, plasmid extraction was performed using a plasmid extraction kit from TransGen Biotech. The Plasmid MiniPrep Kit was used to extract the plasmids, and the extracted products were the recombinant plasmids pXMJ19-TpexyRKU-1 and pXMJ19-GlacS.

[0092] The recombinant plasmids pXMJ19-TpexyRKU-1 and pXMJ19-GlacS were introduced into competent Corynebacterium glutamicum cells (CGMCC1.15647) via electroporation. Target strains were then screened using resistance pressure (containing chloramphenicol, final concentration 30 mg / L) to obtain engineered Corynebacterium glutamicum expressing TpexyRKU-1 (referred to as engineered strain 2-2) and engineered Corynebacterium glutamicum expressing GlacS (referred to as engineered strain 3-2).

[0093] Engineered bacteria 2-2 and 3-2 were individually inoculated into 50 mL of LBB liquid medium (containing 50 mg / L kanamycin sulfate, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L brain heart extract), and then cultured in a bacterial incubator at 30°C and 200 rpm for 16 h to obtain seed culture. The seed culture was then transferred to 5 L of LBB liquid medium and cultured in a bacterial incubator at 30°C and 200 rpm until the bacterial density reached the measured OD value. 600 The range was 0.6. Then, IPTG, the inducer, was added to a final concentration of 1 mM, and the culture was continued for 24 h. Finally, centrifugation was performed, yielding cells 2-2 (containing TpexyRKU-1 glycosidase, Corynebacterium glutamicum) and cells 3-2 (containing GlacS glycosidase, Corynebacterium glutamicum).

[0094] 2) Resuspend bacterial cells 2-2 and 3-2 separately in 50 mM 2-morpholinoethanesulfonic acid buffer (pH 3.5) (or alternatively, 50 mM 2-morpholinoethanesulfonic acid buffer (pH 4.5), 50 mM acetate-sodium acetate buffer (pH 4.5), potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.5), or citrate-sodium citrate buffer), to obtain resuspension 2-2 and resuspension 3-2. Mix resuspension 2-2 and resuspension 3-2 in equal volumes to obtain resuspension mixture 1-2. Dilute the bacterial cells to OD. 600 =30;

[0095] 3) Extract the roots of American ginseng (or Panax notoginseng) by reflux with 6 times the mass of methanol for 30 minutes to obtain extract 2-2;

[0096] 4) Mix the resuspended mixture 1-2 with the extract 2-2 at a volume ratio of 99:1. Place the reaction system in a 65℃ magnetically stirred water bath and run it at 300 rpm for 24 hours to obtain ginsenoside CK.

[0097] Take 0.1 mL of the reaction solution (experimental group 2-2) from step 4) and 0.1 mL of the extract 2-2 obtained from step 3) respectively, add 0.9 mL of methanol to each, vortex for 5 min, centrifuge at 10000 rpm for 5 min, collect the supernatant, filter it through a 0.22 μM filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0098] The liquid phase conditions are the same as in Example 1:

[0099] The yield of CK obtained from the reaction conversion was calculated to be 98.5% according to the method in Example 1.

Claims

1. A method for preparing ginsenoside CK, characterized in that... Includes the following steps: 1) The β-glucosidase gene TpexyRKU-1 and the glycosidase gene GlacS were incorporated into an expression-type host cell to obtain engineered bacteria 1; engineered bacteria 1 was cultured; centrifuged to obtain bacterial cells 1; 2) Add the bacterial cells 1 to a buffer solution with pH = 3.5-6.5 and resuspend them to obtain resuspension 1; 3) Extract ginseng rootlets, American ginseng rootlets, or Panax notoginseng with methanol to obtain extract 1, dry to obtain crude extract 1; dissolve with methanol to obtain methanol solution 1 of crude extract; 4) Mix the resuspended solution 1 with the methanol solution 1 of the crude extract and carry out a biocatalytic reaction to obtain ginsenoside CK.

2. The preparation method according to claim 1, characterized in that, The expressed host cells are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Vibrio natriegens, or Vibrio natriegens.

3. The preparation method according to claim 1, characterized in that... The buffer solution is 2-morpholinoethanesulfonic acid buffer, acetic acid-sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, or citrate-sodium citrate buffer.

4. A method for preparing ginsenoside CK, characterized in that... Includes the following steps: 1) The β-glucosidase gene TpexyRKU-1 was incorporated into the expression-type host cell to obtain engineered bacteria 2; engineered bacteria 2 was cultured; centrifuged to obtain bacterial cells 2; then the glycosidase gene GlacS was incorporated into the expression-type host cell to obtain engineered bacteria 3; engineered bacteria 3 was cultured; centrifuged to obtain bacterial cells 3; 2) Add a buffer solution with pH = 3.5-6.5 to bacterial cells 2 and resuspend them to obtain resuspension 2; add a buffer solution with pH = 3.5-6.5 to bacterial cells 3 and resuspend them to obtain resuspension 3; mix resuspension 2 and resuspension 3 to obtain resuspension mixture 1; 3) Extract ginseng rootlets, American ginseng rootlets, or Panax notoginseng with methanol to obtain extract 2; 4) Add the resuspended mixture 1 to the extract 2 and carry out a biocatalytic reaction to obtain ginsenoside CK.

5. The preparation method according to claim 5, characterized in that, The expressed host cells are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Vibrio natriegens, or Vibrio natriegens.

6. The preparation method according to claim 5, characterized in that... The buffer solution is 2-morpholinoethanesulfonic acid buffer, acetic acid-sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, or citrate-sodium citrate buffer.

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