A kind of β-glucosidase and its application in preparing ginsenosides

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a β-glucosidase and its application in the preparation of ginsenosides. The β-glucosidase of the present invention or a recombinant microorganism expressing the enzyme can generate rare ginsenosides Rg3 and / or Rh1 through whole-cell catalysis, and then the application of the β-glucosidase of the present invention in the preparation of ginsenosides is proposed, and the amino acid sequence of the β-glucosidase is numbered RLF05189.1 on NCBI. The present invention provides a new method for synthesizing ginsenoside Rg3 and / or Rh1, preferably using a whole-cell catalytic method for production, and also has the advantages of short cycle, strong reaction specificity, high yield, low pollution, and simple process.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a beta-glucosidase and an application thereof in the preparation of ginsenosides. Background Art

[0002] Rare ginsenoside Rg3 is a PPD-type triterpenoid saponin with extremely low content in ginseng. It has anti-tumor, anti-viral and immune-enhancing functions (Chinese Journal of Drugs and Clinics, 2011, 11(2): 180-182; Chinese Journal of Traditional Chinese Medicine, 2022, 40(2): 156-159). Rg3 is the main component of some anti-cancer adjuvant drugs. Rg3 has potential application prospects in the fields of medicine and food additives, but the price of pure Rg3 is very expensive. Rare ginsenoside Rh1 is a PPT-type triterpenoid saponin. It exists in trace amounts in plants such as red ginseng, ginseng, Panax notoginseng, and American ginseng. It has significant immunomodulatory activity and can inhibit inflammatory responses (International Journal of Geriatrics, 2022, 43(3): 368-371).

[0003] At present, the methods for preparing rare ginsenosides mainly include traditional solvent extraction (decoction, immersion, reflux, continuous extraction, percolation, etc.), acid hydrolysis, microbial transformation, and enzyme catalysis. For example, the methods for preparing rare ginsenoside Rg3 include: extraction from ginseng root (Chinese patent application number 202110228324.0), hydrolysis (Chinese patent authorization announcement number CN 110229208 B; Chinese patent authorization announcement number CN 102993258 B), microbial transformation (Chinese patent application number 202310626928.X), and enzyme catalysis (Chinese patent application number 201610219176.5; Chinese patent application number 201911117100.1), but in actual applications, there are problems such as difficult control of hydrolysis products, low yield, long microbial transformation cycle, and the conversion rate needs to be improved.

[0004] Therefore, finding new transformation methods that are environmentally friendly, less polluting, highly specific, high in yield, low in cost, and simple in process remains the key to the industrial production and application of rare ginsenosides. Summary of the invention

[0005] One of the purposes of the present invention is to provide a new method for catalytically producing rare ginsenosides, and in particular to provide a method for catalytically producing rare ginsenosides by whole-cell recombinant bacteria.

[0006] In order to achieve this object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides an application of β-glucosidase in the preparation of ginsenosides, wherein the amino acid sequence of the β-glucosidase is numbered RLF05189.1 on NCBI; and the ginsenosides are rare ginsenosides Rg3 and / or Rh1.

[0008] The present invention has been found through research that the beta-glucosidase of thermophilic microorganisms can specifically catalyze substrates to produce rare ginsenosides Rg3 and / or Rh1, and thus the present invention is proposed.

[0009] In a second aspect, the present invention provides a nucleic acid molecule or a biological material containing the nucleic acid molecule, wherein the biological material includes recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, a recombinant microorganism or a transgenic cell line, and the nucleic acid molecule encodes β-glucosidase, and the β-glucosidase is as described above.

[0010] The nucleotide sequence of the nucleic acid molecule in the nucleic acid molecule of the present invention or the biological material containing the nucleic acid molecule is shown as SEQ ID No.1.

[0011] In a third aspect, the present invention provides the use of the above nucleic acid molecules or biological materials containing the above nucleic acid molecules in the preparation of ginsenosides; the ginsenosides are rare ginsenosides Rg3 and / or Rh1.

[0012] After the nucleic acid molecule of the present invention is introduced into the genome of a host microorganism, the obtained recombinant microorganism can express the above-mentioned β-glucosidase, thereby achieving the production of rare ginsenosides by the whole-cell catalytic method.

[0013] In a fourth aspect, the present invention provides a method for producing rare ginsenoside Rg3 and / or Rh1, which uses β-glucosidase or a recombinant microorganism to generate rare ginsenoside Rg3 and / or Rh1 through whole-cell catalysis; the β-glucosidase is as described above, and the recombinant microorganism includes a nucleic acid molecule, and the nucleic acid molecule is as described above.

[0014] In the method of the present invention, when producing rare ginsenoside Rg3, the reaction substrate is ginsenoside Rb1 and / or Rd; when producing rare ginsenoside Rh1, the reaction substrate is Rg1; preferably, when preparing rare ginsenoside Rg3 and / or Rh1, the reaction substrate is Panax notoginseng total saponin extract, so as to save costs and produce the above two rare ginsenosides at the same time.

[0015] In the method of the present invention, when the β-glucosidase is used to generate rare ginsenoside Rg3 and / or Rh1, the reaction temperature is 70-85°C and the pH is 4-7 (preferably, the reaction temperature is 78-82°C and the pH is 5.8-6.2, and more preferably the reaction temperature is 80°C and the pH is 6); the reaction solvent is a PBS solution (50mM) containing 0.8-1.2% Tween 80 or a PBS solution (50mM) containing 8-15% (preferably, 10%) ethanol, or the reaction solvent includes a low eutectic solvent DES, and the volume fraction of the low eutectic solvent in the reaction solvent is 15-25%; the mass ratio of the reaction substrate to the β-glucosidase is 10:1-30:1 (preferably 15:1).

[0016] In the method of the present invention, when the recombinant microorganism is used to generate rare ginsenoside Rg3 and / or Rh1 through whole-cell catalysis, the reaction temperature is 70-85°C and the pH is 4-7 (preferably, the reaction temperature is 78-82°C and the pH is 5.8-6.2, and more preferably the reaction temperature is 80°C and the pH is 6); the reaction solvent is a PBS solution (50 mM) containing 0.8-1.2% Tween 80 or a PBS solution (50 mM) containing 8-15% (preferably, 10%) ethanol, or the reaction solvent includes a low eutectic solvent, and the volume fraction of the low eutectic solvent in the reaction solvent is 15-25%;

[0017] The mass ratio of the reaction substrate to the recombinant microorganism is 20:1-50:1 (preferably 30:1); the recombinant microorganism is induced to express the β-glucosidase by IPTG, preferably, the conditions for the induced expression are: the final concentration of IPTG is 0.1-0.5mM, and the culture is shaken at 37±1°C and 180-220rpm for 4-6 hours. More preferably, the conditions for the induced expression are: the final concentration of IPTG is 0.18-0.22mM, and the culture is shaken at 37±1°C and 180-220rpm for 4.5-5.5 hours.

[0018] The β-glucosidase of the present invention is heat-resistant and can work under a wide range of pH conditions, which is conducive to production and application. Moreover, when the whole-cell catalytic production method is adopted, the reaction is carried out at high temperature, which is conducive to high-temperature cell lysis, so that the enzyme is released from the cell and reacts more fully with the substrate.

[0019] The present invention has also been found through research that when a specific low eutectic solvent is included in the reaction solvent, it is beneficial to maintain the stability of the enzyme of the present invention at high temperatures and improve the conversion rate of the substrate and the solubility of the product.

[0020] In the method of the present invention, the low eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor is choline chloride and / or L-proline, and the hydrogen bond donor is one or more of lactic acid, 1,4-butanediol, D-sorbitol, sucrose, anhydrous ethanol, and glycerol;

[0021] Preferably, the hydrogen bond acceptor is L-proline, the hydrogen bond donor is lactic acid, and the molar ratio of L-proline to lactic acid is (0.8-1.2):(0.8-2.2), more preferably (0.8-1.2):(0.8-1.2); or

[0022] The hydrogen bond acceptor is choline chloride, the hydrogen bond donor is anhydrous ethanol and 1,4-butanediol, and the molar ratio of choline chloride, anhydrous ethanol and 1,4-butanediol is (0.8-1.2):(0.8-1.2):(0.8-1.2); or

[0023] The hydrogen bond acceptor is choline chloride, the hydrogen bond donors are D-sorbitol and 1,4-butanediol, and the molar ratio of choline chloride, D-sorbitol and 1,4-butanediol is (1.8-2.2):(0.8-1.2):(3.8-4.2); or

[0024] The hydrogen bond acceptor is L-proline, the hydrogen bond donors are glycerol and sucrose, and the molar ratio of L-proline, glycerol and sucrose is (3.8-4.2): (9.8-10.2): (0.8-1.2).

[0025] More preferably, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is anhydrous ethanol and 1,4-butanediol, and the molar ratio of choline chloride, anhydrous ethanol and 1,4-butanediol is (0.8-1.2):(0.8-1.2):(0.8-1.2).

[0026] In the method of the present invention, the production of rare ginsenoside Rg3 and / or Rh1 by whole-cell catalysis of the recombinant microorganism comprises:

[0027] (1) Preparing a recombinant microorganism: transforming a host microorganism competent cell with a recombinant vector containing a nucleic acid molecule to obtain a recombinant microorganism; the nucleic acid molecule encodes β-glucosidase, and the β-glucosidase is as described above; preferably, the host microorganism is Escherichia coli;

[0028] (2) after inducing the expression of the β-glucosidase in the recombinant microorganism with IPTG, collecting and resuspending to obtain a whole cell solution of the recombinant microorganism;

[0029] (3) Mixing the whole cell solution of the recombinant microorganism, a substrate and a reaction solvent, and reacting them to produce rare ginsenosides Rg3 and / or Rh1.

[0030] The present invention preferably utilizes whole cells of recombinant bacteria containing the beta-glucosidase gene of the thermophilic microorganism to catalyze the production of rare ginsenosides Rg3 and / or Rh1.

[0031] As a specific embodiment, the method of producing rare ginsenoside Rg3 and / or Rh1 of the present invention comprises:

[0032] The beta-glucosidase gene from the thermophilic microorganism Thermoprotei archaeonr (GenBank Accession No.: RLF05189.1) was fully synthesized and named Thabgl. It was ligated to the pET28a vector by high-fidelity PCR and transformed into Escherichia coli BL21 (DE3) for expression.

[0033] The recombinant bacteria containing Thabgl were cultured at 37°C and 200 rpm with shaking until the OD value reached 0.6, IPTG (final concentration was 0.2 mM) was added, and the culture was continued for 5 hours. The cells were collected by centrifugation at 5000 rpm for 5 minutes, and 50 mM PBS solution at pH 6 was added to resuspend the cells and wash them. The washed cells were collected by centrifugation at 5000 rpm for 5 minutes, and 50 mM PBS at pH 6 was added to prepare a 100 g / L whole cell solution.

[0034] By adding 0.5% whole cell solution into a 20% low eutectic solvent solution containing 15 g / L notoginseng saponins, keeping the temperature at 80°C and reacting for 2 h, the Rb1 substrate in the notoginseng extract can be basically converted into Rg3, and the Rg1 substrate can be basically converted into Rh1.

[0035] The beneficial effects of the present invention are at least:

[0036] The present invention provides a new method for synthesizing rare ginsenoside Rg3 and / or Rh1, preferably using a whole-cell catalysis method to produce the product, which, compared with the classical enzyme catalysis reaction, does not require the separation and purification of the enzyme, and is more cost-effective; compared with the hydrolysis method and the biotransformation method, the whole-cell catalysis at high temperature has higher specificity for the substrate, high yield, less by-products, and a short conversion cycle. More preferably, a low eutectic solvent is used as the reaction system, which can improve the yield, is environmentally friendly, and has less pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis diagram of the amplified Thabgl gene.

[0038] Figure 2 After the Thabgl gene and PET28a ligation product was transformed into BL21 (DE3), six colonies were picked for PCR identification. Lanes 1-6 are the electrophoresis results of the six colonies.

[0039] Figure 3 The results of the induction expression and heat resistance experiments of the Thabgl gene. Lane 1 shows the electrophoresis of total protein of BL21 (DE3) cells; Lane 2 shows the electrophoresis of total protein of BL21-Thabgl recombinant bacterial cells without IPTG; Lane 3 shows the electrophoresis of total protein of BL21-Thabgl recombinant bacterial cells after IPTG induction; Lane 4 shows the electrophoresis of supernatant protein of BL21-Thabgl recombinant bacterial cells after IPTG induction; Lane 5 shows the electrophoresis of supernatant protein of BL21-Thabgl recombinant bacterial cells after IPTG induction and 85°C heat treatment for 10 minutes.

[0040] Figure 4 The results show the effect of pH on enzyme activity in different reaction solutions.

[0041] Figure 5 The results show the effect of temperature on enzyme activity in different reaction solutions.

[0042] Figure 6 The results of THABGL protease solution conversion of ginsenosides Rb1 and Rd by high performance liquid chromatography. In the figure, A is the test result of pure Rd (before reaction); B is the conversion result of pure Rd; C is the test result of pure Rb1 (before reaction); D is the conversion result of pure Rb1; E is the test result of Rg3 standard. The horizontal axis is time (min).

[0043] Figure 7 These are the HPLC detection results of THABGL protease solution converting ginsenoside Rg1. In the figure, A is the detection result of pure Rg1 (before reaction); B is the conversion result of pure Rg1; C is the detection result of Rh1 standard; D is the detection result of 15 g / L Panax notoginseng extract (20% DES2 solution); E is the result of the conversion product of 15 g / L Panax notoginseng extract.

[0044] Figure 8 These are the results of liquid chromatography detection of the conversion of Panax notoginseng extract into rare ginsenosides Rg3 and Rh1 catalyzed by whole cell fluid containing the Thabgl gene. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0047] Example 1

[0048] Experimental reagents and materials:

[0049] Escherichia coli BL21 (DE3) and PET28a vector were purchased from Shanghai Bioengineering Co., Ltd., and restriction endonucleases, ligases, and high-fidelity enzymes were purchased from Takara. HPLC-grade acetonitrile was purchased from Anhui Tiandi High Purity Reagent Co., Ltd., various chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and deionized water was Wahaha purified water.

[0050] The Panax notoginseng extract (containing 80% total saponins) used in the present invention was purchased from Shaanxi Jintai Zhenpin Bioengineering Co., Ltd., and the ginsenosides Rb1, Rd, Rg1 and rare ginsenosides Rg3 and Rh1 were purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0051] The molecular biological methods and reagent formulas used in the present invention are from the Molecular Cloning Experiment Guide (Fourth Edition) .

[0052] 1. Whole gene synthesis of the β-glucosidase gene from the thermophilic microorganism Thermoprotei archaeonr

[0053] The β-glucosidase gene based on Thermoprotei archaeonr was named Thabgl, and the encoded protein was named THABGL protein (GenBank Accession No.: RLF05189.1). The whole gene synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd., and connected to the BamHI site of the vector PUC57, named PUC57-Thabgl. The synthesized whole gene sequence is shown in SEQ ID No.1.

[0054] 2. Amplify Thabgl gene and connect it to PET28a vector

[0055] 1. Amplification of Thabgl gene

[0056] Primers P1 and P2 were designed. A BamHI site (underlined) was introduced into P1, and a HindIII site (underlined) was introduced into P2. The primers were synthesized by Shanghai Bio-Gene Co., Ltd.

[0057] Upstream primer P1 (SEQ ID No. 2): 5′-GT GGATCC ATG AAG CCC GCT ATA GTT-3′;

[0058] Downstream primer P2 (SEQ ID No. 3): 5′-CG AAGCTT TTA CGG GGG GTA TCT AAT CT-3′;

[0059] The pUC57-Thabgl plasmid was used as a template for amplification, and the reaction system was shown in Table 1 below.

[0060] Table 1

[0061] Components Dosage 10×PCR buffer 2.5μl PCR high fidelity enzyme 0.2μl P1 Primer 0.4μl P2 Primer 0.4μl PUC57-Thabgl 1μl <![CDATA[ddH 2 The]]> 20.5μl Total 25μl

[0062] Amplification reaction conditions: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 57℃ annealing for 15s, 72℃ extension for 1min 45s; 35 cycles; final extension at 72℃ for 10min. The electrophoresis results of the PCR reaction products are as follows: Figure 1 shown.

[0063] 2. The obtained PCR product was connected to the PET28a vector (the double restriction sites of the PET28a vector are the BamHⅠ site and the XohⅠ site) after double restriction enzyme digestion and linearization. The connection reaction system is shown in Table 2.

[0064] Table 2

[0065] Components volume PET28a vector 0.5μl SolutionⅠ 5μl High-fidelity PCR products 4.5μl <![CDATA[ddH 2 The]]> Make up to the final volume of 10 μl

[0066] The mixed reaction system was reacted at 16°C for 30 min and placed at 4°C overnight to improve its connection efficiency.

[0067] 3. Transformation of BL21(DE3) competent cells with the ligation product of Thabgl gene and PET28a

[0068] Take out competent cells BL21 (DE3) from -70℃ refrigerator and melt on ice; take 5μL of ligation reaction solution and add it to competent cells BL21 (DE3), mix gently, put it in ice bath for 30min, heat shock it at 42℃ for 60s, and then quickly put it in ice bath for 2min; add 700μL of liquid LB culture medium, shake and culture at 37℃ and 180rpm for 1h; centrifuge at 4000rpm for 5min, remove the supernatant and keep 150μL, gently resuspend the bacteria, and spread the bacterial solution on LB plate containing kanamycin (50μg / ml); culture the plate at 37℃ for 12-16h, randomly pick 6 colonies and inoculate them into 3mL of 5mL LB culture medium containing kanamycin (50μg / ml), shake and culture it at 37℃ overnight at 200rpm, and perform PCR identification with the above P1 and P2 primers. The identification results are as follows: Figure 2The positive recombinants were sent to Shanghai Biotech Co., Ltd. for sequencing, and the correct recombinant strain without mutation was named BL21-Thabgl.

[0069] 4. Inducible expression and thermal stability experiments of Thabgl gene

[0070] The recombinant bacteria BL21-Thabgl were inoculated into 3 mL of LB medium containing kanamycin (50 μg / mL), and cultured at 200 rpm at 37°C overnight; then the culture solution was transferred to 50 mL of fresh LB medium containing kanamycin (50 μg / mL) at a ratio of 1:10, and cultured at 200 rpm at 37°C until the OD value was 0.6; IPTG (final IPTG concentration was 0.2 mM) was added, and the cells were cultured at 200 rpm at 37°C for 5 hours to induce expression. The cells were collected by centrifugation at 4000 rpm for 5 minutes, resuspended and washed with PBS, and then the bacteria were resuspended with PBS for SDS-PAGE protein electrophoresis. The electrophoresis results of each protein are shown in the figure. Figure 3 As shown. The total protein of BL21 (DE3) cells was detected by electrophoresis (lane 1); the total protein of BL21-Thabgl recombinant bacterial cells cultured without IPTG induction was detected by electrophoresis (lane 2). The electrophoresis diagram of the total protein of BL21-Thabgl recombinant bacterial cells after IPTG induction is shown in lane 3. The cells collected after IPTG induction of the above recombinant bacteria BL21-Thabgl were prepared into a 100 g / L cell solution, and the cell solution was ultrasonically disrupted (ultrasonic power 150 W, ultrasonic 1 second, interval 2 seconds, a total of 15 minutes), and the cell solution after disruption was centrifuged at 8000 rpm for 10 minutes to obtain supernatant protein. The electrophoresis diagram of the supernatant protein is shown in lane 4, and obvious soluble THABGL protein is produced at around 80 kDa.

[0071] The cell lysate was further kept at 85°C for 10 min, centrifuged at 8000 rpm for 10 min, and the supernatant was taken for protein electrophoresis. Figure 3 Lane 5 shows an obvious protein band at around 80 kDa, which preliminarily indicates that THABGL protein is a thermostable protein and can be purified and separated from cells by high temperature treatment (85°C for 10 min).

[0072] 5. Effect of pH value of reaction system on the activity of THABGL protease

[0073] The p-NPG method was used to detect the activity of THABGL protease:

[0074] (1) Drawing of the standard curve of p-nitrophenol

[0075] Accurately weigh 0.0139 g of p-nitrophenol standard, dilute to 500 mL with pure water to prepare a 0.2 μmol / mL p-nitrophenol solution, and add 0.1 mol / L Na 2 CO 3 The solution was fixed to volume to prepare p-nitrophenol solutions with concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 μmol / mL, respectively. Pure water was used as a blank, and the absorbance at λmax=395nm was measured. The standard curve of p-nitrophenol was drawn as y=0.00527x-0.00013, R2=0.999.

[0076] (2) The enzyme activity of BGL is defined as: the amount of enzyme that hydrolyzes to produce 1 μmol of p-nitrophenol at 70°C for 1 min is 1 enzyme activity unit (U).

[0077] Method for determining the enzyme activity of BGL: The THABGL protein solution purified and separated by heat treatment (maintained at 85°C for 10 min) in step 4 was diluted 10 times (v / v) with a 50 mM PBS solution, 0.1 mL of the diluted enzyme solution was mixed with 0.9 mL of a 30 mmol / L p-nitrophenyl-β-D-glucoside (p-NPG) solution, and then the mixture was heated at 70°C for 10 min under different pH conditions (the reaction solutions were 50 mM PBS solution containing 1% Tween, 50 mM PBS solution containing 10% ethanol, and a solution containing 20% ​​low eutectic solvent (choline chloride: D-sorbitol: 1,4-butanediol in a molar ratio of 2:1:4)). 1 mol / L Na 2 CO 3 2mL of solution was added to terminate the reaction, and then distilled water was added to make up to 5mL, and the mixture was shaken. Using pure water as blank, the absorbance of the sample was measured at λmax=395nm, and the standard curve of p-nitrophenol was substituted to obtain the amount of p-nitrophenol generated by the reaction, and then the enzyme activity of BGL was calculated. The enzyme activity of the free enzyme was calculated according to formula (1-1). The result is as follows: Figure 4 As shown, it shows that in different reaction solutions, when the pH is 6, the enzyme activity reaches the highest.

[0078]

[0079] Where:

[0080] A: λmax = absorbance of the sample at 395nm;

[0081] N: dilution multiple;

[0082] 0.0527: slope of the p-nitrophenol standard curve;

[0083] 10: reaction time (min);

[0084] 0.1: Volume of enzyme solution added (mL).

[0085] VI. Effect of reaction temperature on THABGL protease activity

[0086] Referring to the method for detecting enzyme activity in step 5, the activity of THABGL protease was detected under different high temperature conditions in a reaction solution of pH 6 (the reaction solutions were 50 mM PBS solution containing 1% Tween, 50 mM PBS solution containing 10% ethanol, and a solution containing 20% ​​deep eutectic solvent (choline chloride: D-sorbitol: 1,4-butanediol in a molar ratio of 2:1:4)). The results are as follows: Figure 5 As shown, in different reaction solutions, the enzyme activity reached the highest when the temperature was 80°C.

[0087] VII. Catalytic reaction of THABGL protease on ginsenosides Rb1, Rd and Rg1: According to the method in step 4, the recombinant bacteria BL21-Thabgl were induced with IPTG and then the bacteria were collected and prepared into a 100 g / L cell solution. The cell solution was ultrasonically disrupted, and the cell disruption solution was kept at 85°C for 10 min. The supernatant solution was taken at 8000 rpm for 10 min to obtain a thermostable THABGL protease solution. Bovine serum albumin (BSA) was gradiently diluted to prepare a standard curve. The protein concentration was determined by Bradford method, and a thermostable THABGL protease solution with a concentration of 0.25 mg / ml was prepared.

[0088] Reaction solutions containing 1 g / L of ginsenosides Rb1, Rd, and Rg1 were prepared respectively (the solvent of the reaction solution was a solution containing 20% ​​DES2 at pH 6, and the composition of DES2 is shown in Table 3).

[0089] The solvent preparation method of the reaction solution is as follows: a series of low eutectic solvents DESs were prepared by heating method, and the components and molar ratios of DESs are shown in Table 3. As shown in Table 3, hydrogen bond acceptors (choline chloride, L-proline) and hydrogen bond donors (lactic acid, 1,4-butanediol, D-sorbitol, sucrose, anhydrous ethanol, glycerol) were accurately weighed in a certain molar ratio, mixed in a round-bottom flask, and heated in a water bath at 80°C constant temperature water bath oscillator at 150 rpm until a clear and transparent liquid was obtained, and the prepared different low eutectic solvents DESs were dissolved in 50 mM PBS buffer at pH 6 at a volume fraction of 20% to prepare a solution containing 20% ​​DESs.

[0090] Table 3 Components and molar ratios of different DES

[0091]

[0092]

[0093] THABGL protease solution (THABGL protease concentration 0.25 mg / ml) was added to the reaction solution (containing DES2) containing 1 g / L of ginsenosides Rb1, Rd, and Rg1 prepared above at 1% (V / V), reacted at 80°C for 2 hours, and then tested by HPLC. The test results are as follows: Figure 6 and Figure 7 As shown. Rb1 and Rd can be almost completely converted into Rg3 ( Figure 6 ), the molar conversion rates were 98.3% and 97.8% respectively; Rg1 was almost completely converted into Rh1 ( Figure 7 A, B, C), the molar conversion rate was 98.1%.

[0094] In this example, 1% (V / V) THABGL protease solution was further added to a 20% DES2 solution containing 15 g / L of Panax notoginseng extract (containing 80% total saponins, of which the content of Rb1 was 43% and the content of Rg1 was 21%). After reacting at 80°C for 2 hours, HPLC detection was performed. The results showed that Rg1 in the Panax notoginseng extract could be converted into Rh1 with a molar conversion rate of 97.6% and an Rh1 content of 2.45 g / L (eg Figure 7 D and E). Rb1 in Panax notoginseng extract can be converted into Rg3, with a molar conversion rate of 97.2%.

[0095] 8. Whole-cell catalytic reactions

[0096] 1. Inoculate 1% of BL21-Thabgl recombinant bacteria into 500 ml of fresh LB medium (final concentration of kanamycin 50 μg / ml), culture at 37 degrees with shaking at 200 rpm until the OD value reaches 0.6, add IPTG to a final concentration of 0.2 mM, culture at 37 degrees with shaking at 200 rpm for 5 hours, and collect the cells in the culture medium by centrifugation at 5000 rpm for 5 minutes.

[0097] 2. Resuspend the cells in pH 6 PBS solution (50 mM) for washing, then centrifuge at 5000 rpm for 5 minutes to collect the washed cells and weigh them.

[0098] 3. Resuspend the washed cells with a pH 6 PBS solution (50 mM) to prepare a 100 g / L bacterial suspension to obtain a BL21-Thabgl recombinant bacterial whole cell solution.

[0099] 4. According to the above enzyme activity detection, the present invention found that the THABGL protease activity was better in the presence of a low eutectic solvent, so the effect of different low eutectic solvents on the reaction was further studied in the catalytic reaction. Specifically, the whole cell solution (100 g / L) was added to each 20% low eutectic solvent DESs prepared in step 7 at 0.5% (v / v) (see Table 3), and the Panax notoginseng extract was used as a substrate to prepare the rare ginsenosides Rg3 and Rh1 by whole cell catalysis in a low eutectic solvent.

[0100] In detail, the 20% low eutectic solvent DESs solution prepared in step 7 was prepared into a reaction solution containing 15 g / L Panax notoginseng extract (containing 80% total saponins, of which the content of Rb1 was 43% and the content of Rg1 was 21%), and 0.5% (v / v) of the whole cell solution containing the Thabgl gene expression product was added for catalytic reaction. The reaction temperature was 80°C and the reaction time was 2 h. The contents of Rg3 and Rh1 in different reaction solutions were detected respectively. The results are shown in Table 4. The results of HPLC detection after the whole cell catalytic reaction containing 15 g / L Panax notoginseng extract and 20% DES solution were as follows: Figure 8 The blank control in the figure is a high performance liquid chromatography detection diagram of a 20% DES2 solution containing 15 g / L of Panax notoginseng extract (without adding whole cell solution for catalytic reaction).

[0101] Among them, the Rg3 content reached 4.47 g / L and the molar conversion rate was 97.9% when whole-cell catalyzed by 20% DES2 (choline chloride: anhydrous ethanol: 1,4-butanediol molar ratio was 1:1:1) containing 15 g / L Panax notoginseng extract (containing 80% total saponins), and the Rh1 content reached 2.45 g / L and the molar conversion rate was 97.7%.

[0102] Table 4 Rg3 content and molar conversion rate generated in different types of DES

[0103]

[0104] The detection and standard curve preparation of ginsenosides (rare ginsenosides) in the present invention are as follows:

[0105] The standard curve was prepared as follows: Ginsenosides (rare ginsenosides) were dissolved in 50% methanol solution to prepare a 0.5 g / ml solution, and each sample was diluted in proportion and subjected to HPLC detection three times to prepare a standard curve. The detection conditions are as follows.

[0106] The reaction solution was diluted with methanol to a certain multiple, and then filtered through a 0.22 μm microporous filter membrane for HPLC analysis. The product content was calculated based on the standard curve.

[0107] The HPLC conditions for determining ginsenosides (rare ginsenosides) are as follows: the mobile phase is acetonitrile (A) and 0.02% (v / v) phosphoric acid aqueous solution (B), binary gradient elution. The elution flow rate is 1.0 mL / min, the injection volume is 20 μL, the gradient elution conditions are: 0-10 min acetonitrile (A) 78%, mobile phase B is 22%, 10-25 min acetonitrile (A) 76%, mobile phase B is 24%, 25-45 min acetonitrile (A) 60%, mobile phase B is 40%, 45-90 min acetonitrile (A) 45%, mobile phase B is 55%, the column temperature is 30°C, and the detection wavelength is 203 nm.

[0108] The molar conversion rate was calculated as: moles of product / moles of substrate × 100%.

[0109] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A use of β-glucosidase in the preparation of ginsenosides, wherein the amino acid sequence of the β-glucosidase is numbered RLF05189.1 on NCBI; the ginsenosides are rare ginsenosides Rg3 and / or Rh1.

2. Use of a nucleic acid molecule or a biological material containing the nucleic acid molecule in the preparation of ginsenosides; the ginsenosides are rare ginsenosides Rg3 and / or Rh1; the biological material includes recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, recombinant microorganism or transgenic cell line; the nucleic acid molecule encodes β-glucosidase, and the β-glucosidase is as described in claim 1.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.

1.

4. A method for producing rare ginsenoside Rg3 and / or Rh1, characterized in that: Producing rare ginsenoside Rg3 and / or Rh1 by whole-cell catalysis using β-glucosidase or recombinant microorganisms; the β-glucosidase is as described in claim 1, and the recombinant microorganism comprises a nucleic acid molecule, and the nucleic acid molecule is as described in claim 2 or 3; When generating rare ginsenoside Rg3 and / or Rh1, the reaction temperature is 70-85° C. and the pH is 4-7; the reaction solvent is a PBS solution containing 0.8-1.2% Tween 80 or a PBS solution containing 8-15% ethanol, or the reaction solvent includes a low eutectic solvent, and the volume fraction of the low eutectic solvent in the reaction solvent is 15-25%.

5. The method according to claim 4, characterized in that When producing rare ginsenoside Rg3, the reaction substrate is ginsenoside Rb1 and / or Rd; when producing rare ginsenoside Rh1, the reaction substrate is Rg1.

6. The method according to claim 5, characterized in that When preparing rare ginsenoside Rg3 and / or Rh1, the reaction substrate is the notoginseng total saponin extract.

7. The method according to any one of claims 4 to 6, characterized in that: When the β-glucosidase is used to generate rare ginsenoside Rg3 and / or Rh1, the mass ratio of the reaction substrate to the β-glucosidase is (10-30):

1.

8. The method according to any one of claims 4 to 6, characterized in that: When the recombinant microorganism produces rare ginsenoside Rg3 and / or Rh1 through whole-cell catalysis, the mass ratio of the reaction substrate to the recombinant microorganism is (20-50):1; the recombinant microorganism is induced by IPTG to express the β-glucosidase.

9. The method according to claim 8, characterized in that The induction expression conditions are: final IPTG concentration of 0.1-0.5 mM, shaking culture at 37±1° C., 180-220 rpm, for 4-6 hours.

10. The method according to claim 4, characterized in that The low eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor is choline chloride and / or L-proline, and the hydrogen bond donor is one or more of lactic acid, 1,4-butanediol, D-sorbitol, sucrose, anhydrous ethanol, and glycerol.

11. The method according to claim 10, characterized in that The hydrogen bond acceptor is L-proline, the hydrogen bond donor is lactic acid, and the molar ratio of L-proline to lactic acid is (0.8-1.2):(0.8-2.2); or The hydrogen bond acceptor is choline chloride, the hydrogen bond donor is anhydrous ethanol and 1,4-butanediol, and the molar ratio of choline chloride, anhydrous ethanol and 1,4-butanediol is (0.8-1.2):(0.8-1.2):(0.8-1.2); or The hydrogen bond acceptor is choline chloride, the hydrogen bond donor is D-sorbitol and 1,4-butanediol, and the molar ratio of choline chloride, D-sorbitol and 1,4-butanediol is (1.8-2.2):(0.8-1.2):(3.8-4.2); or The hydrogen bond acceptor is L-proline, the hydrogen bond donors are glycerol and sucrose, and the molar ratio of L-proline, glycerol and sucrose is (3.8-4.2):(9.8-10.2):(0.8-1.2).

12. The method according to claim 11, characterized in that The hydrogen bond acceptor is L-proline, the hydrogen bond donor is lactic acid, and the molar ratio of L-proline to lactic acid is (0.8-1.2):(0.8-1.2).

13. The method according to claim 8, characterized in that The method of producing rare ginsenoside Rg3 and / or Rh1 by whole-cell catalysis using the recombinant microorganism comprises: (1) Preparing a recombinant microorganism: transforming a host microorganism competent cell with a recombinant vector containing a nucleic acid molecule to obtain a recombinant microorganism; the nucleic acid molecule encodes β-glucosidase, and the β-glucosidase is as described in claim 1; (2) after inducing the expression of the β-glucosidase in the recombinant microorganism with IPTG, collecting and resuspending to obtain a whole cell solution of the recombinant microorganism; (3) Mixing the whole cell solution of the recombinant microorganism, a substrate and a reaction solvent, and reacting them to produce rare ginsenosides Rg3 and / or Rh1.

14. The method according to any one of claims 9 to 12, characterized in that: The method of producing rare ginsenoside Rg3 and / or Rh1 by whole-cell catalysis using the recombinant microorganism comprises: (1) Preparing a recombinant microorganism: transforming a host microorganism competent cell with a recombinant vector containing a nucleic acid molecule to obtain a recombinant microorganism; the nucleic acid molecule encodes β-glucosidase, and the β-glucosidase is as described in claim 1; (2) after inducing the expression of the β-glucosidase in the recombinant microorganism with IPTG, collecting and resuspending to obtain a whole cell solution of the recombinant microorganism; (3) Mixing the whole cell solution of the recombinant microorganism, a substrate and a reaction solvent, and reacting them to produce rare ginsenosides Rg3 and / or Rh1.

15. The method according to claim 13, characterized in that The host microorganism is Escherichia coli.

16. The method according to claim 14, characterized in that The host microorganism is Escherichia coli.

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