Beta-glucosidase, preparation method thereof and application of beta-glucosidase in production of ginsenoside Rg3

Abstract

The invention relates to the technical field of ginsenoside, aims to solve the problem of low conversion rate of ginsenoside Rg3 prepared by adopting a microbial conversion method in the prior art, and particularly discloses beta-glucosidase and a preparation method thereof, and application of the beta-glucosidase in production of ginsenoside Rg3. The preparation method of the beta-glucosidase comprises the following steps: S1, inserting a beta-glucosidase gene into a pET vector to obtain a recombinant plasmid; s2, inoculating the recombinant plasmids into competent cells, culturing the competent cells in an antibiotic-free culture medium and a resistant culture medium in sequence to form monoclonal antibodies, performing IPTG (isopropyl-beta-d-thiogalactoside) induction culture to form monoclonal strains, and storing the monoclonal strains in glycerol to obtain glycerol bacteria; s3, glycerol bacteria are placed in an LB culture medium to be cultured and then transferred into a fermentation culture medium, the fermentation culture medium is placed in a fermentation tank to be fermented, then centrifugal separation, thallus collection and re-suspension are conducted, beta-glucosidase is obtained and used for decomposing ginseng total saponins to produce ginsenoside Rg3, and the conversion rate of main products can reach 98%.

Description

Technical Field

[0001] The present invention relates to the technical field of ginsenosides, and in particular to a beta-glucosidase and a preparation method thereof, and application of the beta-glucosidase in the production of ginsenoside Rg3. Background Art

[0002] Ginsenosides are the main active ingredients of ginseng. Based on differences in their glycoside structure, ginsenosides can be divided into two types: dammarane and oleanane. Dammarane ginsenosides include ginsenosides Rg3, Rb1, Rb2, Rb3, Rc, Rd, and Rh2. Different saponins are found in varying concentrations in ginseng. For example, Rg3, Rh2, CK, F1, F2, and Rh1, which are found in relatively low concentrations, are referred to as rare ginsenosides. Compared to their high-content counterparts, rare ginsenosides share the same sapogenin structure but differ in the number of glycosides. Therefore, rare ginsenosides can be prepared by hydrolyzing high-content ginsenosides. Existing methods for hydrolyzing ginsenoside glycosides primarily include thermal cleavage and acid-base catalysis. However, glycosidases, due to their mildness, efficiency, and specificity, have become effective tools for preparing rare ginsenosides.

[0003] As for ginsenoside Rg3, it is a tetracyclic triterpenoid compound with the molecular formula C 42 H 72 O 13 Ginsenoside Rg3, a protopanaxadiol-type saponin, exhibits significant anti-tumor, immunomodulatory, and cardiovascular protective effects. Therefore, the industrial-scale production of ginsenoside Rg3 is of great significance. Currently, the main methods for producing ginsenoside Rg3 include natural extraction, chemical synthesis, and microbial conversion, but these methods still have significant shortcomings in terms of production efficiency and preparation conditions. In the microbial conversion method for producing ginsenoside Rg3, glycosidases are used to hydrolyze high-glycosyl ginsenosides into ginsenoside Rg3. For example, Jin-Kwang Kim et al., in their paper "Mass production of theginsenoside Rg 3(S) through the combinative use of two glycoside hydrolases," published in Food Chemistry, reported that PPDGM was converted to Rg3 using BglBX10 from Flavobacterium johanssonii UW101T and abf 22-3 from Leuconostoc sp. at pH 6.0 and 37°C, with a conversion rate of 88.3%. Existing microbial conversion methods for preparing ginsenoside Rg3, such as the above-mentioned method, mostly involve attacking the C3 and C20 sugar chains by glycosidases. Since the attacked carbon positions are difficult to precisely control, the product is mixed with various ginsenosides such as Rd and Rh2. The product composition is complex, the main product conversion rate is low, and the product purity is low. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem that in the existing microbial conversion method for preparing ginsenoside Rg3, glycosidase will simultaneously attack the C3 and C20 polysaccharide chains, thereby causing Rd and Rh2 to be mixed into the product.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for preparing β-glucosidase, comprising the following steps:

[0007] S1 recombinant plasmid: insert the β-glucosidase gene into the pET vector to obtain a recombinant plasmid;

[0008] S2 strain construction: The recombinant plasmid is inoculated into competent cells, and the cells are cultured in an antibiotic-free medium and then in an antibiotic-resistant medium to form monoclonal antibodies. The monoclonal antibodies are cultured in LB medium, and then induced with isopropyl-β-D-thiogalactoside. The cells are then cultured continuously to form a monoclonal strain, which is then stored in glycerol to obtain a glycerol strain.

[0009] S3 fermentation treatment: the glycerol bacteria are cultured in LB medium, then transferred to fermentation medium, and placed in a fermenter for fermentation, followed by centrifugation, and the bacteria are collected and resuspended to obtain the β-glucosidase.

[0010] Preferably, in step S1, when the β-glucosidase gene is inserted into the pET vector, BamHI and XhoI are used as the N-terminal and C-terminal restriction sites, respectively.

[0011] Preferably, in step S2, the recombinant plasmid is inoculated into the competent cells, which are then placed in an ice-water bath for 20-40 minutes, heat-shocked at 40-45°C for 80-100 seconds, placed in an ice bath again, and then added to an antibiotic-free culture medium for culture.

[0012] Preferably, in step S2, when culturing in the antibiotic-free medium and the resistant medium sequentially, the temperature is controlled at 35-39°C, and the shaking culture is performed in the antibiotic-free medium for 45-60 minutes, and the culture is performed in the resistant medium for 12-16 hours.

[0013] Preferably, in step S2 and step S3, the LB medium comprises 4-6 g / L yeast extract, 8-12 g / L tryptone and 8-12 g / L sodium chloride, and the LB medium in step S3 is further supplemented with kanamycin at a final concentration of 18-22 μg / mL.

[0014] Preferably, in step S3, the fermentation medium comprises 10-15 g / L tryptone, 22-28 g / L yeast extract, 2-8 ml / L glycerol, 2-2.5 g / L potassium dihydrogen phosphate, 12-13 g / L potassium dihydrogen phosphate and 1-3 g / L magnesium sulfate.

[0015] Preferably, during the fermentation process, when the dissolved oxygen concentration of the fermentation medium increases, feeding is performed, and the feed medium comprises 400-600 g / L glucose and 0.5-2 g / L lactose.

[0016] The beta-glucosidase provided by the present invention is prepared by adopting the above preparation method, and the enzyme molecular weight is 75-85 kDa.

[0017] The present invention provides an application of the above-mentioned β-glucosidase in producing ginsenoside Rg3, wherein the β-glucosidase is used as a catalyst to decompose total ginsenosides to obtain ginsenoside Rg3.

[0018] Preferably, the production method of ginsenoside Rg3 comprises the following steps:

[0019] Add 15-30U / mL of β-glucosidase solution to 60-100g / L of total ginsenosides, place in a 60-100°C water bath for reaction; then add methanol in an amount 1.5-3 times the volume of the total reaction material, let stand, separate, and dry to obtain the ginsenoside Rg3.

[0020] The technical solution of the present invention has the following beneficial effects:

[0021] The present invention addresses the problem of low conversion rate of ginsenoside Rg3 in industrial production. By using beta-glucosidase obtained through high-density fermentation as a catalyst to decompose total ginsenosides, the beta-glucosidase has high specificity and can act more specifically on the C20 sugar chain of the total ginsenosides, thereby converting Rb1 and Rd in the total ginsenosides into ginsenoside Rg3 without generating intermediate by-products. The process has high conversion rate and selectivity, reduces the difficulty of post-processing after conversion between ginsenosides, and efficiently produces ginsenoside Rg3. The conversion rate of the main product of ginsenoside Rg3 can be as high as 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a hydrolysis pathway diagram of β-glucosidase hydrolyzing ginsenosides in the present invention;

[0023] Figure 2 This is the nucleic acid electrophoresis diagram of the pET-28a-Tbbg recombinant plasmid in Example 1;

[0024] Figure 3This is a comparison chart of the enzyme activities of crude glycosidases expressed by multiple monoclonal strains in Example 1;

[0025] Figure 4 This is the SDS-polyacrylamide gel electrophoresis diagram after expression of the β-glucosidase Tbbg in Example 1;

[0026] Figure 5 is the HPLC chromatogram of Rb1 and Rd before conversion in Example 1;

[0027] Figure 6 This is the HPLC chromatogram of Rg3 after 6 hours of conversion in Example 1;

[0028] Figure 7 This is the HPLC chromatogram of the purified Rg3 in Example 1. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed; and where the manufacturers of the instruments, equipment, reagents, and raw materials used are not specified, they are all commercially available conventional products.

[0030] The present invention provides a β-glucosidase, and the preparation method thereof comprises the following steps:

[0031] (1) Recombinant plasmid

[0032] Take the β-glucosidase gene Tbbg, select BamHI and XhoI as the N-terminal and C-terminal restriction sites respectively, insert the β-glucosidase gene Tbbg into the vector pET-28a and connect them to obtain the pET-28a-Tbbg recombinant plasmid.

[0033] The specific operation is as follows: take 0.3 pmol of β-glucosidase gene Tbbg, 0.03 pmol of pET-28a, 1 μL of T4 ligase (provided by Novozymes Biotechnology Co., Ltd., 400 U / μL T4 DNA Ligase), and 1 μL of 10× Ligase Buffer, mix them, and add sterile water to 10 μL, react at 16°C for 12 hours to obtain the pET-28a-Tbbg recombinant plasmid.

[0034] Among them, the β-glucosidase gene Tbbg is from Thermotogota bacterium, with gene accession number (GenBank: MDI3516220.1).

[0035] (2) Construction of strains

[0036] Take 10 μL of 100 ng / μL pET-28a-Tbbg recombinant plasmid and add it to 100 μL of competent cells. Place it in ice water and ice bath for 20-40 minutes. Then heat shock it at 40-45°C for 80-100 seconds and immediately place it on ice for 1-3 minutes. Then add it to 750 μL of antibiotic-free LB medium and place it in a shaker at an ambient temperature of 35-39°C. Incubate it with shaking for 45-60 minutes to obtain antibiotic-free culture medium. Centrifuge the antibiotic-free culture medium, discard the supernatant, mix the remaining culture medium and spread it on kanamycin (kan)-resistant LB solid medium. Incubate it at 35-39°C for 12-16 hours to grow monoclonal colonies in the culture medium.

[0037] Pick multiple monoclonal colonies, place each monoclonal colony in a 50 mL LB medium, culture at 37 ° C to OD = 0.6, then add 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction, cool to 25 ° C, culture for 16 hours, and then detect the enzyme activity of the crude glycosidase enzyme solution; select the monoclonal strain with the highest enzyme activity, store it in 50% glycerol, and control the volume ratio of the monoclonal strain culture solution to glycerol to be 1:1, record it as glycerol bacteria Tbbg, and store it in a -80 ° C refrigerator.

[0038] Among them, LB medium is the full name of Luria-Bertani medium, which refers to a culture medium commonly used for culturing Escherichia coli. Its main components are trypsin, yeast extract and NaCl.

[0039] (3) Fermentation treatment

[0040] Inoculate glycerol strain Tbbg into 80 mL of LB medium containing kanamycin at a final concentration of 18-22 μg / mL at a 1‰ inoculum size. The culture was then shaken at 220 rpm in a shaker at 35-39°C for 12 hours. The LB medium contained 4-6 g / L yeast extract, 8-12 g / L tryptone, and 8-12 g / L sodium chloride.

[0041] The cultured material is transferred into a fermentation medium, and then fermented in a fermenter, with the loading volume in the fermenter being 40% of the total volume of the fermenter. The pH electrode of the fermenter needs to be calibrated before use. After the fermentation is completed, the cells are centrifuged, collected, and resuspended in 20 mM phosphate buffer to obtain β-glucosidase.

[0042] The fermentation medium comprises 10-15 g / L tryptone, 22-28 g / L yeast extract, 2-8 ml / L glycerol, 2-2.5 g / L potassium dihydrogen phosphate, 12-13 g / L dipotassium hydrogen phosphate, and 1-3 g / L magnesium sulfate. During the fermentation process, when the dissolved oxygen concentration (DO) of the medium rebounds rapidly from 30% to 60-70%, feed medium must be immediately added. The feed medium comprises 400-600 g / L glucose and 0.5-2 g / L lactose.

[0043] In the present invention, the β-glucosidase prepared above has an enzyme molecular weight of 75-85 kDa.

[0044] The present invention also provides a method for producing ginsenoside Rg3 using the aforementioned β-glucosidase, wherein β-glucosidase is used as a catalyst to decompose total ginsenosides to obtain the rare ginsenoside Rg3 with high efficiency and high yield. The specific preparation method is as follows:

[0045] To 60-100 g / L of total ginsenosides, add 15-30 U / mL of β-glucosidase solution, place in a 60-100°C water bath, and react for 3-8 hours. After the reaction is completed, add 1.5-3 times the volume of methanol of the total reaction material, stir evenly, and let it stand for more than 10 hours. Then, separate and purify, remove bacterial protein by centrifugation, add an equal volume of water, precipitate crystals, quickly filter with filter paper, and vacuum dry the obtained filter cake to obtain ginsenoside Rg3.

[0046] Example 1

[0047] This example is a specific implementation method for preparing β-glucosidase.

[0048] Step 1: Take the β-glucosidase gene Tbbg (provided by Jinweizhi Biotechnology Co., Ltd., full length 2145bp, encoding a protein of 715 amino acids), select BamHI and XhoI as the N-terminal and C-terminal restriction sites, respectively, insert the β-glucosidase gene Tbbg into the vector pET-28a for ligation, and obtain the pET-28a-Tbbg recombinant plasmid.

[0049] Step 2: Take 10 μL of pET-28a-Tbbg recombinant plasmid and add it to 100 μL of E. coli BL21 (DE3) competent cells (provided by Qingke Biotechnology Co., Ltd., product number TSC-C12), place it in ice water, ice bath for 30 minutes, then heat shock at 42°C for 90 seconds, and then immediately place it on ice for 150 seconds; then add it to 750 μL of antibiotic-free LB medium at 37°C, shake and culture in a shaker at an ambient temperature of 37°C for 50 minutes to obtain antibiotic-free culture medium; centrifuge the antibiotic-free culture medium at 4000 rpm for 2 minutes, discard about 650 μL of supernatant to obtain culture medium, mix the culture medium and apply it to kanamycin-resistant LB solid medium, and culture at 37°C for 10 hours. Ten monoclonal colonies were selected and cultured in test tubes at 37°C, shaking at 180 rpm for 12 h. The cells were then transferred to 50 mL of LB medium containing 50 μL of kanamycin and cultured at 37°C, 200 rpm, until the OD value reached 0.6. 0.2 mM isopropyl-β-D-thiogalactopyranoside was then added for induction, the temperature was lowered to 25°C, and the culture was continued for 16 h. The induced bacterial solution was centrifuged at 10,000 rpm for 10 min in a high-speed refrigerated centrifuge. The supernatant was discarded, and the bacterial pellet was resuspended in 10 mL of 20 mM phosphate buffer (pH 7.0) until the OD value reached approximately 15. The pellet was then placed in an ice bath and disrupted using a cell disruptor at 300 W power, with a cycle time of 3 s and a rest time of 4 s for a total of 5 min. The disrupted solution was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected as the crude glycosidase solution. The crude glycosidase enzyme solution obtained from the 10 monoclonal colonies was assayed for enzyme activity to identify the monoclonal strain with the highest enzyme activity. The monoclonal strain with the highest enzyme activity, Tbbg3, was taken and placed in an equal volume of 50% glycerol to obtain glycerol strain Tbbg3, which was then stored in a -80°C freezer until further use.

[0050] Step 3: Take the glycerol bacteria Tbbg3 and inoculate the glycerol bacteria Tbbg3 into LB medium containing 20 μg / mL kanamycin at an inoculum size of 1‰. The LB medium includes 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride. Then place it in a shaker at an ambient temperature of 7°C and shake at 220 rpm for 12 hours to obtain a seed solution.

[0051] Step 4: Fermentation was performed in a 5L fermentor, with the fermentor loading volume controlled to 2L. The fermentation medium included 12g / L tryptone, 24g / L yeast extract, 4ml / L glycerol, 2.31g / L potassium dihydrogen phosphate, 12.54g / L potassium dihydrogen phosphate, and 1.73g / L magnesium sulfate. Before inoculation, the fermentor temperature was adjusted to 37°C and the pH to 7.0. The dissolved oxygen was then calibrated to 100%. 1‰ kanamycin and 4% seed solution were added by flame inoculation and fermentation was carried out. During the fermentation process, OD600 and residual sugar were monitored to maintain the sugar concentration below 1g / L. When the dissolved oxygen rebounded, feeding was started. The feed medium included 500g / L glucose and 1g / L lactose, and the DO% was controlled within the range of 25-35%. When the OD600 reached approximately 40, induction was started. The IPTG inducer was added at a rate of 10mL / min. After induction, the temperature was lowered to 25°C and the DO% was controlled within the range of 15-20%. The total fermentation time is 24 hours. When OD600 no longer increases, the tank can be removed and centrifuged at 4000 rpm for 25 minutes to collect the bacteria. The bacteria are resuspended with 20 mM phosphate buffer with a pH of about 7.0 until the OD600 of the bacterial suspension is adjusted to about 110 to obtain β-glucosidase.

[0052] Example 2

[0053] This example is a specific implementation method of producing ginsenoside Rg3 using the β-glucosidase in Example 1.

[0054] 80 g / L of ginsenosides was added to the β-glucosidase obtained in Example 1, and the mixture was placed in an 80°C water bath for 7 hours. After the reaction, methanol with a volume twice that of the total reaction material was added, stirred evenly, and allowed to stand for 10 hours for conversion. The mixture was then separated and purified, and the bacterial protein was removed by centrifugation. An equal volume of pure water was added to precipitate crystals, which were quickly filtered using filter paper to obtain a filter cake, which was then vacuum dried to obtain the ginsenoside Rg3 product.

[0055] like Figure 1 The figure shows the hydrolysis pathway of the reaction process. Detected by high performance liquid chromatography, the purity of ginsenoside Rg3 product is >98%.

[0056] Test example

[0057] (1) Take the pET-28a-Tbbg recombinant plasmid in Example 1 and perform nucleic acid electrophoresis. The process is as follows:

[0058] Prepare nucleic acid gel: weigh 1g agarose powder and dissolve it in 100mL TAE buffer (40mM Tris-acetate, 1mM EDTA, pH 8.0). Heat in a microwave until completely dissolved. Cool to approximately 60°C and add the nucleic acid dye GelRed. Pour the gel into a gel mold, insert a comb, and solidify at room temperature for 20-30 minutes.

[0059] Re-prepare the sample: add the plasmid to 1 / 5 volume of buffer.

[0060] Post-electrophoresis settings: Place the solidified gel into the electrophoresis tank, add electrophoresis buffer (the liquid surface covers the gel by 1-2mm), remove the comb, and load the samples in order: add 5μL of marker to the first well and 5-10μL / well of sample to the remaining wells; connect the power supply and set the voltage to 80-120V. After the end, remove the gel and observe the target band under the gel imager.

[0061] The results of the above nucleic acid electrophoresis are as follows Figure 2 As shown, the size of the pET-28a-Tbbg recombinant plasmid is 7479 bp.

[0062] (2) The enzyme activity of the crude glycosidase solution obtained by treating the 10 monoclonal colonies in Example 1 was determined by the pNP colorimetric method. The enzyme activity determination process was as follows:

[0063] First, draw a pNP standard curve; take crude glycosidase enzyme samples respectively, heat them at 80℃ for 5min, centrifuge and take the supernatant, dilute it to 0.2-1mM with 20mM phosphate buffer for testing; take 1.7mL of 20mM phosphate buffer (pH 7.0), 0.2ml of 10mM pNPGlc substrate, and 100mL of enzyme solution, mix them and place them at 85℃ for reaction for 5min, then take them out immediately, add 0.5ml of 0.5M Na2CO3 solution, and finally measure the absorbance at 405nm.

[0064] The enzyme activity was determined by converting 1 μm pNPGlc in 1 min at 85°C. Figure 3 As shown, from Figure 3 It can be seen that the crude enzyme activity of the monoclonal strain TbBg3 obtained by recombination in Example 1 reached 214 U, which has good enzyme activity. Therefore, the monoclonal strain Tbbg3 was selected for subsequent high-density fermentation and other treatments.

[0065] The same method was used to determine the enzyme activity of the enzyme solution after fermentation in step 4 of Example 1. After 24 hours of fermentation, the optical density of the bacterial solution (OD=600) was 120, indicating that the bacterial growth density was high. At this time, the enzyme activity of the enzyme solution reached 1581U, showing strong catalytic activity. This shows that in the preparation method of β-glucosidase proposed by the present invention, the enzyme yield of high-density fermentation is good, and the enzymatic activity of the obtained β-glucosidase is high, which can provide reliable basic data for its production in ginsenoside Rg3.

[0066] (3) Take the β-glucosidase Tbbg in Example 1 and detect the expression of the recombinant protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as follows:

[0067] First, load a 12.5% precast gel (provided by Ziqingke Biotechnology Co., Ltd.) into the gel electrophoresis apparatus and add 1× protein electrophoresis buffer (provided by Sangon Bioengineering Technology Co., Ltd.) to the electrophoresis tank. Transfer the sample to a clean centrifuge tube according to the loading volume, add 2× SDS-PAGE loading buffer according to the proportion, mix well, and denature in a 100°C constant temperature metal bath for 15 min. Place on ice for 5 min.

[0068] Add the prepared sample to the sample loading port and start electrophoresis. First, adjust the voltage to 80V for 30 minutes, then increase the voltage to 120V and run for 90 minutes.

[0069] Remove the glass plate from the electrophoresis tank, place the gel in a staining box, and stain the gel with SDS-PAGE staining solution, shaking at 300 rpm at room temperature for 2 hours; after the staining is completed, recover the staining solution, wash twice with distilled water, and then add SDS-PAGE decolorization solution for decolorization, shaking at 300 rpm at room temperature for 1 hour, and repeat once; after the decolorization is completed, soak it in distilled water for a period of time and take pictures for preservation.

[0070] like Figure 4 This is an SDS-polyacrylamide gel electrophoresis diagram of the above-mentioned β-glucosidase Tbbg after expression, wherein lane M is a protein marker (provided by Sangon Biotechnology Co., Ltd.), lane 1 is the whole fermentation cells after induction, lane 2 is the whole cells after ultrasonic disruption after induction, lane 3 is the supernatant after cell disruption, and lane 4 is the precipitate after cell disruption. The enzyme molecular weight of the prepared β-glucosidase was 79.5 kDa as detected by SDS-PAGE nucleic acid electrophoresis.

[0071] (4) High performance liquid chromatography (HPLC) was used to detect the substrate Rb1, substrate Rd, and product Rg3 in Example 2. The standard products Rb1, Rd, and Rg3 used were all provided by Shanghai Dibo Biotechnology Co., Ltd. The HPLC detection process was as follows:

[0072] The sample purity was determined according to the high performance liquid chromatography (HPLC) method in the Chinese Pharmacopoeia, using a C18 reverse phase column, a detection wavelength of 203 nm, a column temperature of 30°C, and a flow rate of 1.5 ml / min; a 5 mg / ml sample solution was prepared in methanol, and the sample was filtered using a 0.22 μm microporous filter membrane.

[0073] The HPLC process parameters are as follows:

[0074] Table 1 Mobile phase parameters of HPLC detection process

[0075] Time (min) Mobile phase A (%) Mobile phase B (%) 0-20 80 20 20-45 80→54 20→46 45-55 54→45 46→55 55-60 45 55

[0076] The above HPLC test results are as follows Figures 5 to 7 As shown, Figure 5 It is the position of Rb1 and Rd in ginsenosides. Figure 6 is the HPLC chromatogram of the reaction solution 6 hours after conversion, Figure 7 The HPLC chromatogram of Rg3 with a purity greater than 98% after purification is shown in Figure 1. Figures 5 to 7 It can be seen that after Rb1 and Rd in ginsenosides were hydrolyzed at 80℃ for 6h under the action of β-glucosidase, β-glucosidase almost completely acted on the C20 sugar chain of Rb1 and Rd, and Rb1 and Rd in ginsenosides were almost completely converted into Rg3, with a conversion rate of 98%.

[0077] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing β-glucosidase, characterized in that: The steps include: S1 recombinant plasmid: insert the β-glucosidase gene into the pET vector to obtain a recombinant plasmid; S2 strain construction: The recombinant plasmid is inoculated into competent cells, and the cells are cultured in an antibiotic-free medium and then in an antibiotic-resistant medium to form monoclonal antibodies. The monoclonal antibodies are cultured in LB medium, and then induced with isopropyl-β-D-thiogalactoside. The cells are then cultured continuously to form a monoclonal strain, which is then stored in glycerol to obtain a glycerol strain. S3 fermentation treatment: the glycerol bacteria are cultured in LB medium, then transferred to fermentation medium, and placed in a fermenter for fermentation, followed by centrifugation, and the bacteria are collected and resuspended to obtain the β-glucosidase.

2. The method for preparing β-glucosidase according to claim 1, wherein In step S1, when the β-glucosidase gene is inserted into the pET vector, BamHI and XhoI are used as the N-terminal and C-terminal restriction sites, respectively.

3. The method for preparing β-glucosidase according to claim 1, wherein In step S2, the recombinant plasmid is inoculated into competent cells, which are then placed in an ice-water bath for 20-40 minutes, heat-shocked at 40-45°C for 80-100 seconds, placed in an ice bath again, and then added to an antibiotic-free culture medium for culture.

4. The method for preparing β-glucosidase according to claim 1, wherein In step S2, when culturing in the antibiotic-free medium and the resistant medium in sequence, the temperature is controlled at 35-39°C, and the shaking culture is performed for 45-60 minutes in the antibiotic-free medium and 12-16 hours in the resistant medium.

5. The method for preparing β-glucosidase according to claim 1, wherein In step S2 and step S3, the LB medium includes 4-6 g / L yeast extract, 8-12 g / L tryptone and 8-12 g / L sodium chloride, and the LB medium in step S3 is further supplemented with kanamycin at a final concentration of 18-22 μg / mL.

6. The method for preparing β-glucosidase according to claim 1, wherein In step S3, the fermentation medium includes 10-15 g / L tryptone, 22-28 g / L yeast extract, 2-8 ml / L glycerol, 2-2.5 g / L potassium dihydrogen phosphate, 12-13 g / L potassium dihydrogen phosphate and 1-3 g / L magnesium sulfate.

7. The method for preparing β-glucosidase according to claim 6, wherein During the fermentation process, when the dissolved oxygen concentration of the fermentation medium increases, feeding is carried out, and the feeding medium includes 400-600 g / L glucose and 0.5-2 g / L lactose.

8. A β-glucosidase, characterized in that The enzyme is prepared by the preparation method according to any one of claims 1 to 7, and has an enzyme molecular weight of 75-85 kDa.

9. Use of the β-glucosidase according to claim 8 in the production of ginsenoside Rg3, characterized in that: The beta-glucosidase is used as a catalyst to decompose the total ginsenosides to obtain ginsenoside Rg3.

10. Use of the β-glucosidase according to claim 9 in the production of ginsenoside Rg3, characterized in that: The production method of ginsenoside Rg3 comprises the following steps: Add 15-30U / mL of β-glucosidase solution to 60-100g / L of total ginsenosides, place in a 60-100°C water bath for reaction; then add methanol in an amount 1.5-3 times the volume of the total reaction material, let stand, separate, and dry to obtain the ginsenoside Rg3.

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