CN111575333A - Biological preparation method of ginsenoside - Google Patents
Biological preparation method of ginsenoside Download PDFInfo
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Abstract
The invention discloses a biological preparation method of ginsenoside, which comprises the following steps: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious roots after induction culture; (5) and (3) carrying out enzymatic conversion reaction by taking beta-glucosidase as converting enzyme and ginsenoside mixed liquor as a conversion substrate. The invention provides a biological preparation method of combined ginsenoside, which comprises tissue culture, enzyme immobilization and hydrolysis methods to obtain rare ginsenoside. 6 kinds of beta-glycosidases are screened out for enzymatic hydrolysis and are combined for use, and the content of rare ginsenoside in the converted product is obviously improved. The biological preparation method of the invention can replace the method for directly extracting ginsenoside from ginseng.
Description
Technical Field
The invention relates to a method for preparing ginsenoside, in particular to a method for preparing ginsenoside through biotransformation, belonging to the field of biological preparation of ginsenoside.
Background
Ginsenoside is the main pharmacological component of ginseng, especially rare ginsenoside is the molecule which plays the functions of resisting tumor, resisting inflammation, improving immunity and the like. Because wild ginseng has low content of saponin, the source of rare ginsenoside is very limited, and the application of the wild ginseng in functional foods and medicines is limited.
Therefore, there is a great need in the production practice to provide a method for preparing ginsenosides with high efficiency and yield.
Disclosure of Invention
The invention mainly aims to provide a biological preparation method of ginsenoside with high yield;
the above object of the present invention is achieved by the following technical solutions:
a biological preparation method of ginsenoside comprises: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious root after induction culture.
Wherein the method for inducing and producing the ginseng adventitious roots in the step (1) by the tissue culture mode comprises the following steps: (a) inoculating the ginseng root slice to the solution containing 1.0mg L -12,4-D+0.1mg L-1kinetin+30g L-1Inducing callus on the inducing culture medium of cane sugar; (b) callus was transferred to 5.0mg L-1Carrying out callus propagation on an MS culture medium of IBA; (c) in the presence of 3.0mg L-1IBA and 30g L-1The callus was cultured on a sucrose MS solid medium, and the generation of adventitious roots was induced from the callus.
The step (3) of adding methyl jasmonate is to add 200 mu M of methyl jasmonate to induce the content of ginsenoside to be increased; preferably, the ginseng adventitious roots are transferred from the shake flask to the bioreactor and cultured for 50 days, and 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content.
The method for extracting ginsenoside from the adventitious roots of ginseng in the step (4) comprises the following steps: (a) adding Ginseng radix powder into methanol/water mixture at 37 deg.C overnight; (b) extracting with methanol as solvent by ultrasonic instrument: (c) filtering the supernatant of the extractive solution for sterilization, evaporating to remove methanol, and dissolving the residue in water; wherein the methanol/water mixture of step (a) consists of methanol and water in a volume ratio of 4: 1; in the step (b), 80% methanol is used as a solvent to extract for 3 times at room temperature of an ultrasonic instrument, and the extraction time is 0.5h each time.
In order to further improve the content of rare ginsenoside in the adventitious root, the beta-glucosidase is adopted as the invertase, the ginsenoside mixed solution extracted from the adventitious root is used as the conversion substrate to carry out enzymatic conversion reaction, and the content of rare ginsenoside in the conversion product can be obviously improved.
The invention discovers that the content of rare ginsenoside in the converted product can be effectively improved by adding beta-glucosidase into a PPD type ginsenoside mixture for enzymatic reaction; wherein, the beta-glucosidase is preferably any one or any combination of more than one of BglSk, BglPm, Bgp1, BglBX10, Tpebgl3 or Abf 22-3; wherein the enzymatic reaction may be a reaction of a beta-glucosidase solution with a PPD-type ginsenoside mixture in 50mM sodium phosphate buffer (pH8.0) at 37 ℃.
UPLC analysis after the enzymatic reaction is completed detects the relative content of ginsenoside, and compared with untreated control, the results show that Bgpl, BglBX10 and Abf22-3 convert Rd into Rg3, and Tpebgl3 converts Rb1 and Rd into Rg 3. The generation amount of Rg3 in the four reactions is about 15-30% of the PPD type ginsenoside mixture, and the peak value of Tpebgl3 is reached when the concentration is 149.74 +/-5.67 mg. All combined enzyme treatments of BglPm with the other four enzymes with C-20 hydrolytic activity (Bgpl, BglBX10, Tpebgl3 and Abf22-3) produced over 40% Rh2, and over 10% CMc1 and CO. The combination of BglSk with these four enzymes can yield the right amount of Rg3, Gyp75, CMc, CY and Rh2, with a ratio between 10% and 20%. BglPm and BglSk both produce high proportions of CK (277.61 + -4.27 mg, 34.22 + -1.91%). In all transformation processes, 3.0g of PPD-type ginsenoside mixture was hydrolyzed by BglPm + Bgp1 combination to obtain Rh2 with highest yield (326.61 + -7.04 mg) accounting for 10.89 + -0.23% of the PPD-type ginsenoside mixture
The beta-glucosidase BglSk, BglPm, Bgp1, BglBX10, Tpebgl3 or Abf22-3 can be purchased from commercial sources or prepared into recombinant beta-glucosidase by adopting conventional genetic engineering means in the field, and can be applied to the invention.
In order to improve the utilization rate of the enzyme and the separation efficiency of the beta-glucosidase and the product, the invention further immobilizes the beta-glucosidase to obtain the immobilized beta-glucosidase, and the immobilized beta-glucosidase is adopted to carry out enzymatic reaction, so that the utilization rate of the enzyme and the separation efficiency of the enzyme and the product can be effectively improved.
As a reference, the β -glucosidase immobilization method comprises immobilizing enzyme with polysulfone hollow fiber Membrane (MWCO) with molecular weight of 30kDa, equilibrating the membrane in phosphate buffer (pH8.0) for 30min, immobilizing enzyme with pressure-driven filtration, and immobilizing β -glucosidase solution (0.5mg mL)-1) Using peristaltic pump at 10mL min-1Is pumped through the lumen of the membrane, and the circulating 2h β -glucosidase is embedded in an asymmetric, aqueous porous membrane and is encapsulated by 30kDa fibers on the inside surfaceFixing the membrane to obtain the product.
The invention takes the hollow fiber membrane immobilized recombinant His-Bgp1 and His-BglPm as examples to carry out the experiment of converting the PPD type ginsenoside mixture into Rh2, and verifies the effectiveness of the method. The immobilized beta-glucosidase has high repeated utilization rate, and the relative activity of the immobilized beta-glucosidase is kept at about 85% after 9 cycles of repeated catalytic reaction on day 9. The immobilized β -glucosidase maintained 40% of the initial activity even after 15 consecutive cycles of repeated catalytic reactions.
The invention provides a biological preparation method of combined ginsenoside, which comprises tissue culture, enzyme immobilization and hydrolysis methods to obtain rare ginsenoside, wherein 6 β -glycosidases are screened according to glycosidase hydrolysis route for enzymatic hydrolysis and are combined for use, and the yield range of the rare ginsenoside Rg3, Rh2, CK, Gyp75, CMc and CY in the converted product is 5.54-32.66mg L-1. Under the optimized conditions of pH and temperature, the immobilized BglPm and Bgp1 can improve the yield of Rh2 by 7 percent, and the maximum yield can reach 51.17mg L-1(17.06% of the mixture of PPD-type ginsenosides). The biological preparation method provides an efficient way for obtaining various rare ginsenosides, and can replace a method for directly extracting the ginsenosides from ginseng.
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FIG. 1 is a schematic diagram of a rare ginsenoside biotransformation system.
FIG. 2 biotransformation pathway for glycosidase conversion of PPD-type ginsenosides.
FIG. 3 culturing ginseng adventitious roots in a bioreactor for efficient production of ginsenosides; (A) inoculating adventitious roots of ginseng to a solid multiplication culture medium for growth; (B) proliferation of adventitious roots on solid medium for 1 month; (C) further culturing the adventitious roots in a liquid culture medium for 7 days; (D) adventitious roots were expanded to a 10L bioreactor and grown for 2 months; (E) the content of a large amount of ginsenoside in adventitious roots and 5-year-old wild ginseng roots.
FIG. 4 is a graph showing SDS-PAGE results of purified glycosidase.
FIG. 5 is a plot of optimum temperature vs. pH contours for glycosidases.
FIG. 6 UPLC analysis of the conversion of a mixture of glycosidase combination treated PPD-type ginsenosides for 24 hours.
FIG. 7 heatmap of the relative content of PPD-type ginsenosides in glycosidase conversion reactions.
Figure 8 yield of each rare ginsenoside in all glycosidase combinations.
FIG. 9 Loading efficiency and thermostability of immobilized β -glucosidase. (A) Fixing Bglpm and Bgp1 on a hollow fiber column; (B) storage stability determination of immobilized Bgp1 and BglPm.
Fig. 10 UPLC results of combined conversion of PPD-type ginsenoside mixtures by β -glucosidase Bgp1 and BglPm.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
EXAMPLE 1 biological preparation of various rare ginsenosides
1. PPD type ginsenoside mixture is efficiently produced by culturing adventitious roots in bioreactor
The bioreactor culture of ginseng adventitious roots is a three-step scale-up culture (FIGS. 3A-D). Inoculating the root pieces to a solution containing 1.0mgL -12,4-D，0.1mg L-1kinetin and 30g L-1Inducing callus on sucrose induction medium, transferring callus to a medium containing 5.0mg L-1And performing propagation on MS culture medium of IBA. Then 3.0mg L-1IBA and 30g L-1After 4 weeks of culture on sucrose MS solid medium, adventitious roots were induced from callus (FIG. 3A, FIG. 3B).
About 30g of ginseng adventitious roots were collected and transferred to a liquid medium (1/2MS medium containing 5mg L of ginseng) in a rotary shaker-1IBA and 30g L-1Sucrose) at room temperature (25 ℃), avoidingLight, shaking culture at 120rpm for 7 days ((FIG. 3℃) culturing adventitious roots of Ginseng radix by transferring from shake flask to bioreactor (culture medium: 1/2MS, containing 5 mgL)-1IBA and 30g L-1Sucrose, culture conditions: at room temperature (25 ℃), in the absence of light, and in the case of aerobic culture), the biomass of adventitious roots of ginseng did not increase within 4 weeks. From week 5 to week 7, the adventitious root system of ginseng is in a rapid growth phase, and the growth becomes slow after week 8. At day 50, 200 μ M methyl jasmonate was added to induce an increase in ginsenoside content, and at day 56, fresh adventitious roots (785.98 ± 29.43g) were harvested for saponin extraction (fig. 3D).
3g of dry ginseng root powder was added to 30mL of methanol/water mixture (4:1, v/v) and kept at 37 ℃ overnight. Extracting with 80% methanol at room temperature under 240w of power of ultrasonic instrument (PL-S40T, KSJ, China) for 3 times (0.5 h). The supernatants of the respective extracts were mixed together, sterilized by filtration through a 0.45 μm filter, the methanol was evaporated off, and the residue was dissolved in 1mL of distilled water.
Ginsenoside was separated and identified using an ACQUITY-UPLC-BEH-C18 column (2.1mm × 50mm, 1.7 μm) (Watts, USA.) the mobile phases were A (water containing 0.05% phosphoric acid) and B (acetonitrile). gradient elution was started with 82% solvent A and 18% solvent B for 5min at a flow rate of 0.3mL min-1. Then the elution solvent was changed to 20% solvent B and held for 1 minute at a flow rate of 0.2mL min-1After 2 minutes, the mixture was changed to 25% solvent B for 1 minute at a flow rate of 0.3mL min-1Hold for 4 minutes followed by 30% solvent B for 4 minutes at a flow rate of 0.2mL min-1Hold for 5 minutes, then 40% solvent B, hold for 2 minutes, 90% solvent B, 13 minutes, and hold for 2 minutes. The elution solvent was changed to 18% solvent B at a flow rate of 0.3mL min-1. The detection wavelength was 203nm and the amount of sample was 5. mu.L.
The detection result shows that the PPD type ginsenoside mixture in the ginseng adventitious roots cultured in a 10L volume bioreactor is induced by methyl jasmonate to be 0.30 +/-0.02 g L-1Is 3 times higher than that of PPD type ginsenoside mixture cultured by control (0.10 + -0.01 g L)-1)。
Super-efficient liquidAnalysis of the protopanaxadiol major saponins (Rg1, Re, Rb1, Rd, Rc, Rb2) and one of the rare saponins (F2) by phase chromatography (UPLC) showed that the content of all monomeric saponins in the adventitious roots induced by methyl jasmonate was significantly higher than that of the cultured roots without induction treatment, but was similar or slightly lower than that of the 5-year-old wild ginseng roots (fig. 3E). Wherein the content of Rb1 is 0.27 + -0.14 mg g of Ginseng radix-1The dry weight increased to 1.75. + -. 0.01mg g-1Rc content is 0.05 + -0.01 mg g of ginseng root-1The dry weight increased to 1.12. + -. 0.11mg g-1. The Rd content in the adventitious root is 1.29 plus or minus 0.37mg g-1The content of Rd in 5-year-old wild ginseng root (1.49 +/-0.19 mg g)-1) And (4) the equivalent. The content of Rb2 in adventitious root is 0.61 + -0.06 mg g-15-year-old wild ginseng root (1.88 plus or minus 0.27mg g)-1) One third (fig. 3E). The content of rare ginsenoside F2 in adventitious root is only 0.20 + -0.01 mg g-1Similar to 5-year-old wild ginseng root. In conclusion, after 2 months of culturing adventitious roots in a 10L bioreactor, a large amount of ginsenoside is successfully extracted, and the growth content of wild ginseng in 5 years can be partially replaced.
Screening, purifying and property research of beta-glycosidase for PPD type ginsenoside mixture hydrolysis
2.1 screening of beta-glycosidases
In order to increase the content of rare ginsenoside in PPD type ginsenoside mixture, enzyme conversion method is adopted to produce rare ginsenoside.
6 kinds of beta-glycosidase are selected to carry out specific hydrolysis on C-3 glycosyl and C-20 glycosyl of a large amount of ginsenoside, and the rare ginsenoside is synthesized by an enzyme catalysis method. To hydrolyze the C-3 glycosyl groups of Rb1, Rd, Rc and Rb2, BglSk was selected from Klebsiella and BglPm from Bacillus mucosae. Three other beta-glycosidases (Bgp1, BglBX10 and Tpebgl3) were selected to hydrolyze C-20 glycosyl groups of the above-mentioned large amount of ginsenosides, respectively. To hydrolyze the alpha-L-arabinofuranosyl group of Rc, screening experiments were also performed using Abf22-3 from Leuconostoc 22-3.
TABLE 1 sources and Properties of glycosidases
2.2 purification of beta-glycosidase
Coli expression of recombinant glycosidases BglPm, BglSk, Bgp1, BglBX10, Tpebgl3 and Abf22-3 and protein purification preparation the CDS sequence and all primers were synthesized by GENEWIZ technologies, Inc. (Suzhou, China). BglSk, Bgp1 and BglPm were cloned into pET14b vector after PCR amplification, Bglbx10, Tpebgl3 and Abf22-3 were cloned into pCold-SUMO vector, optimal induction conditions were 0.1mM IPTG induction culture at 15 ℃ for 24 hours, then centrifugation at 3000g for 30 minutes at 4 ℃. cells were washed with a solution containing 20 mM-HCl, 0.5M NaCl, 30mM imidazole (pH8.0), then resuspended in the same solution, then sonicated, intact cells and debris were eluted at 4 ℃ with 3000g of NaCl, purified with a column containing 20 mM-HCl, 30mM histidine (pH8.0.0 mM) and protein (SDS-SDS) as protein purification kit, and protein purification kit, protein purification assay kit, protein purification kit2+SDS-PAGE analysis of the protein purified in combination with agarose resin showed that His-Bgp1 had a molecular weight of 95kDa, His-BglPm had a molecular weight of 48kDa, His-BglSk had a molecular weight of 71kDa, His-SUMO-BglBX10 had a molecular weight of 105kDa, His-SUMO-Tpbgl 3 had a molecular weight of 96kDa, and His-SUMO-Abf22-3 had a molecular weight of 70kDa (FIG. 4). The results show that the recombinant protein is successfully expressed in a soluble form, and the possibility of industrial application of the recombinant protein is increased.
2.3 Properties of beta-glycosidases
In order to determine the optimal pH and temperature for the activity of recombinant β -glucosidase, the hydrolytic activity was investigated with pNPG as substrate in buffers at different pH values (4.5-9.5) between 30-55 ℃.
BglPm was more active at all temperatures studied, 7.8-9.5 (FIG. 5). The optimal conditions for enzymatic activity of BglSk require similar pH values as BglPm, but the temperature is limited to 30-40 deg.C (FIG. 5). Bgp1, BglBX10 and Abf22-3 have more than 80% activity under similar pH and temperature conditions: the pH was between 7.5 and 8.5 and the temperature was between 30 and 37 deg.C (FIG. 5). In contrast, Tpebgl3 had higher activity under high temperature conditions of 37 to 55 ℃ (fig. 5). In summary, most β -glucosidases have optimal pH and temperature at pH8.0 and 37 ℃ for subsequent ginsenoside conversion.
3. Preparation of various rare ginsenosides by beta-glycosidase hydrolysis
The concentration is 0.1mg mL-1The purified enzyme solution of (2) was reacted with 3.0g of a mixture of PPD-type ginsenosides in 50mM sodium phosphate buffer (pH8.0) at 37 ℃ for 24 hours.
The following experimental groups included: (1) BglSk, (2) BglPm, (3) Bgp1, (4) BglBX10, (5) Tpebgl3, (6) Abf22-3, (7) BglPm + Bgp1, (8) BglPm + BglBX10, (9) BglPm + Tpebgl3, (10) BglPm + Abf22-3, (11) BglSk + Bgp1, (12) BglSk + BglBglBglX 10, (13) BglSk + Tpebgl3, (14) BglSk + Abf22-3, and (15) BglSk + BglPm.
After completion of the enzymatic reaction, an equal volume of water-saturated n-butanol was added to stop the reaction, and the n-butanol supernatant was evaporated in a water bath at 60 ℃ and then dissolved in methanol and filtered with a 0.45 μm filter. The final filtrate was subjected to UPLC analysis to determine the relative content of ginsenosides, and compared to untreated controls (fig. 6).
The data from the experimental results show that the four major ginsenosides Rb1, Rc, Rb2 and Rd account for 22.95 ± 1.08%, 24.26 ± 0.87%, 20.10 ± 0.93% and 15.68 ± 0.47%, respectively, of the untreated PPD-type ginsenoside mixture (fig. 7). BglPm single enzyme treatment produced large amounts of F2(262.83 + -4.92 mg), 46.12 + -1.39% of PPD-type ginsenoside mixture, 13.72 + -0.42% and 13.39 + -0.40% of CMc1 and CO, respectively (FIG. 7, FIG. 8). BglSk can hydrolyze a large amount of ginsenosides of all types and produce Gyp75, CMc, CY and CK at a ratio of more than 20% (fig. 3D). Bgpl, BglBX10, and Abf22-3 convert Rd to Rg3, Tpebgl3 convert both Rb1 and Rd to Rg3 (fig. 2, fig. 7).
The amount of Rg3 produced in the above four reactions is about 15% -30% of the PPD type ginsenoside mixture, and the peak value of Tpebgl3 is reached at 149.74 + -5.67 mg (FIG. 8). All combined enzyme treatments of BglPm with the other four enzymes with C-20 hydrolytic activity (Bgpl, BglBX10, Tpebgl3 and Abf22-3) yielded more than 40% Rh2, and more than 10% CMc1 and CO (FIG. 7). The combination of BglSk with these four enzymes can yield the right amount of Rg3, Gyp75, CMc, CY and Rh2, respectively, in a ratio between 10% and 20% (fig. 7). Both BglPm and BglSk were able to produce high ratios of CK (277.61 + -4.27 mg, 34.22 + -1.91%) (FIG. 7, FIG. 8). The highest yield of Rh2 (326.61 ± 7.04mg) was obtained by hydrolysis of 3.0g of PPD-type ginsenoside mixture with the BglPm + Bgp1 combination during all transformations, accounting for 10.89 ± 0.23% of PPD-type ginsenoside mixture (fig. 8).
4. Method for improving production efficiency of rare ginsenoside by multi-enzyme immobilized polymeric membrane reactor
In order to improve the utilization rate of enzyme and the separation efficiency of the enzyme and a product, experiments for converting PPD type ginsenoside mixture into Rh2 are carried out by taking hollow fiber membrane immobilized recombinant His-Bgp1 and His-BglPm as examples, and the effectiveness of the strategy is verified.
In order to adapt to the industrial scale up at normal temperature, the ginsenoside conversion reaction based on enzyme immobilization is further carried out under the conditions of pH8.0 and 25 ℃.
Polysulfone hollow fiber Membrane (MWCO) immobilized enzyme with molecular weight of 30kDa is adopted. The membrane was equilibrated in phosphate buffer (pH8.0) for 30min, and immobilized using pressure-driven filtration. The enzyme solution (0.5mg mL)-1) Using peristaltic pump at 10mLmin-1Is pumped into the chamber through the membrane, circulating for 2 h. The enzyme was embedded in an asymmetric, aqueous porous membrane and immobilized by a 30kDa fibrous membrane on the inner surface (FIG. 1). The amount of immobilized biocatalyst was measured by mass balance between the initial solution and the collected fractions at different time points (10, 20, 30, 40, 50, 60, 70, 80, 90 and 120 min).
The immobilized beta-glucosidase was hydrolyzed at 25 ℃ using standard analytical methods and its storage stability was monitored for 15 days. The reaction mixture contained 50mL of 50mM sodium phosphate buffer, pH8.0, 0.5mM pNPG and 50mg of immobilized beta-glucosidase.
5mL of additive was added to the feed tank during each cyclepNPG substrate solution (5mM), then at 25 ℃ at maximum flow rate (5mL min)-1) Circulate in the membrane reactor for 0.5 h. The absorbance of the supernatant at 400nm was measured to determine the concentration of p-nitrophenol. The activity of the immobilized enzyme after the first cycle was defined as control, relative activity was 100%. The remaining activity was calculated by dividing the enzyme activity on the day by the enzyme activity at the start of the immobilization stability test (day 1).
The content of immobilized enzyme rapidly increased in the first 60 minutes and then leveled off in the next 30 minutes (fig. 9A). After 2 hours of loading, the recombinase on the hollow fiber membrane reached a saturation level at about 45mg, indicating the porous region (-50 cm) of the asymmetric hollow fiber membrane2) Has a maximum loading concentration of 0.9mg cm-2(FIG. 9A).
The immobilized beta-glucosidase has high repeated utilization rate, and the relative activity of the immobilized beta-glucosidase is kept at about 85% after 9 cycles of repeated catalytic reaction on day 9. The immobilized β -glucosidase maintained 40% of the initial activity even after 15 consecutive cycles of repeated catalytic reactions (fig. 9B).
The enzymatic kinetic constants of free glycosidase and immobilized glycosidase were studied using mixtures of pNPG and PPD type ginsenosides as substrates, respectively. Wherein Km values of Bgpl and BglPm are respectively 0.85 +/-0.21 mM and 0.40 +/-0.12 mM, which are obviously higher than that of ginsenoside, and the affinity of the ginsenoside to natural substrates is higher. In particular, Rb1 has a Km value of BglPm of 0.04. + -. 0.0003mM, one order of magnitude lower (Table 1). This assay also measured Vmax of recombinant His-Bgp1, calculated kcat of recombinant His-Bgp1 and His-BglPm of recombinant pNPG. The catalytic efficiency (kcat/Km) depends on the enzyme and substrate, BglPm for pNPG (154.08 s)-1mM-1) The catalytic efficiency of (A) is obviously higher, which shows that the catalyst has high activity, Bgp1 is to Rb1(1.88 s)- 1mM-1) The catalytic efficiency of (2) was low. The Vmax value of the immobilized enzyme was about 5 times that of the free enzyme, indicating an increase in hydrolytic activity (Table 2). Km for free and immobilized enzymes were at similar levels, indicating that immobilization did not alter the affinity of the substrate (table 2).
TABLE 2 kinetic parameters of recombinant BglPm and Bgp1
3.0g of PPD-type ginsenoside mixture was added to a reaction cell where Bgp1 and BglPm or their free substances were immobilized, and the conversion efficiency was investigated by analyzing the change in the amount of abundant and rare ginsenosides at different times (FIG. 10). Rb1 and Rd were rapidly consumed within 12 hours, and intermediate Rg3 was accumulated in the initial reaction and then gradually decreased (fig. 6). Other intermediates, including F2, Gyp17, Gyp75 and CK, remained stably at low levels throughout the reaction (fig. 6). In the free enzyme system, Rh2 accumulated to 300.30 + -28.26 mg in 24 hours, which was 10.01% of the PPD-type ginsenoside mixture. Rh2 accumulated to 511.72. + -. 3.04mg in the immobilization system, accounting for 17.06% of the PPD-type ginsenoside mixture (FIG. 10). The final product of ginsenoside PPD gradually increased during hydrolysis (fig. 10). The immobilized glucosidase can promote the recycling of enzyme in the continuous glycosyl hydrolysis process, and rare ginsenoside is produced, so that the cost of the enzyme is reduced; the conversion rate of Rh2 generated by hydrolyzing a PPD-type ginsenoside mixture by using two immobilized glucosidases in combination was 68.32%.
Claims (10)
1. A biological preparation method of ginsenoside is characterized by comprising the following steps: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious root after induction culture.
2. The bioproduction method according to claim 1, wherein the method for inducing production of ginseng adventitious roots by tissue culture in step (1) comprises: (a) inoculating the ginseng root slice to the solution containing 1.0mg L-12,4-D+0.1mg L-1kinetin+30g L-1Induction on sucrose Induction MediumCallus tissue; (b) callus was transferred to 5.0mg L-1Carrying out callus propagation on an MS culture medium of IBA; (c) in the presence of 3.0mg L-1IBA and 30g L-1The callus was cultured on a sucrose MS solid medium, and the generation of adventitious roots was induced from the callus.
3. The biological preparation method according to claim 1, wherein the ginseng adventitious roots in the step (3) are transferred from the shake flask to the bioreactor to be cultured for a period of time, and then 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content; preferably, the ginseng adventitious roots are transferred from the shake flask to the bioreactor and cultured for 50 days, and 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content.
4. The biological preparation method according to claim 1, wherein the method for extracting ginsenoside from adventitious roots of ginseng in the step (4) comprises: (a) adding ginseng root powder to the methanol/water mixture overnight; (b) extracting with methanol as solvent by ultrasonic instrument: (c) filtering the supernatant of the extractive solution for sterilization, evaporating to remove methanol, and dissolving the residue in water; preferably, the methanol/water mixture in step (a) consists of methanol and water in a volume ratio of 4: 1; in the step (b), 80% methanol is used as a solvent to extract for 3 times at room temperature of an ultrasonic instrument, and the extraction time is 0.5h each time.
5. The bioprocess of claim 1 further comprising: carrying out enzymatic conversion reaction by using beta-glucosidase as converting enzyme and ginsenoside mixture extracted from adventitious root as converting substrate.
6. The process of claim 5, wherein the β -glucosidase is selected from any one or any combination of more than one of BglSk, BglPm, bgpp1, bglb10, Tpebgl3, or Abf 22-3.
7. The bioproduction process of claim 6 wherein the β -glucosidase is selected from the group consisting of BglSk, BglPm, a combination of BglSk and bgpp1, a combination of BglSk and BglBX10, a combination of BglSk and Tpebgl3, a combination of BglSk and Abf22-3, a combination of BglPm and bgpp1, a combination of BglPm and BglBX10, a combination of BglPm and Tpebgl3, and a combination of BglPm and Abf 22-3.
8. The biological preparation method according to claim 5, wherein the enzymatic conversion reaction is carried out by reacting a β -glucosidase solution with the extracted ginsenoside mixture in 50mM sodium phosphate buffer at 37 ℃.
9. The biological process of claim 5, wherein the β -glucosidase is an immobilized β -glucosidase.
10. The biological preparation method according to claim 9, wherein the immobilized β -glucosidase is immobilized by polysulfone hollow fiber membrane, preferably by equilibrating polysulfone hollow fiber membrane in phosphate buffer of pH8.0 for 30min, and immobilizing enzyme by pressure-driven filtration, and by peristaltic pump for 10mL min of β -glucosidase solution-1Is pumped through the lumen of the membrane and circulated for 2h, β -glucosidase is embedded in an asymmetric, aqueous porous membrane and immobilized by a 30kDa fibrous membrane on the inner surface.
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| CN117210442A (en) * | 2023-05-30 | 2023-12-12 | 西北大学 | Beta-glycosidase SS-BGL mutant for modifying ginsenoside and application thereof |
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