A beta-glucosidase p03 for rare ginsenoside conversion and a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN NATIONALITIES UNIVERSITY
- Filing Date
- 2026-05-11
- Publication Date
- 2026-06-09
AI Technical Summary
Existing β-glucosidases for the preparation of rare ginsenosides suffer from problems such as poor substrate specificity, harsh catalytic conditions, poor thermal stability, low conversion efficiency, and intellectual property risks, making them unsuitable for large-scale production.
A β-glucosidase PO3 derived from Xanthomonas pseudoepiplocarpa TY3-10 with a unique amino acid sequence (SEQ ID NO.1) was developed. It was expressed in Escherichia coli Origami2 via a recombinant plasmid under mild catalytic conditions and can specifically hydrolyze the C3 and C20 glucosyl groups of ginsenosides to prepare rare ginsenosides F2, CO, C-Mx1, and C-Mc1.
This method achieves efficient conversion of rare ginsenosides, with high yield of target products, mild reaction conditions, suitability for large-scale production, reduced production costs, and avoidance of intellectual property infringement risks.
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Figure CN122168575A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of genetic engineering and biotechnology, specifically relating to a β-glucosidase PO3 for the conversion of rare ginsenosides, its preparation method, and its application. Background Technology
[0002] Ginseng ( Panax ginseng Ginsenosides (CAMeyer) are a traditional and precious Chinese medicinal herb. Modern pharmacological studies have confirmed that ginsenosides are its core active ingredients, possessing significant anti-tumor, anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory activities. Currently, over 300 ginsenoside monomers have been isolated and identified. Among them, the original saponins such as Rb1, Rb2, Rc, Re, and Rg1 are present in relatively high amounts in ginseng, while rare ginsenosides such as F2, CO, C-Mx1, and C-Mc1 are present in extremely low amounts naturally, but their pharmacological activities are significantly superior to those of the original saponins. These rare ginsenosides are currently the core of research in the deep processing and innovative drug development of ginseng.
[0003] The main methods for preparing rare ginsenosides include chemical synthesis, physical degradation, and biotransformation. Chemical synthesis involves complex reaction steps, produces numerous byproducts, causes significant environmental pollution, and easily generates chiral isomers, resulting in extremely high separation and purification costs. Physical degradation (high temperature, acid hydrolysis) produces a mixture of various saponins, making directional transformation impossible and leading to low yields of the target product. Biotransformation methods include whole-cell microbial transformation and enzymatic transformation. Enzymatic transformation offers advantages such as mild reaction conditions, strong catalytic specificity, fewer byproducts, high product purity, and environmental friendliness, making it the mainstream development direction for the large-scale preparation of rare ginsenosides.
[0004] β-glucosidase is the core catalyst for the enzymatic conversion of ginsenosides. It can specifically hydrolyze the glucose groups on the outer side of the sugar chains of proto-ginsenosides, directionally generating rare ginsenosides. Currently reported β-glucosidases for ginsenoside conversion are mostly derived from Aspergillus, Bacillus, and thermophilic bacteria, and generally suffer from the following drawbacks: First, poor substrate specificity, prone to over-hydrolysis, resulting in numerous byproducts and difficulty in separating the target product; second, demanding catalytic conditions, with most thermostable enzymes requiring reactions above 60°C, leading to high energy consumption for industrial production, while room-temperature enzymes have poor thermal stability and are easily inactivated; third, low conversion efficiency of ginsenoside substrates and long reaction times, making them unsuitable for large-scale production; and fourth, most reported enzyme sequences have high homology, lacking innovation and posing a risk of intellectual property infringement.
[0005] Therefore, the discovery of novel β-glucosidases with strong substrate specificity, high catalytic efficiency, and mild reaction conditions is of great theoretical significance and practical application value for the green and large-scale preparation of rare ginsenosides. Summary of the Invention
[0006] The purpose of this invention is to provide a β-glucosidase PO3 for the conversion of rare ginsenosides, its preparation method and application. The β-glucosidase PO3 provided by this invention can not only convert total ginsenosides into rare ginsenosides F2, CO, C-Mx1 and C-Mc1, but also the conversion reaction conditions are mild, green and environmentally friendly, and the yield of the target product is high.
[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a β-glucosidase PO3, which is derived from Xanthomonas pseudoepiplocarpa TY3-10, and its amino acid sequence is shown in SEQ ID NO.1.
[0008] The present invention also provides a nucleic acid molecule encoding the above-mentioned β-glucosidase PO3, the nucleotide sequence of which is shown in SEQ ID NO.2.
[0009] The present invention also provides a recombinant plasmid comprising the above-mentioned nucleic acid molecule and a pET32a or pET28a vector.
[0010] The present invention also provides a recombinant engineered bacterium, which includes the above-mentioned recombinant plasmid, and its host bacterium is Escherichia coli Origami2 (DE3).
[0011] This invention also provides a method for preparing the above-mentioned β-glucosidase PO3, specifically including the following steps: S1. Using the genomic DNA of Xanthomonas pseudoxanthomonas TY3-10 as a template, a PCR amplification reaction was performed to obtain the nucleic acid molecule; S2. The coding gene obtained in step S1 and the pET32a / pET28a vector were double-digested, and the digestion products were recovered and ligated to obtain a recombinant plasmid containing the β-glucosidase P03 coding gene. S3. Transform the recombinant plasmid obtained in step S3 into Escherichia coli Origami2(DE3) competent cells, induce the expression of the target protein with IPTG, and obtain β-glucosidase PO3 after separation and purification.
[0012] Preferably, the nucleotide sequences of the primers used for PCR amplification in step S1 are shown in SEQ ID NO.3-4.
[0013] Preferably, the PCR amplification reaction program in step S1 is as follows: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 43 s, 305 cycles; 72℃ final extension for 5 min; and 4℃ incubation.
[0014] Preferably, the IPTG induction conditions in step S3 are: TB medium, 37°C, 220 rpm incubation until OD. 600 =0.8-1.0, add IPTG to a final concentration of 0.05-0.1 mM, and induce at 18-30℃ and 160-220 rpm for 16-24 h.
[0015] This invention also provides the application of the above-mentioned β-glucosidase PO3 in the preparation of rare ginsenosides.
[0016] Preferably, the β-glucosidase PO3 can convert total ginsenosides into rare ginsenosides F2, CO, C-Mx1 and C-Mc1; Specifically, this includes converting ginsenoside Rb1 to F2, ginsenoside Rb2 to CO, ginsenoside Rb3 to C-Mx1, and ginsenoside Rc to C-Mc1.
[0017] The beneficial effects of this invention are: (1) Strong sequence novelty: The β-glucosidase P03 of the present invention is derived from a novel environmental isolate of Pseudomonas genus that was independently screened. According to NCBIBLAST comparison, the homology of this enzyme with the reported β-glucosidase for ginsenoside conversion is less than 50%, and the homology with the P09 glycosidase of the same source is only 47.92%. It has completely independent intellectual property rights and there is no risk of infringement.
[0018] (2) Excellent substrate specificity: The PO3 enzyme of the present invention can specifically hydrolyze the glucose groups at the C3 and C20 positions of ginsenosides without excessive hydrolysis side reactions. The target product is singular, which significantly reduces the cost of subsequent separation and purification and solves the industry pain point of many byproducts of existing enzymes.
[0019] (3) Excellent catalytic performance: The optimal catalytic pH of the PO3 enzyme of this invention is 8.0, the optimal catalytic temperature is 40℃, the reaction conditions are mild, and the energy consumption for industrial production is low; it can maintain more than 70% enzyme activity in the pH range of 7.5-8.5 and 30-40℃, and has good stability; it has high conversion efficiency for ginsenoside Rb1, and the conversion rate can reach more than 85% under optimal conditions after 12 hours of reaction, and the yield of the target product is ≥80%, which is suitable for the needs of large-scale production.
[0020] (4) High expression efficiency and simple preparation method: The recombinant engineered bacteria constructed in this invention can achieve efficient soluble expression of PO3 enzyme. After induction, the enzyme activity per unit volume of fermentation broth can reach 1.14 U / mL. No complicated purification steps are required, and the crude enzyme solution can be directly used for catalytic reaction, which greatly reduces production costs.
[0021] (5) Broad application prospects: The PO3 enzyme of the present invention can be adapted to a variety of substrates such as total ginsenosides and monomeric saponins. The catalytic products, rare ginsenosides F2, CO, C-Mx1 and C-Mc1, can be widely used in health food, biomedicine, cosmetics and other fields, and have extremely high commercial value. Attached Figure Description
[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0023] Figure 1 The image shows the SDS-PAGE purity of the purified β-glucosidase PO3; lane M is the protein marker, lane 1 is the whole-cell lysate of the host bacteria transformed with the empty vector plasmid, lane 2 is the whole-cell lysate after induced expression, and lane 3 is the purified PO3 protein. Figure 2 The figure shows the results of the enzymatic property determination of β-glucosidase PO3 of the present invention; where a is the result of the optimal reaction temperature determination; b is the result of the temperature stability determination; and c is the result of the optimal reaction pH and pH stability determination. Figure 3 This invention provides a hydrolysis pathway diagram for the conversion of total ginsenosides from ginseng root by β-glucosidase PO3 into rare ginsenosides. Figure 4 The figure shows the HPLC detection results of the conversion of total saponins from ginseng root by β-glucosidase PO3 according to the present invention; where a is the substrate control after 0 h of reaction and b is the product detection result after 12 h of reaction. Detailed Implementation
[0024] This invention provides a β-glucosidase PO3, wherein the amino acid sequence of the β-glucosidase PO3-AA is as shown in SEQ ID NO.1, or has more than 90% homology with the amino acid sequence shown in SEQ ID NO.1; the nucleic acid molecule encoding the amino acid sequence is as shown in the nucleotide sequence SEQ ID NO.2 (PO3), or has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.2 and encodes an amino acid sequence with the same catalytic activity.
[0025] P03-AA:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFMLDQQAGTAHPALWPQLRSPLKPDPRLEARIDALLAKMSPEEKVGQVVQADIASVTPDDLKTYRLGSILAGGSSDPGGQYNAPASAWLALADEFYAASMDTSGGKYTAIPVIFGVDAVHGHNNVVGATLFPHNIGLGATHDPALMGQIAQATAAEMRATGIDWTFAPTLAVPQDDRWGRTYEGYSENPDVVRAYAGPLIQGLQGKPGGQDFLKGAHVVATAKHFLADGGTFEGRDQGDARIDEATLRDVHGAGYPPALKAGVQAVMVSFSSWNGVKMSGNASLLDAVLKQRMGFDGFVVSDWNGHASVPGGSRENCVPAFVAGVDMIMAPDTWKGCYEHLLAAEKGQALPAGRLDEAVRRILRVKFRAGMFEAGKPSTRGVAGRFDLLGSAEHRAIARRAVRESLVLLKNNGGLLPLDPRAKLLVAGDGADDMMMQSGGWTLSWQGTGLKPEDFPHAQTIAAALREQVARAGGQVELAADGHYTRKPDAAVVVFGETPYAEFQGDLKVLAYRPGDDRDLKLLRKLKAEGIPVVAVFLSGRPLWMNREINAADAFVAAWLPGSEGGGVADVLLRDAKGKVQHDFRGTLSYSWPRTAVQTPLNVGQPGYDPQFAYGYGLTYAKPASLPALSEDAGMDLASLGAQTFFERGTPATGWTLRVQSVVGKPRTLSQPSGQRDAPDVAIAPLDYKAQEDAWKITWKQTGEIALLAPRPLDLVRETNGNVMLRVTLRVDAAPSQPGAELFLECGPGCGASLPIDNALAKAPRGTWGTLGIPLKCFAARNAQMGQVTAPLGWRMPAGTVLSIHEVGLGTEAQHVLDCAVKA(SEQID NO.1)。
[0026]
[0027] The β-glucosidase PO3 described in this invention is derived from *Xanthomonas* spp. ( Pseudoxanthomonas sp The optimal catalytic pH for the isolated strain was 8.0, and the optimal catalytic temperature was 40℃. Within the pH range of 7.5-8.5 and the temperature range of 30-40℃, the enzyme activity retention rate was ≥70%. In a system with a final concentration ≤5% methanol and 3% anhydrous ethanol, the enzyme activity retention rate was ≥90%. β-glucosidase PO3 specifically hydrolyzes the glucosyl groups at the C3 and C20 positions of ginsenosides Rb1, Rb2, Rb3, and Rc without excessive hydrolysis side reactions. When p-nitrophenyl-β-D-glucosidase (pNPG) was used as a substrate, the Michaelis constant Km was 1.61 ± 0.08 mmol / L, and the maximum reaction rate Vm was 1.296 mmol / (h·L).
[0028] The present invention describes the use of β-glucosidase PO3 as a catalyst to enzymatically hydrolyze ginsenosides Rb1, Rb2, Rb3, Rc or total ginsenosides under pH 7.5-8.5 and temperature 35-45℃ to prepare rare ginsenosides F2, CO, C-Mx1 and C-Mc1; the enzymatic hydrolysis time is ≥8h and the substrate conversion rate is ≥85%.
[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0030] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.
[0031] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.
[0032] Example 1: Cloning of the β-glucosidase PO3 encoding gene and construction of a recombinant vector 1.1 Template genomic DNA extraction Take 50 mL of fresh culture of *Xanthomonas pseudoepiplo* TY3-10 selected independently, centrifuge at 4000 g for 10 min to collect the bacterial cells, and extract genomic DNA using the CTAB method. The specific steps are as follows: (1) Resuspend the bacterial cells in 9.5 mL TE buffer, add 0.5 mL 10% SDS and 50 μL 20 mg / mL proteinase K, mix well and incubate at 37℃ for 1 h; (2) Add 1.8 mL of 5 mol / L NaCl and 1.5 mL of CTAB / NaCl solution, mix well and incubate at 65℃ for 20 min; (3) Add an equal volume of chloroform / isoamyl alcohol (24:1), mix well, centrifuge at 6000 g for 10 min, and take the upper aqueous phase; (4) Add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix well, centrifuge at 6000 g for 10 min, and take the upper aqueous phase; (5) Add 0.6 times the volume of isopropanol, gently shake until white filamentous DNA precipitate forms, wash twice with 70% ethanol, air dry at room temperature, dissolve in 500 μL TE buffer, and store at -20℃ for later use.
[0033] 1.2 Primer Design and PCR Amplification Based on the gene sequence encoding the PO3 enzyme, specific amplification primers were designed, with EcoRI and NotI restriction sites introduced into the forward and reverse primers, respectively (underlined). The primer sequences are as follows: Upstream primer: 5'-GAATTCATGCTCGATCAGCAGGCGG-3' (SEQ ID NO.3); Downstream primer: 5'-TGCGGCCGCTCATTTCACCGCGCAATCGAG-3' (SEQ ID NO.4).
[0034] Using the extracted genomic DNA as a template, PCR amplification was performed. The reaction system (50 μL) consisted of: 25 μL PrimeSTARMax DNA polymerase, 2 μL upstream primer, 2 μL downstream primer, 1 μL genomic DNA template, and 20 μL ddH2O.
[0035] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 43 s, 305 cycles; 72℃ final extension for 5 min; 4℃ incubation.
[0036] After the PCR product was detected by 1% agarose gel electrophoresis, it was purified using a gel extraction kit to obtain the gene fragment encoding the PO3 enzyme.
[0037] 1.3 Construction of Recombinant Expression Vectors The purified PCR product and pET32a vector were double-digested with EcoRI and NotI, respectively. The digestion system (50 μL) consisted of: 5 μL 10×QuickCut Buffer, 30 μL DNA / vector, 2 μL EcoRI, 2 μL NotI, and 11 μL ddH2O. After reacting at 37°C for 30 min, the digested target gene fragment and linearized vector fragment were recovered by agarose gel electrophoresis.
[0038] The target gene fragment and the linearized vector fragment were mixed at a molar ratio of 3:1 and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated upside down at 37°C for 12–16 h. Single colonies were picked for colony PCR verification. Positive clones were sent for sequencing. Plasmids were extracted from correctly sequenced strains, which became the recombinant expression plasmid pET32a-P03.
[0039] Example 2: Construction and Induced Expression of Recombinant Engineered Bacteria 2.1 Construction of recombinant engineered bacteria The correctly sequenced recombinant expression plasmid pET32a-P03 was transformed into Escherichia coli Origami2(DE3) competent cells, plated on LB agar plates containing ampicillin, and incubated upside down at 37°C for 12 h. Single colonies were picked for colony PCR verification, and positive clones were identified as recombinant engineered bacteria expressing β-glucosidase P03.
[0040] 2.2 Induced Expression A single colony of the recombinant engineered bacteria was picked and inoculated into 5 mL of LB liquid medium containing ampicillin. The culture was carried out at 37°C and 220 rpm for 12 h to prepare the seed culture.
[0041] The seed culture was transferred to 50 mL of TB medium at a 1% inoculation rate and cultured at 37°C with shaking at 220 rpm until OD reached. 600 =0.8-1.0, add 0.1 mM IPTG to a final concentration, and induce at 18℃ and 160 rpm for 24 h. After induction, collect the bacterial cells by centrifugation at 4℃ and 8000 rpm for 15 min, wash twice with 50 mM Tris-HCl buffer (pH 8.0), resuspend, sonicate, centrifuge at 12000 rpm for 10 min, and take the supernatant and precipitate for SDS-PAGE electrophoresis to verify the soluble expression of the target protein.
[0042] The results showed that the recombinant engineered bacteria constructed in this invention can efficiently express β-glucosidase PO3. The target protein is mainly found in the supernatant, with high soluble expression levels and no obvious inclusion bodies.
[0043] Example 3: Preparation and purification of β-glucosidase PO3 3.1 Preparation of crude enzyme solution Following the method in Example 2, 200 mL of recombinant engineered bacteria were cultured. After induction, the bacterial cells were collected by centrifugation. The cells were resuspended in 10 mL of 1×Binding Buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and sonicated on ice (200 W, 3 s sonication, 5 s interval, 30 cycles). The cells were then centrifuged at 4°C and 12000 rpm for 30 min, and the supernatant was collected as the crude enzyme solution.
[0044] 3.2 Ni 2+ Affinity chromatography purification Prepare 1×Wash Buffer (20 mM Tris-HCl, 300 mM NaCl, 60 mM imidazole, pH 8.0) and 1×Elution Buffer (20 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0). Pack 2 mL of Ni-NTA agarose packing material into a column, and equilibrate the column sequentially with 10 mL of sterile water and 10 mL of 1×Binding Buffer. Load the crude enzyme solution onto the column at a flow rate of approximately 1 mL / min. After loading, wash away unbound contaminating proteins with 30 mL of 1×Wash Buffer, and then elute the target protein with 20 mL of 1×Elution Buffer. Collect the elution peak.
[0045] The eluent was desalted by ultrafiltration using a 30 kDa ultrafiltration tube and replaced with 20 mM Tris-HCl buffer (pH 8.0) to obtain purified β-glucosidase PO3. SDS-PAGE electrophoresis confirmed that the purity reached electrophoretic purity. The results are as follows: Figure 1 As shown.
[0046] Example 4: Determination of the enzymatic properties of β-glucosidase PO3 4.1 Enzyme activity assay method Enzyme activity was determined using the pNPG colorimetric method. The reaction system consisted of 200 μL of 20 μL of 5 mM pNPG and 20 μL of appropriately diluted enzyme solution. The reaction was carried out at room temperature for 10 min, and the reaction was terminated by adding 140 μL of 0.2 M Na2CO3. The absorbance at 405 nm was measured using an ELISA reader.
[0047] Enzyme activity unit (U) definition: Under the above standard reaction conditions, the amount of enzyme required to generate 1 μmol of p-nitrophenol (pNP) per minute is 1 enzyme activity unit.
[0048] 4.2 Determination of Optimal Reaction Temperature Within the temperature range of 25-60℃, a temperature gradient of 5℃ was established, and the enzyme activity of PO3 enzyme was measured at each temperature. The highest enzyme activity was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The results are as follows: Figure 2 As shown in (a), the optimal catalytic temperature of the PO3 enzyme is 40℃, and it can maintain more than 70% of the relative enzyme activity in the range of 30-40℃.
[0049] 4.3 Determination of Optimal Reaction pH The activity of PO3 enzyme was measured at 40℃ using citrate-disodium hydrogen phosphate buffer (pH 3.0–6.0), Tris-HCl buffer (pH 6.0–9.0), glycine-NaOH buffer (pH 9.0–10.0), and Na2CO3-NaHCO3 buffer (pH 11.0–12.0). The highest enzyme activity was defined as 100%, and the relative enzyme activity at different pH values was calculated. The results are as follows: Figure 2 As shown in (c), the optimal catalytic pH for PO3 enzyme is 8.0, and it can maintain more than 60% of its relative enzyme activity in the pH range of 5.0-9.0.
[0050] 4.4 Temperature stability determination The purified PO3 enzyme was incubated at 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. Residual enzyme activity was measured at 0, 20, 40, 60, 90, 120, 150, and 180 min, with the enzyme activity of the unincubated enzyme solution considered as 100%. Results are as follows: Figure 2 As shown in (b), the PO3 enzyme retains ≥70% of its activity after being incubated at 30-40℃ for 3 h, demonstrating good room temperature stability.
[0051] 4.5 pH stability determination The purified PO3 enzyme was placed in buffer solutions of different pH values and stored at 4°C for 24 h. The residual enzyme activity was then measured, with the activity of the untreated enzyme solution being taken as 100%. The results showed that the PO3 enzyme retained ≥50% of its activity after being stored at pH 6.0-9.0 for 24 h, demonstrating a wide applicable pH range.
[0052] 4.6 Enzyme Reaction Kinetic Determination Using pNPG as a substrate, an enzyme activity was determined under standard reaction conditions with a substrate concentration gradient ranging from 0.1 to 5.0 mM. The Michaelis constant Km and the maximum reaction rate Vm were calculated using the Lineweaver-Burk double reciprocal method. The results showed that when pNPG was used as a substrate, the PO3 enzyme exhibited a Km value of 1.61 ± 0.08 mmol / L and a Vm value of 1.296 mmol / (h·L), indicating strong substrate affinity and high catalytic efficiency.
[0053] Example 5: Application of β-glucosidase PO3 in the conversion and preparation of rare ginsenosides 5.1 Enzyme Conversion Reaction System The hydrolysis pathway of β-glucosidase PO3 in this invention for converting total ginsenosides from ginseng root into rare ginsenosides is shown in the diagram below. Figure 3 As shown.
[0054] 50 mL reaction system: 50 mM Tris-HCl buffer (pH 8.0) as the reaction medium, ginseng root total saponins final concentration 35 mg / mL, PO3 enzyme final concentration 0.57 U / mL.
[0055] 5.2 Reaction conditions and detection The reaction system was placed in a shaker at 40℃ and 180 rpm. Samples were taken at 0, 3, 6, 12, 18 and 24 h. An equal volume of chromatographically pure methanol was added to terminate the reaction. The enzyme was inactivated by boiling in a water bath for 10 min. The mixture was centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm organic phase filter membrane, and the conversion product was detected by HPLC.
[0056] HPLC detection conditions: Agilent 1260 high-performance liquid chromatograph, C18 column (250 mm × 4.6 mm, 5 μm), detection wavelength 203 nm, column temperature 30℃, flow rate 1.0 mL / min, mobile phase was water (A) and acetonitrile (B), gradient elution was performed as follows: 0 min, 79% A, 21% B; 5 min, 74% A, 26% B; 9 min, 68% A, 32% B; 39 min, 66.5% A, 33.5% B; 42 min, 64% A, 36% B; 44 min, 62% A, 38% B; 59 min, 35% A, 65% B; 64 min, 35% A, 65% B; 69 min, 15% A, 85% B; 75 min, 15% A, 85% B; 80 min, 79% A, 21% B.
[0057] 5.3 Conversion Results The results are as follows Figure 4 As shown in (ab), after 12 h of reaction, the conversion rates of ginsenosides Rb1, Rb2, Rb3, and Rc reached over 85%, and the yields of the target rare ginsenosides F2, CO, C-Mx1, and C-Mc1 reached 80%, with no obvious excessive hydrolysis byproducts, thus achieving the directional and efficient conversion of ginsenosides.
[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A beta-glucosidase P03, characterized in that, The β-glucosidase PO3 was obtained from Xanthomonas pseudoxanthomonas TY3-10, and its amino acid sequence is shown in SEQ ID NO.
1.
2. A nucleic acid molecule encoding the β-glucosidase P03 according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.
2.
3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule of claim 2 and the pET32a or pET28a vector.
4. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria includes the recombinant plasmid described in claim 3, and its host bacteria is Escherichia coli Origami2 (DE3).
5. The method for preparing β-glucosidase PO3 according to claim 1, characterized in that, Includes the following steps: S1. Using the genomic DNA of Xanthomonas pseudoxanthomonas TY3-10 as a template, a PCR amplification reaction was performed to obtain the nucleic acid molecule described in claim 2; S2. The coding gene obtained in step S1 and the pET32a / pET28a vector were double-digested, and the digestion products were recovered and ligated to obtain a recombinant plasmid containing the β-glucosidase P03 coding gene. S3. Transform the recombinant plasmid obtained in step S3 into Escherichia coli Origami2(DE3) competent cells, induce the expression of the target protein with IPTG, and obtain β-glucosidase PO3 after separation and purification.
6. The preparation method according to claim 5, characterized in that, The nucleotide sequences of the primers used for PCR amplification in step S1 are shown in SEQ ID NO.3-4.
7. The preparation method according to claim 5, characterized in that, The PCR amplification reaction program described in step S1 is as follows: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 43 s, 305 cycles; 72℃ final extension for 5 min; 4℃ incubation.
8. The preparation method according to claim 5, characterized in that, The conditions for the IPTG induction in step S3 are: TB medium, 37°C, 220 rpm cultivation to OD 600 = 0.8-1.0, addition of 0.05-0.1 mM IPTG to the final concentration, 18-30°C, 160-220 rpm induction for 16-24 h.
9. The use of the β-glucosidase PO3 of claim 1 in the preparation of rare ginsenosides.
10. The application according to claim 9, characterized in that, The β-glucosidase PO3 can convert total ginsenosides into rare ginsenosides F2, CO, C-Mx1 and C-Mc1. Specifically, this includes converting ginsenoside Rb1 to F2, ginsenoside Rb2 to CO, ginsenoside Rb3 to C-Mx1, and ginsenoside Rc to C-Mc1.