A beta-glucosidase ge000505 for saponin conversion and a preparation method and application thereof

Abstract

The application discloses a kind of β-glucosidase GE000505 for saponin conversion and its preparation method and application, belong to biotechnology field, the amino acid sequence of the β-glucosidase GE000505 as shown in SEQ ID NO.1, it is encoded by the gene with nucleotide sequence as shown in SEQ ID NO.2.This β-glucosidase GE000505 of the application can convert high content ginseng and sanchi saponin into rare saponin R2, Rg3, Rg2 and Rh1, can convert sanchi saponin R1 into R2 entirely, convert ginsenoside Rd into Rg3 entirely, convert ginsenoside Re into Rg2 entirely, convert ginsenoside Rg1 into Rh1 entirely, convert ginsenoside F1 into aPPT entirely, convert ginsenoside F2 into aPPD entirely, solve the problem that existing β-glucosidase is not resistant to alcohol in the conversion of ginsenoside.

Description

TECHNICAL FIELD

[0001] The present application relates to a β-glucosidase, in particular to a β-glucosidase GE000505 for saponin conversion and a preparation method and application thereof. BACKGROUND

[0002] As a traditional precious Chinese medicinal material, ginseng has core active components, ginsenosides, which have multiple pharmacological activities such as anti-tumor, immune regulation, and neuroprotection (Guo J L et al. Chinese Journal of Chinese Materia Medica, 2024, 49(2): 304−314). However, more than 80% of the saponin components in natural ginseng are polysaccharide-based prototype saponins (such as Rb1, Rb2, Rc, Re, etc.), and the content of rare saponins with better pharmacological activities (such as Rg3, Rg2, Rh1, CK, etc.) is extremely low (less than 1%) (Chang K H et al. Journal of Ginseng Research, 2014, 38(1): 47−51).

[0003] Rg3 is a rare ginsenoside with anti-tumor activity as its core function. In past studies, Rg3 has shown pharmacological effects on various breast cancer cells (such as MDA-MB-231, FM3A, MDA-MB-453, MCF-7), and the main mechanisms of action include: inducing apoptosis of breast cancer cells; regulating epithelial-mesenchymal transition in breast cancer; regulating miRNAs in breast cancer cells; regulating long non-coding RNAs in breast cancer cells; affecting methylation in breast cancer cells; regulating ubiquitination and histone modification in breast cancer, etc. (Xu Zichen et al. Chinese Traditional and Herbal Drugs, 2022, 53(20): 6601-6610). It is worth noting that Rg3 can regulate breast cancer cells MCF-7 through multiple mechanisms: Rg3 inhibits the expression of related signaling pathways to promote MCF-7 apoptosis (Xu Zichen et al. Chinese Traditional and Herbal Drugs, 2022, 53(20): 6601-6610); induces G0 / G1 phase arrest of MCF-7 cells to inhibit the proliferation of MCF-7 cells (Icard P et al. Trends Biochem Sci, 2019, 44(6): 490-501); increases the expression of autophagy markers LC3-I / LC3-II, and promotes the autophagy of MCF-7 cells (Chen Kaiyun et al. Contemporary Medicine, 2016, 22(23): 1-3). The regulation of Rg3 on breast cancer cells MCF-7 through multiple mechanisms provides a strong guarantee for Rg3 in breast cancer-related treatment, and Rg3 has the effects of antioxidant, immune enhancement, and adjuvant chemotherapy attenuation. In addition, studies have shown that ginsenoside Rg2 can inhibit the proliferation of various breast cancer cells by activating autophagy (ATG7 / LC3-II pathway) and inducing apoptosis, especially in breast cancer cells MCF-7 (Chung Y et al. Anim Cells Syst, 2018, 22(6): 382−389), and Rg2 focuses on cardiovascular protection, can prevent heart attack / cerebral infarction, improve heart blood supply, and has the characteristics of anti-inflammatory, memory enhancement, and anti-aging.

[0004] Gypenoside R2 can improve obesity-related lipid metabolism disorder by activating the AhR-CYP pathway, inhibiting the expression of FASN and ACC1, and promoting the expression of ATGL (Gypenoside R2 improves obesity-related lipid metabolism disorder by activating the AhR-CYP pathway, Wang Shengchen. Beijing Union Medical College. 2024). Ginsenoside Rh1 exhibits multi-dimensional regulation, including anti-inflammatory, anti-allergic, inhibition of tumor cell proliferation, improvement of obesity by regulating fat metabolism, and inhibition of microglial inflammatory response. Due to the reduction in the number of glycosyl groups or structural modification, this type of rare saponin significantly improves bioavailability and targeting, but the scarcity of natural sources seriously restricts clinical application and industrial development (Research progress of biotransformation and hypoglycemic activity of rare ginsenosides, Journal of Jilin Agricultural University. 2023, 45(6): 674−684).

[0005] Beta-glucosidase can hydrolyze glycosidic bonds to release non-reducing end glucoside residues and corresponding ligands from glycosides or oligosaccharides. It generally acts on beta-(1, 4) glucosidic bonds and can also act on beta-(1, 2), beta-(1, 3), and beta-(1, 6) glucosides. High-content ginseng can be converted into rare saponins by beta-glucosidase. The solubility of ginsenosides in alcohol is much higher than that in water. In order to improve production efficiency, high-concentration ginsenosides are often required for reaction, but enzymes are prone to denaturation and inactivation in alcohol, making alcohol-intolerant beta-glucosidase difficult to convert high-concentration ginsenosides.

[0006] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0007] The purpose of the present application is to provide a β-glucosidase GE000505 for saponin conversion and its preparation method and application. The β-glucosidase GE000505 of the present application can convert high-content ginseng and gypenoside into rare saponins R2, Rg3, Rg2 and Rh1, and saponin aPPT and aPPD, and can tolerate alcohol solvent in the conversion reaction.

[0008] In order to achieve the above purpose, the present application provides a β-glucosidase GE000505 for saponin conversion, the amino acid sequence of which is shown in SEQ ID NO. 1, and the β-glucosidase GE000505 is encoded by a gene with a nucleotide sequence shown in SEQ ID NO. 2.

[0009] The second object of the present application is to provide the coding gene of the beta-glucosidase GE000505, the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0010] The third object of the present application is to provide a recombinant expression vector comprising the coding gene.

[0011] Preferably, the vector is selected from pET-28a(+).

[0012] The fourth object of the present application is to provide a recombinant expression bacterium comprising the coding gene.

[0013] Preferably, the bacterium is selected from BL21(DE3).

[0014] The fifth object of the present application is to provide a preparation method of the beta-glucosidase GE000505, which comprises: connecting the nucleotide sequence shown as SEQ ID NO. 4 sge000505-opt to a vector to obtain a recombinant plasmid; transforming the recombinant plasmid into a bacterium to obtain a recombinant expression bacterium; inoculating the activated bacterium into a liquid culture medium containing Kan, and culturing at 37℃ in a shaker at 180 rpm / min overnight to activate the recombinant strain; inoculating the activated bacterium into a liquid culture medium containing Kan, and culturing at 37℃ in a shaker at 180 rpm / min to an OD 600 of 0.6-1, adding IPTG for induction, and continuing to shake culture at 20℃ in a shaker at 180 rpm / min to induce the production of recombinant protein.

[0015] Preferably, the recombinant strain is inoculated into a liquid Kan-LB culture medium at an inoculation amount of 1‰, and cultured at 37℃ in a shaker at 180 rpm / min overnight to activate the recombinant strain; the activated bacterium is inoculated into a LB liquid culture medium containing 50 μg / mL Kan at an inoculation amount of 1%, and cultured at 37℃ in a shaker at 180 rpm / min to an OD 600 of 0.6-1, adding IPTG at a final concentration of 0.7 mM for induction, and continuing to shake culture at 20℃ in a shaker at 180 rpm / min to induce the production of recombinant protein.

[0016] Preferably, the method further comprises: centrifuging the induced bacterium to collect the bacterial cells; adding a buffer to suspend the bacterial cells, and then ultrasonically breaking the bacterial cells; centrifuging the intracellular concentrated crude enzyme solution to collect the supernatant; and using Nickel-NTA Agarose and imidazole to respectively perform affinity and purification on the target protein to obtain the GE000505 purified enzyme.

[0017] More preferably, the bacteria solution after induction is centrifuged at 4℃, 6000 rpm / min, and the bacteria bodies are collected; the bacteria bodies are suspended in pH=7.0 McIlvaine buffer at 1% of the volume of the bacteria solution, and then the bacteria bodies are broken by ultrasonic waves; the intracellular concentrated crude enzyme solution is centrifuged at 12000 rpm / min to obtain the supernatant, and the target protein is affinity-purified by using Nickel-NTA Agarose and 0-500 mM imidazole to obtain the purified GE000505 enzyme.

[0018] The sixth object of the present application is to provide the application of the β-glucosidase GE000505, the coding gene, the recombinant expression vector or the recombinant expression bacteria in the conversion of ginsenosides and notoginsenosides.

[0019] Preferably, the application comprises any one or more of the following:

[0020] 1) converting notoginsenoside R1 into notoginsenoside R2;

[0021] 2) converting ginsenoside Rd into ginsenoside Rg3;

[0022] 3) converting ginsenoside Re into ginsenoside Rg2;

[0023] 4) converting ginsenoside Rg1 into ginsenoside Rh1;

[0024] 5) converting ginsenoside F1 into sapogenin aPPT;

[0025] 6) converting ginsenoside F2 into sapogenin aPPD.

[0026] The β-glucosidase GE000505 for ginsenoside conversion, the preparation method and the application thereof have the following advantages:

[0027] The β-glucosidase GE000505 of the present application can convert high-content ginsenosides and notoginsenosides into rare ginsenosides R2, Rg3, Rg2 and Rh1, can convert notoginsenoside R1 into R2, can convert ginsenoside Rd into Rg3, can convert ginsenoside Re into Rg2, can convert ginsenoside Rg1 into Rh1, can convert ginsenoside F1 into aPPT, and can convert ginsenoside F2 into aPPD, and can be used in the fields of medicine and functional food.

[0028] The beta-glucosidase GE000505 has an optimum temperature of 37℃, is relatively stable at 30℃, 35℃, 37℃, 40℃ and 45℃, and has more than 80% of the enzyme activity after one hour of resistance, and has a half-life of about 40 min at 50℃. The optimum pH of the beta-glucosidase GE000505 is 5.5, and the enzyme activity after one hour of resistance is more than 60% at pH 5-7. When the beta-glucosidase GE000505 is placed in an ethanol solution with a concentration of 1%-40% (v / v) for one hour, the relative activity of the enzyme after resistance is increased when the ethanol concentration is 5%-15% (v / v). BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Relative activity (a) and relative stability (b) of the beta-glucosidase GE000505 at different pH values.

[0030] Figure 2 Relative activity (a) and relative stability (b) of the beta-glucosidase GE000505 at different temperatures.

[0031] Figure 3 Relative activity and relative stability of the beta-glucosidase GE000505 at different ethanol concentrations.

[0032] Figure 4 HPLC analysis results of the hydrolysis of notoginseng saponin R1 by the beta-glucosidase GE000505; (A) notoginseng saponin R1 standard; (B) hydrolysis of notoginseng saponin R1 by the enzyme GE000505; (C) R2 standard.

[0033] Figure 5 HPLC analysis results of the hydrolysis of ginsenoside Rd by the beta-glucosidase GE000505; (A) ginsenoside Rd standard; (B) hydrolysis of ginsenoside Rd by the enzyme GE000505; (C) Rg3 standard.

[0034] Figure 6 HPLC analysis results of the hydrolysis of ginsenoside Re by the beta-glucosidase GE000505; (A) ginsenoside Re standard; (B) hydrolysis of ginsenoside Re by the enzyme GE000505; (C) Rg2 standard.

[0035] Figure 7 HPLC analysis results of the hydrolysis of ginsenoside Rg1 by the beta-glucosidase GE000505; (A) ginsenoside Rg1 standard; (B) hydrolysis of ginsenoside Rg1 by the enzyme GE000505; (C) Rh1 standard.

[0036] Figure 8HPLC analysis results of ginsenoside F1 hydrolyzed by β-glucosidase GE000505; (A) ginsenoside F1 standard; (B) ginsenoside F1 hydrolyzed by enzyme GE000505; (C) aPPT standard.

[0037] Figure 9 HPLC analysis results of ginsenoside F2 hydrolyzed by β-glucosidase GE000505; (A) ginsenoside F2 standard; (B) ginsenoside F2 hydrolyzed by enzyme GE000505; (C) aPPD standard. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0039] It should be noted that, in the embodiments, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The instruments used are all conventional products that can be obtained by market purchase, and the raw materials and reagents used are all market goods or can be prepared by known methods.

[0040] In the present application, all the features defined in the form of numerical range or percentage range, such as numerical value, quantity, content and concentration, are only for the sake of brevity and convenience. Therefore, the description of numerical range or percentage range should be considered to have covered and specifically disclosed all possible sub-ranges and individual numerical values (including integers and fractions) within the range.

[0041] The features mentioned in the present application can be combined arbitrarily, as long as the combination of the features does not exist contradiction. All possible combinations should be considered as the scope disclosed in the specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless specifically stated, the disclosed features are only general examples of equivalent or similar features.

[0042] Some experimental materials and reagents used in the present application:

[0043] 1) Strains and vectors: Escherichia coli BL21 (DE3) and pET-28a (+) expression vector Escherichia coli BL21 (DE3) was purchased from Yixing Biotech Co., Ltd., and pET-28a (+) expression vector was from Jiangsu Suzhou Hongxun Biotechnology Co., Ltd.

[0044] 2) Enzymes and other biochemical reagents: Kanamycin sulfate (Kan) was purchased from Solario Company; DNA Marker was purchased from Takara Bio Technology Company; IPTG was purchased from Solario Technology Company; p-nitrophenyl-β-D-glucopyranoside (pNPG), Notoginseng Saponin R1, Ginsenoside F1, Ginsenoside F2, Ginsenoside Rg1, Ginsenoside Re and Ginsenoside Rd were purchased from Shanghai Yuan Ye Biological Technology Company, and other reagents were domestic reagents (which can be purchased from ordinary biochemical reagents company).

[0045] 3) Culture medium: LB culture medium: Peptone 10 g, Yeast extract 5 g and NaCl l0 g, add distilled water to 1000 mL, pH natural (about 7).

[0046] Note: The molecular biology experimental methods not specifically explained in the following examples were carried out according to the specific methods listed in the book of "Molecular Cloning Experiment Guide" (third edition) J. Sambrook or according to the reagent kit and product instruction.

[0047] Experimental Example 1 Construction of β-glucosidase GE000505 Expression Vector

[0048] 1) Using bioinformatics analysis means, the amino acid sequence of GE000505 enzyme was selected from the proteome of the strain screened in the early stage of the laboratory (Bacillus subtilis 168) Thomas clavelia ramosa ), the amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the enzyme gene is shown as SEQ ID NO. 2;

[0049] The amino acid sequence of β-glucosidase GE000505 (SEQ ID NO. 1) is as follows:

[0050] Met Arg Asn Ile Lys Arg Ile Ile Lys Gin Met Thr Leu Glu Glu Lys Ala Ser Met Cys Ser Gly Lys Asp Tyr Trp Tyr Leu Lys Gly Ile Glu Arg Leu Gly Ile Pro Glu Val Met Val Ser Asp Gly Pro Ser Gly Ile Arg Lys Gin Val Gin Asp Ser Ser Gin Leu Gly Phe Gly Ser Ile Glu Ala Val Cys Phe Pro Ala Ala Cys Leu Thr Ala Cys Ser Phe Asp Glu His Leu Leu Glu Lys Met Gly Glu Arg Ile Gly Glu Glu Cys Arg Ala Glu Asn Ile Ser Val Ile Leu Gly Pro Ser Ala Asn Ile Lys Arg Ser Pro Leu Cys Gly Arg Asn Phe Glu Tyr Phe Ser Glu Asp Pro Tyr Leu Gly Ser His Met Ala Ala Ala Tyr Val Arg Gly Val Gin Lys Lys Asp Val Ser Ala Cys Val Lys His Phe Phe Ala Asn Asn Gin Glu Tyr Gin Arg Met Thr Cys Ser Ser Asp Met Asp Glu Arg Thr Met Arg Glu Ile Tyr Leu Asn Ala Phe Glu Thr Ile Val Lys Asp Gly Gin Pro Asp Thr Met Met Cys Ser Tyr Asn Lys Ile Asn Gly Thr Tyr Val Ser Glu Asn His Glu Phe Leu Thr Lys Ile Leu Arg Asp Glu Trp Gly Tyr Lys Gly Tyr Val Met Ser Asp Trp Ser Ala Val Asn Asn Arg Val Glu Ala Ile Arg Ser Gly Leu Asp Leu Ala Met Pro Gly Glu Gly GlyTyr Met Asp Glu Glu lie lie Thr Ala Val Arg Ser Gly Thr Leu Ser Glu Glu lie Val Asp Gin Ala Val Glu Arg lie Leu Asn lie lie Phe Lys Tyr Val Asp Ser Asn Gin Thr Gly Thr Phe Asp Lys Gin Leu Asp His Asp Leu Ala Thr Lys Val Ala Thr Glu Ser Met Val Leu Leu Lys Asn Asp Gly Val Leu Pro Leu Pro Lys Lys Gly Lys Lys lie Ala Phe lie Gly Glu Phe Ala Lys Ser Pro Arg Tyr Gin Gly Gly Gly Ser Ala Asn lie Asn Ser Phe Lys Val Val Ser Ala Leu Glu Ala Ala Ser Asp lie Ser Glu lie lie Tyr Ala Gin Gly Tyr Glu Thr lie Glu Asp Arg Lys Asn Gin Glu Leu Phe Asp Glu Ala lie Asp lie Ala Ser Lys Ala Asp lie Ala Val Val Phe Val Gly Leu Pro Glu Ala Phe Glu Ser Glu Gly Tyr Asp Arg Glu His Met Arg Met Pro Asn Cys Gin Asn Glu Leu lie Ser Ser lie Ala Lys Val Gin Lys Asn Thr Val Val lie Leu His Asn Gly Ser Pro Val Glu Met Pro Trp Ala Asp Glu Val Ser Ala lie Leu Glu Ala Tyr Leu Gly Gly Glu Gly Val Gly Thr Ala Glu Val Ser Leu Leu Phe Gly Asp Ala Asn Pro Cys Gly Lys Leu Ala Glu Ser Phe Pro Leu Lys Leu Ser Asp Asn Pro Ser Tyr Leu Phe TyrHis Gly Asn Gly Lys Arg Thr Glu Tyr Arg Glu Gly Val Phe Val Gly Tyr Arg Tyr Tyr Asp Ser Lys Lys Met Asn Val Arg Tyr Pro Phe Gly His Gly Leu Ser Tyr Thr Thr Phe Met Tyr Asp Asn Leu Gln Ile Ser Lys Gly Glu Ile Leu Asp Asn Glu Glu Leu Lys Ile Ser Phe Asp Val Thr Asn Thr Gly Lys Met Ala Gly Lys Glu Thr Val Gin Leu Tyr Ile Ser Asp Lys Thr Asn Leu Ile Glu Arg Pro Glu Lys Glu Leu Lys Asn Phe Val Lys Val Tyr Leu Lys Pro Gly Glu Lys Lys Arg Ile Glu Met Ser Leu Asn Lys Arg Ser Phe Ala Trp Tyr Ser Thr Asp Ile His Asp Trp Tyr Val Gly Thr Gly Glu Tyr Gin Ile Met Val Gly Ser Ser Ser Lys Asp Ile Arg Leu Val Lys Asn Val Arg Ile Val Ser Thr Val Asn Leu Pro Ile Glu Ile Asn Arg Asn Thr Thr Ile His Glu Leu Leu Asn Asn Ser Lys Thr Asn Ser Ile Met Met Ser Val Ile Asp Lys Leu Val Thr Tyr Met Asn Gly Val Gin Lys Glu Gly Asp Thr Val Lys Ala Glu Gin Leu Ile Lys Met Leu Glu Ser Ser Pro Leu Arg Leu Leu His Ser Leu Met Gly Met Ser Phe Glu Asp Ile Asp Lys Leu Ile Ile Gin Phe Gin Asn Thr Phe Lys Glu Asp Asn Ser Leu Asn.

[0051] The nucleotide sequence of the beta-glucosidase GE000505 gene (SEQ ID NO. 2) is as follows:

[0052]

[0053] 2) The sequence encoding ge000505 is codon, GC content, and other gene optimized to obtain the sequence ge000505- opt , as shown in SEQ ID NO. 3. In ge000505-opt , two nucleotides (CC) are added before the start codon, the coding sequence of the 6×His tag (CACCACCACCACCACCAC) is added before the stop codon, and six nucleotides (CTCGAG) are introduced after the stop codon to obtain sge000505-opt The sequence is shown as SEQ ID NO. 4. To avoid introducing redundant nucleotide sequences, the synthesized sge000505-opt is connected to the pET-28a(+) vector by seamless cloning to obtain the recombinant plasmid sge000505-opt -pET-28a(+);

[0054] ge000505-opt The sequence (SEQ ID NO. 3) is as follows:

[0055]

[0056] 3) The recombinant plasmid pET-28a(+) was transformed into E. coli BL21(DE3) by heat shock to obtain the recombinant strain BL21(DE3) / sge000505-opt containing sge000505-opt sge000505-opt

[0057] Preparation of β-glucosidase GE000505

[0058] 1) The recombinant strain BL21(DE3) / sge000505-opt was inoculated into 5 mL liquid Kan-LB medium (the concentration of Kan was 50 μg / mL) at an inoculation amount of 1‰ (v / v) and cultured overnight at 37°C in a shaker at 180 rpm / min to activate the recombinant strain;

[0059] 2) The activated bacterial solution was inoculated into LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1% and cultured at 37°C in a shaker at 180 rpm / min for about 3 h (OD 600 reached 0.6-1), and 0.7 mM IPTG was added for induction. The recombinant protein was induced by further culturing at 20°C in a shaker at 180 rpm / min for about 20 h;

[0060] 3) The induced bacterial solution was centrifuged at 4°C at 6000 rpm / min for 10 min to collect the bacterial cells. The bacterial cells were suspended in McIlvaine buffer (citrate-phosphate buffer) with pH=7.0 at an amount of 1% of the volume of the bacterial solution, and the bacterial cells were broken by ultrasonic wave. The intracellular concentrated crude enzyme solution was centrifuged at 12000 rpm / min for 10 min to obtain the supernatant. The process of centrifugation to obtain the supernatant was repeated three times. The target protein was affinity purified by Nickel-NTA Agarose and 0-500 mM imidazole to obtain GE000505 purified enzyme;

[0061] 4) The obtained GE00505 purified enzyme was loaded into a 14000 kDa dialysis bag and placed in a dialysis solution of pH=7.0 McIlvaine buffer with 100 times the volume of the enzyme. The dialysis was performed in a refrigerator at 4°C, and the dialysis solution was replaced every 2 h, and the dialysis was performed three times.

[0062] Determination of the properties of GE000505 purified enzyme

[0063] ​​GE000505 purified enzyme activity was determined using p-nitrophenol (pNP) method, specifically: p-nitrophenyl-β-D-glucopyranoside (pNPG) was dissolved in optimal pH buffer to a final concentration of 2 mM; the reaction system contained 50 μL enzyme solution and 200 μL substrate-containing buffer; after the substrate was preheated for 5 min at the reaction temperature, the enzyme solution was added and reacted for another 10 min, then 750 μL 1M Na2CO3 was added to terminate the reaction, and after cooling to room temperature, the released pNP was determined at a wavelength of 410 nm; 1 enzyme activity unit (U) was defined as the amount of enzyme required to decompose 1 μmol of substrate to produce pNP per minute.

[0064] 1) GE000505 pH activity and stability determination

[0065] Enzyme activity determination in different pH: the purified β-glucosidase GE000505 was placed in pH 4-8 buffer and subjected to enzymatic reaction at 37°C.

[0066] Enzyme stability determination in different pH: the purified β-glucosidase GE000505 was placed in pH 3-8 buffer and treated at 37°C for 1 h. According to the enzyme activity determination method, the residual enzyme activity was determined at 37°C and pH 5.5 buffer, with untreated enzyme solution as the control.

[0067] The results, as shown in Figure 1 , GE000505 had an optimal pH of 5.5, and the residual enzyme activity was more than 20% between pH 4.5-6.5, and more than 60% after 1 hour of treatment between pH 5-7.

[0068] 2) GE000505 temperature activity and stability determination

[0069] Temperature activity determination of the enzyme: the effect of different temperatures (10-45°C) on β-glucosidase GE000505 was determined at pH 5.5.

[0070] Temperature stability determination of the enzyme: the same amount of enzyme solution was placed in a 30°C, 35°C, 37°C, 40°C, 45°C, and 50°C water bath for 1 h at pH 5.5, and then subjected to enzymatic reaction at pH 5.5 and 37°C.

[0071] The results, as shown in Figure 2 , GE000505 had an optimal temperature of 37°C, and the residual enzyme activity was still more than 80% after 1 hour of treatment at 30°C, 35°C, 37°C, 40°C, and 45°C, and the half-life at 50°C was about 40 min.

[0072] 3) GE000505 ethanol activity and stability assay

[0073] Ethanol activity assay: add ethanol solution (pH=5.5) in the enzymatic reaction system, so that the final concentration of ethanol is 1.0~40.0% (v / v), and the control group is without ethanol. The experimental group and the control group are set in triplicate. The enzymatic reaction is carried out at pH 5.5 and 37°C. The substrate is pNPG. After 10 min of accurate reaction, 1 M Na2CO3 is added to terminate the enzymatic reaction. The OD value is measured at 405 nm. 410 GE000505 activity is determined below.

[0074] Ethanol stability assay: equal amount of enzyme solution is placed in 1.0~40.0% (v / v) ethanol solution, and is tolerated at 37°C for 1 h. The control group is without treatment. The experimental group and the control group are set in triplicate. The enzymatic reaction is carried out at pH 5.5 and 37°C with pNPG as the substrate. After 10 min of accurate reaction, 1 M Na2CO3 is added to terminate the enzymatic reaction. The OD value is measured at 405 nm. 410 GE000505 activity is determined below.

[0075] After measurement, the ethanol activity and stability of GE000505 are as shown in Figure 3 The experimental results show that when the ethanol concentration is in the range of 5%~10% (v / v), the activity of enzyme GE000505 shows an upward trend; when the ethanol concentration is more than 10% (v / v), the activity of enzyme GE000505 begins to gradually decrease; when the enzyme GE000505 is placed in 1%~40% (v / v) ethanol solution for 1 h, when the ethanol concentration is in the range of 5%~15% (v / v), the relative activity of the tolerated enzyme increases; but when the ethanol concentration is greater than 15% (v / v), the relative activity of the tolerated enzyme sharply decreases.

[0076] Experimental Example 4: Effect of GE000505 purified enzyme on hydrolysis of ginsenosides and sanchinosides

[0077] 1. Ginsenoside incubation system

[0078] Incubation: dissolve ginsenoside Rg1, ginsenoside Re, and sanchinoside R1 in citric acid-disodium hydrogen phosphate buffer with pH 5.5 (the concentration of each ginsenoside in the buffer is 2 mg / mL) as the substrate for standby. Dissolve ginsenoside Rd, ginsenoside F1, and ginsenoside F2 in 20% ethanol (diluted with citric acid-disodium hydrogen phosphate buffer with pH 5.5) (concentration is 2 mg / mL) as the substrate for standby.

[0079] Experimental group: GE000505 purified enzyme was dissolved in citric acid-disodium hydrogen phosphate buffer with pH 5.5, and 500 μL of each ginsenoside and sanchinoside prepared above was added to the reaction tube according to the volume ratio of 1:1, so that the final concentration of ginsenoside and sanchinoside was 1 mg / mL, and the reaction tube was incubated at 37°C and 160 rpm for 24 h.

[0080] Control group: citric acid-disodium hydrogen phosphate buffer with pH 5.5 was added instead of enzyme solution, so that the final concentration of ginsenoside and sanchinoside was 1 mg / mL, and the reaction tube was incubated at 37°C and 160 rpm for 24 h.

[0081] 2. Sample extraction

[0082] After incubation in each group, the reaction was terminated with saturated aqueous n-butanol solution, the reaction tube was ultrasonicated for 15 min, and then stood for 30 min. The n-butanol layer was taken, and the process was repeated for 3 times. The n-butanol in the sample was removed by rotary evaporation, and the sample was dissolved with 1 mL of methanol.

[0083] 3. High performance liquid chromatography (HPLC) detection

[0084] The instrument was Shimadzu liquid chromatography-mass spectrometry LCMS-2020; the chromatographic column was RSZG-C18 Plus, 5 μm, 4.6*250 mm; the detection wavelength was 203 nm, the injection amount was 10 μL, the column temperature was 35°C, and the flow rate was 1.0 mL / min.

[0085] About 1 mL of the experimental sample dissolved in methanol was added to the sample bottle. The sample detector was a light emitting diode array detector, the column was YMC PG-C18 (4.6 mm*5.0 um*250 mm), the column oven temperature was 40°C, the flow rate was 1 mL / min, the mobile phase was A: acetonitrile, B: water, gradient elution was performed, the detection wavelength was 203 nm, the elution time was 100 min, and the injection amount was 10 μL each time.

[0086] The results are shown in Figures 4 to 9 HPLC analysis of saponin conversion by enzyme GE000505 hydrolysis. Among them Figure 4 HPLC analysis results of enzyme GE000505 hydrolysis of sanchinoside R1: R1-control is the analysis result of sanchinoside R1 standard (A), R1-505 is the HPLC analysis result of enzyme GE000505 hydrolysis of sanchinoside R1 (B), and R2 standard is the HPLC analysis result of sanchinoside R2 standard (C); Figure 5HPLC analysis results of ginsenoside Rd hydrolyzed by enzyme GE000505: Rd-control is the analysis result of ginsenoside Rd standard (A), Rd-505 is the HPLC analysis result of ginsenoside Rd hydrolyzed by enzyme GE000505 (B), Rg3 standard is the HPLC analysis result of ginsenoside Rg3 standard (C); Figure 6 HPLC analysis results of ginsenoside Re hydrolyzed by enzyme GE000505: Re-control is the analysis result of ginsenoside Re standard (A), Re-505 is the HPLC analysis result of ginsenoside Re hydrolyzed by enzyme GE000505, Rg2 standard is the HPLC analysis result of ginsenoside Rg2 standard (C); Figure 7 HPLC analysis results of ginsenoside Rg1 hydrolyzed by enzyme GE000505: Rg1-control is the analysis result of ginsenoside Rg1 standard (A), Rg1-505 is the HPLC analysis result of ginsenoside Rg1 hydrolyzed by enzyme GE000505 (B), Rh1 standard is the HPLC analysis result of ginsenoside Rh1 standard (C); Figure 8 HPLC analysis results of ginsenoside F1 hydrolyzed by enzyme GE000505: F1-control is the analysis result of ginsenoside F1 standard (A), F1-505 is the HPLC analysis result of ginsenoside F1 hydrolyzed by enzyme GE000505 (B), aPPT standard is the HPLC analysis result of ginsenoside aglycone aPPT standard (C); Figure 9 HPLC analysis results of ginsenoside F2 hydrolyzed by enzyme GE000505: F2-control is the analysis result of ginsenoside F2 standard (A), F2-505 is the HPLC analysis result of ginsenoside F2 hydrolyzed by enzyme GE000505 (B), aPPD standard is the HPLC analysis result of ginsenoside aglycone aPPD standard (C).

[0087] The results show that: when ginsenoside R1 is used as the substrate, β-glucosidase GE000505 converts ginsenoside R1 into ginsenoside R2 completely; when ginsenoside Rd is used as the substrate, β-glucosidase GE000505 converts ginsenoside Rd into ginsenoside Rg3 completely; when ginsenoside Re is used as the substrate, β-glucosidase GE000505 converts ginsenoside Re into ginsenoside Rg2 completely; when ginsenoside Rg1 is used as the substrate, β-glucosidase GE000505 converts ginsenoside Rg1 into ginsenoside Rh1 completely; when ginsenoside F1 is used as the substrate, β-glucosidase GE000505 converts ginsenoside F1 into ginsenoside aglycone aPPT completely; when ginsenoside F2 is used as the substrate, β-glucosidase GE000505 converts ginsenoside F2 into ginsenoside aglycone aPPD completely.

[0088] In conclusion, the beta-glucosidase GE000505 has the ability to convert high content saponins into rare saponins, thereby preparing more kinds of rare saponins with different efficacy. Specifically, the beta-glucosidase GE000505 can convert all notoginseng saponin R1 into notoginseng saponin R2, all ginsenoside Rd into ginsenoside Rg3, all ginsenoside Re into ginsenoside Rg2, all ginsenoside Rg1 into ginsenoside Rh1, all ginsenoside F1 into sapogenin aPPT, and all ginsenoside F2 into sapogenin aPPD. The beta-glucosidase GE000505 has a significant application prospect and economic benefit in the medical, health care and other industries.

[0089] Although the present application has been described in detail by the preferred embodiments, it should be appreciated that the above description should not be considered to be limiting the present application. Various modifications and alterations to this application will be apparent to those skilled in the art upon reading the above description. It is intended that the scope of the application be defined by the following claims.

Claims

1. The application of the gene encoding β-glucosidase GE000505 with the amino acid sequence as shown in SEQ ID NO.1, or the gene encoding β-glucosidase GE000505 with the nucleotide sequence as shown in SEQ ID NO.2, or a recombinant expression vector containing said gene, or a recombinant expression bacterium containing said gene, in the transformation of ginseng and Panax notoginseng saponins, characterized in that, The applications include: 1) Convert notoginsenoside R1 into notoginsenoside R2; 2) Convert ginsenoside Rd into ginsenoside Rg3; 3) Convert ginsenoside Re into ginsenoside Rg2; 4) Convert ginsenoside Rg1 into ginsenoside Rh1; 5) Converting ginsenoside F1 into saponin aPPT; and 6) Convert ginsenoside F2 into saponin aPPD.

2. The application according to claim 1, characterized in that, In applications 1), 3), and 4), β-glucosidase GE000505 was dissolved in citrate-disodium hydrogen phosphate buffer at pH 5.5 and incubated with notoginsenoside R1, ginsenoside Rg1, and ginsenoside Re at a volume ratio of 1:1 in a shaker at 37°C and 160 rpm for 24 h. In applications 2), 5), and 6), β-glucosidase GE000505 was dissolved in citrate-disodium hydrogen phosphate buffer at pH 5.5, and then incubated with ginsenoside Rd, ginsenoside F1, and ginsenoside F2 dissolved in 20% ethanol at a volume ratio of 1:1 for 24 h at 37°C and 160 rpm in a shaker. The 20% ethanol was obtained by diluting with a citrate-disodium hydrogen phosphate buffer solution at pH 5.

5.

3. The application according to claim 1, characterized in that, The recombinant expression vector is selected from pET-28a(+).

4. The application according to claim 1, characterized in that, The recombinant expression bacteria were selected from BL21(DE3).

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