KR102407724B1 - An Enzyme for ginsenoside bioconversion - Google Patents

Abstract

The present invention relates to a bioconversion enzyme for obtaining ginsenoside from ginseng. When ginseng is fermented using the bioconverting enzyme of the present invention, it is possible to obtain more Rh2, which is excellent in treating cancer or cancer-related fatigue, compared to known enzymes.

Description

An Enzyme for ginsenoside bioconversion}

The present invention relates to a bioconversion enzyme for obtaining ginsenoside from ginseng. When ginseng is fermented using the bioconverting enzyme of the present invention, it is possible to obtain more Rh2, which is excellent in treating cancer or cancer-related fatigue, compared to known enzymes.

Ginseng ( Panax ginseng CA Meyer) is a perennial shaggy rooted plant belonging to the genus Panax of the Araliaceae family. The main pharmacologically active ingredient of ginseng is saponin. Saponin is a non-sugar part (aglucan) of glycosides widely present in the plant system composed of several cyclic compounds, and it is known that ginseng contains about 3 to 6%. Unlike saponins that exist in plants such as onions, bellflowers, and garlic, it has a unique chemical structure that is found only in ginseng. Ginsenoside is a representative natural ingredient that has anti-inflammatory effect, antioxidant effect, wrinkle suppression, immunity enhancement, whitening effect, anticancer effect, antibacterial effect, and skin protection effect.

Because saponin itself has a low absorption rate in the body, fermented ginseng is fermented using intestinal microorganisms in an anaerobic condition such as the intestinal environment so that ginseng can be eaten. During the fermentation process, metabolism of saponin occurs and the physiological activity is strengthened, increasing the usability. As the metabolites of fermented red ginseng, as shown in the figure below, more than 40 types of ginsenosides have been isolated and confirmed so far.

(Kwok HH, Guo GL, Lau JK, Cheng YK, Wang JR, Jiang ZH, Keung MH, Mak NK, Yue PY and Wong RN. (2012). Stereoisomers ginsenosides-20(S)-Rg3 and -20(R) -Rg3 differentially induce angiogenesis through peroxisome proliferatoractivated receptor-gamma. Biochemical Pharmacology. 83:893-902)

Among the increased metabolites of the fermentation-amplified extract, especially G-Rg3, G-Rf, or G-Rh2, the effects of red ginseng have been newly reported to show memory improvement effect, erectile dysfunction effect, anti-allergic effect, anti-atopic effect, etc. have. In particular, Rh2 is not contained in ginseng, but only 0.001% in red ginseng and 0.02% in wild ginseng. It is known that anti-cancer agents have anti-cancer activity and immune-enhancing effects. Rg3, which is contained in both ginseng and red ginseng, is known to exhibit cancer cell proliferation inhibitory effects in inhibiting cancer cell metastasis, inhibiting platelet aggregation, antithrombotic action, and inhibiting anticancer drug resistance. Possibilities are being raised that this effect may be due to the effect of ginsenosides, which are present only in red ginseng made by fermenting ginseng.

As the various functions of ginsenoside are scientifically revealed, various studies and applications using ginsenoside are being actively conducted, and consumer interest and demand according to such studies and products are increasing. It is the research and technology development of Ginsenoside that can maximize the value of Korea's excellent ginseng. However, the effective ingredient analysis and extraction technology of ginsenoside is still not clear and incomplete, so its utility value is declining. Among them, protein expression and mechanisms for anti-inflammatory and anticancer-related factors are insufficient and urgent.

Ginsenoside known to have cancer or cancer-related fatigue (CRF) effects so far is ginsenoside Rh2, among which Rh2 is a composition for preventing or treating cancer-related fatigue (Chinese Patent No. 101612159, 2011. 08. 31) .), the fatigue-reducing effect of Rg3 on patients with cancer cells removed (Kou Xiao-ge et al., National Medical Frontiers of China, 2010, Abstract.) and, although not dose-dependent, intranasally ginsenoside Rg3 It has been reported that when administered, it exhibits an anti-fatigue effect (Wenyan et al. 2008).

In order to obtain ginsenosides such as Rg3 or Rh2 from ginseng (panax ginseng), methods such as chemical treatment, heat treatment, and biological treatment such as enzymatic conversion are known. In the case of chemical treatment or heat treatment, there is a limit to mass production due to the release of pollutants during the hydrolysis process, and it is difficult to obtain a desired reactant because the selectivity of the reaction is small. On the other hand, the enzyme conversion method using an enzyme can be tested under environmentally friendly experimental conditions, and it is known that the yield of the desired reactant is high compared to other methods because of the selectivity for a specific reaction site and a specific saccharide. [Kyung Hoon Chang, Mi Na Jo, Kee-Tae Kim, Hyun-Dong Paik, Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3, J Ginseng Res 38 (2014) 47-51]

The present inventors have researched and developed their own ginseng culture enzyme while studying the enzyme conversion reaction of ginseng. Among Aspergillus species, Aspergillus niger, in particular, is known as a safe strain certified as GRAS (Generally Recognized as Safe) by the FDA, and it is said that it is widely used for extracting raw materials for food and pharmaceuticals based on this because it can be mass-produced with a simple process. In addition, compared to other strains, Aspergillus niger releases a large amount of cellulase enzymes that contribute to the reaction to produce the main component of ginsenoside from ginseng, thereby selectively obtaining the desired ginsenoside products such as Rh2 and Rg3. known to be possible. [Fani Ntana, Uffe Hasbro Mortensen, Catherine Sarazin, and Rainer Figge, Aspergillus: A Powerful Protein Production Platform, Catalysts 2020, 10(9), 1064; doi:10.3390/catal10091064]

According to the literature related to the enzymatic reaction, among various cellulase enzymes obtained from Aspergillus niger, β-glucosidase selectively cuts the sugar attached to carbon 3 of ginsenoside Rb1 while not cutting the sugar attached to carbon 20. it is known [Kyung Hoon Chang, Mi Na Jo, Kee-Tae Kim, Hyun-Dong Paik, Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3, J Ginseng Res 38 (2014) 47-51]

As seen in the table on the previous page, the products of ginsenosides to be obtained, such as Rh2 and Rg3, have no sugar structures in both Nos. 3 and 20, so the efficiency of the reaction while selectively cutting off sugars at Nos. 3 and 20 An enzyme was needed to increase the

1) Jose Arnau, Debbie Yaver, and Carsten M. Hjort, Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell FactoriesGrand Challenges in fungal Biotechnology pp179-210 (2020), Chapter 17. 2) Bong-Kyu Song, Kyeng Min Kim, Kang-Duk Choi, and Wan-Taek Im, Production of the Rare Ginsenoside Rh2-MIX (20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3) by Enzymatic Conversion Combined with Acid Treatment and Evaluation of Its Anti-Cancer Activity, J. Microbiol. Biotechnol. (2017), 27(7), 1233-1241 3) Ru Zhang, Xue-Mei Huang, Hui-Juan Yan, Xin-Yi Liu, Qi Zhou, Zhi-Yong Luo, Xiao-Ning Tan, and Bian-Ling Zhang, Highly Selective Production of Compound K from Ginsenoside Rd by Hydrolyzing Glucose at C-3 Glycoside Using β-Glucosidase of Bifidobacterium breve ATCC 15700, J. Microbiol. Biotechnol. (2019), 29(3), 410-418 4) DIA SEPTIANI, HERMAN SURYADI, ABDUL MUN'IM, WIBOWO MANGUNWARDOYO, Production of cellulase from Aspergillus niger and Trichoderma reesei mixed culture in carboxymethylcellulose medium as sole carbon, Biodiversitas 20(12):3539-3544 (2019)

In the present invention, the need for an enzyme reaction that fermentatively decomposes saponin during the manufacturing process of an anticancer drug derived from ginseng (or red ginseng) or a drug for treating cancer-related fatigue (CRF) was investigated, and a trace amount of ginsenoside component Rh2 An attempt was made to develop an enzyme that can effectively obtain After preparing the saponin-degrading enzyme of the present invention, using the saponin-degrading enzyme prepared and hydrolysis with an organic acid, it was confirmed that Rh2 is produced more effectively than when other known enzymes (eg, β-glucosidase or glucoamylase, etc.) are used. The invention was completed.

1. Enzyme for culture fermentation of ginseng.

2. The enzyme according to 1 above, wherein the enzyme produces ginsenoside Rh2.

3. The enzyme according to 1 above, wherein the enzyme cleaves glucose at the 3rd and 20th terminals sequentially or simultaneously from ginsenoside Rb1 to produce ginsenoside F2.

4. The enzyme according to 1 above, wherein the enzyme comprises glucosidase.

5. The enzyme according to 4 above, wherein the glucosidase is β-glucosidase and/or α-glucosidase.

6. The enzyme according to the above 4, characterized in that it contains 5 to 30% of the total enzyme glucosidase.

7. The enzyme according to 1 above, wherein the enzyme includes glucosidase and amylase.

8. The enzyme according to the above 7, wherein the amylase is glucoamylase and/or α-amylase A.

9. The enzyme according to the above 7, wherein amylase is contained in an amount of 10 to 30% of the total enzyme.

10. The enzyme obtained by extraction from the Aspergillus niger strain deposited with the accession number KCCM11451P according to the 1 above.

When ginseng is fermented using the bioconverting enzyme developed in the present invention, it is possible to effectively obtain a large amount of ginsenoside Rh2, which is an active ingredient of an anticancer drug or a therapeutic agent for cancer-related fatigue.

1 is a result of qualitatively confirming F2 and Rh2 and Rg3 after an enzyme reaction using an enzyme of the present invention, a reaction without an enzyme, and a reaction using β-glucosidase and glucoamylase.
2 is a result of qualitative confirmation of F2 and Rh2 and Rg3 after reacting with β-glucosidase 30, 60, 120, and 240 mg, respectively.
3 is a result of qualitatively confirming F2, Rh2, and Rg3 after reacting with 600 mg of β-glucosidase.

[Definition of Terms]

MCB: Abbreviation for Master Cell Bank, a strain that is subcultured in 2% ginseng agar medium from the deposited strain (Korea Microbial Conservation Center KCCM11451P), produced as an ampoule, and stored in a refrigerator.

Strain culture: Step of culturing MCB 3rd passage plate in 8% ginseng agar medium Petridish

Main culture: Inoculation step in case medium to bulk up Aspergillus Niger cultured in Petridish

Harvest: The process of obtaining crude enzymes by separating the medium and enzymes

Ultrafiltration: A process of filtration to obtain an enzyme solution of 100 kDa or more containing enzymes in the culture medium

Alcohol immersion: A process of precipitating the enzyme by adding alcohol to the filtered enzyme solution

Centrifugation: A process to obtain only the precipitate (enzyme) in a state in which alcohol and the precipitate (enzyme) are mixed

Freeze drying (F/D, freeze drying): The process of powdering the precipitate (enzyme)

[Example]

The following reagents and instruments were used.

Raw material and reagent information reagent manufacturer ginseng powder Boguk Pharmaceutical / Ilhwa Agar Powder Junsei / MB cell bran Sajo Dongawon / Youth Smile Intermediate 1 Intermediate product of CRF therapeutic composition Acetic Acid DAE JUNG / Uni-Chemical 95% Ethanol Daehan Liquor Life Co., Ltd. / Alcohol Sales World sodium acetate DAE JUNG / Prime Science

Device and material information Device Model Company Scale AP250D OHAUS Autoclave LAC-5100SD Daehan Lab Tech agitator Lab-stirrer LR400B Yamato Incubator IB39 Vision Science centrifuge Continent 512R plus Hanil Science Industry UPLC Acquity UPLC H-Class Waters 500 mL flask - Schott Duran 500 mL glass column - Pyrex Glass Evaporator custom order Dooyoung Engineering Shaking waterbath LB-SW025 LK LAB Korea HP-20 resin - Samyang Brixmeter PAL-3L Atago pH meter IP54 Mettler Toledo

One. reactions involving enzymes

Among the following reactions from ginseng, some of the steps of preparing an anticancer agent containing ginsenoside such as Rh2 and Rg3 or a drug for treating cancer-related fatigue are as follows. In this reaction, the enzyme of the present invention was denoted as BST101.

Intermediate 1: Starting material before the enzymatic reaction during the manufacturing process of a composition for CRF treatment derived from ginseng

Intermediate 2: Product immediately after enzymatic reaction during the manufacturing process of ginseng-derived CRF treatment composition

In the above reaction, BST101 reacts with Intermediate1 (Rb1) of ginseng raw material to cut the outermost sugar out of the two sugars connected to the 20th carbon position of Rb1 to produce Rd, and then to the 3rd carbon position from Rd again. The outermost one of the two linked sugars is cut to form F2, and Rd and F2 are combined to be called Intermediate 2. Such a process may proceed as a sequential reaction in which Rd is first produced and then F2 is produced. Also, F2 is produced in one step in which the sugars at positions 20 and 3 are simultaneously cleaved from Rb1 without going through Rd. it may become Either way, Intermediate 2 (composed of F2 component and Rd component) is generated, and the generated F2 and Rd undergo acid hydrolysis, respectively, to become final products of Rh2 and Rg3, respectively. In the step of preparing the final composition for CRF treatment, Intermediate 1 (Rb1) is further used in the intermediate acid hydrolysis step to obtain more Rg3, which is not related to the enzymatic reaction step of the present invention, so it will not be mentioned any more. BST101 enzyme can be said to be an essential enzyme for the only reaction ⓛ that generates F2 from Intermediate 1 to produce Rh2.

In the following experimental example, it is investigated whether the process of BST101 is absolutely necessary in the process of generating Rh2 component, which is one of the indicator components of ginseng-derived anticancer drugs or drugs for the treatment of cancer-related fatigue, and the effect of BST101 compared to known single enzymes commercially available It was attempted to determine whether the

2. Contrast experiments with known enzymes

In order to compare the effects of the enzyme of the present invention and the known enzyme, the experiment was designed as follows. Enzyme standards were purchased from Sigma-Aldrich or PubChem and used.

Composition of the enzyme applied in the enzymatic reaction step of each experimental group group configuration amount note Experiment 1 Column 1. BST101
Column 2. Enzyme reaction x
Column 3. β-glucosidase
Column 4. Glucoamylase 0.3g
0
26.4mg
47.4mg

Manufacturer: Sigma Aldrich
Manufacturer: Sigma Aldrich Experiment 2 Column 1. β-glucosidase Column 2. β-glucosidase
Column 3. β-glucosidase
Column 4. β-glucosidase 30mg
60mg
120mg
240mg Manufacturer: Sigma Aldrich
or PubChem Experiment 3 Column 1. β-glucosidase 600mg Manufacturer: Sigma Aldrich

Put 90 mL of 0.02 M sodium acetate buffer solution into a 500 mL Erlenmeyer flask, and add 3 g of intermediate 1 sample. Add each enzyme according to the enzyme composition in Table 3 above.

The reaction was shaken at 32° C. at 100 rpm for 24 hours to proceed with the enzymatic reaction. A portion of the reactant is sampled to check whether the reaction occurs (confirmation of F2 product) by TLC. 270 mL of alcohol was added to the reaction mixture and allowed to stand at room temperature. This solution was centrifuged at 5500 rpm, 4° C., for 5 minutes to completely separate the precipitate and the supernatant. The supernatant is taken and concentrated under reduced pressure at 50°C and 76 cmHg in a vacuum dryer. The concentrate is dried under reduced pressure at 50°C and 76 cmHg in a vacuum dryer. Add 50% acetic acid to the 500mL Erlenmeyer flask above. After adding intermediate 2 dry powder and intermediate 1 dry powder 2 g to the solution, the mixture was stirred at 70° C. for 6 hours. A portion of the reactant is collected and Rg3 and Rh2 are confirmed by TLC. After the reaction was cooled to room temperature, 300 mL of purified water was added. The column filled with 250mL HP20 resin is washed with 2 bead volume (about 500mL) of alcohol, and then washed with 10 bead volume (about 2.5L) of water. Inject the reactants so far into the column above. After all the reactants are loaded, purified water is continuously poured into the column. Pour purified water until the pH of the solution reaches 5.0 or higher. After injecting 625 mL of alcohol into the column adjusted to the pH, the filtrate passing through the column is collected. The collected filtrate is transferred to a vacuum concentrator and concentrated under reduced pressure at 50°C and 76 cmHg. The concentrated under reduced pressure was transferred to a reduced pressure dryer and dried under reduced pressure at 50°C and 76 cmHg. The dried sample is ground in a mortar and weighed to obtain the final ginsenoside.

3. Test method

1) Qualitative Analysis (TLC)

Absorb the sample solution using a thin tube such as a capillary. If the tip of the capillary, which has absorbed the solvent, is dripped onto the TLC plate, the sample solution permeates the plate and creates a spot. Place the lower part of the TLC plate on which the sample solution is dripped into a bottle filled with an appropriate amount of the developing solvent and close the lid. At this time, be careful as the solvent evaporates on the plate if the lid is not closed. Just before the solvent has completely risen, the plate is removed from the solvent and dried. If the dried TLC plate is immersed in 10% sulfuric acid solvent and heated in a hot-plate at a high temperature, the sample on the TLC plate develops color and qualitative analysis is possible.

2) Quantitative analysis (HPLC)

Standards and materials required for HPLC were prepared as follows.

Standard products of ginseng extract are as follows.

- Ginsenoside Rg3 (20S) pure product (Chromadex)

- Ginsenoside Rg3 (20R) pure product (Chromadex)

- Ginsenoside Rh2 (20S) pure product (Chromadex)

- Ginsenoside Rh2 (20R) pure product (Chromadex)

Equipment used:

- Pump and Autosampler System, Waters Alliance e2695

- Photodiode Array Detector, Waters 2998

- Empower 3 chromatography management system

menstruum

- Water (HPLC grade)

- Acetonitrile (HPLC grade)

- Methanol (HPLC grade)

- DMSO (Analytical grade)

- Trifluoroacetic acid (TFA, Analytical grade)

Analysis conditions

- Column: Kinetex C18 250 mm x 4.6 mm, 5 um (Phenomenex) or equivalent.

- Detection Wavelength: 203 nm

- Injection volume: 10 uL

- Column temperature: 25℃

- Autosampler temperature: 25℃

- Flow rate: 1 mL/min

- Mobile phase: Solvent A - Acetonitrile + 50 ppm TFA, Solvent B - Water + 50 ppm TFA

Gradient Concentration Gradient Conditions Time Solvent A (%) Solvent B (%) 0 25 75 32 50 50 50 0 100 55 0 100 62 25 75 65 25 75

Analytical sample

- Blank solution:

Blank solution A: MeOH/Water = 8:2 (V/V)

Blank solution B: DMSO/Blank solvent A = 2:8 (V/V)

- Preparation of standard solution: In a 10 mL volumetric flask, add 1 mg of ginsenoside 20S-Rh2, 20R-Rh2, and 2 mg of 20S-Rg3, respectively, and fill with blank solvent A up to the marked line. Add 2 mg of 20R-Rg3 to a 5 mL volumetric flask, add 2 mL DMSO solvent, dissolve, and fill with blank solution A up to the mark. After ultrasonically vibrating the volumetric flask in which 4 standards were dissolved for 1 minute, filter it using a 0.22 μl PTFE syringe filter, and use this filtrate as a standard solution.

- Preparation of test solution: Put a 25 mg sample in a 10 mL volumetric flask, add 8 mL of blank solution A, vibrate with ultrasonic waves for 5 minutes, and fill with blank solution A up to the mark. This solution is filtered using a 0.22 ㎛ PTFE syringe filter, and this filtrate is used as the test solution.

Experimental method

After setting the column temperature to 25 °C, the initial mobile phase solvent is continuously flowed to equilibrate the HPLC. When the baseline is stabilized, inject the sample in the following order.

① Blank Solution A

② Blank Solution B

③ Ginsenoside Rg3 (20S) reference solution preparation

④ Ginsenoside Rg3 (20R) reference solution preparation

⑤ Ginsenoside Rh2 (20S) reference solution preparation

⑥ Ginsenoside Rh2 (20R) reference solution preparation

⑦ Test solution

content calculation

Ginsenoside Rg3 content (%)

A1 = Ginsenoside S-Rg3 peak area of the test solution

A2 = Ginsenoside R-Rg3 peak area of the test solution

A3 = Ginsenoside S-Rg3 peak area of standard solution

A4 = Ginsenoside R-Rg3 peak area of standard solution

m1 = sample weight (mg)

m2 = weight of standard S-Rg3 (mg)

m3 = weight of standard R-Rg3 (mg)

Ginsenoside Rh2 content (%)

A1 = Ginsenoside S-Rh2 peak area of the test solution

A2 = Ginsenoside R-Rh2 peak area of the test solution

A3 = Ginsenoside S-Rh2 peak area of standard solution

A4 = Ginsenoside R-Rh2 peak area of standard solution

m1 = sample weight (mg)

m2 = weight of standard S-Rh2 (mg)

m3 = Weight of standard R-Rh2 (mg)

[Analysis]

1) Experiment 1

In Experiment 1, the final product produced through the enzymatic reaction with each of the following enzymes in the enzymatic reaction step was analyzed by qualitative analysis (TLC) and quantitative analysis (HPLC), and the presence/absence of BST101 and known enzymes (β-glucosidase or Glucoamylase ) to prove the superiority of the enzyme of BST101 through a comparative experiment.

As a result of qualitative analysis, a dark band of F2 was confirmed in Intermediate 2, a product obtained through an enzymatic reaction using the enzyme BST101 of the present invention, and dark bands of Rg3 and Rh2 were also confirmed in the final product (FIG. 1).

HPLC quantitative analysis result of Experiment 1 BST101 enzymatic reaction x β-glucosidase Glucoamylase Rg3 12.39% 18.38% 19.59% 19.44% Rh2 7.87% 0.29% 0.28% 0.13%

According to the quantitative analysis results, it was confirmed that the contents of Rg3 12.39% and Rh2 7.87% were consistent with the results of TLC. On the other hand, the F2 band could not be confirmed in Intermediate 2, a product that did not undergo enzymatic reaction, and the Rh2 band could not be confirmed even in the final product. As a result of HPLC quantitative analysis, Rg3 was 18.38% and Rh2 was 0.29%, so the production of Rh2 was insignificant in the reaction without enzymatic reaction.

In Intermediate 2 produced when the known enzymes β-glucosidase and Glucoamylase were treated in the enzymatic reaction step, respectively, the F2 band could not be confirmed, and the Rh2 band could not be confirmed even in the final product, CRF treatment composition (Fig. 1) . As a result of HPLC quantitative analysis, Rg3 was 18.38% and Rh2 0.29% in the case of β-glucosidase, and in the case of glucoamylase, Rg3 was 19.44% and Rh2 0.13% The Rh2 content in the composition for CRF treatment was insignificant.

2) Experiment 2

The main mechanism of Rh2 generation is the reaction in which the intermediate F2 is produced by cleaving all the outer glucose chains among the two glucose bonded to the 3rd and 20th carbons of intermediate 1 (ginsenoside Rb1) in the BST101 enzymatic reaction step. In the present invention, it is assumed that BST101 is a complex enzyme in which various enzymes are complexed, and among them, the β-glucosidase component acts to cut glucose in this way and F2 is expected to be produced, and in the enzymatic reaction step of Experiment 1, a single In order to check whether there is a dose dependence on the dose and reaction degree of β-glucosidase, the products were analyzed by treatment with 30, 60, 120, and 240 mg of β-glucosidase, respectively. However, as a result of qualitative analysis (TLC) in Experiment 2, neither F2 nor Rh2 were generated, and in the β-glucosidase reaction, regardless of the concentration of β-glucosidase, the average Rg3 of 19.59% and Rh2 of 0.78% in all the β-glucosidase groups was created

HPLC Results of Experiment 2 β-glucosidase 30mg 60mg 120mg 240mg Rg3 19.75% 19.46% 19.57% 19.57% Rh2 0.77% 0.78% 0.79% 0.76%

3) Experiment 3

As a result of Experiment 2, although the amount of β-glucosidase was increased, the amount of Rh2 produced by β-glucosidase alone was insignificant. Therefore, the content of Rh2 produced by reacting β-glucosidase with a high dose (600 mg) in the enzymatic reaction step was analyzed.

Results of HPLC of Experiment 3 β-glucosidase 600mg Rg3 19.14% Rh2 0.40%

When a high dose of β-glucosidase single enzyme was treated in the enzymatic reaction step, as shown in the qualitative analysis of FIG. 3 and Table 7, Rh2 showed a content of 0.40%, so regardless of the amount of β-glucosidase, Rh2 was produced by a single enzyme reaction knew it wasn't.

Based on the experimental results, the activation mechanism of BST101 is to generate the final product, Rh2, after F2 is generated in Intermediate 2. It was confirmed that F2 and Rh2 resulting from the enzymatic reaction of BST101 of the present invention could be obtained rather than F2 and Rh2 generated from a single enzyme reaction of β-glucosidase or glucoamylase.

4. Preparation of the enzyme of the present invention

The strain (Aspergillus Niger, Accession No. KCCM11451P, Korea Microbial Conservation Center) is aliquoted into Petridish, loaded, put in an incubator, and incubated at 28°C for 72-96 hr. Scrape all of the cultured culture medium to prepare a strain solution, then put it in a drum containing ginseng powder and bran and mix. After flattening it on the main culture tray, incubate at 28°C for 96-144 Hr, and maintain 50-60% humidity. The cultured medium is put in water and stirred for 4 hours, then returned to a dehydrator to separate the liquid and medium. At this time, the resulting culture solution is centrifuged at 5500 rpm for 5 minutes, and the supernatant is filtered with a reduced pressure filter. Enzyme solution of 100 KDa or more is concentrated until it becomes 1/10 of the input amount. After immersing the enzyme by adding alcohol (95% EtOH) in an amount equivalent to 3 times of the concentrated filtrate, centrifugation is performed to obtain the enzyme. In the present invention, 5 LOT samples were prepared up to BST-001, BST-002, BST-003, BST-005, and BST-006 in this way.

The protein of 5 LOT BST101 enzyme prepared in the present invention was analyzed using i) HPLC (SEC and IEX) ii) emPAI iii) Reference protein and Extracted Ion Chromatography and iv) Extracted Ion Chromatography Analysis Results Protein content through peptide content analysis When analyzed by analysis, it was confirmed that it was composed of the following types of enzymes.

Protein identification result of BST101 enzyme

The enzyme of the present invention contains 5 to 30% as glucosidase (alpha-glucosidase and beta-glucosidase), and more specifically, 10 to 30% as amylase (glucoamylase and alpha amylase A) in addition to glucosidase. In the process of culturing using enzymes, numerous sugars such as glucose, amylose, xylose, furanose, galactose, and arabinose can be removed, so such It can be seen that various types of enzymes that remove sugars are included in a complex manner.

To summarize the process of the present invention, when each enzymatic reaction was performed with a single enzyme, F2, an intermediate product of Rh2, was not produced. In order to generate F2, all the sugar bonds connected to the 3rd and 20th carbons of ginsenoside Rb1 must be broken down. The β-glucosidase single enzyme binds the sugars connected to the 3rd carbon of ginsenoside Rb1. It is known that it selectively degrades only the sugar chain, and other cellulase enzymes are known to degrade only the sugar bond linked to carbon 20. The complex enzyme extracted from Aspergillus niger contains various cellulase enzymes, but first, the outer sugar linked to the 20th carbon of ginsenoside Rb1 is cut to produce ginsenoside Rd, and then sequentially added to the 3rd carbon of Rd. It has been reported that F2 is produced by cleaving the linked outer sugar. In the present invention, by developing an enzyme that can carry out such a sequential reaction at once, the sugars connected to the 3-position carbon and the 20-position carbon of ginsenoside Rb1 are cut simultaneously or sequentially to generate F2, and as a result, the gene The present invention was completed by developing a complex enzyme capable of efficiently obtaining Rh2 of senoside.

Korea Microorganism Conservation Center (Overseas) KCCM11451P 20180125

Claims (10)

Aspergillus niger (Aspergillus niger) KCCM11451P containing a protein mixture with a molecular weight of 100KDa or more of the culture supernatant,
The protein mixture comprises glucosidase, amylase, xyloside, xylanase, arabinofuranosidase, cellobiohydrolase and galactosidase, ginsenoside F2 production composition.
The composition of claim 1, wherein the ginsenoside F2 is produced by cleaving the glucose linked to the 3rd and 20th carbons of the ginsenoside Rb1.
The composition of claim 1, wherein the glucosidase is alpha-glucosidase and beta-glucosidase.
The composition of claim 1, wherein the amylase is glucoamylase and alpha-amylase A.
The composition of claim 1, wherein the xylosidase is alpha-xylosidase A and exo-1,4-beta-xylocidase xlnD.
The composition of claim 1, wherein the xylanase is endo-1,4-beta-xylanase A, endo-1,4-beta-xylanase B and endo-1,4-beta-xylanase C. .
The composition of claim 1, wherein the arabinofuranosidase is alpha-L-arabinofuranosidase axhA and alpha-L-arabinofuranosidase B.
The method according to claim 1, wherein the cellobiohydrolase is 1,4-beta-D-glucan cellobiohydrolase A, 1,4-beta-D-glucan cellobiohydrolase B and 1,4-beta-D-glucan A composition that is cellobiohydrolase C.
The composition of claim 1, wherein the galactosidase is alpha-galactosidase A, alpha-galactosidase B and beta-galactosidase A.
Treating ginsenoside Rb1 with the composition of any one of claims 1 to 9 to prepare ginsenoside F2; and preparing ginsenoside Rh2 by acid hydrolyzing the ginsenoside F2.

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