KR20120017733A - Efficient Bioconversion Method of Ginsenoside Albi1 and Its Products - Google Patents

Abstract

The present invention relates to an enzymatic conversion method of ginsenoiside Rb ₁ , a major saponin component of ginseng, and to a ginseng product obtained by using β-glucosides isolated and purified from Aspergillus niger KCCM 11239 strain. It is identified and has an excellent effect of bioconverting ginsenoside Rb ₁ to produce Rd, F ₂ and Rg ₃ .

Description

A bioconversion method of ginsenoside Rb1 and product

The present invention relates to a method for the use of enzymes purified from microorganisms and to a method for enzymatic conversion of Rb ₁ , a major ginsenoside of which various pharmacological activities have been identified using β-glucosidase enzymes derived from Aspergillus niger .

Beta-glucosidase β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) includes enzymes that produce glucose as a final product by hydrolyzing β-glucoside bonds from aryl, alkyl β-glucosides and oligosaccharides do. β-Glucosidase is involved in activating the scent formation of plant hormones and fruits in plants, or in the resistance mechanisms of plant pathogens. In addition, it is distributed in molds, yeasts, bacteria and animal tissues. Perform other functions. β-glucosidase is divided into three groups according to substrate specificity. Class I includes glycosyl β-glucosidase and aryl β-glucosidase. These enzymes hydrolyze on substrates similar to cellobiose, lactose, β-ρ-nitrophenyl-glucoside (β-PNPG), β-ρ-nitrophenylgalactoside (β-PNPGal), and β-ρ-nitrophenylfructoside (β-PNPFru). Have. Class II contains only glcosyl β-glucosidase. Therefore, Class II can only hydrolyze substrates such as cellobiose and lactose. Class III contains only aryl or alkyl β-glucosidases. Class III thus exhibits substrate specificity for substrates similar to β-PNPG.

On the other hand, ginsenoside is the most important component exhibiting various pharmacological effects present in ginseng. To date, about 40 ginsenosides have been identified from Korean ginseng. Major ginsenosides of the relatively high content of ginsenosides include Rb ₁ , Rb ₂ , Rc, and Re, and minor ginsenosides having a very low content or specifically present in red ginseng include Rg ₃ , Rh ₂ , Rk. ₁ , and Compound K.

Such as Rg ₃ and Rg ₅ of minor ginsenosides of ginseng In the conversion, red ginseng is usually treated with organic solvents and subjected to chromatography to treat chickenpox. As a related technique, Korean Patent Nos. 10-228510 and 10-316567 are known.

Meanwhile, the enzymatic conversion technology of ginseng major ginsenoside Rb ₁ to minor ginsenoside Rb ₁ is disclosed in Korean Patent No. 10-424438, which discloses a technique for enzymatic reaction of lactase or β-glucosidase in a solvent.

In recent years, several studies have reported that trace ginsenosides have pharmacological effects such as antitumor, radical scavenging, neurotoxicity inhibition and anti-inflammatory activity. Such ginsenoids continue to make ginseng and ginseng-containing products by using acid treatment, base treatment, heat treatment, microorganisms and enzymes to be converted into new ginsenosides having small but excellent pharmacological efficacy. Among them, the enzymatic conversion method using enzymes does not cause side reactions or environmental pollution such as epimerization, hydroxylation, etc., and the ginsenoside conversion products and their pathways vary depending on the mechanism of action of the enzyme. It can have characteristics. In particular, β-glucosidase hydrolyzes the glycosidic bonds of ginsenosides Rb ₁ and converts them into trace ginsenosides such as ginsenosides Rd, Rg ₃ , F ₂ , Rh ₂ , and compound K. to be. The major ginsenosides Rb ₂ , Rc, and Rd are mass-produced minor ginsenoside Rg ₃ by enzymatic method using A. oryzae- derived lactase enzyme from ginseng saponin mixture. 218552 are each known. As an enzymatic conversion method of ginsenoid F ₂ , ginsenoids Rb ₁ , Rb ₂ ,, Rc, Rd or mixtures thereof are dissolved in a solvent and then structured into one of lactase, cellulase, and β-glucosidase derived from A. oryzae. The method of converting is disclosed in Korean Patent Application Publication No. 2002-58153, and the enzymatic conversion technology of ginsenoside compound K and F1 is an enzyme such as argininase or pectinase derived from penicillium or Aspergillus niger. A method for preparing by reacting after treatment is disclosed in 2003-37005.

On the other hand , Aspergillus filamentous fungus is a useful microorganism that produces enzymes, organic acids and pharmacologically active metabolites, and has been used not only in food industry, liquor industry, medicine industry, but also in agriculture, especially A. oryzae for a long time. Since it has been used in the production of traditional fermented foods has been recognized as a safe strain, has been reported as a high yield strain.

However, no studies of A. niger- derived bio-conversion of specific ginsenosides of ginseng to minor ginsenosides have been conducted.

Accordingly, the present invention has been made in view of the above-mentioned purpose to prepare ginseng products containing minor ginsenoside having various pharmacological effects using β-glucosidase from the physiologically safe genus A. niger KCCM 11239 strain.

The object of the present invention is to isolate and purify β-glucosidase from A. niger KCCM 11239 strain culture medium to characterize the enzyme;

Converting ginsenoside Rb ₁ having the highest content of ginsenosides into minor ginsenosides having various pharmacological efficacy using purified β-glucosidase derived from the strain; This was achieved through an evaluation step.

Aspergillus niger KCCM 11239 strain according to the present invention produces β-glucosidase having a molecular weight of 123 kDa, temperature stability of 20 ~ 80 ℃, pH 5.0 ~ 10.0 and using the enzyme ginsenoside ginsenoside Rb ₁ of the highest conversion rate Rd, F2, there is excellent effect that transition to ₃ Rg.

1 is a time-lapse graph showing cell growth and β-glucosidase enzyme production in A. niger KCCM 11239 strain culture step
Figure 2 is a chromatograph showing the enzyme synthesis on the Sephadex G-100 gel column using a crude enzyme solution containing β-glucosidase enzyme in the metabolite obtained from the culture of A. niger KCCM 11239 of the present invention.
3 is a chromatogram obtained by performing DEAE Sephadex G-100 Column ion exchange chromatography using the β-glucosidase enzyme synthesis fraction of the present invention.
Figure 4 is a diagram showing the results of performing the SDS polyacrylamide gel electrophoresis using the purified enzyme for the purification and molecular weight measurement of the β-glucosidase enzyme of the present invention.
Figure 5 is a diagram showing the pH stability (A) and temperature stability (B) of the β-glucosidase enzyme of the present invention.
Fig. 6 shows the HPLC profile results (A) (B) of the substrate specificity of the β-glucosidase enzyme of the present invention. Fig. 7 shows the ginsenside Rb ₁ by β-glucosidase isolated from the A. niger KCCM 11239 strain of the present invention. HPLC profile showing the change in conversion over time.

Experimental material

The test strain A. niger KCCM 11239 used in the present invention was used as a strain isolated from fermented food and sold at the Korea Microorganism Conservation Center (KCCM, Seoul, Korea), and used as a test material ginsenoside Rb ₁ , Rc, Rd, Re, Rg ₁ , Rg ₃ , F ₂ , Rh ₂ and compound K were purchased from Vitrosys (Korea). Sephadex G-100, DEAE Sephadex, bovine serum albumin (BSA), bicinchoninic acid (BCA), and ρ- nitrophenyl-β-D-glucopyranoside (PNPG) are described by Sigma Chemical Co. Purchased from St. Louis, Mo., USA.

Cultivation of Strains

A. niger KCCM 11239 was inoculated and stored in potato dextrose agar (PDA). The liquid medium for enzyme production was autoclaved 400 mL of PDB at 121 ° C. for 15 minutes, and inoculated with pre-cultured spore suspension (2%, v / v) in aseptic phase for 5 days at 30 ° C. at 200 rpm. Shake incubation for days. Dry cell mass and β-glucosidase activity were measured at 2 days intervals according to the incubation time.

As a result of observing the growth of cells and the production of β-glucosidase according to the incubation time of A. niger KCCM 11239 is shown in FIG. The growth of the cells was centrifuged by taking 5 mL of the culture solution every two days, discarding the supernatant and drying the cells only for 24 hours at 60 ℃ was measured by dry cell weight (DCW). As a result, the cell mass increased steadily until the 12th day of culture, but the increase was not large thereafter, and after 14 days, the cell was stopped. On the other hand, the production of enzyme showed little change until 8 days of cultivation, but rapidly increased after 10 days of cultivation, indicating the maximum production at 16 days of cultivation. Based on the above results, it was found that the β-glucosidase produced by A. niger KCCM 11239 has the maximum β-glucosidase activity after the cell growth reached the stop phase.

Enzyme Activity and Protein Quantitation

Enzyme activity was measured by mixing 1 mL of 5 mM PNPG dissolved in 0.05 M sodium phosphate buffer (pH 5.5) with 0.1 mL of enzyme according to Kohchi's method, reacting at 50 ° C for 10 minutes, and 1 mL of 0.5 M Na ₂ CO _3. After the addition of the reaction was stopped, the absorbance of the yellow color was measured at 400 nm. Free p-nitrophenol (PNP) was quantified in the standard curve, and 1 unit of enzyme activity was defined as the amount of enzyme that produced 1 μmole of PNP for 1 minute.

Protein quantitation was measured using BCA reagent. At this time, the absorbance was measured at 750 nm using bovine serum albumin (BSA) as a standard protein, and the standard curve was prepared and quantified.

Purification of β-Glucosidase

In the present invention, the culture culture shaken for 16 days 14,420 ㅧ g, centrifuged for 20 minutes, the supernatant was fractionated precipitated from 30% to 90% with ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) at 4 ℃ The precipitate obtained by centrifugation at 20,760 ㅧ g for 20 minutes was dissolved in 0.02 M sodium acetate buffer (pH 4.0) and used as coenzyme for the purification of β-glucosidase. Gel chromatography was inflated with Sephadex G-100 in 0.02 M sodium acetate buffer (pH 4.0) for 72 hours at room temperature, charged into the sorting tube (15 ㅧ 300 mm) and equilibrated with 0.02 M acetate buffer (pH 4.0), Saturated with ammonium sulfate (30-90%) eluted partially purified enzyme into the same buffer. At this time, the outflow rate was a natural flow rate, and the eluate was fractionated into each test tube by 3 mL. Each of the eluate was identified as a protein at 280 nm, and then enzyme activity was measured at 400 nm, and then only a large portion of the activity was collected and concentrated in a freeze dryer to be used as an enzyme sample for the next purification process. Ion exchange chromatography was performed with DEAE Sephadex resin, washed with 0.02 M sodium acetate buffer (pH 4.0), charged into fractionation tubes (30x150 mm) and equilibrated with the same buffer. This sorting tube was eluted by adsorbing the enzyme samples collected by Sephadex G-100 column chromatography, and then added with a concentration gradient of 0-0.3 M NaCl. At this time, the elution rate was 1 mL / min, and the eluate was collected in each test tube by 3 mL, and each eluate was measured for protein and enzyme activity. .

Molecular Weight Measurement by SDS-PAGE

In order to measure the purity and molecular weight of the purified enzyme in the present invention, after electrophoresis on 10% SDS-polyacrylamide gel, the molecular weight was calculated by comparing the moving speed with the protein marker. SDS-PAGE was prepared according to the method of Laemmli, using a protein marker (20-140 kDa) of Elpis Biotech.

The supernatant obtained by centrifugation of the culture solution was fractionally precipitated with ammonium sulfate at 4 ° C. at 30-90%, and then the crude enzyme solution having the activity was subjected to Sephadex G-100 gel chromatography. β-glucosidase enzyme activity was shown at fraction number 9-10 and collected and dissolved in a small amount of 0.02 M sodium acetate buffer (pH 4.0) after lyophilization. DEAE Sephadex ion exchange chromatography was performed using this active fraction to obtain the result as shown in FIG. 3. At this time, the enzyme was eluted with NaCl 0-0.3 M concentration gradient in the same buffer. Β-glucosidase activity was eluted at fraction number 27-31 with a concentration of 0.15-0.2 M NaCl. Finally, β-glucosidase enzyme was purified by lyophilization. The purification process of the whole enzyme is shown in Table 1. The final purified enzyme had a 46.5-fold increase in enzyme activity compared to the culture supernatant with a yield of 1.5%.

10% SDS-polyacrylamide gel electrophoresis was performed to confirm the degree of purification and to determine the molecular weight of the purified β-glucosidase. As shown in FIG. The molecular weight of the β-glucosidase produced by A. niger KCCM 11239 was 123 kDa.

The researchers reported that the molecular weight of β-glucosidase from A. niger SFN-416 was 46 kDa, while Bahia et al. Reported that the molecular weight of β-glucosidase produced by Stachybotrys strain was 85 kDa.

 Summary of purification of the β-glucosidase produced by A. niger  KCCM 11239 Purification step Total activity
(U) Total protein
(mg) Specific activity
(U / mg) Recovery
(%) Purification
(fold) Crude extract
(supernatant) 86971.0 8797.0 9.9 100 1.0 (NH ₄ ) ₂ SO ₄
precipitation 22655.0 248.8 91.0 21.5 9.2 Sephadex
G-100 3073.8 13.5 227.1 11.0 22.9 DEAE
Sephadex 356.1 0.7 460.5 1.5 46.5

 Effect of pH and Temperature

Optimum pH measurement of the enzyme of the present invention is 0.1 M citrate buffer (pH 3.0), 0.1 M acetate buffer (pH 4.0-6.0), 0.1 M phosphate buffer (pH 7.0-8.0), 0.1 M glycine-NaOH (pH 9.0), 0.1 M sodium phosphate buffer (pH 10.0) was used. Stability to pH was measured by adding buffers of each pH to the enzyme solution, and remaining enzyme activity after 8 hours at room temperature.

In order to determine the optimum reaction temperature of the enzyme, the reaction was performed for 10 minutes at intervals of 10 ° C. in the range of 20 to 80 ° C. and expressed as the relative activity of the enzyme. Thermal stability was measured by heat treatment of the enzyme solution in units of 2 hours at intervals of 10 ° C. in the range of 20-80 ° C. to measure the residual activity of the enzyme.

Effect of Metal Ions and Inhibitors on Enzyme Activity

Ferrous chloride, copper sulfate, cobalt chloride, potassium chloride, manganase chloride, zinc sulfate, calcium chloride, magnesium chloride, sodium chloride, SDS, EDTA, acetic acid, Dissolve glycerol, Triton X-100, ethanol, and methanol in sodium acetate buffer (pH 6.0) to 10 mM, and add the same amount of enzyme solution to the metal salt solution and the inhibitor, and react for 30 minutes at room temperature. Measured.

The effect of purified β-glucosidase on the pH is shown in FIG. 5 (A). The optimum pH was found to be pH 4.0, which is an acidic condition, and the activity of the enzyme was reduced to less than 20% between pH 7.0 and 8.0, which is neutral. On the other hand, the pH stability for the enzyme was confirmed to be stable in weakly acidic and alkaline conditions to maintain the remaining enzyme activity of more than 70% from pH 5.0 to 10.0.

The effect on the temperature of the enzyme is shown in Figure 5 (B). As a result of examining the optimum temperature, the optimum temperature was found to be 70 ℃, and the temperature stability of the enzyme was left at 20-80 ℃ for 2 hours, and the enzyme activity was measured under the standard conditions of the enzyme. At temperatures exceeding 98% of enzyme activity was lost.

In order to determine the effect of metal salts and inhibitors on the activity of the isolated and purified β-glucosidase, 9 kinds of metal salts and 7 inhibitors were added so that the final concentration was 10 mM. Indicated. As a result, most of the metal salts did not affect the enzyme activity, but the addition of copper sulfate (CuSO ₄ ) decreased the enzyme activity to 79 ㅁ 0.27%. Most inhibitors, including SDS and EDTA, were not affected by enzyme activity, but the addition of acetic acid decreased the residual activity to 86 ± 4.25%.

 Effect of metal ions and reagents on the activity of β- glucosidase from A. niger KCCM 11239 Metal ions or reagents
Relative activity (%) ^a None ^b
FeCl ₂
CuSO ₄
CoCl ₂
KCl
MnCl ₂
ZnSO ₄
CaCl ₂
MgCl ₂
NaCl
Ethanol
Glycerol
Acetic acid
Triton X-100
Methanol
SDS
EDTA 100wh0.00 ^c
92 ㅁ 1.79
79 ㅁ 0.27
88 ㅁ 1.50
97 ㅁ 1.45
97 ㅁ 4.11
94 ㅁ 2.76
101 ㅁ 2.95
87 ㅁ 8.39
101 ㅁ 1.29
97 ㅁ 3.98
95 ㅁ 3.60
86 ㅁ 4.25
110 ㅁ 5.25
94 ㅁ 2.77
95 ㅁ 1.54
97 ㅁ 5.00

Enzyme Substrate Specificity and Ginsenoside Rb _One Conversion

To determine the substrate specificity of the enzyme of the present invention, 0.5 mg of each ginsenoside Rb ₁ , Rb ₂ , Rc, Rd, Re, and Rg ₁ standards were taken, dissolved in 1 mL of distilled water, and the same amount of purified enzyme solution was added at 50 ° C. The reaction was carried out for 30 minutes. The reaction solution extracted the saponin component with the same amount of saturated butanol. Ginsenoside hydrolysis capacity of the purified enzyme was expressed as relative activity (%) by analyzing the content of ginsenosides by HPLC.

In order to determine the conversion process of ginsenoside Rb ₁ with time, the same amount of enzyme solution was mixed with 1 mM ginsenoside Rb ₁ aqueous solution and reacted at 50 ° C. and 150 rpm for 120 hours. Take the reaction mixture at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 hour intervals during the reaction, extract the saponin component with the same amount of saturated butanol, and convert it to HPLC. It was.

In order to determine the substrate specificity of the enzyme, the purified β-glucosidase was purified using 0.5 mg / mL of the ginsenosides Rb ₁ , Rb ₂ , Rc, Rd, Re, and Rg ₁ , which have β-glucoside binding. Hydrolytic resolution was examined. After reacting 6 ginsenoside mixtures with the same amount of β-glucosidase at 50 ° C. for 30 minutes, saponins were extracted using saturated butanol and analyzed by HPLC. Of the six ginsenosides, β-glucosidase causes ginsenoside Rb ₁ to hydrolyze the two molecules of glucose (β-1,6 bond) that are bound to the 20th carbon and are almost completely converted to ginsenoside Rd. lost. On the other hand, purified β-glucosidase did not show hydrolytic activity against ginsenoside Rd, in which two molecules of glucose bound β-1,2 to the third carbon for 30 minutes. In addition, ginsenoside Re and arabinose ( p ) -α- (1 → 6) -glucose-β bonds consisting of rhamnose-α- (1 → 2) -glucosase-β bonds are combined with Rb ₂ and arabionse (f). There was no hydrolysis of) -α- (1 → 6) -glucose-β Rc.

 Relative activities of A.niger KCCM 11239 β-glucosidase on different ginsenosides Ginsenoside
Configuration of glycoside linkage Relative activity (%) ^a Rb ₁
Rb ₂
Rc
Rd
Re
Rg ₁ -glc (2 → 1) glc, -glc (6 → 1) glc
-glc (2 → 1) glc, -glc (6 → 1) arap
-glc (2 → 1) glc, -glc (6 →) araf
glc (2 → 1)
-glc (2 → 1) rha
-glc 100 ㅁ 0.00 ^b
ND ^c
2 ㅁ 2.26
ND
ND
21 ㅁ 0.71

Analysis of Conversion Ginsenosides

Ginsenoside Substrate Specificity and Conversion of Ginsenosides of the Enzyme Purified According to the Invention For the analysis HPLC was used Agilent 1100 series (California, USA), the column was Eclipse C ₁₈ (Agilent, 4.6 ㅧ 150 mm, USA). The mobile phase sequentially increased the ratio of 100% acetonitrile (A) and 14% acetonitrile (B) from A: B = 20: 80 to A: B = 60: 40. The development temperature was room temperature and the flow rate was 1.2 mL per minute. The chromatogram was detected at 203 nm using a UV / VIS detector.

Upon oral administration of ginseng absorption in vivo of the major saponins is very low, Rb _1, Rb ₂ is difficult to substantially decompose in addition to being a very small amount oxygenation by the gastric juice, bioavailability in the gut is a 0.1-4.4% Rb _1, Rb ₂ is 3.7% and Rg ₁ is 1.9% -18.4%, most of which is excreted through the urine. Therefore, in order to increase the efficacy of ginseng saponin, the conversion to minor saponins, which are relatively well absorbed and more effective in major saponins, are required. Currently, ginseng saponin conversion research has a number of methods, but the conversion by enzymes can be carried out at room temperature, normal pressure and near neutral pH, and has the advantage of producing specific saponin due to the substrate specificity of the enzyme.

7 shows the conversion process according to time of ginsenoside Rb ₁ having the highest conversion rate among the six types of ginsenosides in the present invention. The same amount of purified β-glucosidase was added to 1 mM aqueous solution of ginsenoside Rb _{1 and} reacted at 50 ° C. for 120 hours to analyze the change of ginsenoside over time by HPLC. As a result, most of ginsenoside Rb ₁ was converted to Rd within 30 minutes after the reaction between ginsenoside Rb ₁ and the enzyme, and after 1 hour, ginsenoside Rd began to be converted to F ₂ . Over time, the amount of F ₂ further increased. Β-glucosidase also began to become a ginsenoside Rg ₃ binary starting reaction time 12 is detected, that the more the reaction time increased with increasing amounts detected through the above results A. niger KCCM 11239 is produced by β-1,6 glucoside bond In addition to hydrolysis, β-1,2 glucoside bonds showed hydrolysis, indicating that ginsenosides Rb ₁ were converted into Rd, F ₂ and Rg ₃ .

Claims (3)

A biotransformation method of ginsenoside Rb ₁ of ginseng saponin component characterized by using a β-glucosidase enzyme having a molecular weight of 123 kDa derived from Aspergillus niger KCCM 11239 strain and having an optimum temperature stability of 70 ° C. and an optimum PH stability of 4.0. A ginseng product comprising minor ginsenoside obtained by the method of claim 1 as an active ingredient. The method of claim 2,
Ginseng product characterized in that the minor ginsenoside is Rd, F ₂ , Rg ₃ .

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