KR101844975B1 - Method of producing Ginsenoside F1 from the Ginsenoside Rg1 by extracellular enzyme from Fusarium moniliforme var. subglutinans - Google Patents

Abstract

The present invention relates to a method for producing rare ginsenoside F1 using the extracellular enzyme of the microorganism Fusarium moniliforme var . Subglutinans , and more particularly to a method for producing rare ginsenoside F1, There is provided a method for producing ginsenoside F1 by reacting ginsenoside Rg1 as a substrate with a ginsenoside Rg1 in a reaction solvent by using an enzyme of a supernatant in a culture medium of a fungal fungus Moniliformum tocoveglutinans KCTC 6149 .

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a ginsenoside Fp from a ginsenoside aldolase using an extracellular enzyme of a fusarium moniliporum baubulgulinus. subglutinans}

The present invention relates to the use of an extracellular enzyme of Fusarium moniliforme var. Subglutinans KCTC 6149 to produce ginsenoside F1 (Ginsenoside) belonging to the rare ginsenoside which is not present in ginseng More particularly, the present invention relates to a method for producing 6 (Ginsenoside Rg1) of ginsenoside Rg1 which is a substrate represented by the formula (1) using the enzyme of the supernatant in the culture medium of fusarium moniliformum, a fungus fungus fungus. (Ginsenoside F1) represented by the formula (2) which is a rare ginsenoside by selectively hydrolyzing only the glucose of the carbon atom.

Ginsenoside F1 down-regulates the expression of Bcl-2 and Brn-3a from cancer cell proliferation inhibition, the anticancer activity of anti-cancer drugs, the suppression of allergies and apoptosis by irradiation of ultraviolet B It is known that human HaCaT keratinocytes can be protected (Lee EH, et al., J. Invest. Dermatol. 121: 607-13, 2003).

The method of production of ginsenoside F1 until now is known to be produced by intestinal bacteria (Hasegawa, H. et al., Planta Medica, 62: 453 (1996)), And the conversion yield is as low as 10%. Recently, interest has been focused on a method using an enzyme. The enzymatic method includes dissolving ginseng purified saponin in an aqueous solvent such as water or a buffer solution, or a mixture of an aqueous solvent and an organic solvent such as water or a buffer solution, and then adding at least one of Naringin and Pectinase A method of producing ginsenoside F1 from ginseng saponin using a method of producing ginsenoside F1 (Korean Patent Laid-Open Publication No. 2003-37005) and beta-glucosidase of Bacteriodes JY6 Patent Document No. 2002-32038) and a method of reacting ginseng extract or triol saponin with a beta-galactosidase of the genus Penicillium sp. Or Aspergillus sp. (90%) and exhibiting a productivity of 127 to 150 mg / l / h (Korean Patent Laid-Open Publication No. 2003-37005).

Accordingly, the present inventors have conducted extensive research to find a substance capable of producing ginsenoside F1 at a high yield without any byproducts. As a result, it has been found that Fusarium moniliformes, moniliforme there is . subglutinans ) The inventors discovered that the production of ginsenoside F1 from the substrate ginsenoside Rg1 using the extracellular enzyme of KCTC 6149 selectively hydrolyzed only glucose of the 6th carbon of ginsenoside Rg1, Completed.

Therefore, the present invention provides a method for mass production of ginsenoside F1 using an extracellular enzyme derived from a strain of Fusarium moniliforme var . Subglutinans .

The present invention also provides a composition for the production of Ginsenoside F1, which comprises an extracellular enzyme derived from a Fusarium moniliforme var . Subglutinans strain.

The method for producing ginsenoside F1 using the extracellular enzyme of the present invention comprises reacting ginsenoside Rg1 as a substrate with a buffer solution, an aqueous solvent or a mixture of an aqueous solvent and an organic solvent at a reaction temperature of 30 to 70 ° C to form ginsenoside Only the glucose of the 6th carbon of Rg1 is selectively hydrolyzed to convert ginsenoside F1 to 100% in a short period of time to produce a high yield without by-products.

The ginsenoside Rg1 used purely separated ginsenoside Rg1 was purchased from Ambo Institute (Daejeon, Korea).

As the reaction solvent of the enzyme, a buffer solution such as sodium citrate buffer solution or potassium phosphate buffer solution may be used.

The buffer solution of sodium citrate in the buffer solution is preferably prepared by making sodium citrate and citric acid to 50 mM each, mixing them together and pH titration. The potassium phosphate buffer solution is prepared by adding potassium phosphate monobasic and dibasic (KH ₂ PO ₄ , K ₂ HPO ₄ ) to 50 mM each, and then pH is titrated while mixing the two.

In order to obtain the extracellular enzyme, it is preferable to cultivate Fusarium moniliformes for 6 days at 28 DEG C in an oatmeal medium (oatmeal 55 g / L, agar 7 g / L). The mycelia were recovered per flat plate culture plate and cultured for 3 days at 28 ° C and 200 rpm in 250 ml of oatmeal liquid oat medium (oatmeal 55 g / l). Then, 10% of the total volume of the growth medium was grown And cultured at 28 DEG C and 200 rpm for 5 days. The composition of the medium is expressed carboxymethyl cellulose 10 g / ℓ, ( NH 4) SO 4 10g / ℓ, KH ₂ PO ₄ 7.5g / ℓ, MgSO ₄ 2 g / l, CaCl ₂ 0.2 g / l.

The culture broth in the above procedure was filtered using Watman filter paper, and the filtrate was recovered and an extracellular enzyme was obtained by salting out with ammonium sulfate.

The reaction temperature of the enzyme is preferably in the range of 30 ° C to 70 ° C, and more preferably 50 ° C in consideration of thermal stability. When the activity at 50 캜 is assumed to be 100%, the relative activity decreases to less than 20% at less than 30 캜, and the activity decreases to less than 70% when the substrate reacts with the substrate for more than 5 hours at a temperature exceeding 50 캜 to be.

In addition, the reaction between the extracellular enzyme and the substrate is preferably carried out at pH 4.0 to pH 7.0, more preferably at pH 6.0. When the activity at pH 6.0 is assumed to be 100%, the relative activity decreases to less than 40% when the pH is less than 4.0, and the relative activity decreases to less than 60% when the pH exceeds 7.0.

In the above reaction, when the reaction is carried out using 2.5 mg / ml or 5 mg / ml of ginsenoside Rg1, the unit of the extracellular enzyme is preferably 1.2 to 6.7 units / ml, more preferably 6.7 units / ml desirable. The ginsenoside Rg is no longer converted even when the extracellular enzyme is more than 6.7 units / ml, and when it is less than 1.2 units / ml, the conversion is reduced to less than 20%.

According to the method for producing ginsenoside F1 using the extracellular enzyme of the present invention, when ginsenoside Rg1 is used using an extracellular enzyme, the concentration of ginsenosides Can selectively produce ginsenoside F1 from Rg1 and can be industrially useful.

Hereinafter, in order to facilitate understanding of the present invention, test examples and examples will be specifically described, but the present invention is not limited to the examples.

< Test Example  1> Fusarium Moniliforme  bar Suburbin  Growth conditions and Extracellular  Obtain enzyme

In order to prepare the extracellular enzyme, the Fusarium moniliporum baubulugurbinans KCTC 6149 strain was first plated on solid oatmeal medium (oatmeal 55 g / l, agar 7 g / l) at 28 ° C for 6 days And the mycelium was collected and cultured for 1 to 8 days at 28 ° C and 200 rpm on a liquid oatmeal medium (oatmeal 55 g / l).

Nitrophenyl β-D-glucopyranoside (1 mM) as a substrate was added to the culture filtrate at each date to a final concentration of 0.3 mg / ml, and incubated at 37 ° C for 10 minutes. Na ₂ CO ₃ After addition, the activity of the culture filtrate was measured by measuring the absorbance at 450 nm. In addition, the dry cell weight per day was measured to determine the activity (unit / mg DCW) per dry cell weight (FIG. 2). As a result, 5 days were selected as the incubation date because the slope increases steadily until 5 days. At this time, the unit of culture filtrate was calculated to produce 1 nano mole (nmole) of 4-nitrophenol per minute.

561 g of ammonium sulfate was added per 1 L of the culture filtrate cultured for 5 days, followed by elution for 12 hours, followed by centrifugation at 13,000 x g for 20 minutes at 4 占 폚.

The precipitates obtained by centrifugation were dissolved in 50 mM sodium citrate buffer solution and potassium phosphate buffer solution, respectively. Ammonium sulfate was removed through desalting to obtain 15 mg of extracellular enzyme per 1 L culture medium.

< Reference example  1> Extracellular  How to measure enzyme activity

The ginsenoside Rg1 (manufactured by Ambo Institute, Daejeon, Korea) was used for the measurement of the activity of the extracellular enzyme provided in the composition relating to the production of the rare ginsenosides proposed in the present invention. Specifically, 0.6 mg / ml of ginsenoside Rg1, 0.6 ml of extracellular enzyme, 0.5 ml of extracellular enzyme and 50 mM of pH 6.0 were added to the reaction mixture while ginsenoside Rg1 and extracellular enzyme were dissolved in a buffer of 50 mM. Sodium citrate buffer solution to give a final concentration of 0.3 mg / ml of senoside Rg1 and a final concentration of 1 unit / ml of extracellular enzyme, and the total concentration of ginsenoside Rg1, sodium citrate buffer solution and extracellular enzyme And the reaction was allowed to proceed at 60 DEG C for 30 minutes. The reaction was stopped by addition of 1 ml n- butanol. The amount of ginsenoside F1 produced due to the enzyme reaction between the ginsenoside Rg1 and the enzyme reaction was subjected to high pressure liquid chromatography equipped with a column of a VWD detector and a column of YMC-Pack ^™ Pro C ₁₈ ^™ (YMC, Kyoto, Japan) was calculated, YMC-Pack Pro C ₁₈ ^{TM TM} column was to pass acetonitrile from 0% to 1 ㎖ / min at 37 ℃ to 80%. At this time, the unit of the extracellular enzyme was calculated as the amount of extracellular enzyme producing 1 nano mole of ginsenoside F1. The amount of enzyme protein was estimated using the Bradford method based on bovine serum albumin.

< Test Example  2> Extracellular  Investigation of optimal temperature on enzyme activity

To investigate the effect of temperature on the activity of extracellular enzymes, the enzyme reaction was carried out in the presence of 0.5 ml of 0.6 mg / ml of ginsenoside Rg1 dissolved in 50 mM sodium citrate solution both in the ginsenoside Rg1 and extracellular enzyme And 0.5 ml of 2 units / ml of extracellular enzyme to give a final concentration of 0.3 mg / ml of ginsenoside Rg1 and 1 unit / ml of extracellular enzyme. The reaction was carried out at 30 ° C to 70 ° C for 30 minutes at 5 ° C intervals using a 50 mM pH 6.0 sodium citrate buffer solution containing 1 unit / ml of enzyme and 0.3 mg / ml of ginsenoside Rg1. The same experiment was repeated three times and the average value was shown in Fig. The reaction was then stopped by the addition of 1 ml n- butanol and the activity of the enzyme was determined by high pressure liquid chromatography in Reference Example 1. As a result, it was confirmed that the optimum temperature was 60 ° C. (Fig. 3A).

< Test Example  3> Extracellular  Optimal effect on enzyme activity pH  Investigation of effects

In order to investigate the effect of extracellular enzyme activity on the pH, the enzyme reaction was carried out in the same manner as in Example 1, except that both of the ginsenoside Rg1 and the extracellular enzyme were dissolved in a 50 mM sodium citrate buffer solution and 0.5 ml of 0.6 mg / ml ginsenoside Rg1 And 0.5 ml of 2 units / ml of extracellular enzyme to give a final concentration of 0.3 mg / ml of ginsenoside Rg1 and 1 unit / ml of extracellular enzyme. (Test Example 3-1) using 50 mM pH 5.5 sodium citrate buffer solution containing 1 unit / ml of enzyme and 0.3 mg / ml of ginsenoside Rg1 at pH 4.0 to 6.0 at pH 0.5 intervals,

Both the ginsenoside Rg1 and the extracellular enzyme were dissolved in a 50 mM potassium phosphate buffer solution, and 0.5 ml of 0.6 mg / ml of ginsenoside Rg1 and 0.5 ml of 2 units / ml of extracellular enzyme were mixed to give a final concentration of ginsenoside Rg1 0.3 mg / ml, and the extracellular enzyme is 1 unit / ml. The reaction was carried out at 60 ° C for 30 minutes using a 50 mM pH 5.5 ginseng potassium buffer solution containing 1 unit / ml of enzyme and 0.3 mg / ml of ginsenoside Rg1 at a pH of 0.5 to 7.0 from pH 6.0 to 7.0 Example 3-2).

Then, the reaction was stopped by adding 1 ml of n- butanol to Test Example 4-1 and Test Example 4-2, and the activity of the enzyme was measured by high pressure liquid chromatography in Reference Example 1. As a result, it was confirmed that the optimum pH was pH 6.0 (FIG. 3B). 3 shows the activity of the extracellular enzyme according to the pH in FIG. 3B, and FIG. 3B shows a sodium citrate buffer solution (Test Example 3-1) and a phosphate buffer solution (Test Example 3-2).

< Test Example  4> Extracellular  Investigation of Optimum Temperature in Temperature Stability of Enzyme Activity

To measure the temperature stability of the extracellular enzyme activity, 0.5 ml of 2 unit / ml extracellular enzyme dissolved in 50 mM sodium citrate buffer solution was left at 35, 40, 45, 50, 55, 60, and 65 ° C for 5 hours Then, each of these was mixed with 0.5 ml of 0.6 mg / ml of ginsenoside Rg1 to give 0.3 mg / ml of final concentration of ginsenoside Rg1 and 1 unit / ml of extracellular enzyme, which was reacted at 60 ° C for 30 minutes. The reaction was then stopped by the addition of 1 ml n-butanol and the activity of the enzyme was determined by high pressure liquid chromatography in Reference Example 1. The same experiment was repeated three times and the average value was shown in Fig.

As a result, the activity was rapidly decreased to 23% after 5 hours at 60 ° C, which was the highest activity, and the remaining activity of enzyme remaining at 50 ° C for 5 hours was 70% Temperature, and was selected as the application temperature of the ginsenoside F1 production experiment (Fig. 4).

< Test Example  5> Extracellular  Enzyme-assisted Gin Senocide  Optimal enzyme unit of F1 production / ml

When the extracellular enzyme obtained through the above process uses ginsenoside Rg1 as a substrate, the optimum enzyme unit for the production of ginsenoside F1 was investigated.

0.5 ml of 10 mg / ml of ginsenoside Rg1 dissolved in 50 mM sodium citrate buffer solution (pH 6) was mixed with 0.5 ml of 13.4 unit / ml of extracellular enzyme dissolved in 50 mM sodium citrate buffer (pH 6) For 24 hours.

As a result of this reaction, about 4 mg of ginsenoside F1 was obtained at 6.7 units / ml of extracellular enzyme. It was found that the conversion rate of 100% was observed when considering that the 5mg ginsenoside Rg1 used in the reaction was 6.24mM and that the 100% hydrolysis resulted in 6.24mM of ginsenoside F1 (about 3.99mg).

It was confirmed that 100% conversion to ginsenoside F1 at 6.7 units / ml of enzyme and 167 mg / l / h of productivity resulted in 6.7 units / ml of extracellular enzyme in the production of ginsenoside F1 ( 5). In FIG. 5, in the case of the optimal enzyme unit / ml of the extracellular enzyme, (1) represents mg / ml of ginsenoside F1, and () represents the conversion rate (FIG. 5).

< Example  1> Gin Senocide Rg1  2.5 mg / Ml over time Gin Senocide  Production of F1

In order to develop the production method of ginsenoside F1 over time by using the extracellular enzyme, the reaction was carried out at pH 6.0 and 50 ° C, which are optimal temperatures considering the optimal pH and temperature stability of the extracellular enzyme.

Specifically, in the enzyme reaction, 0.5 mg of 5 mg / ml ginsenoside Rg1 and 0.5 ml of 13.4 units / ml of extracellular enzyme were dissolved in 50 mM sodium citrate buffer (pH 6.0), both of ginsenoside Rg1 and extracellular enzyme And reacted at 50 DEG C for 24 hours in such a manner that the final concentration of ginsenoside Rg1 was 2.5 mg / mL and the extracellular enzyme was 6.7 units / mL. Then, 1 ml of n-butanol was added, and the reaction was stopped by a voltex for 1 minute. The mixture was centrifuged at 13000 x g for 1 minute, and the separated organic layer was recovered and evaporated. Then, 1 ml of methanol was added to the reaction solution, and the solution was measured by high pressure liquid chromatography in Reference Example 1 after voltexing.

When the optimum enzyme unit / ml (6.7 units / ml) and 2.5 mg / ml of ginsenoside Rg1 were reacted with each other, the yield was determined to be 100% at 9 hours and 2 mg / ml of ginsenoside F1 Yielding a productivity of 222 mg / l / h (Fig. 6A).

< Example  2> Gin Senocide Rg1  5.0 mg / Ml over time Gin Senocide  Production of F1

In order to develop the production method of ginsenoside F1 over time by using the extracellular enzyme, the reaction was carried out at pH 6.0 and 50 ° C, which are optimal temperatures considering the optimal pH and temperature stability of the extracellular enzyme.

Specifically, in the enzyme reaction, both of the ginsenoside Rg1 and the extracellular enzyme were dissolved in 50 mM sodium citrate buffer (pH 6.0), 0.5 ml of 10 mg / ml ginsenoside Rg1 and 0.5 ml of 13.4 units / ml extracellular enzyme , Followed by reaction at 50 占 폚 for 24 hours in such a manner that the final concentration of ginsenoside Rg1 was 5.0 mg / ml and the extracellular enzyme was 6.7 units / ml. Then, 1 ml of n-butanol was added, and the reaction was stopped by a voltex for 1 minute. The mixture was centrifuged at 13000 x g for 1 minute, and the separated organic layer was recovered and evaporated. Then, 1 ml of methanol was added to the reaction solution, and the solution was measured by high pressure liquid chromatography in Reference Example 1 after voltexing.

When the optimum enzyme unit / ml of 6.7 unit / ml and the 5.0 mg / ml of ginsenoside Rg1 were reacted with each other, the yield was determined to be 100% at 24 hours and 4 mg / ml of ginsenoside F1 Yielding a productivity of 167 mg / l / h (Fig. 6b).

In the production of ginsenoside F1 in Figs. 6A and 6B, &amp;bull; indicates ginsenoside Rg1, and &amp; circ &amp; represents ginsenoside F1.

When the extracellular enzyme is reacted with 2.5 or 5 mg / ml of ginsenoside Rg1 under optimal reaction conditions, the final ginsenoside F1 is produced at 2 or 4 mg / ml and the concentration of 167 to 222 mg / l / h Productivity is highest, showing the highest conversion yield with 100% conversion, which is about 1.5 times higher than the highest reported ginsenoside F1 productivity.

The present invention relates to a method for producing ginsenoside F1 using ginsenoside Rg1 as a substrate using an extracellular enzyme derived from a strain of Fusarium moniliforme var. Subglutinans .

The extracellular enzyme derived from the Fusarium moniliporum herb glubutynin KCTC 6149 strain inhibits proliferation of cancer cells, enhances the anticancer activity of anticancer drugs, inhibits allergies and protects keratinocytes from ginsenoside F1, Due to the high substrate specificity for glucose of Rg1 at position 6, it can be converted to a high yield of 100%.

In addition, it can produce ginsenoside F1 with high yield through optimization of temperature and pH, substrate and enzyme concentration, and can be industrially useful.

Figure 1 shows the conversion of ginsenoside F1 as a result of reaction with ginsenoside Rg1.
FIG. 2 shows the activity of 4-Nitrophenyl β-D-glucopyranoside per dry cell weight, and shows the date of culture.
FIG. 3A shows the activity of the extracellular enzyme according to the temperature.
FIG. 3B shows the activity of the enzyme according to the pH of the extracellular enzyme.
Figure 4 shows the temperature stability of the extracellular enzyme at each temperature.
Figure 5 shows the optimum unit / ml of extracellular enzyme in the production of ginsenoside F1.
Figure 6 shows the production of the ginsenosides F1 by applying the optimal enzyme unit / ml of Figure 5 in the production of ginsenoside F1. (a) was prepared by adding 2.5 mg / ml of ginsenoside Rg1 and 6.7 units / ml of extracellular enzyme, (b) using 5.0 mg / ml of ginsenoside Rg1 and 6.7 units / ml of extracellular enzyme, F1 production.

Claims (6)

Derived from Fusarium moniliforme var. Subglutinans strain, which is a fungus fungus fungus, has an optimum activity temperature of 50 to 60 ° C, an optimum pH of 6.0 to 7.0, and a ginsenoside A composition for the production of ginsenoside F1 comprising an extracellular enzyme having an optimum enzyme unit of F1 production of 1.2 unit / ml to 6.7 unit / ml as an active ingredient,
The extracellular enzyme has an activity of selectively hydrolyzing only glucose of the 6th carbon of ginsenoside Rg1 to convert it to ginsenoside F1,
The extracellular enzyme was obtained by flat plate culture for 6 days at 28 ° C in a solid oatmeal medium (oatmeal 55 g / L, agar 7 g / L) of Fusarium moniliformum buclobultinans KCTC 6149, The cells were cultured for 5 to 8 days at 28 DEG C in a liquid oatmeal medium (oatmeal 55 g / L), and then 561 g of ammonium sulfate was added per 1 L of the culture solution. After elution for 12 hours, the cells were centrifuged at 13,000 x g for 20 minutes And the precipitate obtained by centrifugation is dissolved in 50 mM sodium citrate buffer solution and 50 mM potassium phosphate buffer solution, respectively, and ammonium sulfate is removed through desalting.
2. The composition for the preparation of ginsenoside F1 according to claim 1, wherein the extracellular enzyme has an optimal activation temperature of 50 &lt; 0 &gt; C.
The composition for preparing ginsenoside F1 according to claim 1, wherein the extracellular enzyme has an optimum active pH of 6.0.
Derived from Fusarium moniliforme var. Subglutinans strain, which has an optimum activity temperature of 50 to 60 DEG C and an optimal pH of 1.2 to 1.2, which is an extracellular enzyme having a pH of 6.0 to 7.0 Reacting ginsenoside Rg1 with ginsenoside Rg1 in units / ml to 6.7 units / ml,
(a) Pseudomonas mobilis formum V. subtilis KTTC 6149 strain was plated on solid oatmeal medium (oatmeal 55 g / L, agar 7 g / L) at 28 ° C for 6 days, and mycelia was recovered to obtain liquid oatmeal And cultured at 28 DEG C for 5 to 8 days with a medium (oatmeal 55 g / L). Then, 561 g of ammonium sulfate was added per 1 L of the culture solution and eluted for 12 hours and centrifuged at 13,000 x g for 20 minutes at 4 DEG C, ;
(b) dissolving the precipitate obtained in step (a) in 50 mM sodium citrate buffer solution and 50 mM potassium phosphate buffer solution, respectively, and removing ammonium sulfate through desalting to obtain an extracellular enzyme; And
(c) reacting the extracellular enzyme obtained in step (b) with ginsenoside Rg1 to selectively hydrolyze only the glucose of the 6th carbon of ginsenoside Rg1 to convert to ginsenoside F1; and A method for producing senoside F1.
delete 5. The method according to claim 4, wherein the concentration of ginsenoside Rg1 in step (c) is 2.5 mg / ml or 5.0 mg / ml.

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