KR20040026747A - Method for preparation of rebaudioside A using microorganism. - Google Patents

Abstract

PURPOSE: A method for preparing rebaudioside A using a microorganism is provided. The microorganism produces extracellular enzymes which rapidly produce rebaudioside A, so that high quality rebaudioside A can be rapidly and stably prepared. CONSTITUTION: A method for preparing rebaudioside A comprises digesting a beta-1,3 bond of insoluble beta-1,3 glucan such as pachyman and curdlan by using beta-1,3 glucanase produced from a microorganism Streptomyces sp., preferably Streptomyces matensis NRRL B-2576 to prepare glucose, and binding the glucose to stevioside by using a Streptomyces matensis(NRRL B-25767) specific glucosyltransferase, wherein the enzymes may be produced from E. coli, Streptomyces sp., Bacillus sp., yeast, Pichia sp., Hansenula sp., Chlorella and plants.

Description

Method for preparation of rebaudioside A using microorganism.

The present invention relates to a riboside production method using the microorganism or the enzyme produced by the microorganism. Most suitably as microorganisms using Exo type β-1,3 Glucanase (hereinafter referred to as glucanase) and stevioside specific Glucosyl transferase (hereinafter referred to as glycotransferase) produced by Streptomyces matensis (NRRL B-2576). Efficiently relates to the production of riboside A (hereinafter referred to as riboside) from stevioside. Typically, riboside A and stevioside are produced from the leaves of the perennial Asteraceae Stevia rebaudiana Bertoni. Stevioside Acid resistance, alkali resistance, heat resistance, colorless odorless and non-fermentation, it is used in alcoholic and pickled foods, and the sweetness is 200-300 times of sugar, while calories are used in diet food at 4 cal per gram. Stevioside has been spotlighted as a rather expensive food additive with 155 tons of annual domestic production, about 2.5 billion won in domestic shipments, and $ 1.6 million in exports (as of the end of December, 1998). It is becoming.

Therefore, to overcome this, enzyme-treated glucosyl steviosides have been developed, but still have not removed the bitter taste. Ribodioside A, on the other hand, has a sweetness of 400 times higher than steviosides, has no bitter taste, is closest to sugar, and has the characteristics of low calorie steviosides, making it a high-quality food additive. However, the amount of riboside produced from Stevia plants is about 20%, which is lower than the amount of stevioside, which is 70-80% of the total content, and thus the unit price is high, and there are many technical problems in extracting only riboside. There is an economic problem.

Many researchers have attempted to produce a large amount of riboside, and recently, Japan's INK chemical industry has succeeded in breeding a high content of riboside plant species. Ribodioside high-content plants have been bred, but the plants take about 3 to 6 months from cultivation to production and cover a large area. In particular, the quality and crop conditions vary depending on the weather, and the production cost is determined according to the labor cost of plantation.

The present inventors used Streptomyces matensis NRRL B-2576 strain in order to overcome the above-mentioned difficulties of riboside productivity and have market economy. Β-1,3 glucanase produced by NRRL B-2576 cuts glucose into monosaccharides by cleaving β-1,3 bonds of insoluble β-1,3 glucans such as pachyman and curdlan. Will be made. Glucose produced in this way is specifically linked to steviosides by NRRL B-2576-specific sugar transfer enzymes to produce riboside A.

There are several advantages to using the NRRL B-2576 strain to produce ribosides. First, a large amount of riboside can be produced in a short time. Stevia plants can take five to seven months from sowing to harvest, while NRRL B-2576 requires four to five days. Second, it does not require much space for riboside production. Of course, it requires a considerable amount of cost to equip the production facilities, but once it is input, it is possible to continuously and stably produce the riboside regardless of seasonal or external environmental change. If NRRL B-2576 strain can be used to produce a large amount of riboside in a short period of time, its usage will increase considerably due to the characteristics of riboside, which can be used in the sweetener market to cover the shortcomings of stevioside. Expected. The present invention is a technology for producing riboside in a short time by the enzymatic reaction in stevioside has a significant industrial significance to produce a sweetener superior in quality than the existing stevioside.

Hereinafter, an embodiment for the present invention will be described in detail as follows.

The present inventors used Streptomyces matensis NRRL B-2576 strain in order to overcome the above-mentioned difficulties of riboside productivity and have market economy. Β-1,3 glucanase produced by NRRL B-2576 cuts glucose into monosaccharides by cleaving β-1,3 bonds of insoluble β-1,3 glucans such as pachyman and curdlan. Will be made. Glucose produced in this way is specifically linked to stevioside by NRRL B-2576 specific sugar transfer enzyme to produce riboside A.

FIG. 1 is a diagram showing the measurement of Exo type beta 1,3-glucanase activity by TLC analysis, and it can be seen that only water-insoluble Pakki was decomposed and converted into glucose.

Figure 2 is a measure of the activity of the stevioside-specific sugar transfer enzyme of the strain used in the present invention using TLC, it can be confirmed that the stevioside is converted to riboside by the enzyme.

3 is a diagram showing the best activity at pH 6 as a measure of the acidity of glucanase and sugar transferase.

4 is a measure of the activity according to the temperature of the glucanase and the sugar transfer enzyme showed the highest enzyme activity at 50 to 60 degrees.

The present invention relates to a riboside production method using a microorganism or an enzyme produced by the microorganism. Most suitably as microorganisms, Exo type β-1,3 Glucanase (hereinafter referred to as glucanase) produced by Streptomyces matensis (NRRL B-2576) and stevioside specific Glucosyl transferase (hereinafter referred to as glycotransferase) Efficiently relates to the production of riboside A (hereinafter referred to as riboside) from stevioside.

Typically, riboside A and stevioside are produced from the leaves of the perennial Asteraceae Stevia rebaudiana Bertoni. Stevioside Acid resistance, alkali resistance, heat resistance, colorless odorless, non-fermentation, it is used in alcoholic and pickled foods, sweetness is 200 ~ 300 times of sugar, while calories are 4 cal / g used in diet foods. Stevioside has been spotlighted as a rather expensive food additive with 155 tons of annual domestic production, about 2.5 billion won in domestic shipments, and $ 1.6 million in exports (as of the end of December, 1998). It is becoming. Therefore, to overcome this, enzyme-treated glucosyl steviosides have been developed, but still have not removed the bitter taste.

Ribodioside A, on the other hand, has a sweetness of 400 times higher than steviosides, has no bitter taste, is closest to sugar, and has the characteristics of low calorie steviosides, making it a high-quality food additive. However, the amount of riboside produced from Stevia plants is about 20%, which is lower than the amount of stevioside, which is 70-80% of the total content, and thus the unit price is high.There are many technical problems in extracting only riboside. There is an economic problem.

Many researchers have attempted to produce a large amount of riboside, and recently, Japan's INK chemical industry has succeeded in breeding a high content of riboside plant species. Ribodioside high-content plants have been bred, but the plants take about 3 to 6 months from cultivation to production and cover a large area. In particular, the quality and crop conditions vary depending on the weather, and the production cost is determined according to the labor cost of plantation.

The present inventors used Streptomyces matensis NRRL B-2576 strain in order to overcome the above-mentioned difficulties of riboside productivity and have market economy. Β-1,3 glucanase produced by NRRL B-2576 cuts glucose into monosaccharides by cleaving β-1,3 bonds of insoluble β-1,3 glucans such as pachyman (Pacchiman) and curdlan (Curdlan). Will be made. Glucose produced in this way is specifically linked to steviosides by NRRL B-2576-specific sugar transfer enzymes to produce riboside A.

There are several advantages to using the NRRL B-2576 strain to produce ribosides. First, a large amount of riboside can be produced in a short time. Stevia plants take five to seven months from sowing to harvesting, while NRRL B-2576 requires four to five days. Second, it does not require much space for riboside production. Of course, it requires a considerable amount of cost to equip the production facilities, but once it is input, it is possible to continuously and stably produce the riboside regardless of seasonal or external environmental change.

If NRRL B-2576 strain can be used to produce a large amount of riboside in a short period of time, its usage will increase considerably due to the characteristics of riboside, which can be used in the sweetener market, while expanding its use while covering the shortcomings of stevioside. It is expected. The present invention is a technology for producing riboside in a short time by the enzymatic reaction in stevioside has a significant industrial significance to produce a sweetener superior in quality than the existing stevioside.

Hereinafter, an embodiment for the present invention will be described in detail as follows.

Example 1

Strain Culture and Enzyme Production

Streptomyces matensis was inoculated into 50 ml of sterile medium with 0.5% yeast extract, 2.0% malt-extract, 0.5% glucose, 1.0% cuduran, and pH 7.0 and precultured at 300 rpm at 30 ° C. In the main culture, the whole culture solution was inoculated in 3 L of sterile medium identical to the whole culture medium and cultured in a 5 L fermenter for 3 days. The cultured shake culture was centrifuged at 10,000X g for 10 minutes to obtain 2.4 L of the supernatant. The supernatant was precipitated with 65% ammonium sulfate, centrifuged at 10,000X g for 30 minutes, only the protein was collected, and dissolved in 0.1M potassium phosphate buffer (pH 6.0). The protein solution thus obtained was dialyzed at 4 ° C. for 3 days on 0.1 M potassium phosphate buffer (pH 6.0). The salt-free protein solution was freeze-dried to a powder state. About 1.7 g of enzyme powder was obtained and the total amount of enzyme was about 550 μg / ml.

Example 2

Exo Type β-1,3 Glucanase Activity Measurement by Thin Layer Chromatography (TLC)

The enzyme was dissolved in 0.1M potassium phosphate buffer (pH 6.0) to give final concentrations of 1% and 3%, and pachyman (CALBIOCHEM), an insoluble glucan with a final concentration of 0.5%, was used as a substrate. Enzyme reaction was carried out in 0.1M potassium phosphate buffer (pH 6.0) and the reaction conditions were reacted for 2 hours at 60 ℃ and heat-treated at 90 ℃ for 15 minutes to remove the enzyme activity. 0.5 μl of the reaction solution was loaded on a TLC plate (Slica gel 60 F 254, Layer thickness 0.2 mm, 20 × 20 cm, KODAK) and then developed in iso-propanol / acetic acid / water (67:10:23). The developed TLC plate was well dried and developed using α-naphthol solution (1.57 g α-naphthol, 51 mL ethanol, 4 mL distilled water). As a result, glucose was not seen in the reaction solution reacted with 0.5% pachyman and 3% enzyme that lost enzymatic activity by heat treatment (Fig. 1, lane3). (Fig. 1). Therefore, it was found that Pachyman, an insoluble glucan, was degraded to glucose by β-1,3 glucanase produced by Streptomyces matensis NRRL B-2576 strain. Therefore, NRRL B-2576 strain was found to be able to break down insoluble glucan into glucose, a monosaccharide. Cudlan was also found to be degraded by this enzyme. It is therefore estimated that this enzyme can degrade most of insoluble glucan.

Example 3

Determination of Glycotransferase Activity by TLC

The final enzyme was dissolved in 0.1 M potassium phosphate buffer (pH 6.0) to make final concentrations of 1% and 2.5%, and final concentrations of 1% stevioside (Sigma) and 1% curdran were used. Enzyme reaction was performed on 0.1 M potassium phosphate buffer (pH 6.0) by the same method as β-1,3 globulase activity measurement. The reaction conditions were reacted for 2 hours at 60 ℃ and then heat treated at 90 ℃ for 15 minutes to remove the enzyme activity. 0.5 μl of the reaction solution was loaded on a TLC plate and then developed in chloroform / metanol / water (30: 15: 1). The developed TLC plate was developed by treating α-naphthol solution. As a result, it was found that the sugar conversion from stevioside to riboside by the enzyme (Fig. 2).

Example 4

Measurement of riboside conversion and reaction yield from steviosides by β-1,3 glocanase and sugar transferase

250 mg of powdered enzyme, 100 mg of stevioside (Sigma) and 100 mg of curdlan were dissolved in 10 ml of 0.1 M potassium phosphate buffer (pH 6.0). Enzyme reaction was carried out for 2 hours at 60 ℃ and heat-treated for 15 minutes at 90 ℃ to remove the enzyme activity. In order to purify stevioside and riboside on the reaction solution, the resultant was passed through a synthetic adsorption resin dia ion HP-20 (S.V. is 2) to adsorb stevioside and then desorbed with 95% ethanol. The desorbent was filtered under reduced pressure with ethanol and passed through Envalite IR-120B, a strong acidic ion exchange resin, and Envalite IRA-93 (S.V., 2), a weakly basic ion exchange resin, to remove salts. The desorption liquid from which the salt was removed was lyophilized to obtain 18.4 mg of stevioside and 81.1 mg of riboside. The riboside conversion and reaction yields are shown in Table 1.

Table 1. riboside from stevioside by b-1,3 glucanase and sugar transferase

Conversion and reaction yield.

Reactive enzyme Stevioside and ribodiside amount after the reaction (mg) % Conversion ¹ Reaction yield (%) ² Stevioside Rivodioside b-1,3 glucanase + sugar transferase 18.4 81.1 100 81

% Conversion = (produced amount of riboside / reduced stevioside) x 100

2. Conversion (%) = (produced amount of riboside / stevioside before reaction) x 100

Example 5

pH Safety of β-1,3 Glucanase and Glycotransferase

In order to measure the safety of β-1,3 glucanase and sugar transfer enzymes according to pH, 0.1M glycine HCl (pH 3-5) and 0.1M potassium phosphate buffer (pH 6-8) were used. Enzyme reaction was performed by dissolving the powdered enzyme in each buffer according to pH so that the final concentration was 2.5%. The final concentration was 1% stevioside (Sigma) and 1% curdlan. The reaction conditions were reacted for 2 hours at 60 ℃ and then heat-treated for 15 minutes at 90 ℃ and was separated and purified stevioside and riboside in accordance with Example 4 to determine the riboside conversion according to each pH. As a result, when the pH was 6, the conversion was 100% and the reaction yield was about 70% or more (Fig. 3). The enzyme activity was higher than 80% at pH 5-7, but the enzyme activity decreased rapidly at pH 5 and above 7.

Example 6

Temperature Safety of β-1,3 Glucanase and Sugar Transfer Enzyme

The enzyme was dissolved in 0.1M potassium phosphate buffer (pH 6.0) to give a final concentration of 2.5%. The final concentrations were 1% stevioside (Sigma) and 1% curdlan. In order to measure the temperature stability of β-1,3 glogganase and the sugar transfer enzyme according to the temperature, the reaction was carried out at 30 to 80 ° C. for 2 hours and then heat treated at 90 ° C. for 15 minutes to remove the enzyme activity. Stevioside and riboside were separated and purified according to Example 4 to measure the riboside conversion according to the respective temperatures, and as a result, it had a riboside conversion of 100% at 50 ° C. (FIG. 4). The conversion rate was more than 50% at 20-40 ° C., but the conversion rate decreased from 60 ° C.

According to the present invention, it was found that the Streptomyces NRRL B2576 strain can produce an enzyme capable of producing high-quality riboside A in a short time using water-insoluble glucan and stevioside as a substrate. In particular, both enzymes are secreted out of cells and are found to be easy to purify and extremely stable to the external environment. Therefore, according to the present invention, it was found that high-quality riboside A can be produced using exo type glucanase and stevioside-specific glycotransferases produced by the strain, and the produced riboside is used as food, medicine, It can be used in cosmetics and the like.

Claims (5)

A method for producing riboside A using microorganisms or enzymes produced by microorganisms. The microorganism of claim 1, wherein the microorganism is Streptomyces sp. The microorganism of claim 2 is most preferably Streptomyces matensis NRRL B-2576, but is not limited thereto. 2. The enzyme produced by the microorganism according to claim 1, which is glucanase and glucosyltransferase. When the enzyme of claim 1 is produced in E. coli, Streptomyces microorganism, Bacillus microorganism, baker's yeast, Pichia yeast, Hanserula strain, Chlorella and plants by genetic recombination.