JPH0673468B2 - Manufacturing method of rebaudioside A - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention converts rebaudioside A, which is an excellent sweetener contained in leaves and stems of Stevia rebaudiana bertney belonging to the Asteraceae family, to extremely high conversion. The present invention relates to an enzymatic method of producing stevioside at a rate.

More specifically, in an aqueous solution containing stevioside and a β-1,3-glucosyl sugar compound, β-1,3-
When a glucosyl sugar compound is used as a sugar donor and an enzyme having an activity of transferring sugar to stevioside is caused to act on the conversion reaction to rebaudioside A, an organic solvent and / or exotype β-in the aqueous solution is used. The present invention relates to a method for producing rebaudioside A having a high conversion rate, which is characterized in that 1,3-glucanase is allowed to coexist or separately act to cause a transfer reaction.

(Prior Art) Rebaudioside A (β-1,3-monoglucosyl stevioside) is contained in Stevia leaves and stems at about 2 to 6%,
It has the second highest content after Stevioside (6-12%), which is the main component. Until now, it has been used as a mixture of the two as a stevia sweetener, but while stevioside has a bitterness and has an aftertaste, rebaudioside A has a mellow sweetness with no bitterness and has a sucrose content. The sweetness ratio in comparison is also high. Until now, many methods have been tried to improve the taste quality of stevia sweeteners, such as adding natural sugars and amino acids, but recently sugar was added to stevioside to eliminate bitterness without increasing calories. Stevia sweeteners have been developed. (JP-A-54-50
However, it has a drawback that the sweetening ratio is lower than that of stevioside. For this reason, the development of a stevia sweetener containing rebaudioside A alone or having a high content is eagerly desired. Conventionally, as a method for obtaining rebaudioside A, a method in which recrystallization is repeated after crystallizing and removing stevioside from a mixed state of stevioside and rebaudioside A obtained by extracting and purifying from dried stevia leaves However, since the yield is poor, it is necessary to make rebaudioside A high in content during extraction.

Therefore, the present inventors have already completed the invention aimed at converting stevioside to rebaudioside A by adding glucose to stevioside which is most present in stevia leaf extract by a fermentation method or an enzymatic method, JPA
The patent application has been filed as 58-149697. The invention of the earlier application is stevioside and β-1,3-glucosyl sugar compounds such as curdlan, pakiman, and β-1,3-glucose of laminarin sugar.
In the aqueous solution containing the sugar compound having a bond, these β-
Culture of a microorganism having the C ₃ position in the transition to be active i.e. beta-1,3-glucosyltransferase activity of beta-D-glucose bound from 1,3 glucosyl compound glucose to steviol hydroxyl group of a aglycone stevioside The objective is to produce rebaudioside A by reacting bacterial cells or a treated product of bacterial cells.

However, according to the method of the prior invention, two or more kinds of β-1,3-glucosyl stevioside containing rebaudioside A are produced, but the molar conversion rate to rebaudioside A is 20% or less, which is satisfactory. It wasn't possible.
Therefore, in order to search for a microorganism having the ability to highly convert stevioside to rebaudioside A with remarkable selectivity, the present inventors have isolated many microorganisms from a wide natural soil world, and among them, Streptomyces spp. , Which has a strong β-1,3-dulcosyltransferase activity, and found the ability of the microorganism to convert stevioside to rebaudioside A only at a high molar conversion rate. We applied for it as Sho 59-17996.

However, the conversion rate of rebaudioside A from stevioside by β-1,3-glucosyl glycosyltransferase was limited to 50 to 60% even if the reaction conditions were controlled.

(Means for Solving Problems) Therefore, the present inventors transfer sugar to stevioside,
Diligently researched on a method of producing rebaudioside A at an extremely high conversion rate by suppressing the production of side reaction products by an enzyme having a β-1,3-glucosyl transfer activity for converting to rebaudioside A, The present invention has been completed.

That is, the present invention is a β-produced by a microorganism in an aqueous solution containing a β-1,3-glucosyl sugar compound and stevioside.
By acting an enzyme having a 1,3-glucosyl transfer activity,
In the conversion reaction of stevioside to rebaudioside A, an organic solvent and / or an exotype β-1,3-glucanase coexist or act separately in the aqueous solution to cause a transfer reaction. The present invention provides a method for producing baudioside A.

(Structure) An aqueous solution containing a stevia sweetening component used in the present invention is
The solution is not limited to an aqueous solution of a highly purified or crudely purified Stevia sweetener product, and may be a solution during or after extraction from Stevia leaves.

Β-1,3-gluecosyltransferase used in the present invention is β
Any enzyme, regardless of microorganism, animal or plant, can be used as long as it is an enzyme capable of transferring a sugar to stevioside using a 1,3-glycosyl sugar compound as a sugar donor and converting it to rebaudioside A. It may be from the one.

In the present invention, an enzyme produced by a microorganism belonging to Streptomyces is particularly preferable. Among them, a particularly preferable microorganism is Streptomyces sp. DIC-108.

Further, the present invention, when the enzyme having the β-1,3-glucosyl transfer activity is allowed to act on an aqueous solution containing a β-1,3-glucosyl sugar compound and stevioside, coexist with an organic solvent or exo Type β-1,3-glucanase is allowed to coexist in the transfer reaction, or an organic solvent and exotype β-1,3-glucanase are allowed to coexist, or both are used separately, but an organic solvent It is more preferable to use together with the glucanase. Β- of this exotype
1,3-glucanase decomposes a β-1,3-glycosyl sugar compound to produce glucose as a product, and β-
It may be of any origin as long as it has an action of increasing the conversion rate of stevioside to rebaudioside A by an enzyme having a 1,3-glucosyl transglycosylation activity.
Preferably, it is derived from a microorganism belonging to the genus Streptomyces. A particularly preferred microorganism is Streptomyces.sp DIC-10.
8, but not limited to this.

The mycological properties of Streptomyces sp. DIC-108 have already been described in JP-A-59-17996, but in detail, they have the following properties.

[Mycological properties of Streptomyces sp. DIC-108] (1) Morphological characteristics The growth of vegetative mycelium on the medium used (including ISP medium) is excellent, and inorganic starch salts, oatmeal agar, It forms abundant aerial mycelia on yeast malt agar.

Sporulating aerial hyphae are straight or curved. The spores are ellipsoids with a minor axis x major axis 0.7-0.8μ x 1.0-1.2μ. The surface structure of the spores is warty when observed with a scanning electron microscope.

(2) Growth state in various media 1) Sucrose nitrate agar medium (37 ° C) Gray aerial hyphae are formed on light brown basal hyphae, and no soluble pigment is observed.

2) Glucose / asparagine agar medium (37 ℃) The formation of aerial hyphae is not observed due to the growth of pale yellowish white color. No soluble dye is found.

3) Glycerin / asparagine agar medium (ISP medium-No.
5 37 ℃) Growth of pale yellow color shows no aerial hyphae. In addition, no dissolved raw dye is observed.

4) Starch-inorganic salt agar medium (ISP medium 37 ° C) Green gray aerial mycelium grows on colorless growth, and no soluble pigment is observed.

5) Tyrosine agar medium (ISP medium-7, 37 ° C) On the 7th day of cultivation, aerial hyphae did not grow on light brown growth.
On the day, aerial hyphae of gray color are settled. No soluble dye is found.

6) Nutrient agar medium (37 ° C) Light greenish gray aerial mycelium grows on the growth of greenish gray and no soluble pigment is observed.

7) Yeast malt agar medium (ISP medium-2, 37 ° C) Light greenish gray aerial hyphae are settled on light brown growth. No formation of water-soluble dye was observed.

8) Oatmeal agar medium (ISP medium-3, 37 ° C) Green tea gray aerial mycelium grows on colorless growth, and no water-soluble pigment is formed.

(3) Physiological properties 1) Growth temperature range In a growth test using a yeast extract / malt extract liquid medium,
It grows at 30 ℃ to 50 ℃, but the optimum temperature is 37 ℃ to 45 ℃.

2) Liquefaction of gelatin: negative 3) Coagulation of skim milk and peptization of skim milk: negative 4) Formation of melanin pigment (peptone-yeast-iron agar medium, ISP medium 6): negative 5) Degradation of starch: positive (A white ring is formed in the decomposition zone) 6) Utilization of carbon source (Pridham, Godlieve agar-9, 37 ° C) D-glucose, L-arabinose, D-xylose,
D-fructose, inositol, L-rhamnose, D
-Growth utilizing mannitol and galactose well,
Do not use sucrose, raffinose or salicin.

7) Cell wall composition The sugar composition Type described in ISP belongs to Type I.

This strain has been applied for deposit at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and has been entrusted as Micro-organism Research Institute No. 6593.

A usual solid medium or liquid medium is used for the culture for producing the enzyme according to the present invention, and a β-1,3-glucosyl sugar compound can be used as a carbon source of the medium for liquid culture.

For example, curdlan, pachyman, laminarin, lichenan, yeast cell wall or partial hydrolysates thereof, leucosine, callose, paramylon and the like, and as the nitrogen source, ammonium sulfate, ammonium salt, ammonium phosphate, sodium nitrate, urea, peptone, Both organic nitrogen and inorganic nitrogen such as casein can be used.

Examples of natural nutrient sources include various molasses, corn steep liquor, oatmeal, taste liquid, fish meal, meat extract, yeast, yeast extract, potato extract, malt extract and the like.

Examples of the inorganic substance include dipotassium phosphate, monosodium phosphate, magnesium sulfate, and trace metals. In addition, vitamins and the like can be added if necessary. The use concentration of these is 0.1 to 40% by weight. In addition, in order to suppress foaming during fermentation, 0.
You may add 0001-1.0 weight% of antifoaming agents. As the defoaming agent, a usual defoaming agent such as silicone or soybean oil is used.

The aerobic liquid culture such as shaking culture and aeration culture is suitable as the culture method, and the pH is 5.0 to 8.0 and the culture temperature is 20 ° C to 50 ° C.
It is cultured for about 6 days, preferably at pH 6.5 to 7.5 and 35 to 40 ° C for about 2 days.

Enzymes having β-1,3-glucosyl transfer activity and exotype β-1,3-glucanases are enzymes that are produced outside the cells, so after culturing, filter or centrifuge to eliminate bacteria. , Collect the supernatant. And concentrate if necessary,
Ammonium sulfate, salting out with sodium sulfate, or an organic solvent such as acetone, ethanol, methanol, and isopropanol is added to obtain the enzyme as a precipitate, which is dried and stored.

Β-from the DIC-108 strain preferably used in the present invention
The properties of an enzyme having a 1,3-glucosyl transfer activity (called SGTase) and an exotype β-1,3-glucanase (called β-1,3-glucanase Fi II) are shown below.

SGTase (1) Action: It acts on a β-1,3-glucosyl sugar compound such as curdlan, laminarin, pakiman, yeast cell wall and a partial degradation product thereof to show an endo-type hydrolysis action. The main decomposition products when G ₅ (laminaripentaose) is used as a substrate are G ₂ and G ₃ . (See FIGS. 1 and 2) As a sugar-accepting substrate, any of steviol glycoside, stevioside, rebaudioside C, or rebaudioside A, exhibits a transglycosylation activity.

(2) Working pH range and optimum working pH: It works in the range of pH 3 to 9, and the optimum working pH is around 6.
(See Fig. 3) (3) Working temperature range and optimum working temperature: It works from about 20 ℃ to about 70 ℃, and the optimum working temperature is about 65 ℃. (See FIG. 4) (4) Thermal stability: The activity was reduced by about 50% by heat treatment in 0.1 M phosphate buffer (pH 6.0) for 1 hour.

(5) Purification method This enzyme was recovered from the culture filtrate as 65% saturated ammonium sulfate and recovered as a precipitate, which was then subjected to DEAE-Sephadex A-25 column chromatography (0.01 M Tris-HCl buffer, pH 7.5). Collect the fractions eluted at a concentration of 0.2M. And after desalting CM-
Sephadex C-25 column chromatography (0.01 M acetate buffer, pH 6.0) was performed to collect the unadsorbed fractions, and then D
EAE-Sephadex A-25 column chromatography (0-
Collect active fractions with a 0.25 M linear gradient. Substantially homogeneous enzyme protein is then obtained by G-100 gel permeation chromatography.

(6) Molecular weight: The molecular weight by Sephadex G-100 gel chromatography was estimated to be about 27,000.

β-1,3-glucanase Fi II (1) Action: β-1,3-glucosyl sugar compound such as curdlan, laminarin, pachyman, yeast cell wall and partial hydrolyzate thereof, and exo-type hydrolysis action , But has no transglycosylating action. When G ₅ is used as a substrate, the main decomposition products are glucose and G ₃ . (Refer to FIG. 5) (2) Working pH range and optimum working pH: pH 3 to 9 works, and optimum working pH is 6. (Fig. 6
(3) Working temperature range and optimum working temperature: It works from about 20 ℃ to about 70 ℃, and the optimum working temperature is about 60 ℃. (See Fig. 7) (4) Thermal stability: Completely inactivated by heat treatment at 45 ℃ for 1 hour in 0.01M acetate buffer, but at 60 ℃ in 0.1M phosphate buffer for 1 hour.
Deactivates by about 60% by heat treatment for a long time.

(5) Purification method: This enzyme was recovered from the culture filtrate as a precipitate at 65% saturation with ammonium sulfate and then subjected to DEAE-Sephadex A-25 column chromatography (0.01 M Tris-HCl buffer, pH 7.5) to salify the enzyme. The fractions eluted at a sodium concentration of 0.2M are collected. After desalination
CM-Sephadx C-25 column chromatography (0.01M acetate buffer, pH 6.0) was performed to collect the fractions eluted with 0.3M sodium chloride solution, and then 0.2M phosphate buffer for hydroxyapatite column chromatography. A nearly uniform protein can be obtained by collecting the active fractions eluted.

(6) Molecular weight: The molecular weight by Sephadex G-100 gel chromatography was estimated to be about 44,000.

As the organic solvent used in the present invention, any solvent having a function of increasing the conversion rate of rebaudioside A from stevioside or having a function of inhibiting a stevioside-based sweetener compound other than rebaudioside A from being formed For example, methanol, ethanol, propyl alcohol, butyl alcohol and the like can be mentioned as the alcohols. Examples of the ketones include acetone, alkyl methyl ketone, diethyl ketone, n-butyl ethyl ketone, methyl ethyl ketone and the like.
Examples of the acetic acid esters include methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate and the like.
Examples of the hydrocarbons include hexane and the like.
Examples of phthalates include dimethyl phthalate,
Examples include diethyl phthalate and dibutyl phthalate. The glycols include ethylene glycol, propylene glycol, thioglycol and the like. Other solvents such as dimethylsulfogoxide (DMSO) and dioxane 2-mercaptoethanol are used. One or more kinds of organic solvents selected from these are stevioside and β in a volume ratio.
It is used in an amount of 1 to 50% (V / V), preferably 2 to 30%, with respect to an aqueous solution containing a compound such as -1,3-glucosyl.

Next, a method for converting stevioside to rebaudioside A at a high conversion rate using these enzymes will be described.

First, as a β-1,3-glucosyl sugar compound, for example, 0.1 to
For laminari oligosaccharides containing 30 wt% curdlan or laminaripentaose obtained by decomposing it, 0.
1 to 10 wt% stevioside dissolved in a solution, preferably 2 to 30% (V / V), more preferably 5 to 20% (V / V)
, An organic solvent such as alcohols or ketones,
For example, acetone, ethyl acetate, methyl ethyl ketone, hexane, DMSO, dioxane, thioglycol, propylene glycol, methyl phthalate, methanol, ethanol, etc. are added to act on β-1,3-glucosyltransferase (for example, SGTase). Or exotype β-1,
Act simultaneously with 3-glucanase or β-
After the reaction with 1,3-glucosyltransferase, the exotype β-1,3-glucanase is further allowed to act. By this reaction, the conversion rate from stevioside to rebaudioside A becomes 70 to 100%, and the reaction yield of stevioside to rebaudioside A is 40% or more.

The β-1,3-glucosyl sugar compound used in the method of the present invention may be any one as long as it produces rebaudioside A from stevioside by Streptomyces sp. DIC-108 which is preferably used. , Pakiman, curdlan, laminarin, yeast cell wall or partial hydrolyzate of yeast cell wall, and the yeast referred to here is
For example, Saccharomyces genus, Candida genus, Pichia genus, Schizosaccharomyces genus, Tolulopsis genus, Hysenula genus and the like, further refers to rheucosin (diatomaceous cell wall), callose (higher plant Haseolas cell wall),
Β- such as paramylon (cell wall of unicellular algae), lichenan (moss extract), saccharides obtained from seed endosperm of gramineous plants (the grasses referred to here are rice, barley, wheat, etc.) Examples include 1,3-glucosyl sugar compound-containing materials. Among them, preferable examples include pakiman, curdlan and laminarin, and these are more preferable because they are easily available. In addition, the card run is
Water-insoluble β-based on β-1,3-glucoside bonds
Glucan is a general term for polysaccharides that produce a hard elastic thermo-irreversible gel when their suspension is heated. As for this thing, what was made by the microorganism is marketed at present. (See Agr. Biol. Chem., 29, 757.'65 or Fermentation & Industry 36, 2., '78).

The culture broth and the microbial cells in which Streptomyces sp. DIC-108 preferably used in the present invention may be reacted in a batch system, or the microbial cells are immobilized by a known method to continuously perform a conversion reaction. It can also be done.

The transfer reaction conditions of the present invention are stevioside and β-1,3-
An aqueous solution containing a glucosyl sugar compound is reacted with a culture solution of Streptomyces sp. DIC-108, bacterial cells, treated cells, or an enzyme having a β-1,3-glucosyl transfer activity separated from these. Just do it. In the case of purified stevioside, stevioside used in the reaction has a concentration of stevioside in the reaction solution of about 0.1 to about 10% by weight,
The concentration of the β-1,3-glucosyl sugar compound may be about 0.1 to about 30% by weight. The pH and temperature of these reaction solutions are β-1,3.
-A condition under which an enzyme having a glucosyl transfer activity can react to generate rebaudioside A is usually
pH 3-10, preferably pH 5-8, temperature 20-70 ° C, preferably 30
~ 50 ° C is suitable. The reaction solution thus produced with rebaudioside A can be used as a sweetener as it is. In addition, if necessary, after inactivating the enzyme or bacterial cells by heating, styrene and divinylbenzene polymerization adsorption resin such as Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei), Amberlite XAD-2 (trade name) , Organo) sugar, or ion-exchange resin (for example, H-type strongly acidic ion-exchange resin and OH-type weakly basic ion-exchange resin) for desalting, and concentrating this to obtain a syrupy sweetener. Alternatively, it may be dried or powdered to obtain a powdered sweetener.

Further, the desalted reaction solution was purified to obtain rebaudioside A.
It is also possible to separate and collect to obtain a sweetener. At this time, various methods such as concentration under reduced pressure, membrane concentration, vacuum drying, and spray drying can be freely used for concentration, drying, and pulverization. The sweetness of the rebaudioside A thus obtained varies depending on the measurement conditions of the sweetness, but is generally 1.3 times stronger than the sweetness corresponding to the weight of the solid product of stevioside used in the reaction. The quality of its sweetness is
It has no harshness such as bitterness or astringency, has a mellow sweetness similar to sugar, and has a good residual taste.

This rebaudioside A is an odorless, white powder that has no bitterness, odor, taste, etc., and is soluble in water. Therefore, the coexistence ratio of stevioside and glycyrrhizin, and any coexistence under liquid or powdery conditions. Can be made. In addition, rebaudioside A can be effectively used in its taste characteristics by sharing it with well-known synthetic sweeteners such as saccharin and its salts, sodium cyclamate, dihydrochalcone, and asparatame. By adding and using the present compound to one or more synthetic sweeteners, it is possible to improve the unpleasant taste such as the bitterness and the unpleasant taste peculiar to the synthetic sweetener.

In addition, rebaudioside A is used as an excipient, diluent, adsorbent, sugar, fructose, glucose, lactose, starch syrup, dextrin, starch, etc. Is increased, and it is possible to significantly reduce the usage amount compared to the conventional usage amount.
Furthermore, if this compound is used by adding it to a low-calorie sweetener such as sorbitol, maltitol, mannitol, xylitol, which has a lower sweetness than sugar, it is possible to enhance the sweetness without impairing the advantages of the sweetener, and it is a high-quality low-calorie sweetener. A sweetener is obtained.

It goes without saying that rebaudioside A can be used as a sweetening source for general foods and diet foods, medicines, quasi drugs, cigarettes, feeds and the like.

For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, mayonnaise, dressing, vinegar, three cups vinegar, powdered sushi vinegar, Chinese cabbage, tempura soup, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew sauce, soup A variety of seasonings such as daikon, dashi, mixed seasonings, mirin, new mirin, table syrup, rice crackers, hail, rice cakes, steamed buns, uiro, bean paste, sheep can,
A variety of Japanese sweets such as water sheep can, jelly, castella, candy, bread, biscuits, crackers, cookies, pies, pudding, butter cream, custard cream, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy, etc. Western confectionery, ice cream, sorbet, ice candy and other frozen desserts, fruit syrup pickles, starch syrup and other syrups, flower paste, peanut paste, fruit paste and other pastes, jam, marmalade, syrup pickles, confectionery and other fruits and vegetables Processed foods, Fukujinzuke,
Pickles such as Senmai-zuke and Rakkyo-zuke, meat products such as ham and sausage, meat ham, fish sausage, kamaboko,
Chikuwa, tempura and other fish meat products, sea urchin, salted squid, sardines, various delicacies such as dried puffer fish, seaweed, wild vegetables,
Tsukudani stew, boiled beans, potato salad, kelp rolls, and other soybean foods made of sardines, small fish, shellfish, fish meat, meat, fruits, vegetables in bottles, canned foods, synthetic liquor, fruit liquor, western liquor, etc. Soft drinks such as alcoholic beverages, coffee, cocoa, juice, carbonated drinks, lactic acid drinks, lactic acid bacteria drinks, pudding mix, hot cake mix, instant juice, instant coffee, instant food and drink such as instant shiruko, etc. Can be used for sweetening. Other pharmaceuticals and quasi-drugs include toothpaste, lipstick, lip balm, internal medicine, troches,
It can also be freely used as a sweetener for liver oil drops, mouth cooling agents, mouth sachets, mouthwashes and the like.

(Effect) According to the present invention, stevioside can be converted to rebaudioside A at a higher conversion rate than the conventional method, and thus the method has high productivity and is an industrially excellent method.

Hereinafter, the method of the present invention and the sweetener obtained by the method will be specifically described with reference to Examples.
Is based on weight.

Examples 1 to 3 (1) Preparation of SGTase and β-1,3-glucanase Fi II Streptomyces sp. DIC-108 (Microtechnology Research Institute, Microbiology Contribution No. 6593)
No.) yeast extract 0.2w / v%, polypeptone 0.2w / v% MgSO
_{4 · 7H 2 0 0.1w / v} %, K 2 HPO 4 times locations 3 consisting of 0.2 w / v%
Inoculation was carried out on 5 and 350 g of curdlan which had been separately sterilized was added at the same time, and culturing was performed with aeration and stirring at 70 Jar at 35 ° C for 40 hours. The obtained culture solution 23 was centrifuged, and the supernatant was salted out with 0.65 saturated ammonium sulfate to obtain a crude enzyme preparation (I) containing SGTase and β-1,3-glucanase Fi II. From (I), SGTase and β-
To isolate and purify 1,3-glucanase Fi II, (I)
Was dissolved in 0.01 M Tris-HCl buffer (pH 7.5), dialyzed with the same buffer to desalting, and adsorbed on a DEAE-Sephadex A-25 (330 ml Vol) column equilibrated with the same buffer to give 0.2 Obtain 470 ml of fraction eluted with M NaCl solution. Next, after dialyzing with 0.01 M acetate buffer, column chromatography with CM-Sephadex C-25 (250 ml vol) was carried out.
Β-1,3-glucanase Fi II is obtained in the NaCl solution elution zone. After 800 ml of SGTase fraction was dialyzed and desalted again, it was adsorbed to DEAE-Sephadex A-25 (100 ml vol) again and eluted with a linear concentration gradient method using 0-0.25 M sodium chloride to obtain the SGTase fraction. 100 ml (prot.44 mg) was obtained. The activity at this time was about 5,800 U. After further concentrating this, G-100
A single enzyme can be obtained by purifying by gel filtration chromatography (φ45 × 1800 mm), but in the present invention, the purified enzyme before Zero filtration can be sufficiently used.
Also, 170 ml of β-1,3-glucanase Fi II fraction (prot.
mg) is further subjected to hydroxyapatite column chromatography (vol. 200 ml) and eluted with 0.18 M phosphate buffer to obtain a single enzyme electrophoretically, but in the present invention, It can be sufficiently performed with a purified enzyme before the hydroxyapatite column chromatography. At this time, β-1,3-glucanase Fi II
The activity was about 6,000 U.

One unit of enzyme activity of SGTase here is pH 7.0, 0.1M
0.5% stevioside and 2% in phosphate buffer
Curdlan of No.3 is allowed to act, and an appropriate amount of enzyme is added to it to make 5.0 ml with water, and the mixture is reacted at 45 ° C. The amount of enzyme that produces 1 μM rebaudioside A per hour under these conditions.

In addition, 1 unit of β-1,3-glucanase Fi II enzyme activity means that 1% of laminaripentaose is suspended in 0.01M phosphate buffer (pH 6.0), an appropriate amount of enzyme is added to it, and water is added. Make it 5.0 ml and react at 45 ℃. 1mg per hour under these conditions
It refers to the amount of enzyme that produces the reducing power corresponding to glucose.

(2) Conversion reaction (selective formation reaction of rebaudioside A) Stevioside (purity 98%) 10 g, laminaripentaose (purity 95%) 40 g, SGTase 20 units prepared previously, and β-1,3- Dissolve 1,000 units of glucanase Fi II in 0.01M phosphate buffer (pH 6.0) 1 and at the same time add ethyl acetate 1
00 ml was added and reacted at 40 ° C. for 16 hours. Then 90 ℃
The enzyme was inactivated by heating for 10 minutes. This solution was passed through a synthetic adsorption resin Diaion HP-20 at SV = 2 to adsorb steviosides, and then desorbed with 95% ethanol.
After the ethanol of the desorption solution was distilled off under reduced pressure, Amberlite-IR-120B (H type, trade name, manufactured by Rohm and Haas Company), which is a strongly acidic ion exchange resin, Amberlite-IRA, which is a weakly basic ion exchange resin. -93 (OH type, product name,
It was desalted by passing it through SV = 2 through Rohm and Haas company product). Then, this was concentrated under reduced pressure at 70 ° C. or lower and dried in a vacuum to obtain powder of rebaudioside A (abbreviated as BebA) 6.70 g.
And 3.10 g of stevioside (abbreviated as Stv) were obtained.

Comparative Example 1 The same operation as in Example 1 was carried out except that β-1,3-glucanase Fi II and ethyl acetate were removed, and the obtained Stv and RebA were as shown in Table 1. there were.

Example 4 The same operation as in Examples 1 and 3 was performed except that 15% (V / V) of acetone was used instead of the ethyl acetate used in Examples 1 and 3. As a result, RebA 3.8 g and Stv 5.1 g were obtained by using SGTase and acetone together, and RebA 6.4 g and Stv 3.2 g were obtained by using SGTase, Fi II and acetone together. The conversion rates were 78% and 94%, respectively, and the reaction yields were 38% and 64%, respectively.

Example 5 The same operation as in Examples 1 and 3 was performed except that 10% (V / V) of methyl ethyl ketone was used instead of the ethyl acetate in Examples 1 and 3. As a result, SGTase and methyl ethyl ketone combined use RebA 3.8g, Stv 5.0g, SGTase, F
RebA 6.0g and Stv 3.8g when combined with i II and methyl ethyl ketone
was gotten. The conversion rates were 76% and 96%, respectively, and the reaction yields were 38% and 60%, respectively.

Example 6 The same operation as in Examples 1 and 3 was performed except that ethanol was used instead of ethyl acetate in Examples 1 and 3. As a result, SGTase and ethanol were used together with RebA3.6g, St
v5.0g, RebA6 with SGTase, Fi II and ethanol.
0g and Stv 4.3g were obtained. The conversion rates are 72% and 95% respectively,
The reaction yields were 36% and 60%, respectively.

Example 7 The same operation as in Example 1 was carried out except that 10 g of stevia sweetener (Stv 58%, RebA 14%, RebC 10%) was used instead of stevioside of Example 1, and Stv 22%, Reb
A stevia sweetener of A56% and RebC2% was obtained.

[Brief description of drawings]

FIG. 1 shows a high-performance liquid chromatography (HLC) chart of laminaripentaose, and FIG. 2 shows an HLC chart of laminaripentaose after the SGTase reaction. FIG. 3 is a graph showing the relationship between the action pH and activity of SGTase, and FIG. 4 is a graph showing the relationship between the action temperature and activity of SGTase. FIG. 5 shows an HLC chart of laminaripentaose after β-1,3-glucanase Fi II reaction. Figure 6 is a graph showing the relationship between the action pH and activity of β-1,3-glucanase Fi II, and Figure 7 shows the relationship between action temperature and activity of β-1,3-glucanase Fi II. It is a graph shown.

Claims (2)

[Claims] 1. A β-1,3 produced by a microorganism in an aqueous solution containing a β-1,3-glucosyl sugar compound and stevioside.
-When reacting an enzyme having a glucosyl transfer activity to convert stevioside to rebaudioside A, an organic solvent and / or an exotype β-1,3-glucanase is allowed to coexist or act separately in the aqueous solution. A method for producing rebaudioside A, which comprises carrying out a rearrangement reaction by means of a reaction. 2. The organic solvent present in the reaction solution is acetone, ethyl acetate, methyl ethyl ketone, hexane, dimethyl sulfoxide (DMSO), dioxane, thioglycol, propylene glycol, methyl phthalate,
The method for producing rebaudioside A according to claim 1, which is one or more selected from methanol and ethanol.