JPH07284397A - Production of laminaritriose - Google Patents

Abstract

PURPOSE:To provide a method for efficiently produce laminaritriose directly used for foods and medicines and agrochemicals or used as an intermediate for foods and medicines and agrochemicals. CONSTITUTION:A beta-1,3-glycosylsaccharide compound having 5 or more sugar chains is reacted with an exo type beta-1,3-glycosyl saccharide-decomposing enzyme having ability to isolate glucose and beta-1,3 glycosylsaccharide-decomposing enzyme having ability to isolate 4 or more sugar chains by reaction with the saccharide compound to produce the objective laminaritriose. According to the method, a high-purity laminaritriose can rapidly be obtained by a simple purification method without carrying out a troublesome purification method for mutually separating oligosaccharides.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Food sweeteners, bulking agents, excipients,
It is applicable to foods, pharmaceuticals and various industrial products as a comprehensive drug, and produces laminaritriose useful as an intermediate for medicinal and agrochemical products having antitumor activity, antibacterial infectious disease action, action on the immune system, etc. Regarding things.

[0002]

2. Description of the Related Art Laminaritriose (sugar in which glucose is linked by a β-1,3 glycosidic bond to a trisaccharide) has hitherto been a polysaccharide having a moiety in which glucose is bonded at the β-1,3 position, such as pakiman. And laminarin are hydrolyzed with acid and then chromatographically separated. However, the thus decomposed product becomes an oligosaccharide mixture, and in order to obtain laminaritriose from this, it takes time and effort to separate it by a fractionation method using activated carbon chromatography, which is currently the most effective method. However, there is a disadvantage that the recovery loss is large when trying to obtain the oligosaccharide.

For example, hydrolysis with an acid is disclosed in Japanese Patent Laid-Open No. 2-2.
Even under limiting conditions as disclosed in Japanese Patent No. 43697, it is difficult to make the purity of a single oligosaccharide 20% or more.

On the other hand, a method for producing rebaudioside A by the inventors including the present inventor (Japanese Patent Laid-Open No. 146599/1987).
(Gazette), exotype β-1,3 glucanase is allowed to act on laminaripentaose (a sugar in which glucose is linked to a pentasaccharide by a β-1,3 glycoside bond) to produce glucose and laminaritriose. However, it is not disclosed to collect laminaritriose alone from the laminaritriose, and the method for causing exo-type β-1,3 glucanase to act on laminaripentaose is previously disclosed. It is not efficient because it requires a procedure for obtaining aus and the reaction rate is slow.

As a selective method using an enzyme, a method for producing high-purity laminaribiose (Japanese Patent Laid-Open No. 59-1628).
97) and methods for producing laminaripentaose (Japanese Patent Publication No. 4-37719), but laminaritriose is not produced from these enzymes.

[0006]

The problem to be solved by the present invention is to provide a method for efficiently producing highly pure laminaritriose.

[0007]

As a result of earnest studies on a method for efficiently producing laminaritriose, the present inventors have found that β-1,3 glycosyl sugar compounds having 5 or more sugar chains Exo-type β-1,3 glycosyl glycolytic enzyme is allowed to act after or simultaneously with β-1,3 glucosyl glycolytic enzyme that acts on a sugar compound to produce an oligosaccharide having a sugar chain of 4 or more sugars. And to rapidly accumulate glucose and laminaritriose as final degradation products,
The present invention has been completed by finding that laminaritriose can be separated and purified by a simple method.

That is, the present invention provides β-1, which has a sugar chain of 5 or more sugars,
Exo-type β-1,3 having the ability to act on these glycosyl compounds to liberate glucose
It is intended to provide a method for producing laminaritriose, which is characterized by being obtained by acting a glycosyl glycolytic enzyme and a β-1,3 glycosyl glycolytic enzyme having the ability to release oligosaccharides of 4 or more sugars. .

The present invention will be described in detail below. The β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars in the present invention (hereinafter simply referred to as β-1,3 glycosyl sugar compound) is
Glucose bound at the β-1,3 position (ie β-1,3
If the degree of polymerization of glucose having a glucoside bond is at least 5 sugars or more, preferably 6 sugars or more,
The β-1,3 glycosyl sugar chain may be in the main chain or in the side chain. Examples thereof include oligosaccharides having β1,3-glycosyl bond, polysaccharides, glycosides, glycolipids, glycoproteins and the like, and partially decomposed products of these can also be used. For this purpose, oligosaccharides or polysaccharides having a β-1,3 glycosyl sugar in the main chain or side chain are preferred. As the oligosaccharide, for example, a so-called laminari oligosaccharide consisting only of β-1,3 glucoside sugar chain is more preferable. The polysaccharide may be any polysaccharide as long as it has a β-1,3 glucosyl bond with a degree of polymerization of 5 sugars or more, preferably 6 sugars or more. For example, curdlan, laminarin, pakiman , Β-1, which has a relatively long yeast cell wall,
It is more preferable because it has 3 glucoside bonds.

Next, the enzyme used in the present invention will be described. The exo type β-1,3 glycosyl glycolytic enzyme (hereinafter simply referred to as exo type degrading enzyme) used in the present invention is
Any enzyme may be used as long as it has the ability to act on the β-1,3 glycosyl sugar compound described above to release glucose, and that does not act on laminaritriose or is very difficult to act on laminaritriose, It may be from any source of microorganisms, animals and plants. An enzyme produced by a microorganism belonging to the genus Streptomyces is preferable, and Streptomyces sp. DIC-108 is particularly preferable.
(Streptomyces sp DIC-108)
The enzyme produced by.

[0011] Another preferred method is an artificial mutant strain obtained by artificially mutating the above strain, that is, a mutagenesis treatment by chemical or physical means, or a natural mutant strain. All enzymes are included.

A β-1,3 glucosyl sugar degrading enzyme (hereinafter referred to as an oligosaccharide-forming enzyme) having the ability to act on a β-1,3 glycosyl sugar compound having a sugar of 5 or more sugars to produce an oligosaccharide of 4 or more sugars The term “)” may be endo-type or exo-type as long as it is an enzyme having such an ability, and the present invention can be achieved regardless of the origin of microorganisms, animals and plants. can do. An oligosaccharide degrading enzyme is an enzyme that degrades sugars having a degree of polymerization smaller than that of a β-1,3 glycosyl sugar compound to be degraded, and an enzyme having the ability to release an oligosaccharide having a degree of polymerization close to that of a tetrasaccharide is efficient. Moreover, it is preferable since laminaritriose can be produced. An enzyme that decomposes into pentasaccharide is preferable, and an enzyme produced by a microorganism belonging to the genus Streptomyces is particularly preferable. Among them, Streptomyces sp. D
IC-108 (Streptomyces / sp DI
The enzyme produced by C-108) is particularly preferred.

Further, as another preferred one, it is obtained from an artificial mutant strain obtained by artificially mutating the above strain, that is, a mutagenizing treatment by chemical or physical means, or a natural mutant strain. All enzymes are included.

This Streptomyces SP DI
C-108 (Streptomyces / sp DIC)
-108) is an exo-type glycolytic enzyme suitable for the present invention (hereinafter referred to as exoglucanase ST) and an oligosaccharide-forming enzyme (this enzyme has an ability to release laminaripentaose (G5) by acting on curdlan or the like. It is an enzyme having the following formula (hereinafter referred to as G5 producing enzyme) and is preferably used in the present invention because it is produced outside the bacterial cell at the same time, but the enzyme usable in the present invention is not limited thereto.

The properties of exoglucanase ST and G5 synthase which are particularly preferably used in the present invention are shown below. [Enzymatic properties of exoglucanase ST] (1) Action: β-1,3 glycosyl sugar compound such as curdlan, laminarin, pakiman, yeast cell wall and partial hydrolysates thereof to release glucose . The final products when acting on laminari oligosaccharides are glucose and laminaritriose. (2) Working pH range and optimum working pH; pH 3 to 9
And the optimum working pH is 6. (3) Working temperature range and optimum working temperature;
It works up to about 70 ° C and the optimum working temperature is about 60 ° C. (4) Thermal stability; 45 in 0.01M acetate buffer
It is completely deactivated by heat treatment at 1 ° C. for 1 hour, but is deactivated by about 60% in heat treatment at 60 ° C. for 1 hour in 0.1 M phosphate buffer. (5) Purification method: The present enzyme was concentrated from the culture filtrate after removal of bacterial cells with an ultrafiltration membrane (molecular weight cutoff of 10,000) and then subjected to DEAE cellulofine column chromatography (0.01 M Tris-HCl buffer, pH 7. Perform 5) and collect the fractions eluted with a sodium chloride concentration of 0.1M. C after desalination
M Cellulofine column chromatography (0.01
By carrying out M acetate buffer (pH 6.0) and collecting fractions eluted with a 0.2 M sodium chloride solution, an almost single enzyme can be obtained actively. (6) Molecular weight: The molecular weight by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is about 55,000.

[Enzymatic properties of G5 synthesizing enzyme] (1) Action: Laminaripentaose is produced from β-1,3 glycosyl sugar compounds such as curdlan, laminarin, pakiman, yeast cell wall or partial degradation products thereof. To do. (2) Working pH range and optimum working pH; pH 3 to 9
The optimum pH is around 5. (3) Working temperature range and optimum working temperature: Working up to about 70 ° C, the optimum working temperature is about 60 ° C. (4) Purification method: This enzyme was concentrated from the culture filtrate after removal of bacterial cells with an ultrafiltration membrane (molecular weight cut-off of 10,000) and then DEAE cellulofine column chromatography (0.01 M Tris-HCl buffer, pH 7.5). ) And collect the unadsorbed fraction. Then, CM cellulofine column chromatography (0.01 M acetate buffer, pH 5.0) is performed, and the fractions eluted at 0.1 M are collected to obtain an active single enzyme. (5) Thermal stability; 0.01 M acetate buffer (pH 5.
In 0), it is completely deactivated by heating at 70 ° C. for 90 hours, but at 50 ° C., it is deactivated by 30%. (6) Molecular weight: The molecular weight by SDS-PAGE can be estimated to be about 40,000.

The aforementioned Streptomyces sp. DIC-108 (Strep) which is preferably used in the present invention.
The mycological properties of the S. tomyces.sp DIC-108) strain have already been described in JP-A-59-17996, but in detail, they have the following properties. In addition, this strain was applied for deposit at the Institute of Biotechnology, Institute of Industrial Science and Technology, and was submitted to the Micro Engineering Research Institute No. 253 (FERM BP
-253).

[Streptomyces SP DIC
-108 Mycological properties] (1) Morphological characteristics The growth of vegetative mycelium on the used medium (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 and the size is minor axis × major axis 0.7 to 0.8 μ × 1.0 to 1.
2μ. Observation by a scanning electron microscope shows that the surface structure of spores is warty.

(2) Growth state in various media (a) Sucrose nitrate agar medium (37 ° C.) Gray aerial hyphae are formed on light brown basal hyphae and no soluble pigment is observed. (B) Glucose / asparagine agar medium (37 ° C) The formation of aerial hyphae is not observed in the growth of pale yellowish white color. No soluble dye is found. (C) Glycerin / asparagine agar medium (ISP medium-No. 5, 37 ° C) Growth of pale yellow color shows no aerial hyphae. No soluble dye is found. (D) Starch / inorganic salt agar medium (ISP medium 37
(° C) Greenish gray aerial mycelium grows on colorless growth, and no soluble pigment is observed. (E) Tyrosine agar medium (ISP medium-7 37 ° C.) On light brown growth, aerial hyphae did not settle on day 7 of culture, and 1
On the 4th day, white-gray aerial mycelia settle. No soluble dye is found. (F) Nutrient agar medium (37 ° C) Light greenish gray aerial mycelium grows on greenish gray black growth, and no soluble pigment is observed. (G) Yeast malt agar medium (ISP medium-2 37
℃) A light greenish gray aerial mycelium grows on light brown growth. No formation of water-soluble dye was observed. (H) Oatmeal agar medium (ISP medium-337
(° C) Colorless growth with light green-gray aerial mycelium growing on it, and no water-soluble pigment formation.

(3) Physiological properties (a) Growth temperature range In a growth test using a yeast extract / malt extract liquid medium,
It grows at 30 to 50 ° C, but the optimum temperature is 37 to 45 ° C. (B) Liquefaction of gelatin: negative (c) Coagulation of skim milk and peptization of skim milk: negative (d) Formation of melanin pigment (peptone-yeast-iron agar medium ISP medium 6): negative (e) Degradation of starch Sex: Positive (white ring is formed in the decomposition zone) (f) Utilization of carbon source (Pridham-Gotrieve agar-937C) D-glucose, L-arabinose, D-xylose,
D-fructose, inositol, L-rhamnose, D
-Mannitol and D-galactose are often used for growth, but sucrose, raffinose, and salicin are not used. (G) Cell Wall Composition The sugar composition Type described in ISP is Ty.
belongs to pe1.

For culturing the strain for producing the enzyme used in the present invention, an ordinary solid medium or liquid medium is used, and as the carbon source for liquid culture, for example, glucose or fructose is used. Β as an induced carbon source
A -1,3-glucosyl sugar compound is desirable. Examples thereof include curdlan, pachyman, laminarin, lichenan, yeast cell wall or a partial degradation product thereof, rheucosin, callose, paramylon, etc., and nitrogen sources include ammonium salts of sulfuric acid, hydrochloric acid, phosphoric acid, etc., sodium nitrate and urea, respectively. , Organic nitrogen and inorganic nitrogen such as peptone, peptone and 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 inorganic substances include dipotassium phosphate, monosodium phosphate, magnesium sulfate, and trace metals. In addition, vitamins can be added if necessary. The use concentration of these is 0.1
~ 40% by weight is used. Further, in order to suppress foaming during fermentation, 0.0001 to 1.0% by weight of an antifoaming agent may be added. As the defoaming agent, a usual defoaming agent such as silicone, soybean oil and a surfactant is used. The culture method is shaking culture,
Suitable for aerobic liquid culture such as aeration culture, pH 5
9. Culture at 20 to 50 ° C for 1 to 6 days, preferably at 6 to 8 at 35 to 40 ° C for about 2 days.

Since the enzyme that can be used in the present invention is an enzyme that is produced outside the cell, it is filtered or centrifuged after the completion of the culture to remove the cells, and the supernatant is collected. If necessary, it can be concentrated, purified by ammonium sulfate, salting out with sodium sulfate, or chromatographic techniques such as ion exchange or gel filtration, or can be stored after drying by freeze-drying or the like.

In the production method of the present invention, for example, a polysaccharide such as curdlan having a high degree of polymerization is treated with an oligosaccharide-forming enzyme capable of decomposing into a pentasaccharide and an exo-type decomposing enzyme sequentially or preferably at the same time. It is possible to obtain a reaction solution in which the sugar component consists of glucose and laminaritriose.
Then, the laminaritriose can be efficiently produced by separating and purifying the laminaritriose by a conventional method.

Laminaritetraose can be produced by directly acting only an exo-type degrading enzyme on a polysaccharide having a high degree of polymerization or an oligosaccharide such as laminaripentaose, which is a method for purifying oligosaccharides. It is not preferable because the reaction rate is inferior to that in the case of using in combination. That is, the present invention is characterized by using two kinds of enzymes in combination.

In order to obtain laminaritriose by the production method of the present invention, for example, 1 to 10 of the β-1,3 glucosyl sugar compound having 5 or more sugars as described above is first dissolved in water or a buffer solution.
0.5 to 3 μg of G5 synthase per weight% suspension
After adding ml of the reaction solution, the reaction is conducted under the reaction conditions of pH 3 to 9, preferably 5 to 7, reaction temperature of 30 to 70 ° C., preferably 40 to 60 ° C. for 30 minutes to 50 hours, preferably 10 to 24 hours, Exoglucanase ST1-5μg in the system
/ Addition of reaction solution ml, pH 4-9, preferably 5
7.5, reaction temperature 30-70 ° C, preferably 40-60
It can be obtained by reacting for 3 to 20 days, preferably 10 to 18 days under a reaction condition of ° C. At this time, only glucose and laminaritriose are present as oligosaccharide components.

Preferably, the laminaritriose of the present invention can be produced by simultaneously adding and reacting the above-mentioned two kinds of enzymes. When using another enzyme for the reaction, the temperature and pH optimum for the enzyme may be adjusted.

Since laminaritriose and glucose are present in the reaction solution obtained by the method of the present invention, the subsequent purification need only remove glucose. As a method for removing glucose, there is a method by chromatography, and examples thereof include activated carbon chromatography, ion exchange chromatography, and gel filtration chromatography. Among them, activated carbon chromatography is excellent in that it can process a large amount of sugar. In this case, only laminaritriose is adsorbed on the activated carbon and glucose is not adsorbed, so that it is eluted by washing with water. Laminaritriose adsorbed on activated carbon can usually be eluted and recovered with 10 to 20% ethanol or the like. In addition, a method using a microorganism, for example, a mixture of laminaritriose and glucose is added with an appropriate nitrogen source or a micronutrient source to grow yeast such as Saccharomyces or Kluveromyces, and the like. Since only glucose is utilized and laminaritriose remains as it is, it can be purified by removing components other than sugar by ion exchange purification or the like after removal of bacterial cells.

[0029]

The present invention will be described in detail below with reference to examples.
However, the present invention is not limited to these. (Reference Example 1) [Preparation of exoglucanase ST] Enzyme production using a 90 L jar famentor (manufactured by Maruhishi)
Production culture was performed. That is, glucose 0.5%, polypep
Puton 0.2%, yeast extract 0.2%, K ₂HPO_Four
 0.2%, MgSO_Four・ 7H₂Consists of O 0.1%
Charge 47 liters of liquid medium (pH 7.0), and stir at 121 ° C for 3
After 0 minute sterilization, when the temperature drops to 40 ℃, curdlan
0.5% was added. This is the same medium composition for 2 days, 35
Streptomyces sp. DIC-1 cultured at ℃
Inoculated with 2 L of inoculum of No. 08 (Microtechnology Research Article No. 253), 3
5 ° C, stirring 300 revolutions / min, aeration rate 48L / mi
n. The cells were cultured under the conditions of 40 hours. After completion of the culture,
Minutes with a vortex flow type ultrafiltration membrane device (Maruhishi)
Bacteria are removed with a membrane with a molecular weight cutoff of 100,000 and then the molecular weight cutoff
It was concentrated with an ultrafiltration membrane of 10,000. About 2 L of this crude enzyme solution
About 800 ml (269.7 mg of protein)
EAE cellulofine column chromatography (0.
0.1M Tris-HCl buffer, pH 7.4) was added to
Fractions eluted with 1M NaCl solution (protein amount 125m
Collect g). After desalting with the same outer filter membrane, CM cell
Lurofine column chromatography (0.01M vinegar
Acid buffer solution, pH 5.0), and dissolve in 0.3M NaCl
The fraction eluted with the liquid was collected. The main component in this fraction
It contains exoglucanase ST, and the amount of protein is
It was 75 mg. This enzyme solution has a concentration of 70 μg / ml
Was prepared.

Reference Example 2 Preparation of G5 Gene Enzyme 800 ml of the crude enzyme solution obtained in Reference Example 1 (protein amount 2
69.7 mg) was subjected to DEAE cellulofine column chromatography (0.01 M Tris-HCl buffer, pH 7.4), and the unadsorbed fraction (protein amount 41 m
g) was collected and then subjected to CM cellulofine column chromatography (0.01 M acetate buffer, pH 5.0) to collect fractions eluted with 0.2 M NaCl solution. This fraction contained G5 synthase as a main component, and the amount of protein was 31 mg. 180 this enzyme solution
The concentration was adjusted to μg / ml.

(Example 1) [Production of laminaritriose from curdlan] 500 mg of curdlan was added to a 25 ml sample bottle.
9 ml of 0.1 M phosphate buffer (pH 6.8), 35 μg / 5 exoglucanase ST obtained in Reference Example 1
00 μl and 18 times the G5 synthase obtained in Reference Example 2
After adding μg / 100 μl, the volume was adjusted to 10 ml with distilled water.
This was reacted in a thermostat at 55 ° C. Changes in sugars during the reaction were carried out by the HPLC method.

[HPLC conditions] Column: Amido-80 (4.6φ × 250 mm, manufactured by Tosoh) Solvent: Acetonitrile: Water = 60: 40 Flow rate: 1.0 ml / min. Column temperature: 50 ° C Detector: RI On the first day of the reaction, laminaripentaose (G5), laminaritetraose (G4), laminaritriose (G)
3), the formation of four components of glucose is seen, but reaction 1
On the 5th day, the amount of laminaritriose produced was 22.2 mg.
/ Ml and glucose 27.1mg / ml reaction solution was obtained. At this time, the yield of laminaritriose from curdlan was 44.4%. 8 ml of this reaction liquid was passed through an activated carbon column (vol. 25 ml), and distilled water 1
It was washed with 00 ml. Glucose was detected in the washing solution. Then, the laminaritriose adsorbed with 50 ml of a 10% v / v ethanol solution was eluted, ethanol was distilled off with an evaporator, and then freeze-dried. The obtained laminaritriose had a purity of 98% and a yield of 175 mg (the yield from curdlan was 43.8%).

(Example 2) [Production of Laminaritriose from Curdlan] In a 25 ml sample bottle, 500 mg of curdlan,
After adding 9 ml of 0.1 M phosphate buffer (pH 6.0) and 18 μg / 100 μl of the G5 synthase obtained in Reference Example 2, the volume was adjusted to 9.5 ml with distilled water. This was reacted for 1 day in a thermostat at 55 ° C. When the change of the produced sugar was measured by the method by HPLC shown in Example 1, 30.1 mg / ml of laminaripentaose (G5) was produced. When 35 μg / 500 μl of the exoglucanase ST obtained in Reference Example 1 was added to this reaction system and the reaction was carried out again in a thermostat at 55 ° C., only laminaritriose and glucose were obtained on the 15th day of the reaction. This reaction liquid 8m
l was subjected to activated carbon column chromatography in the same manner as in Example 1 to obtain 170 mg of laminaritriose having a purity of 99%.
(The yield from curdlan was 42.5%).

(Example 3) [Production of Laminaritriose from Advanced Laminarioligosaccharide] In a 25-ml sample bottle, 200 mg of laminarioligosaccharide (37% of 10 sugars, 60% of 11 sugars), 0.1M phosphate buffer solution ( pH 6.0) 9 ml, exoglucanase ST obtained in Reference Example 1 35 μg / 500 μl and G5 synthase 18 μg / 100 μl obtained in Reference Example 2 were added, and then distilled water was added to 10 ml. This was reacted in a thermostat at 55 ° C. The sugar change was measured by the same method as in Example 1. On the second day of the reaction, glucose, laminaritriose and laminaritetraose were 12 mg / ml each,
It was 2.7 mg / ml and 2.7 mg / ml. On the 10th day thereafter, glucose became 14 mg / ml and laminaritriose 3.8 mg / ml. 8 ml of this reaction liquid
Was subjected to activated carbon chromatography in the same manner as in Example 1 to obtain 28.2 mg of laminaritriose having a purity of 99% (yield from laminarioligosaccharide was 17.6%).

(Example 4) [Production of Laminaritriose from Pakiman] In a 25 ml sample bottle, 500 mg of Pakiman (prepared from Bukurou by a conventional method) and 9 ml of 0.1 M phosphate buffer (pH 6.8). After adding 35 μg / 500 μl of the exoglucanase ST obtained in Reference Example 1 and 18 μg / 100 μl of the G5 synthase obtained in Reference Example 2,
The volume was adjusted to 10 ml with distilled water. This was reacted in a thermostat at 55 ° C. When the sugar change was measured by HPLC, a reaction solution containing only laminaritriose and glucose was obtained on the 15th day of the reaction, and the amount of laminaritriose produced was 11.5 m.
It was g / ml. 8 ml of this reaction liquid was subjected to activated carbon chromatography in the same manner as in Example 1 to obtain 90 mg of laminaritriose having a purity of 98.2% (yield from pachyman was 22.
5%) was obtained.

(Example 5) [Production of Laminaritriose from Yeast Cell Wall] In a 25 ml sample bottle, 500 mg of yeast cell wall (prepared from baker's yeast by a conventional method), 0.1 M phosphate buffer (pH 6. 8) After adding 9 ml, 35 μg / 500 μl of the exoglucanase ST obtained in Reference Example 1 and 18 μg / 100 μl of the G5 synthase obtained in Reference Example 2,
The volume was adjusted to 10 ml with distilled water. This was reacted in a thermostat at 55 ° C. When the sugar change was measured by HPLC, a reaction solution containing only laminaritriose and glucose was obtained on the 15th day of the reaction, and the amount of laminaritriose produced was 4.8 mg.
/ Ml. 8 ml of this reaction solution was subjected to activated carbon chromatography in the same manner as in Example 1 to give 37.5 mg of laminaritriose having a purity of 99% (yield from the yeast cell wall was 9.4).
%) Was obtained.

Comparative Example 1 [Production of Laminaritriose from Laminaripentaose] Laminaripentaose 50 was added to a 25 ml sample bottle.
0 mg, 0.1 M phosphate buffer (pH 6.8) 9 m
l, 35 exoglucanase ST obtained in Example 1
After adding μg / 500 μl, the volume was adjusted to 10 ml with distilled water.
This was reacted in a thermostat at 55 ° C. Example 1 for sugar change
When measured by the same method as described above, 1.4 mg / ml of laminaritetraose was still present on the 15th day of the reaction, and it took 24 days on the reaction until the laminaritetraose disappeared. At that time, the amounts of laminaritriose and glucose produced were 25.3 mg / ml and 22.3 mg / ml.
It was ml. 8 ml of this reaction liquid was subjected to activated carbon chromatography in the same manner as in Example 1 to obtain 195 mg of laminaritriose having a purity of 99% (yield from laminaripentaose was 48.8%).

[0038]

EFFECTS OF THE INVENTION According to the production method of the present invention, compared with conventional hydrolysis with an acid, since there is no oligosaccharide component other than glucose and laminaritriose in the reaction system, complicated purification for separating oligosaccharides from each other is performed. High-purity laminaritriose can be obtained by a simple purification method without performing the method. Further, by using the exo-type degrading enzyme and the oligosaccharide-degrading enzyme in combination, laminaritriose can be obtained more quickly than in the case where only the exo-type degrading enzyme is used.

Claims (10)

[Claims] 1. An exo-type β-1,3 glycosyl glycolytic enzyme having the ability to act on these β-1,3 glycosyl sugar compounds having a sugar chain of 5 or more sugars to release glucose, and A method for producing laminaritriose, characterized in that the sugar chain is obtained by acting a β-1,3 glycosyl glycolytic enzyme having an ability to release an oligosaccharide having 4 or more sugars. 2. A β which acts on a β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars to liberate an oligosaccharide of 4 or more sugars.
The method according to claim 1, wherein the -1,3-glycosyl glycolytic enzyme is a β-1,3-glucosyl glycolytic enzyme that releases laminaripentaose. 3. The method according to claim 2, wherein the exo type β-1,3 glycosyl glycolytic enzyme is obtained from Streptomyces. 4. The method according to claim 3, wherein the exo-type β-1,3 glycosyl glycolytic enzyme is obtained from Streptomyces sp. DIC-108 strain (Mikiko Kenjoyori No. 253). . 5. A β having the ability to release an exo-type β-1,3 glycosyl glycolytic enzyme and an oligosaccharide having 4 or more sugars.
The method according to claim 1, wherein the -1,3-glycosyl glycolytic enzyme is obtained from Streptomyces. 6. A β having the ability to release exo-type β-1,3 glycosyl glycolytic enzyme and an oligosaccharide having 4 or more sugars.
-1,3 glycosyl glycolytic enzyme is Streptomyces
SP DIC-108 strain (Microtech Lab.
No. 4). 7. The β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars is an oligosaccharide or a polysaccharide having a β-1,3 glycosyl sugar in its main chain or side chain. The production method according to 1. 8. The β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars is an oligosaccharide or a polysaccharide consisting of only a β-1,3 glycosyl sugar chain, according to claim 1. Manufacturing method. 9. The method according to claim 7, wherein the β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars is curdlan, pachyman, laminari oligosaccharide or yeast cell wall. 10. The production method according to claim 9, wherein the β-1,3 glycosyl sugar compound having a sugar chain of 5 or more sugars is curdlan.