JP3073864B2 - Glucobiose production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to glucobiose, which is expected to be widely used as food and other materials.
and particularly to a novel process for producing kojibiose and nigerose.

[0002]

2. Description of the Related Art Kojibiose is known as O-α-DG-
(1 → 2) -α-DG, nigerose is O-α-D-
It has a structure of G- (1 → 3) -α-DG (G is glucose). Conventionally, as a method for producing these glucobioses, a method of extracting from a natural product, a method of organic synthesis, and a method of synthesizing a carbohydrate-related enzyme are known.

[0003] As a method of extracting from natural products, for example, a method of obtaining from a starch saccharified product [K. Aso et al., Journal of the Japanese Society of Agricultural Chemistry, vol. 29.856-861 (1
955)], a method of obtaining from koji juice [K. Matsuda
Author, Journal of the Japanese Society of Agricultural Chemistry, vol. 33.719-72
3 (1959)], a method of obtaining from honey [T. Watan
abe et al., Journal of the Japanese Society of Agricultural Chemistry, vol. 33.1
054-1058 (1959)] or a method of obtaining from a bead [K. Aso et al., Journal of the Japanese Society of Agricultural Chemistry, vo
l. 35.1073-1078 (1961)].

As a method by organic synthesis, a method by Igarashi et al. (K. Igarashi et al., Carboh
ydr. Res. , Vol. 39.341-343 (1
975)), the method of Takeo et al. (K. Takeo et al., C.
arbohydr. Res. , Vol. 145.307
-311 (1986)), (K. Takeo et al., C.
arbohydr. Res. , Vol. 224.111
-122 (1992)).

As a method of synthesizing a carbohydrate-related enzyme, a method using a yeast α-glucosidase (S. Chiba et al., Agric. Biol. Ch.
em. , Vol. 26.787-793 (196
2)), a method using buckwheat α-glucosidase (M.
Takahashi et al., Agric. Biol. C
hem. , Vol. 33.1339-1410 (196
9)), Saccharomyces sp. Origin α-
Glucosidase, and Rhizopus sp. Using glucoamylase derived from H. Fujinot
o, et al., Agric. Biol. Chem. , Vo
l. 52.1345-1351 (1988)).

[0006]

As described above, various methods are known as a method for producing glucobiose. However, the method of extracting glucobiose from a natural product has a very low content of the target product in a natural material. However, the method using organic synthesis has a complicated process and requires labor and time, and the method based on the synthesis reaction of a carbohydrate-related enzyme has a problem that a sugar reaction product cannot be supplied. Is low. As described above, several methods for producing kojibiose and nigerose are known. However, there is no known example of synthesizing kojiviose and nigerose efficiently by a transfer reaction using sucrose phosphorylase.

[0007]

The present inventors have conducted various studies to develop a method for efficiently obtaining glucobiose using a saccharide-related enzyme, and as a result, have found that sucrose, glucose, and glucose-1 It has been found that significant amounts of kojibiose and nigerose can be efficiently obtained by allowing sucrose phosphorylase to act on phosphoric acid and glucose, glucose-1-phosphate and sucrose, or sucrose. Completed the invention. That is, the present invention allows sucrose phosphorylase to act on sucrose and glucose, glucose-1-phosphate and glucose, glucose-1-phosphate and sucrose, or sucrose to produce and accumulate glucobiose. A process for producing glucobiose.

Hereinafter, the present invention will be described in detail. First, in order to carry out the present invention, sucrose and glucose,
Glucose-1-phosphate and glucose, glucose-1
-Phosphoric acid and sucrose, or sucrose,
Sucrose phosphorylase is allowed to act. And
Dissolve in any combination in water to prepare a mixture.
The amount of the above-mentioned substrate component added to water is expressed as a weight% as a whole.
The concentration is 5 to 100%, more preferably 20 to 80%.

Next, the sucrose phosphorylase used in the present invention typically acts on sucrose in the presence of inorganic phosphorus to produce glucose-1-phosphate and fructose, or catalyzes the reverse reaction. It is an enzyme that does.

[0010] The origin is, for example, Leuconostoc mesenteroides (Leuconostoc)
mesenteroides), Pseudomonas saccharophila (Pseudomonas saccha)
rophila), Pseudomonas putrefacien (Pseudomonas putrefacien)
s), Clostridium pastirianum (Clos)
tridium pasteurianum), Acetobacter xylinum
linum), Pullulia pullulans (Pululul)
aria pullulans) [Biotechnology and Bioengineering (Biotechnol. Bioeng.,) Vo]
l. 29, Pp8-15, 1987], but are not limited thereto.

The amount of sucrose phosphorylase to be added to the mixture is 1 unit or more, preferably 30 to 200 units, per gram of the total weight of the substrate.

One unit is determined according to the method described in JP-A-3-4785, "Method for producing sucrose phosphorylase".

Further, the pH of the enzyme reaction is 5.0 to 8.5,
Preferably, it is 7.0-8.0, and the temperature is 20-5.
0 ° C., preferably 35-45 ° C., and the time is 1-
It is 40 hours, desirably 15 to 30 hours.

From the thus obtained reaction solution, kojibiose and nigerose are separated and collected by removing
Appropriate separation means may be adopted as it is or after previously unreacted sucrose is decomposed and removed by an appropriate decomposing enzyme, for example, invertase. As the separation means, it is possible to easily and efficiently recover the same by using activated carbon, gel filtration, or ion exchange resin. These can be used alone or in combination to separate the desired kojibiose and nigerose. For example, the solution is passed through a column filled with activated carbon resin (manufactured by Takeda Pharmaceutical Company, Shirasagi), and then water is passed through to remove monosaccharides (glucose, fructose) and the like. By doing so, the desired kojibiose and nigerose fractions are obtained.

[0015]

According to the present invention, sucrose phosphorylase is allowed to act on sucrose and glucose, glucose-1-phosphate and glucose, glucose-1-phosphate and sucrose, or sucrose. With simple operations, it is possible to efficiently obtain kojibiose and nigerose, which are expected to be used as foods and other materials.

Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] Sucrose 200 mg and glucose 2
00 mg to 1 ml of 100 mM MES (pH 6.9)
After dissolving in a buffer solution, 30 units of sucrose phosphorylase (manufactured by Kikkoman Co., Ltd.) was added, and the mixture was reacted at 42 ° C. for 15 hours to perform a sugar compound generation reaction.
The mixture was heated at 5 ° C. for 5 minutes to stop the enzymatic reaction, thereby obtaining an enzymatic reaction solution. FIG. 1 shows the result of high performance liquid chromatography (HPLC) analysis of the reaction-terminated liquid.

From the results shown in FIG. 1, it was confirmed that the reaction completed liquid contained kojibiose and nigerose in addition to glucose, fructose and sucrose. Further, according to the peak area measurement by HPLC, 38.91 mg of kodibiose and 21.5 mg of nigerose were determined.
Turned out to be included.

[Conditions for HPLC analysis] Column: Hibar LiChrosorb NH2
, Inner diameter 4.0 mm, length 250 mm flow rate; 1 ml / min mobile phase; acetonitrile: water = 80: 20 detection wavelength; RI

[0019]

Example 2 200 mg of sucrose was added to 1 ml of 10
0 mM MES (pH 6.9) buffer, and sucrose phosphorylase (Kikkoman) 3
0 units were added and reacted at 42 ° C. for 15 hours to carry out a saccharide compound generation reaction. Then, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes to obtain an enzyme reaction treated liquid. FIG. 2 shows the result of HPLC analysis of the reaction-terminated liquid. From the results of FIG. 2, it can be confirmed that the reaction-terminated liquid contains kojibiose and nigerose in addition to glucose, fructose and sucrose. And again, HPLC peak
As a result of the measurement of the surface area, 7.82 mg of kodibiose was obtained.
Nigerose was found to contain 6.21 mg.

[0020]

Example 3 200 mg of glucose and glucose-1-
200 mg of phosphoric acid was added to 1 ml of 100 mM MES (pH
6.9) Dissolve in a buffer, add 30 units of sucrose phosphorylase (manufactured by Kikkoman) and add
The mixture was reacted at 15 ° C. for 15 hours to carry out a sugar compound generation reaction, and then heated at 100 ° C. for 5 minutes to stop the enzyme reaction, thereby obtaining an enzyme reaction-treated solution. FIG. 3 shows the result of HPLC analysis of the reaction-terminated liquid. From the results shown in FIG.
It can be confirmed that kojibiose and nigerose are contained in addition to glucose. And again, HPLC peak
According to the measurement of the surface area, 6.45 mg of kodobiose was obtained.
Nigerose was found to be contained in 3.76 mg.

[0021]

Example 4 200 mg sucrose and glucose
200 mg of 1-phosphoric acid was added to 1 ml of 100 mM MES
(PH 6.9) Dissolved in a buffer, added with 30 units of sucrose phosphorylase (manufactured by Kikkoman), reacted at 42 ° C. for 15 hours to perform a sugar compound generation reaction, and then heat-treated at 100 ° C. for 5 minutes. The enzymatic reaction was stopped to obtain an enzymatic reaction solution. HPL of this reaction end solution
FIG. 4 shows the results of the C analysis. From the results in FIG. 4, it can be confirmed that the reaction termination solution contains kojibiose and nigerose in addition to glucose, fructose and sucrose. Further, peak area measurement by HPLC revealed that 4.23 mg of kodibiose and 2.07 mg of nigerose were contained.

[0022]

Example 5 Sucrose 200 mg and glucose 6
00 mg to 1 ml of 100 mM MES (pH 6.9)
After dissolving in a buffer solution, 30 units of sucrose phosphorylase (manufactured by Kikkoman Co., Ltd.) was added, and a sugar compound generation reaction was performed at 42 ° C. for 33 hours. At each reaction time, a part of the reaction solution was sampled, and HPLC The sugar concentration was analyzed. The result is shown in FIG. The sugar concentration shown on the vertical axis of FIG. 5 is represented by weight% calculated from the peak area of each sugar in the reaction solution by HPLC.

For comparison, 200 mg of glucose was dissolved in 1 ml of 100 mM MES (pH 6.9) buffer, 30 units of sucrose phosphorylase (manufactured by Kikkoman) was added, and the mixture was reacted at 42 ° C. for 15 hours. A compound formation reaction was carried out, followed by heat treatment at 100 ° C. for 5 minutes to stop the enzyme reaction, thereby obtaining an enzyme reaction-treated solution. However, by HPLC analysis of the reaction-completed solution, kojibiose and nigerose could not be detected.

For comparison, 200 mg of glucose-1-phosphate was added to 1 ml of 100 mM MES (pH 6.0).
9) After dissolving in a buffer solution, 30 units of sucrose phosphorylase (manufactured by Kikkoman) were added thereto, and the mixture was added at 42 ° C for 1 hour.
After reacting for 5 hours, a sugar compound formation reaction was performed.
The enzyme reaction was stopped by heating at 00 ° C. for 5 minutes to obtain an enzyme reaction-treated solution.
Kojibiose and nigerose could not be detected.

[0025]

[Effects of the present invention] From the above results, it was found that glucose
When closviolase or nigellose cannot be obtained by the action of sucrose phosphorylase or sucrose phosphorylase on glucose-1-phosphate, sucrose cannot be obtained. When sucrose phosphorylase is allowed to act on a mixed solution of sucrose and glucose, it can be seen that, as the reaction time elapses, kojibiose and nigerose are produced in remarkable amounts. Hitherto, with respect to nigerose, a food material having the function of regulating the physical condition of the human body containing the nigerose has been proposed [Japanese Patent Laid-Open No. 3-229].
58 (1991)]. Kojibiose is also inferred from its structure, and similar effects are expected.According to the present invention, kojibiose and nigerose, which had been difficult to mass-produce so far, are produced in one step, and can be widely used in food materials and others. Expected. Further, depending on the purpose, a purified product can be produced in the form of a mixture mainly with these two components or a mixture with various sugars.

[Brief description of the drawings]

FIG. 1 shows the results of high performance liquid chromatography (HPLC) analysis of a reaction-completed solution obtained by reacting sucrose phosphorylase with sucrose and glucose.

FIG. 2 shows the results of high-performance liquid chromatography (HPLC) analysis of a reaction-finished solution obtained by reacting sucrose with sucrose phosphorylase.

FIG. 3 shows the results of high performance liquid chromatography (HPLC) analysis of a reaction-completed solution obtained by reacting sucrose phosphorylase on glucose and glucose-1-phosphate.

FIG. 4 shows the results of high performance liquid chromatography (HPLC) analysis of a reaction-completed solution obtained by reacting sucrose phosphorylase with sucrose and glucose-1-phosphate.

FIG. 5 shows kojibiose when sucrose phosphorylase is allowed to act on sucrose and glucose.
The change of the accumulated amount of Nigerose and the like over time is shown.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Isao Kusakabe 238-55 Ojin, Dejima-mura, Niigata-gun, Ibaraki Pref. Examiner Mayumi Saito (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 19/00 -19/64 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] A sucrose phosphorylase is allowed to act on sucrose and glucose, glucose-1-phosphate and glucose, glucose-1-phosphate and sucrose, or sucrose to produce and accumulate glucobiose. A process for producing glucobiose. 2. The method for producing glucobiose according to claim 1, wherein the glucobiose is kojibiose or nigerose.