JP2767409B2 - Method for producing heterodisaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a heterodisaccharide, and more particularly to a method for producing various heterodisaccharides having a non-reducing terminal of glucosamine or galactose, which is useful in the fields of the food industry and the pharmaceutical industry.

[0002]

2. Description of the Related Art Heterodisaccharides having a non-reducing terminal of glucosamine or galactose are useful substances expected to be developed as food materials, pharmaceutical materials and the like. However, except for lactose, these hetero 2
Saccharides hardly exist in the natural world, and in addition to lactose and chitobiose, inexpensive synthetic methods have not been established, and thus are not commercially available.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for easily producing the above heterodisaccharide by an enzymatic reaction. The present inventors have studied the production of useful substances by enzymatic reaction, and reacted α-D-glucosamine-1-phosphate or α-D-glucosamine-1-phosphate by reacting chitobiose or lactose with phosphate in the presence of cellobiose phosphorylase. A method for synthesizing a phosphate sugar such as α-D-galactose-1-phosphate was developed. further,
As a result of an investigation to attempt to produce a useful substance using these phosphate sugars, a reaction between the phosphate sugar and a monosaccharide in the presence of the above-mentioned enzyme was carried out to obtain a hetero-2 derivative having a non-reducing terminal of glucosamine or galactose. They have found that saccharides can be obtained, and have reached the present invention.

[0004]

SUMMARY OF THE INVENTION The present invention relates to α-D-glucosamine-1-phosphate and α-D-galactose-1.
-A heterogeneous reaction characterized by reacting a monosaccharide and a phosphate sugar selected from phosphoric acid in the presence of 0.01 to 100 units / ml cellobiose phosphorylase per 1 mM of the phosphate sugar. The present invention relates to a method for producing a disaccharide.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme used in the present invention, cellobiose phosphorylase (EC 2.4.1.20) (hereinafter sometimes abbreviated as CP), is derived from microorganisms and has β-1,4. -Catalyzes the phosphorolysis of glucan.
In the present invention, the objective is to react a monosaccharide with a phosphate sugar selected from α-D-glucosamine-1-phosphate and α-D-galactose-1-phosphate in the presence of this enzyme. Is produced. Note that C
As P, a crude enzyme, a purified enzyme, a microbial cell containing the enzyme, or an immobilized product thereof can be used.

[0006] The raw material phosphoric sugar may be a commercially available product,
Alternatively, one manufactured by the following method can be used. That is, α-D-glucosamine-1-phosphate is
It can be obtained by reacting chitobiose with phosphoric acid in the presence of CP. Further, α-D-galactose-1-phosphate can be obtained by reacting lactose and phosphoric acid in the presence of CP. The phosphoric acid includes orthophosphoric acid and condensed phosphoric acid, and phosphoric acid 1
Sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium trihydrogen phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, potassium trihydrogen phosphate, etc., alone or in combination with two or more phosphate buffers These can be used in combination. In the method of the present invention, these phosphate sugars are used in an amount of 0.01 mM or more and 1000 mM or less, usually 1 to 100 mM, preferably about 2 to 50 mM.

The monosaccharide used in the present invention is glucose, 2
-Amino-2-deoxy-D-glucose (also referred to as D-glucosamine), mannose, 2-amino-2-
It is selected from deoxy-D-mannose (also referred to as D-mannosamine), 2-deoxy-D-glucose, 6-deoxy-D-glucose and D-xylose. These monosaccharides are 0.01 mM or more,
1000 mM or less, usually 1 to 100 mM, preferably about 2 to 50 mM.

The enzyme and CP used in the above reaction are used in an amount of 0.01 to 100 units / m with respect to 1 mM of the starting phosphoric acid sugar.
1, preferably 0.1 to 10 units / ml. If the amount of the enzyme is less than the lower limit, the target reaction does not proceed sufficiently. Further, even if the amount of the enzyme exceeds the upper limit, not only the corresponding amount of the desired product cannot be obtained, but also the enzyme is not completely dissolved in the reaction system and may not be used. The unit of the enzyme activity is cellobiose or phosphoric acid of 1 μmo per minute in the presence of 10 mM glucose-1-phosphate and 10 mM glucose at 37 ° C.
The amount of enzyme generated by le is defined as one unit.

The enzymatic reaction of the present invention is usually carried out by adding a Tris-HCl buffer, a MOPS buffer and other buffers capable of maintaining the pH at 6 to 8, at 20 to 65 ° C., preferably at 37 ° C.
Temperature around pH 5 ° C, pH 5-10, preferably 6-8
It is performed for several hundred hours, usually for 1 to 96 hours, preferably for 4 to 48 hours, more preferably for 10 to 20 hours. Furthermore, the reaction system can be stabilized by adding a stabilizer such as bovine serum albumin, glycerol, and mercaptoethanol to the reaction system.

[0010] The heterodisaccharide obtained by the present invention has a 4-O- [2-amino-] whose non-reducing end is glucosamine.
2-deoxy-β-D-glucosyl] -D-glucose, 2-amino-2-deoxy-4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-glucose (also called chitobiose) ), 4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-mannose, 2-amino-2-deoxy-4-O- [2-amino-2-deoxy-β-D -Glucosyl] -D-mannose, 2-deoxy-4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-glucose, 6-deoxy-4-O- [2-amino-2 -Deoxy-β-D-
Glucosyl] -D-glucose and 4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-xylose and 4-O- in which the non-reducing end is galactose
β-D-galactosyl-D-glucose (also referred to as lactose), 2-amino-2-deoxy-4-O-β-
D-galactosyl-D-glucose, 4-O-β-D-
Galactosyl-D-mannose, 2-amino-2-deoxy-4-O-β-D-galactosyl-D-mannose, 2-deoxy-4-O-β-D-galactosyl-D
-Glucose, 6-deoxy-4-O-β-D-galactosyl-D-glucose and 4-O-β-D-galactosyl-D-xylose.

[0011] Among the hetero disaccharides from the enzyme reaction solution, the use of ion exchange resins is suitable for the separation and purification of disaccharides containing glucosamine. This heterodisaccharide is positively charged and is adsorbed by using an anion exchange resin. Activated carbon column chromatography can be applied to the separation and purification of other heterodisaccharides. Since the method of the present invention is an enzymatic reaction, the generation of by-products is extremely small,
The reaction solution contains almost no components other than the raw material and the target heterodisaccharide. Therefore, the separation and purification of the hetero disaccharide is easy, and the purity of the obtained product is high. Glucosamine, a component of the heterodisaccharide obtained according to the present invention, is an analog of glucose, in which the hydroxyl group at the 2-position of glucose has been changed to an amino group. Therefore, it is similar in properties to glucose. By the way, some enzymes capable of recognizing glucose include those that recognize glucosamine and those that do not. Glucosamine can be used as an index to analyze the characteristics of the enzyme. Furthermore, β-glucosidase that decomposes cellobiose and β-galactosidase that decomposes lactose are of various origins. By examining the action (substrate specificity) of these enzymes on heterodisaccharides, the enzymes can be identified. Is possible.

[0012]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples. Production Example 1 Cellvibrio gilvus (AT
10 g (wet weight) of the cultured cells of CC 13127) was added to 50
The suspension was suspended in 50 ml of a 50 mM phosphate buffer (pH 7.0) and sonicated. Ammonium sulfate was added to the supernatant obtained by centrifuging the treated solution until it reached 20% saturation, and a butyl toyopearl column (1.5 cm diameter × 2) equilibrated with the same solution.
0 cm). Next, after washing the column with the same buffer, CP was eluted with a linear gradient of ammonium sulfate 20% to 10%. The enzymatic activity of the obtained CP was 50 units. This CP was dialyzed against 50 mM Tris-HCl buffer (pH 7.0), and ammonium sulfate was removed therefrom and used as an enzyme.

Preparation Example 2 Dissolve 10 mM chitobiose, 10 mM phosphate buffer and 100 units of CP prepared in Preparation Example 1 in 50 ml of 50 mM Tris-HCl buffer (pH 7.0) (2 units / ml). Then, a reaction solution was prepared. The reaction solution was added to 37
Reaction at 16 ° C. for 2 hours.
Α-D-glucosamine-1-phosphate was produced. This reaction solution was treated with an anion exchange column (Pharmacia, Mono Q) to obtain 25 mg of α-D-glucosamine-1-phosphate.

Preparation Example 3 Dissolve 10 mM lactose, 10 mM phosphate buffer and 100 units of CP prepared in Preparation Example 1 in 50 ml of 50 mM Tris-HCl buffer (pH 7.0) (2 units /
ml) to prepare a reaction solution. The reaction solution was heated at 37 ° C.
Was reacted for 48 hours, and finally 1.8 m
M α-D-galactose-1-phosphate was produced. When this reaction solution was treated with an anion exchange column (Pharmacia, Mono Q), α-D-galactose-1-
20 mg of phosphoric acid were obtained.

Example 1 In 50 ml of 50 mM MOPS buffer (pH 7.0), 2 mM α-D-glucosamine-1-phosphate obtained in Preparation Example 2 and 2 mM D-glucose were prepared in Preparation Example 1. 1
00 units of CP (α-D-glucose-1-phosphate 1 m
1 unit / ml per M) and 0.02% of bovine serum albumin to prepare a reaction solution. The reaction solution was reacted at 37 ° C. for 8 hours.
3 mM 4-O- [2-amino-2-deoxy-β-D
-Glucosyl] -D-glucose. When the reaction solution was treated with a cation exchange column (Pharmacia, Mono S), 4-O- [2-amino-2-deoxy-β
-D-glucosyl] -D-glucose was obtained in an amount of 4 mg.

Example 2 Instead of D-glucose used in Example 1, D-glucose was used.
When the same reaction was carried out using mannose, finally 0.1 mM of 4-O- [2-amino-2-deoxy-
β-D-glucosyl] -D-mannose was produced. When the reaction solution was treated with a cation exchange column, 4-O-
[2-amino-2-deoxy-β-D-glucosyl]-
1.3 mg of D-mannose was obtained.

Example 3 In place of D-glucose used in Example 1, D-glucose was used.
When the same reaction was carried out using xylose, 0.15 mM of 4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-xylose was finally produced. When the reaction solution was treated with a cation exchange column, 4-O-
[2-amino-2-deoxy-β-D-glucosyl]-
2 mg of D-xylose was obtained.

Comparative Example 1 In Examples 1, 2, and 3, 0.5 unit of CP was dissolved (0.1 mM for α-D-glucosamine-1-phosphate 1 mM).
The reaction was carried out in the same manner as in Examples 1, 2, and 3 except that the reaction product was used as the reaction product. The target compound of each reaction, 4-O- [2-amino-2-deoxy −β
-D-glucosyl] -D-glucose, 4-O- [2-
Amino-2-deoxy-β-D-glucosyl] -D-mannose and 4-O- [2-amino-2-deoxy-
The amount of [β-D-glucosyl] -D-xylose produced was small and could not be detected by a differential refraction detector (manufactured by Shimadzu Corporation).

Example 4 1 mM α-D-galactose-1- obtained in Preparation Example 3 in 100 ml of 50 mM MOPS buffer (pH 7.0)
A reaction solution was prepared by dissolving phosphoric acid, 1 mM D-glucose, 100 units of CP (1 unit / ml) prepared in Production Example 1 and 0.02% of bovine serum albumin.
When the reaction solution was reacted at 37 ° C. for 16 hours,
Finally 0.1 mM 4-O-β-D-galactosyl-
D-glucose was formed. When the reaction solution was treated with an activated carbon column, 2.5 mg of 4-O-β-D-galactosyl-D-glucose was obtained.

Example 5 In place of D-glucose used in Example 4, D-glucose was used.
When the same reaction was carried out using mannose, finally, 0.07 mM of 4-O-β-D-galactosyl-D-
Mannose resulted. When the reaction solution was treated with an activated carbon column, 4-O-β-D-galactosyl-D-mannose was obtained in an amount of 0.8 mg.

Example 6 Instead of D-glucose used in Example 4, D-glucose was used.
When the same reaction was carried out using xylose, 0.1 mM 4-O-β-D-galactosyl-D-xylose was finally produced. When the reaction solution was treated with an activated carbon column, 1.3 mg of 4-O-β-D-galactosyl-D-xylose was obtained.

Comparative Example 2 In Examples 4, 5 and 6, 0.5 unit of CP was dissolved (0.005 unit / ml with respect to 1 mM of α-D-galactose-1-phosphate). The reaction was carried out in the same manner as in Examples 4, 5, and 6, except that 4-O-β-D-galactosyl-D-
The production amount of glucose, 4-O-β-D-galactosyl-D-mannose and 4-O-β-D-galactosyl-D-xylose is small and can be detected with a differential refraction detector (manufactured by Shimadzu Corporation). Did not.

[0023]

According to the present invention, a novel hetero disaccharide can be easily produced using specific phosphoric sugars and monosaccharides as raw materials. This hetero disaccharide is useful in fields such as the food industry and the pharmaceutical industry.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hajime Taniguchi 2-194-4 Kariya-cho, Ushiku-shi, Ibaraki (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/12 C12P 19/26

Claims (3)

(57) [Claims] 1. A method according to claim 1, wherein a phosphate sugar and a monosaccharide selected from α-D-glucosamine-1-phosphate and α-D-galactose-1-phosphate are added in an amount of 0.1 to 1 mM of the phosphate sugar. 0
A method for producing a heterodisaccharide, wherein the reaction is carried out in the presence of 1 to 100 units / ml of cellobiose phosphorylase. 2. The monosaccharide is glucose, 2-amino-2.
-Deoxy-D-glucose, mannose, 2-amino-2-deoxy-D-mannose, 2-deoxy-D-
Glucose, 6-deoxy-D-glucose and D-
The method according to claim 1, wherein the method is selected from xylose. 3. The heterodisaccharide is 4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-glucose, 2-amino-2-deoxy-4-O- [2-amino -2-deoxy-β-D-glucosyl] -D-glucose, 4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-mannose, 2-amino-2-deoxy-4- O- [2-amino-2-deoxy-β-D-glucosyl] -D-mannose, 2-deoxy-4-O-
[2-amino-2-deoxy-β-D-glucosyl]-
D-glucose, 6-deoxy-4-O- [2-amino-2-deoxy-β-D-glucosyl] -D-glucose, 4-O- [2-amino-2-deoxy-β-D-glucosyl ] -D-xylose, 4-O-β-D-galactosyl-D-glucose, 2-amino-2-deoxy-
4-O-β-D-galactosyl-D-glucose, 4-
O-β-D-galactosyl-D-mannose, 2-amino-2-deoxy-4-O-β-D-galactosyl-D
-Mannose, 2-deoxy-4-O-β-D-galactosyl-D-glucose, 6-deoxy-4-O-β-
D-galactosyl-D-glucose and 4-O-β-
The method according to claim 1, wherein the method is selected from D-galactosyl-D-xylose.