JPH06343484A - Production of laminarioligosaccharide - Google Patents

Abstract

PURPOSE:To provide a method for readily producing a laminarioligosaccharide from low-cost raw materials. CONSTITUTION:A reaction solution containing >=10mM and <=2M sucrose and <=2M glucose in a molar ratio of sucrose:glucose>=0.1 is treated with (a) sucrose phosphorylase and (b) laminaribiose phosphorylase and/or beta-1,3-oligoglucan phosphorylase in the presence of phosphoric acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel method for producing laminari-oligosaccharides. Laminari oligosaccharides are oligosaccharides in which two or more glucoses are bound by β-1,3 bonds. It is known that β-1,3-glucan, which is a polymer formed by β-1,3 bond, has an antitumor activity, and that its constituent unit, laminari-oligosaccharide, also has various physiological activities. . Recently, there have been reports on AIDS therapeutic agents using laminari-oligosaccharide as a raw material (Biochemical Pharmacology, 43 (1992).
It is also expected to be used as a pharmaceutical raw material.

[0002]

2. Description of the Related Art As a method for producing laminari oligosaccharides,
Method for hydrolyzing the acid moiety of β-1,3-glucan (Met
hods in Carbohydrate Chemistry, 1 (1962) p.330-33
3) and glucose-1-phosphate and glucose with laminaribiose phosphorylase and / or β-1,3
-A method of causing oligoglucan phosphorylase to act (Japanese Patent Laid-Open No. 4-66092) is known.

[0003]

Among the above-mentioned conventional techniques, in the former method, β-1,3-glucan is used as a raw material. However, there is no cheap method for supplying β-1,3-glucan, and as a result, the laminari oligosaccharide produced is extremely expensive. Further, in this method, it is difficult to control the reaction and the yield is not high. In the latter method, it is possible to obtain laminari-oligosaccharides in good yield, but since glucose-1-phosphate, which is one of the raw materials, is expensive, unless a cheap supply method for this is established, the former method is used. As with the case, it is difficult to manufacture at low cost. There is also a problem that phosphoric acid is generated as a by-product of the reaction.

[0004]

As a result of intensive studies to solve these problems, the present inventors have found a method of using sucrose and glucose as a starting material, and completed the present invention. It was That is, the present invention relates to sucrose and 2 at a concentration of 10 mM or more and 2 M or less.
A reaction solution containing glucose at a concentration of M or less and having a molar ratio of sucrose to glucose of 0.1 or more,
The present invention provides a method for producing a laminari oligosaccharide, which comprises treating with (a) sucrose phosphorylase and (b) laminaribiose phosphorylase and / or β-1,3-oligoglucan phosphorylase in the presence of phosphoric acid. The present invention will be described in detail below.

First, sucrose used as one of the raw materials is a compound generally widely used as sugar, and can be obtained in large quantities at low cost. It is also possible to use a sucrose-containing syrup such as molasses as it is.

Regarding the latter two of the above-mentioned three kinds of enzymes used in the present invention, laminaribiose phosphorylase is E as reported by the International Union of Biochemistry Enzymes Committee report.
C2.4.1.31 and β-1,3-oligoglucan phosphorylase have different enzyme numbers from EC 2.4.1.30, but these enzymes all add laminari oligosaccharides. It is acid-decomposed to produce glucose-1-phosphate and a laminari-oligosaccharide having a degree of polymerization one less than that of the starting sugar, and in the present invention, it acts as a substantially equivalent catalyst. Therefore, these can be used alone or as a mixture. On the other hand, the former sucrose phosphorylase is an enzyme classified into EC 2.4.1.7 by the report of the International Union of Biochemistry Enzymes Committee, and sucrose is subjected to phosphorolysis in the presence of phosphate to give glucose- It is a catalyst for a reversible reaction that produces 1-phosphate and fructose.

[0007] Each enzyme used in the present invention may be derived from any organism, or may be produced by a bacterium obtained by genetic recombination. Sucrose phosphorylase is known to be produced by Pseudomonas saccharophira, Leuconostoc mesenteroides, and the like. As the two enzymes of laminaribiose phosphorylase and β-1,3-oligoglucan phosphorylase, it is preferable to use those from the Euglena order known to contain both enzymes in the cells. In addition, these enzymes are purified products,
It may be used in any state such as an unpurified product, an immobilized enzyme immobilized by a known method, or a bacterial cell containing the enzyme. The enzymatic reaction in the present invention is usually carried out in an appropriate solution such as Tris-hydrochloric acid buffer solution or imidazole-hydrochloric acid buffer solution. The activity of each enzyme is 0.001-1,00
0 unit / ml can be used. The unit definition will be described later.

In the present invention, the initial concentration of sucrose is 1
Set to 0 mM or more and 2 M or less. When the initial concentration of sucrose is less than 10 mM, the concentration of laminari-oligosaccharide produced is too thin to be practical. On the other hand, when the initial concentration is 2 M or more, it is difficult to dissolve, so that it is not practical. The initial concentration of glucose, which is another raw material, is usually set to 2M or less. The lower limit is 0.01 mM. If the initial concentration of glucose exceeds 2 M, it is difficult to dissolve, which is not practical.
Further, the molar ratio of the amount of sucrose used to the amount of glucose used is required to be 0.1 or more. If it is less than 0.1, the reaction rate becomes slow, and glucose as a raw material is added to the reaction system. The problem that remains in large quantities arises. The concentration of phosphoric acid is not particularly specified, but is usually 1 mM
The above is performed at 1M or less. Various phosphates may be used as the phosphoric acid. Here, by changing the ratio of the initial concentrations of glucose and sucrose, it is possible to change the degree of polymerization of the laminari-oligosaccharide produced.
That is, as the amount of glucose used is reduced, a laminari oligosaccharide having a higher degree of polymerization is produced. Thus, a polymer having an arbitrary degree of polymerization can be produced, but a polymer having an average degree of polymerization of 2 to 20 is usually obtained.

The reaction temperature for the enzyme treatment may be in the range in which the enzyme is not deactivated, and is usually in the range of 20 to 60 ° C. The pH of the reaction system is in the vicinity of the optimum pH for the activity of the enzyme used and is usually in the range of 5-8.

After the completion of the enzymatic reaction, the laminari oligosaccharide is separated from the reaction solution by an appropriate method. For example, it is possible to separate by adding ethanol to the reaction solution to precipitate the laminari-oligosaccharide. Further, when laminari-oligosaccharides having a constant degree of polymerization are required, they can be obtained by a method such as activated carbon column chromatography or gel filtration.

[0011]

The present invention will be described in more detail with reference to the following examples. The enzyme used in each example was prepared by the following method. <Preparation Example 1> Preparation of sucrose phosphorylase 10 mg of sucrose phosphorylase derived from Leuconostoc mesenteroides, which is commercially available from Sigma, was dissolved in 3 ml of 50 mM Tris-hydrochloric acid buffer solution (pH 7.0). The enzyme activity of the solution was 1.14 units / ml. The unit of enzyme activity is 37 ° C,
It was defined as the amount of enzyme that produced 1 μmol of glucose-1-phosphate and equimolar fructose per minute from 10 mM sucrose and 10 mM phosphoric acid at pH 7.0. <Preparation Example 2> Preparation of laminaribiose phosphorylase and β-1,3-oligoglucan phosphorylase Euglena gracilis Z strain (Euglena gracilis Z)
2 g (wet weight) of the cultured cells of was suspended in 8 ml of 50 mM Tris-hydrochloric acid buffer solution (pH 7.0) and ultrasonically disrupted. The treated solution was centrifuged and the supernatant was used as an enzyme solution. The enzyme activity was 1.11 units / ml. The unit of enzyme activity is 10 mM glucose, 10 mM glucose-1- at 37 ° C. and pH 7.0.
It was defined as the amount of enzyme that produced 1 μmol of phosphoric acid and equimolar laminaribiose per minute from phosphoric acid.

Examples 1 to 5 Sucrose 200 mM, glucose 10 to 200 mM, phosphoric acid 20 mM, sucrose phosphorylase 0.091 unit / ml, laminaribiose phosphorylase and 50 mM Tris-hydrochloric acid buffer (pH 7.0) were added. A reaction solution was prepared so that β-1,3-oligoglucan phosphorylase was 0.089 units / ml, and reacted at 37 ° C for 48 hours. After the reaction, the reaction liquid was treated with invertase to decompose the remaining sucrose, and then the laminari-oligosaccharide was quantified by high performance liquid chromatography. The results are shown in Table 1. The yield in Table 1 represents the yield for sucrose.

Comparative Example 1 1M sucrose and 3 glucose were added to 50 mM Tris-HCl buffer (pH 7.0).
M, phosphate 20 mM, sucrose phosphorylase 0.0
91 units / ml, laminaribiose phosphorylase and β-1,3-oligoglucan phosphorylase 0.0
I tried to prepare the reaction solution so that it would be 89 units / ml.
The reaction liquid could not be prepared because it was difficult to dissolve.

Comparative Example 2 Sucrose 10 mM, glucose 200 mM, phosphoric acid 20 mM, sucrose phosphorylase 0.091 unit / ml, laminaribiose phosphorylase and β-1, in 50 mM Tris-HCl buffer (pH 7.0). 3-Oligoglucan phosphorylase Prepare the reaction solution so that it will be 0.089 units / ml,
The reaction was carried out at 37 ° C for 48 hours. As a result, a laminari oligosaccharide having an average degree of polymerization of 2.1 was obtained with a yield of 75%.
However, the amount of residual glucose in the reaction solution was 10 times or more that of the produced laminari-oligosaccharide.

Comparative Example 3 Sucrose 3M and glucose 1 in 50 mM Tris-HCl buffer (pH 7.0).
M, phosphate 20 mM, sucrose phosphorylase 0.0
91 units / ml, laminaribiose phosphorylase and β-1,3-oligoglucan phosphorylase 0.0
I tried to prepare the reaction solution so that it would be 89 units / ml.
It was difficult to dissolve and the reaction solution could not be prepared.

[0016]

[Table 1]

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily produce laminari-oligosaccharides using inexpensive raw materials. This is difficult to predict due to the known properties of the enzyme used. Moreover, by changing the initial concentration ratio of the raw materials, it is possible to produce laminari-oligosaccharides having different degrees of polymerization. The laminari-oligosaccharide obtained in the present invention is useful in the fields of pharmaceutical raw materials or foods.

Claims (1)

[Claims] 1. A reaction solution containing sucrose at a concentration of 10 mM or more and 2 M or less and glucose at a concentration of 2 M or less and having a molar ratio of sucrose to glucose of 0.1 or more, in the presence of phosphoric acid, ) A method for producing a laminari oligosaccharide, which comprises treating with sucrose phosphorylase and (b) laminaribiose phosphorylase and / or β-1,3-oligoglucan phosphorylase.