JPH089989A - Production of beta-mannosyloligosaccharide - Google Patents

Abstract

PURPOSE:To obtain a beta-1,4-mannooligosaccharide such as beta-1,4-mannobiose, beta-1,4-mannotriose, beta-1,4-mannotetraose or a methyl beta-mannoside useful as a synthetic raw material for biochemical reagents or medicines. CONSTITUTION:This method for producing various beta-mannosyloligosaccharides is to carry out the enzymic reaction in an aqueous solution containing mannose or a mannoligosaccharide or a mannose derivative and a saccharide or an alcohol with a beta-mannosidase or a beta-mannanase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing β-1,4 manno-oligosaccharides by an enzymatic reaction. The compound obtained by the production method of the present invention has β-1,4
Mannobiose, β-1,4 Mannotriose, β-
It covers β-1,4 manno-oligosaccharides such as 1,4 mannotetraose and methyl β-mannoside and related compounds, and these compounds are useful as biochemical reagents and raw materials for synthesizing pharmaceuticals.

[0002]

2. Description of the Related Art An important partial structure of a sugar chain of a human glycoprotein includes β-1,4 manno-oligosaccharide which is a compound in which D-mannose is β-1,4 glycoside-bonded. Furthermore, in recent years, the physiological function of sugar chains has been drawing attention, and its application as a raw material for pharmaceuticals is expected. Further, β-1,4 manno-oligosaccharide is also known as a growth substance of Bifidobacterium (Japanese Patent Laid-Open No. 58-212780).

Β-1,4 manno-oligosaccharides are obtained, for example, by partially hydrolyzing mannan with an enzyme or an acid. However, it is difficult to preferentially obtain an oligosaccharide fraction by a hydrolysis method using an enzyme or an acid, and mannan as a raw material has low water solubility and is difficult to hydrolyze on an industrial scale. Further, natural mannan may have a side chain such as galactose attached to the main chain composed of β-1,4 mannose, in which case it is difficult to be enzymatically decomposed, and an oligosaccharide added such as galactose is by-produced. There is a problem that it ends up. In order to efficiently obtain an oligosaccharide consisting only of mannose, it is necessary to remove the side chain with an enzyme such as galactosidase. As described above, the hydrolysis method using mannan as a raw material has a problem that not only the operation becomes complicated but also the raw material, mannan, cannot be recovered. Further, it is difficult to selectively chemically synthesize useful β-mannooligosaccharides. Therefore, it has been desired to develop an industrial-scale synthetic method for these saccharides and β-mannooligosaccharides as raw materials for pharmaceuticals.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have conducted a condensation reaction or transfer using mannose as a raw material and using β-mannosidase or β-mannanase. Depending on the reaction, various β-
For the first time, we succeeded in producing 1,4 manno-oligosaccharide by a selective and simple method, and completed the present invention. That is, the present invention relates to a method for producing an oligosaccharide having β-mannose at the non-reducing end.

The contents of the present invention will be described in more detail below. In the present invention, β-mannosidase, which is an enzyme that hydrolyzes β-mannosyl bond [EC 3.2.1.2].
5] or β-mannanase [EC 3.2.1.7]
8] can be used. Examples of the origin of the enzyme include Rhizopus niveus and Aspergillus niger. In addition, a commercially available enzyme can be used simply, and β-mannosidase or β-mannanase mixed in a pectinase or hemicellulase preparation can also be used.

The reaction is carried out by condensation reaction or transfer reaction with β-mannosidase or β-mannanase in an aqueous liquid containing mannose or mannooligosaccharide or mannose derivative and sugar or alcohol.

The manno-oligosaccharides include β-1,4 mannobiose, β-1,4 mannotriose and β-1,4.
There are mannotetraose and the like, mannose derivatives include p-nitrophenyl mannoside, o-nitrophenyl mannoside, mannopyranosyl fluoride and the like, and sugars include N-acetylglucosamine and the like.
Alcohol includes methanol and the like.

When mannose is used as a raw material in the condensation reaction, β-1,4 mannobiose and mannothriose are produced. Usually, an enzyme is added to a high-concentration sugar solution to carry out the reaction, but a column method in which columns packed with immobilized enzyme and activated carbon are connected in series and a high-concentration sugar solution is circulated (Japanese Patent Application No. 62-315349). May be used. It is an advantage of the present invention that mannose which is an unreacted raw material can be recovered and used again in the reaction. Further, if mannose and N-acetylglucosamine are used as raw materials, it is also possible to synthesize a hetero mannooligosaccharide such as Manβ1,4GlcNAc, which is an important partial structure of a glycoprotein sugar chain.

Further, in the present invention, mannose derivatives such as p-nitrophenyl mannoside, o-nitrophenyl mannoside or mannopyranosyl fluoride, and various β-1, synthesized by the above condensation reaction, It is also possible to synthesize a hetero mannooligosaccharide by carrying out a transfer reaction using 4-mannooligosaccharide as a mannose donor. In that case, as the mannose receptor, mannose,
Compounds having a hydroxyl group such as mannooligosaccharides, mannose derivatives, various sugars and various alcohols can be used. It is desirable that the mannose donor and the mannose acceptor be used in a molar ratio of 1: 0.5 to 1: 5.

[0010]

【Example】

[0011]

[Production Example] Ammonium sulfate was added to an aqueous solution containing 10 g of a crude enzyme preparation "Sumiteam MC" (trade name, manufactured by Shin Nippon Kagaku Co., Ltd.) of Rhizopus niveus origin so as to be saturated to 75%, and then added. Left overnight at ° C. The resulting precipitate was collected by centrifugation, dialyzed against an acetate buffer of 20 mM, pH 5, and purified using a DEAE-Sepharose column and a Sephacryl S-200 column to obtain a fraction having β-mannosidase and β-mannanase activity. Got a minute.

[0012]

Example 1 To 50 ml of an aqueous solution containing 35 g of mannose, 2.5 U of β-mannosidase prepared in the above Production Example was added and reacted at 37 ° C. for 3 days. After stopping the reaction in a boiling water bath at 100 ° C. for 5 minutes, this reaction liquid was subjected to activated carbon column chromatography, and mannose as a raw material was eluted with water. Next, oligosaccharides were eluted with 2 and 4% aqueous acetonitrile solutions. Lyophilization of each fraction resulted in β-1,4 mannobiose and β-
162 and 65 m of 1,4 mannotriose respectively
g was obtained. Elution pattern of oligosaccharides from activated carbon column chromatography and ^{13 of} β-1,4 mannobiose
C-NMR is shown in FIGS. 1 and 2.

[0013]

[Example 2] To 10 ml of an aqueous solution containing 500 mg of β-1,4 mannobiose, 50 mU of β-mannosidase prepared in the above Production Example was added and reacted at 37 ° C for 4 hours.
After stopping the reaction in a boiling water bath at 100 ° C. for 5 minutes, this reaction solution was subjected to activated carbon column chromatography, and 0 to 1 was used.
The oligosaccharides were eluted with a linear concentration gradient of an aqueous solution containing 0% acetonitrile. As a result of freeze-drying the sugar-eluted fraction detected by the phenol-sulfuric acid method, β-1,4
Mannotriose, β-1,4 mannotetraose and β-1,4 mannohexaose were 97 and 37, respectively.
And 9 mg were obtained. The HPLC of the reaction solution is shown in FIG.

[0014]

Example 3 To 10 ml of an aqueous solution containing 500 mg of β-1,4 mannobiose and 2.5 ml of methanol, 50 mU of β-mannosidase prepared in the above example was added.
The reaction was carried out at 7 ° C for 10 hours. After the reaction was stopped in a boiling water bath at 100 ° C. for 5 minutes, the reaction solution was subjected to activated carbon column chromatography to elute the oligosaccharide with a linear concentration gradient of an aqueous solution containing 0 to 10% concentration of acetonitrile. As a result of freeze-drying the sugar elution fraction detected by thin layer chromatography (TLC), 42 mg of methyl β-mannoside was obtained. HPLC of reaction and product ¹
1 H-NMR is shown in FIGS.

[0015]

As described above, according to the present invention, it is possible to synthesize β1,4-mannooligosaccharide, which is difficult to synthesize organically, by a method capable of producing efficiently and industrially. Becomes

[Brief description of drawings]

1 shows the elution pattern of oligosaccharides in Example 1. FIG.

FIG. 2 shows ¹³ C-NMR of mannobiose.

FIG. 3 shows HPLC of the reaction solution in Example 2.

FIG. 4 shows HPLC of the reaction solution in Example 3.

FIG. 5 shows ¹ H-NMR of methyl β-mannoside.

Claims (3)

[Claims] 1. A method for producing β-mannooligosaccharide, which comprises carrying out an enzymatic reaction with β-mannosidase or β-mannanase in an aqueous liquid containing mannose or mannooligosaccharide or mannose derivative and sugar or alcohol. . 2. The method for producing β-mannooligosaccharide according to claim 1, wherein the enzymatic reaction is a condensation reaction. 3. The method for producing β-mannooligosaccharide according to claim 1, wherein the enzymatic reaction is a transfer reaction.