JPH0228192A - Novel oligosaccaride and production thereof - Google Patents

Abstract

NEW MATERIAL:O-beta-2-Acetamide-2-deoxy-D-glucopyranosyl-(1 6)-O-beta-D- galactopyranosyl-(1 4)-D-glucose expressed by formula I (R1 is N- acetylglucosamine residue; R2 is H) and O-beta-D-galactopyranosyl-(1 4)-{O-beta-2- acetamide-2-deoxy-D-glucopyranosyl-(1 6)}-D-glucose. USE:Capable of utilizing in study of saccharides and as propagation accelerator of Bifidus bacterium. PREPARATION:The mixture of N-acetylchitooligosaccharide with lactic acid is reacted with beta-N-acetylhexosaminidase having sugar transfer ability.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing a novel oligosaccharide and V which can be used for research on sugar chains and as a growth promoter for bifidobacteria.

[Conventional technology]

Recently, it has become clear that glycoproteins, glycopeptides, and glycolipids present on the surface layer of animal cells have essential physiological functions in vivo. For example, m
Some of the glycosphingolipids on the cell surface are involved in blood type specificity, as well as organ/tissue specificity.

Furthermore, it plays an important role as an intercellular recognition site in the development and differentiation of cells and tissues. It is known that certain glycoproteins have the ability to sense aging of the glycoprotein through changes in its sugar chain structure and adjust its turnover.

In addition, since the structure of sugar 1 on the cell surface changes significantly with phosphorylation, it becomes an important marker for distinguishing between phosphorylated cells and normal cells. In this way, as the physiological functions of sugar chains are clarified, research on them is becoming more and more popular.

On the other hand, Bifidobacteria are useful bacteria that enter the human intestines, and their various physiological and nutritional effects on humans have been reported. It is known to inhibit putrefaction, synthesize vitamins B and B2, and affect protein trade metabolism.For this reason, much research has been conducted on substances that promote the growth of bifidobacteria.Recently, It has become increasingly recognized that sugars are important factors for the growth of bifidobacteria in the intestines, and research on the use of various oligo-rings as growth-promoting factors for bifidobacteria is increasing.

[Problem to be solved by the invention]

In view of the current situation as described above, the present inventors discovered a new oligo ring in the process of intensive research on oligo rings.
After separation and purification, the structure was analyzed and the present invention was completed.

The present invention will be explained in detail below.

[Structure of the invention]

The novel oligo ring in the present invention is represented by the following formula (1): -(1→4)-D-glucose and the following formula (
2) O-β-D-77'lactopyranosyl(1→4)-(〇-β-2-7cetamido-2-deoxy-D-glucobyl-7silyl(l→6)LD-glucose) be.

In addition, the method for producing these two types of novel oligo rings is N-
It is characterized by allowing β-N-acetylhexosaminigase to act on a compound of acetyl chito-oligosaccharide and lactose.

The oligo ring with N-acetylglucosamine and 7(, sugar bonded is O-β-D-
Galactopyranosyl-(1→4)-(0-β-2-aceto7mido-2-deoxy-D-glucopyranosyl-(1
→2)) -D-glucose is known, and in this oligo ring, the N-7 cetylglucosamine residue is bonded to the carbon atom at the 2-position of the glucose residue of lactose. The two types of oligo rings of the present invention differ in terms of tχ and hη structure.7 Next, the structure of the present invention will be explained.

(a) N-acetyl chito-oligosaccharide, cellooligo ring, and laminario-oligosaccharide were used as standard substances, and Toyo Pearl HW-40
As a result of the Glue filtration, the two types of oligo rings related to the present invention were estimated to be three types because they showed elution positions that corresponded to both trisaccharide components of the standard oligo ring.

(b) Dead sugars β-N-acenal hexosaminigase was added to each of these oligo rings and reacted at 40°C, and the sugars produced at that time were analyzed by high-performance liquid chromatography over time. As a result, the molar ratio of produced sugar to that is 8.24.
Since the ratio of N-7 cetylglucosamine to lactose is 1:1 at both the time and 48 hours, these two types of oligo rings are both trisaccharides consisting of one molecule of N-acetylglucosamine and one molecule of lactose. It was confirmed that there is.

(c) Distinction between α-linkage and β-linkage These oligosaccharides are processed by β-N-acetylhexosaminidase into N-7 cetylglufusamine and lactose (
Since it was subjected to bisecting angle IF, it was confirmed that it was a β-bond. Further, the C-NMR analysis results shown in Table I also support that it is a β-bond.

(d) Sugar binding mode As a result of conducting methylation analysis on each of these two types of oligosaccharides, the oligosaccharide of formula (1) has 3.4.6-
tri〇-methyl-2(methylacetamide)-D-glucitol, 2.3.4-tri〇-methyl-D-7 lactitol, and 2.3.6-tri〇-methyl-D-
Since three types of fluditol acetate of glucitol were detected, it was confirmed that N-7 cetylglucosamine was bonded to the carbon atom at the 6-position of the lactose residue of lactose.

Further, the oligosaccharides of formula (2) include 2.3, 116-thitra○-methyl-D-galactitol, 3.4.6-tri〇-methyl-2(methylacetamide)-D-glucitol, and V2 . Three types of alk)-acetates, 3-di○-methyl-D-glucitol, were detected, confirming that N-acetylglucosamine is bonded to the carbon atom at position 6 of the glucose residue of lactose. It was done. In addition, 13C-pJMRL:r) shown in Table-1
The analysis results clearly clarified the above results. .

From the structural analysis results described above, the two types of oligosaccharides related to the present invention are 〇-β-2-7 shown in formula (1), respectively.
Cetamido 2-deoxy-D-glucopyranosyl-(
1→6)-〇-β-D-77 Lactoviranosyl (1→
4) -D-glucose and 0-β- shown in formula (2)
D4 Lactopyranosyl-(1→4)-+O-β-2-
It can be identified as 7cetamido 2-deoxy-D-glucobylanosyl(1→6)iD-glucose.

Next, the properties of these oligosaccharides will be shown.

(A) Solubility These oligosaccharides are easily soluble in water, but insoluble in acetone, chloroform, hexane, etc.

(B) Color reaction and ultraviolet absorption 7E/-L sulfuric acid method and alkaline silver nitrate reaction showed positive results, and ultraviolet absorption at 210-220 nm derived from the 7-doamide group of the N-7 cetylglucosamine residue. shows.

(C) Color: When these oligosaccharides are dried and powdered, they all exhibit white color.

(D> Distinction between acidic, basic and neutral All of these oligosaccharides are neutral.

Hereinafter, the method for producing oligosaccharides according to the present invention will be explained.

In the present invention, 't?' between N-acetylchitooligosaccharide and lactose is used.
? : The compound is used as a substrate and β-N-acetylhexosaminigase is applied. The N-acetyl chitooligosaccharides used here are N-7 cetylchitobiose, N-7 cetylchitotriose, N-7 cetyl chitotetraose, N-7
Cetylchitobentaose, N-7 cetylchitohexaose, etc. can be used alone, as a mixture of these, or as a mixture of N-acetylchitooligosaccharides containing the monosaccharide N-7 cetylglucosamine. Also, the substrate used here is a mixture containing 1 to 30% by weight of N-acetylchitooligosaccharide and 2 to 40% by weight of lactose, (11-1 is 3 to 8! β-N-acetylhexosaminigase at 10-60°C
Operate at a temperature of The amount of β-N-acetylhexosaminidase added at this time is 1 to 200 units/
1fi1 is good. Further, the reaction time is preferably selected appropriately, and is usually 3 to 72 hours, when the yield of these glycosyltransfer oligosaccharides is highest. Furthermore, the β-N-7 cetylhexamine saminigase used in the present invention has the effect of transferring N-7 cetylglucosamine residue, which is formed by hydrolyzing N-acetylchito-oligosaccharide, to lactose. It may be of any origin. For example, β-N-7 cetylhexosaminidase derived from microorganisms is mentioned. In addition, β-N-acetylglucosaminiguse and β
-N-acenal〃lactosaminigase may be used as long as it exhibits the above-mentioned action.

In the present invention, N-7 cetylglufusamine is transferred to lactose by the above enzymatic reaction, and a 2R1 compound containing two types of oligosaccharides according to the present invention is produced. This reaction solution is heated at 90° C. or higher for 1 to 2 minutes to quench the enzyme reaction, and then passed through an activated carbon column to adsorb only the oligosaccharides in the reaction solution. This adsorption treatment is carried out in order to remove the 1/1 sugar produced by decomposition in the reaction mixture and obtain only the oligosaccharide components. The activated carbon column on which the oligosaccharides have been adsorbed is washed with a sufficient amount of water to completely remove the monosaccharides, and then the adsorbed oligosaccharides are eluted using a 5-40% ethanol aqueous solution. In addition to the oligosaccharides targeted by the present invention, the eluate thus obtained contains lactose and N-
Since it also contains acetyl chito-oligosaccharide, etc., in the present invention, vi is further produced using the following steps.

After concentrating the above-mentioned eluate under reduced pressure, it was loaded onto a Gello overgrained Biodel P-2 or Biodel P-4 column) and eluted with water. Since it exhibits ultraviolet absorption at 220 nm,
Collect both samples that become positive using these detection methods. Both parts of this elution are concentrated under reduced pressure! After a, Toyo Pearl I-I
Develop on a W-40 column for further purification. Both fractions that are positive by the above detection method are collected and lyophilized to obtain a mixture containing the two types of oligosaccharides according to the present invention.7 These oligosaccharides are further processed using preparative high performance liquid chromatography. The two types of oligosaccharides of the present invention can be obtained by separation and purification.

In the method of the present invention, especially when used as a growth promoter for bifidobacteria, a mixture containing the oligosaccharides of formulas (1) and (2), N-acetylchito-oligosaccharide, and lactose is dried into powder. It is also possible to apply it by converting it into a

〔Effect of the invention〕

The novel oligosaccharide of the present invention can be used for research on WI amount and as a growth promoter for bifidobacteria.

[Example] The present invention will be described in more detail below with reference to Examples, but it goes without saying that such explanation is not limited in any way.

10g of N-7 cetylchitobiots and 9g of lactose at 50+a
β-N-acetylhexosaminidase 2, prepared from Mecardia orientalis IF' 012806, dissolved in M acetic acid enriched solution (p) (4,0)
, 0004'lt was added and reacted at 25°C for 20 hours. Heat the enzyme reaction to stop it, and add 60 m of water to this.
1 was added to dilute the solution.

1,100 ml of this solution was equilibrated with water in advance and passed through an activated carbon-Celiac column (diameter 4 cm, height 50 cm) to adsorb oligosaccharides.

After washing this column with 2.517 liters of water, the adsorbed oligosaccharides were eluted with 2.5 liters of a 20% ethanol aqueous solution. The obtained eluate was concentrated under reduced pressure and then lyophilized to obtain 8 g of white oligosaccharide powder.

Next, this oligosaccharide (1, OR) was dissolved in 10+ ol of water, and then passed through a Biodel P-2 column (diameter: 5 cII 1, height: 184 c) that had been equilibrated with water, and subjected to Dell filtering. At this time, the column was maintained at 55°C using a jacket. Oligosaccharide using water, flow rate 120+
It was eluted with iI/It"/, and the eluate was collected in 10ml portions.

Sugars were detected by the phenol-sulfuric acid method and absorbance measurement at 215n, and both target oligosaccharides were collected. Note that this operation was repeated eight times. Concentrate the eluate under reduced pressure! 2! Thereafter, the mixture was freeze-dried to obtain 3.6 g of white oligosaccharide powder.

Oligosaccharides (0.9 g) were dissolved in 10 u+l of water, and a Toyovar HW-40S column (10 diameter 5
cI@, height 184c+a). Oligosaccharides were eluted with water at a flow rate of 80 ml at 7 hours. ) The volumetric layer was separated into 10 ml portions, sugars were detected by the method described above, and the desired oligosaccharides were collected. Note that this operation was repeated four times. Both oligosaccharide components were concentrated under reduced pressure and then lyophilized to obtain 1.4 g of white oligosaccharide powder.

Furthermore, in order to separate the oligosaccharides of formula (1) and formula (2) coexisting in this oligosaccharide fraction, high performance liquid chromatography was performed under the following conditions.

Column: YMC-Pack PA-43 (2
0X 250 + I1m) Mobile phase: acetonyl: water = 65
:35-70:30 flow rate:9. ! lhl/min detection: differential refractometer sample %l: 50-100+ng This minute glt operation resulted in 0.0% oligosaccharide of formula (1). zg
and oligosaccharide of formula (2) 0.2. I got it.

Claims (2)

[Claims] (1) It is represented by the following general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ where R_1 is an N-acetylglucosamine residue, R
O-β-2-acetamido-2-deoxy-D-glucopyranosyl-(1→6)-O-β-D where _2 is hydrogen
-galactopyranosyl-(1→4)-D-glucose and O-β-D-galactopyranosyl-(1→4) where R_1 is hydrogen and R_2 is an N-acetylglucosamine residue
-{O-β-2-acetamido-2-deoxy-D-glucopyranosyl-(1→6)}-D-glucose. (2) Production of the oligosaccharide according to claim 1, characterized in that β-N-acetylhexosaminidase having transglycosylation ability is allowed to act on a mixture of N-acetylchitooligosaccharide and lactose. Method.