JPS5828880B2 - New oligosaccharides and their production methods - Google Patents

Description

Detailed Description of the Invention The present invention provides a novel oligosaccharide, 〇-α-D glucopyranosyl=(1→3)-〇-α-D-glucopyranosyl -Glucohyranosyl (1→4
)-D-glucose (hereinafter abbreviated as 3-0-α-D-glucopyranosylmal) liose) and its production method. It has been used in large quantities for applications such as sweeteners, pharmaceuticals, enzyme assay substrates, fermentation media, and various drug intermediates.

However, there are currently only a limited number of oligosaccharides for which methods of producing highly pure and efficient oligosaccharides are known, such as sugar, lactose, maltose, and maltotriose.

The present inventors conducted studies on new oligosaccharides, discovered 3-0-α-D-glucopyranosylmaltotriose and a method for producing the same, and completed the present invention.

That is, ercinane, which has a structure in which maltotriose and some maltotetraose are repeatedly polymerized with α-1,3 glucoside bonds (see, for example, JP-A-53-8500), is treated with α-amylase (E, C03,2 ,1,1)
They discovered that the oligosaccharide produced by hydrolysis is the new tetrasaccharide 3-0-α-D-glucopyranosyl maltotriose.

This 3-0-α-D-glucopyranosyl maltotriose can be produced by chemical methods or biochemical methods.

Biochemical methods are needed to easily produce ercinane, especially α
30-α- by hydrolysis by amylase
A suitable method is to generate D-glucopyranosyl maltotriose, purify it, and collect fractions.

The α-amylase used in this case may be one that produces 3-0-α-D-glucopyranosyl maltotriose from ercinane, such as Aspergillus, Rhizopus, Penicillium, Ospora, etc. α-amylase derived from microorganisms such as molds belonging to the genus is suitable.

In addition, as the amylase preparation used, although the purpose of the present invention can be achieved even if it is a crude enzyme, it does not degrade the target product 3-0-α-D-glucopyranosylmaltotriose. Preferably, it does not contain enzymes such as α-glucosidase.

The ercinan hydrolyzate containing 3-0-α-D-glucopyranosyl maltotriose of the present invention can be used as it is as a product, for example, in a fermentation medium.

Further, the 3-0-α-D-glucopyranosyl maltotriose of the present invention can be purified by a known purification method, such as a decolorization method using activated carbon, a deionization method using an ion exchange resin, or a deionization method using an ion exchange resin. It can be easily purified and collected by using various methods alone or in combination, such as fractional precipitation using an organic precipitant such as alcohol or acetone, and fractionation using column chromatography.

The amount of 3-0-α-D-glucopyranosyl maltotriose thus obtained is about 10 to 50% on a solid basis based on the raw material ercinan.

The physicochemical properties of the new oligosaccharide 3-0-α-glucopyranosylmaltotriose in the present invention are as follows.

O elemental analysis: Actual values C=43.18%, H=6.41%, 0=50
．． 41% Calculated value C=43.24%, H=6.36%, 0=50
．． 40% (Chemical formula: C24H4□021) O Molecular weight: 667, Glucose polymerization degree -40 Melting point: Unable to measure due to difficulty in crystallization O Electrophoretic mobility: Electrophoretic mobility when current was applied at a constant voltage of 600 V and 34 mA for 1.5 hours The mobility of D-glucose is 1.00
The value of this product is 0.25, which is almost equal to that of maltose.

O paper chromatography mobility: Using a developing solvent of n-butanol:pyridine:water = 6:4:3, and developing for one day in the descending method, when the glucose mobility was assumed to be 1.00, this product It is 0.34, which is almost equal to that of maltotetraose.

○ Specific optical rotation: [α] +185° (C=1.0, H2
O) O ultraviolet absorption spectrum: Shows no specific absorption.

○Infrared absorption spectrum: Shows absorption near 840 crI'L-, which is specific to α-bonds.

○Solubility: Easily soluble in water, slightly soluble in ethanol, butanol, ether,
Insoluble in chloroform ○Anthrone reaction: Positive ○Silver mirror reaction: Positive O Physical properties: Aqueous solution is colorless, slightly acidic to neutral, powder is white O Composition: (1) Hydrolyzed with acid IN Partially hydrolyzed with sulfuric acid is D-glucose, maltose, nigerose, maltotriose, 3-0
-α-D-glucopyranosyl maltose and the test sugar.

(2) Enzymatic hydrolysis Not degraded by glucoamylase and β-amylase.

(3) Methylation analysis The tetradecamethyl derivative was hydrolyzed with acid, and the resulting mixture was analyzed by gas chromatography.

As a result, 2,3,4,6-chitler O-methylglucose, 2,4,6-toIJ-Q-methylglucose, 2
・3・6-to! The molar ratio of J-0-, luglucose was i,oo:0.96:1.96.

In addition, after reduction with sodium borohydride, methylation was performed, and the resulting tetradecamethyl derivative was hydrolyzed with acid, and the resulting mixture was similarly analyzed by gas chromatography.

As a result, ■.
The molar ratio of 3,6-tIJ-0-,,'-IF-glucose was 0.83:1.00:0.97:1.
It was 06.

In either case, since di-O-methylglucose was not detected, it was not a branched sugar.

From the above results, this product is 3-0-α-D-glucopyranosyl maltotriose, and its structure is as follows.

Next, an example of the production of 3-0-α-D-glucopyranosylmaltotriose of the present invention will be described.

Example Ercinan 20'i! After dissolving 1.51 in hot water, 4
Cool to 0°C and add 5m9 and 10-2M of α-amylase (crystalline Taka amylase A manufactured by Sankyo Pharmaceutical Co., Ltd.).
0.5 M acetate buffer containing calcium chloride (
pH 5, 3) 100rrLl was added and reacted at 45°C for 24 hours.

This reaction solution was kept at 100° C. for 5 minutes, cooled to room temperature, and centrifuged to collect the supernatant.

Twice the volume of ethanol was added to this supernatant, and the supernatant obtained by centrifugation was concentrated to 100 ml.

This concentrated solution was added to a column packed with activated carbon to adsorb sugar, and then eluted using the ethanol concentration gradient method according to a conventional method to collect the 3-0-α-D glucopyranosyl maltotriose fraction. By desalting with an ion exchange resin (H type, OH type), concentrating and drying, 3-〇-α-D-glycopyranosyl maltotriose in the form of a white powder is produced in about 5.
61 samples were collected.

The 3-0-α-D-glucopyranosyl maltotriose thus obtained can be advantageously used, for example, as a substrate for quantitative determination of α-1°3-glucosidase, a fermentation medium, an enzyme inducer, etc. .

In addition, during elution from the activated carbon column, 3-〇-α-
After the D-glucopyranosyl maltotriose fraction, both new heptasaccharides were eluted and similarly purified, concentrated, and dried to yield approximately 4.0'? powder was obtained.

The structure of this new heptasaccharide is
→4) -〇-α-D-glucopyranosyl(1→4)-
〇-α-D-glucopyranosyl = (1→3)-〇-α-
It is estimated to be D-glucopyranosyl(1→4)-〇-α-D-glucopyranosyl(1→4)-D-glucose.

This new heptasaccharide is suitable as a substrate for measuring the activity of amylase derived from living organisms such as blood and saliva.

That is, the new heptasaccharide is easily decomposed by these amylases, and 1 to 2 moles of the new heptasaccharide are 3-0-α-D-
Since glucopyranosyl maltose and 1 mol of glucose are produced, this glucose can be quantified.

Claims (1)

[Claims] 10-α-D-glucopyranosyl-(1→3)〇-α-
An oligosaccharide consisting of D-glucopyranosyl(1→4)-〇-α-D-glucopyranosyl(1→4)-D glucose. 2 Hydrolyze ercinane with α-amylase to form O-
α-D-glycopyranosyl-(1→3)-〇-α-D-
1. A method for producing oligosaccharides, which comprises producing and collecting glucopyranosyl-(1→4)-〇α-D-glucopyranosyl-(1→4)-D-glucose.