JPS63202396A - Production of oligosaccharides having n-acetylglucosamine, glucosamine, mannose or allose at terminal - Google Patents

Abstract

PURPOSE:To efficiently obtain the titled oligosaccharides useful as sweeteners, etc. by reacting a specific enzyme with a blend solution of starch or dextrin and N-acetylglucosamine, glucosamine, mannose or allose. CONSTITUTION:A microorganism, such as Bacillus subtilis, is inoculated into a culture medium containing polypeptone, etc. and aerobically cultivated at about 37 deg.C for 5-48hr to provide microbial cells, which are then subjected to extraction treatment to afford a disproportionating.enzyme (D-enzyme), reactive with maltooligosaccharides and exhibiting saccharide transfer action. The resultant enzyme is subsequently added to a solution obtained by mixing starch, such as corn, or dextrin with N-acetylglucosamine, glucosamine, mannose or allose at 1:1 ratio in 20-30wt.% starch concentration. The solution is then subjected to saccharide transfer reaction at about pH6.5 and about 35 deg.C for about 48hr. The reaction solution is subsequently subjected to filtration and concentration treatment to recover oligosaccharides containing the N- acetylglucosamine, glucosamine, mannose or allose at terminals.

Description

[Detailed Description of the Invention] ■ Industrial Application Field The present invention relates to an enzyme that acts on oligosaccharides to transfer sugars (
Disbrobationating enzyme-4-a
-Glucanotransfe-rase, E, C,
2, 4, 1, 25 (hereinafter abbreviated as one enzyme) is mixed and reacted with starch or dextrin and N-acetylglucosamine, glucosamine, mannose or allose,
The above carbohydrate is α-1,4 at the reducing end of maltooligosaccharide.
The present invention relates to a method for producing new linkage-linked oligosaccharides. All oligosaccharides have a mild sweet taste and are somewhat viscous and syrup-like.

■ Conventional technology D - Enzymes are enzymes that originally use maltooligosaccharides as substrates, cleave the glucosidic bonds, and transfer the cut ends to other oligosaccharides.The reaction can be expressed, for example, as shown in the following equation. Can be done.

G-G-G+G-G-G-+G-G-G-G-G+G In other words, the enzyme cleaves the glucosidic bond of the maltooligosaccharide at an appropriate position, and then converts the cleaved portion into the unreacted oligosaccharide (receptor). metastasizes to. Oligosaccharides are both substrates and receptors.

■ Problems to be solved by the present invention As a result of intensive investigation into what kind of carbohydrates other than oligosaccharides are effective as this receptor, the present inventors found that in addition to maltooligosaccharides, glucose, N-acetylglucosamine ,
n5ferase (CGT-a) has shown that glucosamine, mannose, and even allose can be effective receptors.
se, E, C, 2゜4.1.19>, our research has shown that glucose, xylose, sorbose, or their substituted substances at the 01 position can be effective receptors, but N- It has already been clarified that acetylglucosamine, glucosamine, mannose, allose, etc. cannot be receptors (S, Kitahat-a and
S,Okada,J,Bioche-m,,79 6
41 (1976)) In order to become an effective receptor for a, CGT-ase, the configuration of the OH at the C2, C3, and 04 positions is important, and the configuration of the OH at the C2 and C3 positions is different from glucose. Glucosamine, glucosamine, mannose, and allose cannot be receptors.

However, in the case of D-enzyme, the configuration of the OH at the C4 position is important in order to become an effective receptor, but the configuration of the OH at the C2,
A major difference was found that the configuration of C3 position Of-1 is irrelevant.

(2) Means for Solving the Problems As the enzyme used here, any D-enzyme that acts on maltooligosaccharides and transfers sugars, other than potato-derived enzymes, can be used. For example, higher plants such as sweet potato, sweet corn, mung bean, Bacillus
us 5ubtil-is, Pseudomonas
5tutzer-i, ESC CheriChia
It is an enzyme produced by microorganisms such as coli. In particular, enzymes produced by microorganisms have high receptor specificity, and more recently,
Bacterial strains with high enzyme production have been discovered, and their practicality is increasing.

Next, this enzyme uses maltooligosaccharide as a substrate, but if a receptor is present, higher molecular weight dextrin and starch can also be used as substrates. Therefore, we discovered that when D-H element is applied to a mixed system of polymeric substrates such as starch or dextrin and glucosamine, it is possible to efficiently synthesize the target oligosaccharides containing glucosamine, etc., since maltooligosaccharides are not present in the reaction system. , we were able to complete the present invention.

Industrially, it is desirable to use liquefied starch (decomposition rate 5-20%) obtained by partially decomposing raw starch with acid or enzymes as the starch or dextrin that can be used. Although the rearrangement reaction proceeds even if the starch or dextrin concentration is 1% or less, in the case of industrial production, the starch concentration is 1% or less. [
About 20 to 30% is desirable.

Since this reaction is a transfer reaction, in addition to the concentration of starch or dextrin, the receptor concentration and the mixing ratio of the two are also important. Using the mixing ratio of starch and receptor, 37℃, 5-4
Prepare by aerobic culture for 8 hours. A bacterial cell extract obtained by ultrasonic disruption of cultured bacterial cells is used as a crude enzyme solution, which is further purified to a high degree by various methods. For the crude enzyme solution and the wi enzyme solution, the enzyme activity (the measurement method is as described below) is the same,
In addition to examining the increase in reducing power by acting on maltotriose, the reaction products were examined by paper chromatography. As a result, no increase in reducing power was observed in both reaction solutions, and it was confirmed by paper chromatography that a series of maltooligosaccharides were produced. That is, it has been found that the crude enzyme solution does not contain enzymes that hydrolyze maltotriose (α-amylase, glucoamylase, α-glucosidase), and that the crude enzyme solution can be used as it is as an enzyme agent.

The optimum pH for the action of this enzyme is around 6.5, and the stable pH range is 6.0 to 8.0. Furthermore, the optimum temperature for wood enzyme action is 35°C, and the temperature stability (15 minutes treatment) is up to around 45°C.

Enzyme activity measurement method> Add and react at 40°C for 10 minutes. The amount of glucose produced is determined by NADP, hexokinase, glucose-6-
Enzyme activity was measured by quantifying using phosphate dehydrogenase. The amount of enzyme that produced 1 μq of glucose in 10 minutes under these conditions was defined as 1 unit.

Oligosaccharides containing lucosamine were separated by paper chromatography, and those with Rf values next to N-acetylglucosamine were extracted.

When this substance is hydrolyzed with acid, glucose and N-acetylglucosamine are produced in 1=1 ratio. (2) The degree of polymerization measured by Timmef's method is 2°0. (2) After reduction with sodium borohydride, -acetyl-3,6-di-0-methyl-4-〇-acetylglucosamine is obtained.

From the above results, it was identified as 1,4-α-glucosyl-N-acetylglucosamine.

In addition, when substances that give spots other than 1,4-α-glucosyl-N-acetylglucosamine (G-NAcG) are decomposed with glucoamylase, glucose and G-NAcG
give. When broken down by β-amylase, maltose is produced. Considering these facts and the action specificity of the D-enzyme, it was confirmed that the reaction by the D-enzyme proceeds as shown in the following equation.

ACG Starch G-NACGN-acetylglucosamine→ G-G-NAcGG-GG-
NAcG Next, products containing mannose were also separated by paper chromatography, and those exhibiting an Rf value just below that of mannose were extracted.

The structure of this substance was investigated in the same manner as for N-acetylglucosamine. This substance produces glucose and mannose in a 1:1 ratio through acid hydrolysis. ■T
The degree of polymerization measured by the method of -immel is 2.0. ■Produces borohydride 3,6-tri0-methyl-mannitol. From the above results, 1,4-α-
It was identified as glucosyl-mannose. Furthermore, when oligosaccharides are decomposed with glucoamylase, they produce glucose and 1,4-α-glucosyl-mannose, and when digested with β-amylase, maltose is produced.
It was recognized that it is an oligosaccharide containing mannose at the reducing end.

Regarding oligosaccharides including glucosamine and allose,
A similar experiment was carried out to investigate the structure, and it was confirmed that each of these is a maltooligosaccharide with glucosamine or allose linked to the reducing end via an α-1,4 linkage.

■ Example (Example 1) Polypeptone 1.5%, yeast extract 0.5%, maltose 1%, magnesium chloride 10-3M, ferrous sulfate 2X
After sterilizing 15ff of a medium containing 10''6M, phosphorus!! buffer (PH7゜0>0.1M), E.

E. coli iFo 3806 strain was inoculated and cultured aerobically at 37° C. for 5 hours in a jar fermenter. After collecting the bacteria by centrifugation and washing the cells, the cell extract obtained by ultrasonic disruption is used as a crude enzyme solution.4.
2 x 107 units).

To a solution prepared by dissolving 100 J of soluble starch in 1 Q of water and adding 100 g of N-acetylglucosamine, 10 parts of the above enzyme agent (3.5 x 105 units) was added and reacted at 40°C at pH 7.0 for 48 hours.

After heating at 100° C. for 10 minutes to stop the enzyme reaction, 0.2% activated carbon was added, filtered, and concentrated to obtain a colorless starch syrup-like substance 2109 with a water content of 25%.

1q of this substance was spotted on a filter paper, and after quadruple development with a solvent system with a composition of n-butanol:pyridine:water (6:4:3),
When colored with silver nitrate, N-acetylglucosamine (N
AcG), as well as G-NACG, G-G-NAcG, and G-G-G-NAc, in which one to several glucose molecules are bonded to
A series of spots that appeared to be G, G-G-G-G-NAcG were observed.

(Example 2) 200 ml of commercially available bacterial liquefied α-amylase was added to 100 q of potato starch and dissolved by heating in 112 ml of water. When the decomposition rate was 3%, the enzyme reaction was stopped by boiling to obtain a liquefied starch solution with a decomposition rate of about 5%. Allose 809 was added and dissolved, and the above enzyme solution 10d was added and reacted at 40°C for 48 hours. The reaction solution was purified by treatment with 0.2% activated carbon and ion exchange resin, and concentrated to yield 190 g of a colorless viscous syrup-like substance with a water content of 20%. When the product was examined by paper chromatography, in addition to allose, a series of spots where one to several glucose molecules were bonded to allose could be observed.

(2) Effects of the Invention In this reaction, oligosaccharides with a degree of polymerization of about 2 to 5, such as maltose produced from these starches, can also serve as substrates, but the presence of large amounts of these carbohydrates in the reaction system lowers the yield.

When the amount of starch becomes 1/2 or less of the amount of receptor, the starch is well decomposed and the transfer reaction proceeds, but a large amount of unreacted receptor remains in the reaction solution even in the late stage of the reaction. On the other hand, when the amount of starch is more than twice the amount of receptor, most of the added receptor undergoes a transfer reaction, but starch decomposition becomes insufficient.

Claims (1)

[Claims] A method for producing oligosaccharides containing N-acetylglucosamine, glucosamine, mannose, or allose at their terminals by acting a disproportioning enzyme on a mixture of starch or dextrin and N-acetylglucosamine, glucosamine, mannose, or allose. .