JPH10234393A - Production of oligosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an oligosaccharide useful as a bifidus bacteria proliferation material, etc., in high efficiency by treating an aqueous solution containing a monosaccharide with a β-glucosidase originated from Bifidobacterium breve and condensing the monosaccharide with β1→6 bond. SOLUTION: The objective oligosaccharide such as fucosyl (β1→6) glucose is effective as a substance capable of selectively proliferating bifidus bacteria which are benign intestinal bacteria useful for the host. The oligosaccharide is produced in high efficiency from an inexpensive raw material such as fucose or glucose by treating an aqueous solution containing a monosaccharide such as glucose or fucose with a β-D-glucosidase originated from Bifidobacterium breve and condensing the monosaccharide through β1→6 bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the sugar condensation activity of a hydrolase (the ability to catalyze the reverse reaction of a hydrolysis reaction).
And a method for producing an oligosaccharide directly from a monosaccharide using the method. See Bifidobacterium bleb (Bif
β-D- derived from Idobacteriumbreve)
The present invention relates to a method for producing oligosaccharides directly from monosaccharides by utilizing the saccharide condensation activity of β-D-glucosidase having fucosidase activity.

The oligosaccharide fucosyl (β1 → 6) glucose obtained by the method of the present invention is effective as a substance (bifidus factor) for selectively growing a group of bifidobacteria. Bifidobacterium is known as a beneficial intestinal bacterium that is beneficial to the host together with lactic acid bacteria among intestinal inhabiting bacteria. .

[0003]

2. Description of the Related Art It has been reported that fucosyl glucose composed of fucose and glucose is hydrolyzed only by bifidobacteria among intestinal bacteria and becomes a promising bifidobacterium [N. Sakai et al. , Ag
ric. Biol. Chem. , 53, 313-318
(1989); Nuura et al. , B
iosci. Biotech. Biochem. , 6
0,188-193 (1996)]. However, to obtain fucosyl glucose so far, p-nitrophenyl β-D-fucopyranoside (p-Nitrophen) has been used.
The synthesis was performed only by reacting with glucose, using a very expensive fucosyl group donor such as yl β-D-fucopyranoside as a raw material, and was not practical.

[0004]

An object of the present invention is to provide a method for producing an oligosaccharide such as fucosyl glucose from a less expensive raw material.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, have found that an aqueous solution containing a monosaccharide is added to an aqueous solution containing Bifidobacterium bleb.
The present inventors have found that an oligosaccharide, particularly fucosyl (β1 → 6) glucose, linked by a β1 → 6 bond can be obtained by allowing β-D-glucosidase derived from Bacteriumbreve to act, thereby completing the present invention. That is, the gist of the present invention is that an aqueous solution containing a monosaccharide is added to Bifidobacterium bleb (Bifidoba).
(Cterium breve), and a method for producing an oligosaccharide, which comprises condensing a monosaccharide through a β1 → 6 bond by reacting β-D-glucosidase. Hereinafter, the present invention will be described in detail along with its embodiments.
In the present application, β-D-fucosidase (fucos
β-D-glucosidase having activity (g)
lucidase) (EC 3.2.1.21)
Is hereinafter simply referred to as β-D-glucosidase.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, Bifidobacterium bleb (Bifidobacterium bleb) is used.
breve), which is characterized by using a β-D-glucosidase derived from a microorganism belonging to breve). Examples of microorganisms that produce β-D-glucosidase used in the above method include β-D-glucosidase.
Regardless of whether the D-glucosidase is constitutively expressed, or whether it is inducibly expressed by an inducer or a culture temperature, etc., the cells or culture obtained by culturing in a medium exhibit the enzyme activity. There is no particular limitation as long as it has.

[0007] As such a microorganism, Mitsuo
ka's method [T. Mitsuoka, Zbl. Bact
eriol. , I .; Abt. Orig. , 210, 52
(1969)], can be isolated and obtained from the natural world, or can be selected and obtained from existing stock strains by using the assimilation of β-glucoside and cellobiose as an index. As the microorganism, specifically, Bifidobacterium breb (Bifidobacterium breb)
eve) 203 strains [T. Tochikura, et a
l. , Agric. Biol. Chem. , 50
(9), 2279-2286 (1986)], Bifidobacterium bleb clb strain [K. Sasaki, e
t al. , Agric. Biol. Chem. , 51
(3), 699-705 (1987)].

[0008] In the present invention, β-D-glucosidase refers to any of microorganisms having β-D-glucosidase activity, crushed cells, cell extracts, crude enzymes and purified enzymes. Also means the form of Furthermore, for the purpose of stabilizing the enzyme of the present invention, the above-mentioned microbial cells, their crushed or extracted products, or those obtained by immobilizing a purified enzyme on a carrier can also be used as β-D-glucosidase.

[0009] Further, a genetically modified microorganism obtained by isolating a gene encoding β-D-glucosidase from the above microorganism and expressing the present enzyme in a large amount using an appropriate host-vector system is also used. I can do it. The host and vector are not particularly limited as long as they can express the gene.Specific examples of the host include Escherichia bacteria, Lactobacillus bacteria, and the like, and preferably Escherichia coli. Can be In addition, specific examples of the vector include pBR322, pUC
And plasmid vector such as pUC18.

Cultivation of a microorganism having β-D-glucosidase activity can be carried out in a conventional nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, various vitamins, etc. Examples of the carbon source include glucose, cellobiose, and the like. Sucrose, fructose,
Sugars such as maltose; alcohols such as ethanol and methanol; organic acids such as citric acid; molasses; and the like, and preferably contains at least one of cellobiose and glucose. Cellobiose is particularly preferred from the viewpoint of improving the activity of the enzyme.

Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, casamino acid, corn steep liquor and urea; and inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate. Preferably, polypeptone and yeast extract are used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients of various vitamins such as amino acids such as L-cysteine, ascorbic acid, biotin, and thiamine can be added to the medium.

Culture conditions are usually performed under anaerobic conditions, and it is preferable to perform anaerobic stationary culture. The culture temperature is not particularly limited as long as a microorganism capable of growing a microorganism having β-D-glucosidase activity can be grown.
Is not particularly limited as long as the microorganism can grow. Specifically, the culture is performed at a temperature of 20 to 50 ° C., preferably around 37 ° C., and the pH is 3 to 8, preferably 5 to 5.
Approximately 7 is performed. The pH adjustment during the culture can be performed by adding an acid or an alkali. By collecting cells by centrifugation etc. from the culture thus obtained,
Bacteria containing β-D-glucosidase can be obtained.

As a method for extracting and purifying β-D-glucosidase from microbial cells, any of the known enzyme purification methods can be applied. For example, specifically, as a method for crushing cells, ultrasonic crushing, mechanical crushing using a French press, a homogenizer, or the like, or enzymatic crushing using lysozyme or the like can be used. The thus obtained crushed bacterial cells can be centrifuged to obtain a soluble fraction containing a crude enzyme, and can be used as a crude enzyme fraction of β-D-glucosidase. Further, a purified enzyme obtained by further purifying the crude enzyme fraction may be used. Purification of maleate isomerase from the crude enzyme fraction
Usually, (a) separation by precipitation method, for example, ammonium sulfate precipitation method,
(B) Separation by chromatography, for example, ion exchange chromatography, affinity adsorption chromatography, gel filtration chromatography, etc. (c) Separation by electrophoresis, and any combination of these methods .

The β-D-glucosidase activity is measured by reacting a fraction containing β-D-glucosidase or purified β-D-glucosidase in each of the above purification steps with a reaction solution [composition: 42 mM sodium acetate buffer ( pH5.
5) 1.7 mM p-nitrophenyl glucoside], and incubated at 37 ° C. to release p-nitrophenol (n) released from p-nitrophenyl glucoside as a substrate.
It can be measured as the increase in absorbance at 400 nm.

The purity and molecular weight of the purified β-D-glucosidase are determined by the Davis method [B. J. Davis, A
nn. N. Y. Acad. Sci. , Vol. 121,
p. 404 (1964)] or the Laemmli method [U. K. See Laemmli, Nature, Vol. 2
27, p. 680 (1970)], and the gel is subjected to, for example, Coomassie blue dye solution [composition: 0.2% (v / v) Coomassie brilliant blue R250, 40% (v / v) methanol, 10% % (V / v) acetic acid] or a commercially available silver staining kit (for example, manufactured by Wako Pure Chemical Industries, Ltd.) to detect the protein.

The immobilization of the cells, the disrupted cells, the extract or the purified cells is carried out according to a commonly used method known per se on a suitable carrier such as acrylamide monomer, alginic acid or carrageenan. Method or Ajizaka et al. [N. Ajisaka et al. , C
arbohydr. Res. , 185, 139-146
(1989)] to formyl cellulofine.

The reaction mode of the method of the present invention includes β-D
A method in which the reaction is carried out by coexisting the β-D-glucosidase in an aqueous solution containing glucose and β-D-fucose, or the monosaccharide is contained in a column filled with the immobilized bacterial cells or the immobilized enzyme. A method of passing an aqueous solution is used. The concentration of the monosaccharide serving as a reaction raw material in the aqueous solution is suitably about 0.01 to 2M.

It is also preferable to add a buffer to the reaction system in order to enhance the stability of the enzyme. As the buffer, for example, a phosphate buffer of about 0.02 to 0.2 M (p
H7 to 9). When the cells are used for the reaction as they are, toluene, xylene,
Addition of a nonionic surfactant or the like in an amount of 0.05 to 2% (w / v) is preferable because the substance permeability of the cell membrane of bacterial cells may be increased.

The above enzymatic reaction can be carried out under aerobic conditions or anaerobic conditions, and the reaction temperature and pH are not particularly limited, but are usually 20 to 40 ° C, preferably 37 ° C.
The pH of the reaction solution is suitably around 5 to 10, preferably around 6 to 9, and more preferably around 7. Further, the pH can be adjusted by adding an acid or an alkali. After the completion of the reaction, the reaction solution is passed through an activated carbon column, and the saccharide in the reaction solution is adsorbed on the activated carbon. The target oligosaccharide is eluted from the activated carbon column with an alcoholic solvent such as ethanol and concentrated, whereby the target compound can be isolated.

[0020]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Purification of β-D-glucosidase (1) Culture of β-D-glucosidase-producing bacteria B-clb medium [Composition: 1% polypeptone, 0.5% yeast extract, 0.5% sodium acetate Hydrate, 0.4
% Dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, a mixed solution of L-ascorbic acid and -cysteine hydrochloride (solutions separately dissolved in sterilized water and neutralized with sodium carbonate were used, respectively) Final concentration 0.68%,
0.04%), 1% cellobiose (separately filtered and sterilized)] in a Sakaguchi flask containing 300 ml.
D-glucosidase producing bacterium Bifidobacterium breve c
lb strain (Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto Prefecture, a preserved strain of the Kumagaya Hidehiko Lab., Department of Food Engineering, Kyoto University), carbon dioxide gas was sealed in the gas phase, and the temperature was 3 ° C at 37 ° C.
The culture was allowed to stand for 6 hours.

(2) Extraction and Purification of β-D-Glucosidase The cells obtained by the above-mentioned culture were collected in a cooling centrifuge, and twice with 10 mM potassium phosphate buffer (pH 7.0). After washing, the cells were resuspended in the same buffer. This suspension was sonicated (Kaijo Denki Co., L.).
td. The cells were disrupted by centrifugation (× 75).
00g), and dialyzed in a 10 mM potassium phosphate buffer (pH 7.0, 4 ° C) to obtain a cell extract. The following purification procedure was performed from the obtained bacterial cell extract to isolate β-D-glucosidase.
In addition, the detection of the β-D-glucosidase fraction in each purification process was performed by measuring β-D-glucosidase activity. In the activity measurement, each fraction was subjected to a reaction solution [composition: 42
mM sodium acetate buffer (pH 5.5), 1.7 mM
p-nitrophenyl glucoside], and kept at 37 ° C. for a certain period of time. The enzyme reaction was stopped by adding 1.05 ml of 0.2 M sodium borate buffer (pH 10.5). The measurement was performed by measuring p-nitrophenol released from a certain p-nitrophenyl glucoside at 400 nm. The enzymatic activity unit (U) was defined as 1 U for the amount of an enzyme that produced 1 μmol of p-nitrophenol per minute.

The bacterial cell extract obtained above is subjected to ammonium sulfate precipitation by a conventional method to obtain a 30-70% saturated fraction, and then subjected to dialysis in the potassium phosphate buffer (pH 7.0). Was. Then, DEA equilibrated with the same buffer
E Cellulose column (9 × 110 cm)
The fraction eluted with 8M sodium chloride was collected,
A precipitate was obtained by precipitation with 0% saturated ammonium sulfate, and dialysis was performed in the same manner as in the previous period. Next, 10 mM potassium phosphate buffer (pH
6.0) and applied to a DEAE Cellulofine column (3 × 20 cm) equilibrated in step 2). After collecting the fraction eluted with 0.2 M sodium chloride, the enzyme was precipitated by 50% saturated ammonium sulfate precipitation, and a small amount of 10 mM It was dissolved in a potassium phosphate buffer (pH 7.0). Next, the mixture was applied to a Sephadex G-150 column (2 × 120 cm) and subjected to gel filtration, and the β-D-glucosidase active fraction was collected and 1 mM
Dialysis treatment was performed in a potassium phosphate buffer (pH 7.0). Subsequently, the mixture was applied to a gigapite column (1 × 20 cm) and subjected to linear concentration gradient elution with a 0 to 150 mM potassium phosphate buffer solution to obtain a β-D-glucosidase active fraction at about 40 mM. Then, the same volume of a 2M ammonium sulfate solution was added, and a butyl sepharose column (2 × 3 cm) was added.
The solution was subjected to hydrophobic chromatography, and eluted with a linear concentration gradient using a 1 to 0 M ammonium sulfate solution. The β-D-glucosidase activity was confirmed in the ammonium sulfate fraction of about 0.5M, the active fraction was concentrated by ammonium sulfate precipitation, dialyzed, and again subjected to gel filtration chromatography (Sephadex G-100 super column, 1 × 160 cm 2). ), The purified β
-D-glucosidase was obtained.

Example 2 Production of Fucosyl Glucose by β-D-Glucosidase A 10 mM potassium phosphate buffer (pH 7.0) containing 1 M each of D-glucose and D-fucose was prepared.
0.1 U of β-D-glucosidase purified in Example 1
Was added and incubated at 37 ° C. for 24 hours. The reaction solution after the reaction was introduced into an activated carbon column (1 × 3 cm) to adsorb saccharides, sufficiently washed with water, and eluted with a 7.5% aqueous ethanol solution. The eluted fractions are collected, concentrated by a rotary evaporator, and a part thereof is subjected to thin-phase chromatography [developing solvent: 85% 1-propanol, detection: 20% sulfuric acid-methanol, and sprayed.
At several degrees C. for several minutes].
(Rf: about 0.2) and B (Rf: about 0.05) were confirmed. These products were purified by preparative thin-phase chromatography, compared with Rf values of standard oligosaccharide samples, and treated with β-glucosidase to decompose them into monosaccharides to study the constituent sugars. , Product A is composed of D-glucose and D-fucose, and product B is
-It was found to consist of glucose only; Product B had the same Rf value as gentiobiose, which is glucosyl (β1 → 6) glucose. In addition, mass spectrometry, proton NMR, carbon NMR for both products
As a result of analysis by two-dimensional NMR combining these, the product A is D-fucosyl (β1 → 6) glucose in which the 1-position of D-fucose and the 6-position of D-glucose are β-linked. B is D1-glucose β1 → 6
It was confirmed that the gentiobiose was bound.

Example 3 Continuous Production of Fucosyl Glucose by Immobilized β-D-Glucosidase (1) Immobilization of β-D-glucosidase β-D-glucosidase purified in Example 1 (36.
To 10 ml of 200 mM potassium phosphate buffer (pH 7.0) containing 6 mg) was added 7.7 g of formyl-cellulofine (manufactured by Seikagaku Corporation), and the mixture was slowly stirred at 20 ° C. for 8 hours. , Trimethylamine borane (trimethy)
Lamine borane (65 mg) was added, and stirring was continued at 20 ° C. for another 4 hours. Thereafter, the gel was recovered using a glass filter, and the blocking solution [composition:
200 mM Tris / HCl buffer (pH 7.0), 6.5
mg / ml trimethylamine / borane], and stirred at 20 ° C for 2 hours. Next, the gel was collected by a glass filter, and the gel washed with a 200 mM potassium phosphate buffer (pH 7.0) was used as immobilized β-D-glucosidase.

(2) Continuous Production of Fucosyl Glucose The gel of immobilized β-D-glucosidase prepared as described above was filled in a glass tube (1.5 × 3.5 cm), and an activated carbon column was placed next to the immobilized column. (1.5 x 6.5 cm). The outlet of the activated carbon column was connected to the inlet of the immobilized β-D-glucosidase column, and a 10 mM potassium phosphate buffer (pH 7.0) containing 1 M each of D-glucose and D-fucose was circulated by a peristaltic pump. The produced fucosyl glucose was continuously adsorbed and accumulated on the activated carbon column. After circulating the reaction solution for one week, the activated carbon column was removed, and the column was eluted with 7.5% ethanol water in the same manner as in Example 2.
60 mg of fucosyl (β1 → 6) glucose could be collected.

Example 4 Utilization of Fucosyl (β1 → 6) Glucose by Various Intestinal Bacteria Using fucosyl (β1 → 6) glucose obtained in Example 3 as a carbon source, 21 types of various intestinal bacterial strains were used. The feasibility was examined. For culturing, of the B-clb medium used in Example 1, 1% fucosyl (β1 → 6) glucose was used instead of cellobiose as a carbon source, or 1% glucose was used as a control. Bacterial growth is turbidity at 610 nm (OD
₆₁₀ ). As a result, as shown in Table 1, the fucosyl glucose produced according to the present invention is capable of selectively growing Bifidobacteria without being assimilated into Escherichia coli, enterococci, and the like. It was confirmed to be useful.

[0027]

[Table 1]

[0028]

According to the present invention, fucosyl (β1 → 6) glucose, which is an oligosaccharide useful as a bifidobacterium, can be efficiently produced from inexpensive raw materials such as fucose and glucose.

Claims (4)

[Claims] (1) Bifidobacterium bleb (Bifidobacterium bleb) is added to an aqueous solution containing a monosaccharide.
rev) -derived β-D-glucosidase, and a monosaccharide is condensed through a β1 → 6 bond to produce an oligosaccharide. 2. The method for producing an oligosaccharide according to claim 1, wherein the monosaccharide is glucose and fucose. 3. The method for producing an oligosaccharide according to claim 1, wherein the oligosaccharide is fucosyl (β1 → 6) glucose. 4. A bifidobacterium comprising fucosyl (β1 → 6) glucose obtained by the method according to claim 1.