JP2000245496A - Production of fucosyl oligosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce fucosyl oligosaccharide suitable for food additive in high yield by enzymatic reverse hydrolysis reaction comprising the bonding of fucose to an oligosaccharide by the reverse reaction of the hydrolysis reaction with fucosidase. SOLUTION: The objective fucosyl oligosaccharide containing α-1,2 bond and suitable as a food additive, etc., is produced in high yield by bonding fucose to an oligosaccharide by the reverse reaction of hydrolysis reaction with fucosidase originated from Penicillium multicolor, etc., adsorbing the produced fucosyl oligosaccharide to an activated carbon column and eluting the adsorbed fraction from the column. The production of fucosyl oligosaccharide by the bonding of fucose to an oligosaccharide may be performed by circulating a solution containing fucose and a sugar receptor through a system comprising an immobilized fucosidase column and an activated carbon column connected in series.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing fucosyl oligosaccharide. More specifically, the present invention relates to a method for producing a fucosyl oligosaccharide having an α1-2 bond.

[0002]

2. Description of the Related Art Fucose-containing oligosaccharides, ie, fucosyl oligosaccharides, are widely present not only in cell surface glycolipids and various glycoproteins, but also in breast milk, saliva, erythrocytes, and the like (H.E.
gge, A. Dell, and H. von Nicolai, Archives Biochem
istry and Biophysics, Vol. 224, 235-253 (198
3).). Fucose residues are particularly recognized in their sugar chains,
It is thought to contribute to the expression of important physiological activities such as immunity and adhesion. Fucosyl oligosaccharides are particularly high in breast milk and are said to have a function of preventing infection (DS Newburg, J. Mammary Gland Bio
l. Neoplasia, Vol. 1,271-283 (1996).).

In general, a method for producing an oligosaccharide using a transferase is known (Z. Guo an).
d PG Wang, Applied Biochemistry and Biotechnolog
y, Vol. 68, 1-20 (1997).). However, this method is not only expensive for the enzyme, but also for the sugar nucleotide as a substrate. As another production method, there is a method utilizing a hydrolase. Galacto-oligosaccharides are actually produced using lactose as a raw material and utilizing the transfer activity of β-galactosidase (Y. Matahira, A. Ta
shiro, T. Sata, H. Kawagishi, and T. Usui, Glycoco
njugate Journal, Vol. 12, 664-671 (1995).). For galacto-oligosaccharides, lactose can be used fortunately as a raw material, but in the case of other oligosaccharides, at present, there is no suitable sugar donor. That is, in the case of fucosyl oligosaccharides, fucoidan or its oligosaccharides are conceivable, but these are expensive and difficult to use as raw material saccharides.
In general, a synthetic substrate such as paranitrophenyl-α-L-fucopyranoside is often used as a donor in a glycosyltransfer reaction (K. Ajisaka and M. Shirakabe, Carbo.
hydrate Research, Vol. 224, 291-299 (1992)). This compound is a substance synthesized by an organic synthetic chemistry method, and a product using it as a raw material cannot be used as a food additive. For these reasons, a method for synthesizing fucosyl oligosaccharides from natural materials by an inexpensive method has not been found.

[0004] From such a viewpoint, if fucosyl oligosaccharides can be produced in large quantities at low cost by an enzymatic method, it will be possible to add them to foods, and the nutritional value of the foods will be enhanced. That is, there is a demand for the development of a low-cost production method without using an organic synthetic chemistry technique.

When an oligosaccharide is produced by an enzymatic method, it is desirable to use a hydrolase because transferase is expensive as described above. But,
If a transfer reaction is used in that case, no suitable sugar donor for the production of fucosyl oligosaccharides can be found. As a method for synthesizing oligosaccharides using a hydrolase, a reaction method called a reverse hydrolysis method is known, but the yield is not always high and is not practical. In addition, in the reverse hydrolysis reaction, oligosaccharides of all kinds of bonds are formed, and it is basically impossible to synthesize only α1-2-linked fucosyl oligosaccharides (H. Fujimoto, M.
Isomura, T. Miyazaki, I. Matsuo, R. Walton, T. Sak
akibara, K. Ajisaka, Glycoconjugate Journal, Vo. 1
4, 75-80 (1997).). If only an α1-2-linked oligosaccharide is required, it is necessary to separate it from the reaction solution using a technique such as a column.

[0006]

Accordingly, an object of the present invention is to develop a method for producing a fucosyl oligosaccharide by an enzymatic method, in particular, an α1-2-linked fucosyl oligosaccharide in high yield.

[0007]

Means for Solving the Problems The present inventors have found that fucosyl oligosaccharides can be obtained in high yield by binding fucose to oligosaccharides by a reverse hydrolysis reaction, and completed the present invention. . That is, the present invention provides (1) a method for producing fucosyl oligosaccharides, which comprises connecting fucose to oligosaccharides by a reverse reaction of hydrolysis reaction with fucosidase, and (2) adsorbing the produced fucosyl oligosaccharides on an activated carbon column. The method for producing a fucosyl oligosaccharide according to claim 1, wherein the reaction solution containing fucose and a sugar acceptor is connected in series with an immobilized fucosidase column and an activated carbon column. 3. The method for producing a fucosyl oligosaccharide according to claim 1 or 2, wherein the fucosyl oligosaccharide is circulated through a connected system, (4) the fucosyl oligosaccharide is an α1-2 bond. the method of manufacturing Fukoshiruorigo sugar according to 3, (5) fucosidase manufacture of Fukoshiruorigo sugar according to claim 1, 2, 3, or 4, characterized in that it is derived from Penicillium multicolor Law, (6) (1) to (5) Fukoshiruorigo sugars obtained by any of the manufacturing methods, related.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The reverse hydrolysis reaction is based on the principle that monosaccharides, disaccharides and trisaccharides are in an equilibrium state in an aqueous solution. The time to reach equilibrium can be reduced by adding the enzyme. Therefore, if oligosaccharides can be continuously extracted from a sugar solution in an equilibrium state, the production of oligosaccharides will proceed to maintain the equilibrium. As a method for continuously removing the product oligosaccharide from the reaction solution, the present inventors have previously disclosed a method of circulating the reaction solution through an activated carbon column (K. Ajisaka,
H. Nishida, and H. Fujimoto, Biotechnology Letter
s, Vol. 9, 387-392 (1987).). That is, activated carbon generally has a property of adsorbing disaccharides more strongly than monosaccharides and trisaccharides more than disaccharides. Therefore, if the monosaccharide, which is a raw material of the reverse hydrolysis reaction, is circulated through an activated carbon column connected in series with the immobilized enzyme column, the disaccharide as a product is preferentially adsorbed to the activated carbon column, and Has only a monosaccharide. Thus, the equilibrium will continuously progress in the direction of disaccharide production, which will accumulate on the activated carbon column.

Therefore, the present inventors applied this method to the synthesis of fucosyl oligosaccharide. As a result, as expected, fucosyl oligosaccharides were accumulated on the activated carbon column, and fucosyl oligosaccharides could be obtained with good yield. Also, here, according to the principle described above, if the reverse hydrolysis reaction proceeds, α1-2 bond, α1-3 bond, α1-4 bond,
Many kinds of α1-6 linked disaccharides and trisaccharides having them as receptors should be obtained. However, Penicillium
When the reaction was carried out using a column on which multicolor- derived α-fucosidase was immobilized, it was found that α1-2-linked disaccharide was obtained as a main product. It is known that fucosyl α1-3 glucose or fucosyl α1-3N-acetylglucosamine is synthesized by the transfer reaction of this enzyme (K. Ajisaka, H. Fujimoto and M. Miy).
asato, Carbohydrate Research, Vol. 309, 125-129
(1998)), there is no report that an oligosaccharide can be synthesized by a reverse hydrolysis reaction. The reason that fucosyl α1-2 galactose was regioselectively synthesized is that the rate of disaccharide formation by equilibrium in the reverse hydrolysis reaction depends on the substrate specificity of the enzyme, This is because the use of an enzyme having a high degree of reaction results in the rate at which α1-2 linked disaccharides can be formed is faster than the rate at which α1-3 linked disaccharides are formed. The α1-2-linked disaccharide generated during the passage through the immobilized enzyme column is adsorbed on the activated carbon column bound thereunder. On the other hand, α1-3 linkage or other disaccharides are generated in a small amount during the passage through the immobilized enzyme column due to the slow generation in the immobilized enzyme column. Therefore, it can be explained that the amount accumulated in the activated carbon column was also very small. In other words, here, using an immobilized column of an enzyme having high specificity for α1-2 binding, by using in combination with an activated carbon column, by a reverse hydrolysis reaction,
A method for producing fucosyl oligosaccharides using free fucose as a raw material has been found. Similarly, if an immobilized column of an enzyme having high specificity for α1-3 bond or α1-6 bond is used, synthesis of fucosyl oligosaccharide having α1-3 bond or α1-6 bond, respectively, is possible. The fucose and the acceptor saccharide are dissolved in water or a buffer, the immobilized α-fucosidase is packed in a column, and the column is connected in series with an activated carbon column. The above sugar solution is circulated through the two columns by a peristaltic pump or the like. After a certain period of time, the activated carbon column was washed with water to remove unreacted monosaccharide, and then 0% to 3%.
The fucosyl oligosaccharide is obtained by eluting the oligosaccharide with an aqueous ethanol solution of about 0%.

Here, several percent of an organic solvent may be mixed in the first reaction solution. This reaction does not depend on the method of immobilizing the enzyme. Not only a solid immobilizing agent but also an enzyme dissolved in a semi-permeable membrane or a reaction device using hollow fibers can be used. For elution of the fucosyl oligosaccharide from the activated carbon column, an aqueous ethanol solution is preferable, but depending on the purpose, the solvent is not particularly limited as long as it is a solvent usually used for activated carbon column chromatography, such as acetonitrile and isopropyl alcohol. In some cases, the gradient method is more preferable.

In the practice of this method, although the yield of fucosyl oligosaccharide formed on the raw material fucose is small and the apparent reaction yield is low, the fucose and the saccharide of the acceptor are actually recovered as they are. Therefore, when calculated from the viewpoint of the reaction yield per fucose used, the value is extremely high. In industrial production, in order to normally carry out continuous operation, the recovered sugar is rotated again as it is as a raw material, and it is sufficient to newly supply the reduced amount of sugar used for oligosaccharide production.

[0012]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1 Purification of α-fucosidase P. multicolor culture solution powder Lactase-P (KI Kasei Co., Ltd.)
2 g of 1 mM sodium phosphate buffer (pH 6.8)
0 mL and dissolved in the same buffer 1 using a cellophane membrane (SPECTRUM molecular weight cutoff 12,000-14,000).
After dialysis at 10 ° C. for 12 hours with L, the buffer was exchanged for a new one and dialysis was performed for another 5 hours. One day later, the enzyme solution was
LLIPORE CENTRIPREP ultra-concentration (molecular weight cut-off 1
After adjusting to 2 mL at 3,000, 3,000 rpm, 4 ° C. for 3 hours, column chromatography (column: Econo-Pa)
c CHT-II (Bio-Rad) 5 mL x 5 Eluent: Sodium phosphate buffer (pH 6.8) 1 mM (0 → 100 min) →
400mM (100 minutes → 600 minutes) flow rate: 0.5mL / min)
And purified. Fractions having fucosidase activity were collected from 2 ml as one fraction and subjected to ultraconcentration under the same conditions as above.

[Example 2] Immobilization of α-fucosidase All of α-fucosidase purified in Example 1 was 100 mM.
Dissolve in sodium phosphate buffer (pH 7.5) and total volume 3
It was 3 mL. 1.8g Eupergit C resin (Funakoshi)
The mixture was stirred at 10 ° C. After 60 hours, the resin was filtered off with a glass filter and washed with 100 mL of Milli-Q water. The resulting immobilized α-fucosidase was 8.
It showed an activity of 84 mU.

Example 3 Synthesis of Fucosyl α1-2 Galactose by Reverse Hydrolysis Reaction The immobilized α-fucosidase prepared in Example 2 was packed in a column, and an immobilized enzyme column (φ5.0 cm × 5.0 mm) was used. It was created. Immobilized enzyme column and activated carbon column (φ4.7cm
× 7.0 cm) were connected in series, and the apparatus was kept at 37 ° C. A 100 mM potassium phosphate buffer solution (pH 6.0) containing 5% (w / v) and fucose 10% (w / v) galactose was added to
Circulated through the two columns with a peristaltic pump.
After 4 days, the pump was stopped, and unreacted monosaccharide was poured out from the activated carbon column with 3 L of Milli-Q water. Thereafter, the fucosyl oligosaccharide was eluted from the activated carbon column with a 30% aqueous ethanol solution. The obtained fucosyl oligosaccharide was analyzed by HPLC
Analysis (Dionex Carbopac PA1 column, eluate 50 mM NaO
H, a flow rate of 1 mL / min, and a detection pulsed amperometric detector (Dionex) showed a peak at the same position as that of commercially available fucosyl α1-2 galactose (DEXTRA LABORATORIES). This peak was decreased by adding α1-2 fucosidase from Arthrobacter sp. (Takara Shuzo), which specifically hydrolyzes fucosyl α1-2 bond. In the HSQC spectrum of the eluate from the activated carbon column, Gal-1 position (H: 5.3 ppm, C: 92.5 ppm), Gal-2 position (H: 3.7 pp
m, C: 78.5 ppm), Gal-3 position (H: 4.05 ppm, C: 71 ppm), Gal-6
(H: 3.65 ppm, C: 61.5 ppm), Fuc-1 (H: 5.0 ppm, C: 102p)
pm), Fuc-4th position (H: 3.8ppm, C: 72ppm), Fuc-5th position (H: 4.15pp
(m, C: 68 ppm), etc., and several cross peaks well overlap with the cross peak of the fucosyl α1-2 galactose standard, and it can be understood that fucosyl α1-2 galactose is contained in these. Further, in this spectrum, no cross peak is observed in the region of 80 to 84 ppm (C-13 NMR), which indicates that fucosyl α1-3 galactose and fucosyl α1-4 galactose do not exist (FIG. 1B).

[0016]

According to the present invention, fucosyl oligosaccharide suitable for food addition can be produced from natural fucose and oligosaccharide in good yield.

[Brief description of the drawings]

FIG. 1 shows the HSQC spectra of commercially available fucosyl α1-2 galactose (A) and synthesized fucosyl α1-2 galactose (B, Example 3). The ordinate and abscissa indicate chemical shifts of H-1 NMR and C-13 NMR, respectively.

Claims (6)

[Claims] 1. A method for producing fucosyl oligosaccharides, wherein fucose is bound to oligosaccharides by a reverse reaction of a hydrolysis reaction with fucosidase. 2. The method for producing fucosyl oligosaccharide according to claim 1, wherein the produced fucosyl oligosaccharide is adsorbed on an activated carbon column and removed out of the system. 3. The fucosyl oligosaccharide according to claim 1 or 2, wherein the reaction solution containing fucose and a sugar acceptor is circulated through a system in which an immobilized fucosidase column and an activated carbon column are connected in series. Production method. 4. The method for producing a fucosyl oligosaccharide according to claim 1, wherein the fucosyl oligosaccharide has an α1-2 bond. 5. The method according to claim 5, wherein the fucosidase is Penicillium multicolor.
5. The method of claim 1, 2, 3, or 4, wherein
3. The method for producing a fucosyl oligosaccharide according to 1.). 6. A fucosyl oligosaccharide obtained by the production method according to claim 1.