JPH11253191A - Synthesis of lacto-n-tetaose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enzymatically synthesizing lacto-N-tetraose suitable for use in foods or medicines at practical levels. SOLUTION: This method for synthesizing lacto-N-tetraose comprises allowing UDP-GlcNAc to act on lactose in the presence of β-1,3-N-acetylglucosaminyl transferase to synthesize lacto-N-triose II followed by binding galactose to the lacto-N-triose II in the presence of β-galactosidase. Thereby, the aimed lacto-N-tetraose which is found in human milk but not in cow milk can be efficiently synthesized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc).

[0002]

BACKGROUND OF THE INVENTION Breastfeeding infants are more
It is known to have high resistance to infection and low morbidity and mortality. One of the reasons is that the enterobacteriaceae flora in breastfeeding infants is overwhelmingly superior to bifidobacteria as compared to artificial feeding infants.

[0003] Human milk contains many oligosaccharides (oligosaccharides), which are growth factors of bifidobacteria, in a large amount (more than 30 species). Since it is considered to be an indicator of normal physiological function, it is necessary to synthesize oligosaccharides (oligosaccharides) in human milk and add it to artificial milk (milk powder) in order to bring artificial milk closer to breast milk. Is being done. In particular, lacto-N-tetraose is contained in human breast milk but not in bovine milk, and its physiological role, for example, the growth activity of bifidobacteria (C. Kunz and S. Rudleff, Acta Paediatr., 82, 90
3-912 (1993)) or infection inhibitory activity of Klebsiella pneumoniae (B. Ande
rsson, et al., J. Infections Diseases, 153, 232-237
(1986)) has been attracting attention, and it has been demanded to establish a synthesis technique of the oligosaccharide at a practical level in order to add it to food products such as powdered milk and pharmaceuticals.

[0004] Generally, as a method for synthesizing oligosaccharides,
There are organic synthesis and enzyme synthesis. In the organic synthesis method, a method is employed in which a sugar acceptor in which the hydroxyl group not involved in the reaction is completely protected is reacted with a sugar donor activated at the 1-position (K. Toshima and K. Tatsuta). , C
hem. Rev., 93, 1503-1531 (1993)). Lact-N of the present invention
-Organic synthesis of tetraose has already been reported (T. Takamura, et al., Chem. Pharm. Bull., 28,
1804-1809 (1980)) requires protection and deprotection of hydroxyl groups not involved in the bonding before and after the bonding between sugars, and is not a practical level of synthesis due to the large number of steps. And
Since an organic solvent is used as a reaction solvent, application of the oligosaccharide to foods and pharmaceuticals is not desirable.

On the other hand, the enzyme synthesis method is a synthesis method that can solve these problems. There are two types of enzyme synthesis methods: a method using a hydrolase and a method using a transferase.Each method has advantages and disadvantages.In order to industrially produce long sugar chains using enzymes, there are various methods. Problems to be solved have been pointed out. For example, in the case of a hydrolase, since the substrate specificity is low, it is not suitable for selectively synthesizing a disaccharide having a target bond, or hydrolysis of a product occurs simultaneously with the synthesis. There is a problem that the reaction yield is low. Further, in the case of transferase, since most of the enzymes are derived from animal organs, there is a problem that purification is extremely difficult and is not suitable for use at a practical level.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for synthesizing lacto-N-tetraose by an enzyme synthesis method, which is suitable for use on a practical level in foods and pharmaceuticals.

[0007]

Means for Solving the Problems From the previous studies, it has been found that when an oligosaccharide is synthesized by a transfer reaction using a hydrolase, a binding site that is easily cleaved by the enzyme is also easily formed by the transfer reaction. (Hiroshi Fujimoto et al., Applied Glycoscience, 43, 265-272 (199
6)). From this viewpoint, the present inventors think that in order to enzymatically synthesize a galactosyl oligosaccharide having a β1-3 bond, it is necessary to search for β-galactosidase that hydrolyzes the β1-3 bond with high selectivity. After careful examination, Bacill
us circulans origin of β- galactosidase (JP-A-9-313
177), however, found that β1-3 linked galactosyloligosaccharides can be synthesized selectively and in high yield by a transfer reaction, and could solve the problem of the synthesis method using hydrolase. .

Further, the present inventors have determined that β in bovine blood
-1,3-N-acetylglucosaminyltransferase (β
-GlcNAcT) can be used for the synthesis of lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc) by partial purification using only ammonium sulfate salting-out and succeeded in solving one of the problems related to transferases. did.

By combining such two types of enzyme reactions, a method for synthesizing lacto-N-tetraose at a practical level and easily has been established, and the object of the present invention has been solved. That is, the present invention includes the inventions described in the claims.

[0010]

DETAILED DESCRIPTION OF THE INVENTION UDP-GlcNAc is added to lactose in the presence of crude β-1,3-N-acetylglucosaminyltransferase (β-GlcNAcT) obtained by salting out bovine serum with ammonium sulfate.
By transferring GlcNAc residues, lacto-N-triose II is synthesized. Here, as β-GlcNAcT, those derived from bovine serum are inexpensive and most preferable for the practice of the present invention, but the present invention is not limited to those derived from bovine serum, and human serum, or Gl, such as from pig serum
An enzyme of any origin may be used as long as it has an activity capable of transferring cNAc by β1-3 bond.

Then, a galactose residue of paranitrophenyl-β-D-galactopyranoside (Galβ-pNp) is bound to this lacto-N-triose II in the presence of β-galactosidase by β1-3 binding. By transferring with lacto-N-
Get tetraose. Here, as the galactose donor, not only paranitrophenyl-β-D-galactopyranoside but also an ortho isomer (Galβ-oNp), lactose, Galβ1-3Gal, or the like can be used. Also,
The β-galactosidase is not limited to the above enzyme, and may be any enzyme that hydrolyzes β1-3 linked galactosyl oligosaccharides, such as mammals, for example, pigs, or β from bovine testes. -Galactosidase (JJ Distler and GW Joundian, J. Biol.
Chem., 218, 6772-6780 (1973), T. Murata, et al., J.
Biochem., 120, 851-855 (1996)) or Streptoco
β-galactosidase from ccus 6646K (JH. Yoon and
K. Ajisaka, Carbohydr. Res., 292, 153-163 (1996))
May be used. When these enzymes are used, not only β1-3 bonds but also β1-4 linked tetrasaccharides are produced at the same time.
The β1-4 linked tetrasaccharide is lacto-N-neotetraose (Gal
β1-4GlcNAcβ1-3Galβ1-4Glc) is a sugar contained in breast milk. Furthermore, even if these β-galactosidases are used in an immobilized form, they do not affect the regioselectivity at all.

[0012]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of β-1,3-N-acetylglucosaminyltransferase About 10 l of bovine blood was collected and left at room temperature for about 2.5 hours to coagulate. Collect the supernatant and centrifuge (8,000 rpm, 30 min,
(4 ° C.) to remove the contaminated blood cell components to obtain 2.7 l of serum. Ammonium sulfate was added so as to be 25% saturated and left for 3 hours. Further, ammonium sulfate was added to 50% saturation, and the mixture was left overnight.

The precipitate was collected by centrifugation (8,000 rpm, 30 min, 4 ° C.), dissolved in a small amount of cacodylate buffer (pH 7.0), dialyzed, freeze-dried, and partially purified β-1,3- N-acetylglucosaminyltransferase (β-GlcNAcT)
I got

Example 2 Synthesis of lacto-N-triose II 540 mg of lactose and 90 mg of UDP-GlcNAc were combined with 3 ml of 75 mM
It was dissolved in a cacodylate buffer (pH 7.5), and the β-GlcNAcT obtained in Example 1 was added thereto, followed by a reaction at 40 ° C. for 30 hours. The reaction solution was applied to an activated carbon-celite column (4.4 x 8
7%), a gradient solution of 0% to 30% ethanol was flowed at a flow rate of 150 ml / hr, and fractions of 4 ml each were collected (FIG. 1). By collecting and concentrating the fraction of trisaccharide, 17 mg of lacto-N-triose II (GlcNAc
β1-3Galβ1-4Glc) was obtained.

Example 3 Synthesis of lacto-N-tetraose 1 11.3 mg of lacto-N-triose II as a sugar donor and 10 mg of lactose as a sugar acceptor were added to a 20 mM acetate buffer (p
H5.5) was dissolved in 60 μl, and 7.2 mU of β-galactosidase derived from pig testis was added, followed by a reaction at 40 ° C. for 46 hours.
After the reaction is stopped by boiling, the reaction solution is transferred to a BioLC system (DION
EX company, column: CarboPac PA1,4.6 x 250mm, flow velocity 1m
l / min, and the eluate was subjected to 150 mM NaOH). When detection was performed with a pulsed amperometric detector, lacto-N-
A peak of the transfer product was detected, which coincided with the retention time of the tetraose standard.

Example 4 Synthesis of lacto-N-tetraose 2 60 mg of Galβ-oNp and 36 mg of lacto-N-triose II at 40 mM
Dissolve in 3 ml of acetate buffer (pH 5.5) and add Bacillus circulan
s- derived β1,3-galactosidase (JP-A-9-313177)
After adding 50 mU and reacting at 50 ° C. for 11 hours, the reaction was stopped by heating at 100 ° C. for 5 minutes. When the reaction solution was analyzed by HPLC in the same manner as in Example 3, a peak of a transfer product coincident with the retention time of the lacto-N-tetraose standard product was detected (FIG. 2).

After removing insolubles by centrifugation (5000 × g, 15 min), the supernatant was equilibrated with water containing 5% methanol.
The sample was subjected to ODS Chromatorex DM1020T (manufactured by Fuji Serial Chemical Co.) chromatography (φ1.1 × 5.7 cm) (FIG. 3).
The fraction of the reducing sugar first eluted was concentrated to 15 ml, and activated carbon-
The sample was subjected to celite chromatography (φ2.2 × 28 cm). First water (150ml), then 7.5% ethanol (300m
l) and eluted by a linear gradient method of 7.5% (1 L) to 35% (1 L) of ethanol, as shown in FIG. Two peaks having absorption were detected. The fraction around 20% was presumed to be a mixture of lacto-N-tetraose of the present invention and a product presumed to be the β1-6 isomer,
This fraction is treated with a 50 mM phosphate buffer (pH 7.0) at a substrate concentration of 0.1%.
Diluted with β-galactosidase (Biolact, Daiwa Kasei Co., Ltd.)
a) was added, and the reaction was carried out at 40 ° C. for 21 hours.
For 5 minutes to stop the reaction.

After removing insolubles by centrifugation (5000 × g, 15 min), the supernatant was subjected to activated carbon-celite chromatography (φ2.2 × 10 cm). Wash with water (100 ml), then 7.5% ethanol (60 ml), 7.5% (300 ml) to 35%
As a result of elution of (300 ml) ethanol by the linear concentration gradient method, as shown in FIG. 5, one peak having an absorption of 210 nm was detected near the ethanol concentration of 20%. This was concentrated and freeze-dried to obtain a white powder (7.1 mg). The structure was determined to be lacto-N-tetraose as a result of structural analysis by ¹ H and ¹³ C-NMR (FIGS. 6 and 7).

[0020]

According to the present invention, lacto-N-tetraose which can be used for foods and pharmaceuticals can be easily synthesized at a practical level.

[0021]

[Brief description of the drawings]

FIG. 1 is a view showing elution of sugar from an activated carbon-celite column. The dashed line indicates the ethanol concentration, ○ indicates the absorbance at 210 nm, and □ indicates the absorbance at 485 nm when the saccharide was quantified for each fraction by the phenol-sulfuric acid method. Also, LNT
-II indicates the fraction containing the product, lacto-N-triose II.

FIG. 2 is a view showing an HPLC chart of a reaction solution of Example 4. LNT indicates the product, lacto-N-tetraose.

FIG. 3 is an ODS column chromatogram (absorbance at 210 nm) of a lacto-N-tetraose synthesis reaction solution of Example 4.
FIG. LNT indicates the fraction containing the product lacto-N-tetraose.

FIG. 4 Activated carbon-Celite column chromatogram (210 nm) of a lacto-N-tetraose synthesis reaction solution of Example 4
FIG. LNT indicates the fraction containing the product lacto-N-tetraose.

FIG. 5 is a diagram showing an activated carbon-celite column chromatogram (absorbance at 210 nm) of a selective hydrolysis reaction of a lacto-N-tetraose synthesis reaction solution of Example 4 with β-galactosidase manufactured by Daiwa Kasei Co., Ltd. LNT indicates the fraction containing the product lacto-N-tetraose.

FIG. 6 is a chart showing ¹ H-NMR (270 MHz) of lacto-N-tetraose obtained in Example 4 measured in D ₂ O.

FIG. 7 is a diagram showing ¹³ C-NMR (270 MHz) of lacto-N-tetraose obtained in Example 4 measured in D ₂ O.

Claims (7)

[Claims] 1. Lactose is reacted with UDP-GlcNAc in the presence of β-1,3-N-acetylglucosaminyltransferase to synthesize lacto-N-triose II, and the lactose is further reacted in the presence of β-galactosidase. A method for synthesizing lacto-N-tetraose, wherein galactose is bound to -N-triose II. 2. The method according to claim 1, wherein the β-1,3-N-acetylglucosaminyltransferase is a crude β-1,3-N-transferase derived from mammalian serum.
The method for synthesizing lacto-N-tetraose according to claim 1, which is acetylglucosaminyltransferase. 3. The method for synthesizing lacto-N-tetraose according to claim 1, wherein the β-galactosidase is β1,3-galactosidase derived from Bacillus circulans expressed in Escherichia coli. 4. The method for synthesizing lacto-N-tetraose according to claim 1, wherein the β-galactosidase is β-galactosidase derived from mammalian testis. 5. Use of partially purified β-1,3-N-acetylglucosaminyltransferase and β-galactosidase for the synthesis of lacto-N-tetraose. 6. The method according to claim 5, wherein the crudely purified β-1,3-N-acetylglucosaminyltransferase is derived from bovine. 7. The method according to claim 5, wherein the β-galactosidase is β1,3-galactosidase derived from Bacillus circulans expressed in Escherichia coli.