JPH06253878A - Production of lacto-n-biose i and 2-acetamide-2-deoxy-3-o-beta-d-galactopyranosyl-d-galactopyranose - Google Patents

Abstract

PURPOSE:To easily produce lacto-N-biose I and 2-acetamide-2-deoxy-3O-beta-D- galactopyranosyl-D-galactopyranose existing in the sugar chain of human milk oligosaccharide and complex carbohydrate and utilizable as food additives, pharmaceuticals, reagents, etc., at a low production cost. CONSTITUTION:A substrate containing lactose and N-acetylglucosamine or N-acetylgalactosamine is treated with a beta-galactosidase originated from porcine testes to effect galactose rearrangement reaction. After the reaction, the product is made to react with a beta-galactosidase produced by Bacillus circulans and the isomer produced as a by-product and the unreacted lactose are hydrolyzed.

Description

Detailed Description of the Invention

[0001]

The present invention relates to lacto- N -biose I and 2-acetamide which are contained in sugar chains such as human milk oligosaccharides and complex sugars and can be used as food additives, pharmaceuticals, reagents and the like. It relates to a method for producing 2-deoxy-3- O- β-D-galactopyranosyl-D-galactopyranose.

[0002]

BACKGROUND OF THE INVENTION lacto - N - biose I (Lacto- N -bios
e I, Galβ1-3GlcNAc) is an oligosaccharide in which galactose and N -acetylglucosamine are linked by β-1,3, and is 2-acetamido-2-deoxy-3- O- β-D.
-Galactopyranosyl-D-galactopyranose (Gal
β1-3GalNAc) is an oligosaccharide in which galactose and N -acetylgalactosamine are linked by β-1,3. In the following description, lacto - N - a biose I abbreviated as "Lacto- N -biose I", 2-acetamido -2
-Deoxy-3- O- β-D-galactopyranosyl-D
-Galactopyranose is abbreviated as "Gal β1-3GalNAc".

Lacto- N -biose I and Gal β1-3GalNAc
Is a biochemically important oligosaccharide that is present in the sugar chains of human milk oligosaccharides, blood group glycoproteins, lipopolysaccharides, various glycoproteins and glycolipids, and is used as a functional sugar in the food industry. In addition to the above use, it can also be used as a drug or a reagent such as an enzyme, a substrate or inhibitor of a lectin, or a sugar constituting a living body.

Conventionally, Lacto- N- biose I and Gal β1-3Gal
NAc is produced by chemical synthesis. In recent years, as a simple method, lactose and N -acetylglucosamine or N -acetylgalactosamine are used as substrates, and bovine testes (Bovine testes) and rat mammary glands (Rat mammary glas) are used.
nds) The β-galactosidase of the origin is subjected to a transfer reaction, and then Escherichia coli ( Escherichia coli )
A production method of hydrolyzing isomers of by-products by β-galactosidase produced by A.
rini, A. et al. Biol. Chem. 1955, 220, 71. Hedbys, L. e
t al. Glycoconjugate J.1989,6,161.).

[0005]

However, in the above-mentioned simple method, the bovine testes and rat mammary glands used as the enzyme source of β-galactosidase are both rare and therefore expensive and difficult to obtain.

Therefore, an object of the present invention is to provide Lacto- N- biose I and Gal β1-3GalNAc which are simple and inexpensive to manufacture.
To provide a manufacturing method of.

[0007]

[Means for Solving the Problems] In order to achieve the above-mentioned objects, the present inventors have conducted extensive studies, and as a result, are inexpensive, and are available in large quantities. Lactose and N -acetylglucosamine or N -acetylgalactosamine as a substrate, Lacto- N- biose I or Gal β1-3GalNAc can be efficiently produced, and inexpensive Bacillus circulans commercially available ( Bacillus circulans ) Β- produced by
It was found that galactosidase can selectively hydrolyze the by-product isomer and unreacted lactose, and completed the present invention.

That is, one of the present invention is that a substrate containing lactose and N -acetylglucosamine is reacted with β-galactosidase derived from porcine testes to carry out a galactose transfer reaction, By-products such as isomers and unreacted lactose
Lacto- characterized by being hydrolyzed by β-galactosidase produced by circulans ( Bacillus circulans )
N- biose I manufacturing method.

Another aspect of the present invention is a substrate containing lactose and N -acetylgalactosamine,
After the galactose transfer reaction by acting β-galactosidase of porcine testes (Porcine testes) origin, isomers and unreacted lactose by-products, β produced by Bacillus circulans −
Gal characterized by being hydrolyzed by galactosidase
This is a method for producing β1-3GalNAc.

The present invention will be described in more detail below.

The β-galactosidase derived from the porcine testes of the present invention is, for example, Porcine t
(estes) extract, add solid ammonium sulfate to 20% saturation and remove the precipitate by centrifugation, then add solid ammonium sulfate to 75% saturation, salt out, and use the crude enzyme obtained as precipitate be able to.

[0012] In addition, Bacillus circulans (Bacillu
β-galactosidase produced by s circulans ) is a crude enzyme obtained by adding solid ammonium sulfate to a culture solution in which a microorganism is cultivated so as to be 75% saturated, dissolving, and salting out,
Alternatively, a commercially available enzyme such as "BIOLACTA" (trade name, manufactured by Daiwa Kasei Co., Ltd.) can be used.

The blending ratio of lactose used in the galactose transfer reaction and N -acetylglucosamine or N -acetylgalactosamine is preferably 5: 1 to 1:10 in molar ratio, and all the substrates in the reaction solution are mixed. Concentration 5-40
It is preferable to carry out the reaction as a weight percentage. Further, β-galactosidase derived from porcine testes is preferably added to the reaction system at 0.03 to 0.3 U / ml. The galactose transfer reaction has a pH of 3.5 to 6,
It is preferable to carry out the reaction at a temperature of 30 to 50 ° C and a reaction time of 50 to 15
0 hours is preferred.

Then, the reaction solution is heated to stop the reaction, and then diluted with a phosphate buffer or the like, preferably about 10 times, to obtain Bacillus circulan.
The β-galactosidase produced by s ) is added to the reaction system preferably at 0.50-2 U / ml to hydrolyze the isomer produced as a by-product in the above reaction. This hydrolysis reaction is preferably carried out at a pH of 5 to 8 and a temperature of 30 to 50 ° C., and the reaction time is preferably 2 to 5 hours.

The method for measuring the enzyme activity of β-galactosidase in the present invention was carried out as follows. That is, 0.2 ml of 10 mM ONP-Gal (ortho-nitrophenylgalactoside) solution and 0.1 M citrate buffer solution (pH
4.3) 0.5 ml or 0.1 M phosphate buffer (pH 7.0) 0.5
ml and deionized water 0.2 ml are mixed, 0.1 ml of enzyme solution diluted to an appropriate concentration is added, the reaction is carried out at 40 ° C., the reaction is stopped by adding 0.5 ml of 1M sodium carbonate, and then ortho-nitrophenol is added. Absorbance at 420 nm was measured. Enzyme activity 1 U was defined as the amount of enzyme that liberates 1 μmol of ortho-nitrophenol in 1 minute.

In the transfer reaction of the present invention, lactose and
When N -acetylglucosamine is used as a substrate, La
When cto- N- biose I is produced and lactose and N -acetylgalactosamine are used as substrates, Gal β1-3GalNA
generated by c.

The reaction solution obtained as described above is the Lact formed after the reaction is stopped by boiling for 10 minutes, for example.
O- N- biose I or Gal β1-3GalNAc can be purified by a combination of means such as activated carbon column chromatography, gel filtration, high performance liquid chromatography and the like, if necessary.

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, in the galactose transfer reaction using lactose and N -acetylglucosamine or N -acetylgalactosamine as substrates, β-galactosidase derived from Porcine testes, which is inexpensive and available in large quantities, is available. Is used as an enzyme, and when hydrolyzing an isomer which is a by-product and unreacted lactose, it is commercially available at a low price, Bacillus circulans ( Bacillus
circulans ) produced β-galactosidase, Lacto- N- biose I and Gal β1-3GalNAc,
It can be manufactured inexpensively by a simple method.

[0019]

【Example】

Example 1 20 g of lactose and 5 g of N -acetylglucosamine (molar ratio 2: 1) were dissolved in a 50 mM citrate buffer solution (pH 4.3) to prepare a 100 ml solution. To this solution, add the testicles of Porcine (Porcine
testes) β-galactosidase of 14 U was added, and
The reaction was performed at 100 ° C for 100 hours, and the reaction was stopped by boiling for 10 minutes. As a result of analysis by high performance liquid chromatography, Lacto- N- biose I and N -acetyllactosamine (Gal β1-4GlcNAc) were obtained as main products. The analysis conditions for high performance liquid chromatography are as follows.

Column: Asahipak NH2P-50 (trade name, manufactured by Asahi Kasei Corporation) (4.6 x 250 mm) Mobile phase: acetonitrile: water = 75:25 Flow rate: 1 ml / min Temperature: 40 ° C Detection: UV 210nm sample: 10μl

Then, this reaction solution was mixed with 50 mM phosphate buffer (p
Dilute 10 times with H7.0), add 950 U of β-galactosidase produced by Bacillus circulans , react at 40 ° C for 3.5 hours, and stop the reaction by boiling for 10 minutes. did. By this reaction, the substrate lactose and N -acetyllactosamine (Galβ1
-4GlcNAc), N -acetyl allolactosamine (Gal β1
-6GlcNAc) was selectively hydrolyzed.

The reaction solution thus obtained was applied to an activated carbon column (2.4 × 96 cm) equilibrated with demineralized water. After washing the non-adsorbed part with demineralized water, 3% ethanol solution 16 L (liter)
The substrate, N -acetylglucosamine, was eluted with the following procedure, and then the target product, Lacto- N- biose I, was eluted and separated by a linear concentration gradient of a 3 to 20% ethanol solution. Further, this fraction was concentrated and then crystallized with methanol. As a result, 536 mg of Lacto- N- biose I was produced by this reaction.

Example 2 20 g of lactose and 5 g of N -acetylgalactosamine (molar ratio 2: 1) were dissolved in a 50 mM citrate buffer (pH 4.3) to prepare a 100 ml solution. To this solution, add the testicles of Porcine (Porcine
testes) β-galactosidase of 14 U was added, and
The reaction was carried out at 100 ° C for 100 hours. After stopping the reaction by boiling for 10 minutes, the reaction solution was added to 50 mM phosphate buffer (pH 7.0).
) With Bacillus circulans ( Ba
β-galactosidase produced by cillus circulans )
After adding 950 U, the reaction was carried out at 40 ° C. for 3.5 hours. After the reaction was stopped by boiling for 10 minutes, the obtained reaction solution was subjected to an activated carbon column under the same conditions as in Example 1 to give Gal β1-3Gal.
NAc was separated and purified. As a result, this reaction caused 444 mg.
Gal β1-3GalNAc was produced as a crystal.

[0024]

As described above, according to the present invention,
Lacto- N- biose I and Gal are oligosaccharides that are extremely important in biochemistry and are contained in the sugar chains of human milk oligosaccharides, blood group glycoproteins, lipopolysaccharides, various glycoproteins and glycolipids.
β1-3GalNAc can be produced inexpensively by a simple method. These oligosaccharides can be used as food additives, pharmaceuticals, reagents and the like.

Claims (2)

[Claims] 1. An isomer which is a by-product after a β-galactosidase derived from porcine testes (Porcine testes) is allowed to act on a substrate containing lactose and N -acetylglucosamine to cause a galactose transfer reaction. and lactose of unreacted, Bacillus circulans (Bacillus ci
rculans ) -produced lacto- N -biose I, which comprises hydrolyzing with β-galactosidase. 2. An isomer which is a by-product after a β-galactosidase derived from porcine testes is allowed to act on a substrate containing lactose and N -acetylgalactosamine to cause a galactose transfer reaction. and lactose of unreacted, Bacillus circulans (Bacillus
circulans ) -producing β-galactosidase for hydrolysis of 2-acetamido-2-deoxy-3- O- β-D-galactopyranosyl-D-galactopyranose.