JP2854541B2 - Hydrolysis method of sugar compounds - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for hydrolyzing sugar compounds and a reagent for hydrolyzing sugar compounds, which are useful for analyzing the structure of sugar chains.

[0002]

2. Description of the Related Art In recent years, the structure and function of sugar chains in complex carbohydrates such as glycoproteins and glycolipids derived from higher animals have been studied. Plays an important role. Conventionally, enzymes that release monosaccharides from the non-reducing end of sugar chains have been isolated from various sources and used to study the structure and function of sugar chains. Known enzymes include β-amylase, which releases maltose from starch, and cellobiase, which releases cellobiose from cellulose. However, there is no known enzyme that acts preferentially on hetero-oligosaccharides.

[0003]

At the non-reducing end of the sugar chain, two types of Galβ1-3GlcNAcβ-type 1 type and Galβ1-4GlcNAcβ-type 2 type are often found. Conventionally, analysis methods such as methylation analysis and nuclear magnetic resonance spectroscopy have been used to analyze the sugar chain structure, but have the disadvantage that a large amount of sample must be used. Further, as a method for estimating the structure using a trace amount of a sugar chain sample, Streptococcus 6646K for cleaving type 1 and 2 sugar chains, or β-galactosidase of common bean, and Diprococcus pneumoniae for cleaving only type 2 sugar chain (Diplococcus pneu
Moniae) is known to be used in combination with β-galactosidase.However, both Streptococcus 6646K and bean β-galactosidase rapidly hydrolyze the type 2 sugar chain, and thus completely convert the type 1 sugar chain. Actually, it is difficult to distinguish the type 1 and type 2 sugar chain structures, for example, it is difficult to set the reaction conditions such as a long time reaction using a large amount of enzyme for hydrolysis. Therefore, an object of the present invention is to provide a method for hydrolyzing a sugar compound and a reagent for hydrolyzing the sugar compound, which are useful for analyzing the structure of type 1 and type 2 sugar chains that act on a hetero-oligosaccharide and release a disaccharide from a non-reducing terminal.

[0004]

SUMMARY OF THE INVENTION To summarize the present invention, a first aspect of the present invention is to provide a sugar compound represented by the following general formula (Chemical Formula 1) with a lacto-N-terminal at the non-reducing terminal of the sugar compound. -Selectively and specifically hydrolyzes only biosidic bonds ,
Ctosaminide binding (Galβ1-4GlcNAcβ1-
R) does not hydrolyze, optimal pH 5.5-6.0 and 4 ° C
At pH 4.0 when treated for 16 hours.
Lacto-N- of the sugar compound represented by the general formula (Chemical Formula 1) , wherein the glycosidase of 7.0 is acted on.
The present invention relates to a method for hydrolyzing biosidic bonds.

[0005]

Embedded image Galβ1-3GlcNAcβ1-R

(In the formula, R represents a sugar residue.) In a second aspect of the present invention, only the lacto-N-bioside bond at the non-reducing end of the sugar compound represented by the general formula (Formula 1) is used. Selectively and specifically hydrolyzes, but N-acetyllactosa
The minide bond (Galβ1-4GlcNAcβ1-R)
Does not hydrolyze, optimal pH is 5.5-6.0 and at 4 ° C
PH stability when treated for 16 hours at 4.0-7.0
The present invention relates to a reagent for hydrolyzing a lacto-N-bioside bond of a sugar compound represented by the general formula (Chemical Formula 1), which comprises a certain glycosidase.

In view of the above situation, the present inventors have been searching for a lacto-N-bioside bond-degrading enzyme, and found that certain actinomycetes are specific glycosidases that act only on the lacto-N-bioside bond, ie, exo. The present inventors have found that a type of carbohydrate hydrolase is produced, and have reached the present invention.

Hereinafter, the present invention will be described in detail. The strain used in the present invention may be any strain as long as it has a specific glycosidase-producing ability acting only on the lacto-N-bioside bond, or may be a mutant of these strains. Specific examples of the above-mentioned strain having the ability to produce glycosidase include, for example, Streptomyces S.
P142. This strain is Streptomyces sp 1
No. 42 and deposited at the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry as FERM BP-4569, and its bacteriological properties are as follows.
No. 98583.

(1) Morphological properties The basic mycelia elongate and branch well, but usually do not break. Spores are well formed on oatmeal agar and yeast / malt agar. Observation with a microscope shows that the branching method of the aerial hyphae is simple branching, and no ring-shaped branching is observed. The shape of the aerial mycelium tip is a straight, hooked shape, and rarely a loop shape. The spores usually have 10 to 20 chains, and the surface is smooth. The spores are cylindrical or elliptical, and their size is 0.7-0.8 × 0.9-
1.0 μm. No spores, motile spores, sclerotia, etc. are observed.

(2) Growth on various media Table 1 below shows the results of observation with the naked eye when cultured on various media at 27 ° C for 14 days.

[0011]

[Table 1]

(3) Physiological properties (a) Growth temperature range (yeast / malt agar medium): 10-3
It grows in a temperature range of 7 ° C and grows well at 25 to 30 ° C. (B) Gelatin liquefaction: positive (c) Starch hydrolysis: positive (d) Peptone conversion of skim milk: positive Coagulation of skim milk: negative (e) Melanin-like pigment formation: negative

(4) Utilization of carbon source (cultured on Pridham Gottlieb agar medium at 27 ° C. for 14 days) (a) Utilization: D-glucose, D-fructose,
L-arabinose, D-xylose, L-rhamnose,
D-mannitol, raffinose, L-fucose (b) Not used: inositol, sucrose

From the above properties, this strain belongs to the genus Streptomyces among actinomycetes, and the color of aerial hyphae is "white".
~ "Grey" series, aerial hyphae are straight or key-shaped, spore surface is smooth, and color on the back of colony is white ~
It is summarized as a light brown strain that does not produce melanin-like pigments.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The glycosidase used in the present invention can be obtained, for example, by culturing the above-mentioned bacteria in a nutrient medium and separating the enzyme from the culture. For the culturing, an ordinary method for culturing a microorganism is used. The nutrient source added to the medium may be any one that utilizes the present strain and produces the glycosidase used in the present invention.Examples of the carbon source include glycerol, glucose, galactose,
Maltose, lactose, fucose and the like can be used, and as a nitrogen source, yeast extract, peptone, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like are suitable. In addition, phosphates, potassium salts,
Inorganic and metal salts such as magnesium salts and zinc salts may be added. In culturing the glycosidase-producing bacteria used in the present invention, the amount of production varies greatly depending on the culturing conditions. Generally, the culturing temperature is 20 to 35 ° C.
It is preferable to use 5-8, and aeration and agitation culture for 1-7 days produce glycosidase which can be used in the present invention.
Naturally, the culturing conditions are set so as to maximize the production of glycosidase according to the strain used, the composition of the medium, and the like.

As an example of the glycosidase used in the present invention, exolacto-N produced by the above-mentioned actinomycetes is used.
-Since biohydrolase is mainly present in the cells, the culture is subjected to solid-liquid separation, and the obtained wet cells are subjected to sonication, French press, Dynomill and other various cell disruption means. Destruction of the body or lysis of the cell wall using a cell wall lysing enzyme such as lysozyme gives a cell-free extract. Next, a purified enzyme preparation can be obtained from the extract by a commonly used purification means. For example, salting out, organic solvent precipitation, ion exchange column chromatography, hydrophobic binding column chromatography, gel filtration,
Purification is performed by lyophilization or the like, and a single purified enzyme preparation can be obtained by polyacrylamide gel disk electrophoresis.

The enzymatic and physicochemical properties of the above exolacto-N-biohydrolase are as follows. (1) action: acting on a lacto-N-bioside bond,
Releases lacto-N-biose. (2) Substrate specificity: This enzyme is derived from human milk-derived lacto-N-
Tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4
Glc) to release lacto-N-biose from its non-reducing end, but to obtain lacto-N-neotetraose derived from human milk (Galβ1-4GlcNAcβ1-3Galβ1-4Glc).
Does not work on). Further, the following formula having a lacto-N-bioside bond derived from fetuin, a bovine fetal serum protein:

[0018]

Embedded image

[Hereinafter, G is Gal, GN is GlcNAc,
M represents Man), and liberates lacto-N-biose, but does not act on conjugated sugar chains having no lacto-N-bioside bond. That is, the enzyme is specific for lacto-N-bioside linkage and releases lacto-N-biose from the non-reducing end of the sugar chain. (3) Optimum pH and pH stability: The enzyme has high activity at the optimum pH around 5.5 to 6.0 as shown by the curve in FIG. The pH stability when this enzyme was treated at 4 ° C. for 16 hours at each pH is shown in FIG. As is clear from FIG.
It is stable between 7.0. FIG. 1 is a graph showing the relationship between the pH (horizontal axis) and the relative activity (%, vertical axis) of exolacto-N-biohydrolase of the present invention, and FIG. It is a graph showing the relationship between pH (horizontal axis) and residual activity (%, vertical axis) after treatment at each pH for 16 hours. (4) Optimum temperature: The optimum temperature for the action of the present enzyme is 40 to 55 ° C. as shown by the curve in FIG. FIG. 3 shows the temperature of the exolacto-N-biohydrolase of the present invention (° C.,
It is a graph showing the relationship between relative activity (%, vertical axis) and relative activity (%, vertical axis). (5) Molecular weight: The molecular weight is about 29000 (by a gel filtration method using Toyopearl HW-55S). (6) Measurement of enzyme activity: Measurement of exolacto-N-biohydrolase activity was determined as follows. As a substrate, the following formula (Formula 3)

[0020]

Embedded image Galβ1-3GlcNAcβ1-3 Galβ1-4Glc-PA

A pyridyl aminated sugar chain (manufactured by Takara Shuzo Co., Ltd.) having a structure represented by the following formula: [where PA represents a pyridyl- (2) -amino group] was used. This substrate and the enzymatic reaction product from which lacto-N-biose has been released have a reducing end labeled with a pyridylamino group, so they can be directly qualitatively determined by reversed-phase or amine-based high-performance liquid chromatography equipped with a fluorescence detector. Analysis is possible. Substrate 2 above
6 μl of the enzyme solution was added to 4 μl of a 250 mM potassium phosphate buffer containing 0 pmol, mixed and reacted at 37 ° C. for 20 minutes, and the reaction was stopped by adding 40 μl of a 1% trifluoroacetic acid solution. It was subjected to chromatography. Under these conditions, the amount of the enzyme that produces 1 μmol of pyridylaminated lactose per minute is defined as one unit.

The following items can be clarified by using the present invention. (1) The role of lacto-N-biosyl residue in glycoconjugates can be known. (2) By using this method, the presence or absence of a lacto-N-biosyl group in a glycoconjugate can be directly estimated, and the type 1 sugar chain and the type 2 sugar chain can be directly discriminated. (3) The reducing end of the sugar chain is previously reduced by pyridyl amination [Journal of Biochemistry (J. Bioc.
hem.), 95, 197-203 (1984)]
The above-mentioned enzyme digestion method and 2
Dimensional glycan map method [Analytical Biochem., Vol. 171, p. 73 (198)
8)], it is possible to estimate the entire sugar chain structure including the lacto-N-biosyl group with a sensitivity several hundred times higher than the conventional one. (4) Enzymatic digestion with the present enzyme using a sugar chain labeled with [ ³ H] or an unlabeled sugar chain at the reducing end, and analyzing the enzyme digest by gel filtration chromatography or ion exchange chromatography. Thereby, the sugar chain structure can be estimated.

[0023]

EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited only to the scope of the following examples.

Example 1 (1) Culture of bacteria and preparation of cell-free extract Streptomyces sp 142 (FERM BP-4569)
Of peptone 0.3%, yeast extract 0.05%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.
After culturing at 25 to 27 ° C. for 2 days using 500 ml of a liquid medium (pH 7.0) containing 05% and 1% L-fucose, the culture was centrifuged to obtain a strain. The cells were washed with 10 mM sodium phosphate buffer containing 1 mM ethylenediaminetetraacetic acid, pH 7.0, suspended in the same buffer, sonicated, and the cell-free extract was removed by removing the cell residues by centrifugation. Obtained.

(2) Preparation of enzyme 100 ml of the cell-free extract obtained above was mixed with 10 mM ethylenediaminetetraacetic acid, 1 mM ortho-phenanthroline, and 1 mM
After dialysis against a 50 mM potassium phosphate buffer pH 6.0 containing mM phenylmethanesulfonyl fluoride, the solution was applied to a DEAE-Sepharose CL-6B column (4.0 × 23 cm) equilibrated with the same buffer. The column was washed with three volumes of the same buffer, and the eluted flow-through fractions were collected. The active fraction was concentrated by ultrafiltration (fraction molecular weight: 10,000), then ammonium sulfate was added to a final concentration of 1.5 M, 1.5 M ammonium sulfate, 10 mM ethylenediaminetetraacetic acid, 1 mM ortho-phenanthroline, and 1 mM
A column of phenyl-Sepharose CL-4B (1.0 x 6.0) equilibrated with 50 mM potassium phosphate buffer pH 6.0 containing phenylmethanesulfonyl fluoride.
0 cm). The column was washed with 3 volumes of the same buffer, then 10 mM ethylenediaminetetraacetic acid, 1 mM
The active fraction was eluted with 50 mM potassium phosphate buffer pH 6.0 containing ortho-phenanthroline and 1 mM phenylmethanesulfonyl fluoride. The active fraction was concentrated by ultrafiltration (fraction molecular weight 10,000), and then 50 mM potassium phosphate buffer containing 100 mM sodium chloride, 0.01% sodium azide, and 0.1% Brij-58 (pH 6). The sample was applied to a column (1.5 × 90 cm) of Toyopearl HW-55S equilibrated with a pH of 2.0. Elution with the same buffer,
Exolact-N-biohydrolase of the present invention was obtained from fractions 23-28. The specific activity of the thus obtained exolacto-N-biohydrolase is 3.5 m units / mg.
And was sufficiently usable as a reagent for analyzing a sugar chain structure. The specific activity of the above enzyme was measured by the above-mentioned measuring method.

Example 2 Action on Pyridyl Aminated Sugar Chain As a substrate, the following formula (Formula 4):

[0027]

Embedded image

Using the pyridylaminated sugar chain (manufactured by Takara Shuzo) having the structure represented by the following formula, the present enzyme obtained in Example 1 was allowed to act. 80 mM phosphate buffer pH with 3.3 μM substrate
6.0 was added with 50 µ units of the present enzyme, and reacted at 37 ° C for 16 hours. After completion of the reaction, the reaction solution was analyzed by HPLC, and the following formula (Formula 5) was obtained.

[0029]

Embedded image

It was confirmed that a pyridylaminated sugar chain having the structure represented by the formula was generated. Further, the reaction solution is concentrated to dryness, and a saccharide pyridyl amination device / pulsation (PA
(LSTATION: manufactured by Takara Shuzo Co., Ltd.), followed by HPLC analysis, and the following formula (Formula 6):

[0031]

Embedded image G (β1 → 3) GN-PA

The formation of a pyridylaminated sugar chain having the structure represented by the following formula was confirmed.

[0033]

Industrial Applicability According to the present invention, there are provided a method for hydrolyzing a lacto-N-bioside bond of a saccharide compound and a reagent for hydrolyzing which are useful for elucidating the function of the structure of a glycoconjugate.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the pH and the relative activity of exolacto-N-biohydrolase.

FIG. 2 is a graph showing the relationship between residual activity and pH after treating exolacto-N-biohydrolase at 4 ° C. at each pH for 16 hours.

FIG. 3 is a graph showing the relationship between temperature and relative activity of exolacto-N-biohydrolase.

Claims (2)

(57) [Claims] To 1. A sugar compound represented by the following general formula (Formula 1), although hydrolyzed selectively specifically only lacto -N- Bioshido binding non-reducing end of the sugar compound, N- acetyllactosamine
The minide bond (Galβ1-4GlcNAcβ1-R)
Does not hydrolyze, optimal pH is 5.5-6.0 and at 4 ° C
PH stability when treated for 16 hours at 4.0-7.0
A method for hydrolyzing a lacto-N-bioside bond of a sugar compound represented by the general formula (Chemical Formula 1), which comprises reacting a certain glycosidase. Embedded image Galβ1-3GlcNAcβ1-R (where R represents a sugar residue) 2. The saccharide compound represented by the general formula (Formula 1) selectively and specifically hydrolyzes only the lacto-N-bioside bond at the non-reducing terminal, but the N-acetyllactosaminide bond (Ga
lβ1-4GlcNAcβ1-R) does not hydrolyze, optimal
Treatment at pH 5.5-6.0 and 4 ° C. for 16 hours
A lacto-N-bioside of a sugar compound represented by the general formula (Chemical Formula 1), which comprises a glycosidase having a pH stability of 4.0 to 7.0 when the saccharide is prepared. Reagents for binding hydrolyses.