JP2829810B2 - Carbohydrate hydrolases - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel exo-type carbohydrate hydrolase useful for analyzing the structure of carbohydrate.

[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 have been used to study the structure and function of sugar chains, releasing disaccharides from the non-reducing end of sugar chains. As enzymes, β-amylase which releases maltose from starch and cellobiase which releases cellobiose from cellulose are known. The non-reducing end of the sugar chain often contains Galβ1-3GlcNAc of type 1
Two types of β1-structure and type 2 Galβ1-4GlcNAcβ1-structure are 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. As a method for estimating the structure using a trace amount of a sugar chain sample, Streptococcus 6646K that cuts type 1 and 2 sugar chains or β-galactosidase of common bean, and Diprococcus pneumoniae that cuts only type 2 sugar chain ( Diprococcus pn
eumoniae) -β-galactosidase is also known, but Streptococcus 6646K and bean β-galactosidase all hydrolyze the type 2 sugar chain more quickly, so that the type 1 sugar chain is completely converted. It is difficult to set the reaction conditions, such as a long time reaction with a large amount of enzyme for hydrolysis, and it is actually difficult to distinguish type 1 and type 2 sugar chain structures.
It has been required to provide an enzyme that specifically acts on the hetero-oligosaccharide. The present inventors have found an exo-type saccharide hydrolase that specifically acts on a type 1 sugar chain structure and hydrolyzes a lacto-N-bioside bond in the sugar chain to release lacto-N-biose. The enzyme was named lacto-N-biosidase [Proceedings of the National Academy of Sciences of the
USA (Proceedings of the National Academy of Sci.
ences of the USA), Vol. 89, pp. 8512 (1
992)].

[0003]

However, the enzyme is produced intracellularly and its production is small, and under the same culture conditions, α-fucosidase is produced in a larger amount than lacto-N-biosidase. And had a problem that separation from the α-fucosidase was difficult. An object of the present invention is to provide a novel lacto-N-biosidase which is highly purified easily and is useful as a sugar chain structure analysis reagent suitable for industrial production.

[0004]

SUMMARY OF THE INVENTION In summary, the present invention relates to an exo-type saccharide hydrolase having the following physicochemical properties. (1) Action It acts on a sugar chain represented by the following formula (Formula 1) to cause lacto-N-
Hydrolyzes only biosidic bonds.

[0005]

Embedded image Galβ1-3GlcNAcβ1-R

(R represents a sugar residue) (2) Substrate specificity Acting on a lacto-N-bioside bond, Galβ1-3GlcN
Ac is released, but an N-acetyllactosaminide bond (G
alβ1-4GlcNAcβ1-R). It acts on p-nitrophenyl-β-lacto-N-bioside to produce G
Releases alβ1-3GlcNAc. (3) Optimum pH: around 5.5 (4) Optimum temperature: around 60 ° C. (5) Molecular weight: about 6 × 10 ⁴ (by SDS-polyacrylamide gel electrophoresis)

[0007] The sugar chain represented by the formula (Formula 1) has a lacto-N-biose structure (Galβ1-3) at its non-reducing end.
GlcNAcβ1), and the sugar residue represented by R is, for example, a residue of a saccharide such as a monosaccharide, an oligosaccharide, a polysaccharide, or an oligosaccharide of a glycoprotein or a glycolipid.

In view of the above situation, the present inventors have been searching for a lacto-N-biosiside bond-degrading enzyme suitable for industrial production, and found that certain actinomycetes are specific for lacto-N-bioside bond. The present inventors have found that a novel exo-type saccharide hydrolase is mainly produced extracellularly, 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 lacto-N-biosidase-producing ability, or may be a mutant of these strains. Lact-N- of the present invention
Specific examples of the strain having biosidase-producing ability include, for example, Streptomyces SP142. This strain is designated as Streptomyces sp142, and the Ministry of International Trade and Industry has
Deposited as FERM BP-4569 .

The lacto-N-biosidase of 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. When culturing,
A usual method for culturing microorganisms is used. The nutrient source to be added to the medium may be any one that utilizes the present strain and produces lacto-N-biosidase. Examples of the carbon source include glycerol, glucose, galactose, maltose,
Lactose, fucose, mucin 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. The addition of mucin-type glycoproteins such as gastric mucin and egg white mucin is effective for inducing the enzyme of the present invention. In addition, inorganic and metal salts such as phosphates, potassium salts, magnesium salts, and zinc salts may be added. In culturing the lacto-N-biosidase-producing bacterium of the present invention, the amount of production greatly varies depending on the culture conditions.
In general, the culturing temperature is preferably 20 to 35 ° C., and the pH of the medium is preferably 5 to 8, and the lacto-N-biosidase according to the present invention is produced by aeration and agitation culturing for 1 to 7 days. The culturing conditions are determined according to the strain used, the composition of the medium, etc.
It is natural that the biosidase production is set to be maximized.

The lacto-N-biosidase of the present invention produced by the above-mentioned actinomycete is secreted mainly outside the cells, for example, when porcine stomach mucin is added to a culture medium and cultured, and other exoglycosidases, especially Since almost no α-fucosidase is produced, purification can be performed more easily than the enzyme described in the above-mentioned Proceedings of the National Academy of Sun Enzyes of the USA, which is produced in the cells. That is, the culture solution is subjected to solid-liquid separation, and a purified enzyme preparation can be obtained from the obtained supernatant by a commonly used purification means. For example, purification is performed by salting out, organic solvent precipitation, ion exchange column chromatography, hydrophobic binding column chromatography, hydroxyapatite column chromatography, gel filtration chromatography, lyophilization and the like. A single purified enzyme preparation can be obtained.

The lacto-N-biosidase obtained by the present invention has the following enzymatic and physicochemical properties. (1) Action It acts on a lacto-N-bioside bond to release lacto-N-biose. (2) Substrate specificity This enzyme is derived from human milk-derived lacto-N-tetraose (Gal
acts on β1-3GlcNAc (β1-3Galβ1-4Glc) to release lacto-N-biose from its non-reducing end, but lacto-N-neotetraose (Galβ1-4GlcNAc) derived from human milk
It does not act on β1-3Galβ1-4Glc). Further, the following formula having a lacto-N-bioside bond derived from fetuin, a bovine fetal serum protein:

[0013]

Embedded image

(Hereinafter, G is Gal, GN is GlcNAc, M
Acts on the complex type sugar chain represented by the formula (1) to release lacto-N-biose, but does not act on the complex type sugar chain having no lacto-N-bioside bond. That is,
This 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 optimum pH of the enzyme is determined by the pH as shown by the curve in FIG.
It has a high activity around 5.5. FIG. 2 shows the pH stability when this enzyme was treated at 4 ° C. at each pH for 16 hours. As is clear from FIG. 2, the present enzyme is stable between pH 4.0 and 10.0. FIG. 1 shows the pH of lacto-N-biosidase obtained by the present invention.
A graph showing the relationship between (horizontal axis) and relative activity (%, vertical axis),
FIG. 2 is a graph showing the relationship between pH (horizontal axis) and residual activity (%, vertical axis). In FIG. 2, white circles indicate acetic acid-hydrochloric acid, black circles indicate sodium acetate, and white triangles indicate sodium citrate. ,
The black triangles were measured using sodium phosphate, the white squares were measured using sodium glycine, and the black squares were measured using sodium carbonate. (4) Optimum temperature and thermostability The optimal temperature of action of the present enzyme is 6 as shown by the curve in FIG.
It is around 0 ° C. FIG. 3 is a graph showing the relationship between the relative activity (%, vertical axis) and the reaction temperature (° C., horizontal axis) of the lacto-N-biosidase of the present invention. In addition, as shown in FIG. 4, the present enzyme is stable at 45 ° C. for at least 0.5 hour and at 4 ° C. for at least 6 months. FIG. 4
Is a graph showing the relationship between the residual activity (%, vertical axis) and the processing temperature (° C., horizontal axis) after treating the lacto-N-biosidase of the present invention at each temperature for 0.5 hour. (5) Molecular weight The molecular weight is about 6 × 10 ⁴ (by SDS-polyacrylamide gel electrophoresis).

(6) Measurement of enzyme activity The measurement of lacto-N-biosidase activity was determined as follows. As a substrate, the following formula (Formula 3):

[0017]

Embedded image Galβ1-3GlcNAcβ1-pNP

In the following, a sugar chain having a structure represented by the following formula: pNP means p-nitrophenyl group was used. 35 mM potassium phosphate buffer containing 1 mM of this substrate, pH 6.0
, And reacted at 37 ° C. for 20 minutes in a final volume of 0.1 ml. The reaction was stopped by adding a 1 M sodium carbonate solution, and the absorbance at 405 nm was measured. Under these conditions, the amount of enzyme that produces 1 μmol of p-nitrophenol per minute was defined as one unit.

The following items can be elucidated by using the lacto-N-biosidase of the present invention. (1) The role of lacto-N-biosyl residue in glycoconjugates can be known. (2) The use of the present enzyme makes it possible to directly estimate the presence or absence of a lacto-N-biosyl group in a glycoconjugate, and to directly discriminate type 1 sugar chains from type 2 sugar chains. (3) The reducing end of the sugar chain is previously reduced by a pyridyl amination method [Journal of Biochemistry (Journal of
Biochemistry), 95, 197-203 (19
84)], and using the above-mentioned enzyme digestion method and two-dimensional sugar chain map method (Analytical Biochemistry, Vol. 171,
73 (1988)], 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) Using a sugar chain labeled with [ ³ H] or an unlabeled sugar chain at the reducing end, the enzyme is enzymatically treated with the present enzyme, and the enzyme digest is analyzed by gel filtration chromatography or ion exchange chromatography. Thereby, the sugar chain structure can be estimated.

[0020]

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

Example 1 (1) Culture of bacteria and preparation of culture supernatant and cell-free extract Streptomyces sp 142 (FERM)
BP-4569) containing 0.3% of peptone, 0.01% of yeast extract, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate heptahydrate and 1% of swine stomach mucin 50
25 to 27 ° C. using 0 ml of a liquid medium (pH 7.0)
After culture for 2 days, the culture solution was centrifuged to obtain a culture supernatant and cells. 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. The activity of N-lacto-biosidase produced in the supernatant, ie, extracellularly, was 2.4 mU / ml culture, and the activity of α-fucosidase was 0.01 mU / ml culture. Also,
The activity of the cell-free extract, ie, the N-lacto-biosidase produced in the cells, was 0.5 mU / ml culture, α
-Fucosidase activity was 0.002 mU / ml culture. The activity of α-fucosidase was determined by the journal.
Of Biological Chemistry (Journal
of Biological Chemistr
y), vol. 267, p. 1522 (1992).

(2) Preparation of enzyme 500 ml of the culture supernatant obtained above was mixed with 0.5 mM phenylmethanesulfonyl fluoride and 0.05% bridge (Bri).
j) 50 mM potassium phosphate buffer pH 7.5 containing 58
After dialysis, the mixture was applied to a column of Q-Sepharose (2.5 × 18 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 contains 0.1 mM phenylmethanesulfonyl fluoride and 0.05% bridge 58.
After dialysis against 0 mM sodium acetate buffer pH 5.5,
A column of S-Sepharose equilibrated with the same buffer (1.
5 × 8.5 cm). The column was washed with three volumes of the same buffer and eluted with a NaCl gradient from 0-0.4M. The active fraction was treated with 50% sodium acetate buffer pH 5.5 containing 0.05% bridge 58.
After dialysis, the mixture was applied to a Mono-S column (0.5 × 5.0 cm) equilibrated with the same buffer. Elution is 0-0.5
Performed with a concentration gradient of NaCl up to M. 50 mM potassium phosphate buffer pH 6.8 containing 0.05% bridge 58
After changing the buffer over a P6DG column which had been equilibrated in advance, the mixture was applied to a hydroxyapatite column (0.6 × 4.0 cm) equilibrated with the same buffer. Elution was performed by linearly increasing the concentration of the potassium phosphate buffer from 50 mM to 500 mM. The active fraction was concentrated by ultrafiltration (molecular weight cut off: 10,000) and then 0.05% bridge 58 and 0.2%.
50 mM sodium acetate buffer with M NaCl pH
The mixture was applied to a column (1.5 × 90 cm) of Toyopearl HW-55S equilibrated in 5.5. Elution was carried out with the same buffer, and the active fraction was collected to obtain lacto-N-biosidase of the present invention. The specific activity of the lacto-N-biosidase thus obtained is 8,000 milliunits / mg, and the enzyme produced in the cells (Proceedings of the National Academy of Sciences of the U.
The enzyme preparation was much more purified than the specific activity of SA), 684 milliunits / 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 Pyridylamined Sugar Chain As a substrate, the following formula (Formula 4):

[0024]

Embedded image

The enzyme obtained in Example 1 was reacted with a pyridylaminated sugar chain (manufactured by Takara Shuzo) having a structure represented by the following formula: 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.

[0026]

Embedded image

It was confirmed that a pyridylaminated sugar chain having a structure represented by the following formula was generated. The reaction solution was further concentrated to dryness,
Carbohydrate Pyridyl Amination System / Pulsation (PALSTA
TION: manufactured by Takara Shuzo Co., Ltd.)
HPLC analysis was performed to obtain the compound represented by the formula (5) and the following formula (6):

[0028]

Embedded image G (β1-3) GN-PA

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

[0030]

Industrial Applicability According to the present invention, a novel lacto-N- useful for elucidating the structure and function of a complex sugar chain and suitable for industrial production.
Biosidase was provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between pH and activity of lacto-N-biosidase obtained by the present invention.

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

FIG. 3 is a graph showing the relationship between the relative activity (%, vertical axis) of lacto-N-biosidase of the present invention and the reaction temperature (° C., horizontal axis).

FIG. 4 shows the residual activity (%, vertical axis) after treating the lacto-N-biosidase of the present invention for 0.5 hour at each temperature.
6 is a graph showing the relationship between the temperature and the processing temperature (° C., horizontal axis).

Continuation of front page (56) References Proc. Natl. Acad. Sc i. USA 89 p. 8512-8516 (1992) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 9/24 C12N 9/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] An exo-type saccharide hydrolase having the following physicochemical properties: (1) Action It acts on a sugar chain represented by the following formula (Formula 1) to cause lacto-N-
Hydrolyzes only biosidic bonds. ## STR1 ## Galβ1-3GlcNAcβ1-R (R represents a sugar residue) (2) Substrate Specificity Galβ1-3GlcN acts on a lacto-N-bioside bond.
Ac is released, but an N-acetyllactosaminide bond (G
alβ1-4GlcNAcβ1-R). It acts on p-nitrophenyl-β-lacto-N-bioside to produce G
Releases alβ1-3GlcNAc. (3) Optimum pH: around 5.5 (4) Optimum temperature: around 60 ° C. (5) Molecular weight: about 6 × 10 ⁴ (by SDS-polyacrylamide gel electrophoresis)