JPH10271991A - New saccharide hydrolase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme for synthesis, etc., of sugar chain, having action forming β1-lacto-N-bioside bond from a lactoN-bioside compound derived from a microorganism of the genus Aureobacterium and a sugar receptor and useful for analysis, etc., of sugar chain function. SOLUTION: This new exo type saccharide hydrolase has action forming β1-3 lacto-N bioside bond (Gal β1-3GlcNAc β1-3R) from a lacto-N-bioside compound derived from a microorganism belonging to the genus Aureobacterium and a sugar receptor and is useful for synthesis, etc., of saccharide having β1-3-lacto-N-bioside bond which is sugar chain necessary to elucidate function of sugar chain in sugar protein and sugar lipid. The enzyme is obtained by culturing Aureobacterium sp. L-101 (FERM P-16163), subjecting the culture solution to centrifugal separation, using supernatant as a crude enzyme solution, salting out the crude enzyme solution with ammonium sulfate, separating precipitation and subjecting the precipitation to purification by affinity chromatography with a mucin immobilization column.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel exolacto-N-biohydrolase, and to β1 using the enzyme.
The present invention relates to a method for producing a saccharide having a -3 lacto-N-bioside bond.

[0002]

2. Description of the Related Art In recent years, the importance of sugar chains in glycoproteins and glycolipids has been pointed out in many reports (Kibata Yo, Organic Synthetic Chemistry, Vol. 50, No. 5, 451-451).
463 (1992)). In order to elucidate the functions of such sugar chains, it is necessary to accurately determine the structure of the sugar chain and to synthesize the sugar chain based on the revealed sugar chain structure. come.

Heretofore, as a method for synthesizing a complex sugar chain, a chemical synthesis method has been adopted in which a sugar activated at the 1-position is reacted with an acceptor saccharide in which a hydroxyl group not involved in the reaction is completely protected. (K. Toshima and K. Tatsut
a, Chem. Rev., 93, 1503-1531 (1993)). However, since such a chemical synthesis method has many steps and is not suitable for application to industrial production, a block synthesis method for connecting disaccharide or trisaccharide oligosaccharide blocks has recently been developed. However, even in the block synthesis method, a chemical synthesis method is currently used to connect blocks. In the case of the chemical synthesis method, it is necessary to protect the hydroxyl group not involved in the bond as described above in advance,
It is extremely disadvantageous for industrial production. If the oligosaccharide blocks can be joined together enzymatically, it is expected that this would be a very useful technology in industry, and the discovery of such an enzyme has been demanded.

For example, JP-A-6-153944, JP-A-5-252946 and JP-A-8-53487 report exolacto-N-biohydrolase derived from Streptomyces genus, and more recently, By using exolacto-N-biohydrolase from the genus Streptomyces,
Galβ1-3GlcNAc (lacto-N-biose)
Is reported to be able to transfer the disaccharide block to galactose (Japanese Patent Application No. 8-70892). In this reaction, Galβ1-3GlcNAc was converted to galactose by β1
The -6 linked trisaccharide was obtained as the main product.

[0005] On the other hand, among sugar chains in natural glycoproteins or glycolipids, many sugar chains in which lacto-N-biose is linked by β1-3 bonds are found. However, in the above-described reaction using exolacto-N-biohydrolase derived from Streptomyces sp. 142 strain, lacto-N-biose could not be transferred by β1-3 bond.

[0006]

[Problems to be solved by the invention]
It has been shown that when an oligosaccharide is synthesized by a transfer reaction using a hydrolase, a bond that is easily cleaved by the enzyme is also easily formed by the transfer reaction (Hiro Fujimoto et al., Applied Glycoscience, No. 43 volumes, 265-272 (1
996)). That is, in the case of the above reaction, Galβ1-3
To transfer a disaccharide block called GlcNAc to galactose by β1-3 bond, use Galβ1-3Glc.
Glc on the reducing end side of the trisaccharide named NAcβ1-3Gal
An enzyme that hydrolyzes the NAcβ1-3Gal bond is required. The enzyme used in Japanese Patent Application No. 8-70892 is commercially available Streptomyces sp.
Although it was exolacto-N-biohydrolase from 42 strains, the binding specificity of its hydrolysis was not extremely high. Therefore, the above exolacto-N-biohydrolase binds to a highly reactive primary hydroxyl group,
It seems that the β1-6 bond was mainly generated.

[0007] In view of the above, it was necessary to search for an exolacto-N-biohydrolase that hydrolyzes a β1-3 bond with high selectivity for enzymatic synthesis of a β1-3 bond transfer product.

That is, an object of the present invention is to provide a novel exolacto-N which hydrolyzes the β1-3 bond with high selectivity.
-To provide biohydrolase. Another object of the present invention is to provide a method for producing a saccharide having a β1-3 lacto-N-bioside bond, which comprises using the above exolacto-N-biohydrolase.

[0009]

Means for Solving the Problems The present inventors have developed a lacto-
As a result of intensive search for an exolacto-N-biohydrolase that hydrolyzes an N-bioside bond, a target exolacto-N-biohydrolase was found. In addition, we have used this enzyme to make paranitrophenyl-β-D
-Lacto-N-bioside (Galβ1-3GlcNAc
When β-pNP) is used as a sugar donor for a transfer reaction, Ga
It has been found that lβ1-3GlcNAcβ1-3-linked trisaccharide can be obtained, and the present invention has been completed.

That is, according to the first aspect of the present invention, a β1-3 lacto-N-bioside bond (Galβ1-3GlcNAcβ) is formed from a lacto-N-bioside compound and a sugar acceptor.
An exo-type saccharide hydrolase having an action of forming 1-3R) is provided. According to a second aspect of the present invention, there is provided a method for producing a saccharide having a β1-3 lacto-N-bioside bond, characterized by using the above exo-type saccharide hydrolase.

[0011]

BEST MODE FOR CARRYING OUT THE 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 the ability to produce an enzyme that forms a lacto-N-bioside bond by β1-3 bond, and mutants of these strains may be used. There may be. A strain capable of producing such an enzyme can be isolated by performing screening by measuring exolacto-N-biohydrolase activity. An example of a strain capable of producing such an enzyme is Aureobacterium (Aureobacterium).
terium) L101. This strain is Aureobacte
rium, and entered the Institute of Biotechnology and Industrial Technology at the Institute of Industrial Science and Technology of the Ministry of International Trade and Industry at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture.
Deposited on March 27, 2007 under accession number FERM P-16163.

The exolacto-N-biohydrolase of the present invention can be obtained, for example, by culturing the above-mentioned bacteria in a nutrient medium and isolating the enzyme from the culture. The medium used for culturing the strain is not particularly limited as long as it contains a carbon source, a nitrogen source, an inorganic substance, and a metal salt used for culturing ordinary microorganisms. For example,
Glycerol, glucose, galactose, maltose, lactose, fucose, mucin and the like can be used as the carbon source. As the nitrogen source, yeast extract, peptone, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like can be used.
Further, phosphates, potassium salts, magnesium salts, zinc salts and the like can be used as the inorganic and metal salts.

The culturing temperature is generally 20 to 40 ° C., preferably 25 to 30 ° C., and the pH of the medium is generally 6.0 to 7.5, preferably 6.5 to 7.5. 0,
Culture can be performed by aeration and agitation culture for 1 to 5 days. The culturing conditions may vary depending on the type of the strain, the composition of the medium, and the like.
Preferably, the setting is such that the production of biohydrase is maximized, which is within the ordinary knowledge of a person skilled in the art.

This enzyme is originally an intracellular enzyme, but it can be released into the culture supernatant by adjusting the culture conditions. In this case, the present enzyme can be purified from the culture supernatant by a commonly used purification means. For example,
Purification by salting out, affinity chromatography, ion exchange chromatography, gel filtration, etc.
It can be purified to show a single band in DS electrophoresis.

The enzymatic and physicochemical properties of the exolacto-N-biohydrolase of the present invention are as follows. (1) Action: Acts on an exolacto-N-bioside bond to release lacto-N-biose. (2) Substrate specificity: lacto-N-tetraose (Gal
β1-3GlcNAcβ1-3Galβ1-4Glc)
To release lacto-N-biose and lactose. Acts on paranitrophenyl-β-D-lacto-N-bioside to release lacto-N-biose and paranitrophenol; (3) Molecular weight: about 120, as determined by SDS-PAGE.
000; (4) Optimum pH and pH stability: pH 5.5 to 6.0
It has an optimum pH and is stable between pH 6.0 and 9.0 under the condition of incubating at 30 ° C. for 1 hour. (5) Optimum temperature: The optimum temperature of action is 50 ° C. Yes, pH
30 min incubation at 5.5 is stable up to 37 ° C .; (6) Isoelectric point: 4.0-4.5; (7) Km: 4 mM (using pNP-lacto-N-bioside as substrate)

(8) Measurement of enzyme activity: Exolact-N
-The measurement of biohydrolase activity was performed as follows. 2
mM pNP-lacto-N-bioside (dissolved in 50 mM acetate buffer (pH 5.5)) as a substrate,
50 μl of the enzyme solution is added to 0 μl, and reacted at 37 ° C. for 10 minutes. After the reaction, 0.2 M sodium carbonate solution 1.0
The reaction was stopped by adding 0.1 ml of distilled water, and 2.0 ml of distilled water was further added, and the absorbance at 405 nm was measured. Under such conditions, an enzyme that produces 1 μmol of p-nitrophenol per minute was defined as one unit.

As a sugar donor in a reaction for enzymatically synthesizing an oligosaccharide having a lacto-N-bioside bond using the exo-lacto-N-biohydrolase of the present invention, lacto-N-biose is linked by a β bond. The glycoside compound may be a glycoside compound, and is not limited to the structure on the aglycone side. For example, paranitrophenyl-β-D-lacto-N-bioside, orthonitrophenyl-β-D-
Glycosides such as lacto-N-bioside or G
Galβ1-3GlcNA at the non-reducing end even of a trisaccharide such as alβ1-3GlcNAcβ1-3Gal
The c residue is transferred.

In the oligosaccharide synthesized by the above transfer reaction, a compound in which a Galβ1-3GlcNAc residue is transferred by β1-3 bond is obtained as a product.

Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to only the scope of the following examples.

[0020]

【Example】

Example 1: Screening and isolation screening media of the microorganism (mucin _{(0.5%), NH 4 NO} 3
(0.4%), KH ₂ PO ₄ (0.15%), Na ₂ HP
_{O 4 · 12H 2 O (0.15} %), MgSO 4 · 7H 2 O
_{(0.02%), FeSO 4 ·} 7H 2 O (0.0001
%), CaCl ₂ .2H ₂ O (0.001%), yeast extract (0.05%) and agar (1.5%), pH 7.0.
Apply water or soil diluent to 0)
After culturing for one day, the resulting colonies (120) were isolated. A 0.5% yeast extract activity assay solution containing 1.0 mM p-nitrophenyl (pNP) lacto-N-bioside was dispensed in 50 μl portions into a 96-well microplate, and the isolated colonies were inoculated on the microplate. The productivity of the enzyme was determined by the presence or absence of the generated yellow color. As a result, L-101, L-102, L-104, L-11
Activity was confirmed in 2 and 5 strains of L-118.

The above five strains were inoculated in a liquid medium having the same composition as the screening medium, cultured at 30 ° C. for 28 hours, and the supernatant was separated from the cells. After washing, the cells were suspended in 4 ml of an acetate buffer (pH 6.0), and 0.2 ml (1/20 volume) of toluene was added thereto. The suspension was vigorously suspended for 20 seconds, and the enzyme activity was measured. Table 1 shows the results.

[0022]

[Table 1]

As can be seen from the results shown in Table 1, the above five strains produced enzymes extracellularly and intracellularly. In addition, the L-101 strain was used in the following experiments because it was presumed to be similar from the shape of the colonies of the five strains.
The L-101 strain has the accession number FERM P-1616.
No. 3 is deposited at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.

Example 2 Examination of Culture Conditions of Microorganism (Effect of Yeast Extract on Enzyme Productivity) Mucin (1.0%), NH ₄ NO ₃ (0.4%), KH ₂
PO ₄ (0.15%), Na ₂ HPO ₄ .12H ₂ O (0.
_{15%), MgSO 4 · 7H} 2 O (0.02%), FeS
_{O 4 · 7H 2 O (0.0001} %), CaCl 2 · 2H 2 O
(0.001%) to which a yeast extract (0%, 0.2% or 0.5%) was added to prepare a medium having a pH of 7.0, inoculated, and cultured at 30 ° C. for 22 hours. did. The supernatant and the cells were separated by centrifugation, and the enzyme activities were measured. Table 2 shows the obtained results.

[0025]

[Table 2]

As can be seen from the results in Table 2, L-101
When no yeast extract was added to the strain even when mucin was present, the enzyme productivity was very low, indicating that the yeast extract greatly affected the enzyme production.

Example 3 Identification of L-101 Strain The L-101 strain is a non-spore-forming gram-positive bacillus exhibiting polymorphism, and its bacteriological properties are shown in Table 3 below.
Aerobic, non-mobile, OF test oxidative, cell wall peptidoglycan diamino acid is D-ornithine,
12. Isoprenoid quinone is menaquinone-12. -13. −
11. Since the GC content is 69%,
Aureobacteri was identified as a bacterium belonging to the genus terium.
um sp. L-101.

[0028]

[Table 3]

Example 4: Culture of bacteria Aureobacterium sp. L-101 (FERM P-161)
63) with mucin (porcine stomach) (0.5%), NH ₄ NO
₃ (0.4%), KH ₂ PO ₄ (0.15%), Na ₂ HP
_{O 4 · 12H 2 O (0.15} %), MgSO 4 · 7H 2 O
_{(0.02%), FeSO 4 ·} 7H 2 O (0.0001
%), CaCl ₂ .2H ₂ O (0.001%) and yeast extract (1.0%) in a 3 L liquid medium (pH 7.0).
0) was cultured at 30 ° C. for 24 hours. After the culture, the cells were separated by centrifugation, and the supernatant was obtained as a crude enzyme solution.

Example 5 Purification of Enzyme Ammonium sulfate was added to the crude enzyme solution obtained in Example 4 to a concentration of 65%, and the resulting precipitate was centrifuged to obtain a pellet. Dissolve the pellet in 0.01 M sodium phosphate buffer (pH 7.0, containing 0.5 M NaCl)
After removing insolubles by centrifugation, dialysis was performed against a 0.01 M sodium phosphate buffer (pH 7.0). Then, the enzyme solution was subjected to affinity chromatography using a column prepared from mucin of pig stomach.
That is, mucin (porcine stomach, manufactured by Wako) was used as a ligand, bound to a formyl cellulofine-immobilized carrier, and packed into a column. The column is a 50 mM sodium phosphate buffer (p
After equilibration with H7.0), a dialysate was added, and the column was washed with 5 times the column volume of the same buffer. Subsequently, a fraction containing exolacto-N-biohydrolase was obtained by flowing a 50 mM sodium phosphate buffer solution containing 0 to 0.4 M of sodium chloride. Fig. 1 shows the results of mucin affinity chromatography.

Using the above fraction as a 65% saturated ammonium sulfate solution, the obtained precipitate was dialyzed against a 10 mM sodium phosphate buffer (pH 7.0). This dialysate was added to a MonoQ column that had been equilibrated with 50 mM sodium phosphate buffer (pH 7.0), and the column was eluted with a concentration gradient of sodium chloride from 0 to 0.5 M to prepare an exolacto-N-biohydrolase fraction. Got a minute. The results of the anion exchange chromatography are shown in FIG.

The above fraction was concentrated using an Ultrafree UV15 (Millipore) membrane, and then concentrated in Superdex.
The mixture was added to a 200 HR column and subjected to gel filtration to obtain a highly purified exolacto-N-biohydrolase fraction. This fraction showed a single band on SDS-PAGE (see FIG. 3). In addition, SDS-P
AGE was performed according to the method of Laemmli according to the method of
(Manufactured by Daiichi Kagaku), and the protein was confirmed by silver staining.

Table 4 shows the results showing the degree of purification in each of the above purification steps.

[0034]

[Table 4]

Example 6: Characteristics of exolacto-N-biohydrolase The optimum pH of the enzyme purified in Example 5 was 5.5,
At each pH, the pH stability at 30 ° C. for 1 hour is 6 to 9, the optimum temperature is 50 ° C., and at pH 5.5, each temperature is 30 ° C.
The thermal stability in 37 minutes was 37 ° C. (see FIGS. 4 and 5). The molecular weight was about 12 by SDS-PAGE.
It was 0000. The isoelectric points are 4.0 to 4.5,
The Km value was 4 mM relative to pNP-lacto-N-bioside.
Met. Regarding the inhibition of this enzyme by various metals,
It is shown in Table 5. The enzyme has Zn ² +, Hg ² +, Cu ² + and F ²
Inhibited by e ³ +.

[0036]

[Table 5]

Example 7: Synthesis of trisaccharide by transfer reaction Galβ1-3GlcNAcβ-pNP (100 mg)
And paranitrophenyl-β-D-galactopyranoside (Galβ-pNP) (480 mg) in 12 ml of 4
Dissolved in 0 mM sodium acetate buffer (pH 5.5),
10.4 units of exolacto-N-biohydrolase were added and reacted at 40 ° C. After 100 minutes, the reaction was stopped by heat inactivating the enzyme. The paranitrophenol was removed by washing the reaction solution with diethyl ether. After that, the concentrated reaction solution was
The product was applied to an arl HW-40S column (2.6 × 90 cm), and the product was eluted with 25% ethanol. The elution pattern of the product is shown in FIG. Furthermore, YMC-pack
Further purification was performed using SH-343-5 ODS (20 × 250 mm), and Galβ1-3GlcNAcβ1-
3Galβ-pNP and Galβ1-3GlcNAc
3.8 mg and 4.1 mg of β1-6Galβ-pNP were obtained, respectively.

[0038]

Industrial Applicability The enzyme of the present invention is a novel enzyme, and Galβ1-3GlcNAc is converted to β1
It has become possible to easily synthesize a sugar chain linked by a -3 bond. In addition, the strain producing the enzyme of the present invention has high productivity of the enzyme, has a large amount of extracellular production, and is advantageous for purification.
The cultivation of the strain is also easy.

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern when a solution containing exolacto-N-biohydrolase of the present invention is purified by a mucin affinity column.

FIG. 2 is a view showing an elution pattern when a solution containing exolacto-N-biohydrolase of the present invention is purified by a MonoQ column having a pH of 7.0.

FIG. 3. Final purified exolacto-N of the invention
FIG. 3 shows an SDS electrophoresis pattern of biohydrolase.

FIG. 4 is a graph showing the optimal pH and pH stability of exolacto-N-biohydrolase of the present invention.

FIG. 5 is a graph showing the optimal temperature and temperature stability of exolacto-N-biohydrolase of the present invention.

FIG. 6 shows an elution pattern of a product from a Toyopearl column.

Claims (5)

[Claims] 1. A β1-3 lacto-N-bioside bond (Galβ1-bioside bond) formed from a lacto-N-bioside compound and a sugar acceptor.
An exo-type saccharide hydrolase having an action of forming 3GlcNAcβ1-3R). 2. Aureobacterium
The exo-type saccharide hydrolase according to claim 1, wherein the exo-type saccharide hydrolase is derived from a genus microorganism. 3. The exo-type glycolytic enzyme according to claim 1, which is produced from a microorganism having an accession number of FERM P-16163. 4. The exo-type saccharide hydrolase according to claim 1, which has the following physicochemical properties. (1) action: acting on an exolacto-N-bioside bond to release lacto-N-biose; (2) substrate specificity: lacto-N-tetraose (Gal
β1-3GlcNAcβ1-3Galβ1-4Glc)
To release lacto-N-biose and lactose. Acts on paranitrophenyl-β-D-lacto-N-bioside to release lacto-N-biose and paranitrophenol; (3) Molecular weight: about 120, as determined by SDS-PAGE.
000; (4) Optimum pH and pH stability: pH 5.5 to 6.0
It has an optimum pH and is stable between pH 6.0 and 9.0 under the condition of incubating at 30 ° C. for 1 hour. (5) Optimum temperature: The optimum temperature of action is 50 ° C. Yes, pH
30 min incubation at 5.5 is stable up to 37 ° C .; (6) Isoelectric point: 4.0-4.5 (7) Km: 4 mM (using pNP-lacto-N-bioside as substrate) 5. A β1 comprising using the exo-type saccharide hydrolase according to any one of claims 1 to 4.
A method for producing a saccharide having a -3 lacto-N-bioside bond.