JPH0759584A - Production of disaccharide and new disaccharide - Google Patents

Abstract

PURPOSE:To provide a process for the industrial production of disaccharides useful for pharmaceuticals, foods, cosmetics or enzyme stabilizers and to obtain new disaccharides obtained by the process. CONSTITUTION:This process for the production of disaccharides comprises the condensation reaction of beta-glucose-1-phosphoric acid with a monosaccharide in an aqueous medium in the presence of an enzymatic source having carbohydrate phosphorylase activity and derived from microorganisms belonging to the genus Catellatospora, Kineosporia, Propionibacterium or Enterococcus and the collection of the disaccharides formed in the aqueous medium. This invention also relates to new disaccharides obtained by this process.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing disaccharides. The disaccharide is a substance useful as a stabilizer of an enzyme in synthetic raw materials such as medicines, foods or cosmetics, or clinical test reagents.

[0002]

2. Description of the Related Art As a method for producing a disaccharide having an α, 1-1 glycoside bond, trehalose [glucosyl (α, 1-1) D-glucose] is produced using a microorganism belonging to the genus Nocardia. Method (Japanese Patent Laid-Open No. 50-154485),
Method for producing trehalose by treating maltose [glucosyl (α, 1-4) D-glucose] with maltose phosphorylase (hereinafter abbreviated as MP) and trehalose phosphorylase (hereinafter abbreviated as TP) (Japanese Patent Publication No.
63-60998) and the like are known. Further, as a method for producing a disaccharide having an α, 1-4 glycoside bond, a method for producing a maltose derivative or the like using an extract of Neisseria perflava (Journal of Biological Chemistry (J. Biol . Chem.) 236 ,
2183-2185 (1961)] and the like are known.

However, in the method using a microorganism belonging to the genus Norcadia, the amount of trehalose produced in the culture solution is very small, and in the method of treating maltose with MP and TP, disaccharides other than trehalose are produced. Can not. Further, in the method using the extract of Neisseria perflava, the substrate β-glucose-1-phosphate is obtained by subjecting maltose to phosphorolysis, but in this case, an operation for separating glucose produced as a by-product Is required.

Disaccharides such as trehalose or maltose are known to increase the stability of biopolymers such as enzyme proteins [Bio / Technology (C.
Colaco et al., Bio / Technology), 10 , 1007-1011, (19
92)].

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for industrially producing disaccharides and a novel disaccharide obtained by the method.

[0006]

According to the present invention, the presence of an enzyme source derived from a microorganism belonging to the genus Cateratospora, quineosporia, genus Propionibacterium or genus Enterococcus and having glycosyl phosphate degrading activity , A method for producing a disaccharide characterized by subjecting β-glucose-1-phosphate and a monosaccharide to a condensation reaction in an aqueous medium to collect a disaccharide produced in the aqueous medium, and a novel method obtained by the method Can be provided.

The present invention will be described in detail below. Examples of the enzyme source used in the present invention include a culture of a microorganism having a sugar-phosphorylating activity, a bacterial cell or a treated product of the microorganism, or an enzyme having a sugar-phosphorylating activity. Microorganisms having phosphophosphate degrading activity include genus Cateratospora,
Any microorganism can be used as long as it is a microorganism belonging to the genus Kineosporia, the genus Propionibacterium or the genus Enterococcus and having the ability to produce an enzyme having a glycosyl phosphate degrading activity.・ Ferguinea ( Catellatospo
the ra ferruginea) KY2039 and Kineosuporia-Ouranchiaka (Kineosporia aurantiaca) ATCC 29727, M
As P-producing bacteria Propionibacterium-Freudenberg Reich (Propionibacterium freudenreichii) KY4002 and Enterococcus faecium (Enterococcus faeci
um ) ATCC 10541, respectively.

The mycological properties of the species to which these strains belong are:
For more information on the Cateratospora ferguinea, see the International Journal of Systematic Bacteriology (K. Asano and I. Kawamoto, Int. J. Sys.
tact Bacteriol.), 36 , 512-517 (1986), and for the Kineosporia aurantiaca, see the Berges Manual of Systematic Bacteriology (Berg).
ey's Manual of Systematic Bacteriology), Vol.4, 2
504-2506 (1989), for the Propionibacterium freudenreich, Bergey's Manual of Systematic Bacteriology.
of Systematic Bacteriology), Vol.2, 1346-1350 (198
6) and, regarding Enterococcus faecium, Bergey's Manual of Systematic Bacterio
logy), Vol.2, 1063-1065 (1986), respectively.

[0009] In addition, Cateratospora ferguinea KY20
39 and Propionibacterium freudenreich KY
Based on the Budapest Treaty, 4002 are FEs at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology on June 11, 1993.
RM BP-4329 and FERM BP-4330
Has been deposited as. As a medium used to obtain an enzyme source having a phosphophosphate degrading activity, either a natural medium or a synthetic medium may be used as long as it appropriately contains a carbon source, a nitrogen source, an inorganic substance or other nutrients. be able to.

As the carbon source, various carbohydrates such as glucose, sucrose, trehalose, maltose, starch or molasses, various alcohols such as glycerol, sorbitol or mannitol, or various organic acids such as acetic acid, lactic acid, pyruvic acid or citric acid, etc. Is used. As the nitrogen source, various inorganic or organic ammonium salts such as ammonia, ammonium chloride, ammonium carbonate, ammonium phosphate or ammonium acetate, urea, amino acids, other nitrogen compounds or peptone, NZ-amine, meat extract, corn steep liquor or casein. A nitrogen-containing organic substance such as a hydrolyzate is used.

As the inorganic substance, potassium phosphate, dipotassium phosphate, potassium chloride, sodium chloride, magnesium sulfate or ferrous sulfate can be used. The culture is performed by static culture, aeration-agitation culture, or the like. The temperature is maintained at 25 to 37 ° C., the pH of the medium is maintained at 6.0 to 8.0, and the culture is usually completed in 1 to 7 days. After completion of the culture, the obtained microorganism culture, bacterial cells or treated bacterial cell product may be used as it is for the disaccharide production reaction, or may be used as a crude enzyme or a purified enzyme.

Examples of the treated cells include dried cells, freeze-dried products, surface-activated products, enzyme-treated products, ultrasonically crushed products, mechanically ground products, mechanical pressure-treated products, and bacterial cells. The protein fraction, the immobilized product of bacterial cells or treated bacterial cells, and the like. As the enzyme, either a crude enzyme or a purified enzyme is used, and a method of treating a treated bacterial cell product with an enzyme is generally used, such as salting out, organic solvent precipitation, dialysis, ion exchange column chromatography, gel filtration or lyophilization. It is obtained by using the method.

The enzyme having phosphophosphate degrading activity may be any enzyme as long as it catalyzes the reaction of condensing β-glucose-1-phosphate with a monosaccharide to produce a disaccharide, for example, Examples include TP and MP. The enzyme source having phosphophosphate degrading activity is 1 to 100 g / l, preferably 10 to 50 g / l or 0.1 to 100 unit / ml, preferably 10 to 50 g / l as a wet cell amount in an aqueous medium, preferably Used at 1-10 units / ml.

For example, in the case of TP and MP, the enzymatic activity of an enzyme having a phosphophosphate degrading activity is 37 ° C. for 1 minute in 50 mM phosphate buffer (pH 6.5) using trehalose and maltose as substrates, respectively. 1 μ when performing the reaction
Enzyme activity to produce mol glucose is displayed as 1 unit. Aqueous media include water, phosphates, carbonates, acetates, borates, buffers such as citrate or Tris, alcohols such as methanol or ethanol, esters such as ethyl acetate, ketones such as acetone. And natural media or synthetic media containing amides such as acetamide, carbon sources, nitrogen sources or inorganic salts that can be assimilated by microorganisms. In addition, if necessary, a surfactant such as cetylpyridinium chloride or cetyltrimethylammonium bromide may be added.
0.05 to 1.0% (w / v) or an organic solvent such as toluene or xylene may be added so as to be 1 to 20% (v / v).

The monosaccharide may be a refined product or a crude refined product, for example, D-glucose, D-fucose, D-xylose, D-mannose, D-allose, D-tagatose, D-sorbose, D-glucosamine, 2-deoxy-D- glucose,
N-acetyl-D-glucosamine or L-fucose etc. 1
It is used at -100 g / l, preferably 10-50 g / l. β-glucose-1-phosphate is 1 to 100 g / l, preferably 10 to 50 g / l.
used in l. As β-glucose-1-phosphate, a known commercial product may be used, but maltose is mixed with glucose and β-in an aqueous medium in the presence of an enzyme source having MP activity and an enzyme source having glucose degrading activity. A product obtained by converting glucose to 1-phosphate and decomposing glucose produced in an aqueous medium in the presence of an enzyme source having a glucose decomposing activity may be used.

The enzyme source having MP activity is maltose 1
The amount of wet cells is 10 to 100 g / l, preferably 20.
˜50 g / l or an enzyme amount of 1 to 100 units / ml, preferably 10 to 50 units / ml in the aqueous medium.
The enzyme source having glucose degrading activity may be present in the aqueous medium in an amount sufficient to completely decompose glucose produced by converting maltose by the enzyme source having MP activity.

Examples of the enzyme source having glucose degrading activity include microorganisms having glucose degrading activity, cultures of microorganisms, microbial cells or processed products of microbial cells, and enzymes having glucose degrading activity. As the microorganism having glucose degrading activity, any microorganism can be used as long as it has the ability to produce an enzyme having glucose degrading activity, but yeast is preferably used.

Examples of the enzyme having glucose degrading activity include glucose oxidase, catalase, pyranose oxidase, glucose dehydrogenase,
Either glucokinase or hexokinase can be used, but glucose oxidase or catalase is preferably used. In the production of β-glucose-1-phosphate, the reaction of maltose in the presence of an enzyme source having maltose phosphorylase activity and an enzyme source having glucose degrading activity in an aqueous medium is usually at a temperature of 20 to 60 ° C, preferably 30-50 ° C and pH 5.0-9.0
It is preferably carried out at pH 6.0 to 7.5 for 1 to 72 hours.

After the completion of the reaction, β-glucose-1-phosphate produced in the aqueous medium is used for the condensation reaction with a monosaccharide in the presence of an enzyme source having a phosphohydrolyzing activity for carbohydrates. β
-Glucose-1-phosphate is obtained by removing precipitates such as bacterial cells from an aqueous medium by means such as centrifugation, and the resulting supernatant is subjected to a conventional method such as ion exchange column chromatography or concentration. The collected material may be used in the reaction, or may be used in the reaction as it is in a state of being dissolved in an aqueous medium.

In the production of disaccharides, β in the presence of an enzyme source having sugar-phosphorylating activity in an aqueous medium
-Glucose-1-phosphate and monosaccharides are usually reacted at a normal temperature.
It is carried out at 20 to 60 ° C., preferably 30 to 50 ° C. and pH 5.0 to 9.0, preferably pH 6.0 to 7.5 for 1 to 72 hours. After completion of the reaction, precipitates such as bacterial cells are removed from the aqueous medium by means such as centrifugation, and the resulting supernatant is subjected to a conventional method such as ion exchange column chromatography or concentration to obtain the disaccharide. Can be collected.

According to the present invention, a known disaccharide such as trehalose or maltose or glycosyl (α, 1-1) D-fucose (Glucosyl (α, 1-1) can be used by changing the type of monosaccharide used in the reaction. ) D-fucose), glycosyl (α, 1
-4) D-mannose (Glucosyl (α, 1-4) D-mannose), glycosyl (α, 1-4) D-allose (Glucosyl (α, 1-4) D-allos)
e), glycosyl (α, 1-4) D-tagatose (Glucosyl (α, 1
-4) D-tagatose), glycosyl (α, 1-4) L-fucose (Glu
cosyl (α, 1-4) L-fucose) or glycosyl (α, 1-4)
New disaccharides such as D-sorbose (Glucosyl (α, 1-4) D-sorbose) are obtained.

Among the above disaccharides, trehalose, maltose, glycosyl (α, 1-1) D-fucose or glycosyl (α, 1-4) L-fucose is an enzyme such as (1) solution state of alkaline phosphatase. Stability when stored at
The following are test examples that examined effects on (2) stability during storage in the frozen state, (3) stability during repeated freeze-thawing, and (4) stability during storage in the dry powder state. Test Example 1 A test solution consisting of a solution obtained by diluting alkaline phosphatase (derived from bovine small intestine, manufactured by Boehringer) with distilled water (no addition), or trehalose, maltose, glycosyl (α, 1-1) in the solution. ) Prepare a test solution containing D-fucose or glycosyl (α, 1-4) L-fucose at a concentration of 50 mM, leave it at room temperature (25 ° C) for 24 hours, and then test the alkaline phosphatase activity of the test solution. Was measured.

The activity of alkaline phosphatase was 10 μl of the enzyme solution, 1M diethanolamine buffer (pH 10.2), and 0.
It was incubated at 37 ° C. in 2.0 ml of a reaction solution containing 2 mM magnesium chloride and 5 mM paranitrophenyl phosphate, and the amount of paranitrophenol produced was calculated from the change in absorbance at 405 nm. From the obtained measured value of the enzyme activity, the residual activity was calculated with the activity immediately after the preparation of the test solution as 100%.

The results are shown in Table 1.

[0025]

[Table 1]

Test Example 2 Test solution consisting of a solution obtained by diluting alkaline phosphatase with distilled water (no addition), or trehalose, maltose, glycosyl (α, 1-1) D-fucose or glycosyl ( α, 1-4) L-fucose 50 each
Prepare the test solution added so that it becomes mM, and add the test solution to -20
It was stored frozen at 6 ° C for 6 days. Then, after thawing at room temperature, the alkaline phosphatase activity of the test solution was measured in the same manner as in Test Example 1, and the residual activity was calculated by setting the activity immediately after the preparation of the test solution to 100%.

The results are shown in Table 2.

[0028]

[Table 2]

Test Example 3 A test solution consisting of a solution obtained by diluting alkaline phosphatase with distilled water (no addition), or trehalose, maltose, glycosyl (α, 1-1) D-fucose or glycosyl ( α, 1-4) L-fucose 50 each
A test solution added so as to be mM was prepared, and the test solution was repeatedly frozen by dry ice-ethanol and thawed at room temperature 10 times in total, and then the alkaline phosphatase of the test solution was tested in the same manner as in Test Example 1. The activity was measured, and the activity immediately after the preparation of the test solution was regarded as 100% to calculate the residual activity.

The results are shown in Table 3.

[0031]

[Table 3]

Test Example 4 A test solution (no addition) consisting of a solution obtained by diluting alkaline phosphatase with distilled water, or trehalose, maltose, glycosyl (α, 1-1) D-fucose or glycosyl α, 1-4) L-fucose 50 each
A test solution added so as to be mM was prepared, and the test solution was dried at room temperature using a vacuum pump to completely evaporate water to obtain a dry powder. After storing the obtained dry powder at 50 ° C. for 24 hours, the alkaline phosphatase activity of the test solution was measured in the same manner as in Test Example 1, and the residual activity was calculated by setting the activity immediately after the preparation of the test solution as 100%.

The results are shown in Table 4.

[0034]

[Table 4]

As described above, trehalose, maltose, glycosyl (α, 1-1) D-fucose and glycosyl
(α, 1-4) L-fucose, especially glycosyl (α, 1-1) D-fucose and glycosyl (α, 1-4) L-fucose, has an effect as a stabilizer of alkaline phosphatase.
Hereinafter, the present invention will be specifically described with reference to examples.

[0036]

【Example】

Example 1 50 mM β-glucose-1-phosphate, 50 mM monosaccharides (D-glucose, D-fucose or D-xylose) in 50 mM imidazole-hydrochloric acid buffer (pH 7.0) and from Cateratospora ferguinea 2 units / ml of TP enzyme preparation, 50 mM of β-glucose-1-phosphate, monosaccharide (D-
Glucose, 2-deoxy-D-glucose, D-glucosamine, N-acetyl-D-glucosamine, D-mannose, D-allose, D-tagatose, D-sorbose, L-fucose or D-xylose) at 50 mM and pro A reaction solution was prepared so that the MP enzyme preparation derived from Pionibacterium freudenreich was 2 units / ml and reacted at 30 ° C for 4 hours.

After completion of the reaction, the reaction solution was centrifuged to collect the supernatant, and the resulting supernatant was separated and quantitated by high performance liquid chromatography to identify known substances as they were, Was boiled in 1N sulfuric acid at 100 ° C. for 1 hour to undergo acid hydrolysis or enzymatic decomposition with α-glucosidase, and monosaccharides constituting disaccharides were identified by high performance liquid chromatography. The analysis conditions of high performance liquid chromatography are as follows.

Column: Shim-pack CLC-NH ₂ (6 mm x 15 cm)
(Shimadzu) Mobile phase: 80% acetonitrile-20% water Flow rate: 1 ml / min Detector: Differential refractometer (Shimadzu) The monosaccharide added to the reaction solution, the reaction product, and the yield Shown in the table.

[0039]

[Table 5]

Example 2 50 mM maltose and 50 mg Cateratospora ferguinea KY2039 in 50 mM phosphate buffer (pH 6.5), wet cells / ml,
Propionibacterium freudenreich KY4002 10
mg Wet cells / ml and cetylpyridinium chloride 0.1%
The reaction liquid added so that the above was prepared was reacted at 30 ° C. for 18 hours.

After completion of the reaction, the reaction solution was centrifuged to collect the supernatant, and the obtained supernatant was analyzed by high performance liquid chromatography. As a result, trehalose was produced in a yield of 52%.

Example 3 50 mM maltose in 50 mM phosphate buffer (pH 6.5), M derived from Propionibacterium freudenreich KY4002
A reaction solution was prepared by adding 1 unit / ml of P enzyme preparation and 2 unit / ml of TP enzyme preparation derived from Cateratospora ferguinea KY2039, and reacted at 30 ° C. for 18 hours.

After the reaction was completed, the reaction solution was centrifuged to collect the supernatant, and the obtained supernatant was analyzed by high performance liquid chromatography. As a result, trehalose was produced at a yield of 78%.

Example 4 50 mM maltose, 50 mM maltose, 1 unit / ml MP enzyme preparation derived from Enterococcus faecium ATCC 10541 and Kineosporia aurantiaca in 50 mM phosphate buffer (pH 6.5).
A reaction solution was prepared by adding TP enzyme preparation derived from ATCC 29727 so as to be 2 units / ml, and reacted at 30 ° C. for 18 hours.

After completion of the reaction, the reaction solution was centrifuged to collect the supernatant, and the obtained supernatant was analyzed by high performance liquid chromatography. As a result, trehalose was produced in a yield of 75%.

Example 5 Maltose was added to 50 mM in 50 mM phosphate buffer (pH 6.5) and M derived from Propionibacterium freudenreich KY4002 was used.
Prepare a reaction mixture containing P enzyme preparation at 1 unit / ml, glucose oxidase (Toyobo Co., Ltd.) at 2 unit / ml and catalase (Sigma) at 3000 unit / ml, and then at 6 ℃ at 30 ℃. It is allowed to react for a period of time to produce β-glucose-1-phosphate and glucose from maltose,
The produced glucose was decomposed using glucose oxidase and catalase. 2 units / ml of TP enzyme preparation derived from Cateratospora ferguinea KY2039 and 200 mM of D-fucose were added to the reaction solution, and the mixture was further reacted for 18 hours.

After completion of the reaction, the reaction solution was centrifuged to collect the supernatant, and the obtained supernatant was analyzed by high performance liquid chromatography. As a result, glycosyl (α, 1-1) D-fucose was found to be 29 mM. Was being generated.

Example 6 50 mM maltose in 50 mM phosphate buffer (pH 6.5), M derived from Propionibacterium freudenreich KY4002
2 units / ml of P enzyme preparation and 2 units of glucose oxidase
Unit / ml and catalase were added to 3000 units / ml to prepare a reaction solution, which was allowed to react at 30 ° C for 6 hours to produce β-glucose-1-phosphate and glucose from maltose, and the produced Glucose was digested with glucose oxidase and catalase.
Add L-fucose to the reaction solution to 200 mM,
It was further reacted for 18 hours.

After completion of the reaction, the reaction solution was centrifuged to collect the supernatant, and the obtained supernatant was analyzed by high performance liquid chromatography. As a result, glycosyl (α, 1-4) L-fucose was found to be 38 mM. Was being generated.

Example 7 500 g of activated carbon (manufactured by Nakarai) and 500 g of filter aid Hyflo Supercell (manufactured by Nakarai) were well dispersed in distilled water, and fine particles were removed by decantation. Then, a column having a diameter of 5 cm and a height of 50 cm was used. After washing with about 3 liters of 3% butanol, the column was washed with about 3 liters of water.
Next, 200 ml of the glucosyl (α, 1-1) D-fucose solution obtained in Example 5 was passed through the column, and about 2 liters of distilled water and about
After washing with 2 liters of 0.2% propanol solution, 5%
The desired product was eluted with propanol. As a result of freeze-drying the eluate, about 1 g of glucosyl (α, 1-1) D-fucose was obtained as a white powder.

The physicochemical properties of glycosyl (α, 1-1) D-fucose are shown below. Properties: colorless powder Specific rotation: [α] _D ²⁰ = +189 ° (c = 0.309, H ₂ O),
FABMS spectrum (Negative mode, Matrix: glycerol) without aging (mutation / rotation):
m / z amu 325 (MH) - High resolution FABMS spectrum (Negative mode, Matrix:
glycerol): m / z amu measured value 325.1121 (MH) ^- Calculated 325.1135 IR spectrum as _{C 12 H 21 0 10 (KBr} ): ν max cm -1 3410,2935,1653,141
9,1385,1080,1005,966 ¹³ C NMR spectrum (125MHz, D ₂ O): σppm from TMS9
4.40,94.24,73.41,72.95,72.67,71.94,70.54,69.99,68.
54,67.94,61.37,16.09 ¹ H NMR spectrum (500MHz, D ₂ O): σppm from TMS5.19
(2H, d, J = 3.9 Hz), 4.23 (1H, q, J = 6.5Hz), 4.05 (1H, dd, J = 1
0.4,3.4Hz), 3.91 (1H, dd, J = 10.4,3.9Hz), 3.89 (1H, m), 3.
88 (2H, m), 3.86 (1H, m), 3.80 (1H, dd, J = 11.6,4.8Hz), 3.68
(1H, dd, J = 9.9,3.9Hz), 3.48 (1H, t, J = 9.4Hz), 1.26 (3H,
(d, J = 6.5Hz)

Example 8 Example 8 except that the glucosyl (α, 1-1) D-fucose solution obtained in Example 5 was replaced with the glycosyl (α, 1-4) L-fucose obtained in Example 6. By purifying in the same manner as in 7, about 2 g of glucosyl (α, 1-4) L-fucose was obtained as a white powder.

The physicochemical properties of glycosyl (α, 1-4) L-fucose are shown below. Properties: colorless powder Melting point: 117.0-120.5 ° C Specific rotation: [α] _D ²⁰ = + 39.2 ° (c = 0.335, H ₂ O),
Final value (after 24 hours) FABMS spectrum (Negative mode, Matrix: glycerol):
m / z amu 325 (MH) - High resolution FABMS spectrum (Negative mode, Matrix: gly
cerol): m / z amu measured value 325.1112 (MH) ^- Calculated 325.1135 IR spectrum as _{C 12 H 21 O 10 (KBr} ): ν max cm - 3396,2931,1635,145
6,1417,1362, 1134, 1080,1022 ¹³ C NMR spectrum (100MHz, D ₂ O): σppm from TMS10
2.04,102.00,97.31,93.45,83.17,82.46,74.97,74.15,7
3.77,73.74,73.34,73.30,71.61,71.37,70.57,70.54,69.
85,67.40,61.67,17.45,17.40 ¹ H NMR spectrum (400 MHz, D ₂ O): σppm from TMS5.
27 (d, J = 3.9 Hz), 5.26 (d, J = 4.9Hz), 4.63 (d, J = 7.8Hz), 4.3
0 (q, J = 6.6 Hz), 3.99 (m), 3.98 (m), 3.93 (m), 3.93 (m), 3.92
(m), 3.89 (m), 3.88 (q, J = 6.6Hz), 3.82 (m), 3.81 (dd, J = 11.
8,4.5Hz), 3.75 (dd, J = 10.0,3.2Hz), 3.65 (dd, J = 9.9,3.9H
z), 3.59 (dd, J = 10.0,7.8Hz), 3.46 (t, J = 9.4Hz), 1.33 (d, J =
6.6 Hz), 1.30 (d, J = 6.6Hz)

Reference Example 1 Preparation of Crude Enzyme Solution (1) Kateratospora ferguinea KY2039 was sucrose 3 g / dl, NZ-amine 0.5 g / dl, peptone 0.2 g / dl,
A 2 L Erlenmeyer flask containing 300 ml of a medium (pH 7.0) containing 0.1 g / dl of yeast extract and 0.1 g / dl of meat extract was inoculated and shake-cultured at 30 ° C. for 48 hours. Inoculate 600 ml of the obtained culture into a 30 L jar fermenter containing 15 L of the same composition as the above medium, and incubate at 30 ° C for 3 days.
Aeration-agitation culture was performed.

After completion of the culture, 15 L of the culture solution was centrifuged (12,0
00xg, 20 minutes) and then add the cells to 200 mM phosphate buffer (p
H7.0) was suspended in 1,000 ml, the cells were crushed with Dynomill (WA Bachofen), and then centrifuged (12,000 xg, 20 minutes) to collect a supernatant, which was used as a TP crude enzyme solution. . T. in TP crude enzyme solution of Cateratospora ferguinea KY2039
The productivity of P was 5.0 units / g-wet cells. This is a TP productivity of 0.17 with Euglena gratislis crude enzyme solution.
Unit / g Wet bacterial cells (Japanese Patent Publication No. Sho 63-60998) approximately 30 times.

(2) Kateratospora ferguinea KY20
Kineosporia aurantiaca ATCC 297 instead of 39
A TP crude enzyme solution was obtained by using the same culture and extraction method as in (1) except that 27 was used.

Kineosporia aurantiaca ATCC 297
The productivity of TP in the TP crude enzyme solution of 27 was 5.4 units / g wet cells.

(3) Propionibacterium freudenreich KY4002 with sucrose 2 g / dl, tryptone 1 g / dl
, Yeast extract 1g / dl, dipotassium phosphate 0.5g / dl, magnesium sulfate (7-hydrate) 0.04g / dl, ferric sulfate (7-hydrate) 0.001g / dl, manganese sulfate (4-hydrate) 0.001 g / dl,
An Erlenmeyer flask containing 600 ml of a medium (pH 7.5) containing vitamin C 0.005 g / dl was inoculated, and static culture was carried out at 30 ° C for 2 days.

After the completion of the culture, combine the obtained culture solutions to 1.
Make 2 L, centrifuge this (12,000 xg, 20 minutes) and suspend the obtained bacterial cells in 300 ml of 10 mM phosphate buffer (pH 7.0) .After crushing the bacterial cells with Dynomill, centrifuge (12,000 xg, 20 minutes)
Then, the supernatant was collected and used as an MP crude enzyme solution. The MP crude enzyme solution of Propionibacterium freudenreich KY4002 had a MP productivity of 55.0 units / g wet cells. This is about 1.8 times the productivity of MP with a crude Lactobacillus brevis enzyme solution of 29.9 units / g wet cells (Japanese Patent Publication No. 63-60998).

(4) MP crude enzyme solution was obtained by the same culture and extraction method as in (3) except that Enterococcus faecium ATCC 10541 was used instead of Propionibacterium freudenreich KY4002.

Enterococcus faecium ATCC 10541
The productivity of MP in the crude MP enzyme solution was 50.0 units / g wet cells.

Reference Example 2 Preparation of Enzyme Preparation (1) Reference Example 1 In (1), Cateratospora ferguinea
Ammonium sulfate was added to the crude TP enzyme solution obtained from KY2039 so as to be 50% saturated, and the precipitate was collected. Dissolve the obtained precipitate in a small amount (about 200 ml) of 200 mM phosphate buffer (pH 7.0),
The resulting solution was dialyzed with 5 L of the same buffer for 24 hours, then heat-treated at 65 ° C for 15 minutes, centrifuged (12,000 xg, 20 minutes), and the supernatant obtained was equilibrated with the same buffer gel filtration. Agent Toyopearl HW
The solution was passed through a 65F (Tosoh Corp.) column (1 L, diameter 5 cm). Combine the eluted active fractions, add ammonium sulfate to this to 50% saturation, and centrifuge the precipitate (12,000 rpm, 20
Min) and dissolved in 20 ml of 200 mM phosphate buffer (pH 7.0). The resulting solution was dialyzed against 2 L of the same buffer for 24 hours, and TP
An enzyme preparation (specific activity 100 mU / mg) was obtained. The enzyme preparation has a specific activity 102 times that of the crude enzyme solution, and the yield of enzyme activity is 62%.
% Met.

(2) Kateratospora ferguinea KY20
Instead of the TP crude enzyme solution obtained from 39, Reference Example 1
Reference Example 2 except that the TP crude enzyme solution obtained from Kinesporia aurantiaca ATCC 29727 was used in (2).
Using the same purification method as in (1), the TP enzyme preparation (specific activity
90 mU / mg) was obtained. The enzyme preparation has a higher specific activity than the crude enzyme solution.
It was 90 times and the yield of enzyme activity was 50%.

(3) Reference Example 1 Ammonium sulphate was added to the crude MP enzyme solution obtained from Propionibacterium freudenreich KY4002 in (3) to 80% saturation, and the precipitate was collected in a small amount. (Approximately 20 ml) of 10 mM phosphate buffer (pH 7.0)
Dissolved in. The resulting solution was dialyzed against 2 L of the same buffer for 24 hours, heated at 50 ° C for 15 minutes, and centrifuged (12,000xg, 20 minutes).
The supernatant thus obtained was anion exchange resin DEAE-Sephadex (Pharmacia-LKB) equilibrated with the same buffer solution.
The column (1 L, diameter 5 cm) was passed through the column for adsorption. After washing away the impure protein with the same buffer, 0-1.0 M sodium chloride [10
Elution was performed with a concentration gradient of mM phosphate buffer (pH 7.0). about
Combine the active fractions eluting at 0.5-0.8 M sodium chloride concentration, add ammonium sulfate to this to 80% saturation, and collect the resulting precipitate by centrifugation (12,000 xg, 20 minutes). It was dissolved in 10 ml of 10 mM phosphate buffer (pH 7.0). The obtained solution was dialyzed with 2 L of the same buffer for 24 hours, and MP enzyme preparation (specific activity 50 mU / m
g) was obtained. The specific activity of the enzyme preparation was 48 times that of the crude enzyme solution, and the yield of enzyme activity was 79%.

(4) Instead of the MP crude enzyme solution obtained from Propionibacterium freudenreich KY4002,
Enterococcus faecium ATCC 1 in Reference Example 1 (4)
An MP enzyme preparation (specific activity 45 mU / mg) was obtained using the same purification method as in Reference Example 2 (3) except that the MP crude enzyme solution obtained from 0541 was used. The specific activity of the enzyme preparation was 45 times that of the crude enzyme solution, and the yield of enzyme activity was 75%.

[0066]

Industrial Applicability According to the present invention, it is possible to provide an inexpensive and efficient method for producing a disaccharide and a novel disaccharide obtained by the method.

Claims (9)

[Claims] 1. A genus Cateratospora, a genus Quineosporia,
Β-Glucose-1-in an aqueous medium in the presence of an enzyme source derived from a microorganism belonging to the genus Propionibacterium or the genus Enterococcus and having a glycosyl phosphate degrading activity
A process for producing a disaccharide, which comprises subjecting a disaccharide produced in an aqueous medium to a condensation reaction between phosphoric acid and a monosaccharide. 2. An enzyme having a glycosyl phosphate degrading activity,
The method according to claim 1, which is trehalose phosphorylase or maltose phosphorylase. 3. The enzyme source is a culture of a microorganism, a microbial cell, a treated product of a microbial cell, a crude enzyme or a purified enzyme.
The method described. 4. β-glucose-1-phosphate is obtained by reacting maltose in an aqueous medium in the presence of an enzyme source having maltose phosphorylase activity and an enzyme source having glucose degrading activity. The method according to 1 to 3. 5. The method according to claim 4, wherein the enzyme having glucose degrading activity is glucose oxidase, catalase, pyranose oxidase, glucose dehydrogenase, glucokinase or hexokinase. 6. The enzyme source is a culture of a microorganism, a bacterial cell, a treated product of a bacterial cell, a crude enzyme or a purified enzyme.
The method described. 7. The monosaccharide is D-glucose, D-fucose,
7. D-xylose, D-mannose, D-allose, D-tagatose, D-sorbose, D-glucosamine, 2-deoxy-D-glucose, N-acetyl-D-glucosamine or L-fucose. The method described. 8. Glucosyl (α, 1-1) D-fucose. 9. Glucosyl (α, 1-4) L-fucose.