JPH06197783A - New cyclic isomaltooligosaccharide and its production - Google Patents

Abstract

PURPOSE:To provide a new cyclic isomaltooligosaccharide useful e.g. as a clathrating agent for pharmaceuticals, foods, etc. CONSTITUTION:Cycloisomaltoheptaose having cyclic structure formed by bonding 7 glucose molecules through alpha-1, 6 bonds; cycloisomaltooctaose having cyclic structure formed by bonding 8 glucose molecules through alpha-1, 6 bonds; and cycloisomaltononaose having cyclic structure formed by bonding 9 glucose molecules through alpha-1, 6 bonds. The cyclic isomaltooligosaccharide can be produced by culturing a microbial strain which belongs to the genus Bacillus and capable of producing a cyclic oligosaccharide from dextran [e.g. Bacillus sp. T-3040 strain (FERM BP-4132)] in a medium containing alpha-1, 6 glucan and collecting the saccharide from the cultured product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclic isomaltooligosaccharide and a method for producing the same.

[0002]

2. Description of the Related Art Conventional cyclic oligosaccharides include, for example,
Cyclodextrins in which 6 to 8 glucose molecules are linked by α-1,4 are well known. Cyclodextrin has the ability (inclusion ability) to bind to various hydrophobic substances because the center of the ring is structurally hydrophobic. Therefore, in a wide range of fields such as pharmaceuticals, cosmetics, and foods, application development utilizing this property has been carried out in order to stabilize various substances and improve solubility.
Amylases and Related Enzymes (Ha
ndbook of Amylases and Related Enzymes), Amylase Society of Japan, pp. 233-243, 1988].

Recently, 6 to 8 molecules of fructose are β
-2,1 linked cyclic oligosaccharides have been found and are expected to be used in a wide range of fields as with cyclodextrins [Carbohydrate Research (Carbohyd. Res.)
192, 83-90, 1989]. In addition to these sugars, 17-24 molecules of glucose are β-1, 2-linked cyclosophoraose, 3-4 molecules of glucose are β-1,
6-linked cyclogenthiooligosaccharide, rhamnose 6
Various cyclic oligosaccharides such as cycloawaodrin cyclized with molecules and cyclomannohexaose cyclized with 6 molecules of mannose have been synthesized by enzymatic or chemical synthesis.

However, no cyclic oligosaccharide in which glucose is bound to α-1,6 has been known at all.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel cyclic isomaltooligosaccharide in which glucose is α-1,6 linked, which is expected to be highly useful, and a method for producing the same.

[0006]

Therefore, the present inventors have
As a result of searching soil for a microorganism that produces a cyclic oligosaccharide from dextran, the inventors have found that one strain belonging to the genus Bacillus produces a novel cyclic oligosaccharide in a medium, and completed the present invention.

That is, in the present invention, 7 molecules of glucose are α-
A novel cycloisomaltoheptaose, which forms a cyclic structure with 1,6 bonds, and 8 molecules of glucose are α-
9 molecules of novel cycloisomaltooctaose and glucose formed by forming a cyclic structure with 1,6 bonds are α-
It is a novel cyclic isomaltooligosaccharide selected from the group consisting of a novel cycloisomaltononaose formed by forming a cyclic structure by 1,6 bonds.

Further, the present invention relates to a microorganism belonging to the genus Bacillus and having the ability to produce cyclic isomaltooligosaccharide,
A novel method for producing a cyclic isomaltooligosaccharide, which comprises culturing in a medium containing -1,6-glucan and collecting the cyclic isomaltooligosaccharide from the culture.

The present invention will be described in detail below. First, the physicochemical properties of these substances and the fact that these substances are cyclic oligosaccharides will be explained based on the properties. 1. As a result of elemental analysis, it was found to be as follows, and it was found that they were cyclic heptamers, octamers and 9-mers of glucose. Cyclo iso maltoheptaose; C42H70O35 · 3H ₂
Calculated as O C: 42.43% H: 6.44% Measured value C: 42.78% H: 6.33% Cycloisomaltooctaose; C48H80O40 ・ 4H ₂
Calculated value as O C: 42.11% H: 6.48% Measured value C: 42.37% H: 6.24% Cycloisomaltononaose; C54H90O45 ・ 5H ₂
Calculated as O C: 41.86% H: 6.51% Measured value C: 41.53% H: 6.18%

2. The molecular weight is a mass spectrometer manufactured by Hitachi Ltd.
From the data of the mass spectrum analysis analyzed by 80B, cycloisomaltoheptaose; 1134 cycloisomaltooctaose; 1296 cycloisomaltonononaose; 1458, which were all in agreement with the values estimated from the molecular formula.

3. The melting point was measured by a melting point measuring device manufactured by Yanagimoto Co., Ltd., and it did not melt clearly, and the decomposition temperature was shown because the color changed in the following temperature range, but cycloisomaltoheptaose; 234 to 238, respectively. C. Cycloisomaltooctaose; 238 to 241 ° C Cycloisomaltononaose; 239 to 242 ° C.

4. The ultraviolet absorption spectrum was measured by a spectrophotometer 775 manufactured by Hitachi Ltd. As a result, no characteristic absorption was observed. Therefore, it was suggested that they do not have functional groups such as amino groups and carboxyl groups. 5. The infrared absorption spectrum of the substance was measured by IR from JASCO Corporation.
The result of measurement with a spectrum meter model FT / IR-7300 is shown in FIG. In FIG. 1, A, B, and C respectively indicate a cyclic heptamer, an octamer, and a 9mer of glucose. As a result, 917 ± peculiar to α-1,6 binding
Absorption peaks were shown at 2 cm-1 and 768 ± 1 cm-1. Therefore, it was suggested that the oligosaccharide has an α-1,6 bond.

6. Regarding the solubility in solvents, all oligosaccharides were dissolved in water at room temperature at a concentration of at least 20 mg / ml or more. 7. The color reaction strongly suggested that there was no reducing end, since neither oligosaccharide developed color by the Somogene Nelson method. 8. The substances are all neutral white substances. 9. From the results of 13 C-NMR analysis of the substance by NMR spectrum meter model NM-FX200 manufactured by JEOL Ltd., only 6 signals were recognized in each case, which supported the cyclic structure. Further, the analysis of isomaltheptaose strongly suggested that the binding mode of the substance was α-1,6 binding.

Next, the results of enzymatic analysis of the substance are shown. Ten. As shown in FIG. 2, glucodextranase, which is an exo-type dextranase, was allowed to act on the substance at a concentration of 1% for 24 hours at 40 ° C., but it was not hydrolyzed at all.
Under the same conditions, isomalthexaose and isomaltheptaose were completely hydrolyzed. Therefore, it is suggested that it is not a linear isomaltooligosaccharide. When the endo-type dextranase was allowed to act on the substance at 11.1% concentration, it was decomposed into glucose and isomaltose. Therefore, it is suggested that these oligosaccharides have glucose as the only constituent sugar and consist of only α-1,6 bonds.

From the above results 1 to 11, the substance is
In each case, 7-9 glucose molecules were bound by α-1,6,
It was determined to be a cyclic oligosaccharide. The structural formulas of these cyclic isomaltooligosaccharides are as follows.

[Chemical 1]

As described in detail above, the cyclic oligosaccharide has a property different from that of the conventionally known cyclic oligosaccharide and is a completely novel cyclic oligosaccharide in that it is composed of α-1,6 bonds of glucose. It is an oligosaccharide. Since this substance has a cyclic structure, it has an inclusion action and is useful for stabilizing and solubilizing substances of different sizes from cyclodextrin. And specifically, it is used as an inclusion agent for medicines, foods and the like.

Next, a method for producing this substance will be described.
The microorganism used in the present invention may be any strain as long as it belongs to the genus Bacillus and produces a cyclic oligosaccharide from dextran, for example, T-304.
There are 0 strains. This T-3040 strain is a wild strain obtained from soil. The mycological properties of this strain are shown below. Mycological properties (1) Morphology a. Morphological bacilli b. Motility is observed c. Spores, sporangia, swelling, elliptical position Neutral to sub-edge d. Gram stainability + (2) Growth state a. Meat agar Plate culture Smooth, no pigment production b. Beef broth agar slope culture Smooth, peripheral smoothness, no pigment production (3) Physiological properties a. Reduction of nitrate-b. Denitrification reaction-c. MR test- d. VP test − E. Indole formation − f. Hydrogen sulfide formation − g. Starch hydrolysis + h. Use of citric acid − i. Use of inorganic nitrogen source Nitrate − Ammonia salt + j. Urease − k. Oxidase − l. Catalase + m. Growth range Temperature 10-37 ° C n. Attitude toward oxygen Aerobic o.O.F. test- (no acid production observed) p. Attitude toward sugars Acid production Gas production (1) L -Arabinose- (2) D-xylose- (3) D-glucose +-(4) D-mannose- (5) D-fructose − − (6) D-Galactose + − (7) Maltose + − (8) Sucrose + − (9) Lactose + − (10) Trehalose + − (11) D-Sorbit − − (12) D-Mannitol − − (13) Inosit − − (14) Glycerin − − (15) Starch + −

Since this T-3040 strain is a spore-forming Gram-positive bacillus, it was identified as a bacterium belonging to the genus Bacillus, and Bacillus sp. T-3040.
It was a stock. In addition, Bacillus sp. (Bacillus sp.)
The T-3040 strain has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Article No. 4132 (FERM BP-4132).

The culture of the strain in the method of the present invention is, in principle, the same as the method employed in the aerobic culture of general microorganisms, but usually, it is a shaking culture method using a liquid medium or an aeration stirring culture method. Is used. As the medium, a medium containing a nitrogen source, a carbon source, vitamins, minerals and the like, which are used for culturing ordinary microorganisms, and further containing dextran, which is a raw material of this substance, is used.

The pH may be in any pH range as long as the present bacterium grows, but is usually preferably in the range of 6-8. Culture conditions are, for example, usually 20 to 40 ° C., preferably 30 ° C., and 16
Shaking culture or aeration-agitation culture is performed for about 6 days, preferably 3 days.

The purified product of the present cyclic oligosaccharide is obtained using the culture obtained as described above, for example, by the oligosaccharide collecting step shown below. In order to obtain oligosaccharides from the above-mentioned culture, after sterilizing by centrifugation, membrane concentration, etc., any method can be used as long as it is a usual oligosaccharide separation method. By treating according to the purification method of dextrin, a highly pure cyclic oligosaccharide fraction can be obtained.

Further, if necessary, each high-purity product can be obtained by using a purifying means such as HPLC using a column in a distribution adsorption mode. As a method for purifying cyclic isomalto-oligosaccharide from the sterilized liquid, for example, a known method such as cooling treatment, organic solvent addition treatment, activated carbon treatment, or column chromatography for specifically adsorbing cyclic oligosaccharide, activated carbon column chromatography, etc. Can be separated and removed.

The present invention will be specifically described below with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[0024]

[Example] 3 ml of a liquid medium (using tap water, pH 7.0) consisting of 1% dextran T2000, 1% peptone, 0.5% NaCl and 0.1% yeast extract was placed in a 15 ml test tube and sterilized at 120 ° C for 20 minutes. Was done. In addition, Bacillus sp. T-3040 strain (FERM
BP-4132) Inoculate 1 platinum loop from stored slant at 30 ℃
The cells were cultivated with shaking for 1 day. 3 ml of the main culture broth was inoculated into a 3 liter mini jar containing 2 liter of medium prepared under the same medium composition and sterilization conditions as above, and then 30 ° C, 0.25vvm, 350r.
Aeration-agitation culture was carried out for 2 days under the condition of pm, and after the culture was completed, cells were separated from the culture by centrifugation at 8000 rpm for 20 minutes to obtain a sterilized solution.

The sterilized liquid was passed through an activated carbon column to adsorb cyclic isomalto-oligosaccharides, and then eluted stepwise by 5% each with ethanol. The 20% ethanol-eluted fraction contained most of the target cyclic oligosaccharide. After concentrating this eluate with a rotary evaporator, TSKgel Am
The results of HPLC analysis using an ide80 column (packing column for distribution / adsorption chromatography, manufactured by Toso Corp.) are shown in FIG. 3-A. Next, the crude cyclic isomalto-oligosaccharide solution was concentrated by a rotary evaporator and then concentrated with YMC.
Each cyclic isomalto-oligosaccharide was separated and purified by HPLC using a PA43 column (Yamamura Chemical Co., Ltd. packing column for partition / adsorption chromatography, for fractionation).

After each fraction was concentrated with a rotary evaporator, glucodextranase, an exo-type dextranase, was added to remove linear isomaltooligosaccharides mixed as impurities, and the mixture was added at 40 ° C. overnight. It was made to react. After the reaction was stopped by boiling the reaction solution, the denatured protein was removed by centrifugation, and then YMC PA was added again.
Each cyclic isomaltooligosaccharide was separated and purified by HPLC using 43 columns. After concentrating each fraction with a rotary evaporator, the purity of oligosaccharide in the concentrate was
HPLC using Kgel Amide 80 column (manufactured by Toso Corporation, packed column for partition / adsorption chromatography)
Were analyzed by (Fig. 3-B-D). The purity of each oligosaccharide was 98% or more. Further, these oligosaccharide fractions were freeze-dried to obtain cycloisomaltoheptaose at about 60%.
mg, about 200 mg of cycloisomaltooctaose and about 100 mg of cycloisomaltononaose were obtained.

[0027]

INDUSTRIAL APPLICABILITY According to the present invention, a novel cyclic isomaltooligosaccharide consisting of α-1,6 bond of glucose, which is useful as a clathrate for medicines, foods, etc., and the cyclic isomaltooligosaccharide having a high purity by a Bacillus microorganism It also provides an efficient manufacturing method and is extremely useful industrially.

[Brief description of drawings]

FIG. 1 is a diagram showing infrared absorption spectra of three compounds of the present invention.

FIG. 2 is a diagram showing the results of enzymatic analysis of three compounds of the present invention.

FIG. 3 is a diagram showing an eluate of three compounds of the present invention and the results of HPLC analysis of the produced three compounds.

Claims (2)

[Claims] 1. A novel cycloisomaltoheptaose in which 7 molecules of glucose form a cyclic structure by α-1,6 bonds, and a novel cycloisomaltoheptaose in which 8 molecules of glucose form a cyclic structure by α-1,6 bonds Cycloisomaltooctaose and a novel cyclic isomaltonosaccharide selected from the group consisting of novel cycloisomaltononaose, in which 9 molecules of glucose form a cyclic structure by α-1,6 bonds. 2. A microorganism belonging to the genus Bacillus and having a cyclic isomaltooligosaccharide-producing ability according to claim 1, is α-1,
A novel method for producing a cyclic isomaltooligosaccharide, which comprises culturing in a medium containing 6-glucan and collecting the cyclic isomaltooligosaccharide according to claim 1 from the culture.