JP3075873B2 - Novel cyclic isomaltooligosaccharide and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel cyclic isomaltoligosaccharide and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, cyclic oligosaccharides include, for example,
Cyclodextrins in which 6 to 8 glucose molecules are α-1,4 linked are well known. Cyclodextrin has the ability to bind to various hydrophobic substances (inclusion ability) 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, etc. [Handbook of
Amylases and Rerated Enzyme (Ha
ndbook of Amylases and Related Enzymes), edited by Japan Amylase Research Society, pages 233 to 243, 1988].

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

[0004] However, no cyclic oligosaccharide having α-1,6-linked glucose has been known at all.

[0005]

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

[0006]

Means for Solving the Problems Accordingly, the present inventors have
As a result of searching microorganisms that produce cyclic oligosaccharides from dextran from soil, it was found that one strain belonging to the genus Bacillus produces a novel cyclic oligosaccharide in a medium, and the like, and thus completed the present invention.

That is, according to the present invention, seven molecules of glucose are α-
Novel cycloisomaltoheptaose, which forms a cyclic structure by 1,6 bonds, and 8 molecules of glucose are α-
Novel cycloisomaltooctose and 9 molecules of glucose, which form a cyclic structure by 1,6 bonds, are α-
It is a novel cyclic isomaltoligosaccharide selected from the group consisting of a novel cycloisomaltononanose having a cyclic structure formed by 1,6 bonds.

Furthermore, the present invention relates to a microorganism belonging to the genus Bacillus and having the ability to produce cyclic isomatooligosaccharides,
Cultured in medium containing 1,6-glucan, a method for producing novel cycloisomaltooligosaccharide to recovering said cycloisomaltooligosaccharide from the culture.

Hereinafter, the present invention will be described in detail. First, the fact that these substances are cyclic oligosaccharides will be described based on the physicochemical properties of these substances and their properties. 1. As a result of elemental analysis, the results were as follows, and it was found that the glucose was a cyclic heptamer, octamer, and 9-mer. Cyclo iso maltoheptaose; C42H70O35 · 3H ₂
Calculated O C: 42.43% H: 6.44 % measured value C: 42.78% H: 6.33% cyclo isomalto oct maltoheptaose; C48H80O40 · 4H ₂
Calculated O C: 42.11% H: 6.48 % measured value C: 42.37% H: 6.24% cyclo iso Martenot na ose; C54H90O45 · 5H ₂
Calculated value as O C: 41.86% H: 6.51% Measured value C: 41.53% H: 6.18%

[0010] 2. Molecular weight is mass spectrometer manufactured by Hitachi, Ltd.
From the data of mass spectrometry analyzed by 80B, it was cycloisomaltoheptaose; 1134 cycloisomaltooctaose; 1296 cycloisomaltononaose; 1458, all of which agreed 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. As a result, it did not melt clearly and discolored in the following temperature range, and showed the decomposition temperature. However, cycloisomaltoheptaose; 234 to 238, respectively. Cycloisomaltooctaose; 238-241 ° C Cycloisomaltononaose; 239-242 ° C.

4. The ultraviolet absorption spectrum was measured with a spectrophotometer 775 manufactured by Hitachi, Ltd., and no characteristic absorption was observed. Therefore, it was suggested that it did not have a functional group such as an amino group and a carboxyl group. 5. The external infrared absorption spectrum of the substance was measured by JASCO IR
FIG. 1 shows the results of measurement using a spectrum meter model FT / IR-7300. In FIG. 1, A, B, and C represent cyclic heptamer, octamer, and 9-mer of glucose, respectively. As a result, all were 917 ± unique to α-1,6 linkage.
Absorption peaks were shown at 2 cm-1 and 768 ± 1 cm-1. Therefore, it was suggested that the oligosaccharide had α-1,6 bonds.

6. Regarding solubility in solvents, all oligosaccharides were soluble in water at room temperature at a concentration of at least 20 mg / ml or more. 7. The color reaction showed that none of the oligosaccharides developed color by the Somogyi-Nelson method, strongly suggesting that no reducing end was present. 8. These substances are all neutral white substances. 9. As a result of 13 C-NMR analysis of the substance by an NMR spectrometer model NM-FX200 manufactured by JEOL Ltd., only six signals were observed in each case, and the substance supported a cyclic structure. In addition, analysis of isomaltheptaose strongly suggested that the binding mode of the substance was α-1,6 bond.

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% at 40 ° C. for 24 hours, but was not hydrolyzed at all.
Under the same conditions, isomaltohexaose and isomaltoheptaose were completely hydrolyzed. Therefore, it is suggested that it is not a linear isomaltoligosaccharide. When endo-dextranase was allowed to act on the substance at a concentration of 11.1%, it was degraded to glucose and isomaltose. Therefore, it is suggested that these oligosaccharides have glucose as the only constituent sugar and consist only of α-1,6 bonds.

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

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

Next, a method for producing the 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 bacteriological properties of this strain are shown below. Mycological properties (1) Form a. Form Bacillus b. Motility Recognized c. Spores sporangia Swelling Form Elliptical Position Neutral to subvertebrate d. Gram staining + (2) Growing state a. Juice agar Plate culture Smooth, no pigment production b. Gravy agar slope culture Smooth, peripheral smooth, no pigment production (3) Physiological properties a. Nitrate reduction-b. Denitrification reaction-c. MR test-d. VP test -E. Formation of indole-f. Formation of hydrogen sulfide-g. Hydrolysis of starch + h. Use of citric acid-i. Use of inorganic nitrogen source Nitrate-Ammonia + j. Urease-k. Oxidase-l. Catalase + m. Growth range Temperature 10-37 ℃ n. Attitude to oxygen aerobic o. OF test-(no acid production) p. Attitude to saccharides 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 + −

This T-3040 strain was identified as a bacterium belonging to the genus Bacillus because it is a gram-positive bacillus forming spores, and was identified as Bacillus sp.
Stocks. In addition, Bacillus sp. (Bacillus sp.)
The T-3040 strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as No. 4132 (FERM BP-4132).

The cultivation of the strain in the method of the present invention is basically the same as that employed in aerobic cultivation of general microorganisms, but is usually carried out by a shaking cultivation method using a liquid medium or an aeration stirring cultivation method. Is used. As the medium, a medium containing a nitrogen source, a carbon source, vitamins, minerals, and the like used for culturing an ordinary microorganism, and further containing dextran or the like as a raw material of the substance is used.

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

Using the culture thus obtained, a purified product of the present cyclic oligosaccharide is obtained, for example, by the following oligosaccharide collection step. To obtain oligosaccharides from the above culture, any method may be used as long as it is a normal oligosaccharide separation method after removing bacteria by centrifugation, membrane concentration, etc. By treating according to the dextrin purification method, a high-purity cyclic oligosaccharide fraction can be obtained.

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

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

[0024]

EXAMPLE A 3 ml liquid medium (1% dextran T2000, 1% peptone, 0.5% NaCl and 0.1% yeast extract, using tap water, pH 7.0) was placed in a 15 ml test tube, and sterilized at 120 ° C. for 20 minutes. Was performed. In addition, Bacillus sp. T-3040 strain (FERM)
BP-4132) Inoculate one platinum loop from the stock slant, 30 ℃
For 1 day with shaking. 3 ml of the main culture solution was inoculated into a 3 liter minijar containing 2 liters of the medium prepared under the same medium composition and sterilization conditions as above, and was inoculated at 30 ° C., 0.25 vvm, 350 r.
After culturing for 2 days under aeration and agitation under the conditions of pm, the cells were separated from the culture by centrifugation at 8000 rpm for 20 minutes to obtain a disinfecting solution.

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

After concentrating each fraction with a rotary evaporator, glucodextranase, which is an exo-type dextranase, is added at 40 ° C. overnight to remove linear isomatooligosaccharides contaminated as impurities. Reacted. After the reaction was stopped by boiling the reaction solution, the denatured protein was removed by centrifugation, and then YMC PA
Each cyclic isomaltooligosaccharide was separated and purified by HPLC using a 43 column. After concentrating each fraction on a rotary evaporator, the purity of the oligosaccharide in the concentrated solution was determined by TS
HPLC using Kgel Amide 80 column (manufactured by Toso Corporation, packed column for distribution and adsorption chromatography)
(FIGS. 3-B to D). The purity of each oligosaccharide was over 98%. Further, these oligosaccharide fractions were freeze-dried, and cycloisomaltoheptaose was reduced to about 60%.
mg of cycloisomaltooctaose and about 100 mg of cycloisomaltononaose.

[0027]

Industrial Applicability According to the present invention, a novel cyclic isomatooligosaccharide comprising α-1,6 bonds of glucose, which is useful as a clathrate for pharmaceuticals, foods and the like, and a high purity of the cyclic isomaltoligosaccharide by a microorganism of the genus Bacillus The present invention provides a method for efficiently producing the composition, and is extremely useful in industry.

[Brief description of the drawings]

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

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

FIG. 3 is a diagram showing the eluate of three kinds of the compounds of the present invention and the results of analyzing the three kinds of generated compounds by HPLC.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 19/04-19/18 C08B 37/00-37/02 BIOSIS (DIALOG) CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A novel cycloisomaltoheptaose comprising 7 molecules of glucose forming a cyclic structure by α-1,6 bonds, and a novel cycloisomaltoheptaose comprising 8 molecules of glucose forming a cyclic structure by α-1,6 bonds. A novel cyclic isomaltoligosaccharide selected from the group consisting of a novel cycloisomaltononaose, which comprises a cyclic structure of cycloisomaltooctaose and 9 molecules of glucose formed by α-1,6 bonds. 2. A microorganism belonging to the genus Bacillus and having the ability to produce cyclic isomatooligosaccharides according to claim 1, wherein the microorganism is α-1,
A novel method for producing a cyclic isomatooligosaccharide, comprising culturing in a medium containing 6 glucans and collecting the cyclic isomatooligosaccharide according to claim 1 from the culture.