JPH1036406A - Galactosyl-cycloisomaltooligosaccharide and method for producing the same - Google Patents

Abstract

(57) Abstract: A galactosyl-cycloisomaltooligosaccharide in which a galactosyl group is bonded to an hydroxyl group of a glucosyl group of a cycloisomaltooligosaccharide ring by an α or β bond, and a mixture of a galactosyl sugar compound and a cycloisomaltooligosaccharide A galactosyl-cycloisomaltooligosaccharide by producing a galactosyl-cycloisomaltooligosaccharide. [Effect] Galactosyl-cycloisomaltooligosaccharide can be obtained efficiently. This galactosyl-cycloisomaltooligosaccharide can be widely used in the field of medicine, food, cosmetics, and chemical products.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a galactosyl-cycloisomaltooligosaccharide and a method for producing the same.

[0002]

2. Description of the Related Art Cycloisomaltooligosaccharides are cyclic oligosaccharides in which 7 to 9 glucoses are cyclically linked by α-1,6 bonds. Cycloisomaltooligosaccharides have a cavity inside the molecule, and the interior of the cavity is hydrophobic, like cyclodextrin (hereinafter abbreviated as CD), so that it has an inclusion effect and has various oil properties. It has the property of taking in substances. Since cycloisomaltooligosaccharides have such properties, they are expected to be put to practical use in fields such as the food industry, the chemical industry, and the pharmaceutical industry. Cycloisomaltooligosaccharides have a different property from CD and specifically inhibit the action of glucan synthase produced by caries. As a result, the formation of dental plaque, which causes dental caries, is remarkably suppressed, so that it is expected to be used as an anti-carious agent.

Recently, in the field of the pharmaceutical industry, attention has been paid to the cell recognition of sugar chains in order to reduce the side effects of drugs.
Attempts to utilize sugar chains in so-called drug delivery systems that specifically transport pharmaceuticals to specific organs and organs have been vigorously studied, and various CDs already containing various side chains such as galactose on CDs have been studied. Have been synthesized. However, CD has a strong hemolytic activity, and it is difficult to apply this directly to injections and the like. Further, to overcome this difficulty, a branched CD having a branched chain of glucose or maltose has been developed, but has not yet sufficiently overcome the problem of hemolysis.
Cycloisomaltooligosaccharides, on the other hand, have much lower hemolytic activity than CDs, and thus can be sufficiently used for injections and the like.

[0004]

An object of the present invention is to provide a galactosyl-cycloisomaltooligosaccharide and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors aimed to provide a new transport carrier for a drug delivery system by binding galactose having affinity for hepatocytes to cycloisomaltooligosaccharides. An attempt was made to synthesize a galactosyl-cycloisomaltooligosaccharide in which a galactosyl group was directly transferred to the cycloisomaltooligosaccharide ring. As a result, a commercially available α-galactosyltransferase converts an α-galactosyl sugar compound from an α-galactosyl sugar compound to a hydroxyl group of one glucosyl group of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononanose by α-bonding to a galactosyl group. It has been found that transfer-linked galactosyl-cycloisomaltooligosaccharides are produced. In addition, the inventors have found that a commercially available β-galactosyltransferase similarly produces a galactosyl-cycloisomaltooligosaccharide having a galactosyl group transfer-bonded with a β bond, and completed the present invention based on this finding.

That is, the present invention relates to a galactosyl group comprising a galactosyl group bonded to the hydroxyl group of a glucosyl group of a cycloisomaltooligosaccharide ring by an α or β bond.
It is a cycloisomaltooligosaccharide. Furthermore, the present invention provides a method for producing a galactosyl-cycloisomaltooligosaccharide, which comprises reacting a mixture of a galactosyl sugar compound and a cycloisomaltooligosaccharide with a galactosyltransferase to produce the galactosyl-cycloisomaltooligosaccharide. Is the law. The galactosyl-cycloisomaltooligosaccharide of the present invention can be represented by the following structural formulas I to IV.

[0007]

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[0008]

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[0009]

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[0010]

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Cl: Cl-7, Cl-8, Cl-9 The galactosyl-cycloisomaltooligosaccharide of the present invention can be widely used in the fields of foods, cosmetics, chemicals, etc. in addition to the field of pharmaceuticals. First, as the cycloisomaltooligosaccharide to be used as a raw material of the present invention, any kind of cyclic body can be used as long as glucose is a cyclic body having a cyclic body composed of α-1,6 bonds, for example, a derivative, a substituted body, Branched cyclics and the like, preferably cycloisomaltoheptaose, cycloisomaltooctaose, cycloisomaltonononaose and the like are exemplified. These may be used alone or as a mixture.

The method for obtaining the cyclic body may be any method as long as it is a method for producing the cyclic body.
A method for culturing a microorganism that produces the cyclic form from dextran in a medium containing dextran and separating and purifying the cyclic form from the culture, an enzyme produced by the action of cycloisomaltooligosaccharide synthase that produces the cyclic form from dextran After specifically producing from dextran, a method of separating and purifying a cyclic body from a reaction solution may be used. The physicochemical properties of cycloisomaltooligosaccharide used in the present invention, for example, cycloisomaltoheptaose are as follows.

1. The elemental analysis is as follows. _{C 42 H 70 O 34 · 3H} 2 O Calculated C: 42.43% H: 6.44% measured value C: 42.78% H: 6.33% 2. The molecular weight was 1134 based on mass spectrum analysis data analyzed by a mass spectrometer 80B manufactured by Hitachi, Ltd. 3. As a result of measuring the melting point with a melting point measuring device manufactured by Yanagimoto Co., the melting point was not clearly melted, and the decomposition temperature was 234 to 238 ° 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 infrared absorption spectrum was measured with an IR spectrum meter model FT / IR-7300 manufactured by JASCO Corporation. Figure 1 shows the results
Shown in In FIG. 1, 917 ± unique to α-1,6 bond
Absorption peaks are shown at 2 cm-1 and 768 ± 1 cm-1.
Therefore, it was suggested that the oligosaccharide had α-1,6 bonds.

6. Solubility in solvents should be at least 2 at room temperature
It was dissolved in water at a concentration of 0 mg / ml or more. 7. The color reaction did not develop color by the Somogyi-Nelson method, which strongly suggests that no reducing end is present. 8. The substance is a neutral white substance. 9. The NMR spectrum was measured with an NMR spectrometer model NM-FX200 manufactured by JEOL Ltd., and only six signals were recognized from the result of 13 C-NMR analysis, supporting the cyclic structure. In addition, analysis of isomaltheptaose strongly suggested that the binding mode of the substance was α-1,6 bond.

Next, the behavior of cycloisomaltoheptaose with respect to each enzyme is as follows. 10. As shown in FIG. 2, glucodextranase, which is an exo-type dextranase, was added to the substance at a concentration of 1%.
After acting at 40 ° C. for 24 hours, it was not hydrolyzed at all. Under the same conditions, isomaltohexaose and isomaltoheptaose were completely hydrolyzed. 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 results of 1. to 11. above, it can be understood that the substance is a α-1,6-linked cyclic oligosaccharide.

Next, as an example of a method for preparing cycloisomalto-oligosaccharide, a method for preparing the above cycloisomaltoheptaose will be described. The cycloisomaltoheptaose can be produced by a microorganism, and as a microorganism to be used, belongs to the genus Bacillus,
For example, a T-3040 strain that produces cycloisomaltoheptaose from dextran is used. This T-3040 strain is a wild strain obtained from soil, and the following shows the mycological properties of the present strain.

Bacteriological properties (1) Form a. Form Bacillus b. Motility observed c. Spores sporangia Swelling type Oval position Neutral to subvertical d. Gram staining +

(2) Growth state a. Gravy agar plate culture Smooth, no pigment production b. Gravy agar slope culture Smooth, peripheral smooth, no pigment production

(3) Physiological properties a. Reduction of nitrate-b. Denitrification reaction-c. MR test-d. VP test-e. Formation of indole-f. Formation of hydrogen sulfide-g. Hydrolysis of starch + H. Utilization of citric acid-i. Utilization of inorganic nitrogen source nitrate-ammonium salt + 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-Mannit--(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 that forms spores, and was identified as Bacillus sp. T-3040.
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 FERM BP-4132.

This Bacillus sp.
T-3040 strain is used in a culture medium containing a nitrogen source, a carbon source, vitamins, minerals and the like used for cultivation of a normal microorganism, and further containing dextran and the like as a raw material of the present substance, pH 6 to
8. Cycloisomaltooligosaccharides can be obtained in the culture by shaking culture or aeration and stirring culture at a temperature of 20 to 40 ° C., preferably 30 ° C. for 16 hours to 6 days, preferably 3 days.

To obtain cycloisomaltooligosaccharides from the above culture, the bacteria are removed by centrifugation, membrane concentration, etc.
Any method may be used as long as it is a normal oligosaccharide separation method.In the case of the present production method, a high-purity cycloisomalto-oligosaccharide fraction is obtained by treating the sterilized solution according to a known cyclodextrin purification method. Can be done. Next, an example of the biosynthesis of cycloisomaltoheptaose will be described.

Biosynthesis Example 1% Dextran T2000, 1% Peptone, 0.5% N
3 ml of a liquid medium (using tap water, pH 7.0) consisting of aCl and 0.1% yeast extract was placed in a 15 ml test tube, and
Sterilization treatment was performed at 20 ° C. for 20 minutes. In addition, Bacillus
SP. T-3040 strain (FERM BP-4132)
One loopful of platinum was inoculated from the stock slant and cultured with shaking at 30 ° C for 1 day. 3 ml of the main culture was inoculated into a 3 liter mini-jar containing 2 liters of the medium prepared under the same medium composition and sterilization conditions as above, and then inoculated at 30 ° C., 0.25 vvm, and 350 rpm.
Perform aeration and agitation culture for one day.
Separate the cells by centrifugation at 00r.pm for 20 minutes,
A sanitizing solution was obtained.

The sterilized solution is passed through an activated carbon column to adsorb cycloisomaltooligosaccharide, and the solution is added with ethanol to a concentration of 5%.
Eluted step by step. TSKgel Amide80
FIG. 3-A shows the results of analysis by HPLC using a column (packed column for distribution and adsorption chromatography, manufactured by Tosoh Corporation). The target cyclic oligosaccharide was contained most in the fraction eluted with 20% ethanol. Next, the crude cycloisomaltooligosaccharide solution was concentrated by a rotary evaporator, and then concentrated on a YMC PA43 column (manufactured by Yamamura Chemical Co., Ltd .;
Each crude cycloisomal oligosaccharide was separated and purified by HPLC using a packed column for 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 cycloisomaltooligosaccharide was separated and purified by HPLC using 43 columns. After concentrating each fraction with a rotary evaporator, the purity of the oligosaccharide in the concentrated solution was determined by HP using a TSKgel Amide 80 column (manufactured by Tosoh Corporation, packed column for distribution and adsorption chromatography).
Analyzed by LC (FIG. 3-B). The purity of each oligosaccharide was over 98%. Further, these oligosaccharide fractions were freeze-dried, and cycloisomaltoheptaose was reduced to about 60%.
mg was obtained. Other cycloisomaltoligosaccharides, such as cycloisomaltooctaose, cycloisomaltononaose, etc., are similarly prepared.

Next, the α-galactosyl-cycloisomaltooligosaccharide of the present invention is reacted, for example, by adding an α-linked galactosyl sugar compound and an α-galactosyltransferase to a solution containing cycloisomaltooligosaccharide. Can be obtained. On the other hand, when obtaining a β-linked galactosyl-cycloisomaltooligosaccharide, for example, a solution containing cycloisomaltooligosaccharide,
It can be obtained by adding and reacting a β-linked galactosyl sugar compound and a β-galactosyltransferase.

The α-galactosyl sugar compound used in the present invention may be any α-linked galactosyl sugar compound, such as melibiose, phenyl-α-galactoside, paranitrophenyl-α-galactoside, α-galactosyl sugar compound. -Galacto-oligosaccharides and the like. On the other hand, the β-galactosyl sugar compound may be any compound as long as it is a β-linked galactosyl sugar compound, such as lactose, phenyl-β-galactoside, paranitrophenyl-β-galactoside, β-galacto-oligosaccharide, and the like. Is mentioned. These may be used alone or as a mixture.

Next, the α-galactosyltransferase used in the present invention is characterized in that when it is allowed to act on a solution containing an α-galactosyl sugar compound and cycloisomaltooligosaccharide, a sugar donor is decomposed, and The group is directly transferred to the cycloisomaltooligosaccharide by an α bond, and the α-
Any substance may be used as long as it synthesizes galactosyl-cycloisomaltooligosaccharide. Examples include enzymes derived from plants such as coffee beans, α-galactosyltransferases derived from microorganisms such as Aspergillus niger and Escherichia coli. These are commercially available from Sigma.

Similarly, as a β-galactosyltransferase, when it acts on a solution containing a β-galactosyl sugar compound and cycloisomaltooligosaccharide, a sugar donor is decomposed, and the β-galactosyl group is converted into a cyclodextrin. Β-galactosyl-transferred directly to isomaltooligosaccharides by β-linkage.
If it synthesizes cycloisomaltooligosaccharide,
Any type of enzyme may be used, such as enzymes derived from plants such as coffee beans, and β-galactosyltransferases derived from microorganisms such as Penicillium and Bacillus, similar to α-galactosyltransferase. These are commercially available from KI Kasei.

In the reaction system of the present invention, the concentration of cycloisomaltooligosaccharide is 0.2% to 150% (W / V), and the concentration of sugar donor is 0.1% to 100% (W / V). Preferably, and the ratio of the sugar donor to cycloisomaltooligosaccharide is in the range of 0.1 to 100 times, preferably 0.1.
The range is 2 to 5 times. Also, if necessary, methanol,
An organic solvent such as ethanol and ethylene glycol may be added.

The pH of the reaction solution varies depending on the optimum pH of the enzyme to be used. For example, the reaction is carried out in the range of pH 2.5 to 11.0, and the temperature is preferably 10 to 90 ° C., preferably 30 to 60 ° C. It is. The reaction time depends on the amount of enzyme added,
The reaction is carried out for a period of from minutes to 7 days, preferably from about 3 hours to 48 hours. As a method for separating and purifying the galactosyl-cycloisomalto-oligosaccharide produced by the above-mentioned reaction, a normal oligosaccharide purification method is used.
Precipitation with an organic solvent, an activated carbon column, an ODS resin column, etc. are performed alone or in appropriate combination.
Separation and purification are performed using HPLC and a column for HPLC.

[0033]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[0034]

【Example】

Example 1 3.16 g of melibiose and 2 g of cycloisomaltooctaose (CI-8) were dissolved in 14.4 ml of 50 mM phosphate buffer (pH 6.5), and 2.4 mg of α-galactosidase (manufactured by Sigma) derived from coffee beans ( 1 ml) and reacted at 40 ° C. for 48 hours.
A part of the reaction solution is transferred to a YMC Pack Polyamine II column (150 ×
Fig. 4 shows the results of analysis by HPLC using 4.6 mm id).
Shown in After completion of the reaction, the enzyme was inactivated by heat and insolubles were removed by centrifugation. From the supernatant, two types of α-galactosyl-cycloisomaltooctaose A and B
Using an ODS column (DAISO PAK; 250 × 20 mm id)
110 mg and 100 mg, respectively, were isolated by PLC operation.

Example 2 lactose 3.16 g, cycloisomaltooctaose (C
I-8) 2 g was dissolved in 14.4 ml of a 50 mM acetic acid solution (pH 4.5), and Penicillium multicolor was used.
Derived β-galactosidase (KI Kasei Co., Ltd.) 50 mg (1 m
l) was added and reacted at 40 ° C. for 3 hours. Part of the reaction solution is YM
FIG. 5 shows the results of analysis by HPLC using a C Pack Polyamine II column (150 × 4.6 mm id). After completion of the reaction, the enzyme was inactivated by heat and insolubles were removed by centrifugation. From the supernatant, two kinds of β-galactosyl-cycloisomaltooctaose C and D were converted to an ODS column (DAI
180m by HPLC operation using SOPAK; 250 × 20 mm id)
g and 120 mg were isolated.

Example 3 The transfer products (AD) isolated in Examples 1 and 2 were subjected to TOF-MS
Analysis shows that all of these transfer products have a molecular weight of 14
58 (degree of polymerization 9), that is, it was found that one molecule of galactose was bound to CI-8. Next, NMR spectra were measured and all signals were assigned by HH COSY and CH COSY. As a result, α-galactosyl CI-8
In (A and B), the signals of C-3 and C-2 of one glucose of CI-8 are largely down-field shifted, and therefore,
A is 3-O-α-D-galactopyranosyl-CI-8, B is 2
It was found to be -O-α-D-galactopyranosyl-CI-8 (FIGS. 6A and 6B).

Similarly, β-galactosyl CI-8 (C
And D), C-3 and C-2 of glucose in CI-8 were also downfield shifted by one each, so that C was 3-O-β-D-galatopyranosyl-CI-8, and D was It was determined as 2-O-β-D-galactopyranosyl-CI-8 (FIGS. 7C and D).

[0038]

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[0039]

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[0040]

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[0041]

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Example 4 A 50 mM acetic acid solution (pH 4.5) 5 adjusted to a concentration of 0.8 M lactose and 0.2 M cycloisomaltoheptaose (CI-7)
Add 0 μl to Penicillium multicolor
color-derived β-galactosidase (KI Kasei) 50mg (1
ml) was added thereto and reacted at 40 ° C. for 1 hour. A part of the reaction solution was analyzed by HPLC using a TSKgel Amide 80 column (250 × 4.6 mm id), and 3-0-β-D-galactopyranosyl-CI-7 and 2-7 were analyzed by TOF-MS and NMR spectrum. It was confirmed that -0-β-D-galactopyranosyl-CI-7 was produced.

Example 5 0.8M lactose and 0.2M cycloisomaltononaose
(CI-9) 50 mM acetic acid solution (pH 4.5) 50 prepared to a concentration of 50
μl to Penicillium multicolor
lor-derived β-galactosidase (KI Kasei) 50mg (1m
l) was added thereto and reacted at 40 ° C for 1 hour. A part of the reaction solution was analyzed by HPLC using a TSKgel Amide80 column (250 x 4.6 mm id), and analyzed by TOF-MS and NMR spectrum.
It was confirmed that 3-0-β-D-galactopyranosyl-CI-9 and 2-0-β-D-galactopyranosyl-CI-9 were produced.

[0044]

According to the present invention, the hydroxyl group of the glucosyl group of the cycloisomaltooligosaccharide ring is converted to α-1,2-hydroxyl group by utilizing the transglycosylation of α-galactosyltransferase or β-galactosyltransferase. Bond, α-1,3-bond or
A galactosyl-cycloisomaltooligosaccharide having a galactosyl group bonded by β-1,2-bond or β-1,3-bond can be obtained efficiently. The galactosyl-cycloisomaltooligosaccharide of the present invention can be used in the food field,
It can be widely used in cosmetics and chemical products.

[Brief description of the drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of cycloisomaltoheptaose.

FIG. 2 is a diagram showing the results of enzymatic analysis of cycloisomaltoheptaose.

FIG. 3 is a diagram showing the eluate of cycloisomaltoheptaose and the results of HPLC analysis of the produced compound.

FIG. 4 shows the results of analysis of α-galactosyl-cycloisomaltooligosaccharides A and B by HPLC.

FIG. 5 shows the results of HPLC analysis of β-galactosyl-cycloisomaltooligosaccharides C and D.

FIG. 6 shows the results of ¹³ C-NMR analysis of transfer products A and B.

FIG. 7 shows the results of ¹³ C-NMR analysis of transfer products C and D.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Satoru Kitao 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (2)

[Claims] 1. A galactosyl-cycloisomaltooligosaccharide comprising a galactosyl group bonded to an hydroxyl group of a glucosyl group of a cycloisomaltooligosaccharide ring by an α or β bond. 2. A galactosyl-cycloisomaltooligosaccharide according to claim 1, wherein a galactosyltransferase is allowed to act on a mixture of the galactosyl sugar compound and cycloisomaltooligosaccharide. Sugar production method.