JPS61287902A - New branched beta-cyclodextrin and its production - Google Patents

Abstract

PURPOSE:To obtain a branched beta-cyclodextrin having strong power of clathrate formation and excellent solubility, by reacting maltose with beta-cyclodextrin in the presence of pullulanase. CONSTITUTION:Pullulanase is added to a solution containing beta-cyclodextrin and 1-7pts.wt., per pt.wt. beta-cyclodextrin, maltose and having a substrate concentration of 40-85%, and the resulting solution is allowed to react at 40-80 deg.C and a pH of 4.0-7.0 for 1-6 days to obtain dimaltosyl-beta-cyclodextrin having the following physical and chemical properties: molecular formula of C66H110O55, MW of 1,783, m.p. of 275.0 deg.C (amorphous, decomposed), specific rotation [alpha]D<20> of + 174 deg. (C=0.1, in H2O), paper chromatography giving one spot in each case when it is developed on a paper by using a developer comprising 1-butanol, 1-propanol and water (3:5:4) and either colored with an iodine solution or pretreated with glucoamylase and colored with silver nitrate; and being easily soluble in water and hardly soluble in ethanol.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel branched β-cyclodextrin and a method for producing the same, and more particularly, to a novel branched β-cyclodextrin and a method for producing the same.
-Regarding cyclodextrin and its manufacturing method.

Obedience! Oral distortion - Cyclodextrins have glucose residues in β-1,4-
Oligosaccharides bonded in a cyclic manner through bonds, such as α-cyclodextrin consisting of 6 glucose residues, β-cyclodextrin consisting of 7 glucose residues, and γ-cyclodextrin consisting of 8 residues, are generally known.

Cyclodextrin has voids inside due to its structure, and since the inside of these voids is a lipophilic region, it can take in various oily substances.

Therefore, various uses are being considered for utilizing these properties, such as stabilizing unstable substances, retaining volatile substances, masking off-odors, and solubilizing difficult or insoluble substances.
These cyclodextrins are generally expensive, and this is a major factor hindering the expansion of their use. However, among these, β-cyclodextrin is relatively inexpensive compared to other cyclodextrins, and also has the strongest inclusion effect, so it is considered promising in terms of use.

However, such β-cyclodextrin
The drawback is that the solubility in water at low temperatures (below room temperature) is extremely low (1.55%, 20°C), and improvements in this point have been awaited.

Regarding α-cyclodextrin, there are already known branched cyclodextrins in which glucose or maltose is bound to the 06 position of the glucose residue through an α-1,6-bond. is known to have higher solubility in water than non-branched species.

The present inventors focused on this point and conducted various studies based on the idea that if a branch could be introduced into β-cyclodextrin, its solubility could be improved. The inventors came up with the idea that β-cyclodextrin and maltose could be combined, and as a result of further studies, they arrived at the present invention.

In order to improve the quality of the product, the present invention allows pullulanase to act on a mixture containing maltose and β-cyclodextrin to produce dimaltosyl-β.
- A method for producing dimaltosyl-β-cyclodextrin, which comprises producing cyclodextrin and separating and collecting the produced dimaltosyl-β-cyclodextrin from a reaction solution, and dimaltosyl-β-cyclodextrin thus obtained. .

Dimaltosyl-β-cyclodextrin obtained by the present invention is a new compound having the following physical and chemical properties.

1) Molecular formula C, 6H, l. 0.5 2) Molecular weight 1783 3) Melting point 275.0°C (amorphous; decomposed) 4) Specific optical rotation [α]2. '+174° (C-0,1; H2O
) 5) Paper chromatography 1-butanol: 1-la-lovanol: water = 3:5
: After development on paper using a developing solvent of 4, one spot is shown for each color development using an iodine solution and color development using silver nitrate after pretreatment with glucoamylase.

6) Thin layer chromatography Using a developing solvent of 1-butyl/ethanol:ethanol:water=5:5:2, a thin layer plate (DC-FertiHplatt
One spot is shown for each color development using iodine solution and phosphomolybdic acid/sulfuric acid.

7) High performance liquid chromatography (conditions) Column size: 6φ×50+am Support: Nucleosil-N13 (manufactured by Nadel)
Solvent Niacetonitrile:Water = 65:35 Flow rate: 2.0
ml/min Detector: Differential spectrometer ERC7520 model (manufactured by Elma Optical Co., Ltd.) This product shows one peak under the above conditions.

8) Solubility Easily soluble in water, slightly soluble in ethanol.

9) Sexual condition The powder is white and the aqueous solution is colorless.

10) Infrared absorption spectrum (see Figure 1) C=3,4
00cm-1, 2,930cm-1, 1,150cm-1
1. Absorption is observed at 1.030 cm-'.

11) 1C nuclear magnetic resonance spectrum (see Figure 2) δ(
020) 68.3 (C6 of 1-6 bond) 78.9 (C< of 1-4 bond of maltosyl residue) 10
0.0 (C+ of 1-6 bond) 12) Methylation analysis After methylation according to the Hakomori method, hydrolysis is performed to form a methylated product, and the resulting hydrolyzate is reduced and acetylated to derive aldidol-acetate. , when identified by gas chromatography, 2,3,4,6-tetra-
The molar ratio of 0-methylglucose, 2,3.6-tri-0-methylglucose, and 2.3-di-0-methylglucose was 1.9 nia, 0:1.8.

(Conditions for gas chromatography) Equipment: Hitachi C163 (newly manufactured by Hitachi) Column: 3% E
CN5S-M (3 φ are produced in a ratio of 2:1 (molar ratio).

Dimaltosyl-β-cyclodextrin, which has the above-mentioned physicochemical properties, has a structure in which two maltosyl residues are a-1°6-bonded to the glucose residue constituting β-cyclodextrin at different positions. This was demonstrated by methylation analysis and enzymatic digestion using pullulanase.

According to the present invention, such dimaltosyl-β-cyclodextrin is produced as follows.

That is, a predetermined amount of pullulanase is added to a solution containing maltose and β-cyclodextrin with a substrate concentration of 40 to 85%,
Maintaining the temperature, pH, etc. of the solution within the preferred action range of the enzyme,
It is produced by reacting for 1 to 6 days to produce dimaltosyl-β-cyclodextrin, and then, if desired, separating and collecting it from the reaction solution by a method such as chromatography.

The pullulanase used in the present invention not only hydrolyzes the ff-1,6-glucoside bond of the sticky polysaccharide pullulan, but also hydrolyzes the 6-116-glucosidic bond of amylopectin and glycogen.
It is an enzyme that has the ability to also cleave glucosidic bonds, and is mainly used in Aerobacter
ter aerogenes), Pasillas 5p (Ba
It is obtained from microorganisms such as Cillus sp. The amount of pullulanase used varies slightly depending on the quality of the substrate and the method of reaction, but in normal cases,
10 or more units are used per gram of β-cyclodextrin. The enzymatic activity of this pullulanase is measured by the following method. That is, 0.5% pullulan solution (
Pullulan was added to 505M sodium acetate buffer, pH 5.0.
Dissolved in ) 200μm and enzyme solution 50μ! (dissolved in the same buffer) and allowed to react with the enzyme at 50°C for 10 minutes. After the reaction, reducing sugars produced in the reaction solution are measured by the Somohyi-Nelson method. The enzyme unit is 1μmo per minute under these conditions.
The amount of enzyme that generates the reducing power equivalent to a)) liose is defined as 1 unit.

Maltose and β-cyclodextrin used as raw materials in the present invention can be commercially available products as they are, but it is advantageous to use ones with high purity in view of the number of steps involved in separating and purifying the product. The amount of maltose and 1β-cyclodextrin used is usually 1 to 7 times, preferably 4 to 5 times, the amount of maltose relative to β-cyclodextrin.

In addition, since the method of the present invention utilizes the condensation reaction of pullulanase, it is generally preferable that the concentration of the raw material substrate is higher, and therefore, the substrate concentration in the present invention is 60 to 85%. It is preferable to use

In the method of the invention, the reaction is carried out in a manner adapted to the operating conditions of pullulanase. Therefore, the reaction temperature, 9
Although there are differences in H and the like depending on the species M (origin) of the enzyme used, it is generally preferable to carry out the reaction at 40 to 80°C and at pH 4.0 to 7.0.

To separate the produced dimaltosyl-β-cyclodextrin from the reaction solution, for example, Toyopearl HW-4O8
This can be easily carried out by using column chromatography using a Whatman 17 chromium or paper chromatography using Whatman 17 chromium, but for cost reasons, ion exchange resin chromatography, large-scale del filtration separation, etc. are used industrially. is advantageous.

The novel branched cyclodextrin obtained by the present invention has a strong adhesion force comparable to that of the known β-cyclodextrin, and has significantly superior solubility, so it can be used in pharmaceuticals, foods, etc. This has greatly contributed to the development of applications for cyclodextrin in cosmetics and other general chemical industry fields.

EXAMPLES The present invention will be explained in more detail and concretely with reference to Examples and 19.

Example 1 Maltose (Nippon Starp Industry KKI!!, purity 99%) 2
．． 00. and β-cyclodextrin (Nihon Shokuhin Kako K)
K! ! , purity 98%) to 0.40g, pH 5.0°50
Add 0.63+al of mM sodium acetate buffer and dissolve by heating in a boiling bath. After cooling, Bacillus sp (B
acillus sp) thermostable pullulanase (Novo.
Manufactured by Industry Japan, 200 units/6) 40
Add 0a+g and react at 60°C for 72 hours.

After completion, the reaction solution was separated and purified by gel filtration chromatography using a column (4,5 x 100 cm: 2 columns) packed with Toyopearl HW-4OS. Collect the 7-lakusilone that was eluted 13 to 14 hours after loading the sample. , concentrated and dried using a rotary evaporator to obtain 134 aa white powder of dimaltosyl-β-cyclodextrin.
get g.

The elemental analysis values of this product were as follows.

Elemental analysis value (CssH+-+.06.) Calculated value C=44
．． 46%1 (=6.22%0=49.38% actual value C=
44.35%H=6.24% Also, this thing is 275℃
It was disassembled.

This obtained powder was subjected to paper chromatography (1-butanol:1-propanol:water=3:
Using a 5:4 developing solvent, color development using an iodine solution and color development using silver nitrate after once treating the paper with glucoamylase) and thin layer chromatography (1-butanol:ethanol:water) = 5:
Using a 5:2 developing solvent, color development using an iodine solution and color development using phosphomolybdic acid/sulfuric acid was performed, and one spot was obtained for each, confirming that it was a single substance. .

In addition, 511 g of the powder obtained above and A erobacter aeroge
nes) pullulanase (manufactured by Novo Ingestry Japan, 200 units/6) 1 unit is 985.0.50
Dissolved in 500 μm of mM sodium acetate buffer and incubated at 50°C.
When the mixture was reacted overnight, it was confirmed by thin layer chromatography and high performance liquid chromatography that maltose and β-cyclodextrin were produced in a ratio of 2:1.

Furthermore, the powder 3,8 trH obtained above was dissolved in 2 ml of dimethyl sulfoxide, 0,5 ml of methylsulfinyl carbanion was added, and the mixture was reacted for 4 hours at room temperature in a nitrogen stream, and then iodide was added to the reaction solution. Methyl 1
．． 5 ml was added and reacted at 20°C or lower for 1 hour. After completion of the reaction, extraction with chloroform, Sephadex LH-2
The methylated sample was purified using 0. This sample was dissolved in 0.1161 sulfuric acid and reacted at 4°C for 1 hour, and then 0.81% distilled water was added and reacted at 100°C for 4 hours.

The reaction solution was neutralized with barium carbonate, the centrifuged supernatant was desalted with DOWEX 50W (H"), sodium borohydride was added thereto, and after standing overnight, the centrifuged supernatant was desalted with DOWEX 50W (H").
) and concentrated under reduced pressure with methanol. This was followed by 0.11 pyridine and 0.1 acetic anhydride.
ml was added and reacted at 100° C. for 2 hours, and the reaction solution was concentrated to dryness together with water under reduced pressure. When this product was dissolved in chloroform and analyzed by gas chromatography, it was found that 2.3,4.8-tetra-0-methylglucose, 2,3.6-tri-0-methylglucose, 2.3
-di-〇-methylglucose 1.9nia, 0:1,8
(molar ratio).

From the above results, it was confirmed that the above substance was dimaltosyl-β-cyclodextrin.

Examples 2 to 8 Reactions were carried out using the same procedure as in Example 1, but with various substrate concentrations, enzyme amounts, reaction temperatures, and reaction times.
The results shown in the following table were obtained.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of dimaltosyl-β-cyclodextrin, and FIG. 2 shows the 1″C nuclear magnetic resonance spectrum of dimaltosyl-β-cyclodextrin.

Claims (1)

[Scope of Claims] (1) Dimaltosyl-β- having the following physical and chemical properties
cyclodextrin. 1) Molecular formula C_6_6H_1_1_0O_5_5 2) Molecular weight 1783 3) Melting point 275.0°C (amorphous; decomposed) 4) Specific optical rotation [α]^2^0_D+174° (C=0
．． 1; H_2O) 5) Paper chromatography After developing on paper using a developing solvent of 1-butanol: 1-propanol: water = 3:5:4, color development using an iodine solution and pretreatment with glucoamylase were performed. When coloring is performed using silver nitrate, 1
Indicates a spot. 6) Thin layer chromatography Using a developing solvent of 1-butanol:ethanol:water=5:5:2, a thin layer plate (DC-Fertigplatt)
One spot is shown for each color development using iodine solution and phosphomolybdic acid/sulfuric acid. 7) High performance liquid chromatography (conditions) Column size: 6φ x 50mm Support: Nucleosil-NH_2 (manufactured by Nagel) Solvent: acetonitrile:water = 65:35 Flow rate: 2.0ml/min Detector: Differential spectrometer ERC7520 type ( (manufactured by Elma Optical Co., Ltd.) This product shows one peak under the above conditions. 8) Solubility: Easily soluble in water, poorly soluble in ethanol. 9) Properties The powder is white and the aqueous solution is colorless. 10) Infrared absorption spectrum (see Figure 1) ν: 3,4
00cm^-^1, 2,930cm^-^1, 1,15
Absorption is observed at 0cm^-^1 and 1,030cm^-^1. 11)^1^3C nuclear magnetic resonance spectrum (see Figure 2) δ(D_2O) 68.3 (C_6 of 1-6 bond) 78.9 (C_4 of 1-4 bond of maltosyl residue) 100.0( 1-6 bond C_1) 12) Methylation analysis After methylation according to the Hakomori method, the methylated product is hydrolyzed, and the resulting hydrolyzate is reduced and acetylated to lead to alditol-acetate, which is then subjected to gas chromatography. 2,3,4,6-tetra-
The molar ratio of 0-methylglucose, 2,3,6-tri-0-methylglucose, and 2,3-di-0-methylglucose is 1.9:7.0:1.8. 13) Composition (enzymatic decomposition product) Decomposed by pullulanase to produce maltose and β-cyclodextrin at a ratio of 2:1 (molar ratio). (2) A method for producing dimaltosyl-β-cyclodextrin, which comprises reacting maltose and β-cyclodextrin in the presence of pullulanase, and separating and collecting dimaltosyl-β-cyclodextrin from the reaction solution.