JPS61236801A - Novel branched alpha-cyclodextrin and its preparation - Google Patents

Abstract

NEW MATERIAL:Maltotriosyl-alpha-cyclodextrin. USE:Useful in such fields as drugs, foods and cosmetics, because of improved solubility to water at low temperatures (below room temperature) and having high clathration ability. PREPARATION:Pullulanase is allowed to act, normally at 40-70 deg.C and a pH 4.0-6.0, on a solution with a substrate concentration pref. 50-80%, containing both maltotriose and alpha-cyclodextrin to induce condensation to effect bonding of the maltotriose to the alpha-cyclodextrin followed by separation and collection of the resultant maltotriosyl-alpha-cyclodextrin(molecular formula: C54H90O45, molecular weight: 1459).

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial Application Field The present invention relates to a novel branched α-cyclodextrin and a method for producing the same.
The present invention relates to a-cyclodextrin and a method for producing the same.

b. Conventional technology cyclodextrin has glucose residues α-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.

Due to their structure, these cyclodextrins each have a void inside each molecule of 6 molecules for α-cyclodextrin, 8 A for β-cyclodextrin, and IOA for γ-cyclodextrin, and the inside of this void is a lipophilic region. Therefore, various oily substances can be taken in. Therefore, various uses are being considered by utilizing these properties, such as: 1) stabilizing unstable substances, 2) retaining volatile substances, 2) masking off-odors, and 4) solubilizing difficult to dissolve or insoluble substances.

However, since α-cyclodextrin and β-cyclodextrin have low solubility in water at low temperatures (below room temperature), improvements in this respect have been awaited.

Problems to be solved by the C0 invention Already, in a-cyclodextrin, glucose or maltose is present at the 06 position of the glucose residue.
Branched cyclodextrins bonded by f-1,6-bonds are known, and these are also known to have higher solubility in water than those without branches.

The present inventors focused on the above-mentioned properties of branched a-cyclodextrin, and after conducting various studies, found that by utilizing the condensation reaction of pullulanase, it was possible to combine malto)liose instead of glucose, maltose, etc. The present invention was arrived at as a result of further study and the knowledge that this is a cram school.

Means for Solving the d0 Problems The present invention is directed to producing maltotriosyl-a-cyclodextrin by treating a mixture containing mal)) liose and a-cyclodextrin with pullulanase. - It relates to maltotriosyl-a-cyclomaltotriosyl-a-cyclodextrin, which consists of separating and collecting cyclodextrin from a reaction solution.

Maltotriosyl-a-cyclodextrin obtained by the present invention is also called 6-0-a-maltotriosylcyclohexaose and has a molecular formula of CS4HI. 0
45 This is a new compound with a molecular weight of 1459 and the following physical and chemical properties.

1) Specific rotation [α] 2D, '+171° (C=0.2, H2O
)2) Thin layer chromatography plate a DC-Fertigplatten Kie
selgel 60 (manufactured by Merck & Co.) Developing solvent: (^) 1-butanol: ethanol: water = 5
:5:2 (B) Two 1-butanol:pyridine = 6:4:3 Rf=0.1 (in the case of (^)) 0.4 (in the case of (B)) This product uses an iodine solution. The product was colored using phosphomolybdic acid/sulfuric acid and was confirmed to be Nispot.

3) Paper chromatography 1-butanol: 2 bottles of 1-propanol = 3:5:
The sample was developed three times at 55° C. using the developing solvent No. 4.

This product is colored using an iodine solution, pretreated with glucoamylase, and then colored using silver nitrate.
It was confirmed that the amount was 1 sbo/ ).

4) High performance liquid chromatography (conditions) Column size 26φ x 5011III Support: LiC
hrosorb-NHZ (manufactured by Merck & Co.) Solvent: 2 acetonitrile = 70:30 flow rate: 2. Oml/a+in Detector: Differential refractometer model ERC7520 (manufactured by Elma Optical Co., Ltd.) This product had one peak under the above conditions.

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

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

7) Infrared absorption spectrum @ i see figure 13) IC nuclear magnetic resonance spectrum see figure 2 δ (D20) 68.7 (C5 of 1-8 bond) 78.
5 (0 of glucose bonded α-1,6 at C,
4) 100.1 (CI of 1-6 bond) 9) Composition of α-cyclodextrin and maltotriose Decomposed into maltotriose and α-cyclodextrin by pullulanase (manufactured by Hayashibara Biochemical Research Institute, crystal product), As a result of determining the composition ratio by paper chromatography and high performance liquid chromatography, it was found that liose:α-cyclodextrin=1:1.

Pullulanase (manufactured by Novo Industry Japan)
It was decomposed into maltotriose and a-cyclodextrin, and the composition ratio thereof was determined by paper chromatography and high performance liquid atomography, and it was found that maltotriose: a-cyclodextrin = 1:1.

After decomposition with glucoamylase (manufactured by Seikagaku Corporation, crystalline product) and isolation by paper chromatography, 2 moles of glucose and 1 mole of sugar (glucosyl-a-cyclodextrin), which shows a yellow-orange color when colored with iodine, were obtained. .

According to the present invention, such maltotriosyl-a-cyclodextrin is produced as follows.

That is, a predetermined amount of pullulanase is added to a solution containing maltotriose and α-cyclodextrin with a substrate concentration of 40 to 80%, and the temperature and pH of the solution are maintained within the enzyme's preferred action range and allowed to react for 1 to 6 days. , maltotriosyl-a-
It is produced by producing 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 is an enzyme that not only hydrolyzes the a-its-glucosidic bonds of the sticky polysaccharide pullulan, but also has the ability to cleave the α-1,6-glucosidic bonds of amylopectin and glycogen. Aerobacter aerobac
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 a-cyclodextrin. The enzymatic activity of this pullulanase is measured by the following method. That is, 0.5% pullulan solution (
Pullulan was added to 50mM 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 Somogyi-Nelson method. The enzyme unit is 1 μ5 ole per minute under these conditions.
)) The amount of enzyme that generates the reducing power equivalent to liose is defined as 1 unit.

In the present invention, both liose and Oa-cyclodextrin, which are used as raw materials, can be used as they are as commercially available products. is advantageous. The amount of a-cyclodextrin used is usually 2 to 7 times, preferably 3 to 4 times, the amount of α-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. Therefore, the concentration of the substrate in the present invention is 50 to 80%. 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, although the reaction temperature, pH, etc. vary depending on the species M (origin) of the enzyme used, it is generally preferable to carry out the reaction at 40 to 70°C and pH 4.0 to 6.0.

To separate the generated maltotriosyl-α-cyclodextrin from the reaction solution, for example, Toyopearl HW-4
This can be easily carried out using column chromatography using O8 or paper chromatography using Whatman 17 chromium, but from an industrial perspective, ion exchange resin chromatography, large-scale gel filtration, etc. It is advantageous to use

e0 Effects of the Invention The novel branched a-cyclodextrin obtained by the present invention has a strong adhesion force comparable to that of known a-cyclodextrins, and has much better solubility, so it can be used as a pharmaceutical, This will greatly contribute to the development of applications for cyclodextrins in food, cosmetics, and other general chemical industry fields.

r, Examples Next, examples will be shown to further explain the present invention in detail and concretely.
) Explain in a J-like manner.

Example 1゜Mar)) Liose (manufactured by Nihon Shokuhin Kako KK, purity 99%)
To 5.00 g of i s, oOg and a-cyclodextrin (manufactured by Nihon Shokuhin Kako KK, purity 98%), pH 5.0,
Add 50-M sodium acetate buffer 7.701111 and dissolve by heating in a boiling bath. After cooling, add Bacillus*s
Heat-stable pullulanase of Bacillus sp. (manufactured by Bo Industry Japan, 200 units)
g) Add 2.5g and react at 60°C for 48 hours.

After completion, the reaction solution is subjected to Delfiltration chromatography using columns (2.5 x 100 cm x 2 columns) packed with Toyopearl HW-4OS for separation and purification. 7 Lacrayons eluted 24 to 26 hours after loading the sample were collected and concentrated and dried using a rotary evaporator to obtain a white powder of malt-triosil-a-cyclodextrin.
Get 7 mg.

The elemental analysis values of -2 were as follows.

Elemental analysis value (Cs<L.Ol,) Actual value C=44.47%H=6.20% Also, this product decomposed at 277°C.

Next, this powder was mixed with 070% 1-propanol ■1-butanol:pyridine:water = 6:4:3 ■1-butanol:1-propanol:water = 3:5:4 as a developing agent After development on paper using iodine solution, the paper was treated once with glucoamylase and then silver nitrate color was developed, which gave one spot and confirmed that it was a single substance.

Next, 5 mg of the dead substance obtained above and Aerobacter aerogenes were added.
When 1 unit of pullulanase (manufactured by Taishu Biochemistry Institute, crystal product 40 units/a+g) of es) was dissolved in 500 μm of pH 6, 0, 50 mM sodium acetate buffer and reacted overnight at 30°C, it was found that liose It was confirmed by paper chromatography and high performance liquid chromatography that a-cyclodextrin and a-cyclodextrin were produced in a 1:1 ratio.

Furthermore, the substance 51 obtained above and Rhizopus de1/?
- (Rhizopus delemer) glucoamylase (manufactured by Seikagaku Corporation, crystalline product 30 units/erg)
When 10 units were dissolved in pH 5, 0, 50oM sodium acetate buffer and reacted overnight at 30°C, paper chromatography and high performance liquid analysis revealed that glucose and glucosyl-α-cyclodextrin were produced in a 2:1 ratio. Confirmed by chromatography.

From the above results, the above substance is maltotriosyl-α-
It was confirmed that it was cyclodextrin.

Examples 2 to 13 Reactions were carried out in the same manner as in Example 1, but with various substrate concentrations, enzyme amounts, reaction temperatures, and reaction times, and the results shown in the following table were obtained.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of maltotriosyl-α-cyclodextrin, and FIG. 2 shows the I''C nuclear magnetic resonance spectrum of maltotriosyl-α-cyclodextrin.

Claims (2)

[Claims] (1) Maltotriosyl-α-cyclodextrin (2) Maltotriosyl-α-cyclodextrin, characterized in that maltotriose and α-cyclodextrin are reacted in the presence of pullulanase, and maltotriosyl-α-cyclodextrin is separated and collected from the reaction solution. manufacturing method.