JPH044877B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel method for producing branched β-cyclodextrin, and more particularly to a method for producing dimaltosyl-β-cyclodextrin. Conventional technology Cyclodextrin has glucose residues α-
Oligosaccharides cyclically bonded through 1,4-bonds, such as α-cyclodextrin consisting of 6 glucose residues, β-cyclodextrin consisting of 7 residues, and γ-cyclodextrin consisting of 8 residues, are generally known. ing. 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 by utilizing these properties, such as stabilizing unstable substances, retaining volatile substances, masking off-odor, and solubilizing difficult-to-dissolve and insoluble substances.However, these cyclodextrins are generally Cyclodextrins are expensive, and this is a major factor preventing them from expanding their use. However, among these, β-
Cyclodextrins are relatively inexpensive compared to other cyclodextrins, and
Since it also has the strongest inclusion effect, it is considered promising in terms of usage. However, such β-cyclodextrin has the drawback of extremely low solubility in water (1.55%, 20°C) at low temperatures (below room temperature), and improvements in this point have been awaited. Problems to be Solved by the Invention Regarding β-cyclodextrin, branched cyclodextrin is already known in which glucose or maltose is bonded to the _C6 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 of β-cyclodextrin could be introduced, it would be possible to improve its solubility. We came up with the idea that β-cyclodextrin and maltose could be combined by doing so, and as a result of further study, we arrived at the present invention. Means for Solving the Problems The present invention involves treating a mixture containing maltose and β-cyclodextrin with pullulanase to produce dimaltosyl-β-cyclodextrin, and separating the produced dimaltosyl-β-cyclodextrin from the reaction solution. consisting of taking
The present invention relates to a method for producing dimaltosyl-β-cyclodextrin. Dimaltosyl-β-cyclodextrin obtained by the present invention is a new compound having the following physical and chemical properties. (1) Molecular formula C ₆₆ H ₁₁₀ O ₅₀ (2) Molecular weight 1783 (3) Melting point 275.0℃ (amorphous; decomposed) (4) Specific rotation [α] ²⁰ _D +174° (C=0.1; H ₂ O) ( 5) Paper chromatography 1-butanol: 1-propanol: water =
One spot is shown in each case, after development on paper using a 3:5:4 developing solvent, color development using an iodine solution, and color development using silver nitrate after pretreatment with glucoamylase. (6) Thin layer chromatography 1-butanol:ethanol:water=5:5:
A thin layer plate (DC-
Fertigplatten Kieselgel 60 (manufactured by Merck & Co., Ltd.)) and color development using iodine solution and phosphomolybdic acid/sulfuric acid, one spot is shown for each. (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 refractometer ERC7520 Type (manufactured by Elma Optical Co., Ltd.) This product shows one peak under the above conditions. (8) Solubility Easily soluble in water (compared to β-cyclodextrin, about 50 times more soluble at 20℃ and 13 times more soluble at 60℃), slightly soluble in ethanol. (9) Properties The powder is white and the aqueous solution is colorless. (10) Infrared absorption spectrum (see Figure 1) ν=3400cm ^-1 , 2930cm ^-1 , 1150cm -1 , 1030
Absorption is observed at cm ^-1 . (11) ¹³ C nuclear magnetic resonance spectrum (see Figure 2) δ (D ₂ O) 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 was hydrolyzed, and the resulting hydrolyzate was reduced and acetylated to lead to alditol acetate, which was analyzed by gas chromatography. When identifying, the molar ratio of 2,3,4,6-tetra-O-methylglucose, 2,3,6-tri-O-methylglucose, and 2,3-di-O-methylglucose is 1.9:7.0 : It was 1.8. (Conditions for gas chromatography) Equipment: Hitachi GC163 (manufactured by Hitachi, Ltd.) Column: 3% ECNSS-M (3φ x 2000 mm) Column temperature: 185°C Carrier gas: _N2 flow rate: 50 ml/min Detector: FID (13) Configuration (Enzymatic decomposition product) Decomposed by pullulanase to produce maltose and β-cyclodextrin at a 2:1 molar ratio
Generate at a ratio of Note that dimaltosyl-β-cyclodextrin having the above-mentioned physicochemical properties has two maltosyl residues in the glucose residue constituting β-cyclodextrin, and α-1,
It was shown to consist of a 6-linked structure 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%, the temperature and pH of the solution are maintained within the enzyme's preferred action range, and the reaction is carried out for 1 to 6 days. It is produced by producing -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 is an enzyme that has the ability to hydrolyze α-1,6-glucosidic bonds in the sticky polysaccharide pullulan, and also to cleave α-1,6-glucosidic bonds in amylopectin and glycogen. , mainly obtained from microorganisms such as Aerobacter aerogenes and Bacillus sp. Although the amount of pullulanase used varies somewhat depending on the quality of the substrate and the method of reaction, it is usually 10 units or more per gram of β-cyclodextrin. The enzymatic activity of this pullulanase is measured by the following method. That is, 50μ of an enzyme solution (dissolved in the same buffer) is added to 200μ of a 0.5% pullulan solution (pullulan dissolved in a 50mM sodium acetate buffer, pH 5.0), and an enzyme reaction is performed 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 1μmole per minute under these conditions.
The amount of enzyme that generates the reducing power equivalent to maltotriose is defined as one 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 β-cyclodextrin to be used is usually 1 to 7 times, preferably 2 to 7 times, and more preferably 3 to 5 times the amount of maltose 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. 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, pH, etc. vary depending on the type (origin) of the enzyme used, but generally it is 40-80℃,
It is preferable to carry out the reaction at a pH of 4.0 to 7.0. The produced dimaltosyl-β-cyclodextrin can be easily separated from the reaction solution by, for example, column chromatography using Toyopearl HW-40S or paper chromatography using Watmann 17 chromium. Industrially, it is advantageous to use ion exchange resins by chromatography, large-scale gel filtration separation methods, etc., especially for cost reasons. Effects of the Invention 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, and cosmetics. It also greatly contributes to the development of applications for cyclodextrins in other general chemical industry fields. Examples Next, examples will be shown to explain the present invention in more detail and concretely. Example 1 Maltose (manufactured by Nippon Starch Industries KK, purity 99%)
2.00 g of β-cyclodextrin (manufactured by Nihon Shokuhin Kako KK, purity 98%), 0.63 ml of 50 mM sodium acetate buffer, pH 5.0, was added and dissolved by heating in a boiling bath. After cooling, this is infused with Bacillus sp.
sp) thermostable pullulanase (manufactured by Novo Industries Japan, 200 units/g) was added,
Incubate at 60°C for 72 hours. After completion, the reaction solution is separated and purified by gel filtration chromatography using columns (4.5 x 100 cm: 2 columns) packed with Toyopearl HW-40S. The fractions eluted 13 to 14 hours after loading the sample are collected and concentrated and dried using a rotary evaporator to obtain 134 mg of a white powder of dimaltosyl-β-cyclodextrin. The elemental analysis values of this product were as follows. Elemental analysis value (C ₆₆ H ₁₁₀ O ₅₅ ) Calculated value C = 44.46% H = 6.22% O = 49.36
% Actual value C=44.35% H=6.24% Also, this product decomposed at 275°C. This obtained powder was subjected to paper chromatography (using a developing solvent of 1-butanol: 1-propanol: water = 3:5:4), color development using an iodine solution, and treatment of the paper once with glucoamylase. Color development using silver nitrate) and thin layer chromatography (using a developing solvent of 1-butanol:ethanol:water=5:5:2, color development using an iodine solution, and using phosphomolybdic acid/sulfuric acid) When color development was carried out, one spot was obtained for each substance, confirming that they were a single substance. In addition, 5 mg of the powder obtained above and 1 unit of Aerobacter aerogenes pullulanase (manufactured by Novo Industries Japan, 200 units/g) were dissolved in 500μ of 50mM sodium acetate buffer, pH 5.0. When reacted overnight at 50°C, 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. Further, 3.8 mg of the powder obtained above was dissolved in 2 ml of dimethyl sulfoxide, 0.5 ml of methyl sulfinyl carbanion was added, and the mixture was reacted for 4 hours at room temperature in a nitrogen stream. Then, 1.5 mg of methyl iodide was added to the reaction solution. ml was added and reacted for 1 hour at 20°C or lower.
After the reaction was completed, the mixture was extracted with chloroform and purified using Sephadex LH-20, which was used as a methylated sample. This sample was dissolved in 0.1 ml of sulfuric acid and reacted at 4°C for 1 hour, and then 0.8 ml of distilled water was added and reacted at 100°C for 4 hours. Neutralize the post-incubation solution with barium carbonate and centrifuge the supernatant using Dowex 50W.
Desalt with (H ⁺ ), add sodium borohydride, leave overnight, and then add Dowex 50W (H ⁺ ) again.
and concentrated under reduced pressure with methanol.
Next, add 0.1 ml of pyridine and acetic anhydride to this.
0.1 ml of the mixture 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 2,3,4,6-tetra-O-methylglucose, 2,3,6-tri-O-methylglucose, 2,3-dimethylglucose, -O-methylglucose was produced in a ratio of 1.9:7.0:1.8 (molar ratio). From the above results, the above substance is dimaltosil-
It was confirmed that it was β-cyclodextrin. Examples 2 to 8 Reactions were carried out according to the same procedure 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.

【table】

【table】 [Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of dimaltosyl-β-cyclodextrin, and Figure 2 shows the infrared absorption spectrum of dimaltosyl-β-cyclodextrin.
^13C nuclear magnetic resonance spectrum is shown.

Claims (1)

[Claims] 1. A solution containing 2 to 7 parts by weight of maltose and a substrate concentration of 40 to 85% per 1 part by weight of β-cyclodextrin is reacted in the presence of pullulanase, and dimaltosyl-β-cyclodextrin is extracted from the reaction solution. A method for producing dimaltosyl-β-cyclodextrin, which comprises separating and collecting dextrin. 2. The production method according to claim 1, characterized in that a solution containing 3 to 5 parts by weight of maltose and a substrate concentration of 60 to 85% per 1 part by weight of β-cyclodextrin is reacted in the presence of pullulanase. .