JPH042237B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing branched cyclodextrins.

Cyclodextrin (hereinafter abbreviated as "CD") is an oligosaccharide in which six or more glucose units are α-1,4-linked, with α-CD consisting of six glucose units and β-CD consisting of seven glucose units. −
CD, γ-CD consisting of eight glucose units, is mainly known.

Due to its structure, CD has a cavity in its molecule, and since this cavity is hydrophobic, it has the property of absorbing various oily substances.

Because CDs have such properties, they have a very wide range of uses, and are attracting attention in fields such as the pharmaceutical industry, cosmetics industry, fragrance industry, and food industry.

However, the solubility of CD is low, 14 for α-CD and β
-CD is about 2, and γ-CD is about 23. Especially β-
The solubility of CD is extremely low, and for practical use,
It is a disadvantageous property.

Recently, the present inventors have promoted research on branched cyclodextrins and clarified their properties (Kobayashi et al., Starch Science, 30, 231-239 (1983)).
For example, in terms of solubility, branched CDs are 10 times more soluble than the original CDs.

Therefore, a method for producing branched CDs from starch has been developed, and various branched CDs have been obtained. This method involves involving the branch portions of starch molecules to carry out the cyclization reaction, and has the advantage that various branched CDs can be obtained. However, this method is disadvantageous in obtaining a single branch CD.

Mix CDs and oligosaccharides, apply pullulanase to the mixture, and use a retrosynthetic reaction to branch into various types.
Generating CD has already been reported by French et al. (M.
Abdullah, D.French, Nature, 210, No.5052,
p.200 (1966)). but,
Their report was that pullulanase retrosynthesis was observed on paper chromatography, but the details were unknown.

The present inventors have diligently studied the retrosynthetic reaction of debranching enzymes such as pullulanase, and have completed a practical method for producing branched cyclodextrin.

The present invention relates to a method for producing branched CD, which is characterized in that a mixture of β-CD or γ-CD and maltose is treated with pullulanase,
A mixture of CD and maltose, ethyl alcohol,
Branching characterized by adding an alcohol selected from the group consisting of propyl alcohol and isopropyl alcohol or a glycol selected from the group consisting of ethylene glycol and propylene glycol, and then allowing pullulanase to act.
Method for producing CD and β-CD or γ-CD
A method for producing glucosyl-CD, which comprises reacting a mixture of Taka amylase and glucoamylase and yeast having the ability to assimilate glucose and maltose with a reaction product obtained by reacting pullulanase with a mixture of maltose and maltose. It provides:

In the present invention, as the pullulanase, not only ordinary pullulanase but also heat-resistant and acid-resistant pullulanase can be used. When a thermostable enzyme is used, the solubility of the substrate can be increased, making the retrosynthesis reaction easier. In addition, the enzyme can also be made continuous as a bioreactor by using an immobilized enzyme.

In the first aspect of the present invention, since a mixture of β-CD or γ-CD and maltose is used, it is sufficient to allow pullulanase to act alone, and branched CD can be obtained by treating this reaction product with column chromatography. Maltosyl-CD can be separated.

Further, in another embodiment, alcohol or glycol is used, and the addition of these compounds allows the retrosynthetic reaction by the debranching enzyme to proceed effectively, thereby improving the production rate of branched CDs.
In this case, the amount of alcohol or glycol added is such that the concentration of these compounds in the reaction system is between 10 and 40.
%, preferably 25 to 30%. If the amount added is too small, the above effects will not be fully expressed, and if the amount added is too large, not only will the corresponding effect not be obtained, but the production rate of branched CD will be lower than in the case of no addition. Sometimes. Note that when adding alcohol or glycol to the reaction system, CD and maltose should each be added at high concentrations (approximately
10-15%) to perform the enzymatic reaction.

Furthermore, in the second aspect of the present invention, glucosyl-CD is produced by reacting the reaction product with a mixed enzyme of Takaamylase and glucoamylase and yeast.

In the method of the present invention described above, the retrosynthetic reaction using a debranching enzyme is generally performed at a pH of 4.5 to 6.0 and a temperature of 30 to 50°C.
It may be carried out under the conditions of 24 to 72 hours.

According to the method of the present invention described above, branched CDs consisting mainly of maltosyl-CDs are obtained. Since the first invention uses maltose, branched CDs can be produced more efficiently. Also,
When alcohol or glycol is added to the reaction system, the reaction proceeds effectively and the production rate of branched CDs increases.

When it is desired to obtain a glycol-CD as a branched CD, as in the second invention, a mixed enzyme consisting of Taka amylase and glucoamylase is added to the reaction product by the above method, and yeast having the ability to assimilate glucose and maltose is used. It is sufficient if they act together. Suitable yeasts used in this case include Satucharomyces cerevisiae, Satucharomyces salmon, and Satucharomyces diastateicus. These branched CDs can ferment and remove coexisting glucose and maltose without decomposing CDs.

Next, in order to separate the produced branched CDs such as maltosyl-CDs from the reaction system containing unreacted sugars, in addition to the method described above, the reaction products are heated at a low temperature of 2 to 10°C for 20
It may be left for ~100 hours, preferably 24-72 hours. Alternatively, a precipitate forming agent such as trichloroethylene, tetrachloroethane or bromobenzene may be added to the reaction product and the mixture may be shaken at 5 to 10°C for about 10 to 20 hours. Next, branched CDs can be obtained by performing operations such as centrifugation. These separation methods can also be applied to the separation of branched CDs from mixtures of branched CDs other than maltosyl- or glucosyl-CDs and other sugars, and can also be applied to the separation of branched CDs from mixtures of other sugars, such as carbon,
It is also possible to carry out the method in combination with a method using an exchange resin, a method using a difference in molecular weight such as Sephadex, a separation method using a membrane, and the like.

The branched CD obtained by the present invention can be purified and used as a pure product, and depending on the intended use, the reaction product of the above-mentioned retrosynthesis reaction can also be commercialized as it is. These branch CDs include pharmaceuticals, cosmetics,
It can be widely used for solubilizing fragrances, foods, etc.

Next, the present invention will be explained using test examples and examples.

Test Example 1 γ-CD and oligosaccharides from glucose (G ₁ ) to maltohexaose (G ₆ ) are mixed at a concentration of 20% each (40% total sugars), and commercially available pullulanase crude enzyme is added to this mixture for all groups. Add 200IU per mass (g),
Branched γ-CD was obtained by reacting at 40°C at pH 5.5 for 48 hours. Table 1 shows the production rate of branched γ-CD by various oligosaccharides (percentage relative to the starting substrate as 100).

Table 1 G ₁ G ₂ G ₃ G ₄ G ₅ G ₆ 0 12.5 8.8 6.2 4.1 3.8 Analysis of branched CD was performed using high performance liquid chromatography (HPLC) and paper chromatography. The HPLC conditions were JASCO's "Tri Rotor", 60% and 65% acetonitrile elution, flow rate 2ml/min, detection RI, Attenuation 8X, column: precolumn (diameter: 4.6mm, length: 5cm) and main column (diameter: 4.6 mm, length: 25 cm), column carrier: Fine sil-NH ₂ (10 μ), and the retention time (min.) of each sugar under these conditions is as shown in Table 2. In the table, for example, G ₁ -α-CD is glucosyl-α-CD, G 2 -α-CD is maltosyl-α-CD, and G ₂ -α-CD is maltosyl-α-CD.
It means α-CD.

Table-2 G ₁ 4.5 G ₂ 5.2 α−CD 8.1 β−CD 11.0 G ₁ −α−CD 12.8 γ−CD 14.3 G ₂ −α−CD 16.2 G ₁ −β−CD 17.1 G ₂ −β−CD 21.4 G ₁ −γ−CD 24.9 G ₂ −γCD 28.6 G Branched CDs with ₂ or more branches are
-40" (Super fine) 2.6 x 100 cm column, and then paper chromatography.
Analyzed by HPLC. Furthermore, the same analysis was performed using pullulanase at a substrate concentration of 1% or less. In addition, the sugar produced by the retrosynthesis reaction of pullulanase is 1
It is a branched CD with branches.

Test Example 2 Table 3 shows the production rate (%) of maltosyl-γ-CD when equal amounts of γ-CD and maltose (G ₂ ) were mixed to increase the concentration. In the table, for example, 1
% indicates that each of γ-CD and _G2 is contained at 1%, which is 2% as total sugar.

Table 3 Concentration (%) 1 2 5 10 15 20 40 0.2 0.8 2.6 8.2 11.6 12.5 26.3 Example 1 Mix 200 mg of β-CD and 200 mg of maltose (G ₂ ), and add 80 mg of crude enzyme pullulanase (21 U/mg)
was dissolved in 1 ml of 0.1 M acetate buffer (PH5.5), 0.5 mg of the supernatant obtained by centrifugation was added, and 0.5 ml of water was further added and reacted at 40°C for 48 hours with stirring to obtain G ₂ -β.
-A reaction product containing CD and unreacted sugar was obtained.

This reaction product was divided into two parts, one part was left at 4℃ for 48 hours, and the other reaction product was added with bromobenzene.
200μ was added, shaken overnight at 10°C, and then centrifuged (5000 rpm, 20 minutes) to obtain a supernatant, and the amount of G ₂ -CD in the supernatant was analyzed. In other words, in the supernatant
G ₂ −mol% of CD when the total number of moles of CD is 100
I asked for it as. The results are shown in Table 4.

Table 4 Raw material reaction product left at low temperature Precipitating agent treatment β-CD 1.6 38.6 64.2 Example 2 Mix 300 mg of β-CD and 300 mg of G ₂ and prepare 400 IU of heat-resistant, acid-resistant pullulanase (750 μl of buffer station with pH 4.5 to 5.5)
(dissolved in) 750μ and add ethanol 250μ
As a result of reaction at 70°C for 48 hours, the production rate of G ₂ -β-CD reached 21%. Note that the optimum concentration of ethanol is 25 to 30%, and when the amount added is 45% or more, the production rate was lower than when no addition was made.

Example 3 After heat inactivating 1 ml of the reaction solution of Example 2, the pH was reduced to 4.
After adjusting the sugar concentration to ~5 and adding water to make the total sugar concentration 20%, 2 mg of commercially available crystalline glucoamylase, 1 mg of Taka amylase, and 50 mg of yeast (wet bacterial cells of Satucharomyces cerevisiae) were added and reacted at 30°C for 48 hours. After that, centrifuge to obtain a supernatant, which is concentrated.
_G1 -β-CD was obtained. The purity of this specimen was 78%, and the recovery rate was 65% of the produced G ₂ -β-CD.

In addition, the reaction solution of Example 2 was mixed with glucoamylase,
Taka amylase, passed through yeast immobilization column
G ₁ -β-CD can also be produced.

[Brief explanation of drawings]

Figure 1 is a chromatogram of the fraction eluted with 10% ethanol.

Claims (1)

[Scope of Claims] 1. A method for producing branched cyclodextrin, which comprises causing pullulanase to act on a mixture of β-cyclodextrin or γ-cyclodextrin and maltose. 2. To a mixture of β-cyclodextrin or γ-cyclodextrin and maltose, add an alcohol selected from the group consisting of ethyl alcohol, propyl alcohol and isopropyl alcohol or a glycol selected from the group consisting of ethylene glycol and propylene glycol, and then The manufacturing method according to claim 1, wherein pullulanase is allowed to act. 3. Applying a mixed enzyme of Taka amylase and glucoamylase to a reaction product obtained by applying pullulanase to a mixture of β-cyclodextrin or γ-cyclodextrin and maltose using yeast having the ability to assimilate glucose and maltose. A method for producing glucosyl-cyclodextrin, characterized by: