JPS6211701A - Recovery method of α-cyclodextrin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for recovering α-CD from a mixture containing α-cyclodextrin and other cyclodextrins (hereinafter referred to as rcDJ).

Conventional technology CDs have 6 to 12 glucose molecules α-1,4
- A non-reducing maltoori linked in a cyclic manner through glucoside bonds is a type of sugar. Utilizing its ability to include hydrophobic compounds based on its unique molecular structure, it is widely used in technical fields such as food, medicine, cosmetics, and agricultural chemicals.

These CDs are generally obtained by allowing cyclodextrin glucanotransferase (hereinafter referred to as "CGTase J") to act on starch and/or starch partial hydrolyzate, and include α-CD and β-CD.

It is produced as a mixture in which γ-CD, maltodext, etc. coexist. Methods for separating and collecting each CD from this mixture include methods using various organic solvents (for example, Japanese Patent Application Laid-open No. 51-12941, Japanese Patent Publication No. 46-2380, etc.), and methods using enzymes such as dextrin in the reaction mixture. A decomposition treatment (see Japanese Patent Publication No. 52-43897) is known, which converts β-CD into α-CD by separating and recovering β-CD, which has relatively low water solubility. In addition to the method, as a method for separating and collecting the produced α-CD from the reaction mixture, C
A method in which unreacted β-CD and r-CD are decomposed into glucose, maltose, etc. by inactivating GTaa and then allowing saccharifying α-amylase obtained by culturing Bacillus bacteria to act on the reaction mixture. is also known (Japanese Patent Publication No. 58-18074). However, β-CD
Because it shows resistance to α-amylase decomposition, it is necessary to use a large amount of enzyme or to carry out a long reaction time.

Problems to be Solved by the Present Invention The present invention aims at separating and collecting α-CD from a mixture containing α-CD and other CDs. β-CD, r-CD and various chain molecules reduce the influence of sugars and improve the recovery rate of α-CD. .

As a result of intensive research to solve the above problems, the present inventor discovered that α-CD in a mixture containing α-CD and other CDs
By making amylases act with CGTaae, which has a low affinity for They discovered that scales can be efficiently decomposed into short-chain dextrins, such as glucose, maltose, or maltotriose, and completed the present invention. That is, since a mixture containing α-CD and other CDs can be made into a reaction solution of α-CD and short-chain oligosaccharides by the method of the present invention, a collection method known per se,
For example, α-
CD can be separated and collected.

In the present invention, the mixture containing α-CD and other CDs refers to α-1β-1γ-CD consisting of 6, 7, or 8 α-1,4-′ glycosidic bonded glucose molecules.
It refers to a mixture consisting of α-CD and one or more types of so-called branched CDs, which have α-1,6-glucosidically bonded side chains, and other higher CDs, and α-CDs. The ore may contain sugar. Specific examples include various reaction solutions containing α-9β- and/or γ-CD, etc., which are produced by reacting starch and/or starch hydrolysates with CGT e, and , β-CD, or γ-CD, but all are included as long as they contain α-CD and can achieve the effects of the present invention.

Next, DTasa, which has a low affinity for α-CD, refers to a type of CGTase that preferentially produces β-CD.
ohb@n51m) derived from CGTass (JP-A-4
9-124285), 9 Chirus megatherium (B.

m@gat@rlum)'s CGTaae (% Kaisho 4
8-40996), alkaliphilic bacillus (Ba
cillus) lip.

A38-2, same black 135, wholesaler 169 CGTase (
(Japanese Patent Publication No. 53-31223). Among these, CG derived from Bacillus ohbena1g
Since Tass has extremely low affinity for α-CD,
Suitable for use in the present invention.

Also, amylases include α-amylase (E, C).

3.2.1.1. ), β-amylase (E, C, 3,
2,1,2) Glucoamylase (E, C, 3,2,1,
3) etc., regardless of its origin. Among these, β-amylase and glucoamylase are preferably used.

The conditions under which amylases and CGTaae, which have a low affinity for α-CD, are allowed to act on a mixture containing α-CD and other CDs depend on the composition or composition ratio of the mixture, the type of CGTase used, or the type of amylase. Although it is different and not critical, in the coexistence of CGTase and amylases, the reaction temperature is generally -5 to 8, and the reaction temperature is 30 to 60.
The reaction may be carried out at a temperature of about 1 to 24 hours.

In this way, other CDs other than α-CD are not present in the resulting reaction solution, and since the sugars present are glucose or maltose, α-CD can be efficiently separated using the above-mentioned known recovery method. Can be collected.

Effect of the invention The effects of the present invention will be explained more specifically using experimental examples.

Experimental Example 1 A buffer solution (pH 5.5 or pH 7.0
) 4 Go, C from Bacillus ohbsnsig
A solution containing 0.5 M of GTass solution and each amylase solution Q and 5 d shown in Table 1 was reacted at 60° C. for 5 hours, and the CD composition and the amount of glucose and maltose after the reaction were determined by liquid chromatography. CGTase used in reaction
km Q u /ml, amylases each 10 u
/d, β-amylase (“Amano”, Amano Pharmaceutical)
In the reaction using P) (7 buffer), other amylase, α-amylase (Taka amylase, Sankyo Co., Ltd.)
and glucoamylase (NL-3, Amano Pharmaceutical Co., Ltd.)
So p! (A buffer solution of 5.5 was used. Regarding α-amylase, the concentration of addition was also investigated in the presence and absence of CGTass. As a blank, no enzymes were added, and each enzyme was allowed to act alone. The results are shown in Table 1.

Table 1 α-1β-9γ-CD amylases and CGT
Behavior when using CGTage in combination with amylase As is clear from Table 1, by using CGTage in combination with amylases, CDs other than α-CD are efficiently and completely degraded with almost no effect on α-CD, making it difficult to use them. It has been shown that the remaining linear sugars can be simplified depending on the type of amylase used.

In the target experiment, when α-amylase was used, if an extremely large amount of enzyme was used, most CDs other than α-CD could be degraded, but a trace amount of β-CD remained, and α-C
D Problems remain during collection.

For reference, a case will be described in which CGTase, which has high affinity for α-CD, is similarly used. Bacillusma
CGTa from the culture solution of P. cerans IAM 1227
aa was purified and isolated, and experiments similar to those in Table 1 were conducted.

Bacillus macerans CGTase has a high affinity for α-CD, so when used in combination with other amylases to act on CDs, α-CD is preferentially eliminated, β-CD is also considerably reduced, and γ -CDs are completely gone. Therefore, it was shown that CGTam, which has a high affinity for α-CD, cannot be used for the recovery of α-CD.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited in any way by this example.

Example 1 Peripheral starch 100NK Neospitase (Nagaushi Sangyo [
(manufactured by Co., Ltd.) was o, osy was added, the total amount of water was 11,
After gelatinization by heating at 80°C for 20 minutes, 120°C.

Autoclaved for 10 minutes. Next, cool to 50℃,
Bacillus mae@rans lA11l! 1
500 u of CGTam* obtained from No. 227 was added thereto, and a CD production reaction was carried out at pH 6, 0, and 50° C. for 24 hours. After treating the reaction solution at 100°C for 15 minutes, a portion of it was measured using a liquid chromatograph to measure the amount of CD produced.
-CD is 31.0 units (wt/'wt), β-CD is 12
．． 0 width (wt/vt), r-CD is 3.2% (w
t/Wt).

Bacillus ohb@n51m was added to this CD-containing solution.
CGTas@500 u and β-amylase obtained from
Amine” (manufactured by Ohno Pharmaceutical Co., Ltd.) 0.0625 mj (50
0 u) was added and reacted for 5 hours at 50°C and pH 6°O. The reaction solution composition was α-CD31.0 (wt/
wt), β-CDO, γ-CDO, maltose 56
．． 2 tons, maltriose 2.1%, maltotetraose 10.21%. This reaction solution was concentrated under reduced pressure until the total volume was 3
When the temperature reached 50-, add a small amount of ethanol and
The α-CD was left to stand overnight at ℃, and the crystals were collected in an oven and dried, resulting in a pure α-CD product of 21. I got ON.

This sample was free from any contamination with other CDs and was satisfactory in terms of quality. Glukdeim NL-3.
Add 500u of Amano Pharmaceutical Co., Ltd. and adjust the pHs, s, so
CGTas of Baeillua ohbenais at °C
The reaction was carried out for 5 hours in the presence of *. When the reaction solution composition was quantified by liquid chromatography, it was found that α-CD was 30.8 units (wt/vt), β-CDO was 0, glucose was 61.2 units (wt/wt), and maltose was 7. ．．
6th person (wt/wt) and mourning. This reaction solution was concentrated and crystallized in the same manner as in Example 1 to obtain crystal α-CD22.311◎
This α-CD crystal powder also did not contain any other CDs and was of good quality.

Claims (1)

[Claims] 1. When separating and collecting α-cyclodextrin from a mixture containing α-cyclodextrin and other cyclodextrins, a cyclodextrin glucanotransferase having a low affinity for α-cyclodextrin is added to the mixture. 1. A method for recovering α-cyclodextrin, which comprises reacting it with amylases. 2. Claim 1, wherein the cyclodextrin glucanotransferase with low affinity for α-cyclodextrin is an enzyme having the ability to preferentially produce β-cyclodextrin when it acts on a substrate. the method of. 3. The method according to claim 1 or 2, wherein the amylase is an enzyme capable of cleaving α-type glucoside bonds.