JP3009944B2 - Method for producing branched cyclodextrin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for producing a branched cyclodextrin by facilitating fractionation by preferentially enzymatically decomposing cyclodextrin in a mixture of a branched cyclodextrin and a cyclodextrin.

[0002]

2. Description of the Related Art Cyclodextrin (hereinafter abbreviated as "CD") is composed of 6 to 12 glucose molecules of α-1,4.
A cyclic non-reducing maltooligosaccharide linked by a glucopyranosidic bond, industrially 6 glucose molecules, 7 glucose molecules
Α-, β- and γ-CDs, each of which has been bound to one or eight, are considered to be useful.

[0003] These CDs are produced by bacteria of the genus Bacillus or Klebsiella.
D-forming enzyme (Cyclomaltodextrin glucanotransferase,
(Hereinafter abbreviated as CGTase) on starch,
It is produced through purification operations such as an organic solvent precipitation method and various types of chromatography.

[0004] CD is capable of changing various physicochemical or physiochemical effects of a guest compound by incorporating various organic compounds as a guest compound into a cavity in the molecule to form an inclusion compound. , Widely used in industry.

A series of linear or branched maltooligosaccharides containing glucose linked to this CD via an α-1,6-glucopyranoside bond is a branched CD.

[0006] This branched CD forms an inclusion compound similarly to CD. However, while CD is difficult to dissolve in water or an organic solvent, it is easily dissolved in water or an organic solvent, and particularly easy to dissolve in ethanol. Therefore, it is expected to be used for stabilizing perfumes, pharmaceuticals, and the like by inclusion, and solubilization in water and organic solvents.

[0007] Branched CD is produced as a by-product when CDT is produced by allowing CGTase to act on starch (Carbohyd
r. Res., 131, 175-179, 1984, 153, 55
P. 67, 1986, vol. 173, vol. 173, p. 324-331, 1988), as well as reverse synthesis of starch debranching enzymes such as pullulanase and isoamylase using a mixture of high concentration maltooligosaccharide and CD as a substrate. (Nature, 210
Volume, 200, 1966, Arch.Biochem.Biophys., 137
Volume, 483-493, 1970, Carbohydr. Res., 154,
81-92, 1986, Vol.159, 303-313, 1987,
168, 285-294, 1987, 192, 223-23
Page 1, 1989, Agric. Biol. Chem., 159, 303-31
3, 1987, Vol. 52, 1655-1659, 1988, 53
Volume, 2181-2188, 1989, 54, 2585-2591, 19
90 years).

[0008] Based on these findings, the branched CD
Many patents have been filed for the production method of
61-70996, 61-92592, 61-197602, 61-212
No. 297, No. 61-236801, No. 61-236802, No. 61-293395
No., JP-A-2-20502, JP-A-2-21890, Tokuhei 3-20121
No. 3-20122).

In the above method for producing a branched CD, the method described in the above (1) produces a by-product of CD. In the method (2), when the debranching enzyme described in the above-mentioned literature and patent publication is used, the used CD is not used. All of them cannot be converted into branched CDs, and the conversion rate is about 20 to 70%, so that a large amount of CD and maltooligosaccharide remain in the reaction solution. Therefore, the branched CD must be isolated and purified from a mixed solution of the branched CD, CD, and maltooligosaccharide. It is easy to fractionate maltooligosaccharides from these mixtures, but it is difficult to fractionate branched CDs and CDs because they have similar molecular weights and physical properties.

[0010] Some of these fractionation methods are described in the above-mentioned patent publications and publications. For example, JP-A-61-70996, JP-A-61-92592, JP-A-61-197602
No. 61-236801, "Toyopearl HW-40S"
(Trade name, manufactured by Tosoh Corporation) is described. Also, JP-A-61-236
No. 802, Japanese Patent Publication No. 3-20121, and Japanese Patent Publication No. 3-20122 describe that the treatment is carried out by a conventional purification method such as ion exchange chromatography, reverse phase column chromatography such as ODS, or solvent precipitation (however, , Are not specifically described in the examples). Furthermore, Japanese Patent Application Laid-Open No. 2-20502 describes a method using an organic solvent which forms an inclusion compound with CD. Further, the aforementioned Carbohydr. Res., 192
Volume, pp. 223-231, 1989, and Monthly Food Chemical,
5, 27-36, 1989, Kobayashi, Hara et al.
A method for fractionation by adsorption chromatography using a ctadesylated silica) carrier has been proposed. This method
This is a method in which a branched CD and a CD are fractionated from maltooligosaccharides by adsorbing them on a carrier, and then eluted with a 5% ethanol solution to fractionate the branched CD and the CD.

On the other hand, the enzyme that hydrolyzes CD
Several studies have been made on what effect D has. For example, Agric. Biol. Chem., 49, 3391-3
398, 1985, Aspergill
illus oryzae) produced by α-amylase (also referred to as Taka-amylase A) and Thermoactionmyces vulgaris α-amylase on CD and maltosyl CD. Volume 38, Issue 1, Pages 17-22, 1991,
"Fungamyl 800L" (product name, manufactured by Novo Nordisk), which is an industrial Taka-amylase A derived from Aspergillus oryzae, and maltogenase (made by Novo Nordisk) derived from Bacillus subtillis. , CD, glucosyl CD.

[0012]

However, the above-described fractionation by gel chromatography is generally very complicated in operation and low in productivity. Branch C
The molecular weight difference between D and CD was small, and it was practically impossible to fractionate them from each other.

In the fractionation method using ion exchange chromatography or ultrafiltration membrane, fractionation of maltooligosaccharide and CD is possible, but fractionation of branched CD and CD is almost impossible. .

Further, in the method using the ODS carrier, a small amount of CD and branched CD that can be adsorbed on the ODS carrier, and a large amount of ethanol used for fractionation of CD and branched CD are required. There was a problem that the cost was too high.

On the other hand, as described above, various enzymes are
D, how it acts on branched CDs has also been reported, but the enzyme described in Agric. Biol. Chem.
Both enzymes are reported to hydrolyze without discrimination, and the enzymes described in starch science are reported to have a significant difference in the rate of hydrolysis between CD and glucosyl CD for both enzymes. It did not preferentially hydrolyze only the branched CDs and the CDs in the mixture of maltooligosaccharides.

That is, in the conventional method, the branch CD
Are difficult to manufacture on their own, branch CDs and CDs
After the production as a mixture with the above, only the branched CD must be fractionated, but this fractionation treatment is also a complicated and costly method and has a poor yield.

The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for easily and economically producing a branched CD from a mixture of CD and branched CD, that is, a method for producing a mixed CD. To provide a method for producing a branched CD in a high yield by hydrolyzing a CD of the above with an enzyme that preferentially degrades the CD into low-molecular maltooligosaccharides and fractionating the CD by a simple and economical method. It is in.

[0018]

Means for Solving the Problems To achieve the above object, a method for producing a branched CD according to the present invention comprises the steps of: reacting a mixture of branched CD and CD with an enzyme that preferentially hydrolyzes CD; It is characterized by performing fractionation processing.

In a preferred embodiment of the present invention, the enzyme that preferentially hydrolyzes CD is α-amylase TF.
-35 is used.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments.

In the present invention, the CD in the mixture of the branched CD and the CD is preferentially hydrolyzed into a low-molecular maltooligosaccharide, and then the low-molecular maltooligosaccharide and the branched CD are fractionated. By doing so, a branched CD is manufactured.

Examples of the mixture of the branched CD and the CD include, for example, a mother liquor remaining after removing most of the CD by crystallization or other methods after a CGTase is acted on starch, or a high concentration malto-oligosaccharide. A mixture obtained by performing a reverse synthesis reaction with a starch branching enzyme such as pullulanase, isoamylase or the like using a mixture of and CD as a substrate is used. However, the present invention is not limited to these, and can be widely applied to the case of separating a branched CD from a mixture containing a branched CD, a CD, and in some cases, a maltooligosaccharide or the like.

The term "enzyme which preferentially hydrolyzes CD" used in the present invention means an enzyme which degrades CD more selectively than branched CD. The enzyme which decomposes most is used. Preferred examples of such enzymes include, for example,
Α-amylase TF-35 described in No. 3886. This α-amylase TF-35 is an enzyme collected from a culture of Thermomonospora viridis TF-35 (No.
The production method and the physicochemical properties are described in the above-mentioned patent publication, and thus the description thereof is omitted.

The amount of the enzyme used varies depending on the amount, type, substrate concentration, and reaction time of CD in the mixture. In general, this enzyme has a property that it is difficult to decompose α-CD, it is easy to decompose γ-CD, and it degrades β-CD at an intermediate rate between them. Thus, for example, α-CD
Is required to be used in a larger amount than when other types of CDs are decomposed.

The reaction conditions are preferably pH 5 to 7 and temperature of about 40 to 70 ° C., and the reaction time is preferably about 5 to 100 hours.

When α-amylase TF-35 is allowed to act on a mixture of branched CD and CD under the above conditions, CD
Is decomposed into low-molecular-weight maltooligosaccharides containing maltose as a main component, and becomes a mixture of branched CD and maltooligosaccharides.

The method of fractionating the branched CD from this mixture may be a conventionally used method, such as an ultrafiltration membrane treatment, a method using a strongly acidic cation exchange resin, etc. You can fractionate well by the method,
A method using a resin carrier for gel filtration can also be adopted. The ultrafiltration membrane preferably has an average molecular weight cut-off of about 700 to 1500, and more preferably about 1,000.

[0028]

In the present invention, an enzyme that preferentially hydrolyzes CD is allowed to act on a mixture of branched CD and CD, so that branched CD is hardly decomposed, and most of CD is decomposed, and It becomes a molecular maltooligosaccharide and becomes a mixture of a branched CD and a maltooligosaccharide. The mixture of the branched CD and the maltooligosaccharide can be sufficiently fractionated by a simple and economical method such as ultrafiltration membrane treatment and a method using a strongly acidic cation exchange resin. That is, since the molecular weight and the physical properties are similar, fractionation is difficult, and even if complicated and costly fractionation treatment is performed, the branched CD, which could not be sufficiently fractionated, and CD It can be conveniently and economically fractionated, and the branched CD can be obtained in high yield.

Therefore, it is possible to produce a branched CD by an industrially advantageous method, and it is expected to be used for stabilizing by incorporating a fragrance, a medicine, etc., and solubilizing in water and an organic solvent. Can be easily and inexpensively obtained.

[0030]

【Example】

Example 1 Pullulanase "DB-250" (product) was added to 10 L (liter) of a saccharide mixture (A) having a pH of 4.5 containing 1.9 kg of α-CD, 5.6 kg of maltose and 3 mM of calcium chloride. Name, manufactured by Amano Pharmaceutical Co., Ltd.), and reacted at 65 ° C. for 72 hours to obtain α-CD 19.8% by weight.
And maltosyl α-CD 10.3% by weight, and dimaltosyl α
-A saccharide mixture (B) containing 2.7% by weight of CD and 67.2% by weight of other oligosaccharides and containing 30 W / V% solids was obtained.

Next, 50 units of α-amylase T were added to the obtained saccharide mixture (B) per 1 g of the solid content in the mixture.
F-35 (manufactured by Nippon Shokuhin Kako Co., Ltd., 2700 U / mL) was added, and the mixture was reacted at pH 6.5 and 60 ° C. for 70 hours to obtain a saccharide mixture (C).

Comparative Examples 1 and 2 For comparison, 20 units of α derived from Aspergillus oryzae were added to 1 g of the solid content in the saccharide mixture (B) for comparison. -Amylase (trade name “Sumiteam L”, manufactured by Shin Nippon Chemical Co., Ltd., 32
00 U / g) or 1.0 unit of Bacillus subt. (Bacillus subt.) Per 1 g of the solid content in the saccharide mixture (B).
ilis) -derived α-amylase (manufactured by Daiwa Kasei Co., Ltd., 52
000 U / g), and carbohydrate mixtures (D) and (E) were reacted at pH 5.5 in the same manner. These were respectively referred to as Comparative Example 1,
Let it be 2.

The saccharide mixture before decomposition (B), the saccharide mixture of Example 1 (C), the saccharide mixture of Comparative Examples 1 and 2 (D),
For (E), the respective sugar compositions were measured, and the results are shown in Table 1. The sugar composition was measured by high performance liquid chromatography under the following conditions. Column: “Asahi Pack NH2P-50” (trade name, manufactured by Asahi Kasei Corporation) Mobile phase: 65% by weight acetonitrile Flow rate: 0.8 mL / min Detection: IR detector

[0034]

[Table 1]

From the results in Table 1, it can be seen that α-amylase TF-35 was allowed to act on a saccharide mixture comprising α-CD, maltosyl α-CD, dimaltosyl α-CD, and other oligosaccharides. In Example 1, most of 99.5% of α-CD is hydrolyzed, but maltosyl α-CD and dimaltosyl β-CD, which are branched α-CDs,
Only 17.5% and 3.7% are decomposed, the majority of which is 82.5
% And 96.3% are not decomposed. On the other hand, in Comparative Examples 3 and 4, the branch α
-It turns out that CD is also decomposed | disassembled.

Subsequently, "SD-100" (trade name, manufactured by Sumitomo Bakelite Co., Ltd.), which is an ultrafiltration membrane having an average molecular weight cut off of 1,000, was added to the saccharide mixture (C) of Example 1 while adding water. Let pass, maltosyl α-CD 75.3% by weight,
A component composed mainly of branched α-CD, having a composition of 23.0% by weight of dimaltosyl α-CD, 0.6% by weight of α-CD, and 1.1% by weight of other maltooligosaccharides was obtained.

EXAMPLE 2 10 L of a saccharide mixture (F) containing 1.25 kg of β-CD, 6.25 kg of maltose and 3 mM calcium chloride and having a pH of 4.5 was prepared by adding:
Pullulanase was added and reacted as in Example 1.
5.5% by weight of β-CD and 6.6% by weight of maltosyl β-CD
Then, a saccharide mixture (G) containing 30% w / v solids, comprising 1.6% by weight of dimaltosyl β-CD and 86.3% by weight of other oligosaccharides, was obtained.

Next, 20 units of α-amylase T were added to the obtained saccharide mixture (G) per 1 g of solids in the mixture.
F-35 was added and reacted under the same conditions as in Example 1 to obtain a saccharide mixture (H).

Comparative Examples 3 and 4 For comparison, as in Comparative Examples 1 and 2, Aspergillus oryza was added to the above saccharide mixture (G).
e) derived α-amylase (trade name “Sumiteam L”, manufactured by Nippon Chemical Co., Ltd., 3200 U / g), or α-amylase derived from Bacillus subtilis (Daiwa Chemical Co., Ltd., 52000 U / g) g) was added thereto, and the mixture was reacted in the same manner to obtain saccharide mixtures (I) and (J). These are Comparative Examples 3 and 4, respectively.

The saccharide mixture (G) before decomposition, the saccharide mixture (H) of Example 1, the saccharide mixture (I) of Comparative Examples 1 and 2,
For (J), the respective saccharide compositions were measured under the same conditions as in Example 1, and the results are shown in Table 2.

[0041]

[Table 2]

From the results in Table 2, it can be seen that α-amylase TF-35 was allowed to act on a saccharide mixture comprising β-CD, maltosyl β-CD, dimaltosyl β-CD, and other oligosaccharides. In Example 2, most of 98.2% of β-CD is hydrolyzed, but maltosyl β-CD, a branched β-CD, is degraded only 19.7%, and dimaltosyl β-CD is not decomposed at all. It can be seen that most or all of them, 80.3% and 100%, are not decomposed. On the other hand, in Comparative Examples 3 and 4, the branched β-CD was decomposed together with the β-CD.

Example 3 10 L of a saccharide mixture (K) containing 1.9 kg of γ-CD, 5.6 kg of maltose and 3 mM of calcium chloride and having a pH of 4.5 was prepared by adding:
Pullulanase was added and reacted as in Example 1.
12.9% by weight of γ-CD and 12.8% by weight of maltosyl γ-CD
And a sugar mixture (L) containing 5.2% by weight of dimaltosyl γ-CD and 69.1% by weight of other oligosaccharides and containing 30 W / V% solids.

Then, 1.0 unit of α-amylase TF-35 was added to 1 g of the solid content in the obtained saccharide mixture (L), and the mixture was reacted under the same conditions as in Example 1. Thus, a saccharide mixture (M) was obtained.

Comparative Examples 5 and 6 For comparison, as in Comparative Examples 1 and 2, Aspergillus oryza was added to the above saccharide mixture (L).
e) derived α-amylase (trade name “Sumiteam L”, manufactured by Nippon Chemical Co., Ltd., 3200 U / g), or α-amylase derived from Bacillus subtilis (Daiwa Chemical Co., Ltd., 52000 U / g) g) was added and reacted in the same manner to obtain a saccharide mixture (N), (O). These are referred to as Comparative Examples 5 and 6, respectively.

The saccharide mixture before decomposition (L), the saccharide mixture of Example 1 (M), the saccharide mixture of Comparative Examples 1 and 2 (N),
For (O), the respective saccharide compositions were measured under the same conditions as in Example 1, and the results are shown in Table 3.

[0047]

[Table 3]

From the results in Table 3, it can be seen that α-amylase TF-35 was allowed to act on a saccharide mixture containing γ-CD, maltosyl γ-CD, dimaltosyl γ-CD, and other oligosaccharides. In Example 3, 97.7% of the majority of γ-CD was hydrolyzed, but maltosyl γ-CD and dimaltosyl γ-CD, which are branched γ-CDs, respectively,
Only 64.1% and 28.8% were decomposed, and 35.9% and 71.2% were not decomposed. On the other hand, in Comparative Examples 5 and 6, it can be seen that the branched γ-CD was decomposed together with the γ-CD.

Examples 4 to 6 Carbohydrate mixtures (B), (G) comprising the branched CD prepared in Examples 1, 2 and 3, a CD and an oligosaccharide and having a solid content concentration of 30 w / v%, To (L), 2 units of glucoamylase (trade name "Gluczyme Special", manufactured by Amano Pharmaceutical Co., Ltd., 3000 units / g) were added to 1 g of the solid content in the mixed solution, and the pH was 5 to 5.5 and 55 ° C. And reacted for 30 hours under the conditions described above to convert maltosyl CD and dimaltosyl CD into
Glucosyl CD and diglucosyl CD were used, respectively.

Each of the saccharide mixtures (B '), (G') and (L ') obtained above was mixed with the solid content 1 in the mixture.
1.0 unit of α-amylase TF-35 was added to g, and the mixture was reacted under the same conditions as in Example 1 to obtain saccharide mixtures (P), (Q), and (R). These are referred to as Examples 4, 5, and 6, respectively.

After the glucoamylase treatment described above, α
-A saccharide mixture (B ') before amylase TF-35 treatment,
(G ′), (L ′) and the saccharide mixture (P), (Q) of Examples 4, 5, and 6 after the treatment with α-amylase TF-35
For (R), the sugar composition was measured under the same conditions as in Example 1, and the results are shown in Table 4.

[0052]

[Table 4]

From the results in Table 4, it can be seen that the branched CD is glucosyl C
D, even if it is a diglucosyl CD, a branched CD and a CD
And α-amylase TF-35 act on the mixture with
It can be seen that CD is hydrolyzed with little hydrolysis of glucosyl CD and diglucosyl CD.

[0054]

As described above, according to the present invention,
An enzyme that preferentially hydrolyzes CD is allowed to act on a mixture of branched CD and CD to decompose CD into low-molecular-weight maltooligosaccharides. It can be obtained in yield. That is, branch C
D can now be produced in an industrially advantageous manner. Therefore, the use of the branched CD for applications such as stabilizing perfumes and pharmaceuticals by inclusion and solubilization in water and organic solvents is expected.

Claims (2)

(57) [Claims] 1. A process for producing a branched cyclodextrin, comprising reacting a mixture of a branched cyclodextrin and a cyclodextrin with an enzyme that preferentially hydrolyzes the cyclodextrin, followed by fractionation. 2. The method for producing a branched cyclodextrin according to claim 1, wherein the enzyme is α-amylase TF-35.