JP3124356B2 - Dextran production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for converting dextran (α-1,6-glucan) used in the field of medicine or biochemistry from starch or a starch hydrolyzate having an α-1,6 bond into enzyme chemistry. To a method for efficient and efficient production.

[0002]

2. Description of the Related Art Dextran has been conventionally produced from sucrose as a raw material, Leuconostoc mesent, a bacterium belonging to the genus Leuconostoc.
It is industrially produced using eroides or dextran sucrase (hereinafter, referred to as DSase) derived therefrom, and is used in fields such as medicine and biochemistry.
DSase produces dextran using only glucose, which is a constituent sugar of sucrose. At this time, fructose, which is also a constituent sugar, is released without being used at all for dextran production. Due to such an action mechanism, when dextran is produced from sucrose using DSase, the yield based on sugar never exceeds 50%. 37% actually
Only moderate yields are obtained.

[0003] Dextrin dextranase (hereinafter referred to as DD)
About 40 years ago, Gluconobacter oxydans ATCC 11894 strain, which is the causative agent of stringing beer,
(Gluconobacter oxydans; American Type Culture Coll
Section Strain No. 11894), it has already been reported that dextran is produced from maltooligosaccharides having an α-1,4 bond having a degree of polymerization of 3 or more (Journal of Japan).
Biological Chemistry, 192, 161-174 (195
1)). According to this report, DDase is α-1,4 with a degree of polymerization of 3 or more.
Dextran is produced by transferring bound linear amylose (including maltooligosaccharides) and glucose residues on the non-reducing end of hydrolysates such as starch. However, even when DDase is allowed to act on various gelatinized starches, no dextran is produced. The yield of dextran from soluble starch is only about 20%. This is because DDase cannot act in the vicinity of the branched portion present in structures such as soluble starch, that is, the structure having α-1,6-linked glucose as a side chain, and many unreacted portions remain. Conceivable. This fact is confirmed by the fact that β-amylase-limited dextrin is not a substrate for DDase.

[0004] As described above, the production yield of dextran by DSase or DDase is low. Therefore, despite the widely accepted safety of dextran, it becomes expensive and is hardly used for food. . If dextran can be supplied at low cost, it can be expected to be used as a highly safe and excellent food polysaccharide. For that purpose, it is necessary to efficiently produce dextran from inexpensive raw materials at a higher yield.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the conventional problems, and it is an object of the present invention to convert dextran used in the field of medicine or biochemistry into starch or α-1. It is an object of the present invention to provide a method for efficiently producing enzymatically from an inexpensive raw material such as a hydrolyzed starch having 6 bonds.

[0006]

The process for producing dextran of the present invention comprises the steps of: providing starch or a starch hydrolyzate having an α-1,6 bond;
A step of allowing a debranching enzyme and dextrin dextranase to act, thereby achieving the above object.

In a preferred embodiment, the debranching enzyme is isoamylase or pullulanase.

The dextrin dextranase (DDase) used in the method for producing dextran of the present invention is Gluconobacter oxydans ATCC 11894 strain (Gluconoba
cteroxydans; American Type Culture Collection stra
in No.11894), Gluconobacter oxydans ATCC
11895 strain (Gluconobacter oxydans; American Type
It is conventionally known as an enzyme present in bacteria such as Culture Collection strain No. 11895). The pathogenicity and the like of these acetic acid bacteria having DDase are not known, and therefore, it is safe to use DDase derived from these bacteria in the processing of food materials. In the present invention, preferably,
The DDase used is purified by the purification method of the present inventors (described in detail in Japanese Patent Application No. 3-69590).

The method for producing and purifying the DDase used in the present invention is, for example, as follows.

First, Gluconobacter oxydans AT
CC11894 strain (Gluconobacter oxydans; American T
ype Culture Collection strain No. 11894), Gluconobacter oxydans ATCC 11895 strain (Gluconoba
cter oxydans; American Type Culture Collection str
ain No. 11895) or other DDase-producing bacteria belonging to Gluconobacter by a conventional method. After the culture, the collected cells are suspended as they are or after cell disruption. As the buffer, it is desirable to use an aqueous solution such as an acetate buffer having a pH at which DDase can stably exist. By removing the cells as necessary, a crude enzyme solution can be obtained.

The crude enzyme solution is purified by the above-mentioned purification method of the present inventors as follows. First, the above crude enzyme solution (which may contain bacterial cells) is extracted with a low-polarity organic solvent and removed. After collecting the aqueous layer and dialysis,
Subject to hydrophobic chromatography. Next, the adsorbed protein is eluted with a buffer containing an alcohol (e.g., ethylene glycol) or the like which can be easily mixed with water. Serve and collect. The low-polarity organic solvent is defined as 5 when the polarity of hexane is 0 and the polarity of water is 9.
It refers to something lower. For example, n-butanol, chloroform and the like are preferably used. When the enzyme adsorbed on the hydrophobic chromatography is eluted, for example, an organic solvent having a polarity of 5 or more is suitably used. Examples of such an organic solvent include alcohols (for example, n-butanol, ethylene glycol and the like), acetone and the like.

The purified DDase that can be used in the present invention has the following features.

1) Action The present enzyme acts on maltooligosaccharides having a degree of polymerization of 3 or more, including amylose, and produces dextran by transferring glucose residues on the non-reducing terminal side of these substrates. This action proceeds until the substance used in the reaction becomes maltose (degree of polymerization = 2). The action of this DDase is the same even when glucose located at the reducing end of maltooligosaccharide is modified by hydrogenation or the like.

2) Optimum pH and stable pH The optimal action pH of DDase is 4.0 to 4.5, and is stable at pH 2.5 to 6.0 when left for 30 minutes under various pH conditions.

3) Optimal temperature and stable temperature The optimal operating temperature of DDase is 37 to 45 ° C, and is stable at 45 ° C or less when left for 30 minutes under various temperature conditions. This enzyme is 55
Active up to ° C.

4) Molecular weight The molecular weight of DDase determined by electrophoresis is about 300,000.

5) Titer measurement method A reduced and low-hydrolyzing starch is used as an enzyme reaction substrate, dextran produced by the reaction is decomposed with dextranase, and the reducing power generated at this time is measured to determine the amount of dextran produced. Ask for. The titer is 1 U of the enzyme when 1 μmol of glucose unit is converted into dextran per minute.
And

The debranching enzyme used in the present invention is an enzyme that cleaves an α-1,6 glucosyl bond existing in the structure of starch, and includes, for example, isoamylase or pullulanase. The starch used in the present invention is an undecomposed starch that has not been decomposed. The term "hydrolyzate of starch" as used herein means an acid hydrolyzate of starch or a hydrolyzate obtained by treatment with an enzyme such as α-amylase, which has α-1,6 bonds in the molecule. The decomposition limit of the starch degradation product is not particularly limited.

In order for the debranching enzyme and DDase to act enzymatically on the starch or starch hydrolyzate, the usual conditions for enzymatic reaction can be employed. For example, the conditions of the enzymatic reaction used in the present invention are preferably pH 3.5 to 5.5, temperature 20 to 4
5 ° C. and reaction time 3 to 72 hours. The debranching enzyme and DDase used at this time may be crudely purified, but preferably, a purified enzyme is used. When a part of the raw material starch or the starch decomposed product having an α-1,6 bond remains unreacted, starch liquefied α-amylase or the like may be further added to decompose it. However, if the reaction conditions are appropriate and the amount of the enzyme and the reaction time are sufficient, the raw material starch or the starch decomposed product having an α-1,6 bond does not remain unreacted, and usually does not remain, such as an ethanol precipitation method. Dextran of high purity can be efficiently obtained only by the method for separating and obtaining polysaccharides.

[0020]

[Effect] DDase acts on α-1,4 bonds in starch molecules, but does not act on α-1,6 bonds. However, it acts on a polysaccharide having only α-1,4 bonds, which is a decomposition product thereof, and produces dextran. When DDase is allowed to act on starch or a starch hydrolyzate having an α-1,6 bond, the α-1,4 linked non-reducing terminal glucose residue present there is converted to α-1,6 to form a dextran molecule. Although the reaction is carried out in a binding manner, the reaction stops near the branched portion present in the structure of the starch hydrolyzate. Therefore, linear α-1,4 oligosaccharides having no α-1,6 bond are good raw materials for dextran production, and the best raw materials are short chains obtained by degrading starch with a debranching enzyme. It is considered amylose. The inventors proceeded further with research and found that dextran can be efficiently produced in a short time by using DDase and a debranching enzyme in combination with starch or a degradation product having an α-1,6 bond and an α-1,4 bond. I found something to do. In the present invention, DD
ase and a debranching enzyme simultaneously act on starch or a starch hydrolyzate having an α-1,6 bond; or It breaks the -1,6 bond, which is then acted upon by DDase. For example, when a DDase and an isoamylase are used, while a short-chain amylose is produced from starch or a starch degradation product having an α-1,6 bond by the isoamylase, DDase acts on the produced amylose to produce dextran. Generate. At this time, if excess isoamylase is used in the reaction, short-chain amylose is generated during the reaction and may precipitate, but this short-chain amylose is finally converted to dextran. Therefore, dextran can be produced at a high yield without any particular problem. Generally, it is recommended to use an excess of isoamylase. On the other hand, when the amount of isoamylase used is small, the time required for the production of dextran increases, but the yield of dextran does not decrease.

Among the debranching enzymes, pullulanase also acts on starch or on the α-1,6 bond of a starch degradation product having an α-1,6 bond, and its action site is on the branching portion existing outside the starch molecule. Limited, does not act on internal branches. Therefore,
It is said that pullulanase alone does not act on starch. When this pullulanase and DDase are used in combination, pullulanase acts on α-1,6 bond existing outside the starch molecule to generate short-chain amylose. The outer straight-chain portion of the formed short-chain amylose and debranched starch is DDase
Is converted to dextran. As the outer straight chain portion of the starch is converted to dextran and shortened, the branched portion existing inside the starch is exposed,
The α-1,6 bond in this part is cleaved by the action of pullulanase, resulting in a short-chain amylose and a straight-chain part, in which DD
ase acts. By this repetition, starch completely disappears and dextran can be produced in high yield. This mechanism is similar to the fact that when starch is used in combination with β-amylase and pullulanase in the production of maltose, pullulanase efficiently acts as a starch debranching enzyme.

As described above, the present invention provides a method for obtaining dextran from starch in a high yield by using a debranching enzyme and DDase.
And the simultaneous action of DDase and the action of DDase during the action of the debranching enzyme. According to the method of the present invention, for example, using a soluble starch as a raw material as a degradation product of starch, the yield of dextran when a debranching enzyme is acted on simultaneously with DDase is the same as the yield of dextran when only DDase is acted on. By comparison, the combined use of DDase and debranching enzyme can produce dextran at a much higher yield. To date, little research has been done on the action of DDase, and the reaction is stopped near the α-1,6 bond in starch, or this part is cleaved by isoamylase or pullulanase.
It has not been revealed that ase works again. These facts were first clarified by the present invention.

Hereinafter, the present invention will be described with reference to examples.

[0024]

【Example】

Example 1 Japanese Patent Application No. 3-69 for 0.75 ml of 4% corn starch
1U / ml DDase 0.75ml and 12,500U obtained by the method of No.590
5 μl / ml isoamylase (manufactured by Amano Pharmaceutical Co., Ltd., trade name “isoamylase“ Amano ””, 1,250,000 U / ml), and add pH
Reaction was carried out at 30 ° C. for 30 hours at 4.8. This reaction solution is
After heating in boiling water for 5 minutes, 4.5 ml of ethanol was added, the mixture was allowed to stand at 4 ° C. for 1 hour, and the precipitate was collected by centrifugation. The precipitate obtained at this time was dried to obtain 19.5 mg of dextran. The yield was 65.0%. Table 1 shows the results of this example. Table 1 also shows the results of Examples 2 to 5, Comparative Examples 1 and 2, and Reference Example 1.

Example 2 0.75 ml of 4% corn starch
0.75 ml of 1 U / ml DDase and 10 μl of 400 U / ml pullulanase (manufactured by Hayashibara Biochemical Laboratories, trade name “Pullulanase”, 40 U / mg protein) as in Example 1 were added thereto, and pH 4.8 was added at 30 ° C. for 30 hours. Reacted. After heating this reaction solution in boiling water for 15 minutes, 4.5 ml of ethanol was added and 4
The mixture was left at room temperature for 1 hour and centrifuged to collect a precipitate.
The precipitate obtained at this time was dried to obtain 10.2 mg of dextran. The yield was 68.0%.

Comparative Example 1 To 2 ml of 2% corn starch, 2 ml of 0.3 U / ml DDase as in Example 1 was added,
The reaction was performed at 0 ° C. for 48 hours. After heating the reaction solution in boiling water for 15 minutes, 8 ml of ethanol was added, the mixture was allowed to stand at 4 ° C. for 1 hour, and the mixture was centrifuged to collect a precipitate. The precipitate obtained at this time was dissolved in 4 ml of distilled water, and 200 U / ml was added thereto.
Liquefied α-amylase (trade name “Fuctamylase”, manufactured by Ueda Chemical Industry Co., Ltd., 300,000 U / g) was added and reacted at 40 ° C. for 1 hour. The reaction solution was left at 4 ° C. for 2 hours and centrifuged. As a result, no dextran precipitate was obtained.

Example 3 0.2 ml of 10 U / ml DDase as in Example 1 and 5 μl of 5,900,000 U / ml isoamylase as in Example 1 were added to 1.8 ml of 30% soluble starch, and pH 4.8 was added.
At 40 ° C. for 66 hours. After heating the reaction solution in boiling water for 15 minutes, 6 ml of ethanol was added and 4
The mixture was left at room temperature for 1 hour and centrifuged to collect a precipitate.
The precipitate obtained at this time was dried to obtain 280 mg of dextran. The yield was 51.9%.

Example 4 0.2 ml of 10 U / ml DDase as in Example 1 and 20 μl of 400 U / ml pullulanase as in Example 2 were added to 1.8 ml of 30% soluble starch, pH 4.8 and 40 ° C.
For 66 hours. After heating the reaction solution in boiling water for 15 minutes, 6 ml of ethanol was added, and the mixture was allowed to stand at 4 ° C. for 1 hour, and centrifuged to collect a precipitate. The precipitate obtained at this time was dried to give dextran 28.6.
mg was obtained. The yield was 52.9%.

Comparative Example 2 To 1 ml of 2% soluble starch, 1 ml of 0.2 U / ml DDase as in Example 1 was added and reacted at 30 ° C. for 16 hours at pH 4.8. After heating the reaction solution in boiling water for 15 minutes, 6 ml of ethanol was added, and the mixture was allowed to stand at 4 ° C. for 1 hour, and centrifuged to collect a precipitate. This precipitate
Dissolve in 0.5 ml of distilled water and add 100 U /
Then, 50 μl of liquefied α-amylase was added, and the mixture was reacted at 37 ° C. for 30 minutes. After heating the reaction solution in boiling water for 15 minutes, 1.7 ml of ethanol was added, and the mixture was left at 4 ° C. for 1 hour.
This was centrifuged to collect the precipitate. The precipitate thus obtained was dried to obtain 4.3 mg of dextran.
The yield was 21.4%.

Example 5 To 3.6 ml of 40% soluble starch, 30 μl of 400 U / ml pullulanase as in Example 2 was added.
After reacting at 40 ° C. for 2 hours with H4.8, 3.6 ml of 1 U / ml DDase as in Example 1 was added thereto, and the reaction was further continued for 60 hours. After heating the reaction solution in boiling water for 15 minutes, 20 ml of ethanol was added, the mixture was left at 4 ° C. for 1 hour, and the precipitate was collected by centrifugation. The precipitate obtained at this time was dried to obtain 745 mg of dextran. The yield was 51.7%.

(Reference Example 1) To 1.5 ml of 4% short-chain amylose, 1.5 ml of 1 U / ml DDase similar to that in Example 1 was added.
The reaction was performed at 0 ° C. for 6 hours. After heating the reaction solution in boiling water for 15 minutes, 9 ml of ethanol was added, and the mixture was heated at 4 ° C. for 1 hour.
This was left for a time and centrifuged to collect the precipitate. The precipitate obtained at this time is dried to obtain dextran.
42 mg were obtained. The yield was 70.0%.

[0032]

[Table 1]

[0033]

According to the present invention, dextran can be produced from starch or a starch hydrolyzate having an α-1,6 bond by a simple operation in a short time.

Claims (1)

(57) [Claims] 1. A starch or a starch hydrolyzate having an α-1,6 bond selected from isoamylase and pullulanase.
A method for producing dextran, comprising the step of reacting a debranching enzyme to be used and dextrin dextranase.