JP2002065291A - Method for manufacturing glucan having cyclic structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for manufacturing a cyclic structure- containing glucan having excellent properties higher in solubility in water when compared with a known starch, low in the viscosity of a solution prepared by dissolving the glucan, free from the aging which is observed in a common starch and useful as an alternative substance to starch. SOLUTION: An α-glucan is cyclized by using an enzyme which cuts an α-1,4-glucoside bond and forms a new α-1,4-glucoside bond by a transfer reaction, and can not use glucose as a receptor of the transfer reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is useful as a raw material in the starch processing industry, a composition for food and drink, a composition for food addition, an inclusion or adsorbate forming agent, or a substitute for starch for biodegradable plastics. , Glucan or a derivative thereof. More specifically, the present invention relates to a method for producing a glucan having one cyclic structure having at least 11 α-1,4-glucoside bonds in a molecule, or a derivative thereof.

[0002]

BACKGROUND OF THE INVENTION (Problems of conventional starch and cyclic glucan) Conventionally, starch is maltose, starch syrup,
Alternatively, it is a polymer material used as a raw material for production of cyclodextrin or the like, a composition for eating and drinking, a composition for adding food, an inclusion or adsorbate forming agent, or a material for a biodegradable plastic. However, existing starches suffer from poor solubility, aging, and high viscosity. Specifically, starch generally has low solubility in water. Therefore, in order to dissolve the starch, it is necessary to perform a heat treatment or a treatment with an organic solvent, an acid, an alkali, or the like. The dissolved or gelatinized starch rapidly ages and forms an insoluble precipitate. Aging of starch changes the viscoelasticity of the starch solution, the physical properties such as the adhesiveness of the starch, or in foods containing starch,
It causes problems such as reduced water retention, shape retention, freezing resistance, or digestibility.

[0003] Further, gelatinized starch has a high viscosity. This is due to the fact that amylopectin in starch is a very long molecule with a large number of tufted structures. When starch is used as a raw material for producing maltose or cyclodextrin due to its high viscosity, there is a problem that handling is difficult. For example, when gelatinized starch having a certain concentration or more is used, a transport pipe may be clogged during production.

[0004] Such properties (low solubility, aging, and high viscosity) of existing starches have limited the use of starch in food and other industries. Therefore, studies have been made to improve the solubility and aging resistance by reducing the molecular weight of these starches, and it has become possible to prevent aging to some extent. However, it is difficult to suppress an excessive decrease in the molecular weight, and there has been a problem that the inherent properties of the original starch, which is a polymer, are lost. Furthermore, in these methods, the reducing power of starch increases, and when mixed with proteins or amino acids, the starch is colored by the reaction with these substances when heated. I was On the other hand, studies have been conducted to improve the solubility of these starches without reducing their molecular weight, cutting the α-1,4-bond of starch and synthesizing α-1,6-bond by a transfer reaction. Water-soluble starch has been obtained by reacting an enzyme (Q-enzyme, EC 2.4.1.18), a branching enzyme, with starch (JP-A-60-75295). But,
The water-soluble starch obtained by this method is a high-molecular substance having the same molecular weight as the starch used as a raw material, and has not solved the above-mentioned problems of starch.

On the other hand, it is conceivable to use a cyclic polysaccharide composed of D-glucose, that is, a cyclic glucan, as an alternative to the above-mentioned starch. As this cyclic glucan, cyclodextrins having a degree of polymerization of 6 to 8 are already known, and their use as an inclusion compound-forming agent has been particularly developed. For example, it is used in various industrial fields as an inclusion compound forming agent for the purpose of stabilizing volatile substances, emulsifying or solubilizing hardly soluble substances, and preventing oxidation of unstable substances. On the other hand, cyclic glucans having a higher degree of polymerization have also been developed (JP-A-8-134104, JP-A-8-3104).
11103). These cyclic glucans are highly water-soluble, the aqueous solution does not age, and has low viscosity, so it is a glucan having no problems inherent in starch, and it is a composition for eating and drinking, a composition for adding food, and a clathrate. Alternatively, it is used as an adsorbate-forming agent or a material for biodegradable plastics. Furthermore, since it is a polymer higher than cyclodextrin, it can be applied to a compound that cannot be included by cyclodextrin, particularly as an inclusion or adsorbate-forming agent. Conventionally, the following three methods have been used as a method for producing cyclic glucan having a higher molecular weight than cyclodextrin (hereinafter, simply referred to as cyclic glucan).

(Conventional Method for Producing Cyclic Glucan and Problems Thereof) (Method Using D Enzyme) Method for Producing Cyclic Glucan Using D Enzyme (also called Dispositioning Enzyme or Amylomaltase; EC 2.4.1.25) Are known (JP-A-8-311103, JP-A-11-46780, etc.). This cyclic glucan production process is shown in FIG. That is, when the D enzyme acts on α-glucan such as amylose or starch, a part of the substrate glucan chain is cyclized. After this cyclization step, the D enzyme is inactivated by heat treatment or the like. Then, exo-amylases such as glucoamylase and β-amylase, and debranching enzymes such as isoamylase and pullulanase act to decompose unreacted substrates, and purify cyclic glucan by operations such as membrane treatment and ethanol precipitation. can do.

[0007] In the method using the enzyme D, cyclic glucan can be obtained at a very high yield, particularly when high-molecular amylose is used as a substrate. For example, Thermus aquaticus
It has been disclosed that the yield of cyclic glucan can reach 80% or more when amylose having a molecular weight of 110,000 is used as a substrate by using a D enzyme derived from the enzyme (Terada et al., Appl. Envionment. Micr.
obiol. 1999 Vol 65, p.910-915). Further, at the time of this reaction, the molecular weight of the cyclic glucan can be controlled by controlling the amount of addition of the D enzyme and the reaction time. For example, when the reaction is stopped by a heat treatment or the like at the beginning of the reaction, a macromolecular cyclic glucan having a degree of polymerization of about 50 or more can be produced. On the other hand, by allowing the reaction to proceed sufficiently, a relatively low-molecular cyclic glucan having a degree of polymerization of about 20 can be produced.

However, when starch or dextrin, which is a cheaper substrate, is used, the yield of cyclic glucan is low. For example, using a potato-derived D enzyme, when using waxy corn starch as a substrate, the yield of a cyclic glucan consisting of only α-1,4-linkage is 15.2%, α-1,
It is disclosed that the yield was 31.7% even when a cyclic glucan containing a 6-linkage was added (Takaha et al., Biochem. Bioph.
ys. Res. Commun. 1998 Vol 247, p. 493-497). Also,
In this case, there is also a problem that it is difficult to produce a macromolecular cyclic glucan having a degree of polymerization of about 50 or more. That is, it is shown that the degree of polymerization of the cyclic glucan obtained under any reaction conditions is about 20.

Further, as a factor affecting the yield of cyclic glucan, a purification step after the cyclization step can be mentioned. In this purification step, exo-amylases such as glucoamylase and β-amylase and debranching enzymes such as isoamylase and pullulanase act to decompose unreacted substrates, but before this, the D enzyme is completely inactivated. You need to keep it. That is, when a small amount of the active D enzyme is present, a cyclic glucan ring-opening reaction using glucose or the like as an acceptor generated in the purification step occurs, and the yield of cyclic glucan is extremely reduced. About this ring opening reaction
Takaha et al., J. Biol. Chem. 1996 Vol. 271, p. 2902-29.
08 mag. Therefore, it is necessary to completely inactivate the D enzyme by an operation such as heating the reaction solution after the cyclization step.

(Method using CGT) A method for producing a cyclic glucan using CGT (cyclodextrin glucanotransferase; EC 2.4.1.19) is also known (Japanese Patent Laid-Open No. 9-2).
89, etc.). That is, CGT is known as an enzyme for producing cyclodextrin, which is a low-molecular cyclic sugar, but also generates a cyclic glucan having a degree of polymerization of 16 or more, particularly in the early stage of the reaction. In the production of cyclic glucan using this CGT, there is a problem that the yield from starch and the like is low, a problem that a complete inactivation step is required, and a problem described above in the method using the above-mentioned D enzyme. Another problem is that fractionation from the coming cyclodextrin is required.

(Method using branching enzyme) Branching enzyme (also called Q-enzyme, branching enzyme. EC 2.4.
1.18) is also known for the production of cyclic glucan
(For example, JP-A-8-134104). However, the cyclic glucan obtained by this production method has an outer branched structure, and when it is intended to obtain a glucan having only a cyclic structure, the yield is, for example, when waxy corn starch is used as a substrate. About 5% (Takata et al., Carbohydr. Res. 1996, V
295, p. 91-101), which is as low as about 15% even when amylose is used as a substrate (Takata et al., J. Bacteriol. 1
996, Vol. 178, p. 1600-1606). As described above, the conventional method for producing cyclic glucan has various problems, and a simple method for producing cyclic glucan has been desired.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems. Compared to existing starches, it has higher solubility in water, lower viscosity of the dissolved solution, and excellent properties such that the aging observed in ordinary starch does not occur. It is intended to provide a new manufacturing method.

[0013]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, glycogen debranching enzyme, which has never been considered to be able to produce cyclic glucan, has been hitherto considered. (EC 2.4.1.25/EC
3.2.1.33, hereafter referred to as GDE), which led to the present invention by finding that α-glucan can be cyclized with high efficiency.

That is, as shown in FIG. 2, GDE was originally considered to work with glycogen phosphorylase to degrade glycogen. As expected from this physiological function, the best substrate for GDE is GDE
It is a glycogen sufficiently decomposed by P and is not considered to act well on amylose or starch, and was never expected to catalyze the cyclization reaction. Furthermore, the enzyme activity having the activity of cleaving the α-1,4-glucosidic bond and synthesizing a new α-1,4-glucosidic bond by a transfer reaction as in GDE, and transferring glucose. It was not thought that enzymatic activity that cannot be used as a receptor for the reaction (hereinafter simply referred to as α-1,4-transferring activity) could be used to produce cyclic glucan, and was not used.

The present inventors cyclized α-glucan using α-1,4-transferring activity to obtain a polymerization degree of 50%.
It has been found that it is easy to produce a high-molecular cyclic glucan of a degree or higher, and that even if glucose is present in the reaction system, the yield of the cyclic glucan does not decrease, that is, the above-mentioned problem can be solved, and the present invention was completed. Things. Therefore, the present invention is a method for producing a glucan having at least one cyclic structure comprising at least 11 α-1,4-glucosidic bonds in a molecule, comprising a saccharide having an α-1,4-glucosidic bond, A method comprising the step of reacting with an enzyme having α-1,4-transfer activity. Further, the present invention is a method for producing a glucan having one cyclic structure in a molecule comprising at least 11 α-1,4-glucoside bonds, and a saccharide having an α-1,4-glucoside bond, reacting with an enzyme having α-1,4-transfer activity and an enzyme having an activity of hydrolyzing α-1,6-glucosidic bond (hereinafter referred to as α-1,6-degrading activity). Is the way.

[0016] In a preferred embodiment showing the characteristics of GDE as an enzyme in the following, the α-1,4-transfer activity is an activity by GDE. In a preferred embodiment, α-1,6-
Degradation activity is amylo-1,6-glucosidase activity. In a preferred embodiment, the α-1,6-degrading activity is
It is the activity of an enzyme capable of releasing glucose from glucosyl-cyclodextrin. In a preferred embodiment,
α-1,4-transfer activity and α-1,6-degrading activity are enzyme activities derived from a single polypeptide. In a preferred embodiment, the α-1,4-transfer activity and α-1,6-degrading activity are enzyme activities derived from GDE. In a preferred embodiment, the GDE
Is derived from a fungus such as a filamentous fungus or yeast. In a preferred embodiment, the GDE is from yeast. In a preferred embodiment, the GDE is from Saccharomyces cerevisiae.

Hereinafter, the present invention will be described in more detail. The enzyme having α-1,4-transfer activity used in the present invention includes, for example, GDE. GDE is known to be widely distributed in mammals, filamentous fungi and yeast, and it can be used as it is or purified to the extent that it does not contain an enzyme that cleaves α-1,4-glucosidic bond in endo form. Can be As the GDE, preferably, Saccharomyce such as baker's yeast and brewer's yeast
GDE from S. cerevisiae can be used. GDE is known as an enzyme having two active centers (Gillard
Et al., J. Biol. Chem. (1980) 255, p. 8451-8457). That is, one active center catalyzes the α-1,4-transfer activity,
The other active center catalyzes α-1,6-degrading activity. It is known that enzymes of this type can be cleaved into two polypeptides having their respective activities (eg, Ara et al., Biosci. Biotechnol. Biochem. (1996) 60
Volume, p634-639, α-1,6-glucosidic bond decomposition activity,
An example is described in which two polypeptides having respective activities were obtained from one polypeptide having α-1,4-glucosidic bond decomposing activity). Therefore, by reducing the size of GDE using a proteolytic enzyme or using genetic engineering techniques, the resulting GDE-derived protein having only α-1,4-transfer activity can also be used. . Further, as an enzyme having α-1,4-transfer activity,
Any enzyme that can not use glucose as a receptor for the transfer reaction can be used. For example, Thermotoga
4-alpha-glucanotransferase from maritima or
4-α-glucanotransferase from Bacillus subtilis (Takaha and Smith Biotechno).
l. Genet. Eng. Rev. (1999) 16: pp. 257-280).

GDE is also an enzyme having α-1,6-degrading activity, which is advantageous for producing cyclic glucan. However, GDE is isoamylase (EC 3.2.1.68) and pullulanase (EC 3.2.
1.41) and other α-1,6-glucoside bond-degrading enzymes,
Other enzymes having amylo-1,6-glucosidase activity can also be suitably used.

As a raw material used in the present invention, a saccharide having an α-1,4-glucoside bond can be used. Such saccharides include starch. As the starch, any starch which is generally commercially available can be used. For example, underground starch such as potato starch, sweet potato starch, waste starch, tapioca starch and above-ground starch such as corn starch, wheat starch and rice starch can be used. Starch can include amylopectin, amylose. A partially decomposed product of starch can also be suitably used. Examples of the partially decomposed products of starch include those obtained by partially hydrolyzing the above-mentioned starch with an enzyme, an acid, or the like, and branched products of starch. In addition, as the raw material, derivatives such as the above-mentioned starch or partially decomposed products of starch may be used. For example, at least one of the alcoholic hydroxyl groups of the starch is glucosylated,
Alkylated, acetylated, carboxymethylated, sulfated or phosphorylated derivatives and the like can also be used. Further, a mixture of two or more of these can also be used as a raw material. As a raw material of the present invention, the above-mentioned starch, a saccharide having only an α-1,4-glucoside bond, α-
A saccharide having a 1,4-glucoside bond and an α-1,6-glucoside bond can be used. Examples of the saccharide having only an α-1,4-glucoside bond include amylose, partially decomposed products of starch, branched products of starch, amylose synthesized by phosphorylase, and maltooligosaccharides. α-1,4
-As a saccharide having a glucoside bond and an α-1,6-glucoside bond, starch, partially degraded starch, amylopectin, glycogen, waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolyzate, phosphorylase Saccharides having an α-1,6-branched structure such as enzyme-synthesized amylopectin.

In the method for producing a glucan or a derivative thereof according to the present invention, the step of reacting the above-mentioned raw material with an enzyme having α-1,4-transfer activity is carried out by using a glucan or a glucan having at least one cyclic structure in the molecule. The reaction can be carried out by selecting reaction conditions such as pH, temperature, amount of enzyme and the like which can produce the derivative. The concentration of the raw material (substrate concentration) can also be determined in consideration of the reaction conditions. When GDE is used as the enzyme having α-1,4-transfer activity, the amount of the enzyme to be used is generally about 0.01 to about 1000 units per gram of the substrate, preferably about 0.1 to about 5 units.
00 units, more preferably about 0.5 to about 500 units. The pH during the reaction is usually about 2 to about 10. Reaction rate, efficiency,
From the viewpoint of the stability of the enzyme, preferably about 3 to about 9,
More preferably, from about 4 to about 8. The reaction temperature is about 20
C. to about 105.degree. C., preferably from about 30.degree. C. to 90.degree. C., more preferably about 40.degree. C. to 70.degree. C., from the viewpoints of reaction rate, efficiency, enzyme stability and the like.

By the above reaction, glucan having one cyclic structure in the molecule can be obtained. Here, the fact that cyclic glucan is produced by the method of the present invention will be specifically described. FIG. 3 shows the change over time in the amount of cyclic glucan and the change in iodine coloration of the reaction solution when GDE was allowed to act on enzyme-synthesized amylose. The amount of cyclic glucan in the reaction solution was determined by measuring the amount of glucose not decomposed by glucoamylase. Further, after the product at each time point in FIG. 3 was treated with glucoamylase, the product was analyzed by a sugar analysis system (solution sending system: DX300, detection end: PAD-2, column: CarboPacPA100) manufactured by Dionex. Elution was performed at a flow rate of 1 ml / min and a NaOH concentration of 1
50 mM, sodium nitrate concentration: 0 min-8 mM, 2 min-
8 mM, 2.1 min-8 mM, 13 min-26 mM (Gra
dient curve No. 3), 35 minutes-70 mM
(Gradient curve No. 4), 50 minutes-
200 mM (Gradient curve No.
7) for 52 minutes-200 mM.

As shown in FIG. 4, cyclic glucans having a degree of polymerization of at least 50 or more are mainly produced in the initial stage of the reaction. As the reaction proceeds, low-molecular cyclic glucan increases, and finally, cyclic glucan having a degree of polymerization of 11 or more accumulates. In the present invention, the raw material may be mixed with glucose. After completion of the reaction, the enzyme which degrades glucan from the non-reducing end and an enzyme which cleaves the α-1,6-glucoside bond but does not cleave the α-1,4-glucoside bond are reacted with each other to form α-1. , 4-
A glucan consisting only of a cyclic structure having a glucoside bond can be produced. In the present invention, it is not always necessary to add a step of inactivating the enzyme having α-1,4-transfer activity after the completion of the reaction. Examples of enzymes that decompose glucan from the non-reducing end include β-amylase and glucoamylase. cleaves the α-1,6-glucosidic bond,
Enzymes that do not cleave α-1,4-glucosidic bonds include isoamylase and pullulanase. The glucan having a cyclic structure obtained as described above is then known to those skilled in the art such as precipitation using a common solvent (eg, methanol, ethanol), membrane separation, chromatographic separation (eg, gel filtration chromatography, HPLC) and the like. It can be purified by a separation method. The present invention also relates to a method for producing a glucan derivative using an enzyme having α-1,4-transfer activity.
As the raw material saccharide used in the above method, at least one of the alcoholic hydroxyl groups of the raw material saccharide is glucosylated, hydroxyalkylated, alkylated, acetylated, carboxymethylated, sulfated, or phosphorylated. The glucan derivative can be produced by using an enzyme having α-1,4-transfer activity.
Various known methods can be used for derivatization. For example, phosphorylated glucan can be obtained by reacting the glucan obtained above with phosphorus oxychloride in dimethylformamide.

The fact that the glucan obtained by the production method of the present invention has a cyclic structure can be confirmed by the following properties (1) to (5). (1) Neither the reducing terminal nor the non-reducing terminal can be detected. (2) Not degraded by β-amylase (manufactured by Seikagaku Corporation) or glucoamylase (manufactured by Toyobo Co., Ltd.), which is an exo-type amylase that hydrolyzes the α-1,4-glucosidic bond at the non-reducing terminal. . 3) Decomposition by combined use of isoamylase and pullulanase (both manufactured by Hayashibara Biochemical Laboratory) which hydrolyze α-1,6-glucosidic bond in starch, or combined use of isoamylase, pullulanase and β-amylase Not done. (4) It is completely degraded by α-amylase (manufactured by Nagase Seikagaku Co., Ltd.), which is an endo-type amylase that hydrolyzes α-1,4-glucoside bonds in starch molecules. (5) Bacterial saccharification type α-amylase (Nagase Biochemical Co., Ltd.)
Hydrolysis and HPLC analysis yield only glucose, maltose, and some maltotriose. That is, there is no bond other than the α-1,4-glucoside bond.

The quantification of the number of reducing ends was determined by Hizukuri et al., Ca.
According to the modified Park Johnson method of rbohydr. Res. 94: 205-213 (1981), quantification of non-reducing ends was determined by Hizukuri et al., Carboh.
ydr. Res. 63: 261-264 (1978). Degradation by β-amylase and glucoamylase, which are exo-type amylase, or isoamylase, pullulanase, or α-amylase, which is an endo-type amylase, is, for example, the cyclic α-amylase of the present invention.
After dissolving -1,4-glucan in distilled water to a concentration of 0.1% (W / V), take 100 μl, add an appropriate amount of each of the above-mentioned degrading enzymes, and react at 30-45 ° C. for several hours.
The reaction product was subjected to a sugar analysis system manufactured by Dionex (liquid sending system: DX300, detector: PAD-2, column:
Carbo Pac PA100).
For elution, for example, the flow rate is 1 ml / min, and the NaOH concentration is 1
50 mM, sodium nitrate concentration: 0 min-8 mM, 2 min-
8 mM, 2.1 min-8 mM, 13 min-26 mM (Gra
dientcurve No. 3), 35 minutes-70 mM
(Gradient curve No. 4), 50 minutes-
200 mM (Gradient curve No.
7), performed for 52 minutes-200 mM, and the degree of polymerization and the resulting sugar can be analyzed. The degree of polymerization of cyclic α-1,4-glucan can be measured using chromatography. Already, the structure and chromatographic behavior of cyclic glucan obtained using CGT have been studied in detail (Koizumi et al., J. Chromatogr. A. (1999) 852,
p. 407-416), the degree of polymerization of the cyclic polysaccharide can be determined by comparing the elution positions of the respective molecules.
Also, by using laser ionization TOF-MS, it is possible to determine the degree of polymerization and to prove that the compound is cyclic (Koizumi et al., J. Chromatogr. A. (1999) 852, p. 407).
-416).

The GDE activity was measured using 60 μl of 30 mM
Glucosyl-β-cyclodextrin solution (20 mM
(Adjusted to pH 6.0 with sodium acetate buffer) into a thermostat set at 30 ° C. in advance, and then add 60 μl of GDE solution to this solution to start the reaction. 3
After the reaction for 0 minutes, the amount of glucose produced in the reaction solution is quantified by the glucose oxidase-peroxidase method. At this time, a blank to which GDE previously inactivated by heating was added is prepared as a blank, and the same operation is performed. One unit of the activity was defined as the amount of an enzyme that produced 1 μmol of glucose per minute under these conditions. When an enzyme other than GDE is used as the enzyme having α-1,4-transfer activity, the activity can be adjusted using the change in iodine coloration as an index. This is because, as shown in FIG. 3, there is an inverse phase relationship between the amount of cyclic glucan synthesized and the degree of iodine coloration.

[0026]

EXAMPLES (Example 1) (Preparation of enzyme having α-1,4-transfer activity) In this example, GDE derived from Saccharomyces cerevisiae was used. Sac
As charomyces cerevisiae, a raw yeast block commercially available for bread making was used. Add 2 kg of raw yeast block to 4 L of 10 mM Tris-HCl buffer (pH 7.5, 1 mM Phe
Suspend in nylmethylsulfonyl fluoride and use Dynomill (600ml grinding container, 40m
The cells were disrupted using a continuous operation at 1 / min). The collected cell lysate was centrifuged, and ammonium sulfate was added to the collected supernatant so as to be 30% saturated. After leaving at 4 ° C. for 2 hours, the supernatant was recovered, ammonium sulfate was further added to 70% saturation, and the mixture was left overnight at 4 ° C., and the precipitate was recovered. Precipitate 10 mM Tris-H
It was dissolved in a Cl buffer (pH 7.5), dialyzed, and then heat-treated at 50 ° C for 20 minutes. After removing insolubles, 20 mM Tris-HCl buffer (pH
7.0) Equilibrated Q-Sepharose column (diameter 5 cm, length 20 cm column, Amersham Pharmacia Biotech)
Was washed with the same buffer containing 0.1 M NaCl. Thereafter, the NaCl concentration in the buffer was increased linearly to 0.5 M, and the eluted GDE-active fraction was collected. The active fraction was dialyzed against 20 mM phosphate buffer (pH 6.8),
Hydrox equilibrated with 10 mM phosphate buffer (pH 6.8)
The sample was applied to a yapatite column (1.5 cm diameter, 10 cm length column, manufactured by Nippon Bio-Rad Laboratories). The phosphate buffer concentration was increased linearly to 160 mM and GDE
The active fraction was collected. Ammonium sulfate was added to the active fraction to a final concentration of 0.5 M, and a Phenyl-TOYOPEARL column (1.6 cm in diameter, length 1.6 mm) equilibrated with 50 mM Tris-HCl buffer (pH 7.0) containing 0.5 M ammonium sulfate GDE activity was eluted by applying a 10 cm column (manufactured by Tosoh Corporation) to linearly reduce the concentration of ammonium sulfate in the buffer to 0M. The obtained active fraction was subjected to 20 mM Tr
Dialyze against is-HCl buffer (pH 7.0) and purify about 53 units
Got GDE.

(Example 2) (Preparation of cyclic glucan having only α-1,4-glucosidic bond using GDE) Enzymatically synthesized amylose AS-30 with 0.2% (average molecular weight 30,000, Nakano vinegar Co., Ltd.) 5)
ml and 2.3 units of GDE prepared in Example 1 were allowed to act for 24 hours at 40 ° C. After heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. Ten units of glucoamylase were added to 5 ml of the supernatant, and reacted at 40 ° C. for 18 hours to remove non-cyclic amylose. After the reaction solution was again heated at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation, and a 9-fold amount of ethanol was added to the supernatant, followed by centrifugation. The obtained precipitate was dissolved in distilled water and freeze-dried.
In this way, 5.2 mg of cyclic glucan having only an α-1,4-glucoside bond was obtained. The cyclic glucan thus obtained was analyzed using a sugar analysis system (Dionex Co., Ltd .: DX300, detector: PAD-2, column: CarboPacPA100).
Elution: flow rate: 1 ml / min, NaOH concentration: 150 m
M, sodium nitrate concentration: 0 min-8 mM, 2 min-8 m
M, 2.1 min-8 mM, 13 min-26 mM (Gradi
ent curve No. 3), 35 minutes-70 mM (G
radiant curve No. 4), 50 minutes-20
0 mM (Gradient curve No. 7), 5
The test was performed for 2 minutes at -200 mM. The result is shown in FIG. From this comparison, the peak of the product obtained by GDE completely coincided with the peak of the degree of polymerization of 11 or more among the cyclic glucans obtained by CGT. From the above, it was found that the peak of the product by GDE was a cyclic glucan having only an α-1,4-glucoside bond, and its polymerization degree was 11 or more.

(Example 3) (Confirmation that the substance of Example 2 is a cyclic glucan having only an α-1,4-glucoside bond) Quantification of reducing end and non-reducing end Obtained in Example 2 (1981) Carbohydr. Res: 94: p.
205-213 Park Johnson modified method. Quantification of non-reducing ends was determined by Hizukuri et al. (1978) Carbohydr. Res.
: 63: p. 261-264 by rapid Smith decomposition method. As a result, neither reducing terminal nor non-reducing terminal could be detected. Digestion with Exo Enzyme and α-1,6-Glucoside Bond Degrading Enzyme The powder obtained in Example 2 was dissolved in distilled water so as to have a concentration of 0.1% (w / v). Add 1 unit of each of the following starch-degrading enzymes and add 2 units at 40 ° C.
Allowed to react for hours. The reaction product was analyzed under the same conditions as in Example 2 using a sugar analysis system manufactured by Dionex. The powder obtained in Example 2 was obtained from β-amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.), which are exo-type amylases that hydrolyze α-1,4-glucosidic bonds at the non-reducing terminals of starch molecules. Was not decomposed. Further, a combination of isoamylase (manufactured by Hayashibara Biochemical Laboratories) and pullulanase (manufactured by Hayashibara Biochemical Laboratories) which hydrolyzes the α-1,6-glucoside bond in starch, or isoamylase is used. It was not degraded by the combined use of pullulanase and β-amylase. However, this powder was completely decomposed by α-amylase (manufactured by Nagase Seikagaku Kogyo Co., Ltd.), which is an endo-type amylase that hydrolyzes α-1,4-glucosidic bonds inside starch molecules. (3) Digestion with endo-type enzyme The lyophilized sample obtained in Example 2 was dissolved in distilled water so as to have a concentration of 0.1% (V / V), and 100 μl of this aqueous solution was added to bacterial saccharified α-amylase ( 1 unit of Nagase Seikagaku Corporation)
The reaction was performed at 0 ° C. for 2 hours. Analysis of the reaction by HPLC showed only glucose, maltose, and some maltotriose. From this, it was proved that the powder obtained in Example 2 had no bond other than the α-1,4-glucoside bond, and the obtained lyophilized sample was identified as α-1,4-glucoside. It was found to be a cyclic glucan having only a bond.

(Example 4) (Preparation of cyclic glucan using various substrates)
2% enzymatically synthesized amylose AS-5,10,30,7
0, 110, 320 (average molecular weight: 5,000, 10,
000, 30,000, 70,000, 110,000, 320,000, manufactured by Nakano Vinegar Co., Ltd.), short-chain amylose EX-I (average molecular weight 2,700,
Example 1 with 5 ml of Hayashibara Biochemical Laboratory Co., Ltd.) or amylopectin (potato-derived, Type III, Sigma)
2.3 units of GDE prepared in the above was allowed to act for 24 hours at 40 ° C. After heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. Add 10 units of glucoamylase to 5 ml of the supernatant, and add
For 18 hours to remove acyclic amylose. After the reaction solution was again heated at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation, and a 9-fold amount of ethanol was added to the supernatant, followed by centrifugation. The obtained precipitate was dissolved in distilled water and freeze-dried. The obtained cyclic glucan was analyzed under the same conditions as in Example 2 using a sugar analysis system manufactured by Dionex. As shown in FIG. 6, it was found that a cyclic glucan having a degree of polymerization of 11 or more was generated from any of the substrates.

(Example 5) (Amount of cyclic glucan produced using starch as a substrate and molecular weight of cyclic glucan when GDE is used) 0.1% low-resolution dextrin (starch is slightly reduced by α-amylase. Disassembled product name: Paindex # 10
0, manufactured by Matsutani Chemical Co., Ltd.) and GD prepared in Example 1.
E 2.3 units were incubated at 40 ° C. After sampling over time and heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. Supernatant 5m
To this were added 100 units of glucoamylase and 944 units of isoamylase and reacted at 40 ° C. for 18 hours to remove acyclic amylose. After the reaction solution was heated again at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation, and a 9-fold amount of ethanol was added to the supernatant, followed by centrifugation to prepare a cyclic glucan. As shown by the black circle curve in Fig. 7, the yield of cyclic glucan
It rose to 6 hours and reached about 60%. The cyclic glucan obtained from the reaction solution at that time was analyzed under the same conditions as in Example 2 using a sugar analysis system manufactured by Dionex. As shown in FIG. 8A, cyclic glucan having a degree of polymerization of 40 or more was significantly detected, and cyclic glucan having a degree of polymerization of 50 or more was clearly detected. The distribution pattern of the degree of polymerization did not change much from the beginning to the end of the reaction.

Comparative Example 1 (Amount of Cyclic Glucan Produced Using Starch as Substrate and Molecular Weight of Cyclic Glucan When Using Conventional Method) In order to compare with Example 5, a conventional method was used. A cyclic glucan was synthesized from the same substrate as in Example 5. As a cyclizing enzyme, the yield of cyclic glucan is the best among known enzymes (Takaha and Smith, (1999), Biotechnology an).
d Genetic.Engineering Reviews, vol. 16, p. 257-280)
A potato-derived D enzyme is used.
4104. Reaction is 3500 units of D
Performed at 30 ° C. using enzymes. As shown by the black square curve in FIG. 7, when the enzyme D was used, the yield of cyclic glucan was at most about 15%. Furthermore, to increase the yield of cyclic glucan, 5900 units of isoamylase were allowed to act simultaneously with the D enzyme as a debranching enzyme. As shown by the black diamond curve in FIG. 7, the yield of cyclic glucan increased, but was at most about 38%. Further, cyclic glucan obtained from the reaction solution after 6 hours was analyzed under the same conditions as in Example 2 using a sugar analysis system manufactured by Dionex. As shown in FIGS. 8B and 8C, regardless of the presence or absence of the debranching enzyme, the pattern of the degree of polymerization did not change, and only a very small amount of cyclic glucan having a degree of polymerization of 40 or more was produced. Also,
No cyclic glucan having a degree of polymerization of 50 or more was detected at all. The distribution pattern of the degree of polymerization did not change much from the beginning to the end of the reaction. From the results of Example 5 and Comparative Example 1, the use of an enzyme having α-1,4-transferring activity, particularly GDE, provides a high yield from inexpensive starch and a relatively high molecular weight as compared with the conventional method. It was found that cyclic glucan was obtained.

(Example 6) (Preparation of cyclic glucan in the presence of glucose)
0.1% enzyme synthesized amylose AS-30 (average molecular weight
30,000, manufactured by Nakano Vinegar Co., Ltd.)
%, 0.01% or 0.1%, and 5 units of GDE prepared in Example 1 was heated at 40 ° C.
For 1 hour or 2 hours. Reaction solution is 10
After heating at 0 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. 100 units of glucoamylase was added to 10 ml of the supernatant and reacted at 40 ° C. for 18 hours to remove acyclic amylose. Again, the reaction solution was
After the heating, the denatured enzyme protein was removed by centrifugation, and a 9-fold amount of ethanol was added to the supernatant, followed by centrifugation to prepare a cyclic glucan. As shown in FIG. 9, the yield of cyclic glucan was not affected at all by the presence or absence of glucose.

(Comparative Example 2) (Preparation of cyclic glucan in the presence of glucose using conventional method) To compare with Example 6, the synthesis of cyclic glucan was performed in the same manner as in Example 6 using the conventional method. went. As the cyclizing enzyme, the D enzyme prepared in Comparative Example 1 was 3
A similar experiment was performed using 50 units and a reaction temperature of 30 ° C.
As shown in FIG. 10, the presence of a very small amount of glucose drastically reduced the yield of cyclic glucan. From the results of Example 6 and Comparative Example 2, an enzyme having α-1,4-transfer activity
In particular, it was found that when GDE was used, cyclic glucan could be efficiently synthesized from a substrate mixed with glucose, and the step of inactivating the cyclizing enzyme before the step of purifying cyclic glucan could be omitted.

[0034]

According to the method for producing glucan of the present invention, at least 11 α-1,4-α-glycans can be very easily prepared using GDE, which was conventionally considered unusable.
Glucan having one cyclic structure having a glucoside bond in the molecule or a derivative thereof can be produced. In that case, not only amylose as a substrate, it is possible to produce cyclic glucan in high yield using cheaper starch as a substrate,
In addition, the production of relatively high-molecular cyclic glucan becomes easy.
Furthermore, it is possible to efficiently synthesize cyclic glucan from a substrate mixed with glucose, and it is possible to omit the step of inactivating the cyclizing enzyme before the step of purifying cyclic glucan, which is conventionally required.

[0035]

[Brief description of the drawings]

FIG. 1 is a view showing a conventional cyclic glucan production process.

FIG. 2 is a schematic diagram showing a GDE reaction conventionally considered.

FIG. 3 is a graph showing the change over time in the cyclic glucan yield and iodine coloration when GDE is applied to amylose having an average molecular weight of 30,000.

FIG. 4 is a diagram showing the results of analysis when cyclic glucan produced during each reaction time was applied to a sugar analysis system manufactured by Dionex when GDE was allowed to act on amylose having an average molecular weight of 30,000.

FIG. 5 is a diagram showing the results of examining the degree of polymerization of cyclic glucan obtained from amylose having an average molecular weight of 30,000 using GDE using a sugar analysis system manufactured by Dionex.

FIG. 6 is a diagram showing analysis results when cyclic glucans obtained from various substrates using GDE were applied to a sugar analysis system manufactured by Dionex.

FIG. 7 is a graph showing the time-dependent change in cyclic glucan yield by GDE when starch is used as a substrate. For comparison with the conventional method for producing cyclic glucan, changes over time in the yield of cyclic glucan when the D enzyme or the D enzyme and the branching enzyme are used in combination are also shown.

FIG. 8 is a diagram showing the results of examining the distribution of the degree of polymerization of the cyclic glucan obtained using each of GDE, D enzyme, or D enzyme and a branching enzyme using a sugar analysis system manufactured by Dionex. is there.

FIG. 9 is a graph comparing yields when cyclic glucans are produced using GDE in the presence of various concentrations of glucose.

FIG. 10. D in the presence of various concentrations of glucose.
It is a figure which compared the yield at the time of producing a cyclic glucan using an enzyme.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B050 CC01 DD04 FF04E FF05E FF09E FF11E LL02 LL05 4B064 AF12 AF15 CA21 CB07 CB30 CC01 CD19 CD30 DA10

Claims (11)

[Claims] 1. An enzyme having the activity of cleaving an α-1,4-glucosidic bond and synthesizing a new α-1,4-glucosidic bond by a transfer reaction, and a receptor for the transfer reaction of glucose. Enzyme activity that cannot be used as
A process for cyclizing α-quantan using the term “transfer activity”). 2. The method according to claim 1, wherein the α-1,4-transfer activity is an activity derived from a glycogen debranching enzyme.
The method described in 3. An enzyme activity for hydrolyzing an α-1,6-glucosidic bond (hereinafter referred to as α-1,6-transferase) in addition to the α-1,4-transfer activity.
3. The method according to claim 1, wherein 4. The method according to claim 1, wherein the α-1,6-degrading activity is amylo-1,6-glucosidase activity. 5. The method according to claim 3, wherein the α-1,6-degrading activity is an activity of an enzyme capable of releasing glucose from glucosyl-cyclodextrin. 6. The method according to claim 1, wherein the α-1,4-transfer activity and α-1,6-degrading activity are enzyme activities derived from a single polypeptide. 7. The method of claim 6, wherein said single polypeptide is a glycogen debranching enzyme. 8. The method according to claim 7, wherein the glycogen debranching enzyme is derived from a fungus such as a filamentous fungus or yeast. 9. The method according to claim 7, wherein the glycogen debranching enzyme is derived from yeast. 10. The method according to claim 1, wherein the α-glucan is amylose. 11. The method according to claim 1, wherein the α-glucan is starch and the yield of cyclic glucan is 40% or more.