JP2000232878A - Fructosyltransferase and fractional production of 1- kestose and nystose using the enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new fructosyltransferase derived from a microorganism belonging to the genus Eurotium, having activity producing a fructooligosaccharide having a specific composition from sucrose, having growth stimulating activity on Bacillus bifidus, useful for producing 1-kestose and nystose, or the like. SOLUTION: This new fructosyltransferase is produced by a microorganism belonging to the genus Eurotium and has an activity for producing a fructooligosaccharide as a reaction product, whose composition ratio is expressed by the formula: fructosylnystose/(1-kestose + nystose)<0.05, when sucrose of 0.1 M or more is made to react as a substrate. The enzyme is suitable for industrial production of a fructooligosaccharide by an enzymatic method. The enzyme is made to react on fructose in a reaction tank 1, the products are separated by chromatographic separation towers 2 and 3, unreacted matters, the enzyme and the like are placed in a recycling tank 4 to recycle them, and thus 1-kestose and nystose are obtained. The enzyme can be obtained by culturing Eurotium repens.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel fructosyltransferase which is an enzyme useful for producing 1-kestose and nystose as oligosaccharides having an effect of promoting the growth of bifidobacteria, and 1 The present invention relates to a method for separately producing kestose crystals and nystose crystals.

[0002]

2. Description of the Related Art 1-Kestose is a trisaccharide having a molecular structure in which one fructose is translocated to the first carbon atom of the fructosyl group of sucrose, and nystose is one more fructose translocated to 1-kestose. Tetrasaccharide. Further, one in which fructose is further translocated to the nystose is fructosyl nystose. These fructooligosaccharides, when ingested by humans, are difficult to digest in the stomach and reach the intestine, and are useful as food materials as fructooligosaccharides that have the effect of promoting the growth of bifidobacteria, and have low cariogenicity. (See, for example, Japanese Patent Publication No. 59-53434).

[0003] These fructooligosaccharides are generally a mixture of various fructooligosaccharides,
Since it was difficult to crystallize each individual component, amorphous powders obtained by spray-drying or freeze-drying a mixture of various fructooligosaccharides, and those obtained by pulverizing solidified molasses containing crystals were mainly produced (for example, JP-A-60-1
No. 49596), all of these products have strong hygroscopicity and are not suitable for long-term storage. For example, special measures such as using a moisture-proof and moisture-absorbing packaging material as a packaging form are required. In addition, care was required for handling fructooligosaccharides after opening.

As a solution to the problem of hygroscopicity of 1-kestose and nystose, a technique of crystallizing each of these fructooligosaccharides has been proposed, and Japanese Patent Publication No. 6-700.
No. 75 discloses a 1-kestose crystallization technique, and JP-A-6-165700 discloses a nystose crystallization technique. However, regarding the 1-kestose crystallization technique, it is necessary to prepare an aqueous solution containing 1-kestose having a purity of 70% or more (for industrial purposes, a purity of 80% or more), and the crystallization of the nystose is required. The technology has a purity of more than 40% (purity of 7% for industrial purposes).
0% or more) must be prepared.
In order to obtain such a high-purity oligosaccharide solution, as shown in the publication, a product-specific production bacterium capable of producing only a specific fructooligosaccharide with high purity is used, or It was necessary to carry out a pretreatment to increase the purity by such an efficient separation method.

[0005] For example, as a method of using a production bacterium having a high product specificity, the method described in JP-B-6-70075 and JP-B-4-41600 can be mentioned.
According to the official gazette, high purity 1-kestose is obtained as a fermentation product by a cultivation method using Scolariopsis brevicauris, but the culture of the microorganism itself has low enzymatic activity. The method using a so-called enzyme reaction method in which a substrate is reacted has poor productivity, and the enzyme derived from the microorganism is inactivated at 45 ° C. or higher, so its application is disadvantageous.

As an example of the efficient separation method, various chromatographic separation methods using a cation ion-exchange resin are suitable for industrial separation of fructooligosaccharides and methods for increasing the purity of fructooligosaccharides. However, the chromatographic separation method has a drawback that a mixture of fructooligosaccharides having a large difference in the molecular weight ratio is easily separated, but a mixture having a molecular weight ratio closer to 1 is difficult to separate.
For example, the monosaccharide and disaccharide have a molecular weight ratio of 1: 2,
The ratio is 1: 1.5 for disaccharides and trisaccharides, 1: 1.3 for trisaccharides and tetrasaccharides, and 1: 1: for tetrasaccharides and pentasaccharides.
The molecular weight ratio approaches 1 as the number of constituent sugars increases, and there is a problem that the chromatographic separation becomes difficult in the order listed above.

For these reasons, techniques for separating oligosaccharides using a chromatographic separation method include, for example, JP-B-63-1988.
As disclosed in Japanese Patent No. 5100, monosaccharides, disaccharides,
Separation of three or more trisaccharides has been performed, but at present, the technology of separating trisaccharides and tetrasaccharides, and furthermore, separation of tetrasaccharides and pentasaccharides or more has not been advanced. The biggest reason is that in conventional oligosaccharide-producing enzymes derived from microorganisms, besides kestose and nystose, in addition to kestose and nystose, fructosyl nystose, which is a pentasaccharide with a large number of constituent sugars, is not negligible. It was produced, so that the relative large amount of fructosylnystose had an effect, so that kestose and nystose could not be separated cleanly.

[0008] Explaining with specific examples, microorganisms such as Asperigius niger (ATCC 20611) and Aureobasidium pullulans (AHU 9549) are conventionally known as fructooligosaccharide-producing microorganisms. The composition of fructooligosaccharides of various microbial origins, including fructosylnystose /
It is about 10 to 15% (about 5 to 7.5% by weight) as (1-kestose + nystose). The pentasaccharide fructosylnystose hindered the fractionation and crystallization of the trisaccharide 1-kestose and the tetrasaccharide nystose.

[0009]

Therefore, the present invention is suitable for industrial production of fructooligosaccharides by an enzymatic reaction method,
And a fructosyltransferase which is suitable for efficient separation of 1-kestose and nystose by a chromatographic separation method and which can be formed into crystals having no hygroscopicity as a final product, in which the amount of fructosylnystose produced is very small. It is an object of the present invention to provide a fructosyltransferase having the characteristics described above, to provide a method for fractionating and producing 1-kestose and nystose using the enzyme, and to provide a method for producing 1-kestose crystal and nystose crystal. And

[0010]

Accordingly, the present inventors have searched for a fructo-oligosaccharide-producing enzyme that does not produce pentasaccharide fructosylnystose, or that produces only a small amount, from microorganisms existing in nature. As a result, sucrose acts as a substrate on cultured cells of Eurotium repens , which is a kind of "Katsuobushi" -producing bacterium, and on a culture solution or an enzyme extracted and purified therefrom, to convert 1-kestose and nystose into a substrate. Mainly produced and found to have the effect of producing almost no fructosylnystose, and established a method for producing high-purity 1-kestose and high-purity nystose and a method for producing each crystal thereof using a chromatographic separation technique. . Also,
From the chromatographic separation liquid, not only 1-kestose and nystose were obtained as crystalline products, but also a method for producing 1-kestose and nystose with a small production loss was established by subjecting the collected honey to the enzymatic reaction again. Thus, the present invention has been completed.

That is, the fructosyltransferase of the present invention is characterized by being produced by a microorganism belonging to the genus Eurotium, and as a reaction product when reacted with sucrose of 0.1 M or more as a substrate. The fructo-oligosaccharide has a composition ratio of the following formula (1), fructosyl nystose / (1-kestose + nystose) <0.05: a fructosyl transferase having an ability to generate fructooligosaccharides represented by the formula (1): There is a feature.

The microorganism belonging to the genus Eurotium which can produce the fructosyltransferase of the present invention may be any microorganism which belongs to the genus Eurotium and produces the fructooligosaccharide represented by the above formula (1). Is, for example, Eurotium lipens.
um response ) (alias: Eurotium s )
p. HS-1005 ) (FERM P-17199) can be used.

The method of the present invention for producing fructooligosaccharides mainly comprising 1-kestose and nystose is selected from the group consisting of microbial cells belonging to the genus Eurotium, cultures of the microorganisms, and enzymes extracted and purified therefrom. The method is characterized in that fructooligosaccharides are produced in a reaction solution by using sucrose as a substrate using the biocatalyst to produce fructooligosaccharides represented by the formula (1).

In the method for producing fructooligosaccharide according to the present invention, the sucrose as a substrate to be reacted with the enzyme reaction is adjusted to 0.1 M or more so that 1-kestose and nystose having a composition ratio represented by the above formula (1) are obtained. Can be obtained.

[0015] 1-Kestose and nystose can be separated from the reaction solution containing these fructooligosaccharides by a chromatographic separation method. The chromatographic separation can be composed of a first-stage chromatographic separation and a second-stage chromatographic separation. The first-stage chromatographic separation divides the first fraction containing reducing sugar as a main component, the second fraction containing unreacted sucrose as a main component, and the third fraction containing kestose and nystose as main components. 1-kestose and nystose are separated from the third fraction by a second-stage chromatographic separation.

1-kestose obtained in the above step and / or
Alternatively, 1-kestose crystals and / or nystose crystals having low hygroscopicity can be produced by crystallizing nystose by a known method.

The process for producing fructooligosaccharides of the present invention comprises:
The above process can be applied repeatedly, for which purpose the second fraction containing the unreacted sucrose as a main component is collected in a recycle tank, and the 1-kestose section is crystallized and shaken with 1-kestose crystal. Condensate, collect the condensate in a recycle tank, and crystallize the nystose section to obtain a confinement with the nystose crystal, collect the consolidation in the recycle tank, and apply the mixture collected in the recycle tank to the recycle tank. The sucrose concentration is adjusted by further adding sucrose so that the sucrose as a substrate is 0.1 M or more, and the sucrose concentration adjusted mixture obtained in the above step is used as a sucrose substrate. The method is characterized in that the process is repeated by applying the biocatalyst again as a raw material.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Microorganism used The microorganism producing the fructosyltransferase of the present invention has been isolated from the site of the Shimizu Sugar Factory of the Hokuren Agricultural Cooperative Association, Shimizu City, Kamikawa-gun, Hokkaido. The microbiological properties of the microorganism are described below.

(1) Colonies grew on Tupec agar medium and reached 2-3 cm in diameter on the 20th day. 20%
On Tuapec agar containing sucrose, it reaches 6-7 cm on day 14 at 25 ° C. The color of the colonies is yellow-green to greenish-grey due to the conidial head and vegetative mycelium, but sometimes yellowish-orange to reddish-brown.

(2) Spherical to subspherical angiosperms (75-100 μm) on Tupec agar medium containing 20% sucrose
Are abundantly formed, forming an orange-yellow layer, around which a network of yellow-orange mycelia surrounds.

(3) Ascomycetes are spherical to subspherical (10 to 12)
μm), the chains appear to have gathered. Ascospores are lens-shaped (4.8-5.6 μm in diameter, 4.0-4.5 μ in minor axis)
m) and the outside is smooth, but slightly uneven and traces of the groove can be observed near the equator.

(4) The conidial head is 100-200 μm in diameter
It is.

(5) The conidiophores bulge at the vertices with a length of 200 to 800 μm to form peaks with a diameter of 20 to 40 μm.

(6) Conidia 7 to 10 μm × 3.5
It occurs at the conidia top at a size of 4.5μ.

(7) Conidia are slightly spherical to elliptical, densely covered with fine spines, and have a diameter of 5 to 6.5 μm.

(8) The optimum temperature range for growth is 25 to 30 ° C., and it does not grow at 40 ° C. or higher.

The above-mentioned mycological properties are described in Food Fungi Handbook, translated by Shunichi Udagawa et al., Medical and Dental Business Publishing (1984) No. 32
Page and Comopendium of Soil F
ungi, Vol. 1 [K. H. A. Ponsch et al., A
Cademic press (1980) P293-2
95], the bacterium was found to be Eurotium repense (alias: Eurotium sp. HS-1005) (FER
MP-17199).

E. fructosyl transfer from C. repens
Enzyme (1) Action and Substrate Specificity In the presence of sucrose of 0.03 M or less, only hydrolysis is mainly performed, and in the presence of 0.1 M or more of sucrose, fructooligosaccharides are mainly produced. 0.8M sucrose as a substrate,
When reacted for 24 hours, the production of 1-kestose and nystose was observed, and the ratio was 15.7 with 1-kestose being 100. The km value for sucrose is 1.53M.

The fructosyltransferase of the present invention produces only a small amount of fructosyl nystose, and when reacted with sucrose of 0.1 M or more as a substrate, the composition ratio of fructooligosaccharide as a reaction product is as follows: The following formula is obtained: fructosylnystose / (1-kestose + nystose) <0.05.

(2) Optimum pH and stable pH range The optimum pH is 6.8. In addition, it is 18
After holding for a time, the stable range when the residual activity is measured at pH 6.8 is 5.5 to 7.5.

(3) Titration method The enzyme reaction is carried out at 45 ° C. using 0.1 M sucrose as a substrate.
The generated 1-kestose is subjected to liquid chromatography (H
PLC). The enzyme unit is the amount of enzyme that produces 1 μmol of 1-kestose per minute under the above conditions.
Unit. From the above measurements, the specific activity of the enzyme purified by the method described in (6) below was 1,260 units / m
The recovery in g was 15.4%.

(4) Range of action temperature The optimum temperature of the present enzyme is 45 ° C, and after maintaining at various temperatures of 25 ° C or higher for 60 minutes and measuring the residual activity at 45 ° C, the stable range is 45 ° C. C. and above, that is, 20% inactivation at 50.degree. C. and complete inactivation at 60.degree. However, in the presence of a high concentration of sucrose, its stability increases. For example, in the presence of 1.8M sucrose, the optimum temperature is 60 ° C, the stable temperature range is up to 65 ° C,
% Inactivation, complete inactivation at 75 ° C.

FIG. 1 is a graph showing the relative enzyme activity (%) when the fructosyltransferase of the present invention is maintained at various temperatures. The measurement conditions of the enzyme activity in FIG.
After placing 4 units of the enzyme in the buffer solution of No. 8 and holding at various temperatures for 60 minutes, the residual activity was measured at 45 ° C. In the activity measurement in the presence of sucrose, the amount was calculated by subtracting the amount of 1-kestose generated when a temperature stress was applied. In the graph of FIG. 1, curve A shows the case without sucrose, and curve B shows the case with 1.8 M sucrose.

(5) Deactivation conditions depending on pH, temperature, etc. At pH 4.5, about 40% of the deactivation occurs, and at pH 3.5, it is completely deactivated. Approximately 30% is deactivated at pH 8.5 and completely deactivated at pH 10.

[0035] (6) purification method E. repens (FERM P-17199) to 30
Using 500 ml of a medium containing weight% sucrose, 30 ° C., 7
The cells were shake-cultured for one day to obtain wet cells, and the wet cells were subjected to ultrasonic treatment. The obtained cell extract was used as a crude enzyme solution in various column chromatography operations (DEAE-
Sepharose CL-6B, Toyopearl HW-55S and Toyopearl HW-65S, and phenyl-Toyopearl 650S) are used as purified enzymes.

(7) Molecular Weight The purified enzyme purified in the above (6) was confirmed to be uniformly purified by polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this enzyme is calculated to be 77,000. The isoelectric point by isoelectric focusing (IEF) is pH4.
8

[0037] The method of manufacturing fructooligosaccharides then the E. A method for producing a fructooligosaccharide-containing saccharide composition using a repens- derived fructosyltransferase will be described. First, E. repens (FERM P-171
A method for obtaining a crude enzyme from 99) will be described.

(1) Preparation of Crude Enzyme Solution In the culture medium, 5 to 40% by weight, preferably 2
0 to 30% by weight, and 0.5% of yeast extract as a nitrogen source.
-3% by weight, preferably 1.0-2.0% by weight,
Further, phosphates, magnesium salts and the like, for example, inorganic salts such as potassium phosphate, magnesium sulfate, etc.
To 1.0% by weight, preferably 0.2 to 0.5% by weight. The pH of the medium is adjusted to 4.5-7.5, preferably 5.0-6.0. Culture of the above microorganisms,
The culture is carried out by incubating the culture with shaking in advance for 24 to 48 hours into the above-mentioned medium, and culturing with aeration and agitation at 25 to 35 ° C, preferably 28 to 30 ° C, for 24 to 72 hours, preferably 30 to 48 hours.

The fructooligosaccharide-producing enzyme obtained by this culture is present in both the cells and the culture solution, and the cells, the culture solution, or the whole culture can be used as a crude enzyme. When the titer producing 1 μM 1-kestose per minute is 1 unit, for example, the titer per 1 ml of the main culture has a property of 30 to 40 units at pH 6.0 and 55 ° C.

(2) Properties of Crude Enzyme Solution As a substrate, sucrose was adjusted to a 70% (w / v) solution, and 5 g of wet cells per liter of the substrate solution was added as a crude enzyme. Reaction at pH of 16 hours,
The amount of 1-kestose produced was measured, and the results obtained are shown in FIG. FIG. 2 shows that the pH suitable for producing 1-kestose of the present fructosyltransferase is 5.0 to 6.0.

Further, sucrose is used as a substrate at a concentration of 70% (w
/ V) adjusted to a solution, 5 g per liter of the substrate solution
The wet cells were added as a crude enzyme, reacted at various temperatures at pH 6.0 for 24 hours, and the amount of 1-kestose produced was measured. The results obtained are shown in FIG. According to FIG. 3, it is found that the temperature suitable for producing 1-kestose of the crude fructosyltransferase of the present invention is 50 to 60 ° C.

(3) Production of Fructooligosaccharide-Containing Material The crude enzyme solution prepared in the above (1) was subjected to pH 4.5 to 7.5.
0, preferably 30-80% adjusted to 5.0-6.0
(W / v), preferably 5 to 30 times, preferably 10 to 20 times the amount of a sucrose solution of 60 to 70% (w / v), and the reaction temperature is 40 to 60 ° C, preferably 50 to 50%. ~ 55
C. and the reaction is carried out with slow stirring for 24-72 hours, preferably 30-48 hours. As a result, as a reactive sugar composition, reducing sugar 20-35%, sucrose 15-35%,
A fructooligosaccharide composition containing 20 to 35% kestose, 10 to 25% nystose, and 1% or less fructosyl nystose is obtained.

For comparison with the fructooligosaccharide, which is a reaction product of the fructosyltransferase of the present invention,
If the oligosaccharide composition produced by a conventional fructooligosaccharide-producing bacterium, for example, Asperigius niger (ATCC 20611) is shown, reducing sugar 35%, sucrose 10%,
-25% kestose, 25% nystose, 5% fructosyl nystose. As described above, the present fructosyltransferase has a feature that the productivity of fructosylnystose is extremely low, and this feature is an advantage for easily performing chromatographic separation as described below.

FIG. 4 shows that 3.3 liters of the crude enzyme solution prepared in the above (1) was allowed to act on 100 liters of a 70% (w / v) sucrose solution under optimal conditions (pH 6.5, 55 ° C.). The relationship between the sucrose concentration, the 1-kestose concentration, the nystose concentration, and the fructosyl nystose concentration with respect to the reaction time when the enzyme reaction was performed is shown as a graph.

(4) Chromatographic separation of fructooligosaccharide Next, the fructooligosaccharide composition of the present invention obtained in the above (3) is chromatographed by the following method. The feature of the present chromatographic separation method is that a total of two chromatographic separation steps are performed, and each of the chromatographic separation steps is performed by a different method and purpose as described in detail below. First, in the first stage of the chromatographic separation step, the fructooligosaccharide composition of the present invention obtained in the above (3) is subjected to chromatographic separation treatment using a cation ion exchange resin as a separating agent, and a reducing sugar as a main component. , A second fraction containing unreacted sucrose as a main component, and a third fraction containing kestose and nystose as main components. This is the stock solution to be used for the next chromatographic separation. For separation of such various kinds of sugars, for example, a new MCI method which is a known semi-continuous chromatographic separation method, specifically, a method disclosed in JP-A-63-158105 is preferably used. Available.

The new MCI system is a chromatographic separation system in which a front end and a rear end of a packed bed filled with an adsorbent such as an ion exchange resin can be circulated with a fluid connected by a fluid passage.
After supplying a raw material fluid containing a plurality of components having different affinities to the adsorbent, and flowing through the packed bed in the direction from the front end to the rear end of the packed bed to form an adsorption zone having a concentration distribution of each component, In the method of separating into two or more fractions, a first step of extracting a fraction enriched with any one component from a packed bed while supplying a raw material fluid from a packed bed front end, and a desorbent fluid from a packed bed front end And the second step of extracting other component-enriched fractions from the rear end of the packed bed, and supplying the fluid to the packed bed and extracting the fluid from the packed bed without removing the fluid from the packed bed. And the components extracted in the first step and each step of the third step of moving the zone in which other components are mixed to the front end of the packed bed in any order, and repeating the above steps And a chromatographic separation method.

The first fraction containing the reducing sugar as a main component according to the new MCI method may be eliminated or concentrated, and may be separately recovered as a sweetener. The second fraction containing the unreacted sucrose as a main component is returned to the reaction solution,
The enzyme reaction may be performed again. Since the third fraction is composed mainly of kestose and nystose,
It is used as a stock solution for the second-stage chromatographic separation step to be performed next.

To explain a more preferred embodiment of the first-stage chromatographic separation step, preferably, a sodium-type cation ion exchange resin (average particle size: 220 μm) is used as the cation ion exchange resin, and the concentration of the stock solution is reduced. 5
0-70% (w / w), operating load 0.05-0.1
0, preferably 0.07 to 0.08 (liter / hour.
Resin), the water usage ratio is 2-3 (v / v), preferably 2.
The chromatographic separation step is performed with the setting being 7 to 2.8 (v / v).

The three fractions obtained in the first stage of the chromatographic separation step are as follows: the first fraction has a sugar composition of 95 to 98% of reducing sugar purity, the second fraction has a sugar composition of 40 to 50% sucrose, Fructooligosaccharides 35-4
The sugar composition of 0%, reducing sugar 10-20%, and third fraction is fructooligosaccharide 95-97% (internal fructosylnystose 3% or less) and sucrose 3-5%.

In order to achieve the object of the present invention, in addition to the above-described chromatographic separation step, chromatographic separation by a batch method using a single column is also possible, but the amount of resin used and the amount of water used are larger than those in the above method.

Next, the second-stage chromatographic separation will be described. The purpose of the second-stage chromatographic separation is to obtain kestose and nystose with high purity from the third fraction obtained by the first-stage chromatographic separation as a stock solution. Therefore, as a chromatographic separation method, known simulated moving bed type chromatographic separation, for example, Journal of Sugar Manufacturing Technology,
Vol. 41, p. 29-36 (1993) can be suitably applied.

The chromatographic separation by the simulated moving bed method refers to a moving bed method (filling while supplying an eluent to one end of a column) without actually moving a packed bed filled with a filler such as an ion exchange resin. At the same time as the stock solution containing two components is continuously supplied in the middle of the layer, the filler is moved in the opposite direction to the flow direction of the eluent, and each component in the stock solution is moved in the opposite direction to each other to continuously make each component. This is a method for obtaining the same effect as that of the method for separating. Specifically, the packed bed is divided into several columns, and the system is configured to be connected in a ring shape.The supply position and the withdrawal position of the supply liquid are intermittently moved in the opposite direction, and apparently the packing With the effect of moving layers,
This is a method of separating two components.

The first fraction obtained by the second-stage chromatographic separation is a fraction mainly containing kestose, and the second fraction is a fraction mainly containing nystose.

It is advantageous to employ a sodium-type cation ion-exchange resin as the separating agent used in the second-stage chromatographic separation column, as in the first-stage, in order to efficiently separate the two fractions. It is.

In the first-stage chromatographic separation and the second-stage chromatographic separation used in the method of the present invention for separating and producing 1-kestose and nystose, a sodium-type cation ion is preferably used as a separating agent. An exchange resin (average particle size 220 microns) can be employed.

In the second stage of chromatographic separation employing the simulated moving bed system, to separate 1-kestose and nystose, the concentration of the stock solution supplied to the second stage of chromatographic separation apparatus is 50 to 70% (w / W), the operating load is 0.02
０．０0.03 (liter / hour · resin), preferably 0.
025 to 0.028 (liter / hour resin), eluent water use ratio 1.5 to 2.5 (v / v), preferably 1.9
By setting to -2.2 (v / v), the 1-kestose purity is 80-85 from the second-stage chromatographic separation.
% (1% or less of inner fructosylnystose) and a second fraction having a nystose purity of 70 to 80% (4% or less of inner fructosylnystose).

The crystallization of each fructooligosaccharide from each of the high-purity 1-kestose solution and nystose solution obtained in the above step can be carried out by a known crystallization method, for example,
It can be produced by a method for producing 1-kestose crystal disclosed in Japanese Patent Application Laid-Open No. 70075/1995 and a method for producing a nistose crystal disclosed in Japanese Patent Application Laid-Open No. 6-165700.

FIG. 5 is a flow chart showing the steps of the method for producing 1-kestose crystal and nystose crystal of the present invention described in detail above. 1 is a reaction tank for causing the fructosyltransferase of the present invention to act on sucrose to carry out an enzymatic reaction. The fructooligosaccharide mixture obtained by the enzymatic reaction in the reaction tank 1 is introduced into the first-stage chromatographic separation tower 2 and fractionated into a sucrose section, a reducing sugar section, and a fructooligosaccharide section. The sucrose section is sent to a recycle tank 4 described later. The reducing sugar category,
Separately, it can be used for sweeteners and the like.

The fructooligosaccharide fraction is introduced into the second-stage chromatographic separation column 3, where it is fractionated into 1-kestose and nystose. A crystallization step is performed on each of the separated 1-kestose and nystose to produce a 1-kestose crystal and a nystose crystal, respectively. In each of these 1-kestose and nystose crystallization steps, shaking after collecting crystals is as follows:
It is collected in the recycle tank 4 and mixed with the collected sucrose section. The mixture collected in the recycling tank 4 is used as a sugar composition as reducing sugars 5 to 7%, sucrose 60 to 70%, 1-kestose 17 to 22%, nystose 6 to 8%, fructosyl nystose 0. 2-0.5
%.

This sugar mixture was returned to the reaction tank 1 again,
Maintained at 55 ° C, sucrose concentration 70% (w / v),
The pH was adjusted to 5.0 to 6.0, and the culture of the fructosyltransferase-producing bacterium belonging to the genus Eurotium of the present invention was added again as described above and reacted for 26 to 31 hours to obtain a reactive sugar composition. , Reducing sugar 29-31%,
A sugar composition comprising 18 to 22% of sucrose, 31 to 34% of 1-kestose, 15 to 17% of nystose and 1% or less of fructosyl nystose can be obtained.

As described above, the method for producing 1-kestose crystals and nystose crystals according to the present invention provides the sucrose section generated in the first-stage chromatographic separation, the shrinkage generated in the 1-kestose crystallization step, and the sucrose generated in the 1-kestose crystallization step. Thickness can be completely reused, and a highly efficient manufacturing cycle without waste can be realized.

[0062]

EXAMPLES Example 1 Production of Oligosaccharides Using Enzyme Solution (1) Production of Crude Enzyme Solution Eurotium repens (alias: Euroti)
um sp. HS-1005) (FERM P-171)
99) from a slant of a potato dextrose agar medium to a 10 cm diameter Tupec agar medium (sucrose 20%
) And cultured at 30 ° C for 14 days.
Spores were collected from the resulting colonies with sterile water, sucrose 100 g / l, yeast extract 15 g / l,
100 cc of a culture solution containing 0.3 g / liter of calcium phosphate and 0.3 g / liter of magnesium sulfate (pH
6.0) and shaking culture at 30 ° C. and 120 rpm for 24 hours.

[0063] A 5 liter liquid medium comprising 200 g / liter of sucrose, 15 g / liter of yeast extract, 0.3 g / liter of calcium phosphate, 0.3 g / liter of magnesium sulfate, and 1.5 g / liter of calcium carbonate was obtained. (PH 6.0), 48 hours, 3
The culture was performed at 0 ° C., aeration rate of 1/2 vvm, and stirring speed of 400 rpm. The oligosaccharide-producing activity (1-kestose-producing activity) of the obtained culture was 31.8 units / 1 ml culture. This culture solution was used as a crude enzyme solution.

(2) Production of Oligosaccharide by Enzyme Reaction 100 liters of a sucrose aqueous solution adjusted to pH 6.0 with a sucrose concentration of 70% (w / v) was kept at 55 ° C. 3.3 liters of the crude enzyme solution obtained in the step 1) was added. While stirring slowly at the above temperature 4
Hold for 3 hours. Thereafter, the solution was filtered to obtain a solution containing the sugar composition as a reaction product. The sugar composition of the sugar composition solution is shown in Table 1 below.

[0065]

[Table 1]

As shown in Table 1 above, the sucrose was subjected to the enzymatic reaction according to the present invention, whereby 1-kestose (trisaccharide) and nystose (trisaccharide) having very low content of the pentasaccharide fructosylnystose were obtained. It can be seen that fructooligosaccharides mainly composed of (4 monosaccharides) were obtained.

Example 2 Purification of Oligosaccharide (First Chromatographic Separation) 100 liters of the saccharide composition solution obtained in Example 1 was subjected to three-component separation by chromatographic separation using the new MCI separation method. Then, the following three categories of reducing sugar category (Table 2), sucrose category (Table 3), and oligosaccharide category (Table 4) were separated and obtained. The chromatographic separation apparatus (Mitsubishi Diaion UBK530Na type) used for the chromatographic separation was charged with a total resin volume of 11 liters, and the operating load (0.075 liter / h · resin liter, water use ratio 2.75 (v /
v)). Table 2 below shows the distribution ratio of each component in each section at that time, and the ratio of each component in each section.
It is shown in Table 4.

[0068]

[Table 2]

[0069]

[Table 3]

[0070]

[Table 4]

According to Table 4 above, the oligosaccharide classification was 5
It can be seen that an oligosaccharide composition containing 1-kestose (a trisaccharide) and nystose (a monosaccharide) as main components, which has a very low content of the monosaccharide fructosylnystose, was obtained.

Example 3 Separation of 1-Kestose and Nystose (Second-stage Chromatographic Separation) The oligosaccharide fraction obtained in Example 2 was reduced to 70% (w /
Concentrated to w) to obtain 35 liters. It was separated into a 1-kestose section and a nystose section by two-component separation using a simulated moving bed system. The chromatographic column is a chromatographic separator with a total resin volume of 9 liters (Mitsubishi Diaion UBK53).
0Na type) (operating load 0.027 liter / h
Resin liter and water use comparison 2.0 (v / v)). At that time, the distribution ratio of each component in each section and the ratio of each component in each section are as shown in Tables 5 and 6 below.

[0073]

[Table 5]

[0074]

[Table 6]

The 1-kestose fraction was subjected to crystallization of 1-kestose described in Example 4 below, and the nystose fraction was subjected to crystallization of nistose described in Example 5 below.

Example 4 Crystallization of 1-Kestose The 1-kestose fraction obtained in Example 3 (1-kestose purity: 83.7%, about 2% as a 56% (w / w) solution)
0 liter) was concentrated to 90% (w / w) by an evaporator, and this was used as a crystal mother liquor. Seed slurry of 1-kestose at 80 ° C (20 ml ethanol suspension)
, And the temperature was gradually lowered from 80 ° C. to 65 ° C. over 17 hours with gentle stirring under atmospheric pressure, whereby crystals having a uniform particle diameter (0.1 to 0.2 m in diameter) were deposited. . The crystals were separated by centrifugation (3,000 rpm) under the condition of 65 ° C. to obtain 6.2 kg of 97% pure 1-kestose crystals.

Since the purity of the honey was 73.4%, it was again concentrated to 90% (w / w), and a 1-kestose seed crystal slurry (10 ml, ethanol suspension) was added. By gradually lowering the temperature from 80 ° C to 65 ° C over 20 hours while stirring under atmospheric pressure,
Crystals having a uniform particle size were deposited again. The crystals were separated by centrifugation (3,000 rpm) at 65 ° C.
2.5 kg of 6% 1-kestose crystals were obtained. The composition of the sugar beet sugar is 1-kestose 64%, nystose 25
%, Sucrose 9%, and F-nystose 2%, and the solid content was 5.3 kg. This was returned to the enzyme reaction solution described in Example 6 below and reused.

Example 5 Crystallization of Nistose The nystose fraction obtained in Example 4 (Nistose purity: 76.0%, about 15 liters as a 56% (w / w) solution) was 80% (w / w) by an evaporator. / W), which was used as a crystal mother liquor. At 65 ° C., a nystose seed crystal slurry (40 ml ethanol suspension) was charged, and the mixture was slowly stirred at atmospheric pressure for 24 hours.
By gradually lowering the temperature from 200 ° C. to 20 ° C., crystals having a uniform length (0.2 to 0.4 mm) were deposited. The crystals were separated by centrifugation (3,000 rpm) at room temperature to obtain 5.1 kg of 96% pure nystose crystals.

The purity of nystose of the honey is 58.2%.
Was concentrated again to 80% (w / w),
A nystose seed crystal slurry (20 ml, ethanol suspension) was charged at 65 ° C., and the temperature was gradually lowered from 65 ° C. to 20 ° C. over 24 hours while gently stirring under atmospheric pressure, so that uniform particles were formed again. Diameter (0.1-0.3m
The crystals of m) precipitated. Centrifuge at room temperature (3,000
The crystals were separated by rpm) to obtain 1.7 kg of 94% pure nystose crystals. The composition of this nectose is 42% nystose, 47% 1-kestose, 5% sucrose, and 6% fructosyl nystose, and has a solid content of 3.7 kg.
Therefore, this was returned to the enzyme reaction solution described in Example 6 below and reused.

[Example 6] Sugar composition by enzymatic reaction mixed with recycle section In place of the undiluted reaction solution in Example 1, the sucrose section in Example 2 and 1-kestose crystallized honey in Example 4 When the nystose crystallized honey of Example 5 was mixed, the weight of the solid content was 3%.
6.9 kg, and add 33.1 kg of sucrose to this,
The reaction solution was set to 100 liters, and a reaction solution having a total sugar amount of 70% (w / v) was prepared. In this, E. coli prepared in Example 1 was used . re
3.3 liters of pens culture solution was added, the mixture was controlled at 55 ° C., and reacted for 29 hours while stirring slowly. The sugar composition before the reaction and the sugar composition after the reaction at this time are shown in Table 7 below.

[0081]

[Table 7]

From the above results, when the above-mentioned recycle category is used, the enzymatic reaction proceeds smoothly as in the case where pure sucrose is used, and the sugar composition shown in Example 1 can be settled in a short time. Do you get it.

[0083] [Example 7] E. Purification of fructosyltransferase derived from C. repens (1) Preparation of crude enzyme Eurotium repens (FERM P-17
199) was placed in a flask together with 500 ml of the medium, and shaking culture was performed at 30 ° C. for 7 days. The cells were separated from the culture and washed to obtain a washed cell with a wet weight of 63 g. 43 g of the suspension was suspended in 200 ml of 20 mM phosphate buffer (pH 7.0). 200 ml of the supernatant obtained by sonication and centrifugation was used as a crude enzyme preparation.

(2) DEAE-Sepharose CL6B column treatment DE equilibrated with 10 mM phosphate buffer (pH 7.0)
The crude enzyme having an activity of 179 units was applied to an AE-Sepharose CL6B column (diameter 30 mm, length 270 mm) and washed with the same buffer. The adsorbed enzyme protein was eluted at a flow rate of 40 ml per hour by linearly increasing the salt concentration from 0 to 1M. One fraction was 10 ml, and the active fractions (fractions 72 to 76) were collected and concentrated by ultrafiltration.

(3) Toyopearl HW-55S column treatment The enzyme solution concentrated by ultrafiltration in step (2) was
Toyopearl HW-55S dialyzed against Tris-HCl buffer (50 mM, pH 7.0) and equilibrated with the same buffer
The solution was applied to a column (diameter: 26 mm, length: 420 mm), and 5 ml of each fraction was fractionated with the same buffer. 49-59 showing activity
Fractions were collected and concentrated by ultrafiltration.

(4) Toyopearl HW-65S column treatment The enzyme solution concentrated by ultrafiltration in step (3) was
The solution was applied to a Toyopearl HW-65S column (diameter 26 mm, length 420 mm) equilibrated with a Tris-HCl buffer (50 mM, pH 7.0), and fractionated with the same buffer at 5 ml each. The active 54-62 fractions were collected and concentrated by ultrafiltration.

(5) Phenyl-Toyopearl 650S column treatment The enzyme solution concentrated by ultrafiltration in step (4) was
Phenyl-Toyopearl 650S column (diameter 16 mm, length 1) equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 2 mM mercaptoethanol and 50% saturated ammonium sulfate
65 mm). The adsorbed enzyme protein was eluted by linearly decreasing the ammonium sulfate concentration from 50% to 0% saturation. The flow rate was 30 ml / h, and one fraction 2.5
When expressed in ml, activity was observed in fractions 61 to 64.

The purified enzyme concentrated by ultrafiltration of the fraction was applied to a phenyl-Toyopearl 650S column again. Elute under the same conditions, collect the activity categories,
It was concentrated by ultrafiltration. Thereafter, the purified enzyme was assayed by polyacrylamide electrophoresis (SDS-PAGE) to confirm that the enzyme was purified to a single band.

The results (total amount of protein, total activity, specific activity, degree of purification, yield) obtained by the above procedure are shown in Table 8 below.

[0090]

[Table 8]

According to Table 8, the specific activity of the final purified product was increased to 1260 units / mg, the activity recovery was 15.4%, and the purification was performed 18300 times from the crude enzyme. Although the activity is increased at the step, it is presumed that this was probably due to reversible inhibition for some reason in the purification operation at the second step.)

[0092]

The fructosyltransferase of the present invention has the above formula (1) when sucrose is used as a substrate.
As shown by, the amount of fructosyl nystose produced is smaller than that of 1-kestose and nystose.
-In kestose and nystose separation, the effect of fructosyl nystose can be eliminated, and efficient separation can be performed. Therefore, it is suitable for industrial production of fructooligosaccharides by an enzymatic reaction method.

The method for producing 1-kestose crystals and nystose crystals of the present invention comprises the steps of: sucrose division produced in the first stage of chromatographic separation;
The compaction generated in the nystose crystallization process can be completely reused, and a highly efficient manufacturing cycle without waste can be realized.

1-Kestose and each characteristic possessed by nystose (for example, 1-kestose has the highest bifidobacterial activity among fructooligosaccharides, and nystose has an action of incorporating water of crystallization and is useful as an edible desiccant. ) Can be provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the relative enzyme activity (%) when the fructosyltransferase of the present invention is maintained at various temperatures.

[FIG. 2] Sucrose is adjusted to a 70% (W / V) solution as a substrate, and 5 g of wet cells per liter of the substrate solution is added as a crude enzyme of fructosyltransferase of the present invention.
The reaction was carried out at 5 ° C. and various pHs for 16 hours to produce 1-
The results obtained by measuring the amount of kestose are shown as a graph.

[FIG. 3] Sucrose as a substrate is adjusted to a 70% (W / V) solution, and 5 g of wet cells per liter of the substrate solution are added as a crude enzyme of fructosyltransferase of the present invention.
The results obtained by reacting with H6.0 at various temperatures for 24 hours and measuring the amount of 1-kestose produced are shown as a graph.

FIG. 4: Crude enzyme solution of fructosyltransferase of the present invention
3 liters of the sucrose concentration were calculated based on the reaction time when an enzymatic reaction was performed by reacting 100 liters of a 70% (W / V) sucrose solution under optimal conditions (pH 6.5, 55 ° C.). The relationship between kestose concentration, nystose concentration, and fructosyl nystose concentration is shown as a graph.

FIG. 5 is a flowchart showing steps of a method for producing a 1-kestose crystal and a nystose crystal of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Reaction tank 2 First-stage chromatographic separation tower 3 Second-stage chromatographic separation tower 4 Recycling tank

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme Court ゛ (Reference) C12R 1: 645) (72) Inventor Yutaka Koga 71, Shimizu-cho, Kamikawa-gun, Hokkaido No. 71, Hokuren Agricultural Cooperative Association Inside the Federation Association's Shimizu Sugar Factory (72) Inventor Shiomi ▲ Toku ▼ Upper Hokkaido 2-chome 2-chome 1-2-29, Sapporo City, Hokkaido (72) Inventor Shuichi Onodera 7-23 Kita 1-chome Asahi, Shiroishi-ku, Sapporo Heiwa Building 303 (72) Inventor Hiroshi Sasaki 4-2-1-11 Nishioka, Toyohira-ku, Sapporo, Hokkaido F-term (reference) 4B050 DD03 LL02 LL05 4B064 AF04 CA05 CA21 CE10 CE15 CE10

Claims (10)

[Claims] 1. The composition ratio of fructo-oligosaccharide, which is a fructosyltransferase produced by a microorganism belonging to the genus Eurotium and reacted as sucrose of 0.1 M or more as a substrate, is represented by the following formula: A fructosyltransferase having the ability to produce fructooligosaccharides, which can be represented by: fructosylnystose / (1-kestose + nystose) <0.05. 2. The microorganism belonging to the genus Eurotium,
Eurotium repens
2. The fructosyltransferase according to claim 1, which is an enzyme. 3. Using a biocatalyst selected from the group consisting of a microbial cell belonging to the genus Eurotium, a culture solution of the microorganism, and an enzyme extracted and purified therefrom, using sucrose of 0.1 M or more as a substrate. Act to produce fructooligosaccharides in the reaction solution, wherein the composition ratio of the fructooligosaccharides is represented by the following formula: fructosylnystose / (1-kestose + nystose) <0.05 Method for producing fructooligosaccharide. 4. The microorganism belonging to the genus Eurotium,
Eurotium repens
The method according to claim 3 1-kestose and mixtures of nystose according a ense). 5. The reaction solution according to claim 3, which contains a fructooligosaccharide having a composition represented by the following formula: fructosylnystose / (1-kestose + nystose) <0.05.
1. A method for producing 1-kestose and nystose by separating 1-kestose and nystose into respective fructooligosaccharides by chromatographic separation. 6. The chromatographic separation comprises a first-stage chromatographic separation and a second-stage chromatographic separation, and the first-stage chromatographic separation comprises a first fraction containing a reducing sugar as a main component, and a second fraction. The second fraction containing reactive sucrose as a main component and the third fraction containing 1-kestose and nystose as main components are separated. The third fraction is separated into 1-kestose and nystose by the second-stage chromatographic separation. 6. The 1-fructooligosaccharide according to claim 5, wherein
Separation production method of kestose and nystose. 7. The method for producing 1-kestose and nystose according to claim 6, wherein the second-stage chromatographic separation is of a simulated moving bed type. 8. A method for producing 1-kestose crystals, characterized by crystallizing 1-kestose obtained in claim 5, 6 or 7. 9. A method for producing nystose crystals, characterized by crystallizing the nystose obtained in claim 5, 6 or 7. 10. A bacterium selected from the group consisting of microbial cells belonging to the genus Eurotium, a culture solution of the microorganism, and a fructosyltransferase extracted and purified therefrom, using sucrose of 0.1 M or more as a substrate. To produce fructooligosaccharides in the reaction solution, and (2) separating the obtained reaction solution into a first fraction mainly composed of reducing sugars and unreacted sucrose by the first-stage chromatographic separation. (2) a second fraction mainly composed of 1-kestose and nystose and a third fraction mainly composed of 1-kestose and nystose; (3) collecting the second fraction mainly composed of the unreacted sucrose in a recycling tank; The third fraction mainly composed of 1-kestose and nystose is separated into a 1-kestose section and a nystose section by a second-stage chromatographic separation. (5) crystallizing the 1-kestose section obtained in the above step to obtain a 1-kestose crystal, and (6) crystallizing the nistose section obtained in the above step, and shaking with the nystose crystal. (7) collecting the condensate generated in the 1-kestose crystallization step and the consolidation generated in the nystose crystallization step in the recycle tank, and (8) using the mixture collected in the recycle tank as a substrate. So that the sucrose is 0.1M or more,
Further, sucrose is added to adjust the sucrose concentration. (9) The mixture with the adjusted sucrose concentration obtained in the above step is again applied to the biocatalyst as a sucrose raw material as a substrate, A method for separating and producing 1-kestose crystals and nystose crystals, wherein the steps are repeated.