JP3200129B2 - Method for producing α-glucosyl sugar compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an oligosaccharide, and more particularly, to a cyclomaltodextrin glucanotransferase (hereinafter, referred to as a "substrate solution") in which an α-glucosyl sugar donor and a monosaccharide or an oligosaccharide coexist. C
Abbreviated as GTase. ) To produce an α-glucosyl sugar compound.

[0002]

2. Description of the Related Art CGT
ase acts on starch to produce cyclodextrin (hereinafter referred to as CD).
Abbreviated. ) Is famous as an enzyme that synthesizes). However, this enzyme has a function of transferring a maltooligosyl group to itself when a suitable receptor is present (coupling reaction) in addition to the CD synthesizing action, and also has a disproportionation reaction between oligosaccharides. Do. Therefore, various transfer products can be produced by utilizing the coupling reaction which is an effect of CGTase.

[0003] Among the substances produced by utilizing the transfer function of CGTase, there is maltooligosyl sucrose (coupling sugar) in which a glucosyl group is linked to sucrose by an α-1,4 bond. This carbohydrate is sweet and sticky, but inhibits the synthesis of insoluble glucan from sucrose by Streptococcus mutans, which is the causative fungus of caries, and has anti-cariogenic properties.
Moreover, since it is a non-reducing saccharide, it has a characteristic that it does not brown when heated with amino acids, proteins, and the like, and is currently used in various foods.

However, Bacillus megaterium (Bacillu
s megaterium) and Bacillus macerans (B. macerans), in order to be effective receptors in the glycosyltransferase of CGTase, the sugar forms a pyranose ring in an aqueous solution, and its C2-, C3-, C4- It is said that OH is indispensable, and that their conformation needs to be all equatorial, like D-glucose. That is, D-glucose, L-sorbose, D
-Xylose, 6-deoxy-D-glucose, etc.
Although excellent as a receptor, D-glucose is C2-
D-mannose, C2-
D the OH is replaced with -NH ₂ or -NHCOCH ₃
L-rhamnose, which differs from N-glucosamine and N-acetyl-D-glucosamine and 6-deoxy-D-glucose only in the configuration of C2-OH, cannot be any receptor. Similarly, with respect to the C-3 position, D-xylose differs from D-xylose only in the configuration of C3-OH.
Ribose cannot be an acceptor, and for position C-4, D
D-galactose which differs from glucose only in the configuration of C4-OH, and D-xylose is C4-OH
It has been reported that L-arabinose, which differs only in the configuration, cannot almost become a receptor.

[0005]

DISCLOSURE OF THE INVENTION In view of the above-mentioned current situation, the present inventors have proposed a saccharide D which cannot be the above-mentioned receptor.
-Mannose, D-glucosamine, N-acetyl-D-
Glucosamine, D-galactose, L-arabinose,
Intensive research has been conducted to efficiently synthesize an α-glucosyl sugar compound having a glucosyl group transferred to D-ribose or the like.
As a result, B. stearothermophilus (B. stear
othermophilus) and Bacillus circulans (B. cir
culans), the transfer reaction is carried out using CGTase derived from D-mannose, D-glucosamine,
N-acetyl-D-glucosamine, D-glucuronic acid,
D-ribose, D-galactose, D-arabinose,
L-arabinose, D-fucose, L-fucose, L-
It has been found that an α-glucosyl sugar compound having a glucosyl group transferred to rhamnose, D-galactosamine or the like can be obtained, and the present invention has been completed.

[0006] The present invention relates to an α-glucosyl sugar donor and D-
Galactose , D-mannose, D-ribose, D-glucosamine, N-acetyl-D-glucosamine, D-arabinose, L-arabinose, D-glucuronic acid, D
-Fucose, L-fucose, L-rhamnose and D-
A cyclomaltodextrin glucanotransferase derived from Bacillus circulans or Bacillus stearothermophilus is allowed to act on a substrate solution containing at least one monosaccharide or oligosaccharide selected from galactosamine. And a method for producing an α-glucosyl sugar compound.

The α-glucosyl sugar donor used in the present invention,
That is, a solution containing an α-glucose residue donor and a monosaccharide or oligosaccharide, that is, a solution containing an α-glucose residue acceptor is a so-called substrate solution.
Is an aqueous solution containing a substance serving as a donor of an α-glucose residue and a substance serving as an acceptor of an α-glucose residue in the glucosyl transfer reaction by the above method. The mixing ratio (molar ratio) of the donor and the acceptor in the solution is about 1:50 to 50: 1.
And the substrate concentration is preferably about 5 to 50 W / W%.

In the present invention, examples of the substance serving as a donor of an α-glucose residue include starch, partially decomposed products of the starch, and cyclodextrin. These substances may be used alone or in combination of two or more. It is used as a mixture of the above. Here, the α-glucose residue means a dextrin having a glucosyl group, a maltooligosyl group or a branch formed by decomposition of the donor.

Next, the monosaccharide or oligosaccharide used in the present invention becomes a receptor for the transfer reaction of CGTase.
Monosaccharides or oligosaccharides capable of producing glucosyl sugar compounds. As specific examples of these, D-galacto
Scan, D- mannose, D- ribose, D- glucosamine, N- acetyl -D- glucosamine, D- arabinose, L- arabinose, D- glucuronic acid, D- fucose, L- fucose, L- rhamnose, D- galactosamine These may be used alone or in combination of two or more.

[0010] The CGTase is an enzyme capable of decomposing a donor such as starch when transferred to the above-mentioned substrate solution and transferring the generated glucosyl group or maltooligosyl group to an acceptor to generate an oligosaccharide. I just need. Specific examples of such enzymes include CG derived from various microorganisms such as B. circulans, B. stearothermophilus, and the like.
There is Tase. These enzymes may be in a pure form or in a crude enzyme state. Further, these enzymes may be used in a suspended state in the solution or may be used in an immobilized state.

The reaction conditions for producing the α-glucosyl sugar compound are as follows: CGTase is added to a substrate solution in which the above-mentioned donor and acceptor coexist with pH 4 to 8, preferably 5 to 5.
6. It may be operated at a temperature of 20 to 70 ° C, preferably 45 to 55 ° C. For example, when using CGTase derived from Bacillus circulans, a pH of 5.6 and a temperature of 45 ° C. are preferable, and CGT derived from Bacillus stearothermophilus is preferred.
When Tase is used, pH 6.0 and a temperature of 50 ° C. are preferred.

In order to separate and purify the α-glucosyl sugar compound thus produced from the reaction solution, a known method may be applied. For example, the reaction solution is heated at 100 ° C. for 10 minutes to inactivate the enzyme, and then filtered, centrifuged or the like.
A liquid separation means is applied, and then a transfer reaction product is fractionated by a purification means such as activated carbon column chromatography and high performance liquid chromatography to obtain a target α-glucosyl sugar compound.

[0013]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited by these examples.

Example 1 100 mg of soluble starch as a donor and D- as an acceptor
1 ml of a substrate solution (pH 5.6) containing 100 mg of any of galactose, D-ribose, D-mannose , D-arabinose, L-arabinose, D-fucose, L-fucose, L-rhamnose and D-glucosamine.
CGTas derived from Bacillus stearothermophilus, Bacillus circulans or Bacillus macerans
e was added in 700 units and reacted at 45 ° C. for 17 hours.
As a control, CGTase was also allowed to act on a substrate solution containing only a donor without any acceptor.

Thereafter, a part of the reaction solution was analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) under the following conditions.

HPLC analysis column Asahipak NH2P-50 (4.6 × 250 mm) Solvent 70% acetonitrile Flow rate 1.0 ml / min Temperature 40 ° C. Detector Differential refractometer TLC analysis Plate MERCK Kieselgel-60 (20 × 20 cm) Solvent Ethyl acetate: acetic acid : Water (3: 1: 1) Coloring sulfuric acid: methanol (1: 1)

FIG. 1 shows the results of TLC. In the figure, R is the development of various sugars used for reference, G1 is glucose, G2 is maltose, G3 is maltotriose, G4
Represents maltotetraose, respectively. B shows the case where only the acceptor used in each test was developed, and S, C and M show cases where CGTase derived from Bacillus stearothermophilus was used as the donor and the acceptor used in each test, respectively (S ), When using CGTase derived from Bacillus circulans (C), CGT derived from Bacillus macerans
This is the expansion of (M) when ase is used. Furthermore, (1)
Is the case where only the donor is used without adding the acceptor, (2) is the case where D-galactose is used as the acceptor, (3) is the case where D-ribose is used as the acceptor, and (4) is the case where D-ribose is used as the acceptor
When D-mannose was used, ( 5 ) was D
-When using arabinose, ( 6 ) when using L-arabinose, ( 7 ) when using D-fucose,
( 8 ) shows the case where L-fucose was used, ( 9 ) shows the case where L-rhamnose was used as the receptor, and ( 10 ) shows the case where D-glucosamine was used as the receptor.

As is apparent from this figure, CGTase derived from Bacillus stearothermophilus and Bacillus circulans produced a large amount of a transfer product for any of the receptors. Also, HPLC Figure 2-5
The result is shown. As is clear from the figure, when any of the receptors was used, in addition to the unreacted receptor molecules, peaks ( A1, A2, B1,
B2, C1, C2, C3) were detected (Figure 2 If only donor without addition of receptor, FIG 3 is D- gas as acceptor
4 shows the case where lactose was used, FIG. 4 shows the case where D-mannose was used as the receptor, and FIG. 5 shows the case where D-arabinose was used as the receptor. ).

After heating the reaction solution at 100 ° C. for 10 minutes to inactivate the enzyme, A1, A2, B1, B2,
C1, C2, C3 were isolated by preparative HPLC.

Next, A1, A2, B1, B2, C1,
1 mg each of C2 and C3 was dissolved in 50 μl of 50 mM, pH 4.5 acetate buffer, and α-glucosidase was added.
For 30 minutes. As a result of analyzing the reaction solution by HPLC, products A1, A2, B1, B2, C1, C2,
C3 is hydrolyzed, and A1, B1 and C1 are glucose and D-galactose used as an acceptor, respectively.
Scan, D- mannose, was equimolar generate the D- arabinose. In addition, A2, B2, and C2 produce glucose used twice as much as the receptor used and the receptor, and C3
Produced three times as much glucose as the receptor used and the receptor used.

Therefore, considering the action specificity of CGTase, the products A1, A2, B1, B2, C1,
C2 and C3 are α-glucosyl-galactose ,
It is apparent that α-maltosyl-galactose, α-glucosyl-mannose, α-maltosyl-mannose, α-glucosyl-D-arabinose, α-maltosyl-D-arabinose, α-maltotriosyl-D-arabinose. is there.

Example 2 A substrate solution containing 100 mg of α-cyclodextrin as a donor and 100 mg of any one of D-galactose , D-mannose, D-glucosamine, D-arabinose and L-arabinose as an acceptor (pH 5. 6) 1
CGTas derived from Bacillus stearothermophilus in ml
500 units of e were added and reacted at 50 ° C. for 17 hours.
As a control, CGTase was allowed to act on a substrate solution containing only α-cyclodextrin without adding any receptor. Thereafter, a part of the reaction solution was analyzed by HPLC in the same manner as in Example 1.

[0023] The results are shown in FIGS. 6-11. As is clear from the figure, when any of the receptors was used, in addition to the unreacted receptor molecules, peaks ( a1, a2, b1, b2, c1, c2 ) considered to be transfer products to the receptor. , D1, d
2, d3, e1, e2, e3) were detected (Fig. 6 If only α- cyclodextrin without adding receptors, 7
8 shows a case where D-galactose is used as a receptor, FIG. 8 shows a case where D-mannose is used as a receptor, FIG. 9 shows a case where D-glucosamine is used as a receptor, and FIG. 10 shows a case where D-arabinose is used as a receptor. FIG. 11 shows the case where L-arabinose was used as the receptor. ).

After the reaction solution was heated at 100 ° C. for 10 minutes to inactivate the enzyme, a1, a2, b1, b2 and
c1, c2, d1, d2, d3, e1, e2, e3 were isolated by preparative HPLC.

Next, a1, a2, b1, b2, c1,
1 mg each of c2, d1, d2, d3, e1, e2, e3 was dissolved in 50 μl of 50 mM, pH 4.5 acetate buffer, and α
-Glucosidase was added and reacted at 40 ° C for 30 minutes. As a result of analyzing the reaction solution by HPLC, product a
1, a2, b1, b2, c1, c2, d1, d2, d
3, e1, e2, and e3 are all hydrolyzed ;
In b1, c1, d1, and e1 , glucose and D-galactose , D-mannose,
D-glucosamine, D-arabinose and L-arabinose were produced in equimolar amounts. A2, b2, c2, d2,
e2 produced twice as much glucose as the used receptor and its receptor, and d3 and e3 produced three times as much glucose as the used receptor and its receptor.

Therefore, considering the specificity of the action of CGTase, the products a1, a2, b1, b2, c1,
c2, d1, d2, d3, e1, e2, e3 are respectively
α-glucosyl-galactose , α-maltosyl-galactose, α-glucosyl-mannose, α-maltosyl-mannose, α-glucosyl-glucosamine, α-
Maltosyl-glucosamine, α-glucosyl-D-arabinose, α-maltosyl-D-arabinose, α-maltotriosyl-D-arabinose, α-glucosyl-
It is clear that they are L-arabinose, α-maltosyl-L-arabinose, α-maltotriosyl-L-arabinose.

[0027]

According to the present invention, an α-glucosyl sugar compound can be produced efficiently by allowing cyclomaltodextrin glucanotransferase to act on a substrate solution in which an α-glucosyl sugar donor and a monosaccharide or an oligosaccharide coexist. can do. The α-glucosyl sugar compound is expected to be used in fields such as the food industry and the pharmaceutical industry, and is expected to be used as, for example, a sweetener, an anti-cariogenic food, a bifidobacterial proliferative food, and the like.

[Brief description of the drawings]

FIG. 1 shows the results of thin layer chromatography.

FIG. 2 shows a high performance liquid chromatogram in Example 1 in the case of using only a donor without adding an acceptor.

FIG. 3 shows a high performance liquid chromatogram when D-galactose was used as a receptor in Example 1.

FIG. 4 shows a high performance liquid chromatogram when D-mannose was used as a receptor in Example 1.

FIG. 5 shows a high performance liquid chromatogram when D-arabinose was used as a receptor in Example 1.

FIG. 6 shows a high performance liquid chromatogram in Example 2 in the case of using only a donor without adding an acceptor.

FIG. 7 shows a high performance liquid chromatogram when D-galactose was used as a receptor in Example 2.

FIG. 8 shows a high performance liquid chromatogram when D-mannose was used as the receptor in Example 2.

FIG. 9 shows a high performance liquid chromatogram when D-glucosamine is used as a receptor in Example 2.

FIG. 10 shows a high performance liquid chromatogram when D-arabinose was used as the receptor in Example 2.

FIG. 11 shows a high performance liquid chromatogram when L-arabinose was used as a receptor in Example 2.

──────────────────────────────────────────────────続 き Continued on the front page (72) Nobuhiro Kuwahara 13-46 Oguro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Inside Shimizu Port Refining Co., Ltd. (72) Sumio Kitahata 621-440 Noda, Kumatori-cho, Sennan-gun, Osaka Prefecture (72) Inventor Hirofumi Nakano 3-14-3 Hattori Nishimachi, Toyonaka City, Osaka Prefecture (56) References JP-A-63-39597 (JP, A) Sumio Kitahara, Shigehara Fukuda, Shi getaka Okada, Science and Industry , 65 (9), 404-409 (1991) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 19/00-19/64

Claims (1)

(57) [Claims] 1. An α-glucosyl sugar donor and D-galacto
Over scan, D- mannose, D- ribose, D- glucosamine, N- acetyl -D- glucosamine, D- arabinose, L- arabinose, D- glucuronic acid, D- fucose, L- fucose, L- rhamnose and D- A substrate solution containing at least one monosaccharide or oligosaccharide selected from galactosamine is added to a Bacillus.
A method for producing an α-glucosyl sugar compound, which comprises causing cyclomaltodextrin glucanotransferase derived from Circulans or Bacillus stearothermophilus to act.