JPH04222594A - Production of glucotetrasaccharide - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the title compound useful as a humectant by treating dextran with dextranase I or dextranase II derived from Arthrobacter globifomis to form glucotetrasaccharide. CONSTITUTION:Arthrobacter globifomis W31 strain is subjected to liquid culture at 30 deg.C for 3 days by using a jar fermentor, the culture solution thereof is subjected to high-speed cooling continuous centrifuging, a supernatant liquid after removal of molds is subjected to salting-out with 90% saturated aqueous solution of ammonium sulfate, salting-out precipitate is dissolved in 20mM acetic cid buffer solution (pH 6.0) and purified by gel filtration or ion exchange column chromatography to give dextranase I or dextranase II. Dextran is treated with dextranase I or dextranase II in 20mM acetic acid buffer solution containing Ca ion to give 3-O-alpha-D-glucosyl-D-isomaltotriose shown by the formula, a glucotetrasaccharide.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to the glucotetrasaccharide 3-
The present invention relates to a method for enzymatically producing O-α-D-glucosyl-D-isomaltotriose.

0002

[Prior Art] Glucotetrasaccharide, 3-O-α-D-glucosyl-D-isomaltotriose (3-O-α-D
-glucosyl-D -isomaltotrio
se) is the dextran Arthrobacter globiformis (Arthrobacter globiformis).
Mis) It is known as an enzymatic decomposition product of isomaltodextranase derived from T6 (Biochemica
l and Biophysical Research
hi Communicatios Vol. 70N
o. 2, 459-464 (1976)). However, no enzymatic production method using dextranase I or dextranase II is known.

0003

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for enzymatically producing glucotetrasaccharide represented by formula (I).

0004

[Means for solving the problems] The glucotetrasaccharide of the present invention is
Formula (I):

[0005]

3-O-α-D
-Glucosyl-D-isomaltotriose (3-O-
α-D-glucosyl-D-isomalto
triose) or O-α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranosyl-(1
→6)-O-α-D-glucopyranosyl-(1→6)
-D-glucose, (O-α-D-Glcp-(1
→3)-O-α-D -Glcp-(1→6)-O-α
-D-Glcp-(1→6)-D-Glc).

[0007] The method for producing glucotetrasaccharides of the present invention involves injecting dextran such as Dextran T2000 (manufactured by Pharmacia) into Arthrobacter globiformis (Arthrobacter globiformis).
Acter globiformis) W31 strain (N
RRL B-4428) consists of an enzymatic reaction using the original dextranase I or dextranase II. As the dextran used as a substrate in the present invention, Dextran T2000 manufactured by Pharmacia, Meito 70 manufactured by Meito Sangyo, etc. can be used.

[0008] Dextranase I and Dextranase II are disclosed in JP-A-1-228466 and JP-A-1-228467, respectively, together with their production methods. In the enzymatic reaction, both dextranase I and II enzymes were used at a reaction temperature of 30 to 55°C and a pH of
5 to 7, and dextran T2000 concentration is preferably 2 to 8%. However, the reaction time is dextranase I
For Dextranase II, the time is preferably 0.5 to 2 hours or more, and for Dextranase II, it is preferably 1 to 3 hours or more.

The reaction product obtained in the above manner is
In addition to glucotetrasaccharide (I), it contains isomaltotriose, isomaltotetraose, etc., and can be purified to some extent by ordinary separation and purification means, such as column chromatography, but preferably earth Lobacter globiformis (Arthrobacter)
globiformis) I42 strain (NRRL B-
4427, IAM 12102) By-products such as isomaltotriose and isomaltotetraose are hydrolyzed to glucose using the original glucodextranase and removed.

[0010] Glucotetrasaccharide (I
) is useful as a humectant in the food industry due to its high moisturizing properties. Preparation example Preparation of dextralanase I and II (1)
Arthrobacter globiformis (Arthr)
obacter globifomis) W31 strain (N
RRL B-4428) was cultured in liquid at 30°C for 3 days using a jar fermenter, 3L of the culture solution was subjected to high-speed refrigerated continuous centrifugation (10,000 × g), and the supernatant obtained after removing the bacterial cells was Salting out was carried out with a 90% saturated ammonium sulfate aqueous solution. The salting out precipitate was then treated with 5mM Ca2+
20 mM acetate buffer (pH 6.0) containing
The solution was dissolved in the same buffer and dialyzed against the same buffer, and then applied to a column (4.4 x 70 cm) packed with DEAE-cellulose that had been sufficiently equilibrated with the same buffer. In this column chromatography, 0.2 M NaC
The active fraction eluted with the same buffer containing 1. (2) After concentrating the obtained active fraction, it was applied to a column (1.8 x 120 cm) packed with Bio-Gel P-150 sufficiently equilibrated with the same buffer solution and subjected to gel filtration.

Two active fractions are obtained by this gel filtration, and the first eluted fraction is collected, concentrated, and then applied to a column (1. 2 × 50 cm), 0
The enzyme was eluted using a linear concentration gradient from M to 0.3 M in NaCl aqueous solution. A single active fraction eluted with an aqueous NaCl solution around 0.14 M was collected,
Concentrate and dialyze to obtain a purified sample of dextranase I (titer:
30.6 units/mg protein) was obtained. (3) On the other hand, among the two active fractions obtained by the gel filtration described above, the fraction eluted later was collected, concentrated, and then column filled with DEAE-Sepharose sufficiently equilibrated with the above buffer. (1.2 × 50 cm)
The enzyme was eluted using a linear concentration gradient from 0M to 0.3M NaCl aqueous solution. A single active fraction eluted with an aqueous NaCl solution around 0.16 M was collected,
A purified sample of dextranase II (titer: 59.2 units/mg protein) was obtained by concentration and dialysis. (4) Table 1 shows the physicochemical and enzymatic properties of dextranase I and II obtained as described above.

0012

[0013]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these in any way.

[0014]

[Example 1] (1) Dextran T manufactured by Pharmacia
15 g of 2000 in 50 ml of 20 mM acetate buffer (pH 6.0) containing 5 mM Ca2+
26 mg of dextranase I obtained in the preparation example was added to 1
The mixture was placed in a 00ml Erlenmeyer flask and reacted at 35°C for 24 hours. FIG. 1 shows a silver nitrate-stained chromatogram showing the change over time in the sugar produced by the reaction. In Figure 1, Std is standard carbohydrate, IG2 is isomaltose, IG3 is isomaltotriose, IG4 is isomaltotetraose, IG5 is isomaltopentaose, IG6 is
is isomaltohexaose, X1 is the above (I)
The formula glucotetrasaccharide is shown. (2) After the reaction was completed, the resulting sugar mixture was applied to a carbon-celite column. First, 10
After eluting isomaltotriose (near fraction tube number 115 in Figure 2) using % ethanol, elution with 15% ethanol yields isomaltotetraose (near fraction tube number 195 in Figure 2), formula Glucotetrasaccharide (X1) of (I) (near fraction tube number 205 in Figure 2) and isomaltopentaose (near fraction tube number 220 in Figure 2) were eluted in sequence.

The results are shown in FIG. Gluco 4 of formula (I)
The fraction containing sugar (X1) still contains a small amount of isomaltotriose and isomaltotetraose, so in order to remove these, Arthroba with high substrate specificity was used.
Glucodextranase originating from S. cter globiformis I42 strain was allowed to act at 35° C. for 24 hours to hydrolyze it to glucose.

[0016] Through this reaction, only the glucotetrasaccharide of formula (I) remains as an oligosaccharide, and all of the small amounts of impurities mentioned above are converted to glucose. The fraction of the substance obtained by this operation is designated as X-3. This fraction X-3 was further purified by paper chromatography. This fraction was
shall be.

Fraction X-4 contains various enzymes with high substrate specificity and well-known mechanisms of action, including A. g
isomaltodextranase (indicated as “ID” in Fig. 3) originating from P. lobiformis T6 strain (Ag
ric. Biol. Chem. , Vol. 52 (
2), 495-501, 1988; Agric.
Biol. Chem. , Vol. 52(3), 8
29-836, 1988), A. globifor
Glucodextranase originating from mis I42 strain (
Indicated as “GD” in Fig. 3) (Agric. Bi
ol. Chem. , Vol. 52(9), 216
9-2176, 1988; Agric. Biol
．． Chem. , Vol. 53(1), 223-2
28, 1989), Rhizopus niveu
Crystalline glucoamylase originating from s (in Figure 3, “G
A) (BIO-CHEMICALS, PRO
CTS FOR LIFE SCIENCE, 1
989/1990 General Catalog 35 pages (Seikagaku Kogyo Co., Ltd. Publishing), Glucoamylase (Rhi
zopus niveus)). In FIG. 3, P and IP represent standard carbohydrates panose and isopanose, respectively.

These enzyme reaction samples were subjected to chromatography to examine the sugars produced by the reaction. The results are shown in Figure 3. Both glucodextranase and glucoamylase cannot hydrolyze the glucotetrasaccharide produced sugar. As a result, the non-reducing end of X1 is α-1
, 6-, α-1,4-glucosidic bond, and if X1 is a glucotetrasaccharide, the second bond from the non-reducing end is an α-1,6-glucosidic bond. It was speculated that.

[0019] Furthermore, 1 of the glucotetrasaccharide X1 of formula (I)
3C-NMR spectrum (dew water, room temperature)
As shown in the figure. From the sugar binding method and position based on the enzyme reaction results and the 13C-NMR analysis results, the above (I) of the present invention
The glucotetrasaccharide of the formula is 3-O-α-D of the formula (I)
-glucosyl-D-isomaltotriose.

[0020]

[Example 2] Dextran T2000 manufactured by Pharmacia
15 g of 20 mM containing 5 mM Ca2+
Add 30 mg of dextranase II obtained in the preparation example to 50 ml of acetate buffer (pH 6.0).
The mixture was placed in a ml Erlenmeyer flask and reacted at 35°C for 24 hours. FIG. 1 shows a silver nitrate-stained chromatogram showing the change over time in the sugar produced by the reaction.

[0021] Hereinafter, the same process as in Example 1 was carried out to obtain the formula (I).
A glucotetrasaccharide represented by was obtained.

[0022]

[Example 3] 15 g of dextran 70 manufactured by Meito Sangyo
Dextranase I obtained in the preparation example was added to 20 mM acetate buffer (pH 6.0) containing 5 mM Ca2+.
25 mg of was added to a 100 ml Erlenmeyer flask and reacted at 35°C for 24 hours. Thereafter, the same procedure as in Example 1 was carried out to obtain the glucotetrasaccharide represented by formula (I).

[0023]

[Example 4] 15 g of dextran 70 manufactured by Meito Sangyo
Dextranase I obtained in the preparation example was added to 20 mM acetate buffer (pH 6.0) containing 5 mM Ca2+.
35 mg of I was added to a 100 ml Erlenmeyer flask and reacted at 35°C for 24 hours. Thereafter, the same procedure as in Example 1 was carried out to obtain the glucotetrasaccharide represented by formula (I).

[0024]

According to the present invention, 3-O-α-D-glucosyl-D-isomaltotriose, which is a glucotetrasaccharide useful as a humectant, can be efficiently produced.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a silver nitrate-stained chromatogram showing changes over time in reaction-produced sugars.

FIG. 2 is a diagram showing the elution pattern of carbon-celite column chromatography.

FIG. 3 is a diagram showing the results of chromatography of samples obtained by treating the glucotetrasaccharide of the present invention with various purified enzymes.

FIG. 4: 13C-NM of glucotetrasaccharide represented by formula (I)
It is a figure showing an R spectrum.

Claims (1)

[Claims] Claim 1: Dextran containing Arthro-bacter globiformis (Arthro-bacter globiformis)
gluco-4 of the following formula (I) by acting with dextranase I or dextranase II derived from
A method for producing glucotetrasaccharide, which is characterized by producing sugar.