JPH0725891A - New isomaltooligosaccharide and enzyme for producing the same and method therefor - Google Patents

Abstract

PURPOSE:To provide a new isomaltooligasaccharide excellent in functionalities such as low sweetness, low calorific value, nonfermentability and bifidus growth factor, thus useful as a health food material. CONSTITUTION:The objective isomaltooligosaccharide; which can be obtained by making (A) an enzyme capable of hydrolyzing alpha-1,4-glucoside linkage in the presence of glucose (pref. an alpha-amylase capable of producing panose from pullulan) act on (B) pullulan or panose.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel isomaltooligosaccharide industrially useful as a highly functional food material, and an enzyme and a method for producing the same.

[0002]

2. Description of the Related Art In contrast to maltooligosaccharides consisting only of α-1,4-glucoside bonds, isomaltooligosaccharides having α-1,6-glucoside bonds are called branched sugars, and in recent years, as functional foods. It is a sugar that has been drawing attention. Originally, sugars have been used as a sweetening source and a nutritional source, but sugars having functionalities such as low sweetness, low calories, non-fermentability, bifidobacterial growth factor, and difficult caries have been developed and used as a health food material. It's being used. Particularly, isomaltooligosaccharides such as isomaltose, panose (isomaltosyl glucose, isomaltotriose) and isomaltosyl maltose (isomaltotetraose) have been attracting attention as oligosaccharides having excellent functionality.

To date, isomaltose, panose, isomaltosylmaltose and the like are known as isomaltooligosaccharides. These isomaltooligosaccharides are α-1,6-derived from amylopectin when starch is hydrolyzed with α-1,4-glucoside bonds by a usual α-amylase.
Since the glucoside bond is less susceptible to hydrolysis, α-1,
6-Glucoside bonds remain and are generated. Alternatively, isomaltose or the like is produced by the reverse reaction of glucoamylase.

However, the novel isomaltooligosaccharide of the present invention (isomaltosyl isomaltose, hereinafter referred to as "IMI"
M ”. ), Two isomaltose are α-
No oligosaccharides linked by 1,4-glucoside bonds are known at all.

[0005]

DISCLOSURE OF THE INVENTION The present inventor has studied in detail the enzymatic action of amylase, and found that α-1, 6-
The present inventors have succeeded in obtaining IMIM, which is a novel isomalto-oligosaccharide, which is a homo-oligosaccharide composed of four glucoses having two glucoside bonds, and completed the present invention.

That is, according to the present invention, among the oligosaccharides attracting attention in the food industry where functionality is important, isomalt oligosaccharides, which are known to show particularly excellent functionality, have two α- 4 having a 1,6-glucoside bond
It is an object of the present invention to provide IMIM, which is a novel isomaltooligosaccharide consisting of individual glucose, and novel α-amylase, and a method for producing the novel isomaltooligosaccharide using a hydrolase.

[0007]

The present invention includes the following inventions. (1) The following formula (I):

[0008]

[Chemical 4]

A novel isomaltooligosaccharide IMI represented by
M. (2) Physicochemical properties shown below: Action (a) Maltose is mainly produced from starch. (B) Panose is produced from pullulan.

(C) Pullulan is hydrolyzed in the presence of glucose to give the following formula (I):

[0011]

[Chemical 5]

Isomalto-oligosaccharide represented by the following formula (II):

[0013]

[Chemical 6]

An isomalto-oligosaccharide represented by is produced. Substrate specificity Hydrolyzes α-1,4-glucoside bonds such as starch and pullulan to produce maltose and panose. The produced panose is not decomposed, and in the presence of glucose, the above formula (I)
To produce the isomaltooligosaccharide represented by and the isomaltooligosaccharide represented by the formula (II). The isomaltosyl maltose represented by the formula (II) is decomposed.

Although the activity against starch is stronger than that against pullulan, the thermoactinomycete Thermoactinomyces vulg
α-amylase produced by aris R-47 (enzyme mainly producing maltose from starch, hereinafter referred to as "TVAI")
The activity against pullulan is stronger than that of. Optimum pH and pH stability With pullulan as a substrate, there is an optimum pH between 6 and 7,
It is stable at 9.

Optimum temperature and thermal stability With pullulan as a substrate, the optimum temperature is 45-55 ° C, and 50 ° C
Is stable up to. The molecular weight is about 60,000 (by SDS polyacrylamide gel slab electrophoresis). Α-amylase having (3) The method for producing the novel isomaltooligosaccharide IMIM according to (1) above, wherein pullulan or panose is caused to act on an enzyme that hydrolyzes an α-1,4-glucoside bond in the presence of glucose.

The hydrolase used in the production method of the present invention is not particularly limited as long as it is an enzyme that hydrolyzes α-1,4-glucoside bonds, but α-amylase which produces panose from pullulan is preferable. Can be mentioned. Examples of such enzymes include Thermoactinomyces
vulgaris R-47 (M. Shimizu et al., Agric. Biol. Che
m., 42 , 1681 (1978)), that is, the novel α-amylase of the present invention.

Shimizu et al. Thermoactinomyces vu
It was found that the α-amylase TVAI produced by lgaris R-47 decomposes pullulan to produce panose. Next, the present inventor conducted a detailed study on the enzymatic action of TVAI and clarified its property (Y. Sakano et.
al., Agric. Biol. Chem., 46 , 1121 (1982); Y. Sakan
o et al., Agric. Biol. Chem., 46 , 1423 (1982); Y. S
akano et al., Agric. Biol. Chem., 47 , 2211 (1983);
Y. Sakano et al., Agric. Biol. Chem., 49 , 3391 (198
Five)) .

Furthermore, the present inventor has found that α-amylase (TVAII) different from TVAI is produced in Escherichia coli cloned by collecting a plasmid from the gene of this bacterium by the sit-gun method (Y. Sakano). et al., Biosci.
Biotech. Biochem., 57 , 395 (1993)). Then TVA
A high-purity enzyme that constructs an expression system that enhances the protein synthesizing ability of II and greatly increases the production of this enzyme in Escherichia coli, and at the same time, easily crystallizes by culturing E. coli and then performing extraction operations such as heating and sonication. Has developed a production method.

TVAII is an α-amylase which differs from TVAI in the nucleotide sequence and amino acid sequence of the gene and differs in physicochemical properties from conventional enzymes (Y. Sakano et al., Bios.
ci.Biotech.Biochem., 57 , 395 (1993)). The nucleotide sequence and amino acid sequence of the TVAII gene are shown in FIG. The present inventor investigated the enzymatic action of TVAII, and found that when it was allowed to act on pullulan in the presence of glucose, two oligosaccharides having a shorter developing distance than panose were formed by thin layer chromatography (FIG. 2). . Both are oligosaccharides consisting of 4 glucoses, one of which is developed at the same position as known isomaltosyl maltose (hereinafter referred to as "IMM"), and the other unknown sugar has a development distance larger than that of IMM. It was a short sugar and was clearly different from IMM. Therefore, when an unknown sugar is isolated and NMR is measured,
^{In 13} C-NMR, peaks showing the signals of α-1,4- and α-1,6-glucoside bonds were obtained at almost the same height (FIG. 3), and the signals of α-1,6-bond were two. It had broken up into a peak. Therefore, the glucosidic bond is α-1, 4-
It was revealed that there was a ratio of bond: α-1, 6-bond = 1: 2. Then, maltose and isomaltose were produced when IMM was acted with isomaltdextranase, and only isomaltose was produced when the same enzyme was acted on an unknown sugar (FIG. 4). 2 and 4
In the thin layer chromatography shown in 1), 1-butanol: ethanol: water = 5: 5: 3 was used as the developing solvent, and silica gel G60 manufactured by Merck was used as the thin layer.

From these experimental facts, it was found that the unknown oligosaccharide is IMIM represented by the above formula (I) and is a completely novel isomalt oligosaccharide which has not been found so far. The IMIM of the present invention can be easily produced by dissolving pullulan or panose and glucose to act an enzyme that hydrolyzes an α-1,4-glucoside bond.

The enzyme used here includes, for example, the above-mentioned TVAII, but in addition, neopullulanase (N. Kuriki et al., J. Bacteriol., 170 , 1554 (198)
8); N. Kuriki et al., J. Bacteriol., 173 , 6147 (199
An α-amylase that produces panose from pullulan such as 1)) can be used. For example, when TVAII is used, both pullulan and glucose are dissolved in 1.5 L of sodium phosphate buffer (pH 6.5) at a concentration of 5% to give 60 mg of TVAII.
In addition, the mixture is reacted at 40 ° C for 72 hours. When this reaction solution was sampled with time and subjected to thin layer chromatography, IMIM and IMM were produced as shown in FIG. At this time, IMM
Is reduced by TVAII and therefore decreases after 24 hours of reaction.

One substrate for the reaction is pullulan or panose, and starch, amylopectin, etc., which is a raw material for panose, also serves as a substrate. Regarding the concentration of pullulan or panose, the higher the concentration, the better, but the range of 1 to 30% is preferable. Of course, the concentration of IMIM produced by adding pullulan or panose during the reaction can be increased. Glucose as the other substrate is usually added to the reaction solution from the beginning of the reaction, but it may be added during the reaction.
That is, since TVAII cannot hydrolyze panose and the glucose transfer reaction proceeds even in the presence of panose, glucose is added to increase the IMIM concentration after the high-concentration pullulan solution undergoes enzymatic decomposition to reduce the viscosity. It can also be higher. The enzyme concentration varies depending on the type of enzyme, but is usually 0.0001 to 1.0%.

The reaction conditions for producing IMIM may be any conditions as long as they are in the pH range and temperature range of action of the enzyme, preferably pH 4.0 to 7.0 and temperature 20 to 80 ° C. Further, an organic solvent or the like can be added if necessary. The reaction time is appropriately set depending on the intended use of the product, but in most cases, 30 minutes to 72 hours is preferable. The IMIM-containing sugar solution obtained by the reaction can be directly used as a syrup after purification operations such as decolorization with activated carbon and desalting with an ion exchange resin according to a conventional method. Further, it can be used as sugar alcohol if subjected to reduction treatment.

Further, if necessary, high-purity IMIM is prepared by using gel filtration chromatography, carbon column chromatography, ion exchange resin column chromatography, or by using a membrane fractionation method or a crystallization method. be able to.

[0026]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited by the following examples. (Example 1) Thermoactinomyces vulgaris R-47 was prepared by the method of Shimizu et al. (M. Shimizu et al., Agric. Biol. Chem., 4
2 , 1681 (1978)) and cultured by Marmur's method (J. Marmu
r, J. Mol. Biol., 3 , 208 (1961)), genomic DNA was prepared from cultured cells of this bacterium. This genomic DNA was partially digested with the restriction enzyme Sau3AI and the resulting 4-10 kb DNA was obtained.
The fragment was introduced into the BamHI site of pUC119 plasmid. With the recombinant plasmid thus obtained, E. coli JM83
The strain was transformed.

The search for Escherichia coli having the TVAII gene was carried out by using the presence or absence of iodine starch reaction as an index. From about 30,000 strains, 5 strains of Escherichia coli carrying the TVAII gene were obtained, and the shortest 4 kb gene was named pTO1, and a mass production system of TVAII was constructed according to the procedure shown in FIG. First, unnecessary parts were deleted. pTO1 TV
A 2.8 kb PstI-PstI fragment of the AII gene portion was added to pUC11.
TVAII activity was observed in the plasmid pTO124 subcloned in 9. Next, exonuclease II
By the method using I and mung bean nuclease, p
PTO424 with 0.2 kb downstream of the TVAII gene portion of TO124, and pT with 0.5 kb further upstream
N1 was constructed and TVAII for these plasmids
It was confirmed that activity was observed. 2.1k thus obtained
As a Shine-Dalgarno ribosome binding sequence of the plasmid pTN1 containing the TVAII gene of b, the Shine-Dalg derived from the TVAII gene is directly under the promoter derived from β-galactosidase derived from pUC119.
The arno ribosome binding sequence was constructed by the site-directed mutagenesis method using the Kunkel method. Escherichia coli MV1184 strain was transformed with the obtained plasmid pTN302-10. This transformant produced 100 mg or more of TVAII per liter of culture solution.

The transformant was Escherichia coli
NK699311 (FERM P-13717) has been deposited at the Institute of Biotechnology, National Institute of Industrial Science on July 2, 1993. The transformant 1 platinum loop was placed in a medium (1% peptone,
0.5% yeast extract and 0.5% sodium chloride), and incubate at 37 ℃
Seed culture was performed by culturing for 16 hours. Obtained seed culture solution 1
mL 1.6g peptone, 1.0g yeast extract, 0.5g salt, 5mg
Add 100 mL of medium containing ampicillin, and shake in a 500 mL Sakaguchi flask at 37 ° C for 16 hours reciprocal shaking to perform main culture (medium 100
mL × 10). In addition, 0.5M isopropyl β for 5 hours of culture
0.1 mL of -D-thiogalactoside was added to the medium. The bacterial cells were collected by centrifuging the culture solution (2,000g, 20 minutes) and collecting 5mM.
The cells were suspended in 100 mM Tris-HCl buffer solution (pH 7.5) containing CaCl ₂ . The suspended bacterial cells were heat-treated at 80 ° C. for 30 minutes, then disrupted by ultrasonication, and the supernatant was recovered by centrifugation (7,500 g, 10 minutes). The obtained supernatant was used as a crude enzyme solution of TVAII and used for the reaction. The enzyme protein was measured by the method of Lowry.

20% pullulan and glucose at 5% concentration
Dissolve in 2 L of mM sodium phosphate buffer (pH 6.5) and add T
85 mg of VAII was added and reacted at 40 ° C. for 72 hours to obtain a sugar solution containing IMIM. 1/10 of the obtained sugar solution is Toyopearl H
Separation was performed using a W40S column (10 cm x 2 m). Flow rate is 10 ml
/ Min, the differential refractometer was used for detection. The pattern when separated is shown in FIG. Was collected and concentrated to dryness with an evaporator. IIM containing no IMM
0.5 g was obtained. It was found by thin layer chromatography that some of the fractions contained some IMM. The amount of crude IMIM including this slight amount of IMM is about 2 g.
Met.

[0030]

Industrial Applicability According to the present invention, a novel isomaltooligosaccharide can be provided. IMIM of the present invention, which is a novel isomalto-oligosaccharide, is one of oligosaccharides which has been attracting attention as a functional food in recent years, and has low sweetness, low calorie, non-fermentability, bifidobacterial growth factor, carious caries, etc. It is a sugar that has functionality and is highly useful as a health food material. Therefore, the IMIM of the present invention is an oligosaccharide having a high utility value in the food industry where functionality is required.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence and amino acid sequence of the TVAII gene.

FIG. 2 is a diagram showing thin layer chromatography of a reaction solution obtained by allowing pullulan to act on TVAII in the presence of glucose.

[Explanation of symbols]

 Degradation product of pullulan by PTV AII MOS Series of maltooligosaccharides

FIG. 3 is a view showing a ¹³ C-NMR spectrum of IMIM.

FIG. 4 is a diagram showing thin layer chromatography of a reaction solution obtained by allowing IMIM to act with isomaltdextranase.

[Explanation of symbols]

MOS A series of maltooligosaccharides 1 isomaltose 2 IMM 3 1% IMM dissolved in 20 mM acetate buffer (pH 5.3) 90 μl of isomaltodextranase (5 U
nit / ml) 10 μl and reacted at 40 ° C. for 1 hour 4 IMIM 5 1% IMIM dissolved in 20 mM acetate buffer (pH 5.3) 90 μl against isomaltodextranase (5
Unit / ml) was added at 10 μl and reacted at 40 ° C for 1 hour

FIG. 5 is a diagram showing a procedure for constructing a mass production system of TVAII.

FIG. 6 is a diagram showing a pattern when a sugar solution is separated by column chromatography.

Claims (5)

[Claims] 1. The following formula (I): A novel isomalto-oligosaccharide represented by. 2. Physicochemical properties shown below: (1) Action (a) Maltose is mainly produced from starch. (B) Panose is produced from pullulan. (C) Pullulan is hydrolyzed in the presence of glucose to give the following formula (I): And an isomaltooligosaccharide represented by the following formula (II): To produce the isomalto-oligosaccharide. (2) Substrate specificity The α-1,4-glucoside bond of starch and pullulan is hydrolyzed to produce maltose and panose, respectively.
The produced panose is not decomposed to produce the isomaltooligosaccharide represented by the formula (I) and the isomaltooligosaccharide represented by the formula (II) in the presence of glucose. The isomaltosyl maltose represented by the formula (II) is decomposed. (3) Optimum pH and pH stability With pullulan as a substrate, there is an optimum pH between pH 6 and 7,
It is stable at 9. (4) Optimum temperature and thermal stability With pullulan as a substrate, the optimum temperature is 45-55 ℃, and 50 ℃
Is stable up to. (5) The molecular weight is about 60,000 (by SDS polyacrylamide gel slab electrophoresis). Α-amylase having 3. In the presence of glucose, α-1,
The method for producing a novel isomaltooligosaccharide according to claim 1, wherein an enzyme that hydrolyzes a 4-glucoside bond is allowed to act on pullulan or panose. 4. The method according to claim 3, wherein the enzyme that hydrolyzes the α-1,4-glucoside bond is α-amylase that produces panose from pullulan. 5. The method according to claim 3, wherein the enzyme that hydrolyzes an α-1,4-glucoside bond is the α-amylase according to claim 2.