JP3537464B2 - Novel isomaltoligosaccharides and enzymes for producing them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel isomaltooligosaccharide which is industrially useful as a food material having excellent functionality, and an enzyme and a method for producing the same.

[0002]

2. Description of the Related Art Isomaltooligosaccharides having an α-1,6-glucosidic bond are called branched sugars, whereas maltooligosaccharides having only an α-1,4-glucosidic bond have been used as functional foods in recent years. It is a carbohydrate that has attracted attention. Originally, carbohydrates have been used as sources of sweetness and nutrients, but sugars with low sweetness, low calorie, non-fermentative properties, growth factors for bifidobacteria, intractable caries, etc. have been developed and used as health food materials. It's being used. In particular, isomalto-oligosaccharides such as isomaltose, panose (isomaltosylglucose, isomalttriose) and isomaltosylmaltose (isomaltotetraose) have been attracting attention as oligosaccharides having excellent functionality.

To date, isomaltose, panose, isomaltosyl maltose and the like are known as isomaltooligosaccharides. These isomaltoligosaccharides are obtained by hydrolyzing starch with an ordinary α-amylase at α-1,4-glucosidic bond, thereby obtaining α-1,6-derived amylopectin.
Glucosidic bonds are less susceptible to hydrolysis, so α-1,
A 6-glucosidic bond remains to form. Alternatively, isomaltose and the like are produced by the reverse reaction of glucoamylase.

However, the novel isomaltooligosaccharides of the present invention (isomaltosyl isomaltose, hereinafter referred to as "IMI")
M ”. )), Two isomaltose are α-
Oligosaccharides linked by 1,4-glucosidic bonds are not known at all.

[0005]

The present inventors have studied in detail the enzymatic action of amylase.
The inventors succeeded in obtaining IMIM, which is a homo-oligosaccharide composed of four glucoses having two glucosidic bonds and is a novel isomaltoligosaccharide, and completed the present invention.

That is, the present invention relates to isomalto-oligosaccharides which are known to exhibit excellent functionality among the oligosaccharides which have attracted attention in the food industry in which the importance of functionality is important. 4 having a 1,6-glucosidic bond
It is an object of the present invention to provide a novel isomaltooligosaccharide comprising IMIM, which is a novel isomaltooligosaccharide comprising glucose, and a 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]

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Novel isomaltooligosaccharide IMI represented by
M. (2) Physicochemical properties shown below: Action (a) Mainly produces maltose from starch. (B) Generate panose from pullulan.

(C) hydrolyzing pullulan in the presence of glucose;

[0011]

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An isomatooligosaccharide represented by the following formula (II):

[0013]

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The isomalt oligosaccharide represented by the formula is produced. It hydrolyzes α-1,4-glucosidic bonds such as substrate-specific starch and pullulan to produce maltose and panose. The produced panose is not decomposed, and in the presence of glucose, the above-mentioned formula (I)
And the isomaltooligosaccharide represented by the formula (II). Decomposes the isomaltosyl maltose represented by the formula (II).

Although the activity on starch is stronger than that on pullulan, the thermoactinomyces vulg
α-Amylase produced by aris R-47 (enzyme that mainly produces maltose from starch, hereinafter referred to as “TVAI”)
The activity on pullulan is stronger than that of. Optimum pH and pH stability Using pullulan as a substrate, there is an optimum pH between pH 6 and 7,
9 and stable.

Optimum temperature and heat stability Pullulan is used as a substrate.
Until stable. The molecular weight is about 60,000 (by SDS polyacrylamide gel slab electrophoresis). Α-amylase having the formula: (3) The method for producing a novel isomatooligosaccharide IMIM according to (1), wherein an enzyme that hydrolyzes an α-1,4-glucosidic bond is allowed to act on pullulan or panose 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 an α-1,4-glucosidic bond. Preferably, α-amylase that generates panose from pullulan is used. No. Such enzymes include, for example, high-temperature actinomycetes Thermoactinomyces
vulgaris R-47 (M. Shimizu et al., Agric. Biol. Che.
m., 42 , 1681 (1978)), ie, the novel α-amylase of the present invention.

Shimizu et al., Thermoactinomyces vu
It has been found that α-amylase TVAI produced by lgaris R-47 degrades pullulan to produce panose. Next, the inventor conducted detailed studies on the enzymatic action of TVAI, and clarified its properties (Y. Sakano et. Al.
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 separating a plasmid from the gene of this bacterium by the sit-gun method (Y. Sakano). et al., Biosci.
Biotech. Biochem., 57 , 395 (1993)). After that, TVA
A high-purity enzyme that constructs an expression system that enhances the protein synthesis ability of II and greatly increases the production of this enzyme in Escherichia coli, and at the same time, cultivates Escherichia coli and then easily crystallizes by extraction operations such as heating and ultrasonic treatment. Developed a production method.

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

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

As the enzyme used herein, for example, the above-mentioned TVAII can be mentioned. In addition, neoplulanases (N. Kuriki et al., J. Bacteriol., 170 , 1554 (198)
8); N. Kuriki et al., J. Bacteriol., 173 , 6147 (199
Α-Amylase that produces panose from pullulan such as 1)) can be used. For example, when using TVAII, both pullulan and glucose are dissolved in 1.5 L of sodium phosphate buffer (pH 6.5) at a concentration of 5%, and 60 mg of TVAII is dissolved.
In addition, the reaction is carried out at 40 ° C. for 72 hours. The reaction solution was collected over time and subjected to thin layer chromatography to produce IIM and IMM, as shown in FIG. At this time, IMM
Is reduced by TVAII, and decreases after 24 hours of the reaction.

One substrate of the reaction is pullulan or panose, and starch, amylopectin, etc., which are raw materials of panose, also serve as substrates. The concentration of pullulan or panose is preferably as high as possible, but is preferably in the range of 1 to 30%. Of course, pullulan or panose can be added during the reaction to increase the concentration of the generated IMIM. The other substrate, glucose, is usually added to the reaction solution from the beginning of the reaction, but may be added during the reaction.
That is, TVAII cannot hydrolyze panose, and the transfer reaction of glucose proceeds even in the presence of panose. Therefore, after a high-concentration pullulan solution undergoes enzymatic degradation and its viscosity decreases, glucose is added to reduce the IMIM concentration. It can be higher. The enzyme concentration varies depending on the type of the enzyme, but is usually 0.0001 to 1.0%.

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

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

[0026]

The present invention will be described in more detail with reference to the following examples.
As will be described, the technical scope of the present invention is
It is not limited. (Example 1) Thermoactinomyces vulgaris R-47 was added to Shimizu
(M. Shimizu et al., Agric. Biol. Chem.,Four
Two, 1681 (1978)) and the method of Marmur (J. Marmu
r, J. Mol. Biol.,Three, 208 (1961))
From the genomic DNA. Restrict this genomic DNA
4-10kb DNA obtained by partial digestion with the enzyme Sau3AI
The fragment was introduced into the BamHI site of the pUC119 plasmid.
Was. Escherichia coli JM83
The strain was transformed.

The search for Escherichia coli having the TVAII gene was carried out using the presence or absence of an iodine starch reaction as an index. Five strains of E. coli having the TVAII gene were obtained from about 30,000 strains, and the shortest 4 kb gene was named pTO1, and a large-scale TVAII production system was constructed according to the procedure shown in FIG. First, unnecessary parts were deleted. TV of pTO1
The 2.8 kb PstI-PstI fragment of the AII gene was replaced with pUC11.
The plasmid pTO124 subcloned in No. 9 exhibited TVAII activity. Next, exonuclease II
By using I and mung bean nuclease, p
PTO424 from which 0.2 kb downstream of the TVAII gene portion of TO124 was deleted, and pT4 from which 0.5 kb was further deleted upstream.
N1 was constructed and TVAII was also used for these plasmids.
Activity was confirmed. The 2.1k thus obtained
b, the promoter of the plasmid pTN1 containing the TVAII gene, and the Shine-Dalgarno ribosome binding sequence immediately below the pUC119-derived β-galactosidase-derived promoter, the TVAII-derived Shine-Dalg
The arno ribosome binding sequence was constructed by site-directed mutagenesis 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.

The transformant was obtained from Escherichia coli
NK699311 (FERM P-13717) has been deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on July 2, 1993. One loopful of the transformant was placed in a medium (1% peptone,
0.5% yeast extract and 0.5% salt)
For 16 hours to perform seed culture. Seed culture solution 1 obtained
mL 1.6g peptone, 1.0g yeast extract, 0.5g salt, 5mg
In addition to 100 mL of medium containing ampicillin, reciprocally shake in a 500 mL Sakaguchi flask at 37 ° C for 16 hours to perform main culture (100 mL of medium).
mL × 10). In addition, 0.5M isopropyl β
0.1 mL of -D-thiogalactoside was added to the medium. The cells were collected by centrifugation (2,000 g, 20 minutes) of the culture, and 5 mM
The suspension was suspended in a 100 mM Tris-HCl buffer (pH 7.5) containing CaCl ₂ . The suspended cells were heat-treated at 80 ° C. for 30 minutes, disrupted by sonication, 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 measurement of the enzyme protein was performed by the method of Lowry.

Both pullulan and glucose were added at a concentration of 5% to 20%.
Dissolve in 2 L of mM sodium phosphate buffer (pH 6.5)
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 used for Toyopearl H
Separation was performed using a W40S column (10 cm × 2 m). Flow rate is 10ml
/ Min, detection was performed using a differential refractometer. FIG. 6 shows the pattern when separated. Was collected and concentrated to dryness by an evaporator.
0.5 g was obtained. Was found to contain some IMM by thin layer chromatography. The amount of crude IMIM containing this slight amount of IMM is about 2 g
Met.

[0030]

According to the present invention, a novel isomaltooligosaccharide can be provided. IMIM of the present invention, which is a novel isomalt oligosaccharide, is one of the oligosaccharides that has recently attracted attention as a functional food, and has low sweetness, low calorie, non-fermentation, bifidobacterial growth factor, intractable 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 a highly useful oligosaccharide in the food industry where functionality is required.

[Brief description of the drawings]

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

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

[Explanation of symbols]

Pullulan Decomposed Product by PTVAII MOS series of maltooligosaccharides

FIG. 3 shows a ¹³ C-NMR spectrum of IMIM.

FIG. 4 is a diagram showing thin-layer chromatography of a reaction solution obtained by allowing isomalt-dextranase to act on IMIM.

[Explanation of symbols]

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

──────────────────────────────────────────────────続 き Continuation of front page (56) References Jpn. Soc. Starch Sci. , 1983, Vol. 2, pages 191-8 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/14-9/46 JSPlus (JOIS) CA (STN) REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] (1) The following formula (I): A novel isomaltoligosaccharide represented by 2. The following physicochemical properties: (1) action (a) mainly producing maltose from starch; (B) Generate panose from pullulan. (C) hydrolyzing pullulan in the presence of glucose to give the following formula (I): And an isomaltooligosaccharide represented by the following formula (II): To produce the isomaltooligosaccharide represented by (2) Substrate specificity Hydrolyze the α-1,4-glucosidic bond between starch and pullulan to produce maltose and panose, respectively.
The generated panose is not decomposed, and produces an isomaltooligosaccharide represented by the above formula (I) and an isomaltooligosaccharide represented by the above formula (II) in the presence of glucose. Decomposes the isomaltosyl maltose represented by the formula (II). (3) Optimum pH and pH stability Using pullulan as a substrate, there is an optimum pH between pH 6 and 7,
9 and stable. (4) Optimum temperature and heat stability There is an optimum temperature of 45-55 ° C using pullulan as a substrate,
Until stable. (5) The molecular weight is about 60,000 (by SDS polyacrylamide gel slab electrophoresis). Having, from actinomycetes belonging to Thermoactinomyces genus
Is α- amylase.