JP2000316581A - Production of cyclic glucan by highly thermotolerant branching enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing cyclic glucan which has cyclic structure by glucose in its molecule and is useful for improving foods and so on by cyclizing α-glucan using a branching enzyme having a specific optimal temperature. SOLUTION: This is a method for effectively and efficiently producing cyclic glucan which has cyclic structure by glucose molecule in its molecule and is useful as an additive and an improving agent for foods such as boiled rice, Japanese-style confections, snack sweets, breads, noodles, pastry cases for chaozu and shao-mai, fishery milled products, processed foods for chilled or freezed circulation, weaning foods, baby foods, feeds for animals, drinks, foods for sports, and nutritional auxiliaries by cyclizing α-glucan using a branching enzyme having an optimal reaction temperature at 65-70 deg.C where starch begins to become paste or higher.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a cyclic glucan using a highly heat-resistant blanching enzyme,
And a method for modifying food.

[0002]

2. Description of the Related Art A branching enzyme (EC. 2.4.1.
18) is an enzyme that acts on the α-1,4-glucosidic bond of the α-glucan molecule to produce an α-1,6-glucosidic bond by a transglycosylation reaction, a so-called branching reaction (see FIG. 1). 1). It is known that this enzyme is widely distributed in microorganisms such as Escherichia coli, Bacillus bacteria and yeast, plant tissues such as potato tubers, corn endosperm and spinach, and animal tissues such as muscle, liver and brain. In recent years, it has been found that branching enzymes can catalyze the cyclization of α-glucan (FIG. 2). For example, a branching enzyme can catalyze the intramolecular transfer reaction of amylopectin to synthesize a cyclic glucan having one cyclic structural part and one or more non-cyclic structural parts bonded thereto in the molecule. (Japanese Patent Laid-Open No. Hei 8-311103)
issue). Such cyclic glucans have very high solubility in water, and have the property that the solution is stable without causing cloudiness, and are expected to be applied to foods, pharmaceuticals, paper products, and the like. . Cyclic glucans are particularly useful as a raw material in the starch processing industry, food and beverage compositions, food additive compositions, infusions, adhesive compositions, starch aging inhibitors, or starch substitutes for biodegradable plastics. (See JP-A-8-311103).

Until now, branching enzymes have been found in animal tissues such as rabbit muscle, rabbit liver and brain, and in various plant tissues such as potato tubers, spinach green leaves, corn endosperm and rice endosperm. I have. Furthermore, branching enzymes have been found in mesophilic microorganisms such as yeast, Escherichia coli, and Bacillus megaterium, and also in thermophilic microorganisms such as Bacillus stearothermophilus and Bacillus calditicus.

[0004] Branching enzymes can be applied to various industrial applications. As one example, branching enzymes can be utilized in α-glucan processing, such as in the production of cyclic glucans. When an enzyme is used industrially, it is desirable to carry out the reaction at as high a temperature as possible, preferably at about 60 ° C. or higher. This is to prevent contamination of the reaction system by various bacteria. Further, among α-glucans, when relatively inexpensive starch is used as a substrate, 65-70 ° C.
It is desirable to carry out the above reaction. This is because the temperature at which the crystallinity of starch as a substrate starts to disappear, that is, the gelatinization start temperature is 65 to 70 ° C. (Carbohydrat edited by A.-C. Eliasson).
e in Food, Marcel Dekker, pp. 431-503 (1996)),
This is because the enzyme is very unlikely to act on ungelatinized starch. In particular, since the gelatinization start temperature of corn starch, which is most frequently used industrially, is about 70 ° C., it is desirable that the reaction of the branching enzyme be carried out at 70 ° C. or higher. However, among the branching enzymes mentioned above,
Plant, animal and mesophilic microorganisms
It is an enzyme with high activity in the middle temperature range of about 35 ° C. For example, the optimal reaction temperature of a branching enzyme derived from corn endosperm is about 25 to 35 ° C. (Takeda et al.,
rbohydr. Res. Vol. 240, pp. 253-263, (1993)), and the optimal reaction temperature of a branching enzyme derived from Escherichia coli is about 30.
° C (Guan et al., Arch. Biochem. Biophys., Vol. 34).
2, pp. 92-98 (1997)). On the other hand, in order to obtain an enzyme having an optimum temperature in a high temperature range, searching for an enzyme derived from a thermophilic bacterium is widely performed. The branching enzyme was also searched from Bacillus stearothermophilus, a moderately thermophilic bacterium with an optimum growth temperature of 60 to 65 ° C. As a result, a branching enzyme having an optimal reaction temperature of about 50 ° C. was obtained (Kiel et al., Mol. Gen. Genet. Vol. 230,
pp. 136-144, (1991), and Takata et al., Appl.Environ.
Microbiol. Vol. 60, pp. 3096-3104, (1994)). This branching enzyme rapidly deactivates at 65 ° C,
Heating at 60 ° C. for 30 minutes had a residual activity of 80% or more. Generally, the enzyme has an optimum temperature for the reaction at the upper limit temperature in the stable region or at a temperature slightly higher than the upper limit temperature. From this, it was inexplicable to have a relatively low optimal temperature of 50 ° C.

Further, the optimum growth temperature is about 70 ° C.
Bacillus calditicus, a highly thermophilic bacterium that can grow at 0 ° C (Heinen and Lauwers, Arch. Microbiol. Vol. 1
29, pp. 127-128, (1981)), a branching enzyme was obtained, but the optimal reaction temperature of this enzyme was only about 39 ° C. (Kiel et al., DNA Seq.-J. DNA Seq.). . Map.Vol.
3, pp. 221-232, (1992)). With respect to branching enzymes derived from mesophilic organisms, the stability and optimal reaction temperature of Bacillus megaterium-derived enzymes have been investigated. Even in the case of this enzyme, the optimum temperature for the reaction was about 25 ° C., although it was stable up to about 40 ° C. (Okada et al., Starch Chemistry Vol. 30, pp. 223-230, (1983)). In addition, three branching enzymes derived from corn endosperm also exhibited lower optimal reaction temperatures than expected from their thermostability (Takeda et al., Carbohydr. Res. Vol. 24
0, pp. 253-263, (1993)).

[0006] As described above, a phenomenon in which a branching enzyme has an optimum reaction temperature lower than that predicted from its thermal stability or from the optimum growth temperature of the organism from which it is derived is generally observed. . And the highest reaction optimum temperature found so far is only about 50 ° C.

As an explanation for such inconsistency,
The following hypothesis has been made. That is, in order for the branching enzyme to catalyze the reaction, it is important that the substrate adopt a certain conformation, and that the conformation is more stable at low temperatures (Borovsky et al., FE
BS Lett., Vol. 54, pp. 201-205 (1975) and Borovsk
y et al., Eur. J. Biochem., Vol. 62, pp. 307-312 (197
6)). As its higher-order structure, a parallel double helix structure of a glucan chain is assumed, and this structure is basically the same as that found in the crystal structure of starch (Imberty).
J. Mol. Biol. Vol. 201, pp. 365-378 (1988)).
Therefore, the branching enzyme has two parallel glucan chains, even if the enzyme itself is stable at that temperature.
It was considered that the compound had little or no activity at a high temperature at which a double helix structure could not be obtained. As a specific example of such a high temperature, a temperature of 65-70 ° C. or higher, in which the strong crystal structure in the starch starts to be broken, can be particularly envisaged. This suggests that in the cells of a thermophilic bacterium that grows at 65-70 ° C or higher, another factor (such as a special intracellular environment such as a hydrophobic environment, Protein) may be required. From the above, the branching enzyme is not suitable for use in inexpensive α-glucan processing of starch and the like on an industrial scale, especially for use at 65 to 70 ° C. or higher, which is the gelatinization start temperature of starch. Has been considered. As described above, the conventional problem that it is difficult to efficiently produce a cyclic glucan from inexpensive starch while preventing contamination by various bacteria has not been sufficiently solved. Therefore, development of a method capable of producing cyclic glucan at a temperature of 65 to 70 ° C. or higher has been expected.

[0008] As another example of the application of branching enzymes to industrial applications, branching enzymes can be used directly to modify starch-rich foods. Starch is a major component in cereals and is found in large quantities in very many foods. However, starch has the properties of being easily aged, highly viscous, and poorly soluble. Therefore, for foods high in starch, the quality (eg, physical properties and texture)
May decrease. As a result, the shape retention of food,
There is a problem that the digestibility and the decrease in freezing or refrigeration resistance are accompanied and the commercial value can be significantly impaired. Therefore, a technique for suppressing the aging of starch in food is strongly desired, and conventionally, two methods have been used.

A first method is to add a substance having an effect on preventing starch aging and reducing viscosity to food. To date, many methods have been tried to add saccharides, polysaccharides, sugar alcohols, proteins, and fatty acid esters. Furthermore, as mentioned above,
It has been reported that aging of starch was prevented by adding cyclic glucan to starch (Japanese Patent Laid-Open No. 8-31).
See 1103). The second method is a method in which an enzyme acting on starch is directly added to a food material to reduce the molecular weight of starch. The enzymes conventionally used in this method are mainly amylase which hydrolyze starch. For example, a method using a thermostable β-amylase (JP-A-62-7974)
No. 5 and JP-A-62-79746) and a method using maltose-forming α-amylase (European Patent Application Publication No. 4).
No. 94233). However, since amylase is a hydrolase, its use is limited in that it produces low-molecular-weight molecules having a sweet taste. Therefore, amylase is often unsuitable for modifying starch-containing foods.

[0010] The use of glucanotransferases, such as branching enzymes, for modifying foods has also been studied (see, for example, Japanese Patent Publication No. 58-22182 or PCT Application W097 / 41736). This is because the branching enzyme lowers the molecular weight of starch by a cyclization reaction to produce a cyclic glucan having an antiaging effect. However, as described above, the branching enzyme can hardly act at a starch gelatinization start temperature of 65 to 70 ° C. or higher, and its use range is significantly limited. Therefore, the gelatinization start temperature of starch is 65-70.
There is a demand for the development of a method for allowing a branching enzyme to act at a temperature of at least ℃ and utilizing it for modifying foods.

[0011]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has the following objects. (1) The starting temperature of starch gelatinization: 65 to To provide a method for producing a cyclic glucan with high effect and high efficiency by allowing a branching enzyme to act at 70 ° C or more (2) Acting a branching enzyme at 65 to 70 ° C or more, which is the starch gelatinization start temperature To provide a method for modifying or producing starch-containing foods

[0012]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, it has been found that a certain type of branching enzyme, contrary to the conventional hypothesis, has a temperature at the onset of starch gelatinization. It has been found that it has high activity at a certain 65 ° C. or higher. Furthermore, they have found that this branching enzyme has particularly excellent properties for producing cyclic glucan in the same kind of enzymes discovered so far and for modifying foods, and completed the present invention. The highly heat-resistant branching enzyme used in the present invention is α-
Using glucan as a substrate, it has the effect of generating a cyclic glucan by an intramolecular transfer reaction, has an optimal reaction temperature of 65 ° C or higher, and has the property of maintaining activity at 65 ° C or higher for 10 minutes or longer.

In one embodiment, the above branching enzyme is derived from a hyperthermophilic bacterium having an optimum growth temperature of 80 ° C. or higher. In one embodiment, the branching enzyme is derived from a bacterium belonging to the order Thermotogales or a bacterium belonging to the order Aquificales. In one embodiment, the above branching enzyme is derived from Aquifex aeolicus VF5, and its amino acid sequence is from Met at position 1 of SEQ ID NO: 1 to Gly at position 630 in the sequence listing. In one embodiment, the above branching enzyme is recovered from a microorganism transformed with a vector containing a gene encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing. That these highly thermostable branching enzymes can catalyze the α-1,6-glucosidic bond synthesis reaction at a temperature of 65 ° C. or higher without the intervention of other factors, and furthermore, the α-1,6-glucosidic bond The fact that the glucan can be cyclized by synthesizing is completely unpredictable from the mechanism of action of the branching enzyme that has been conventionally considered.

[0014] The present invention provides a cyclic compound comprising a step of cyclizing α-glucan at a temperature of 65 ° C. or higher using any of the above-mentioned highly thermostable branching enzymes, and a step of recovering and purifying the cyclic glucan. It relates to a method for producing glucan. In one embodiment, the α-glucan is treated with α-amylase before the cyclization step described above. In one embodiment, in the cyclization step, amylomaltase or D-enzyme is further used. In one embodiment, cyclodextrin glucanotransferase is further used in the cyclization step.

Further, the present invention is a step of adding any of the above-mentioned highly heat-resistant blanching enzyme to a food material before or immediately after cooking the material, wherein the blanching enzyme is added to the food material. The present invention relates to a method for producing a food, comprising a step of purifying cyclic glucan from starch in a material. In one embodiment, the food is cooked rice, Japanese confectionery, snack confectionery, bakery,
Noodles, dumplings and shrimp skins, fishery products, frozen or chilled processed foods, baby foods, baby foods,
It is selected from the group consisting of pet food, animal feed, beverages, sports foods, and dietary supplements. In one embodiment, the food is a Japanese sweet. In one embodiment, the food product is a snack.

The present invention also relates to a food material, a food additive, and a food modifier characterized by containing any of the above-mentioned highly heat-resistant blanching enzymes.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the present specification, “α-glucan” is α-1,4-glucan (polysaccharide having a chain structure having maltose as a disaccharide unit), or α-1 having an α-1,6-branched structure. , 4-glucan and includes amylose, amylopectin, starch, and glycogen, as well as waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolyzate, and enzyme synthesis amylopectin by phosphorylase. As used herein, “cyclic glucan”
Is a glucan having one cyclic structure in the molecule,
Its cyclic structure consists of both α-1,4-glucosidic bonds and α-1,6-glucosidic bonds. Additionally, one or more non-cyclic structural moieties may be attached to the cyclic structural moiety.

As used herein, the term "branching enzyme activity" refers to 10 mM sodium phosphate (pH 7.5), 0.06
% (w / v) The activity measured by coloring a reaction product with iodine after allowing a branching enzyme to act at 70 ° C. for 10 minutes in a composition solution composed of amylose.
The activity of reducing the absorbance at 660 nm by 1% per minute is defined as one unit. In the present specification, “quantification of a cyclic structure portion” means to quantify only a cyclic structure portion of a cyclic glucan excluding a non-cyclic structure portion. The amount of the cyclic structure portion is measured as the amount of glucan that is not decomposed by glucoamylase when the cyclic glucan is digested with glucoamylase. As used herein, "maintaining the activity at 65 ° C for 10 minutes or more" refers to a 10 mM sodium phosphate buffer.
When the branching enzyme is treated at 65 ° C. for 10 minutes in (pH 7.5), no decrease in activity is observed.

As used herein, the term “optimal reaction temperature” refers to 0.06% in 10 mM sodium phosphate buffer (pH 7.5).
(w / v) 10% at each temperature under conditions where amylose is present
The temperature at which the activity is highest when the branching enzyme is allowed to act for a minute. As used herein, the term "thermophilic bacterium" refers to a microorganism whose optimal growth temperature is 50 ° C or higher and hardly grows at 40 ° C or lower. Among these, microorganisms having an optimum growth temperature of 50 to 70 ° C are referred to as “moderate thermophiles”, and microorganisms having a temperature of 70 ° C or higher are referred to as “extreme thermophiles”. Among them, microorganisms whose optimum growth temperature is 80 ° C or higher are called “hyperthermophiles”. As used herein, the term “mesophilic bacterium” refers to a microorganism having a growth temperature in a normal temperature environment, and particularly refers to a microorganism having an optimum growth temperature of 20 to 40 ° C.

(Branching enzyme and purification thereof) The highly heat-resistant branching enzyme used in the method of the present invention is characterized by being heat-resistant and having a high optimal reaction temperature as described above. The enzyme preferably remains active after heating at 60 ° C. for 10 minutes, more preferably at 70 ° C. for 10 minutes, preferably at 65 to 100 ° C., more preferably at 70 to 95 ° C., even more preferably at 70 to 85 ° C. It has an optimum reaction temperature in ° C. In addition, even if the optimal reaction temperature is 65 ° C or lower, it can be used in the method of the present invention as long as it has sufficient activity at 65 to 70 ° C or higher.

A highly heat-resistant branching enzyme having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing in Table 1,
And 1 of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing
Or higher amino acid deletion, substitution, or highly thermostable branching enzyme containing mutation by addition,
An enzyme that can be used in the method of the present invention. The latter highly thermostable branching enzyme containing the mutation has the same or higher activity as the former enzyme not containing the mutation, and exhibits the same heat resistance and optimal reaction temperature. Mutations such as those described above
It can occur naturally or by the action of a mutagen, or artificially by introducing site-specific or site-unspecific mutations. Techniques for site-specific or site-unspecific mutation are well known in the art (eg, Nucl. Acid Research, Vol. 10, pp. 6487-65).
00 (1982), Kuipers, Methods Mol Biol. Vol. 57, pp.
351-356 (1996)). As used herein, the term "deletion, substitution, or addition of one or more amino acids" refers to a number of deletions, substitutions, or additions that can be introduced by site-specific or site-unspecific mutation. . One skilled in the art can easily select a branching enzyme mutant having the desired properties.

The corresponding highly thermostable branching enzyme gene for obtaining the highly thermostable branching enzyme used in the method of the present invention can be obtained as follows. A gene encoding the amino acid sequence of the branching enzyme of a hyperthermophilic bacterium, for example, Aquifex aeolicus VF5 (described in SEQ ID NO: 1 in Sequence Listing 1) is chemically synthesized by a method well known to those skilled in the art. To both ends of this gene, various restriction enzyme cleavage sites and sequences serving as transcription and translation signals can be added to facilitate subsequent gene expression or subcloning. Alternatively, the branching enzyme gene can be directly amplified using polymerase chain reaction (PCR) using genomic DNA of Aquifex aeolicus VF5 strain as a template.

Further, for example, Aquifex pyrophilus DSM68
Bacteria belonging to the order Aquificales such as 58, and Thermotoga ma
Using genomic DNA of a bacterium belonging to the order Thermotogales such as ritima MSB8, a branching enzyme gene of each bacterium can be obtained by Southern hybridization using a gene encoding a branching enzyme of Aquifex aeolicus VF5 as a probe. . This is Aquifex aeolicus VF5 and Aquifex pyrophilus DSM
Bacteria belonging to the order Aquificales such as 6858, and Thermotoga
This is because bacteria belonging to the order Thermotogales such as maritima MSB8 are closely related genetically to each other (for example, see Pace, Science, Vol. 276, pp. 734-740 (1997)). It is well known to those skilled in the art that closely related species have functionally and structurally similar enzymes and their genes, and one gene may be used to identify another structurally similar gene, or a portion thereof. It is also well known to those skilled in the art that they can be detected by performing Southern hybridization under stringent conditions. As used herein, the term “stringent conditions” refers to conditions that hybridize to a specific sequence but do not hybridize to a non-specific sequence. The setting of stringent conditions is well known to those skilled in the art and is described, for example, in Sambrook et al., Molecu.
lar Cloning: A Laboratory Manual (1989).

It is noted that even if a bacterium other than a hyperthermophilic bacterium, for example, a bacterium having an optimum growth temperature of less than 80 ° C.
If it can grow as above, it can be a donor of a highly thermostable branching enzyme gene. Expression of the highly thermostable branching enzyme gene obtained as described above can be performed by constructing an appropriate expression vector into which the gene fragment has been inserted, introducing the expression vector into a microorganism, and culturing and producing a recombinant microorganism. Achieved by recovery. These methods are well known to those skilled in the art. Microbial hosts for production of highly thermostable branching enzymes include prokaryotes and eukaryotes. Preferred prokaryotic hosts include mesophiles such as E. coli and Bacillus subtilis.

(Cyclization reaction with highly heat-resistant branching enzyme) Using the highly heat-resistant branching enzyme obtained as described above, cyclic glucan can be produced using α-glucan as a substrate. At this time, a highly thermostable branching enzyme can be used as the immobilized enzyme. Cyclic glucan production is achieved at the appropriate concentration of α
-It can be carried out by reacting a highly thermostable branching enzyme in a solution containing glucan. As α-glucan, linear α-glucan having a degree of polymerization of about 100 or more,
Alternatively, a branched α-glucan having a degree of polymerization of about 200 or more is preferable. Amylopectin, glycogen, starch, waxy starch, high amylose starch and the like are particularly preferred. By the method of the present invention, a cyclization reaction proceeds smoothly, and typically, a cyclic glucan having a degree of polymerization of 50 or more and a degree of polymerization of a cyclic structure portion of 10 to 200 can be produced. In particular, the degree of polymerization is about 50 to 5000, and the degree of polymerization of the cyclic structure portion is
Preferably, between 10 and 100 cyclic glucans are produced.

In order to reduce the viscosity of α-glucan as a substrate and increase the efficiency of cyclic glucan production, α-glucan is treated with α-amylase prior to addition of a highly thermostable branching enzyme. It is also possible. Typically, the reducing sugar ratio is 10
It is preferable to stop the reaction of α-amylase within a range not exceeding%. Also, highly thermostable branching enzymes can be used with other glycosyltransferases to produce cyclic glucans with more complex structures. For example, with a branching enzyme, amylomaltase (EC.2.4.
1.25) or cyclodextrin glucanotransferase (EC. 2.4.1.19). The reaction temperature is at least 65 ° C, preferably 65 to 110 ° C, particularly preferably 70 to 100 ° C. As a result, α-glucan as a substrate is gelatinized to increase the efficiency of the cyclization reaction, and at the same time, aging of the glucan and contamination of the reaction system by various bacteria are prevented. The properties of the cyclic glucan produced in the above-described method, such as aging, viscosity, anti-aging effect of starch, digestibility, and energy conversion efficiency, can be evaluated using any method known to those skilled in the art. For example, a test method described in JP-A-8-311103 can be used.

(Use of highly heat-resistant blanching enzyme for modifying foods)
Acts on starch in foods to catalyze the cyclization of starch. As a result, starch in foods is degraded to produce cyclic glucan. As described above, the cyclic glucan produced by this reaction has a very low viscosity, is excellent in solubility, and has an effect of inhibiting starch aging. Thus, a food can be modified using a branching enzyme. Typically, a branching enzyme acts on starch in food to accumulate cyclic glucan, thereby improving the physical properties (especially, stability during storage) and texture of the food. Further, the cyclic glucan produced by the cyclization of starch has a structure composed of α-1,4-glucosidic bonds and α-1,6-glucosidic bonds. This cyclic glucan
It is easily broken down into glucose by enzymes present in the organism. Therefore, the cyclic glucan also has characteristics of being excellent in digestibility and having high energy conversion efficiency.
The highly heat-resistant branching enzyme has extremely excellent heat resistance as described above, and has high activity even at 65 to 70 ° C or higher. Therefore, in using the branching enzyme for modifying food, there is an advantage that it is possible to add the enzyme before or immediately after cooking the food material.

[0028] The highly thermostable branching enzyme can be used to modify a wide range of foods (ie, any food containing starch). Foods rich in starch include cooked rice (eg, onigiri, sushi rice, and rice for lunch), Japanese confectionery (eg, bracken, manju, mochi, uiro, and rice), snack confectionery (eg, rice cracker, oyster, potato) Chips, and other snacks), bakeries (eg, bread, pie, pizza, cake, cookies, biscuits, and crackers), noodles (eg, udon, buckwheat, and ramen)
And pasta such as spaghetti and macaroni) dumplings and shrimp skins, fish paste products (eg, chikuwa and kamaboko), processed foods frozen or refrigerated, baby foods, baby foods, animal feeds, beverages (eg sports drinks) And dietary supplements. Boiled rice, Japanese sweets,
Snacks, bakeries and noodles are examples of foods preferred as objects of the present invention. Frozen or refrigerated processed foods, for which stability at low temperatures is important, are another example of a preferred food. In the present invention, a food material refers to any material for producing the above-mentioned food and any preparation in the course of processing into food.

It can be confirmed that the food treated with the highly heat-resistant blanching enzyme has been modified by examining that the starch in the food has been reduced in molecular weight and cyclic glucan has been produced. In the present invention, when the formation of cyclic glucan is observed in a food to which a highly heat-resistant blanching enzyme is added, the food is said to be modified. Food materials, food additives, and food modifiers containing highly heat-resistant blanching enzymes are useful for producing foods modified as described above. The amount of enzyme to be added can be appropriately selected by those skilled in the art. Food additives include seasonings (eg, soy sauce, sauce, sauce, stock, stew, curry, mayonnaise, dressing, ketchup, and complex seasonings). Examples of the food modifier include a starch aging inhibitor, a cooked rice modifier and the like.

[0030]

The present invention will be described in detail with reference to the following examples.
The present invention is not limited to these examples. (Example 1: Recombinant production of highly thermostable branching enzyme) (A) Preparation of Aquifex aeolicus VF5 branching enzyme gene A gene encoding the amino acid sequence of Sequence Listing 1 was chemically synthesized. An SD sequence was provided upstream of the translation initiation codon of the gene, and a BamHI site was further provided upstream thereof. An EcoRI site was provided downstream of the translation stop codon. This synthetic gene was cut with BamHI and EcoRI, and
Plasmid pUC19 cut by coRI (Takara Shuzo Co., Ltd.)
Was ligated with T4-DNA ligase to obtain plasmid pAQBE1. (B) Expression of highly thermostable branching enzyme gene in Escherichia coli Escherichia coli TG-1 strain transformed with the recombinant plasmid pAQBE1 was prepared using 0.2 l of a final concentration of 100 μg / ml ampicillin.
After culturing at 37 ° C. in an L medium (1% tryptone (Difco), 0.5% yeast extract (Difco), 1% NaCl, pH 7.5) until mid-log phase (about 3 hours), the final concentration is 0.1 mM
IPTG (Isopropyl-β-D-thiogalactopyranoside)
Was added. After further culturing at 37 ° C. for 21 hours, the cells were collected by centrifugation. The obtained cells were added to 50 ml of buffer A (1
After washing with 0 mM sodium phosphate buffer (pH 7.5) and then dispersing in 20 ml of buffer A, the cells were disrupted by ultrasonication. The lysate was heated at 70 ° C. for 30 minutes to denature Escherichia coli-derived protein, which was used as an enzyme solution. This enzyme solution and a solution obtained by treating Escherichia coli having no pAQBE1 in the same manner were subjected to SDS-polyacrylamide gel electrophoresis, and their patterns were compared, whereby the branching enzyme gene was expressed and the protein encoded by this gene was expressed. It was confirmed that it was produced.

(Example 2: Activity of highly thermostable branching enzyme) The branching enzyme activity of the enzyme solution obtained in Example 1 was measured as follows. 50 μl
50 μl in 0.12% (w / v) amylose (Sigma Type III)
Was allowed to act at 70 ° C. for 10 minutes. Amylose solution
1.2% (w / v), prepared immediately before use by adding 200 μl of a 50 mM potassium phosphate solution and 700 μl of distilled water to 100 μl of a stock solution dissolved in dimethyl sulfoxide, and stirring well to prepare a solution immediately before use. . After the action, the reaction was stopped by adding 1 ml of 0.4 mM hydrochloric acid.
1 (% (w / v) potassium iodide, 0.005% (w / v) iodine, 1.54 mM hydrochloric acid) was added, and the absorbance at 660 nm was measured. As a control, a solution obtained by adding the enzyme solution after adding 0.4 mM hydrochloric acid was used. The activity which reduced the absorbance at 660 nm by 1% per minute was defined as 1 unit, and the activity was calculated.
The branching enzyme activity of the enzyme solution was 9,500 units / ml. The decrease in absorbance due to iodine does not necessarily mean the synthesis of α-1,6-glucosidic bond. That is, the absorbance of iodine may be reduced by cleavage of α-1,4-glucoside bond of amylose by hydrolysis reaction with α-amylase or the like, or synthesis of α-1,4-glucoside bond by amylomaltase or the like. Therefore, in order to confirm the α-1,6-glucoside bond synthesizing activity of the protein in the enzyme solution, the product from amylose was analyzed as follows. 5 mg of amylose (AS-1000, manufactured by Azinoki Co., Ltd.) was added to 900 μl of 10 mM sodium phosphate buffer (pH 7.5).
, And 100 μl of the enzyme solution was added thereto, followed by incubation at 70 ° C. for 60 minutes. This was heated at 110 ° C. for 10 minutes to stop the reaction.
Aliquot 0 μl, and add 5 μl of 1 M sodium acetate buffer (pH 3.
5) and 5 μl of an isoamylase solution (0.05 mg / ml; manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) were added, and the mixture was kept at 37 ° C. for 16 hours. The obtained solution was subjected to a sugar analysis system manufactured by Dionex (liquid sending system: DX300, detector: PAD-2, analysis column:
CarboPacPA100). For elution, flow rate: 1 ml /
Min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 m
M, 2min-50mM, 37min-350mM (Gradient curve No.3),
The measurement was performed under the conditions of 45 minutes-850 mM (Gradient curve No. 7) and 47 minutes-850 mM. As shown in FIG. 3, when no isoamylase treatment was performed, almost no glucan having a short chain length was detected.
Glucans with short chain lengths of 6-30 were detected. Isoamylase is an enzyme that specifically recognizes α-1,6-glucosidic bond of α-glucan and hydrolyzes it. This confirmed that the enzyme can generate α-1,6-glucoside bond at a high temperature of 70 ° C., and that it was not necessary to intervene with other hyperthermophilic bacteria-derived factors.

(Example 3: Properties of highly thermostable branching enzyme) The enzymatic properties of the branching enzyme of Example 1 were analyzed using a method well known to those skilled in the art. The optimal reaction temperature of this enzyme is 75-80 ° C (Fig. 4),
Even after the heat treatment at 85 ° C. for 10 minutes, it had almost 100% residual activity, and almost no deactivation was observed (FIG. 5). In addition, it was confirmed in the same manner as in Example 2 that it actually had α-1,6-glucoside bond synthesizing activity at 65 to 90 ° C.

(Example 4: Production of cyclic glucan by highly heat-resistant blanching enzyme) (Production of cyclic glucan) 20 g of waxy corn starch (manufactured by Sanwa Starch Industry Co., Ltd.) was added to 100 ml of 10 mM sodium phosphate buffer ( pH 7.5) and gelatinized by heating. Then, 6,000 units of the enzyme obtained in Example 1 were added,
The reaction was carried out at 16 ° C for 16 hours. After the reaction solution was heated at 100 ° C. for 20 minutes, it was filtered through filter paper, and further passed through a membrane having a pore size of 5 μm. An equal amount of ethanol was added to this solution to precipitate glucan, which was then freeze-dried to obtain 12 g of a powder containing cyclic glucan. (Molecular weight analysis of glucan) The average molecular weight of the obtained glucan was measured by a multi-angle laser light scattering photometer (DAWN DSP, manufactured by Wyatt Technology) and a differential refractometer (Showa Denko KK)
High Performance Liquid Chromatography (HPLC) System with Shodex RI-71 (manufactured by Showa Denko KK, OHPA)
(SB-806MHQ). 30 mg of powder, 10 ml of 100 mM
It was dissolved in an aqueous solution of sodium nitrate, passed through a membrane having a pore size of 0.5 μm, and 50 μl of the solution was injected into the HPLC system. As shown in FIG. 6, glucan was eluted as a single peak from about 8.5 minutes to 10.5 minutes, indicating that the weight average molecular weight was about 174,000 (approximately 1,070 in terms of polymerization degree). The horizontal axis shows the column retention time, and the vertical axis shows the molecular weight and the differential refractometer response. (Analysis of cyclic structure portion in glucan) It was shown by the following method that the above-mentioned glucan has a cyclic structure.
Glucoamylase is an enzyme that hydrolyzes α-1,4-glucosidic bonds from the non-reducing end of glucan such as starch. It is known that α-1,6-glucosidic bonds can be hydrolyzed from the non-reducing end, though at a slow rate. It has been shown by Takata et al. That when a glucoamylase is acted on a cyclic glucan having a cyclic structure portion and a non-cyclic structure portion in a molecule, only the cyclic structure portion can be isolated (Carbohydrate Research, Vol. 295, Vol. pp. 91
-101 (1996)). In addition, the same paper indicates that no such ring-shaped structure can be obtained from waxy corn starch. After dissolving 0.25 g of glucan in 5 ml of distilled water, 0.25 ml of 1 M sodium acetate buffer (pH
5.5) and 0.25 ml of a 500 unit / ml glucoamylase (manufactured by Toyobo Co., Ltd.) solution were added, and the mixture was kept at 37 ° C. overnight. This solution was heated at 100 ° C. for 10 minutes, and then centrifuged to precipitate denatured protein. A 5-fold amount of acetone was added to the supernatant, and the circular structure was recovered as a precipitate by centrifugation. The non-cyclic structure portion was completely removed by another glucoamylase treatment to obtain 13.5 mg of the cyclic structure portion. By subjecting this cyclic structure part to the above-mentioned HPLC system,
Weight average molecular weight is about 8,600 (weight average polymerization degree is about 5
3). Furthermore, the reducing power is modified by the modified Park Johnson method (Hizukuri et al., Starch, Vol., 35, pp. 3
48-350, (1983)), it was hardly detected (less than 0.1% based on the total amount of sugars), and it was confirmed that this cyclic structure was indeed cyclic and had no reducing end. Was.

(Example 5: Use of highly heat-resistant blanching enzyme for food) In a pot, 90 g of commercially available bracken flour (main component: sweet potato starch; manufacturer: Mitsushi Yamamoto)
And 360 ml of water were mixed well. The pot was put on the heat with constant stirring, and when the bracken powder became transparent, the pot was removed from the fire and the stirring was continued. The temperature of the pot
When the temperature dropped to about 80 ° C., 30,000 units of the enzyme obtained in Example 1 were added, and then stirring was continued at about 70 ° C. for 15 minutes. The rice cake thus obtained was poured into a flat container, and cooled in a refrigerator to produce bracken rice cake. As a control, Bracken beetle to which no enzyme was added was similarly prepared. 1 g of the obtained bracken beetle was heated and dissolved in 20 ml of water to extract starch. 20 mg of the extracted starch was dissolved in 1 ml of 100 mM aqueous sodium chloride solution, and 250 μl of the dissolved starch was Superose6 (Φ1 cm × 30 cm,
Amersham Pharmacia Biotech) and Superdex30
(Φ1 cm × 30 cm, manufactured by Amersham Pharmacia Biotech) was injected into the connected column. 100m in this column
M sodium chloride aqueous solution was sent at a rate of 1 ml / min, and the detection was performed with a differential refractometer. The results shown in FIG. 7 were obtained. It can be seen that the starch of Bracken beech to which the enzyme was added was reduced in molecular weight by the cyclization reaction. Further, the same experiment as in Example 4 was performed, and it was confirmed that the low-molecular-weight glucan had a cyclic structure.

[0035]

According to the present invention, contrary to the general understanding so far, even when a certain heat-resistant branching enzyme is used at a temperature of 65 ° C. or more, which is the temperature at which gelatinization of starch starts, α-α is obtained without intervening other factors. It has been proved that it has the ability to catalyze the 1,6-glucosidic bond formation reaction. That is, the highly heat-resistant blanching enzyme has an optimum reaction temperature of 75 to 80 ° C, hardly deactivates even after heating at 85 ° C, and can be sufficiently used at 90 ° C. Therefore, in producing cyclic glucan, it is possible to overcome the problems of contamination of various bacteria caused by a low reaction temperature and the problem of aging of α-glucan, and to carry out the reaction at a gelatinization starting temperature of inexpensive starch or higher. Therefore, it has become possible to efficiently produce cyclic glucan. In addition, in the modification of food, it has become possible to reduce the molecular weight of starch in the food to produce cyclic glucan by adding an enzyme before or immediately after cooking the food.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 630 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence of SEQ ID NO: 1 Met Lys Lys Phe Ser Leu Ile Ser Asp Tyr Asp Val Tyr Leu Phe Lys 5 10 15 Glu Gly Thr His Thr Arg Leu Tyr Asp Lys Leu Gly Ser His Val Ile 20 25 30 Glu Leu Asn Gly Lys Arg Tyr Thr Phe Phe Ala Val Trp Ala Pro His 35 40 45 Ala Asp Tyr Val Ser Leu Ile Gly Asp Phe Asn Glu Trp Asp Lys Gly 50 55 60 Ser Thr Pro Met Val Lys Arg Glu Asp Gly Ser Gly Ile Trp Glu Val 65 70 75 80 Leu Leu Glu Gly Asp Leu Thr Gly Ser Lys Tyr Lys Tyr Phe Ile Lys 85 90 95 Asn Gly Asn Tyr Glu Val Asp Lys Ser Asp Pro Phe Ala Phe Phe Cys 100 105 110 Glu Gln Pro Pro Gly Asn Ala Ser Val Val Trp Lys Leu Asn Tyr Arg 115 120 125 Trp Asn Asp Ser Glu Tyr Met Lys Lys Arg Lys Arg Val Asn Ser His 130 135 140 Asp Ser Pro Ile Ser Ile Tyr Glu Val His Val Gly Ser Trp Arg Arg 145 150 155 160 Val Pro Glu Glu Gly Asn Arg Phe Leu Ser Tyr Arg Glu Leu Ala Glu 165 170 175 Tyr Leu P ro Tyr Tyr Val Lys Glu Met Gly Phe Thr His Val Glu Phe 180 185 190 Leu Pro Val Met Glu His Pro Phe Tyr Gly Ser Trp Gly Tyr Gln Ile 195 200 205 Thr Gly Tyr Phe Ala Pro Thr Ser Arg Tyr Gly Thr Pro Gln Asp Phe 210 215 220 Met Tyr Leu Ile Asp Lys Leu His Gln Glu Gly Ile Gly Val Ile Leu 225 230 235 240 Asp Trp Val Pro Ser His Phe Pro Thr Asp Ala His Gly Leu Ala Tyr 245 250 255 Phe Asp Gly Thr His Leu Tyr Glu Tyr Glu Asp Trp Arg Lys Arg Trp 260 265 270 His Pro Asp Trp Asn Ser Phe Val Phe Asp Tyr Gly Lys Pro Glu Val 275 280 285 Arg Ser Phe Leu Leu Ser Ser Ala His Phe Trp Leu Asp Lys Tyr His 290 295 300 Ala Asp Gly Leu Arg Val Asp Ala Val Ala Ser Met Leu Tyr Leu Asp 305 310 315 320 Tyr Ser Arg Lys Glu Trp Val Pro Asn Ile Tyr Gly Gly Lys Glu Asn 325 330 335 Leu Glu Ala Ile Glu Phe Leu Arg Lys Phe Asn Glu Ser Val Tyr Arg 340 345 350 Asn Phe Pro Asp Val Gln Thr Ile Ala Glu Glu Ser Thr Ala Trp Pro 355 360 365 Met Val Ser Arg Pro Thr Tyr Val Gly Gly Leu Gly Phe Gly Met Lys 370 375 380 380 Trp Asn Met G ly Trp Met Asn Asp Thr Leu Phe Tyr Phe Ser Lys Asp 385 390 395 400 400 Pro Ile Tyr Arg Lys Tyr His His Glu Val Leu Thr Phe Ser Ile Trp 405 410 415 Tyr Ala Phe Ser Glu Asn Phe Val Leu Pro Leu Ser His Asp Glu Val 420 425 430 Val His Gly Lys Gly Ser Leu Ile Gly Lys Met Pro Gly Asp Tyr Trp 435 440 445 Gln Lys Phe Ala Asn Leu Arg Ala Leu Phe Gly Tyr Met Trp Ala His 450 455 460 Pro Gly Lys Lys Leu Leu Phe Met Gly Gly Glu Phe Gly Gln Phe Lys 465 470 475 480 Glu Trp Asp His Glu Thr Ser Leu Asp Trp His Leu Leu Glu Tyr Pro 485 490 495 Ser His Arg Gly Ile Gln Arg Leu Val Lys Asp Leu Asn Glu Val Tyr 500 505 510 Arg Arg Glu Lys Ala Leu His Glu Thr Asp Phe Ser Pro Glu Gly Phe 515 520 525 Glu Trp Val Asp Phe His Asp Trp Glu Lys Ser Val Ile Ser Phe Leu 530 535 540 Arg Lys Asp Lys Ser Gly Lys Glu Ile Ile Leu Val Val Cys Asn Phe 545 550 555 560 Thr Pro Val Pro Arg Tyr Asp Tyr Arg Val Gly Val Pro Lys Gly Gly 565 570 575 Tyr Trp Arg Glu Ile Met Asn Thr Asp Ala Lys Glu Tyr Trp Gly Ser 580 585 590 590 Gly Met Gly A sn Leu Gly Gly Lys Glu Ala Asp Lys Ile Pro Trp His 595 600 605 Gly Arg Lys Phe Ser Leu Ser Leu Thr Leu Pro Pro Leu Ser Val Ile 610 615 620 Tyr Leu Lys His Glu Gly 625 630

[0036]

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing an α-1,6-glucoside bond synthesis reaction by a branching enzyme. The carbon atoms constituting the six-membered ring structure of each glucose residue are omitted. The dotted line indicates the direction in which glucose residues continue.

FIG. 2 is a conceptual diagram showing a cyclization reaction by a branching enzyme. Each glucose residue is indicated by ○, and α-1,4-
Glucosidic bonds are indicated by solid lines, and α-1,6-glucosidic bonds are indicated by vertical arrows. The dotted line indicates the direction in which glucose residues continue.

FIG. 3 shows the results of analysis using a sugar analysis system manufactured by Dionex before (A) and after (B) the treatment of amylose with a highly thermostable branching enzyme and treating the product with isoamylase. FIG.

FIG. 4 is a graph showing an optimum reaction temperature of a highly heat-resistant blanching enzyme.

FIG. 5 is a graph showing the heat resistance of a highly heat-resistant branching enzyme. 10 mM sodium phosphate buffer (pH
In 7.5), the residual activity after heating at the indicated temperature for 10 minutes is shown.

FIG. 6 is a view showing the results of analyzing the molecular weight of cyclic glucan produced by a highly thermostable branching enzyme. The solid line shows the response of the differential refractometer, and the black dots show the molecular weight of glucan at each elution position.

FIG. 7 is a diagram showing that cyclic glucan was produced in Bracken beetle (B) to which an enzyme was added. As a control, Bracken beetle to which no enzyme was added was used (A).

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) (C12N 9/10 C12R 1:01) F term (reference) 4B024 AA05 BA10 CA05 CA06 DA06 EA04 GA11 HA01 4B035 LC03 LG20 LG51 LP51 LP01 LP21 4B050 CC01 CC03 DD02 LL02 4B064 AF12 AG01 CA02 CA19 CB30 CC24 CD19 DA10 4B065 AA01Y AA26X AB01 AC14 BA02 CA29 CA41

Claims (11)

[Claims] 1. A method for producing a cyclic glucan having a cyclic structure with a glucose molecule in a molecule, comprising a step of cyclizing an α-glucan using a branching enzyme having an optimal temperature of the reaction at 65 ° C. or higher. 2. A method for producing a cyclic glucan having a cyclic structure with a glucose molecule in a molecule, comprising a step of cyclizing an α-glucan using a branching enzyme having an optimal temperature of 70 ° C. or higher. 3. The method according to claim 1, wherein the branching enzyme is a branching enzyme derived from a hyperthermophilic bacterium having an optimum growth temperature of 80 ° C. or higher. 4. The method according to claim 1, wherein the branching enzyme is Thermoto.
3. The method according to claim 1, wherein the branching enzyme is a bacterial-derived branching enzyme belonging to the order Gales or Aquificales. 5. The method according to claim 5, wherein the branching enzyme is Aquifex.
The method according to claim 1 or 2, which is a branching enzyme derived from aeolicus VF5. 6. The method according to claim 6, wherein the branching enzyme is Aquifex.
The method according to claim 1 or 2, which is a branching enzyme derived from pyrophilus DSM6858. 7. The method according to claim 1, wherein the branching enzyme is Thermoto.
The method according to claim 1 or 2, which is a branching enzyme derived from ga maritima MSB8. 8. The method according to claim 1, wherein the branching enzyme is the following branching enzyme (a), (b) or (c). (A) Amino acid sequence from Met at position 1 to Gly at position 630 of SEQ ID NO: 1 in the sequence listing (GenBank base sequence database, ACC
ESSION No., a branching enzyme having an amino acid sequence obtained by translating the nucleotide sequence from 7449 to 9341 of AE000704) (b) In the amino acid sequence (a), one or more amino acid sequences are deleted, substituted, Or a branching enzyme having an added amino acid sequence and having an optimal reaction temperature of 65 ° C. or higher. (C) GenBank base sequence database, ACCESSION number,
A branching enzyme encoded by a DNA having the nucleotide sequence described in AE000704, or a DNA that hybridizes under stringent conditions with a part thereof, wherein the optimal reaction temperature is 65 ° C or higher. Enzyme 9. A step of adding the branching enzyme according to any one of claims 1 to 8 to a food material before or immediately after heat treatment of the material, wherein the branching enzyme is added to the food material. Producing a cyclic glucan from starch in the food material. 10. The food is cooked rice, Japanese confectionery, snack confectionery, bakery, noodles, gyoza and shumai peel, fishery kneaded product, frozen or refrigerated processed food,
Baby food, baby food, animal feed, beverages, sports foods, and selected from the group consisting of dietary supplements,
The method according to claim 9. A food material, a food additive, and a food modifier, comprising the branching enzyme according to any one of claims 1 to 8.