Panose-degrading enzyme, its manufacturing method, and uses

Abstract

The present invention addresses the problem of providing: an enzyme useful for the production of isomaltose or isomalto-oligosaccharide; and a method for efficiently producing isomaltose or isomalto-oligosaccharide using the enzyme. The problem can be solved by providing: a panose-degrading enzyme having the below-mentioned substrate specificity properties (1) and (2), and a method for producing the enzyme; a microorganism capable of producing the enzyme; DNA encoding the enzyme, and recombinant DNA and a transformant each harboring the DNA; and a method for producing isomaltose or a method for producing isomalto-oligosaccharide, in which the enzyme is utilized. (1) The enzyme can hydrolyze panose to produce isomaltose and D-glucose; and (2) the enzyme cannot react with isomaltotriose, dextran and pullulan.

Description

[Technical Field]

[0001] The present invention relates to a panose-degrading enzyme, a method for producing the same, and its uses, and more specifically, to a novel panose-degrading enzyme and a method for producing the same, a microorganism capable of producing the enzyme, DNA encoding the enzyme, recombinant DNA containing the same, and a transformant, and further to a method for producing isomaltose and isomaltoligosaccharide using the enzyme. [Background technology]

[0002] Isomaltose (6-O-α-D-glucosyl-D-glucose) is a reducing disaccharide with a structure in which two D-glucose molecules are linked via an α-1,6-glucosidic bond. It is a poorly crystalline carbohydrate with excellent moisturizing properties. Isomaltose is found in trace amounts in fermented foods and has traditionally been used in various foods and cosmetics in the form of mixtures with D-glucose, maltose, panose, etc.

[0003] One known method for producing isomaltose involves decomposing starch with β-amylase to obtain maltose, which is then treated with α-glucosidase (also known as "transglucosidase") derived from Aspergillus to produce isomaltose, isomalttriose, panose, isomalttetraose, and other isomaltoligosaccharides (Patent Document 1). This is then chromatographically fractionated to extract isomaltose. However, the isomaltose content in isomaltoligosaccharide-containing carbohydrates obtained by enzymatic reactions is usually only about 26% by mass per solid, and isomaltose isolation from isomaltoligosaccharide mixtures is not easy. Furthermore, even if isomaltose is produced by treating the isomaltoligosaccharide-containing carbohydrate with isomaltodextranase (EC 3.2.1.94), the isomaltose content in the reaction solids is usually low, typically less than 40% by mass. On the other hand, another known method for producing isomaltose involves partially decomposing dextran with acid and then treating it with isomaltodextranase (Patent Document 2). However, although this method using dextran as a raw material yields a high yield of isomaltose, the production and availability of dextran, a special α-1,6 glucan, are not easy, and therefore isomaltose has not been industrially produced.

[0004] In Patent Document 3, the same applicant as the present application established and disclosed an efficient method for producing isomaltose, characterized by combining a novel enzyme, 6-α-glucosyltransferase (also known as α-isomaltosylglucoglucopropylase), with isomaltodextranase and simultaneously acting on starch or a partially hydrolyzed starch product, which is the raw material. The 6-α-glucosyltransferase is an enzyme that acts on starch or a partially hydrolyzed starch product and has the activity to produce branched α-glucan having a branched structure in which D-glucose is α-1,6 bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4 glucan chain. The branched α-glucan obtained by acting on starch or a partially hydrolyzed starch product is a carbohydrate having a branched structure in which D-glucose is α-1,6 bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4 glucan chain, that is, a carbohydrate having an isomaltose structure at the non-reducing end of the α-1,4 glucan chain. When isomaltodextranase is applied to this, the α-1,4 bond to which the isomaltosyl group is attached is specifically hydrolyzed, releasing and producing isomaltose. By repeating the reactions of these two enzymes alternately, isomaltose can be efficiently produced from starch or partially hydrolyzed starch. According to this production method, a sugar composition containing approximately 63% by mass of isomaltose per solid content is obtained from partially hydrolyzed starch by an enzymatic reaction combining 6-α-glucosyltransferase and isomaltodextranase. Furthermore, by using a starch debranching enzyme in combination with these enzymes, a sugar composition containing approximately 70% by mass of isomaltose per solid content is obtained, thus enabling the efficient production of isomaltose. However, this method using isomaltodextranase has several drawbacks, including the fact that increasing the concentration of raw starch or partially hydrolyzed starch during the enzymatic reaction to industrial production levels of 30% by mass or higher reduces isomaltose production, and even when using a starch debranching enzyme in combination, the isomaltose content per unit of solid matter in the reaction solution drops to 55% by mass or less, and that the enzymatic reaction requires a large amount of isomaltodextranase. For these reasons, it has not yet been put into practical use.

[0005] In the method for producing isomaltose in combination with the above-mentioned 6-α-glucosyltransferase, if a new enzyme can be found that can substitute for isomaltodextranase and has superior isomaltose production ability, isomaltose can be produced more efficiently using starch or partially hydrolyzed starch as a raw material. Furthermore, it is believed that if this enzyme is used in combination with other specific glycosyltransferases possessed by the applicant, efficient production of isomaltoligosaccharides will also be possible.

[0006] Among the carbohydrates having an isomaltose structure at the non-reducing end of the aforementioned α-1,4-glucan chain, the lowest molecular weight carbohydrate is panose (6 2 It is O-α-D-glucosyl-maltose. The isomaltodextranase is an enzyme that hydrolyzes panose into isomaltose and D-glucose. Besides isomaltodextranase, isopullulanase (EC 3.2.1.57, Non-Patent Document 1) is also known as an enzyme that catalyzes this reaction, but no other enzymes are known besides these two. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 61-219345 [Patent Document 2] Japanese Patent Publication No. 63-216493 [Patent Document 3] International Publication No. 2002 / 088374 Pamphlet [Non-patent literature]

[0008] [Non-Patent Document 1] Sakano et al., Biochemistry Journal (Biochem.J.), Vol. 323, pp. 757-764 (1997). [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of this invention is to provide an enzyme useful for the production of isomaltose or isomaltoligosaccharide, and an efficient method for producing isomaltose or isomaltoligosaccharide using the enzyme. [Means for solving the problem]

[0010] The present inventors focused on enzymes that have a similar effect on panose as isomaltodextranase, as useful enzymes for the production of isomaltose or isomaltoligosaccharides, and have diligently continued screening microorganisms using the activity of hydrolyzing panose into isomaltose and D-glucose as an indicator. In the process, they discovered a fungal strain U4520 that produces a completely novel panose-degrading enzyme that hydrolyzes panose into isomaltose and D-glucose, but differs from isomaltodextranase in that it does not act on isomalttriose or dextran, and also differs from the known isoplulanase in that it does not act on pullulan. The inventors established this novel panose-degrading enzyme, a method for producing it, a microorganism capable of producing the enzyme, the DNA encoding the enzyme, recombinant DNA containing it, and transformants, and established a method for producing isomaltose or isomaltoligosaccharides by combining the enzyme with other specific glycosyltransferases, thereby completing the present invention.

[0011] In other words, the present invention solves the above problems by providing a panose-degrading enzyme having the substrate specificity described in (1) and (2) below, a method for producing the same, a microorganism capable of producing the enzyme, DNA encoding the enzyme, recombinant DNA containing the same, and a transformant, as well as a method for producing isomaltose or isomaltoligosaccharide using the enzyme: (1) Hydrolyze panose to produce isomaltose and D-glucose; and (2) It does not act on isomalttriose, dextran, and pullulan. [Effects of the Invention]

[0012] The panose-degrading enzyme of the present invention specifically hydrolyzes the α-1,4 bond to which the terminal isomaltosyl group in saccharides such as panose, in which D-glucose is linked to the 6-position hydroxyl group of the non-reducing terminal glucose residue of maltooligosaccharide via an α-1,6 bond, to release isomaltose. Therefore, similar to isomaltodextranase having the same activity, by combining with other specific glycosyltransferases, isomaltose can be produced more efficiently using starch or a partially degraded starch product as a raw material. Further, the panose-degrading enzyme of the present invention specifically hydrolyzes the α-1,4 bond of a saccharide having a structure in which D-glucose is linked to the 1-position of the reducing terminal glucose of an isomaltoligosaccharide (α-1,6 glucan) in which D-glucose is linked by an α-1,6 bond, to release isomaltoligosaccharide. Therefore, by combining with other specific glycosyltransferases, isomaltoligosaccharide can be efficiently produced using starch or a partially degraded starch product as a raw material.

Brief Description of Drawings

[0013] [Figure 1] It is a TLC chromatogram of a reaction solution obtained by allowing the culture supernatant of U4520 strain to act on panose, isomaltotriose, pullulan or dextran. [Figure 2] It is a micrograph (magnification: ×600) showing the morphology of U4520 strain. [Figure 3] It is an SDS-polyacrylamide gel electrophoresis diagram of a purified preparation of panose-degrading enzyme derived from Saccharomyces cerevisiae U4520. [Figure 4] It is a diagram showing the optimum pH of the panose-degrading enzyme derived from U4520 strain. <00​​​​​​​​​​​ [Figure 9] This is a schematic diagram illustrating the action of panose-degrading enzymes derived from strain U4520 on various substrates. [Figure 10] This is a TLC chromatogram of a reaction solution obtained by reacting the culture supernatant of Acremonium microorganisms with panose, isomalttriose, pullulan, or dextran. [Figure 11] This is a schematic diagram illustrating the isomaltose production reaction from starch or partially hydrolyzed starch. [Figure 12] This is a schematic diagram illustrating the reaction for the production of isomaltoligosaccharides from starch or partially hydrolyzed starch. [Figure 13] These are TLC chromatograms of each reaction product obtained by reacting a partially hydrolyzed starch product with a combination of α-glucosyltransferase, panose-degrading enzyme, isoamylase, and α-amylase. [Figure 14] This is an HPLC chromatogram of a carbohydrate composition obtained by treating a partially hydrolyzed starch with a combination of α-glucosyltransferase, panose-degrading enzyme, isoamylase, and α-amylase, and then further treating the resulting reaction product with glucoamylase. [Modes for carrying out the invention]

[0014] The present invention relates to a panose-degrading enzyme having the following substrate specificity: (1) and (2) (1) Hydrolyze panose to produce isomaltose and D-glucose; and (2) It does not act on isomalttriose, dextran, and pullulan.

[0015] The panose-degrading enzyme of the present invention is a novel enzyme previously unknown, possessing the characteristics of (1) hydrolyzing panose to produce isomaltose and D-glucose, and (2) not acting on isomalttriose, dextran, and pullulan. The panose-degrading enzyme of the present invention can be clearly distinguished from known panose-degrading enzymes in the respect of (2) above. The known panose-degrading enzyme isomaltodextranase (EC 3.2.1.94) has the activity to hydrolyze isomalttriose to produce isomaltose and D-glucose, to hydrolyze dextran, an α-1,6-glucan, from the non-reducing end in isomaltose units to produce isomaltose, and to hydrolyze pullulan to produce isopanose (6-O-α-maltosyl-D-glucose). Similarly, the known panose-degrading enzyme isoplulanase (EC 3.2.1.57), although it does not have the activity to hydrolyze isomalttriose and dextran, does have the activity to hydrolyze pullulan to produce isopanose. Therefore, it is completely different from the panose-degrading enzyme of the present invention, which does not have the activity to hydrolyze isomalttriose, dextran, and pullulan, i.e., does not act on isomalttriose, dextran, and pullulan. Panose-degrading enzymes are included in the panose-degrading enzymes of the present invention, without being limited by their source, form, or degree of purification, such as whether they are crude or purified enzymes, as long as they are enzymes having the substrate specificity described in (1) and (2) above.

[0016] The enzymatic activity of the panose-degrading enzyme of the present invention can be measured as follows: Dissolve panose as a substrate in 50 mM phosphate buffer (pH 7.0) to a concentration of 1.0% (w / v) to prepare a substrate solution. Add 0.2 mL of enzyme solution, diluted with the same buffer, to 2.0 mL of the substrate solution and start the reaction at 30°C. At 0.5 minutes and 20.5 minutes of the reaction, 0.5 mL of the reaction solution is sampled and heated in a 100°C water bath for 10 minutes to inactivate the enzyme and stop the reaction. Then, the amount of D-glucose in each solution is quantified using the conventional glucose oxidase-peroxidase method (GOD method). The amount of D-glucose produced in 20 minutes of reaction is calculated by subtracting the amount of D-glucose at 0.5 minutes from the amount of D-glucose at 20.5 minutes. One unit (U) of panose-degrading enzyme activity is defined as the amount of enzyme that produces 1 μmol of D-glucose per minute under the above conditions.

[0017] Specific examples of the panose-degrading enzyme of the present invention include, for example, a panose-degrading enzyme having the following physicochemical properties. (a)Molecular weight In SDS-polyacrylamide gel electrophoresis, it exhibits a value of 85,000 ± 5,000 Daltons; (b) Optimum pH Under conditions of 30°C and 20 minutes of reaction, the pH is 5.0 to 5.6; (c) Optimum temperature Reaction conditions: pH 5.5, 20 minutes, 35°C; (d) pH stability Under conditions of being held at 4°C for 24 hours, it is stable in the pH range of 4.5 to 11.5; and (e) Temperature stability Under conditions of pH 5.5 and holding for 1 hour, Ca 2+ Stable up to 30°C in the absence of ions, 5 mM Ca 2+ Stable up to 35°C in the presence of ions.

[0018] Furthermore, the panose-degrading enzyme of the present invention typically has a predetermined amino acid sequence, one example being the amino acid sequence shown in Sequence ID No. 11 in the sequence listing or an amino acid sequence homologous thereto. Examples of mutant enzymes having an amino acid sequence homologous to the amino acid sequence shown in Sequence ID No. 11 in the sequence listing include those having an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in Sequence ID No. 11, while maintaining the enzymatic activity of hydrolyzing panose to produce isomaltose and D-glucose. Preferably, the amino acid sequence has an amino acid sequence that has homology of 70% or more, preferably 80% or more, and even more preferably 90% or more, to the amino acid sequence shown in Sequence ID No. 11. The amino acid sequence shown in Sequence ID No. 11 in the sequence listing is the amino acid sequence encoded by the structural gene of the panose-degrading enzyme (the amino acid sequence listed together with the base sequence shown in Sequence ID No. 10 in the sequence listing), and includes an amino acid sequence consisting of 24 amino acid residues that are presumed to be a signal peptide sequence for secretion.

[0019] The panose-degrading enzyme of the present invention is not limited by its source, but preferred sources include microorganisms, and in particular, strain U4520 or its mutant strains isolated by the inventors from soil are preferably used. Examples of mutant strains include mutant strains in which the culture characteristics are improved compared to the parent strain U4520 by introducing mutations artificially, enzyme-high-productivity mutant strains in which the panose-degrading enzyme production ability is improved compared to the parent strain U4520, and mutant strains that produce panose-degrading enzymes with higher activity.

[0020] As mentioned above, strain U4520, a microorganism capable of producing panose-degrading enzymes, was newly isolated by the inventors from soil. As described in Experiment 2 below, the homology with that of known fungi was examined based on the rRNA (rDNA) base sequence, and the species was identified by observing its morphology under a microscope. It was determined that strain U4520 is a fungus and was identified as Sarocladium kiliense. Based on these results, the inventors named strain U4520 the novel microorganism Sarocladium kiliense U4520 and deposited it with the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, and it was accepted on June 23, 2020, with accession number NITE BP-03236.

[0021] The microorganisms possessing panose-degrading enzyme production ability of the present invention include not only the above-mentioned bacteria, but also their mutant strains, and furthermore, microorganisms belonging to other genera and species possessing panose-degrading enzyme production ability, as well as their mutant strains, isolated and selected from nature by the screening method used herein, in which a culture medium is used as a crude enzyme solution to react with the substrate panose and the production of isomaltose and D-glucose. For example, microorganisms of the genus Sarocladium or Acremonium and their mutants are preferably used, more preferably Sarocladium and its mutants, and even more preferably Sarocladium killiense and its mutants. Here, while not particularly limited, examples of microorganisms belonging to the genus Sarocladium include Sarocladium bacillisporum, Sarocladium hominis, Sarocladium killiense, and Sarocladium orizae, and examples of microorganisms belonging to the genus Acremonium include Acremonium ochraceum, Acremonium implicatum, Acremonium butyri, and Acremonium furcatum. It should be noted that both the genera Sarocladium and Acremonium are classified as fungi, and in recent years, their classification has been revised, with some species of Acremonium being moved to the genus Sarocladium (Giraldo et al., Persoonia 34, pp. 10-24, 2015).

[0022] The present invention also relates to the base sequence encoding the panose-degrading enzyme according to the present invention, as described above, and to DNA having a base sequence complementary to said base sequence. The DNA of the present invention may be of natural origin or artificially synthesized, as long as it has a base sequence encoding the panose-degrading enzyme. Examples of natural sources include microorganisms of the genus Sarocladium, including Sarocladium kylience U4520, and microorganisms of the genus Acremonium. By preparing mRNA from cultures of these microorganisms and treating them with reverse transcriptase by a conventional method, cDNA encoding the panose-degrading enzyme according to the present invention can be obtained. To artificially synthesize the DNA of the present invention, for example, it can be chemically synthesized based on the amino acid sequence shown in Sequence ID No. 11 in the sequence listing. Alternatively, PCR synthesis can be advantageously carried out using cDNA containing the DNA as a template and chemically synthesized DNA as a suitable primer.

[0023] An example of DNA according to the present invention is DNA having the base sequence shown in Sequence ID No. 10 in the sequence listing, or a base sequence homologous thereto, or a base sequence complementary thereto. DNA having a base sequence homologous to the base sequence shown in Sequence ID No. 10 in the sequence listing includes DNA having a base sequence in which one or more bases are deleted, substituted, or added to the base sequence shown in Sequence ID No. 10 in the sequence listing, within the range that maintains the activity of the encoded panose-degrading enzyme. Preferably, the base sequence has homology (sequence identity) of 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more to the base sequence shown in Sequence ID No. 10 in the sequence listing. Furthermore, in DNA encoding panose-degrading enzymes, based on the degeneracy of the gene code, DNA in which one or more bases are substituted with other bases without changing the amino acid sequence of the panose-degrading enzyme each encodes, or DNA having a base sequence complementary thereto, is also included in the DNA of the present invention.

[0024] The DNA of the present invention can also be advantageously incorporated into a suitable autonomously replicating vector to produce recombinant DNA. Recombinant DNA typically consists of DNA and an autonomously replicating vector, and if DNA is available, it can be prepared relatively easily using conventional recombinant DNA techniques. Examples of such vectors include plasmids, phages, or cosmids, which can be appropriately selected depending on the cells to be introduced or the method of introduction. The specific type of vector is not particularly limited; any vector that can be expressed in host cells should be appropriately selected. Depending on the type of host cell, an appropriate promoter sequence can be selected to reliably express the above gene, and this sequence and the above gene can be incorporated into various plasmids, etc., and used as an expression vector. Examples of such expression vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosome vectors, episome vectors, and virus-derived vectors (e.g., bacterial plasmids, bacteriophages, yeast episomes, yeast chromosome elements, and viruses (e.g., baculovirus, papovavirus, vaccinia virus, adenovirus, tripoxvirus, pseudorabies virus, herpesvirus, lentivirus, and retrovirus)) and vectors derived from combinations thereof (e.g., cosmids and phagemids).

[0025] Preferred vectors for use in eukaryotes include pPICZαA, pWLNE0, pSV2CAT, pOG44, pXT1, and pSG; as well as pSVK3, pBPV, pMSG, and pSVL. Preferred vectors for use in bacteria include, for example, pRSET A, pQE-70, pQE-60, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH6a, pNH18A, and pNH46A; as well as ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5.

[0026] To insert the DNA of the present invention into such a vector, methods commonly used in this field are employed. Specifically, first, the gene DNA containing the target DNA and the autonomously replicating vector are cleaved using restriction enzymes and / or ultrasound, and then the resulting DNA fragments and vector fragments are ligated together. The recombinant DNA thus obtained can be introduced into a host to create a transformant, which can then be cultured to replicate indefinitely.

[0027] The recombinant DNA obtained in this way can be introduced into suitable host microorganisms, including yeast, Escherichia coli, Bacillus subtilis, and actinomycetes. To obtain transformants, colony hybridization can be applied, or they can be cultured in a nutrient medium and selected to produce panose-degrading enzymes.

[0028] The culture medium used for culturing microorganisms, including the transformants of the present invention that have the ability to produce panose-degrading enzymes, can be any nutrient medium on which microorganisms can grow and produce panose-degrading enzymes, and may be either a synthetic medium or a natural medium. As a carbon source, any substance that microorganisms can utilize for growth is acceptable, such as starch and its partial decomposition products, plant-derived starch and phytoglycogen, animal or microbial-derived glycogen and pullulan, as well as their partial decomposition products, sugars such as glucose, fructose, lactose, sucrose, mannitol, sorbitol, and molasses, and organic acids such as citric acid and succinic acid, and alcohols such as methanol and ethanol. The concentration of these carbon sources in the culture medium can be appropriately selected depending on the type of carbon source. As a nitrogen source, inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, and meat extract can be appropriately used. Furthermore, as inorganic components, salts such as calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, and cobalt salts can be used as appropriate. In addition, amino acids and vitamins can be used as appropriate, if necessary.

[0029] Culturing is usually carried out aerobically under conditions selected from a temperature of 15 to 37°C and a pH of 5.5 to 10, preferably a temperature of 20 to 34°C and a pH of 5.5 to 8.5. The culturing time should be any time required for the microorganisms having panose-degrading enzyme production ability of the present invention to grow, preferably 10 to 150 hours. There are no particular restrictions on the dissolved oxygen concentration of the culture medium under the culturing conditions, but it is usually preferably 0.5 to 20 ppm. To this end, appropriate measures such as adjusting the aeration rate or stirring are employed. The culturing method may be either batch culturing or continuous culturing.

[0030] After culturing the microorganisms in this manner, the culture containing the enzyme of the present invention is recovered. Panose-degrading enzyme activity is mainly observed in the disinfection solution of the culture, and the disinfection solution can be collected as the crude enzyme solution, or the entire culture can be used as the crude enzyme solution. Known solid-liquid separation methods are used to remove microbial cells from the culture. For example, methods such as centrifuging the culture itself, filtration separation using a pre-coated filter, or separation by membrane filtration using a flat membrane or hollow fiber membrane can be used as appropriate. Although the disinfection solution can be used directly as the crude enzyme solution, it is generally concentrated before use. Concentration methods that can be used include ammonium sulfate salting-out, acetone and alcohol precipitation, and membrane concentration methods using flat membranes, hollow membranes, etc.

[0031] Furthermore, panose-degrading enzymes can be immobilized using known methods with disinfectant solutions and their concentrates that possess panose-degrading enzyme activity. For example, methods such as binding to ion exchangers, covalent bonding and adsorption with resins and membranes, and encapsulation methods using polymeric substances can be appropriately employed.

[0032] As described above, the panose-degrading enzyme of the present invention can be used as is or concentrated from the crude enzyme solution, but it can also be further separated and purified by known methods as needed. For example, the crude enzyme standard obtained by ammonium sulfate precipitation of the culture solution and concentration can be dialysis-treated and then purified using anion exchange chromatography with a "DEAE-Toyopearl 650S" gel (manufactured by Tosoh Corporation), hydrophobic chromatography with a "Phenyl-Toyopearl 650M" gel (manufactured by Tosoh Corporation), or gel filtration chromatography with a "Superdex 200pg" gel (manufactured by GE Health Sciences), thereby obtaining a purified enzyme that has been purified to a single electrophoretic level.

[0033] By using the natural or recombinant panose-degrading enzymes obtained by the above method in combination with other glycosyltransferases having specific activity, isomaltose, isomaltoligosaccharides, and carbohydrates containing them can be efficiently produced from starch or partially hydrolyzed starch as raw materials.

[0034] When producing isomaltose or isomaltoligosaccharides by combining the panose-degrading enzyme of the present invention with other glycosyltransferases, suitable raw material substrates include, for example, above-ground starches such as corn starch, rice starch, and wheat starch, underground starches such as potato starch, sweet potato starch, and tapioca starch, and their partial hydrolysates (partially hydrolyzed starch products). The aforementioned partially hydrolyzed starch products can usually be obtained by suspending the above-ground or underground starch in water to a starch milk with a concentration of 10% by mass or more, more preferably 15% to 65% by mass, and even more preferably 20% to 50% by mass, heating it to gelatinize it, and then liquefying (partially hydrolyzing) it with acid or heat-resistant α-amylase. The degree of liquefaction is preferably set relatively low, and is usually less than DE (Dextrose Equivalent, glucose equivalent) 15, preferably less than DE 10, and more preferably in the range of DE 9 to 0.1. When liquefying with acid, for example, the material is liquefied with an acidic agent such as hydrochloric acid, phosphoric acid, or oxalic acid, and then typically neutralized to the desired pH using an alkaline agent such as calcium carbonate, calcium oxide, or sodium carbonate.

[0035] When the panose-degrading enzyme of the present invention is combined with other glycosyltransferases and reacted with a substrate raw material, the substrate concentration is not particularly limited, but a substrate concentration of 40% by mass or less is preferable, and isomaltose and isomaltoligosaccharides can be advantageously produced under these conditions. The reaction temperature should be up to the temperature at which the reaction proceeds, i.e., around 45°C. Preferably, a temperature of around 30°C is used. The reaction pH is usually adjusted to a range of 4.0 to 6.0, preferably a range of pH 5.0 to 5.5. The amount of enzyme used and the reaction time are closely related, and the amount of enzyme used and the reaction time should be appropriately adjusted according to the progress of the desired enzymatic reaction.

[0036] <Method for producing isomaltose> The present invention also relates to a method for producing isomaltose, comprising the steps of: combining a 6-α-glucosyltransferase (also known as α-isomaltosylglucosaccharide-producing enzyme) which has the activity to act on starch or a partially hydrolyzed starch product to produce a branched α-glucan (also known as α-isomaltosylglucosaccharide) having a branched structure in which D-glucose is bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4 glucan chain via an α-1,6 bond; and the panose-degrading enzyme of the present invention, and acting on starch or a partially hydrolyzed starch product to produce isomaltose; and collecting the produced isomaltose.

[0037] The present invention relates to a method for producing isomaltose, which is disclosed in Patent Document 3 by the same applicant as the present application, and involves combining 6-α-glucosyltransferase (also known as α-isomaltosylglucoglucopropylase) and isomaltodextranase, but with the panose-degrading enzyme of the present invention used instead of isomaltodextranase. Isomaltodextranase has the activity to hydrolyze isomalttriose to produce isomaltose and D-glucose, the activity to hydrolyze dextran, an α-1,6 glucan, from the non-reducing end in isomaltose units to produce isomaltose, the activity to hydrolyze panose into isomaltose and D-glucose, and the activity to hydrolyze pullulan to produce isopanose (6-O-α-maltosyl-D-glucose). In contrast, the panose-degrading enzyme of the present invention is the same as isomaltodextranase in that it has the activity to hydrolyze panose into isomaltose and D-glucose, but it is a decisively different enzyme from isomaltodextranase in that it does not hydrolyze the α-1,6 bond and therefore does not have the activity to hydrolyze isomalttriose or dextran. When 6-α-glucosyltransferase and panose-degrading enzyme are combined and applied to starch or a partially hydrolyzed starch product, the 6-α-glucosyltransferase enzyme produces a branched α-glucan with a branched structure in which D-glucose is α-1,6 bonded to the hydroxyl group at the 6th position of the non-reducing end glucose residue of the α-1,4-glucan chain. In other words, a carbohydrate with an isomaltose structure at the non-reducing end of the α-1,4-glucan chain (also known as α-isomaltosylglucosaccharide) is produced. Subsequently, the panose-degrading enzyme specifically hydrolyzes the α-1,4 bond to which the isomaltosyl group at the end is attached, producing isomaltose. This reaction is repeated, resulting in the accumulation of isomaltose in the reaction solution.

[0038] Examples of 6-α-glucosyltransferases (also known as α-isomaltosylglucoglucotransferases) that can be suitably used in the method for producing isomaltose of the present invention include enzymes derived from Bacillus globisporus C9, Bacillus globisporus C11, or Bacillus globisporus N75, and enzymes derived from Arthrobacter globiformis A19 or Arthrobacter ramosus S1, as disclosed by the same applicant in International Publication No. 2002 / 010361. Bacillus globisporus is now classified as Paenibacillus filicis based on the homology (identity) of the 16S rDNA base sequence.

[0039] The enzyme activity of the 6-α-glucosyltransferase used in the method for producing isomaltose of the present invention can be measured by the following method disclosed in Patent Document 3 by the same applicant as the present application. Specifically, maltotriose is dissolved in 100 mM acetate buffer (pH 6.0) to a concentration of 2% (w / v) to prepare a substrate solution, 0.5 mL of enzyme solution is added to 0.5 mL of the substrate solution, the enzyme reaction is carried out at 35°C for 60 minutes, the reaction solution is boiled for 10 minutes to stop the reaction, and the isomaltosylmaltose (6) mainly produced in the reaction solution is measured. 3 Of the two components, maltose (α-D-glucosylmaltotriose) and maltose, is quantified by high-performance liquid chromatography (HPLC). HPLC is performed using a YMC Pack ODS-AQ303 column (manufactured by YMC Corporation), deionized water as the eluent, at a column temperature of 40°C and a flow rate of 0.5 mL / min. The produced sugar is detected using a differential refractometer RI-8012 (manufactured by Tosoh Corporation). One unit (U) of 6-α-glucosyltransferase activity is defined as the amount of enzyme that produces 1 μmol of maltose per minute under the above conditions.

[0040] Furthermore, in the step of producing isomaltose in the isomaltose production method of the present invention, it is advantageous to also use, if necessary, one or more enzymes selected from starch branching enzymes such as isoamylase and pullulanase, α-amylase, cyclomaltodextrin glucanotransferase (CGTase), and glucoamylase in combination. In particular, starch branching enzymes are enzymes that specifically hydrolyze (de-branch) the α-1,6 bonds in the branched structure via α-1,6 bonds in the raw material starch or starch hydrolysate, and are therefore commonly used to increase the yield of the target oligosaccharide when producing various oligosaccharide products using starch or starch hydrolysate as a raw material. By using starch branching enzymes in combination, the amount of isomaltose produced can also be increased in the isomaltose production method of the present invention, which combines 6-α-glucosyltransferase and panose-degrading enzyme.

[0041] Patent Document 3 (International Publication No. 02 / 088374) discloses a method for producing isomaltose by combining the 6-α-glucosyltransferase and isomaltodextranase. In this method, which also uses a starch debranching enzyme, the isomaltose content per reaction solid of the reactant obtained from the partially hydrolyzed raw starch is approximately 70% by mass at a substrate concentration of 5% by mass, approximately 55% by mass at a substrate concentration of 30% by mass, and approximately 50% by mass at a substrate concentration of 40% by mass. It is stated that increasing the substrate concentration significantly reduces the production of isomaltose. In contrast, when isomaltodextranase is replaced with the panose-degrading enzyme of the present invention, as shown in the experimental section described later, the isomaltose content per reaction solid of the reactant reaches 70% by mass or more even at a substrate concentration of 30 to 40% by mass, allowing for more efficient production of isomaltose compared to isomaltodextranase.

[0042] Furthermore, the present invention relates to a method for producing isomaltose, further comprising the steps of reducing isomaltose by hydrogenation to convert it to isomaltitol and collecting the converted isomaltitol, to the above-mentioned method for producing isomaltose. Isomaltitol or isomaltitol-containing products can be obtained in high yield by further hydrogenation and reduction of the isomaltose or isomaltose-containing carbohydrate obtained by the above-mentioned method for producing isomaltose under a reducing catalyst. Specifically, for example, a Raney nickel catalyst is added to an aqueous solution containing isomaltose with a solid content of 40 to 60%, this is placed in a pressure-resistant container, hydrogen is filled into the container, pressurized, and hydrogenation is carried out by stirring at a temperature of 100 to 120°C until no more hydrogen is consumed. At this time, isomaltose is reduced and converted to isomaltitol, and other reducing carbohydrates that may be contained in the isomaltose-containing product, such as D-glucose, maltose, maltotriose, and other reducing starch partial hydrolysates are also reduced to sugar alcohols. The obtained isomaltitol-containing solution is separated from the Raney nickel catalyst, decolorized with activated carbon according to a conventional method, desalted with H-type, OH-type ion exchange resin, etc., purified, concentrated to obtain a syrup, or further dried to obtain a powder. If necessary, further purification can be performed using one or more methods, such as fractionation and crystallization by column chromatography (e.g., ion exchange column chromatography, activated carbon column chromatography, silica gel column chromatography), fractionation using organic solvents such as alcohol and acetone, or membrane separation, to obtain high-purity isomaltitol. It should be noted that isomaltitol is known to exist in crystal form, and even higher purity products can be produced by crystallization.

[0043] <Method for producing isomaltoligosaccharide> The present invention also relates to a method for producing isomaltoligosaccharide, comprising the steps of: combining an α-glucosyltransferase having the activity to act on starch or a partially hydrolyzed starch product to produce a branched α-glucan having a branched structure in which D-glucose or α-1,6-glucan with a degree of polymerization of 2 or more of glucose is bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain via an α-1,6 bond; and the panose-degrading enzyme of the present invention, and acting on starch or a partially hydrolyzed starch product to produce isomaltoligosaccharide; and collecting the produced isomaltoligosaccharide.

[0044] The present invention provides a novel isomaltoligosaccharide production method that combines an α-glucosyltransferase disclosed by the same applicant in International Publication No. 2002 / 010361 with the panose-degrading enzyme of the present invention. When the α-glucosyltransferase and panose-degrading enzyme are combined and applied to starch or a partially hydrolyzed starch product, the α-glucosyltransferase produces a branched α-glucan having a branched structure in which D-glucose or α-1,6-glucan with a glucose polymerization degree of 2 or higher is α-1,6-linked to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain. Subsequently, the panose-degrading enzyme specifically hydrolyzes the isomaltosyl group at the terminal or the α-1,4 linkage to which α-1,6-glucan with a glucose polymerization degree of 3 or higher is attached, thereby producing isomaltoligosaccharide with a glucose polymerization degree of 2 or higher.

[0045] Examples of α-glucosyltransferases that can be suitably used in the method for producing isomaltooligosaccharides of the present invention include enzymes derived from Bacillus circulans PP710 and Arthrobacter globiformis PP349, disclosed by the same applicant in International Publication No. 2008 / 136331.

[0046] The enzymatic activity of the α-glucosyltransferase used in the method for producing isomaltooligosaccharide of the present invention can be measured by a method disclosed in International Publication No. 2008 / 136331 by the same applicant as this application, which uses maltose as a substrate and quantifies the amount of D-glucose remaining after the α-glucosyltransferase transfers the glucosyl group on the non-reducing end of maltose. Specifically, maltose is dissolved in 20 mM acetate buffer (pH 6.0) to a final concentration of 1% (w / v) to prepare a substrate solution. 0.5 mL of the enzyme solution is added to 5 mL of the substrate solution, and the enzymatic reaction is carried out at 40°C for 30 minutes. 0.5 mL of the reaction solution is mixed with 5 mL of 20 mM phosphate buffer (pH 7.0), and the reaction is stopped by heating in a boiling water bath for 10 minutes. The amount of glucose in the reaction solution is then measured by the glucose oxidase-peroxidase method according to a conventional method, and the amount of D-glucose produced by the enzymatic reaction is calculated. One unit (U) of α-glucosyltransferase activity is defined as the amount of enzyme that produces 1 μmol of D-glucose per minute under the above conditions.

[0047] Furthermore, in the step of producing the isomaltoligosaccharide in the method for producing isomaltoligosaccharide of the present invention, as described in the section on the method for producing isomaltose, it is also advantageous to use in combination, if necessary, one or more enzymes selected from starch debranching enzymes such as isoamylase and pullulanase, α-amylase, CGTase, and glucoamylase.

[0048] As shown in the experimental section described later, in the method for producing isomaltoligosaccharides of the present invention, which combines α-glucosyltransferase and the panose-degrading enzyme of the present invention, and further uses starch debranching enzyme and α-amylase, the total content of isomaltoligosaccharides from DP2 isomaltose to DP8 isomaltoctaose per unit of reaction solids of the reactant obtained from the partially hydrolyzed raw starch reaches at least 73% by mass. Therefore, according to the method for producing isomaltoligosaccharides of the present invention, isomaltoligosaccharides can be produced with significantly greater efficiency than conventional methods, using starch or partially hydrolyzed starch as raw materials.

[0049] Furthermore, the present invention relates to a method for producing isomaltoligosaccharide alcohol, which further adds to the above-described method for producing isomaltoligosaccharide a step of reducing the isomaltoligosaccharide by hydrogenation to convert it into isomaltoligosaccharide alcohol, and a step of collecting the converted isomaltoligosaccharide alcohol. The isomaltoligosaccharide or isomaltoligosaccharide-containing carbohydrate obtained by the above-described method for producing isomaltoligosaccharide can be converted into isomaltoligosaccharide alcohol, i.e., a mixture of isomaltitol, isomalttriitol, isomalttetraitol, isomaltopentitol, etc., by hydrogenation and reduction under a reducing catalyst, similar to the case of isomaltose described above.

[0050] The reaction products or their reduced products (hydrogenated products) obtained by the isomaltose production method or isomaltoligosaccharide production method of the present invention described above can be used as isomaltose-containing sugar solution, isomaltoligosaccharide-containing sugar solution, or sugar alcohol-containing solutions thereof, but generally, they are further purified before use. As for the purification method, any conventional method used for purifying sugars and sugar alcohols may be used as appropriate. For example, decolorization with activated carbon, desalting with H-type or OH-type ion exchange resins, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, or silica gel column chromatography, fractionation with organic solvents such as alcohol and acetone, separation with a membrane having appropriate separation performance, and furthermore, purification methods such as fermentation treatment by microorganisms that assimilate and decompose impurities without using isomaltose, isomaltoligosaccharides, or their sugar alcohols, such as yeast, can be used as appropriate.

[0051] In particular, ion exchange column chromatography is preferred as a mass production method. For example, by using column chromatography with a strongly acidic cation exchange resin disclosed in Japanese Patent Publication No. 58-23799 and Japanese Patent Publication No. 58-72598, contaminating sugars can be removed, and isomaltose-containing carbohydrates, isomaltoligosaccharide-containing carbohydrates, or sugar alcohol-containing products thereof, with an improved content of the target substance, can be advantageously produced. In this case, any of the following methods can be used: fixed bed, moving bed, or pseudo-moving bed.

[0052] The aqueous solutions containing isomaltose, isomaltoligosaccharides, or their sugar alcohols (hydrogenated) obtained in this manner can usually be concentrated to form a syrup product. This syrup product can optionally be further dried to form an amorphous solid product or an amorphous powder product.

[0053] The powdered products of isomaltoligosaccharide-containing carbohydrates, isomaltose-containing carbohydrates, or their sugar alcohols (hydrogenated) obtained by the manufacturing method of the present invention may be used as is, or mixed with fillers, excipients, binders, etc. as needed, and molded into various shapes such as granules, spheres, short rods, plates, cubes, tablets, etc.

[0054] The isomaltoligosaccharide-containing carbohydrates, isomaltose-containing carbohydrates, or their sugar alcohols (hydrogenated) obtained by the production method of the present invention can be advantageously used in combination with other components as sweeteners, flavor enhancers, quality enhancers, stabilizers, discoloration inhibitors, excipients, etc., in various compositions such as food and beverages, stimulants, animal feed, vegetarian feed, cosmetics, pharmaceuticals, and industrial products.

[0055] The method for incorporating isomaltoligosaccharide-containing carbohydrates, isomaltose-containing carbohydrates, or their sugar alcohols (hydrogenated) obtained by the manufacturing method of the present invention into the above-mentioned compositions is to incorporate them during the process until the product is completed. For example, known methods such as mixing, kneading, dissolving, melting, immersion, penetration, spraying, coating, coating, atomizing, injection, crystallization, and solidification can be appropriately selected. The amount is usually 0.1% by mass or more, preferably 1% by mass or more.

[0056] The present invention will be described in detail below through experiments. The activity of the panose-degrading enzyme in the following experiments is expressed as the activity that produces D-glucose from the substrate panose, as determined by the activity measurement method described above.

[0057] <Experiment 1: Screening of panose-degrading enzyme-producing bacteria from soil-isolated bacteria> 950 microbial strains isolated from soil were each inoculated into a liquid culture medium (pH 6.8) consisting of 15 g / L of partially hydrolyzed starch (product name "Pinedex #4", sold by Matsutani Chemical Industry Co., Ltd.), 1.0 g / L of yeast extract (product name "Yeast Extract SH", sold by Nippon Pharmaceutical Co., Ltd.), 5.0 g / L of peptone (product name "High Polypeptone", sold by Nippon Pharmaceutical Co., Ltd.), 1.0 g / L of dipotassium phosphate, 0.6 g / L of monosodium phosphate heptahydrate, 0.5 g / L of magnesium sulfate heptahydrate, 0.01 g / L of ferrous sulfate heptahydrate, 0.01 g / L of manganese sulfate pentahydrate, 3.0 g / L of calcium carbonate, and water, with 3 mL of the medium placed in a test tube. The culture medium was then sterilized in an autoclave at 121°C for 20 minutes and the cultured cells were incubated at 27°C at 240 rpm with shaking for 3 days. To the obtained culture medium, 100 μL of 6 mg / mL lysozyme solution and 100 μL of 3% surfactant (product name "Triton X100") solution were added, and the mixture was shaken at 27°C for 2 hours to lyse the cells and obtain a crude enzyme solution. Next, the obtained crude enzyme solution was mixed in equal volumes with a substrate solution containing 3% (w / v) panose, 1 mM acarbose, 50 mM acetate buffer (pH 5.5), and 120 ppm preservative, and the mixture was reacted at 40°C for 24 hours. The resulting reaction solution was subjected to TLC analysis under the following conditions to investigate whether or not there was an effect on panose, and if there was an effect, the product was examined.

[0058] <TLC analysis conditions> TLC plate: Silica gel aluminum plate (trade name 'Silica gel 60F254' , 10×20 cm, manufactured by Merck) Developing solvent: n-butanol: pyridine: water mixture (volume ratio 6:4:1) Developing method: Ascending method, single development Detection method: Sulfuric acid-methanol method

[0059] When acting on the substrate panose, the microorganism that produces an enzyme that clearly shows isomaltose and D-glucose as products in TLC analysis was only one strain of U4520. The U4520 strain was selected, and its culture supernatant was used as a crude enzyme and allowed to act on isomaltotriose, pullulan, or dextran in the same manner as panose, and an attempt was made to identify the enzyme from its reactivity. The TLC chromatograms of the reaction solutions obtained by allowing the crude enzyme derived from the U4520 strain to act on panose, isomaltotriose, pullulan, and dextran are shown in Fig. 1. As can be seen in Fig. 1, the crude enzyme derived from the U4520 strain hydrolyzes panose into isomaltose and D-glucose (reference numeral 8 in Fig. 1), while it did not act on any of isomaltotriose (reference numeral 9 in Fig. 1), pullulan (reference numeral 10 in Fig. 1), and dextran (reference numeral 11 in Fig. 1). Although isomaltodextranase (EC 3.2.1.94) and isopullulanase (EC 3.2.1.57) are known as enzymes that hydrolyze panose into isomaltose and D-glucose, isomaltodextranase is an enzyme that hydrolyzes isomaltotriose, pullulan, and dextran, and isopullulanase is an enzyme that does not act on isomaltotriose and dextran but hydrolyzes pullulan to produce isopanose. Therefore, it was found that the panose-degrading enzyme derived from the U4520 strain is a novel enzyme that is different from both isomaltodextranase and isopullulanase and has not been known before.

[0060] <Experiment 2: Identification of the panose-degrading enzyme-producing bacterium, strain U4520> Figure 2 shows a microscopic image of strain U4520, a panose-degrading enzyme-producing fungus isolated during soil screening. As can be seen in Figure 2, microscopic observation revealed that strain U4520 is a fungus, as it exhibits hyphae, conidiophores, and conidia. Furthermore, it was found that numerous conidia formed clusters at the head of the conidiophores. In this experiment, the rRNA (rDNA) base sequence was determined, and the species identification of the microorganism U4520 was performed based on this base sequence information and the morphology of the fungus observed under a microscope.

[0061] <Experiment 2-1: Preparation of rDNA from strain U4520> A culture medium (pH 6.8) consisting of 1.5 g / L of commercially available dextrin (product name "Pinex #4", sold by Matsutani Chemical Industry Co., Ltd.), 0.2 g / L of yeast extract (product name "Yeast Extract SH", sold by Nippon Pharmaceutical Co., Ltd.), 1.0 g / L of polypeptone (product name "High Polypeptone", sold by Nippon Pharmaceutical Co., Ltd.), 1.0 g / L of dipotassium phosphate, 0.6 g / L of monosodium phosphate heptahydrate, 0.5 g / L of magnesium sulfate heptahydrate, 0.01 g / L of ferrous sulfate heptahydrate, 0.01 g / L of manganese sulfate pentahydrate, 20.0 g / L of agar, and water was sterilized in an autoclave at 121°C for 20 minutes, then dispensed into petri dishes and cooled to prepare agar plates. Next, strain U4520 was inoculated into these plates and incubated statically at 27°C for 5 days to form single colonies.

[0062] The U4520 strain, which had formed a single colony as described above, was isolated and suspended in 50 μL of a commercially available rapid DNA extraction reagent (product name "MightyPrep reagent for DNA", sold by Takara Bio Inc.). After treatment at 95°C for 10 minutes, the supernatant containing genomic DNA was collected by centrifugation at 15,000 rpm for 2 minutes. PCR was performed on the collected genomic DNA of the U4520 strain using the sense primer "ITS" with the nucleotide sequence shown as SEQ ID NO: 1 in the sequence listing, and the antisense primer "LR7" with the nucleotide sequence shown as SEQ ID NO: 2 in the sequence listing. Agarose electrophoresis was performed on the PCR amplification product, and a PCR amplification product of approximately 2 kbp was observed, which was collected by ethanol precipitation and identified as rDNA.

[0063] <Experiment 2-2: Determination of rDNA base sequence> The rDNA sequence of the U4520 strain obtained in Experiment 2-1 was determined by a standard method and found to have the nucleotide sequence (1,658 bp) shown as Sequence ID No. 3 in the sequence listing.

[0064] <Experiment 2-3: Identification of Microorganism U4520 Strain> The rDNA base sequence determined in Experiment 2-2 was subjected to a homology search in a base sequence database using the base sequence homology search program "BLASTN". To remove uncertain information, a comparison was made using type material from the NCBI Taxonomy database.

[0065] Table 1 shows the results of a homology search performed on the D1 / D2 region of the 26S rDNA of strain U4520, which is used for fungal species identification. Table 2 shows the results of a homology search performed on the ITS1·ITS2 region located between the 18S rDNA and 26S rDNA, which is also used for fungal species identification.

[0066] [Table 1]

[0067] [Table 2]

[0068] As shown in Tables 1 and 2, the rDNA sequence of strain U4520 showed 99.67% homology (identity) with Sarocladium kiliense in the D1 / D2 region and 100% homology with Dictyosporium digitatum. Furthermore, the rDNA sequence of strain U4520 showed 100% homology with Sarocladium kiliense in the ITS1-ITS2 region and 95.52% homology with Sarocladium hominis. Generally, it is said that if there is 99% or more homology in the rDNA sequence of bacteria, there is a high probability that they are the same species. Furthermore, the aforementioned microscopic observations revealed that strain U4520 was morphologically similar to Sarocladium kyliens and significantly different from Dictyosporium digitatum. Based on the rDNA sequence and microscopic morphological observations, strain U4520 was identified as Sarocladium kyliens and named Sarocladium kyliens U4520.

[0069] Salocladium kyliens U4520 was deposited with the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, and was accepted on June 23, 2020, under accession number NITE BP-03236.

[0070] <Experiment 3: Crude panose-degrading enzyme obtained by culturing Salocladium kyliens U4520> Preparation of enzyme solution A liquid culture medium (pH 6.8) consisting of 45g / L of commercially available dextrin (product name "Pinex #4", sold by Matsutani Chemical Industry Co., Ltd.), 1.0g / L of yeast extract (product name "Yeast Extract SH", sold by Nippon Pharmaceutical Co., Ltd.), 15g / L of polypeptone (product name "High Polypeptone", sold by Nippon Pharmaceutical Co., Ltd.), 1.0g / L of dipotassium phosphate, 0.6g / L of monosodium phosphate heptahydrate, 0.5g / L of magnesium sulfate heptahydrate, 0.01g / L of ferrous sulfate heptahydrate, 0.01g / L of manganese sulfate pentahydrate, and water was prepared by placing 100mL of this mixture into 500mL Erlenmeyer flasks, and 70 flasks were prepared. These flasks were sterilized in an autoclave at 121°C for 20 minutes, and then Salocladium kyliens, which had been cultured in seed culture medium beforehand, was prepared. A 1.0% (v / v) seed culture solution of U4520 was added aseptically, and the culture was incubated at 27°C for 72 hours while stirring at 240 rpm. After incubation, the culture solution was centrifuged, and 6,900 mL of the resulting culture supernatant was collected as crude enzyme solution. The total activity of panose-degrading enzyme in the crude enzyme solution was 1,656 U.

[0071] <Experiment 4: Purification of panose-degrading enzyme> Ammonium sulfate was added to 6,900 mL of the crude enzyme solution obtained in Experiment 3 to achieve 80% saturation and dissolved. After standing overnight, salting-out was performed, and the resulting precipitate was collected by centrifugation and dialyzed against 10 mM phosphate buffer (pH 7.0). Insoluble matter in the dialysate was removed by centrifugation to obtain 420 mL of dialyzed enzyme solution. The dialyzed enzyme solution was subjected to anion exchange column chromatography (gel volume 83 mL) using an anion exchanger (product name "DEAE-Toyopearl 650S", manufactured by Tosoh Corporation) that had been pre-equilibriumized with 10 mM phosphate buffer (pH 7.0). The panose-degrading active fraction was adsorbed onto the anion exchanger. After washing the column with the same buffer, elution was performed using a linear gradient from 0 M to 0.5 M sodium chloride concentration. The panose-degrading active fraction eluted at a sodium chloride concentration of approximately 0.22 M. The panose-degrading active fraction was recovered from the eluted fraction and dialyzed with 10 mM phosphate buffer (pH 7.0). Ammonium sulfate was dissolved in the resulting dialysate to a final concentration of 1.5 M, and the mixture was subjected to hydrophobic column chromatography (gel volume 11 mL) using a hydrophobic chromatographic support (product name "Phenyl-Toyopearl 650M", manufactured by Tosoh Corporation) pre-equilibrium with 10 mM phosphate buffer (pH 7.0) and 1.5 M ammonium sulfate. The panose-degrading active fraction was adsorbed onto the hydrophobic chromatographic support. After washing the column with the same buffer, elution was performed using a linear gradient from 1.5 M to 0 M ammonium sulfate, and the panose-degrading active fraction eluted at an ammonium sulfate concentration of approximately 0.75 M. The panose-degrading active fraction was recovered from the eluted fraction, concentrated to 3 mL via membrane chromatography, and then subjected to gel filtration column chromatography (gel volume 120 mL) using a gel filtration carrier (product name "Superdex 200pg", manufactured by GL Healthcare Life Sciences) pre-equilibriumized with 10 mM phosphate buffer (pH 7.0) with a sodium chloride concentration of 0.4 M. Elution was performed with the same buffer, and the panose-degrading active fraction was recovered from the eluted fraction. Next, the recovered active fraction was dialyzed and subjected to anion exchange column chromatography (gel volume 1 mL) using an anion exchanger (product name "Resource Q", manufactured by GL Healthcare Life Sciences) pre-equilibriumized with 10 mM phosphate buffer (pH 7.0). The panose-degrading active fraction was adsorbed onto the anion exchanger.After washing the column with the same buffer, elution was performed using a linear gradient from 0 M to 0.5 M sodium chloride. The panose-degrading fraction eluted at approximately 0.23 M sodium chloride. The eluted panose-degrading fraction was used as the purified panose-degrading enzyme standard. The purification process is summarized in Table 3.

[0072] [Table 3]

[0073] As shown in Table 3, the specific activity of the purified panose-degrading enzyme preparation was 70.3 U / mg protein. Compared to the specific activity of the culture supernatant used as the raw material for purification, this purification process resulted in a purification level of approximately 500 times. The purity of the obtained purified panose-degrading enzyme preparation was tested by SDS-polyacrylamide gel electrophoresis using a 5 to 20% (w / v) concentration gradient gel. As shown in lane 2 of the electrophoresis diagram in Figure 3, the purified preparation showed a nearly single protein band, indicating high purity.

[0074] <Experiment 5: Properties of Panose-degrading enzymes> The molecular weight and enzymatic properties of the panose-degrading enzyme were investigated using the purified panose-degrading enzyme preparation obtained by the method in Experiment 4.

[0075] <Experiment 5-1: Molecular Weight> The purified panose-degrading enzyme prepared by the method in Experiment 4 was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 5 to 20% (w / v) concentration gradient), and its molecular weight was measured by comparing it with the mobility of a molecular weight marker (manufactured by Bio-Rad Laboratory) that was simultaneously electrophoresed. The molecular weight of the panose-degrading enzyme was found to be 85,000 ± 5,000 daltons. Furthermore, the same purified prepared was subjected to gel filtration column chromatography using a gel filtration carrier (product name "Superdex 200pg", manufactured by GL Healthcare Life Sciences), and the molecular weight was measured from the relationship between the elution time and the elution time of the molecular weight marker (manufactured by Bio-Rad Laboratory). The molecular weight of the panose-degrading enzyme in gel filtration chromatography was found to be 67,000 ± 5,000 daltons.

[0076] <Experiment 5-2: Optimal pH and Optimal Temperature> Using the purified panose-degrading enzyme standard obtained by the method of Experiment 4, the effects of pH and temperature on panose-degrading activity were investigated according to the activity measurement method. These results are shown in Figure 4 (optimal pH) and Figure 5 (optimal temperature). The optimal pH of the panose-degrading enzyme of the present invention was pH 5.0 to 5.6 under reaction conditions of 30°C for 20 minutes. Furthermore, it was found that the optimal temperature was 35°C under reaction conditions of pH 5.5 for 20 minutes.

[0077] <Experiment 5-3: pH stability and temperature stability> The pH stability and temperature stability of the panose-degrading activity were investigated using the purified panose-degrading enzyme standard obtained by the method of Experiment 4. pH stability was determined by holding the enzyme in 20 mM Britton-Robinson buffer at 4°C for 24 hours at each pH, ​​then adjusting the pH to 5.5 and measuring the remaining enzyme activity. Temperature stability was determined by holding the enzyme solution at each temperature for 1 hour using 20 mM Britton-Robinson buffer (pH 5.5) with or without 5 mM calcium chloride, then cooling with water and measuring the remaining D-glucose-producing activity. These results are shown in Figure 6 (pH stability) and Figure 7 (temperature stability). As is clear from Figure 6, the activity of the panose-degrading enzyme of the present invention was found to be stable in the pH range of 4.5 to 11.5. Also, as is clear from Figure 7, the activity of the panose-degrading enzyme of the present invention is stable in the Ca 2+ In the absence of ions (indicated by "●" in the diagram), it is stable up to 30°C, and with 5 mM Ca 2+ It was found that the substance is stable up to approximately 35°C in the presence of ions (indicated by "○" in the diagram).

[0078] <Experiment 5-4: N-terminal amino acid sequence> When the purified panose-degrading enzyme sample obtained by the method in Experiment 4 was subjected to N-terminal amino acid sequence analysis by the conventional Edman degradation method, the N-terminal amino acid sequence could not be obtained. From this, it was inferred that the N-terminal amino acid residue of this panose-degrading enzyme had undergone some modification, making it impossible to analyze the amino acid sequence.

[0079] <Experiment 5-5: Internal partial amino acid sequence> 250 μg of the purified panose-degrading enzyme standard obtained by the method in Experiment 4 was concentrated to 260 μL by membrane treatment, with the buffer replaced with 10 mM Tris-phosphate buffer (pH 9.0), and then the enzyme protein was thermally denatured by heating at 100°C for 10 minutes. 5 μg of lysyl endopeptidase (distributed by Wako Pure Chemical Industries, Ltd.) was added to the resulting denatured product, and the enzyme protein was hydrolyzed by holding at 30°C for 20 hours. The hydrolyzed product was then subjected to a reverse-phase HPLC column (product name "μBondashere C") that had been pre-equilibriumized with 0.1% (v / v) trifluoroacetic acid. 18The solution was injected into a 3.9 mm diameter x 150 mm length chamber (manufactured by Waters), and a linear gradient of acetonitrile concentrations from 0.1% (v / v) trifluoroacetic acid to 0.1% (v / v) trifluoroacetic acid - 40% (v / v) acetonitrile solution was passed through it over 100 minutes at a flow rate of 0.9 mL / min and room temperature to elute and fractionate the peptide fragments. Elution of the peptide fragments was detected by measuring the absorbance at a wavelength of 210 nm. Four peptide fragments P1, P2, P3, and P4 eluted at retention times of approximately 42 minutes, 58 minutes, 76 minutes, and 86 minutes were separated, and the amino acid sequences of each were analyzed from the N-terminus (5 residues each). They were found to have the amino acid sequences shown in Sequence ID No. 4, Sequence ID No. 5, Sequence ID No. 6, and Sequence ID No. 7 in the sequence listing.

[0080] <Experiment 6: Cloning of cDNA encoding panose-degrading enzyme and preparation of recombinant DNA containing it and transformants> The cDNA encoding the panose-degrading enzyme of the present invention was cloned from Sarocladium kyriens U4520, and recombinant DNA capable of autonomous replication was produced, the base sequence of the cDNA encoding the panose-degrading enzyme was determined, and transformants were prepared.

[0081] <Experiment 6-1: Cloning and sequencing of cDNA encoding panose-degrading enzyme> Since Salocladium kyliens U4520 is a fungus and its genomic DNA was thought to contain introns, mRNA was prepared from this strain, and cDNA encoding a panose-degrading enzyme was prepared.

[0082] The cells of Salocladium kylience U4520 obtained by culturing in the liquid medium used in Experiment 3 for 24 hours were disrupted using cell disruption beads (product name "NucleoSpin Bead Tubes Type A" (Takara Bio Inc.)), and then RNA was extracted using an RNA extraction kit (product name "NucleoSpin RNA" (Takara Bio Inc.)). The obtained RNA was subjected to agarose (1%) electrophoresis, and the purity was confirmed by confirming the rRNA band. Then, cDNA was synthesized using a cDNA synthesis kit (product name "PrimeScript II 1st strand cDNA Synthesis Kit" (Takara Bio Inc.) with oligo dT primers. Next, primers were designed with the sequences indicated by SEQ ID NOs.8 and SEQ ID NOs.9 in the sequence listing outside the start codon and stop codon, respectively. After amplifying the target region, the sequence of the panose-degrading enzyme protozoan was determined using a DNA sequencer.

[0083] The decoded 1,949 bp base sequence contained an open reading frame that began with methionine and encoded an amino acid sequence containing all four internal partial amino acid sequences of the panose-degrading enzyme identified in Experiment 5-5 (amino acid sequences indicated by SEQ ID NOs. 4 to 7 in the sequence listing). This revealed that the full-length gene of the target panose-degrading enzyme was present in this DNA. From this result, it was inferred that the obtained cDNA was the cDNA encoding the target panose-degrading enzyme. Based on this finding, the base sequence of the panose-degrading enzyme gene and the amino acid sequence of the panose-degrading enzyme encoded by it were determined. As a result, it was found that the structural gene of the panose-degrading enzyme from Salocladium kyriense U4520 has a base sequence with a chain length of 1,872 bp, indicated by SEQ ID NO. 10 in the sequence listing, and encodes an amino acid sequence consisting of 624 residues listed alongside that base sequence. The four internal partial amino acid sequences revealed in Experiment 5-5 (amino acid sequences shown as Sequence IDs 4 to 7 in the sequence listing) were all found in the amino acid sequences listed alongside the base sequence shown as Sequence ID 10 in the sequence listing (amino acid sequences shown as Sequence ID 11 in the sequence listing), and they were in complete agreement with the amino acid sequences at positions 46 to 50, 588 to 592, 234 to 238, and 397 to 401, respectively, in those amino acid sequences.

[0084] Furthermore, using the signal peptide prediction software "SignalP-5.0," we predicted the signal peptide sequence for secretion from the amino acid sequence of the panose-degrading enzyme indicated by SEQ ID NO: 11 in the sequence listing, as determined above. The amino acid sequence consisting of 24 amino acid residues at the N-terminus was predicted to be the signal peptide sequence. The molecular weight calculated from the amino acid sequence indicated by SEQ ID NO: 11 in the sequence listing was 69,335 daltons, and the molecular weight excluding the predicted signal peptide sequence of 24 amino acid residues was calculated to be 66,849 daltons. This value was in good agreement with the molecular weight of the panose-degrading enzyme from Salocladium kyriense U4520 obtained in Experiment 5-1, which was 67,000 ± 5,000 daltons, but it differed significantly from the 85,000 ± 5,000 daltons obtained by SDS-PAGE. The reason why the molecular weight calculated from the amino acid sequence differed significantly from the molecular weight determined by SDS-PAGE is thought to be that the panose-degrading enzyme actually produced underwent modifications such as sugar chains, which affected its mobility on SDS-PAGE.

[0085] <Experiment 6-2: Construction of a recombinant panose-degrading enzyme expression vector and preparation of transformants>

[0086] The gene sequence of panose-degrading enzyme, excluding the signal peptide, was amplified using cDNA prepared from Sarocladium kylience U4520 as a template. PCR was also performed using the expression plasmid vector 'pPICZαA' as a template to amplify the target gene sequence. Subsequently, infusion reactions, transformation into E. coli XL10 Gold, colony PCR, and plasmid extraction were carried out. After linearization of the obtained plasmids with restriction enzymes, agarose gel electrophoresis was performed to confirm the correct size of each plasmid. The obtained recombinant expression DNA 'pPICZαA-P' is shown in Figure 8. As can be seen in Figure 8, in this recombinant expression DNA, the panose-degrading enzyme gene is expressed using the promoter of the methanol-inducible alcohol oxidase gene AOX1, and the resulting recombinant panose-degrading enzyme is designed to be secreted using the secretion signal peptide of the yeast conjugation factor α-factor. Next, after linearizing 'pPICZαA-P' with restriction enzymes, the yeast Pichia pastoris KM71H was used as the host, and recombinant DNA was introduced by electroporation to transform it, yielding the transformant 'PICZαA-P'.

[0087] <Experiment 6-3: Expression of recombinant panose-degrading enzyme in transformants> The transformant 'PICZαA-P' obtained in Experiment 6-2 was inoculated into one 500 mL Erlenmeyer flask containing 200 mL each of YPD medium (1.0% yeast extract, 2.0% polypeptone, 2.0% D-glucose) with 20 μg / mL of the antibiotic phleomycin D1 as the main component (trade name 'Zeocin', sold by Invitrogen), and cultured with shaking at 30°C for 24 hours. The resulting culture was centrifuged according to a standard method to recover the yeast cells. Next, the cells were washed with sterile water and inoculated into 40 mL of YP medium (1.0% yeast extract, 2.0% polypeptone) containing 20 μg / mL of the same antibiotic and 1.0% methanol. The culture was maintained at 25°C for 72 hours, with methanol added every 24 hours to reach a final concentration of 1.0%, to induce the expression of the panose-degrading enzyme gene. After culturing, the culture supernatant was collected by centrifugation and sterilized by filtration through a 0.22 μm filter to obtain a recombinant panose-degrading enzyme solution with a panose-degrading activity of 30.3 U / mL. When this enzyme solution was subjected to SDS-polyacrylamide gel electrophoresis, a nearly single recombinant panose-degrading enzyme protein band was observed.

[0088] The enzymatic properties of the obtained recombinant panose-degrading enzyme preparation were investigated according to the method shown in Experiment 5. The optimal pH of the recombinant panose-degrading enzyme was pH 5.0 to 5.5 under conditions of reaction at 30°C for 20 minutes, the optimal temperature was 35°C under conditions of reaction at pH 5.5 for 20 minutes, the pH stability was stable in the range of approximately 4.7 to 12.0 under conditions of holding each pH at 4°C for 24 hours, and the temperature stability was stable under conditions of holding each temperature at pH 5.5 for 1 hour, Ca 2+ The enzyme was stable up to 30°C in the absence of ions. These physicochemical properties were substantially identical to those of the natural panose-degrading enzyme prepared in Experiment 4. These results indicate that the panose-degrading enzyme of the present invention can also be successfully produced as a recombinant enzyme.

[0089] <Experiment 7: Substrate specificity of panose-degrading enzymes> The purified panose-degrading enzyme obtained by the method in Experiment 4 was reacted with various carbohydrates, and its substrate specificity was investigated.

[0090] The substrate specificity of panose-degrading enzyme was examined using 31 kinds of carbohydrates shown in Table 4 below. Each carbohydrate was dissolved in 20 mM Britton-Robinson buffer (pH 5.5) to a final concentration of 1% as a substrate, and 1 U or 10 U of panose-degrading enzyme was added per 1 gram of the substrate solid matter, followed by reaction at 30 °C for 24 hours. After the reaction, the reaction products generated from each substrate were subjected to the same TLC analysis as used in Experiment 1 to confirm the presence or absence of enzymatic action on each carbohydrate and the generated carbohydrates. In TLC, those with spots of reaction products other than the substrates used were determined as "acting" (+), and those without observable reaction products were determined as "not acting" (-). For the substrates with observable action, the reaction products were identified. The results are shown in Table 4. Also, the substrate specificity of the panose-degrading enzyme clarified from the results of this test, that is, the substrates on which the panose-degrading enzyme acts and those on which it does not act, and regarding the structure of those substrates and the bonds to be hydrolyzed for the substrates on which it acts, are summarized in Figure 9.

[0091]

Table 4

[0092] As is clear from the results in Table 4 and Figure 9, the panose-degrading enzyme of the present invention not only hydrolyzes the α-1,4 bond of panose to produce isomaltose and D-glucose, but also 2 hydrolyzes the α-1,4 bond of 6 3 -α-isomaltosyl maltose (α-isomaltotriosyl-(1→4)-D-glucose) to produce isomaltotriose and D-glucose. Also, the panose-degrading enzyme of the present invention 4 6 5It was found that when the panose-degrading enzyme of the present invention is applied to a series of carbohydrates having a structure in which D-glucose is α-1,6 linked to the hydroxyl group at the 6th position of the non-reducing end glucose residue of a maltooligosaccharide, such as -α-glucosylmaltopentaose, it specifically hydrolyzes the α-1,4 linkage to which the isomaltose structure present at the non-reducing end is linked, producing isomaltose and the remaining maltose, maltotriose, and maltotetraose, respectively. On the other hand, the panose-degrading enzyme of the present invention hydrolyzes α-1,6 glucans such as isomalttriose and dextran, as well as pullulan, 6 2 -α-maltosylmaltose and 6 3 It was found that the effect does not extend to carbohydrates in which maltose or maltotriose is α-1,6 linked to the hydroxyl group at the 6th position of the non-reducing end glucose residue of maltooligosaccharides, such as -α-maltotriosylmaltotriose.

[0093] Furthermore, as is clear from Table 4, the panose-degrading enzyme of the present invention did not act on maltose and a series of maltooligosaccharides, isomaltose and a series of isomaltoligosaccharides linked only via α-1,6 bonds, nor did it show any effect on various disaccharides, amylose, starch, glycogen, pullulan, dextran, and other polysaccharides.

[0094] Based on the substrate specificity described above, the panose-degrading enzyme of the present invention hydrolyzes panose to produce isomaltose and D-glucose, but differs from the known enzyme isomaltodextranase in that it does not hydrolyze the α-1,6 bond and does not act on isomalttriose, isomalttetraose, or dextran. It also differs from the known enzyme isoplulanase in that it does not act on pullulan, making it a novel enzyme previously unknown.

[0095] <Experiment 8: Production of panose-degrading enzyme by microorganisms of the genus Acremonium> With the aim of obtaining panose-degrading enzyme-producing microorganisms other than the fungal strain U4520, we screened for panose-degrading enzyme-producing bacteria from strains of the genus Acremonium, which is related to the genus Sarocladium, using the same procedure as shown in Experiment 1. The culture supernatant containing the crude enzyme solution from one strain of Acremonium microorganism used in the screening was reacted with panose, isomalttriose, pullulan, and dextran, respectively, as in Experiment 1. The TLC chromatograms of the resulting reaction solutions are shown in Figure 10. As can be seen in Figure 10, the culture supernatant containing the crude enzyme solution from the Acremonium microorganism hydrolyzed panose into isomaltose and D-glucose (indicated by 8 in Figure 10), but did not react with any of the isomalttriose (indicated by 9 in Figure 10), pullulan (indicated by 10 in Figure 10), or dextran (indicated by 11 in Figure 10). Thus, it was confirmed that microorganisms of the genus Acremonium also produce panose-degrading enzymes with similar enzymatic activity to those produced by microorganisms of the genus Sarocladium.

[0096] <Experiment 9: Production of isomaltose from partially hydrolyzed starch by combining 6-α-glucosyltransferase and panose-degrading enzyme> The purified panose-degrading enzyme obtained by the method in Experiment 4 was combined with 6-α-glucosyltransferase derived from Bacillus globisporus N75, as disclosed in the republished Japanese Patent Publication No. 02 / 088374, and each was applied to a partially hydrolyzed starch product to attempt to produce isomaltose. A schematic diagram of the isomaltose production reaction from the partially hydrolyzed starch product using the combination of 6-α-glucosyltransferase and panose-degrading enzyme is shown in Figure 11.

[0097] <Experiment 9-1: Combined effects of various enzymes in the isomaltose production reaction> Using partially decomposed starch (trade name: "Bindex #100", sold by Matsutani Chemical Industry Co., Ltd.) as the raw material substrate, it was dissolved in a 20 mM acetate buffer (pH 5.5) containing 1 mM calcium chloride to a final concentration of 5% by mass to obtain a substrate solution. Then, 1 U of 6-α-glucosyltransferase (product prepared by Hayashibara Co., Ltd.) per 1 g of substrate solids, 8 U of panose-degrading enzyme, 1,0 FU of isoamylase (product prepared by Hayashibara Co., Ltd.), and 0.5 U of cyclomaltodextrin glucanotransferase (CGTase, product prepared by Hayashibara Co., Ltd.) were added in the combinations shown in Table 5 respectively. Furthermore, hinokitiol was added as a preservative to a final concentration of 60 ppm, and then the reaction was carried out at 30 °C for 72 hours. After the reaction was completed, the enzyme was inactivated by heating at 100 °C for 10 minutes. Then, the sugar composition of each reaction solution was measured by HPLC under the following conditions, and the isomaltose content of the reaction solution was determined. The results are shown in Table 5.

[0098] <HPLC Conditions> Column: 'MCIgel CK04SS' (manufactured by Mitsubishi Chemical Corporation) Two columns were connected in series and used; Eluent: Ultra-pure water Column temperature: 80 °C Flow rate: 0.4 mL / min Detection: Differential refractometer RID-10A (manufactured by Shimadzu Corporation)

[0099]

Table 5

[0100] As shown in Table 5, when 6-α-glucosyltransferase and panose-degrading enzyme were applied to partially hydrolyzed starch products individually, almost no isomaltose was produced. However, when 6-α-glucosyltransferase and panose-degrading enzyme were applied in combination, isomaltose was efficiently produced, reaching an isomaltose content of 66.5% by mass per solid reaction solution. Furthermore, when isoamylase, a starch branching enzyme, was added to this combination of enzymes, the isomaltose content per solid reaction solution reached 76.7% by mass, showing a significant increase in isomaltose yield of approximately 10% by mass. This result indicates that the branched structure (branches) via α-1,6 bonds inherently present in partially hydrolyzed starch products hindered the isomaltose production reaction using 6-α-glucosyltransferase and panose-degrading enzyme, and that hydrolysis of this branched structure by the starch branching enzyme further promoted the isomaltose production reaction. Furthermore, it was found that by adding CGTase to this combination of enzymes, the isomaltose content per solid reaction solution could be further increased to 79.5% by mass, although this is only about 3% by mass.

[0101] <Experiment 9-2: Effect of substrate concentration on isomaltose production> The isomaltose production reaction was carried out under the same conditions as in Experiment 9-1, except that the substrate concentration used in the isomaltose production reaction was varied to 5, 10, 20, 30, or 40% by mass, and all four types of enzymes used in Experiment 9-1 were used. The isomaltose content per unit of solid matter in each reaction solution was measured by the HPLC analysis described above. In addition, the reaction temperature was also tested at 40°C only under the condition of a substrate concentration of 30% by mass. The results are shown in Table 6.

[0102] [Table 6]

[0103] As shown in Table 6, at a reaction temperature of 30°C, the isomaltose content of the reaction solution reached approximately 79% by mass per solid matter when the substrate concentration was in the range of 5 to 10% by mass. Under conditions of substrate concentrations of 20%, 30%, and 40% by mass, the isomaltose content of the reaction solution was 76.1%, 72.5%, and 70.0% by mass per solid matter, respectively, and the isomaltose content in the reactant gradually decreased as the substrate concentration increased. On the other hand, at a reaction temperature of 40°C and a substrate concentration of 30% by mass, the isomaltose content of the reaction solution was 72.5% by mass per solid matter, which was equivalent to that at a reaction temperature of 30°C.

[0104] The results of Experiment 9 demonstrate that, in the isomaltose production method of the present invention, which combines 6-α-glucosyltransferase and panose-degrading enzyme, by using starch debranching enzymes and CGTase in combination, a reaction product with an isomaltose content of 70% by mass or more can be obtained even under relatively high substrate concentration conditions, indicating that isomaltose can be efficiently produced from partially hydrolyzed starch.

[0105] <Experiment 10: Production of isomaltoligosaccharide from starch by combining α-glucosyltransferase and panose-degrading enzyme> The purified panose-degrading enzyme preparation obtained by the method in Experiment 4 was combined with α-glucosyltransferase derived from Bacillus circulans PP710, as disclosed in International Publication No. 2008 / 136331, and applied to a partially hydrolyzed starch product to attempt the production of isomaltoligosaccharides. A schematic diagram of the reaction for the production of isomaltoligosaccharides from a partially hydrolyzed starch product using the combination of α-glucosyltransferase and panose-degrading enzyme is shown in Figure 12.

[0106] <Experiment 10-1: Combined effects of various enzymes in the isomaltoligosaccharide production reaction> The starch partial degradation product (trade name: “Bindex #100”, sold by Matsutani Chemical Industry Co., Ltd.) was used as a raw material substrate and dissolved in 20 mM acetate buffer (pH 5.5) containing 1 mM calcium chloride to a final concentration of 10% by mass to prepare a substrate solution. Next, 10 U of α-glucosyltransferase (product prepared by Hayashibara Co., Ltd.), 10 U of panose-degrading enzyme, 1,000 fu of isoamylase (product prepared by Hayashibara Co., Ltd.), and 1.0 U of α-amylase (trade name: “Cristase E5CC”, sold by Amano Enzyme Inc.) per 1 g of the substrate solid were added in the combinations shown in Table 7, respectively. Further, hinokitiol was added as a preservative to a final concentration of 60 ppm, and then the reaction was carried out at 30 °C for 72 hours. After the reaction was completed, the enzyme was inactivated by heating at 100 °C for 10 minutes, and then each reaction solution was subjected to thin-layer chromatography (TLC) under the following conditions to preliminarily analyze the saccharides contained in each reaction solution. The TLC chromatogram is shown in Fig. 13.

[0107] <TLC analysis conditions> TLC plate: Silica gel aluminum plate (trade name: “Silica gel 60F254”, 10 × 20 cm, manufactured by Merck) Developing solvent: n-butanol:pyridine:water mixture (volume ratio 6:4:1) Developing method: Ascending method, developed twice Detection method: Sulfuric acid-methanol method

[0108] As shown in Figure 13, in the TLC chromatograms of the partially hydrolyzed starch (lane 0 in Figure 13), the α-glucosyltransferase reaction product (lane 1 in Figure 13), and the panose-degrading enzyme reaction product (lane 2 in Figure 13) used as raw materials, no clear sugar spots were observed in any of them except at the origin where the sample was spotted. When compared with the chromatograms of the maltooligosaccharide marker (lane labeled "Gn" in Figure 13) and isomaltoligosaccharide marker (lane labeled "IGn" in Figure 13), which were simultaneously subjected to TLC, it was found that at least maltooligosaccharides with a glucose polymerization degree of 10 or less and isomaltoligosaccharides with a glucose polymerization degree of 4 or less were substantially absent. On the other hand, the chromatograms of the reaction products obtained by treating partially hydrolyzed starch with α-glucosyltransferase and panose-degrading enzyme (lane 3 in Figure 13), the reaction product obtained by treating with α-glucosyltransferase, panose-degrading enzyme and isoamylase (lane 4 in Figure 13), and the reaction product obtained by treating with α-glucosyltransferase, panose-degrading enzyme, isoamylase and α-amylase (lane 5 in Figure 13) showed spots of D-glucose, isomaltose, isomalttriose, and isomalttetraose. Furthermore, no spots of maltooligosaccharides with a degree of polymerization (DP) of 10 or less were observed in these samples. From these results, it was concluded that maltooligosaccharides with a DP of 10 or less are substantially absent in these three reaction products.

[0109] Furthermore, the sugar composition of each reaction solution was measured by HPLC under the aforementioned conditions, and the total content of isomaltoligosaccharides DP2 to DP8 was determined. In addition, for the reaction solutions obtained by treating with all four enzymes, 10 U of glucoamylase (derived from the genus Rhizopus, sold by Fujifilm Wako Pure Chemical Industries, Ltd.) was added per gram of solid matter, and the mixture was treated at pH 5.0 and 50°C for 18 hours. The sugar composition and the total content of isomaltoligosaccharides DP2 to DP8 were similarly determined for the resulting glucoamylase-treated product. The results are shown in Table 7.

[0110] [Table 7]

[0111] As shown in Table 7, in the partially hydrolyzed starch (control), α-glucosyltransferase reaction product (sample 1), and panose-degrading enzyme reaction product (sample 2) used as raw materials, carbohydrates with a DP of 11 or higher accounted for nearly 90% or more by mass, while carbohydrates with a DP of 10 or lower were present in very small amounts. On the other hand, in the reaction product obtained by treating the partially hydrolyzed starch with α-glucosyltransferase and panose-degrading enzyme (sample 3), the amount of carbohydrates with a DP of 11 or higher was reduced to 37.1% by mass, and the production of carbohydrates with a DP of 1 to 10 was observed, totaling 62.9% by mass. Isomaltooligosaccharides (isomaltose to isomaltoctaose) with a DP of 2 to 8 accounted for a total of 51.1% by mass. Furthermore, in the reaction product (Sample 4) obtained by treating partially hydrolyzed starch with α-glucosyltransferase, panose-degrading enzyme, and isoamylase, a starch branching enzyme, the amount of carbohydrates with DP11 or higher was reduced to 15.0% by mass, with carbohydrates with DP1 to DP10 accounting for a total of 85.0% by mass, and isomaltoligosaccharides (isomaltose to isomaltoctaose) with DP2 to DP8 accounting for a total of 68.7% by mass. Moreover, in the reaction product (Sample 5) obtained by treating partially hydrolyzed starch with α-glucosyltransferase, panose-degrading enzyme, isoamylase, and isoamylase, the amount of carbohydrates with DP11 or higher was reduced to 5.8% by mass, with carbohydrates with DP1 to DP10 accounting for a total of 94.2% by mass, and isomaltoligosaccharides (isomaltose to isomaltoctaose) with DP2 to DP8 accounting for a total of 77.1% by mass.

[0112] Furthermore, Figure 14 shows the HPLC chromatogram of the reaction product (Sample 6) obtained by further glucoamylase treatment of the reaction product (Sample 5), which was obtained by treating a partially hydrolyzed starch with α-glucosyltransferase, panose-degrading enzyme, isoamylase, and α-amylase, and Table 7 also shows the sugar composition obtained by analysis. As can be seen in Figure 14, the HPLC chromatogram shows a series of peaks separated by the difference in the degree of glucose polymerization of carbohydrates from DP1 D-glucose to DP10, and the sugar composition is as shown in the column for Sample 6 in Table 7. Compared to the sugar composition before glucoamylase treatment (Sample 5), the sugar composition after glucoamylase treatment (Sample 6) showed no significant change, although D-glucose increased by 2.8 mass%. Even if a small amount of unreacted maltooligosaccharides derived from partially hydrolyzed raw starch were present in sample 5, these would be broken down by glucoamylase in sample 6. Therefore, all carbohydrates other than D-glucose in sample 6 can be considered isomaltooligosaccharides. The total value of isomaltooligosaccharides DP2 to DP8 in sample 6 was 75.7% by mass.

[0113] The results of this experiment demonstrate that by combining α-glucosyltransferase and panose-degrading enzyme and applying them to starch or partially hydrolyzed starch, a series of isomaltoligosaccharide mixtures can be produced. Furthermore, by using isoamylase and α-amylase in combination, an isomaltoligosaccharide mixture containing at least 75% by mass of isomaltoligosaccharides ranging from DP2 isomaltose to DP8 isomaltoctaose can be efficiently produced using starch or partially hydrolyzed starch as a raw material.

[0114] <Experiment 10-2: Effect of substrate concentration on isomaltoligosaccharide production> The enzymatic reaction was carried out under the same conditions as in Experiment 10-1, except that the substrate concentration used in the isomaltoligosaccharide production reaction was varied to 5, 10, 20, or 30% by mass, and all four types of enzymes used in Experiment 10-1 were used. The sugar composition of each reaction solution was measured by the HPLC analysis described above, and the total value of isomaltoligosaccharides from DP2 (isomaltose) to DP8 (isomaltooctaose) was calculated, as in Experiment 10-1. The results are shown in Table 8.

[0115] [Table 8]

[0116] As shown in Table 8, even when the substrate concentration was changed to 5, 10, 20, or 30% by mass, the sugar composition of the product did not change significantly, and an isomaltoligosaccharide mixture was obtained containing approximately 11% by mass of D-glucose, approximately 16% by mass of isomaltose, approximately 16% by mass of isomalttriose, approximately 14% by mass of isomalttetraose, approximately 11% by mass of isomaltopentaose, approximately 9% by mass of isomalthexaose, approximately 7% by mass of isomaltheptaose, and approximately 5% by mass of isomaltoctaose. Furthermore, the total value of isomaltose from DP2 to isomaltoctaose from DP8 was 77-79% by mass.

[0117] The results of Experiments 10-1 and 10-2 demonstrate that, according to the present invention's method for producing isomaltoligosaccharides using a combination of α-glucosyltransferase and panose-degrading enzyme, even under conditions of relatively high substrate concentration, a sugar composition can be obtained with a total content of isomaltoligosaccharides from DP2 isomaltose to DP8 isomaltoctaose per unit solid, by appropriately using other enzymes. This indicates that isomaltoligosaccharide mixtures can be efficiently produced using starch or partially hydrolyzed starch as raw materials.

[0118] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited in any way by these examples. [Examples]

[0119] <Preparation of panose-degrading enzyme> Approximately 20 L of the liquid culture medium used in Experiment 3 was placed in a 30 L jar fermenter, sterilized at 120°C for 20 minutes, and then 2% (v / v) of Sarocladium kyliens U4520 seed culture solution was aseptically added. The culture was incubated at 27°C for 60 hours with aeration and stirring. Approximately 18 L of the culture solution was centrifuged to remove the bacterial cells, and the culture supernatant with a panose-degrading activity of 0.28 U / mL was obtained as a crude enzyme solution. This crude enzyme solution was concentrated approximately 30 times using an ultrafiltration membrane according to a conventional method to obtain a concentrated enzyme preparation with a total activity of approximately 5,000 U. This product can be used as a panose-degrading enzyme preparation in combination with 6-α-glucosyltransferase to produce isomaltose, and can also be used in combination with α-glucosyltransferase to produce isomaltoligosaccharide. [Examples]

[0120] <Preparation of panose-degrading enzyme> Approximately 20 L of YPD medium liquid medium containing 20 μg / mL of the antibiotic used in Experiment 6-3 (trade name "Zeocin," sold by Invitrogen) was placed in a 30 L jar fermenter, sterilized at 120°C for 20 minutes, and then 1% (v / v) of the seed culture solution of the transformant "PICZαA-P" obtained in Experiment 6-2 was aseptically added. The culture was incubated at 27°C for 48 hours with aeration and stirring. The resulting culture was centrifuged to collect the yeast cells, washed with sterile water, and reinoculated into 7.5 L of YP medium (1.0% yeast extract, 2.0% polypeptone) containing 20 μg / mL of the same antibiotic and 1.0% methanol. The culture was maintained at 25°C for 24 hours, with methanol added every 6 hours to reach a final concentration of 1.0%, to induce the expression of the panose-degrading enzyme gene. After culturing, approximately 7.3 L of the culture medium was centrifuged to remove the bacterial cells, and the culture supernatant with a panose-degrading activity of 32.1 U / mL was obtained as a crude enzyme solution. This crude enzyme solution was concentrated approximately 30 times using an ultrafiltration membrane according to a conventional method to obtain a concentrated enzyme preparation with a total activity of approximately 240,000 U. This product can be used as a recombinant panose-degrading enzyme preparation in combination with 6-α-glucosyltransferase to produce isomaltose, and can also be used in combination with α-glucosyltransferase to produce isomaltoligosaccharide. [Examples]

[0121] <Preparation of panose-degrading enzyme> Approximately 20 L of the liquid culture medium used in Experiment 3 was placed in a 30 L jar fermenter, sterilized at 120°C for 20 minutes, and then 2% (v / v) of the seed culture solution of the Acremonium strain used in Experiment 8 was aseptically added. The culture was incubated at 27°C for 72 hours with aeration and stirring. Approximately 17 L of the culture solution was centrifuged to remove the bacterial cells, and the culture supernatant with a panose-degrading activity of 0.35 U / mL was obtained as a crude enzyme solution. This crude enzyme solution was concentrated approximately 20-fold using an ultrafiltration membrane according to a conventional method to obtain a concentrated enzyme preparation with a total activity of approximately 5,500 U. This product can be used as a panose-degrading enzyme preparation in combination with 6-α-glucosyltransferase to produce isomaltose, and can also be used in combination with α-glucosyltransferase to produce isomaltoligosaccharide. [Examples]

[0122] <Production of isomaltose from starch> Tapioca starch was prepared as a starch milk with a concentration of approximately 30% (w / v), to which 0.1% calcium carbonate was added to adjust the pH to 6.5, and 0.3% heat-resistant α-amylase (product name "Termamyl 60L", manufactured by Novo National Corporation) was added per gram of starch. The mixture was reacted at 95°C for 15 minutes, then autoclaved at 120°C for 20 minutes, and then rapidly cooled to approximately 35°C to obtain a starch liquefaction solution with a DE of approximately 4. To this solution, Bacillus globisporus, as described in International Publication No. 2002 / 010361, was added. N75-derived 6-α-glucosyltransferase (α-isomaltosylglucoglucotransferase) was added at a rate of 1.0 U per gram of starch solids, the panose-degrading enzyme obtained by the method of Example 1 was added at a rate of 8 U per gram of starch, isoamylase (Hayashibara Corporation) was added at a rate of 1,000 fu per gram of starch, and cyclomaltodextrin glucanotransferase (CGTase, Hayashibara Corporation) was added at a rate of 0.5 U per gram of starch. The mixture was reacted at pH 5.5 and a temperature of 35°C for 72 hours. The reaction solution was held at 95°C for 30 minutes to inactivate the enzymes, then cooled and filtered. The resulting filtrate was decolorized with activated carbon, desalted and purified using H-type and OH-type ion exchange resins according to a conventional method, further concentrated, dried, and powdered to obtain a high-isomaltose carbohydrate in a yield of approximately 95% by mass per solid. This product contained 4.7% by mass of D-glucose, 72.5% by mass of isomaltose, and 22.8% by mass of other carbohydrates on a solid basis. Because this product has excellent moisturizing properties, low sweetness, osmotic pressure regulating properties, shape-forming properties, gloss-granting properties, viscosity, carbohydrate crystallization prevention properties, resistance to fermentation, and starch retrogradation prevention properties, it can be advantageously used in various foods and beverages, health foods, animal feed, feed, cosmetics, pharmaceuticals, and luxury goods. [Examples]

[0123] <Isomaltose syrup> The isomaltose-rich powder obtained by the method of Example 4 is dissolved in water, and a strong acid cation exchange resin (product name "Amberlite CR-1310") and Na are used. +Column chromatography was performed using a (model, manufactured by Organo Corporation) 10 jacketed stainless steel columns with an inner diameter of 12.5 cm were packed with the resin, and these columns were connected in series to form a total resin layer of 16 m. While maintaining the column temperature at 40°C, the solution was added at a concentration of 1.5% (v / v) relative to the amount of resin, and the mixture was fractionated by passing 40°C warm water through it with an SV of 0.2. The sugar composition of the eluate was monitored by HPLC, and the fraction with a high isomaltose content was collected and purified to obtain a high isomaltose content solution. The yield of isomaltose per unit solid was approximately 80%. This solution was decolorized, desalted, and concentrated according to conventional methods to obtain isomaltose syrup with a concentration of approximately 75%. This product contained approximately 95.7% isomaltose per unit solid. This product is poorly crystallized and possesses excellent moisture retention, low sweetness, osmotic pressure regulation, shape-forming properties, gloss-enhancing properties, viscosity, sugar crystallization prevention, fermentation resistance, and starch retrogradation prevention properties, making it advantageous for use in various foods and beverages, health foods, animal feed, feed, cosmetics, pharmaceuticals, and luxury goods. [Examples]

[0124] <Production of isomaltose from starch> Corn starch was prepared as a starch milk with a concentration of approximately 30% (w / v), to which 0.1% calcium carbonate was added to adjust the pH to 6.5, and heat-resistant α-amylase (product name "Termamyl 60L", manufactured by Novo National Corporation) was added at a rate of 0.3% per gram of starch. The mixture was reacted at 95°C for 15 minutes, then autoclaved at 120°C for 20 minutes, and then rapidly cooled to approximately 35°C to obtain a starch liquefaction solution with a DE of approximately 5. To this solution, Bacillus globisporus, as described in International Publication No. 2002 / 010361, was added. N75-derived 6-α-glucosyltransferase (α-isomaltosylglucoglucotransferase) was added at a rate of 1.0 U per gram of starch solids, the panose-degrading enzyme obtained by the method of Example 3 was added at a rate of 8 U per gram of starch, isoamylase (manufactured by Hayashibara Corporation) was added at a rate of 1,000 fu per gram of starch, and cyclomaltodextrin glucanotransferase (CGTase, manufactured by Hayashibara Corporation) was added at a rate of 0.5 U per gram of starch. The mixture was reacted at pH 5.5 and a temperature of 35°C for 72 hours. The reaction solution was held at 95°C for 30 minutes to inactivate the enzymes, then cooled and filtered to obtain a filtrate which was decolorized with activated carbon, desalted with H-type and OH-type ion exchange resins according to a conventional method, purified, further concentrated, dried, and powdered to obtain a high-isomaltose-containing carbohydrate in a yield of approximately 95% by mass per solid. This product contained 4.5% by mass of D-glucose, 71.8% by mass of isomaltose, and 23.7% by mass of other carbohydrates on a solid basis. Because this product has excellent moisturizing properties, low sweetness, osmotic pressure regulating properties, shape-forming properties, gloss-granting properties, moisture retention, viscosity, carbohydrate crystallization prevention, resistance to fermentation, and starch retrogradation prevention properties, it can be advantageously used in various foods and beverages, health foods, animal feed, feed, cosmetics, pharmaceuticals, and luxury goods. [Examples]

[0125] <Production of crystalline isomaltitol> Activated Raney nickel was added to the isomaltose syrup obtained in Example 5, and after hydrogenation by a conventional method, the Raney nickel was removed, decolorized with activated carbon, desalted and purified using H-type and OH-type ion exchange resins, and an isomaltitol-high syrup containing 1.2% by mass of sorbitol, 96.0% by mass of isomaltitol, and 2.8% by mass of other sugar alcohols was obtained in a yield of approximately 90% per solid. This isomaltitol-high syrup was concentrated to a concentration of approximately 75% by mass, and this concentrate was placed in a crystallization vessel. Crystallized isomaltitol powder was added as seed crystals at a temperature of 25°C for approximately 20 hours, and the isomaltitol was crystallized. Subsequently, the molten material was separated using a centrifuge, and crystalline isomaltitol was recovered. The isomaltitol crystals were vacuum-dried at 80°C for 20 hours to obtain crystalline isomaltitol. This product contained 0.2% by mass of sorbitol, 99.3% by mass of isomaltitol, and 0.5% by mass of other sugar alcohols, per solid portion. Because this product is non-reducing, non-hygroscopic, low in sweetness, osmotic pressure regulating, excipient, gloss-enhancing, moisturizing, viscosity-enhancing, carbohydrate crystallization prevention, fermentation resistance, and starch retrogradation prevention, it can be advantageously used in various foods and beverages, health foods, health supplements, animal feed, feed, cosmetics, pharmaceuticals, and luxury goods. [Examples]

[0126] <Production of isomaltoligosaccharide from starch> Tapioca starch was prepared as a starch milk with a concentration of approximately 30% by mass. 0.1% by mass of calcium carbonate was added to this, and the pH was adjusted to 6.5. 0.3% by mass of heat-resistant α-amylase (product name "Termamyl 60L", manufactured by Novo National Corporation) was added per unit of starch, and the mixture was reacted at 95°C for 15 minutes. Then, it was autoclaved at 120°C for 20 minutes, and further cooled to approximately 40°C to prepare a starch liquefaction solution with a DE of approximately 4.5. The liquefied starch solution was adjusted to pH 5.5, and 10 U of α-glucosyltransferase (derived from Bacillus Circulans PP710, manufactured by Hayashibara Pharmaceutical Co., Ltd.), 10 U of panose-degrading enzyme (prepared by the method of Example 1), 1,000 fu of isoamylase (manufactured by Hayashibara Pharmaceutical Co., Ltd.), and 1.0 U of α-amylase (product name "Kleistase E5CC," sold by Amano Enzyme Co., Ltd.) were added per gram of starch solids. Sodium pyrosulfite was then added as a preservative to a final concentration of 0.01% by mass, and the mixture was reacted at 30°C for 72 hours. After the reaction was complete, the enzyme was deactivated by heating at 96°C for 30 minutes. The sugar composition of the reaction solution was then measured by HPLC and found to be 11.3% by mass of D-glucose, 16.0% by mass of isomaltose, 15.8% by mass of isomalttriose, 13.8% by mass of isomalttetraose, 11.5% by mass of isomaltopentaose, 9.1% by mass of isomalthexaose, and 22.5% by mass of other sugars including isomaltheptaose. This isomaltoligosaccharide-containing sugar solution can be decolorized, desalted, and concentrated to be used as a high-isomaltoligosaccharide-containing syrup, which can be advantageously used as a sweetener, a carbon source for fermentation, a reagent, a chemical, a raw material and intermediate for pharmaceuticals, etc. [Examples]

[0127] <Isomaltooligosaccharide alcohol> Activated Raney nickel was added to the isomaltoligosaccharide-rich syrup obtained by the method of Example 8, hydrogenation was performed by conventional means, the Raney nickel was removed, decolorized with activated carbon, desalted and purified using H-type and OH-type ion exchange resins, and an isomaltoligosaccharide alcohol syrup containing 23.3% by mass of the following sugar alcohols per solid: sorbitol 11.1% by mass, isomaltitol 15.9% by mass, isomalttriitol 15.6% by mass, isomalttetraitol 13.7% by mass, isomaltopentitol 11.4% by mass, isomalthexythol 9.0% by mass, and isomaltoheptitol, obtained in a yield of approximately 90% per solid. This isomaltoligosaccharide alcohol syrup was concentrated to a concentration of approximately 75% by mass, filled into cans, and used as the product. Because this product possesses properties such as non-reducing, non-hygroscopic, low sweetness, osmotic pressure regulating, shape-forming, gloss-enhancing, moisturizing, viscosity-enhancing, carbohydrate crystallization prevention, resistance to fermentation, and starch retrogradation prevention, it can be advantageously used in various foods and beverages, health foods, health supplements, animal feed, feed, cosmetics, pharmaceuticals, and luxury goods. [Examples]

[0128] <Sweeteners> Eight parts by mass of powdered isomaltose-rich carbohydrate obtained by the method of Example 4 were uniformly mixed with 2 parts by mass of trehalose dihydrated crystal-containing powder (registered trademark "Treha", sold by Hayashibara Co., Ltd.), 0.1 parts by mass of α-glycosylstevioside (product name "αG Sweet", sold by Toyo Sugar Refining Co., Ltd.), and 0.1 parts by mass of L-aspartyl-L-phenylalanine methyl ester (product name "Aspartame"), and then processed in a granulation machine to obtain a granular sweetener. This product has excellent sweetness quality and is about twice as sweet as sugar. This product is a sweetener composition containing isomaltose, which is poorly crystalline and has excellent moisture retention properties. Furthermore, this product is stable and does not show any concerns about deterioration or degradation when stored at room temperature. [Examples]

[0129] <Chocolate> 40 parts by mass of cocoa paste, 10 parts by mass of cocoa butter, and 50 parts by mass of powdered isomaltose-rich sugar obtained by the method of Example 4 were mixed. The resulting mixture was passed through a refiner to reduce its viscosity, then placed in a conche and kneaded at 50°C for two days and nights. During this time, 0.5 parts by mass of lecithin was added and thoroughly dispersed. Next, the temperature was adjusted to 31°C using a temperature controller, and just before the butter solidified, the mixture was poured into a mold. After removing bubbles with a vibrator, it was passed through a 10°C cooling tunnel for 20 minutes to solidify. This was then cut out and packaged to obtain chocolate. This product is non-hygroscopic, has good color and luster, a good internal structure, melts smoothly in the mouth, and is a chocolate with an elegant sweetness and mellow flavor. [Examples]

[0130] <Cosmetic cream> Two parts by mass of polyoxyethylene glycol monostearate, five parts by mass of self-emulsifying glycerin monostearate, two parts by mass of crystalline isomaltitol obtained by the method of Example 7, two parts by mass of α-glucosylrutin (trade name "αG Rutin", sold by Toyo Sugar Refining Co., Ltd.), one part by mass of liquid paraffin, ten parts by mass of glycerin trioctanoate, and an appropriate amount of preservative were heated and dissolved according to a conventional method. Two parts by mass of L-lactic acid, five parts by mass of 1,3-butylene glycol, and 66 parts by mass of purified water were added to this mixture, and the mixture was emulsified using a homogenizer. An appropriate amount of fragrance was then added and stirred to produce a cosmetic cream. This product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin beautifier, and skin whitening agent. [Examples]

[0131] <Injury treatment ointment> To obtain 100 parts by mass of isomaltose syrup and 300 parts by mass of maltose obtained by the method of Example 5, 50 parts by mass of methanol in which 3 parts by mass of iodine was dissolved was added and mixed, and then 200 parts by mass of 10% (w / v) pullulan aqueous solution was added and mixed to obtain a wound treatment ointment exhibiting appropriate spreadability and adhesion. This product is a highly valuable ointment with minimal deterioration over time, as the volatilization of iodine and methanol is prevented by isomaltose. Furthermore, this product not only has a bactericidal effect due to iodine, but also acts as an energy supply agent to cells due to maltose, thus shortening the healing period and allowing wounds to heal cleanly. [Examples]

[0132] <Tablets> After thoroughly mixing 10 parts by mass of aspirin with 60 parts by mass of powdered crystalline isomaltitol obtained by the method of Example 7 and 4 parts by mass of corn starch, tablets with a thickness of 5.25 mm and a weight of 680 mg per tablet were produced by a tablet press according to a conventional method. This product utilizes the excipient properties of crystalline isomaltitol powder, is non-hygroscopic, has sufficient physical strength, and disintegrates very well in water. [Examples]

[0133] <Water yokan> One agar bar was placed in 400 mL of tap water, and after bringing it to a boil, 25 g of brown sugar, 50 g of refined sugar, and 20 g of isomaltoligosaccharide-rich syrup prepared by the method of Example 8 were added as solids. Then approximately 300 g of strained bean paste was added, and the mixture was simmered over medium heat, skimming off the foam. After boiling for 5 minutes, the heat was turned off, an appropriate amount of salt was added, and the mixture was stirred well. The mixture was then poured into a mold, left at room temperature for 30 minutes to solidify, and then refrigerated to make the water yokan. This product is a water yokan containing isomaltoligosaccharide and has a refined sweetness. [Examples]

[0134] <Lactic acid drink> 100g of isomaltoligosaccharide-rich syrup prepared by the method of Example 8 was taken, 100mL of milk was added to it, and the mixture was heated to a boil. The heat was then removed, the scum was skimmed off, and the mixture was allowed to cool naturally. After confirming that the temperature of the mixture had fallen below 37°C, appropriate amounts of citric acid, lactic acid, lactic acid essence, and lemon essence were added and the mixture was thoroughly stirred. The mixture was then bottled, sealed, and refrigerated to prepare a lactic acid beverage. This product is a lactic acid beverage containing isomaltoligosaccharide with reduced sweetness and calories. [Examples]

[0135] <Emulsion> The emulsion was prepared based on the following formulation. (Ingredients) Mass% Squalane 5.0 Olive oil 5.0 Jojoba oil 5.0 Cetyl alcohol 1.5 Glycerin monostearate 2.0 Polyoxyethylene (20) cetyl ether 3.0 Polyoxyethylene (20) sorbitan monooleate 2.0 1,3-Butylene glycol 1.0 Glycerin 2.0 Isomaltooligosaccharide-rich syrup obtained by the method of Example 8 7.0 Preservative (parahydroxybenzoic acid ester) appropriate amount Fragrance (appropriate amount) Purified water = 100, remainder [Industrial applicability]

[0136] The panose-degrading enzyme of the present invention is a novel enzyme previously unknown. When the panose-degrading enzyme of the present invention is used in combination with a specific glycosyltransferase, isomaltose and isomaltoligosaccharides can be efficiently produced on an industrial scale using starch or partially hydrolyzed starch as a raw material. Therefore, establishing the panose-degrading enzyme of the present invention, and methods for producing isomaltose and isomaltoligosaccharides using this panose-degrading enzyme in combination with other glycosyltransferases, will have great significance not only for the sugar industry but also for related food, cosmetics, and pharmaceutical industries. [Explanation of Symbols]

[0137] In Figure 1, M: Maltooligosaccharide marker 1: Isomaltose standard 2: Isopanose standard 3: Panos standard product 4: Isomaltotriose standard 5: Pullulan standard product 6: Dextran Standard Product 7: Crude enzyme only from strain U4520 8: Reaction solution obtained by reacting the crude enzyme of strain U4520 with panose. 9: Reaction solution obtained by reacting crude enzyme from strain U4520 with isomalttriose. 10: Reaction solution obtained by reacting pullulan with crude enzyme from strain U4520. 11: Reaction solution obtained by reacting the crude enzyme of strain U4520 with dextran. In Figure 3, 1: Molecular weight marker 2: Purified panose-degrading enzyme preparation 3: Molecular weight markers In Figure 7, ●:Ca 2+ In the absence of ions 〇: 5mMCa 2+ In the presence of ions In Figure 8, α-factor: The nucleotide sequence encoding the secretion signal peptide of α-factor, a conjugation factor in baker's yeast. Panose-hydrolysing enzyme: Panose-degrading enzyme gene Stop: Stop codon 5'AOX1: Promoter of the methanol-inducible alcohol oxidase gene AOX1 AOX1 TT: Transcription termination signal of the methanol-inducible alcohol oxidase gene AOX1 PTEF1: Promoter of TEF1, a transcription elongation factor for inducing zeosin resistance gene expression. PEM7: Promoter for inducing zeosin resistance gene expression Zeocin: Zeosin resistance gene CYC1TT: Transcription termination region of the cytochrome C gene pUC ori: pUC replication origin Bgl II: Restriction enzyme site Bam HI: Restriction enzyme site In Figure 10 M: Maltooligosaccharide marker 1: Isomaltose standard 2: Isopanose standard 3: Panos standard product 4: Isomaltotriose standard 5: Pullulan standard product 6: Dextran Standard Product 7: Crude enzymes only from microorganisms of the genus Acremonium 8: Reaction solution obtained by reacting panose with crude enzyme from a microorganism of the genus Acremonium. 9: Reaction solution obtained by reacting crude enzymes from microorganisms of the genus Acremonium with isomalttriose. 10: Reaction solution obtained by reacting pullulan with crude enzyme from a microorganism of the genus Acremonium. 11: Reaction solution obtained by reacting dextran with crude enzyme from a microorganism of the genus Acremonium. In Figure 13, G1: D-glucose G2: Maltose G3: Maltotrios G4: Maltotetraos G5: Maltpentaous G6: Maltohexaous G7: Maltheptaos G8: Maltooctaos IG2: Isomaltose IG3: Isomalttriose IG4: Isomalttetraose Gn: Maltooligosaccharide marker IGn: Isomaltooligosaccharide marker Lane 0: Partially hydrolyzed starch (raw material substrate) Lane 1: Reaction product obtained by treating partially hydrolyzed starch with α-glucosyltransferase. Lane 2: Reaction product obtained by treating partially hydrolyzed starch with panose-degrading enzyme. Lane 3: Reaction product obtained by treating partially hydrolyzed starch with α-glucosyltransferase and panose-degrading enzyme. Lane 4: Reaction product obtained by treating partially hydrolyzed starch with α-glucosyltransferase, panose-degrading enzyme, and isoamylase. Lane 5: Reaction product obtained by treating partially hydrolyzed starch with α-glucosyltransferase, panose-degrading enzyme, isoamylase, and α-amylase. In Figure 14, G1: D-glucose IG2: Isomaltose IG3: Isomalttriose IG4: Isomalttetraose IG5: Isomaltopentaose IG6: Isomalthexaose IG7: Isomalheptaous IG8: Isomaltoctaos IG9: Isomaltononaos IG10: Isomaltodecaose

Claims

1. A panose-degrading enzyme having the substrate specificity described in (1) and (2) below, and exhibiting a molecular weight of 85,000 ± 5,000 daltons in SDS-polyacrylamide gel electrophoresis, wherein the amino acid sequence is either the amino acid sequence shown in Sequence ID No. 11 in the sequence listing, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in Sequence ID No. 11 in the sequence listing: (1) Hydrolyze panose to produce isomaltose and D-glucose; and (2) When isomalttriose, dextran, and pullulan are reacted under the conditions of pH 5.5, 30°C, and 24 hours of reaction, no degradation products of isomalttriose, dextran, and pullulan are produced.

2. Furthermore, the panose-degrading enzyme according to claim 1 having the following physicochemical properties (a) to (d): (a) Optimal pH Under conditions of 30°C and a reaction time of 20 minutes, the pH is 5.0 to 5.6; (b) Optimal temperature Under conditions of pH 5.5 and a reaction time of 20 minutes at 35°C; (c) pH stability Under conditions of being held at 4°C for 24 hours, it is stable in the pH range of 4.5 to 11.5; and (d) Temperature stability Under conditions of pH 5.5 and being maintained for 1 hour, Ca ２＋ Stable up to 30°C in the absence of ions, 5 mM Ca ２＋ Stable up to 35°C in the presence of ions.

3. The panose-degrading enzyme according to claim 1 or 2, which is derived from a microorganism of the genus Sarocladium.

4. The panose-degrading enzyme according to claim 3, wherein the microorganism of the genus Sarocladium is Sarocladium kiliense U4520 strain (National Institute of Technology and Evaluation, Patent Microorganism Depositary Center, accession number NITE BP-03236) or a mutant strain thereof having the ability to produce the panose-degrading enzyme according to claim 1 or 2.

5. Sarocladium kiliense U4520 strain (National Institute of Technology and Evaluation, Patent Microorganism Depositary Center, Accession No. NITE BP-03236) having the ability to produce the panose-degrading enzyme described in claim 1 or 2, or a mutant strain thereof having the ability to produce the panose-degrading enzyme described in claim 1 or 2.

6. DNA encoding the panose-degrading enzyme according to claim 1 or 2.

7. The DNA according to claim 6, having a base sequence that is 90% or more identical to the base sequence that is 10% identical to the base sequence that is 10% identical to the base sequence that is 10% identical to the base sequence in the sequence listing, or a base sequence that is complementary to them.

8. The DNA according to claim 7, obtained by substituting one or more bases in the base sequence indicated by sequence number 10 in the sequence listing with other bases, without changing the encoded amino acid sequence, based on the degeneracy of the gene code.

9. Recombinant DNA comprising the DNA described in any one of claims 6 to 8 and an autonomously replicating vector.

10. A transformed organism obtained by introducing the recombinant DNA described in claim 9 into a suitable host cell.

11. A method for producing a panose-degrading enzyme, characterized by culturing a microorganism having the ability to produce the panose-degrading enzyme described in claim 1 or 2 in a nutrient medium to produce the panose-degrading enzyme described in claim 1 or 2, and then collecting it.

12. A method for producing a panose-degrading enzyme according to claim 11, wherein the microorganism is a microorganism belonging to the genus Sarocladium.

13. A method for producing a panose-degrading enzyme according to claim 12, wherein the microorganism of the genus Sarocladium is Sarocladium kiliense U4520 (National Institute of Technology and Evaluation, Patent Microorganism Depositary Center, accession number NITE BP-03236) or a mutant strain thereof having the ability to produce panose degradation according to claim 1 or 2.

14. A method for producing recombinant panose-degrading enzyme, characterized by culturing the transformant described in claim 10 and collecting the panose-degrading enzyme described in claim 1 or 2 from the culture.

15. A method for producing isomaltose comprising the steps of: combining a 6-α-glucosyltransferase having the activity to act on starch or a partially hydrolyzed starch product to produce a branched α-glucan having a branched structure in which D-glucose is α-1,6 bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain, with the panose-degrading enzyme described in any one of claims 1 to 4, and acting on starch or a partially hydrolyzed starch product to produce isomaltose; and collecting the produced isomaltose.

16. A method for producing isomaltose according to claim 15, wherein the 6-α-glucosyltransferase, which acts on the starch or partially hydrolyzed starch and has the activity to produce a branched α-glucan having a branched structure in which D-glucose is α-1,6 bonded to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain, is an enzyme derived from a microorganism of the genus Bacillus or Arthrobacter.

17. The method for producing isomaltose according to claim 15 or 16, wherein in the step of producing the isomaltose, one or more enzymes selected from starch debranching enzyme, α-amylase, cyclomaltodextrin glucanotransferase, and glucoamylase are used in combination.

18. A method for producing isomaltose, comprising the steps of: further reducing isomaltose by hydrogenation to convert it to isomaltitol; and collecting the converted isomaltitol, to the method for producing isomaltose according to any one of claims 15 to 17.

19. A method for producing isomaltoligosaccharide, comprising the steps of: combining an α-glucosyltransferase having the activity to act on starch or a partially hydrolyzed starch product to produce a branched α-glucan having a branched structure in which D-glucose or α-1,6-glucan with a glucose polymerization degree of 2 or higher is α-1,6-linked to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain, with the panose-degrading enzyme described in any one of claims 1 to 4, and acting on starch or a partially hydrolyzed starch product to produce isomaltoligosaccharide; and collecting the produced isomaltoligosaccharide.

20. A method for producing isomaltoligosaccharide according to claim 19, wherein the α-glucosyltransferase that acts on the starch or partially hydrolyzed starch and has the activity to produce a branched α-glucan having a branched structure in which D-glucose or α-1,6-glucan with a glucose polymerization degree of 2 or higher is α-1,6-linked to the hydroxyl group at the 6th position of the non-reducing terminal glucose residue of the α-1,4-glucan chain is an enzyme derived from a microorganism of the genus Bacillus or Arthrobacter.

21. A method for producing isomaltooligosaccharide according to claim 19 or 20, wherein in the step of producing the isomaltooligosaccharide, one or more enzymes selected from starch debranching enzyme, α-amylase, cyclomaltodextrin glucanotransferase, and glucoamylase are used in combination.

22. A method for producing isomaltooligosaccharide alcohol, comprising the steps of: reducing the isomaltooligosaccharide by hydrogenation to convert it into isomaltooligosaccharide alcohol; and collecting the converted isomaltooligosaccharide alcohol, to the method for producing isomaltooligosaccharide according to any one of claims 19 to 21.

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