WO2005056811A1 - β−1,4−グルカンをα−グルカンに変換する方法 - Google Patents
β−1,4−グルカンをα−グルカンに変換する方法 Download PDFInfo
- Publication number
- WO2005056811A1 WO2005056811A1 PCT/JP2004/018416 JP2004018416W WO2005056811A1 WO 2005056811 A1 WO2005056811 A1 WO 2005056811A1 JP 2004018416 W JP2004018416 W JP 2004018416W WO 2005056811 A1 WO2005056811 A1 WO 2005056811A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glucan
- phosphorylase
- reaction
- glucose
- cellobiose
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
Definitions
- the present invention relates to a method for producing hi-glucan from ⁇ -1,4-glucan.
- a-Glucan is widely used not only in the food industry but also as a raw material in the pharmaceutical, cosmetics, chemical industries, papermaking, textiles, etc., and is a very useful substance.
- amylose is expected to be used in a wide range of fields because of its abundant functions.
- ⁇ -glucan cannot be directly converted to a-glucan.
- a method for synthesizing G-1-P and dalcocellulose cellobiose by the action of cellobiose phosphorylase (CBP) is known, and cellodextrin phosphorylase (CBP) is known.
- CBP cellobiose phosphorylase
- a method of synthesizing a cell oligosaccharide having a degree of polymerization of n + 1 from G-1 P and cellooligosaccharides (degree of polymerization n) by the action of CDP) is also known.
- alpha 1, 4-by the action of glucan phosphorylase error zero a method of synthesizing a high molecular weight a-glucan is known from G-1-[rho and low molecular weight a-glucan.
- the reactions catalyzed by enzymes are often reversible reactions, so the present inventors considered the reaction catalyzed by CBP to be a method for degrading cellobiose, and Proceeding to produce G-1P, and considering the ability to synthesize ⁇ -glucan from the resulting G-1P, consider constructing a method for synthesizing darcan from ⁇ -1,4-glucan did.
- j8-glucan is phosphorolyzed using cellobiose phosphorylase (CBP) or cellodextrin phosphorylase (CDP) to obtain G-1P (first step).
- ⁇ -glucan is synthesized by glucan phosphorylase (GP) as a raw material (Step 2)
- GP glucan phosphorylase
- Step 2 This is a two-step method.
- the inorganic phosphoric acid In order to inhibit the synthesis reaction of H-glucan using G-1 ⁇ as a raw material, the inorganic phosphoric acid must be removed after the completion of the reaction in the first step.
- the cost of the purification step is one of the drawbacks.
- Non-Patent Document 1 sucrose phosphorylase (SP) Discloses a technology for efficiently converting sucrose to cellobiose by acting on sucrose. Further, Fujii et al. (Patent Document 1) disclose a technique for efficiently converting sucrose to amylose by simultaneously acting SP and GP.
- Glucose is involved in the enzymatic reaction for producing ⁇ -glucan from ⁇ -1,4-glucan. Therefore, it is considered that controlling the glucose concentration makes it possible to carry out the target enzyme reaction efficiently.
- Kitaoka et al. (Non-Patent Document 2) reported that in a system for synthesizing sucrose force cellobiose, the concentration of glucose, which is an essential raw material as an axceptor, in the reaction system was required to advance the reaction toward the cellobiose synthesis side. Insist that it is important to keep it low! . Therefore, fructose generated by the action of SP is converted to glucose using xylose isomerase, thereby allowing the reaction to proceed without adding glucose from outside the system and increasing the yield of cellobiose. Kitaoka et al.
- the present invention includes a reaction for degrading cellobiose, which is a substrate thereof, using CBP. Based on the above findings, those skilled in the art consider that a high glucose concentration is a favorable condition for cellobiose degradation by inhibiting the cellobiose synthesis reaction.
- Patent Document 1 International Publication No. 02Z097107 pamphlet
- Non-Patent Document 1 Kitaoka et al., Denpun Kagaku, vol. 39, No. 4, 1992, pp. 281-283
- Patent Document 2 Kitaoka et al., Trends in Glycoscience and Glycotechnology, vol. 14, No. 75, 2002, pp. . 35—50
- the present invention aims to solve the above-mentioned problems, and provides a method for efficiently converting j8-1,4 glucan, which cannot be used as food, to ⁇ -dalkan without going through a complicated manufacturing process.
- the purpose is to provide.
- the present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the present inventors have found that ⁇ -glucan is phosphorolyzed in the presence of ⁇ -1,4-glucan phosphorylase to produce glucose-1-phosphate. By coupling the reaction of synthesizing acid with the reaction of synthesizing a-glucan by reacting glucose 1-phosphate with a primer in the presence of a-glucan phosphorylase, a-dulcan is converted from ⁇ -1,4-glucan. They found that the synthesis was efficient, and based on this, completed the present invention.
- the method of the present invention is a method for producing ⁇ -glucan from j8-1,4-glucan, comprising j8-1,4-glucan, a primer, a phosphate source, and j8-l, 4-glucan. Includes the step of reacting a solution containing phosphorylase with a-1,4-glucan phosphorylase to produce higlucan.
- the j8-1,4-glucan can be cellobiose
- the 131,4-gnorecan phosphorylase can be cellobiose phosphorylase
- the j8-1,4-glucan can be a cellooligosaccharide having a degree of polymerization of 3 or more, and the j8-1,4-glucan phosphorylase can be cellodextrin phosphorylase.
- the j8-1,4-glucan may be a cellooligosaccharide having a degree of polymerization of 3 or more
- the j8-1,4-glucan phosphorylase may be cellobiose phosphorylase and cellodextrin phosphorylase. obtain.
- the production step may further include a step of removing glucose by-produced simultaneously with the production of the a-dalcan from the solution.
- the solution may further include glucose isomerase or glucose oxidase.
- the solution may further include glucose oxidase and mutarotase.
- the solution may further include catalase or peroxidase.
- the phosphate source may be inorganic phosphate, glucose monophosphate, or a mixture of inorganic phosphate and glucose-1-phosphate.
- the concentration of the phosphate source may be ImM-50mM.
- the method of claim 1 wherein the ⁇ -glucan amylose.
- non-digestible cellulose can be efficiently converted into digestible food.
- FIG. 1 shows an outline of a reaction occurring in the production method of the present invention.
- FIG. 2 shows an outline of the reaction that occurs in the production method of the present invention when cellobiose is used as ⁇ -1,4-glucan and cellobiose phosphorylase is used as ⁇ -1,4-glucan phosphorylase. Show.
- FIG. 3 shows changes in amylose yield when the concentration of cellobiose phosphorylase was changed.
- FIG. 4 shows a change in amylose yield when the concentration of phosphoric acid was changed.
- FIG. 5 shows changes in amylose yield when the cellobiose concentration was increased while the ratio of cellobiose concentration, primer concentration, and phosphate concentration was kept constant.
- FIG. 6 shows a change in amylose yield when glucose isomerase (GI) or dalcos oxidase (GOx) + mutarotase (MT) + peroxidase (POx) was added in the production method of the present invention. .
- GI glucose isomerase
- GOx dalcos oxidase
- MT mutarotase
- POx peroxidase
- SEQ ID NO: 1 is the base sequence of synthetic DNA primer 1.
- SEQ ID NO: 2 is the base sequence of synthetic DNA primer 2.
- a-glucan refers to a saccharide having D-glucose as a constituent unit and having at least two or more saccharide units linked by a-1,4 darcoside bonds.
- ⁇ -glucan can be a linear, branched or cyclic molecule. Linear ⁇ -glucan and ⁇ -1,4-glucan are synonyms. In a linear ⁇ -dalcan, saccharide units are linked only by ⁇ -1,4 darcoside bonds. An a-glucan containing one or more 6-darcoside bonds is a branched a-glucan. The a-glucan preferably contains some linear parts. Unbranched linear ⁇ -dalkans are more preferred.
- the ⁇ -glucan produced in the present invention is preferably amylose, glucan having a cyclic structure or glucan having a branched structure, and more preferably amylose.
- the number of sugar units contained in one molecule of ⁇ -dalcan is called the degree of polymerization of ⁇ -glucan.
- the ⁇ -glucan preferably has a small number of branches (ie, a number of 6 darcoside bonds).
- the number of branches is typically 0 to 10000, preferably ⁇ 0 to 1000, more preferably ⁇ 0 to 500, and still more preferably ⁇ 0 to 100, and more preferably. Is 0-50, more preferably 0-25, and even more preferably 0.
- the ratio of the number of a-1,4 darcoside bonds to the number of 6 darcoside bonds when the number of ⁇ 1,6 darcoside bonds is 1 is as follows: It is preferably 1 to 10000, more preferably 10 to 5000, further preferably 50 to 1000, and still more preferably 100 to 500.
- the a-1, 6-darcoside bond may be distributed randomly or uniformly in the ⁇ -glucan. Preferably, the distribution is such that five or more linear moieties are formed in the a-glucan per saccharide unit.
- the a-glucan may be composed of only D-glucose, Derivatives modified to a certain extent without impairing the quality may be used. It is preferable to be qualified. Modifications that do not impair the properties of ⁇ -Dalkan include, but are not limited to, esterification, etherification, cross-linking, and the like. These modifications can be made according to methods known in the art.
- the ⁇ -glucan is typically about 1 X 10 3 or more, preferably about 5 X 10 3 or more, more preferably about IX 10 4 or more, further preferably about 5 X 10 4 or more, and more preferably about 5 X 10 4 or more. preferably has a molecular weight of over about IX 10 5 or more.
- the ⁇ -glucan typically has a molecular weight of about 1 ⁇ 10 6 or less, preferably about 5 ⁇ 10 5 or less, more preferably about 1 ⁇ 10 5 or less.
- a person skilled in the art can appropriately set the amount of the substrate (eg, primer, ⁇ -1,4-glucan, etc.), the amount of the enzyme, the reaction time, etc., to be used in the production method of the present invention, to thereby obtain the desired molecule. It is easy to see that an amount of ⁇ -Dalkan is obtained.
- a solution containing j8-1,4-glucan, a primer, a phosphate source, ⁇ -1,4-glucan phosphorylase, and ⁇ -1,4-glucan phosphorylase is used.
- 8-1,4-glucan phosphorylase, ⁇ -1,4-glucan phosphorylase And a buffer and a solvent in which these are dissolved are used as main materials. These materials are generally added at the start of the reaction. During the reaction, any of these materials may be added and added.
- phosphate source refers to a molecule capable of providing phosphate to the catalytic reaction of CBP, and includes inorganic phosphates (eg, NaHPO, NaHPO, KHPO and
- Inorganic phosphates such as K HPO
- organic phosphates e.g.,
- the solution may further contain glucose isomerase or glucose oxidase.
- glucose oxidase When using glucose oxidase, mutarotase may be further included. If glucose oxidase is used, the solution of the present invention may also contain a potentiase or a peroxidase.
- a debranching enzyme a branching enzyme
- An enzyme selected from the group consisting of ⁇ -dalcanotransferase and glycogen debranching genzyme can be used.
- An enzyme selected from the group consisting of a debranching enzyme, branching genzym, 4a dalcanotransferase, and glycogen debranching genzym is selected according to the structure of the target ⁇ -glucan according to the present invention.
- the initial force of the production method may be added to the solution, or the intermediate force may be added to the solution.
- j8-1,4-glucan is a saccharide having D-glucose as a constituent unit and has at least two saccharide units linked by ⁇ -1,4 darcoside bonds.
- ⁇ -1,4-glucan can be a linear molecule.
- Linear j8-glucan, j8-1,4-glucan and cellulose are synonyms.
- linear j8-glucan the sugar units are linked only by ⁇ -1,4 darcoside bonds.
- the number of sugar units contained in one molecule of / 3-1,4-glucan is called the degree of polymerization of
- the degree of polymerization of ⁇ -1,4-glucan is preferably about 2 to about 10, more preferably about 2 to about 8, and more preferably about 2 to about 5.
- J8-1,4-glucan having a degree of polymerization of about 2 to about 10 is also called cellooligosaccharide.
- J8-1,4-glucan with a degree of polymerization of 2 is particularly called cellobiose.
- a j8-1,4-glucan with a weight of 3 is called cellotriose.
- J8-1,4-glucan with a degree of polymerization of 4 is called cellotetraose.
- ⁇ -1,4-glucan is present in all plants.
- ⁇ -1,4-glucan can be isolated from a plant and unmodified, but also obtained by subjecting a plant with isolated plant power to a daniological or enzymatic treatment. You may.
- ⁇ -1,4-glucan may also be waste-regenerated cellulose such as waste paper, building materials, used cloth, or prepared from it. For example, by reacting cellulose with a high molecular weight cellulose isolated from a plant, a lower molecular weight cellooligosaccharide can be obtained. Methods for producing large quantities of cell oligosaccharides from plants are known in the art.
- JP-A-2001-95594 is an example of such a document.
- the ⁇ -l, 4-glucan may be provided as a step in the generation of a shift from a plant crushed liquid containing 13-1,4-glucan to purified ⁇ -1,4-glucan.
- the j8-l, 4-glucan used in the method of the present invention is pure Preferably, it is However, any other contaminants may be included as long as the action of the enzyme used in the present invention is not inhibited.
- the concentration of ⁇ -1,4-glucan contained in the solution is typically about 0.1% to about 40%, preferably about 0.5% to about 30%, and more preferably about 0.5% to about 30%. Is about 1% to about 20%, particularly preferably about 2% to about 15%, and most preferably about 3% to about 12%. Note that, in this specification, the concentration of j8-1, 4-gunorecan is WeightZVolume, that is,
- the molar concentration of ⁇ -1,4-glucan in a solution is obtained by dividing the molar concentration of inorganic phosphate and the molar concentration of glucose 1-phosphate in a reaction solution.
- the resulting ratio is referred to as the ⁇ -1,4-glucan: phosphate ratio. That is:
- the j8-1,4-glucan: phosphate ratio at the start of the reaction may be any ratio, preferably about 0.01 or more, more preferably about 0.03 or more, and even more preferably about 0.03 or more. 06 or more, particularly preferably about 0.1 or more, and most preferably about 0.1 to about 0.6.
- the primer used in the method of the present invention refers to a molecule that acts as a starting material for adding a glycoside residue during the synthesis of ⁇ -glucan.
- a glycoside residue and a glucose residue can be used interchangeably.
- the primer can also be referred to as a molecule that acts as an receptor for the glycoside residue of G-1--1-.
- Primers should have at least one free moiety to which a saccharide unit can bind via an ⁇ -1,4 darcoside bond. If so, other parts may be formed by parts other than sugar.
- one glycoside residue is transferred by an ⁇ -1,4 bond to a primer contained at the start of the reaction, and thus ⁇ -glucan having a degree of polymerization one greater than that of the primer is formed. Is done.
- the ⁇ -glucan formed can again act as an receptor in the same solution.
- glycoside residues are sequentially bonded to the primer by ⁇ 1,4 darcoside bonds, and ⁇ -dalcan having an arbitrary degree of polymerization is synthesized.
- Primers include any sugar to which a sugar unit can be added by glucan phosphorylase.
- the primer may be any one that can act as a starting material for the reaction of the present invention.
- the ⁇ -glucan synthesized by the method of the present invention may be used as a primer to produce a 4-darcoside chain by the method of the present invention. Can be extended again.
- the primer may be a 4-glucan containing only an ⁇ -1,4 darcoside bond, or may partially have an ⁇ -1,6-darcoside bond.
- One skilled in the art can easily select an appropriate primer according to the desired glucan.
- primers include maltooligosaccharides, amylose, amylopectin, glycogen, dextrin, pullulan, coupling sugar, starch, and derivatives thereof.
- Maltooligosaccharides are substances formed by the dehydration condensation of about 2 to about 10 glucoses, and refer to substances linked by four bonds. Maltooligosaccharides preferably have about 3 to about 10 saccharide units, more preferably about 4 to about 10 saccharide units, and more preferably about 5 to about 10 saccharide units. Examples of maltooligosaccharides include maltose, manoletotriose, manoletotetraose, manoletopentaose, manoletohexaose, manoletoheptaose, manoletoctaose, manoletononaose, manoletodeose, etc.
- the maltooligosaccharide is preferably maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose, more preferably maltotetraose, maltopentaose, maltopentaose, maltopentaose or maltopentaose. It is orthohexaose or maltoheptaose, more preferably maltotetraose.
- the maltooligosaccharide may be a single product or a mixture of a plurality of maltooligosaccharides.
- the mixture of maltooligosaccharides contains at least one of maltotriose, maltose and glucose, in addition to maltooligosaccharides having a degree of polymerization equal to or higher than the degree of polymerization of maltotetraose.
- maltooligosaccharide having a degree of polymerization of maltotetraose or higher refers to a maltooligosaccharide having a degree of polymerization of 4 or higher.
- the oligosaccharide may be a linear oligosaccharide or a branched oligosaccharide. Oligosaccharides may have a cyclic moiety in the molecule. In the present invention, linear oligosaccharides are preferred.
- Amylose is a straight-chain molecule composed of glucose unit forces connected by ⁇ - 1,4 bonds. Amylose is contained in natural starch.
- Amylopectin is a branched molecule in which glucose units are linked by ⁇ -1,6 bonds to glucose units linked by ⁇ -1,4 bonds. Amylopectin is contained in natural starch.
- amylopectin for example, picorn corn starch consisting of 100% amylopectin can be used.
- amylopectin having a degree of polymerization of about 1 ⁇ 10 5 or more can be used as a raw material.
- Glycogen is a type of glucan composed of glucose and is a glucan having a high frequency of branching. Glycogen is widely distributed in granular form in almost all cells as animal and plant storage polysaccharides. Glycogen is present in plants, for example, in the seeds of maize corn. Glycogen is typically present at a rate of about 1 every 3 units of glucose relative to the glucose 4-linked sugar chain, with 0; -1,4-linked glucose having an average degree of polymerization of 12-18. Are linked by ⁇ -1,6-linkage. Similarly, the 4-linked sugar chain of glucose is also linked by a 1,6-bond to the branch linked by an ⁇ -1,6-linkage. Therefore, glycogen forms a network
- the molecular weight of glycogen is typically about 1 X 10 5 - about 1 X 10 8, preferably about 1
- Pullulan is composed of ⁇ -1,6-linked maltotriose in a regularly stepped manner, with a molecular weight of about 100,000 to about 300,000 (eg, about 200,000) glucans.
- Pullulan is produced, for example, by culturing black yeast Aureobasidium pullulans using starch as a raw material. Pullulan can be obtained, for example, from Hayashibara.
- Coupling sugar is a mixture containing sucrose, darcosyl sucrose, and maltosyl sucrose as main components. Coupling sugar is produced, for example, by allowing a cyclodextrin glucanotransferase produced by Bacillus megaterium or the like to act on a mixed solution of sucrose and starch. Coupling sugar can be obtained, for example, from Hayashibara Corporation.
- Starch is a mixture of amylose and amylopectin.
- any starch which is usually commercially available and can be used can be used.
- the ratio of amylose to amylopectin contained in starch varies depending on the type of plant producing starch. Most of the starches contained in barley corn and waxy corn are amylopectin. On the other hand, starch consisting only of amylose and containing no amylopectin cannot be obtained from ordinary plants.
- Starch is classified into natural starch, starch degradation products, and modified starch.
- Natural starch is divided into potato starch and cereal starch depending on the raw material.
- potato starch include potato starch, tapio starch, sweet potato starch, waste starch, and bracken starch.
- cereal starches include corn starch, wheat starch, rice starch, and the like.
- An example of a natural starch is a noamylose starch (eg, no, iamylose cornstarch) with an amylose content increased to 50% -70% as a result of breeding of the plant producing the starch.
- Another example of a natural starch is an amylose-free waxy starch as a result of breeding of a starch-producing plant.
- the soluble starch refers to a water-soluble starch obtained by subjecting natural starch to various treatments.
- Modified starch is a starch obtained by subjecting a natural starch to a treatment such as hydrolysis, esterification, or a-formation so as to have more easily usable properties.
- a treatment such as hydrolysis, esterification, or a-formation so as to have more easily usable properties.
- modified starches with various combinations of gelatinization start temperature, paste viscosity, paste transparency, aging stability, etc. are available.
- modified starch There are various types of modified starch.
- An example of such a starch is that starch molecules are cut by immersing the starch particles in an acid at or below the gelatinization temperature of the starch. The particles are destroyed! / What is starch.
- the starch hydrolyzate is an oligosaccharide or polysaccharide obtained by subjecting starch to a treatment such as an enzyme treatment or hydrolysis and having a smaller molecular weight than before the treatment.
- a treatment such as an enzyme treatment or hydrolysis and having a smaller molecular weight than before the treatment.
- examples of the starch hydrolyzate include starch debranching enzyme hydrolyzate, starch phosphorylase hydrolyzate and starch partial hydrolyzate.
- a starch debranching enzyme degradation product is obtained by allowing a branching enzyme to act on starch.
- a starch debranching enzyme degraded product in which the branched portion (ie, ⁇ -1,6-darcoside bond) is cleaved to an arbitrary degree can be obtained.
- the degraded enzymatic degraded product include a degraded product having 20 ⁇ - 1, 6-darcoside bonds out of 4-10000 saccharide units, ⁇ - 1, 6-having 3 to 500 saccharide units Degradants without any darcoside bonds, maltooligosaccharides and amylose are mentioned.
- the molecular weight distribution of the obtained hydrolyzate may differ depending on the type of the degraded starch.
- the starch debranching enzyme hydrolyzate can be a mixture of sugar chains of various lengths.
- the starch phosphorylase hydrolyzate is obtained by allowing glucan phosphorylase (also referred to as phosphorylase) to act on starch.
- Glucan phosphorylase transfers glucose residues from the non-reducing terminal of starch to other substrates one saccharide unit at a time.
- Glucan phosphorylase cannot break the ⁇ -1,6-darcoside bond. Therefore, when glucan phosphorylase is allowed to act on starch for a sufficiently long time, a portion of the ⁇ -1,6-darcoside bond may be formed. A degraded product that has been cut is obtained.
- the number of sugar units of the starch phosphorylase hydrolyzate is preferably ⁇ 10 to about 100,000, more preferably ⁇ 50 to about 50,000, and even more preferably about 100 to about 50,000. 10,000.
- the starch phosphorylase hydrolyzate may have a different molecular weight distribution of the resulting degradation product depending on the type of starch degraded.
- the starch phosphorylase hydrolyzate can be a mixture of sugar chains of various lengths.
- Dextrin and partially hydrolyzed starch refer to degraded products obtained by partially decomposing starch by the action of acids, alkalis, enzymes and the like.
- the number of sugar units contained in the dextrin and the starch partial hydrolyzate is preferably about 10 to about 100,000, more preferably about 50 to about 50,000, and still more preferably about 100 to about 50,000. 10,000.
- the degradation obtained by the type of degraded starch The distribution of molecular weights of the products may be different.
- the dextrin and starch partial hydrolyzate can be a mixture of sugar chains having various lengths.
- Starch is selected from soluble starch, xy-starch, naphthia, iamylose starch, degraded starch branching enzyme, degraded starch phosphorylase, partially hydrolyzed starch, modified starch, and their derived body strengths. It is preferred to be.
- the above-mentioned various sugar derivatives can be used as primers.
- a hydroxyalkylated, alkylated, acetylated, carboxymethylated, sulfated, or phosphorylated derivative of at least one alcoholic hydroxyl group of the sugar can be used.
- a mixture of two or more of these derivatives can be used as a raw material.
- a phosphate source such as inorganic phosphoric acid refers to a substance capable of providing a phosphate substrate in a CBP reaction.
- the phosphate substrate refers to a substance that is a raw material of a phosphate moiety (moiety) of glucose monophosphate.
- inorganic phosphate is thought to act as a substrate in the form of phosphate ions.
- this substrate is conventionally referred to as inorganic phosphoric acid, and thus the substrate is referred to herein as inorganic phosphate.
- Inorganic phosphoric acid includes phosphoric acid and inorganic salts of phosphoric acid.
- inorganic phosphoric acid is used in water containing cations such as alkali metal ions.
- phosphoric acid, phosphate, and phosphate ions are in an equilibrium state, it is difficult to distinguish phosphoric acid from phosphate. Therefore, for convenience, phosphoric acid and phosphate are collectively referred to as inorganic phosphoric acid.
- the inorganic phosphoric acid is preferably any metal salt of phosphoric acid, and more preferably an alkali metal salt of phosphoric acid.
- Preferred specific examples of the inorganic phosphoric acid include sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, phosphoric acid (H
- one or more inorganic phosphoric acids may be contained.
- the inorganic phosphoric acid is, for example, polyphosphoric acid (for example, pyrophosphoric acid, triphosphoric acid and tetraphosphoric acid).
- a phosphoric acid condensate or a salt thereof may be provided by adding a solution obtained by decomposing the phosphoric acid condensate or a salt thereof by a physical, chemical or enzymatic reaction or the like to a reaction solution.
- glucose-1-phosphate is glucose-1-phosphate (CHOP)
- Glucose monophosphate is preferably any metal salt of glucose monophosphate (CHOP) in a narrow sense, more preferably glucose monophosphate (CHH).
- CHOP glucose monophosphate
- CHH glucose monophosphate
- O P is any alkali metal salt.
- Preferred examples of glucose-1-phosphate are glucose-1-phosphate
- glucose-1-sodium phosphate examples thereof include glucose-1-sodium phosphate, glucose-1-dipotassium phosphate, glucose-1-phosphate (CHOP), and the like.
- glucose-1-sodium phosphate examples thereof include glucose-1-sodium phosphate, glucose-1-dipotassium phosphate, glucose-1-phosphate (CHOP), and the like.
- a chemical formula is written in parentheses.
- Unmarked glucose monophosphate refers to glucose monophosphate in a broad sense, ie, glucose-1-phosphate (CHOP) and its salts in a narrow sense.
- the ratio between phosphoric acid and glucose 1-phosphate in the reaction solution at the start of the reaction may be any ratio.
- the total of the molar concentration of inorganic phosphoric acid and the molar concentration of glucose monophosphate contained in the reaction solution is typically about 0. ImM to about 1000 mM, preferably about ImM to about 500 mM, and more preferably. Is from about ImM to about 50 mM, and even more preferably from about 5 mM to about 30 mM. If the amounts of the inorganic phosphoric acid and glucose monophosphate are too large, the reaction itself occurs, but the yield of ⁇ -dalcan may decrease. If these amounts are too small, synthesis of ⁇ -dalcan may take a long time.
- the content of inorganic phosphoric acid in a solution in the method of the present invention can be determined by a method known in the art.
- the content of glucose monophosphate in this solution can be determined by a method known in the art.
- the total content of inorganic phosphoric acid and glucose monophosphate may be measured by an atomic absorption method.
- Inorganic phosphoric acid is obtained, for example, as a phosphate ion by the following method.
- a solution containing inorganic phosphoric acid (200 ⁇ l) was mixed with 800 ⁇ l of molybdenum reagent (15 mM ammonium molybdate, 100 mM zinc acetate), followed by 200 ⁇ l of 568 mM ascorbic acid (200 ⁇ l). pH 5.0) was added and stirred to obtain a reaction system. After keeping the reaction system at 30 ° C. for 20 minutes, the absorbance at 850 nm is measured using a spectrophotometer. Measure absorbance in the same manner using inorganic phosphoric acid of known concentration, and create a standard curve. Apply the absorbance obtained for the sample to this standard curve to determine the inorganic phosphoric acid in the sample. In this assay, the amount of inorganic phosphate is determined, not the amount of glucose monophosphate.
- Glucose monophosphate can be quantified, for example, by the following method.
- 300 ⁇ l measurement reagent 200 mM Tris-HCl (pH 7.0), 3 mM NADP, 15 mM magnesium chloride, 3 mM EDTA, 15 ⁇ Gnorecose-1,6-diphosphate, 6 / z gZml Phosphognorecomtase, 6 ⁇ g / ml glucose-6-phosphate dehydrogenase
- add a solution 6001 containing appropriately diluted glucose-1-phosphate add a solution 6001 containing appropriately diluted glucose-1-phosphate and stir to obtain a reaction system. After keeping the reaction system at 30 ° C.
- the absorbance at 340 nm is measured using a spectrophotometer. Measure absorbance in the same manner using sodium glucose monophosphate with a known concentration, and create a standard curve. The absorbance obtained from the sample is applied to this standard curve to determine the concentration of glucose 1-phosphate in the sample. Usually, 1 minute is defined as the activity to produce L mol of glucose monophosphate per minute. In this assay, only glucose monophosphate is quantified, not the amount of inorganic phosphate.
- j8-1,4-glucan phosphorylase refers to any enzyme that performs phosphorolysis by transferring the non-reducing terminal glucose residue of 13-1,4-glucan to a phosphate group.
- ⁇ -1,4-glucan phosphorylase can also catalyze the
- the reaction catalyzed by ⁇ -1,4-glucan phosphorylase is described by the following equation:
- the ⁇ -1,4-glucan phosphorylase is preferably cellobiose phosphorylase (EC: 2.4.1.20) or cellodextrin phosphorylase (EC: 2.4.4.149).
- Cellobiose phosphorylase refers to an enzyme that transfers a glucose residue on the non-reducing terminal side of cellobiose to a phosphate group to perform phosphorolysis.
- the reaction catalyzed by cellobiose phosphorylase is described by the following equation:
- Cellodextrin phosphorylase is an enzyme that transfers a non-reducing terminal-glucose residue of cellooligosaccharide having a degree of polymerization of 3 or more to a phosphate group to perform phosphorolysis. Cellooligosaccharides are also called cellodextrins.
- the reaction catalyzed by cellodextrin phosphorylase is represented by the following formula:
- cellobiose phosphorylase it is preferable to use as 4-glucan phosphorylase!
- cellodextrin phosphorylase is preferably used as ⁇ -1,4 glucan phosphorylase.
- j8-1,4-glucan is a cellooligosaccharide, ⁇ -1
- cellobiose phosphorylase and cellodextrin phosphorylase are used as the 4-glucan phosphorylase.
- glucose 1-phosphate generated by cellooligosaccharide degradation by the action of cellodextrin phosphorylase is used for the synthesis of glucan, and cellobiose finally produced can be degraded by cellobiose phosphorylase.
- the synthesis rate of hidalkan from cellooligosaccharides is faster.
- ⁇ -1,4-glucan phosphorylase is included in various organisms in nature.
- organisms that produce j8-l, 4-glucan phosphorylase include organisms of the genus Clostridium (eg, For example, Clostridium thermocellum and Clostridium sterocorarium), organisms of the genus Cellvib rio (e.g., Cellvibrio gilvus), organisms of the genus Thermotoga (e.g., Ther motoga neapolitana and Thermotoga maritima), organisms of the genus Ruminococcas (e.g., Ruminococcas flavofaciens), Forties (For example, Forties a nnos), organisms of the genus Cellulomonas and organisms of the genus Erwinia.
- Clostridium eg, Clostridium thermocellum and Clostridium sterocorarium
- Organisms producing ⁇ -1,4 glucan phosphorylase are preferably Clostridium thermocellum, Clostridium sterocorarium ⁇ ellvibrio gilvus ⁇ Thermotoga neapolitana, Thermotoga maritima ⁇ Ruminococcas flavofaciens ⁇ Forties annos, Cel lulomonas sp., Erwinia sp. Selected.
- the j8-l, 4-glucan phosphorylase may be of plant origin.
- Cellobiose phosphorylase is included in various organisms in nature.
- organisms that produce cellobiose hostrophylase include organisms of the genus Clostridium (eg, Clostri dium thermocellum and Lostridium sterocorarium; organisms of the genus Cellviorio (eg, Cellvibrio gilvus), and organisms of the genus Thermotoga (eg, Thermotoga neapolitana and Thermotoga maritima), organisms of the genus Ruminococcas (eg, Ruminococcas flavofaciens), organisms of the genus Forties (eg, Forties annos), organisms of the genus Cellulomonas, and organisms of the genus Erwinia.
- Clostridium eg, Clostri dium thermocellum and Lostridium sterocorarium
- organisms of the genus Cellviorio eg, Cellvibri
- Cellobiose phosphorylase may be of plant origin.
- Cellodextrin phosphorylase is included in various organisms in nature.
- organisms that produce cellodextrin phosphorylase include Clostridium organisms (eg, C1 ostndium thermocellum and Clostridium sterocorarium), ellvibrio organisms (eg, Cellvibrio gilvus), and Thermotoga organisms (eg, Thermoto ga neapolitana and Thermotoga maritima), organism of RuminococcasJ3 ⁇ 4 (row f Examples include Ruminococcas flavofaciens), organisms of the genus Forties (for example, Forties annos), organisms of the genus Cellulomonas, and organisms of the genus Erwinia.
- Clostridium organisms eg, C1 ostndium thermocellum and Clostridium sterocorarium
- ellvibrio organisms eg, Cellvibrio gilvus
- the organism producing cellodextrin phosphorylase is preferably selected from the group consisting of Clostridium thermocellum, Clostrldium sterocorarium, Cellvibno gilvus, Tnermotoga neapolitana, Ther motoga maritima, Ruminococcas flavofaciens ⁇ Forties annos ⁇ Cellulomonas sp., Erwinia sp.
- it is Clostridium the rmocelium or i Cellulomonas sp., Most preferably Tana 3; Cellodextrin phosphorylase phosphorylase is of plant origin.
- ⁇ -1,4-glucan phosphorylase (preferably cellobiose phosphorylase or cellodextrin phosphorylase, most preferably cellobiose phosphorylase) is a 13-1,4 glucan phosphorylase (preferably cellobiose phosphorylase or cellodextrin phosphorylase, most preferably cellobiose). Phosphorylase).
- ⁇ -1,4-glucan phosphorylase preferably has a certain degree of heat resistance. -1,4-glucan phosphorylase is more preferable as the heat resistance is higher.
- j8-1,4-glucan phosphorylase when j8-1,4-glucan phosphorylase is heated at 55 ° C for 20 minutes in a 50 mM phosphate buffer (pH 7.5) containing 1.4 mM 2 mercaptoethanol, ⁇ -1 It is preferable that it retains 50% or more of the activity of 4-glucan phosphorylase.It is more preferable that it retains 60% or more of the activity.It retains 70% or more of the activity. It is more preferable that the compound has an activity of 80% or more, and it is particularly preferable that the compound has an activity of 85% or more.
- 4-glucan phosphorylase is preferably selected from the group consisting of Clostridium thermocellum, C1 ostndium sterocorarium, Cellviono giivus, Thermotoga neapolrcana, T hermotoga maritima, Ruminococcas flavofaciens ⁇ Forties annos ⁇ Cellul omonas sp., Erwinia sp. It is derived from bacteria.
- cellobiose phosphorylase can be used in the form of cellobiose phosphorylase [ma, preferably ⁇ ma Clostridium thermocellum; It is derived from a bacterium selected from the group consisting of maritima, Ruminococcas flavofaciens, Forties annos, Cellulomonas sp. and Erwinia sp., more preferably from Clostridium thermocellum or Cellvibrio gilvus, and most preferably from Clostridium thermocellum.
- ma preferably ⁇ ma Clostridium thermocellum
- maritima Ruminococcas flavofaciens
- Forties annos Cellulomonas sp.
- Erwinia sp. more preferably from Clostridium thermocellum or Cellvibrio gilvus, and most preferably from Clostridium thermocellum.
- the cellobiose phosphorylase can be selected from the group consisting of cellobiose phosphorylase; , Forties annos, Cellulomonas sp., Erwinia sp. Derived from a bacterium, more preferably from Clostridium thermocellum, or more preferably from Cellulomonas sp., Most preferably from 3; and more preferably from Clostridium thermocellum. (?is there.
- the term "derived from” an organism does not only mean that the enzyme is directly isolated from the organism, but that the enzyme is obtained by utilizing the organism in some way. That can be done. For example, when a gene encoding the obtained enzyme is introduced into E. coli and the E. coli enzyme is isolated, the enzyme is said to be "derived from” the organism.
- the j8-l, 4-glucan phosphorylase used in the present invention can also be directly isolated from the biological ability to produce ⁇ -1,4-glucan phosphorylase which exists in nature as described above.
- the j8-l, 4-glucan phosphorylase used in the present invention is a microorganism (for example, a bacterium) that has been genetically modified using a gene encoding
- the ⁇ -1,4-glucan phosphorylase used in the method of the present invention can be prepared, for example, as follows. First, a microorganism (for example, bacteria, fungi, etc.) that produces / 3-1,4-glucan phosphorylase is cultured. This microorganism may be a microorganism that directly produces ⁇ -1,4-glucan phosphorylase. We also cloned a gene encoding j8-1,4-glucan phosphorylase, and genetically modified the resulting gene into microorganisms (eg, bacteria, fungi, etc.) that are advantageous in expressing j8-1,4-glucan phosphorylase.
- microorganism for example, bacteria, fungi, etc.
- j8-l, 4-glucan phosphorylase may be obtained from the resulting microorganism.
- Microorganisms used for genetic recombination with the ⁇ -1,4-glucan phosphorylase gene include easy expression of ⁇ -1,4-glucan phosphorylase, easy cultivation, rapid growth, and safety. It can be easily selected in consideration of various conditions such as. Since ⁇ -1,4-glucan phosphorylase preferably does not contain amylase as a contaminant, a microorganism (eg, a bacterium, a fungus, etc.) that does not produce amylase or has low levels and does not express force is genetically engineered. It is preferably used instead.
- ⁇ -1,4-glucan phosphorylase For gene recombination of ⁇ -1,4-glucan phosphorylase, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis.
- the j8-l, 4-glucan phosphorylase produced using a microorganism that does not produce amylase or has a low level and does not express force eg, a bacterium, a fungus, etc.
- Gene recombination of a microorganism with the cloned gene can be performed according to a method well known to those skilled in the art.
- a cloned gene it is preferred that this gene be operably linked to a constitutive or inducible promoter.
- “Operably linked” means that the promoter and the gene are linked so that expression of the gene is regulated by the promoter.
- the culturing is preferably performed under inducing conditions.
- Various inducible promoters are known to those of skill in the art.
- a base sequence encoding a signal peptide can be linked to this gene so that the produced ⁇ -1,4-glucan phosphorylase is secreted outside the cells.
- the nucleotide sequence encoding the signal peptide is known to those skilled in the art.
- culturing microorganisms for example, bacteria, fungi, and the like
- culturing microorganisms for example, bacteria, fungi, and the like
- Media suitable for culturing microorganisms, induction conditions suitable for each inducible promoter, and the like are known to those skilled in the art.
- the expressed j8-1,4-glucan phosphorylase when the expressed j8-1,4-glucan phosphorylase accumulates in transformed cells, the transformed cells are cultured under appropriate conditions, and then the culture is centrifuged or filtered. The cells are then recovered and suspended in a suitable buffer. Next, after crushing the cells by ultrasonic treatment or the like, the supernatant is obtained by centrifugation or filtration.
- expressed j8-l, 4-glucan phosphorylase is secreted outside the transformed cells If so, the transformed cells are cultured in this manner, and then the culture is centrifuged or filtered to separate the cells to obtain a supernatant.
- the supernatant containing ⁇ 8-1,4-glucan phosphorylase thus obtained is usually used.
- Concentration is performed using the above method (eg, salting-out method, solvent precipitation, ultrafiltration) to obtain a fraction containing j8-l, 4-glucan phosphorylase.
- the fraction is subjected to filtration, centrifugation, desalting and other treatments to obtain a crude enzyme solution.
- the crude enzyme solution is further appropriately combined with ordinary enzyme purification means such as lyophilization, isoelectric focusing, ion exchange chromatography, and crystallization to obtain a crude enzyme or a purified enzyme with improved specific activity.
- the crude enzyme can be used as it is, for example, for the production of ⁇ -glucan.
- the amount of ⁇ -1,4-glucan phosphorylase contained in the solution at the start of the reaction is typically about 0,1 with respect to j8-l, 4-glucan in the solution at the start of the reaction.
- 01-1, OOOU / g ⁇ -1,4-glucan preferably about 0.05-500 U / g ⁇ -1,4-glucan, more preferably about 0.1 l-100U / g ⁇ -1, 4-glucan, particularly preferably about 0.5-50 UZ g j8-1,4-glucan, and most preferably about 117 UZ j8-1,4-glucan.
- the weight of j8-1,4-glucan phosphorylase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself may occur, but the glucan yield may decrease.
- the ⁇ -1,4-glucan phosphorylase may be purified or unpurified. ⁇ -1,4-glucan phosphorylase may or may not be immobilized. ⁇ -1,4-glucan phosphorylase is preferably immobilized.
- the method of immobilization may be a method known to those skilled in the art, such as a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a crosslinking method or an entrapping method (lattice type or microcapsule type). A method can be used.
- the ⁇ -1,4-glucan phosphorylase is preferably immobilized on a carrier.
- ⁇ -1,4-glucan phosphorylase (EC: 2.4.1.1) is ⁇ -1,4-glucan (polymerization Ex-1 and 4-glucan (degree of polymerization n1) and ⁇ -D glucose
- Glucan phosphorylase is a reverse reaction of phosphorolysis. ⁇ -1,4-glucan (degree of polymerization ⁇ -1) and ⁇ -D-glucose monophosphate are converted to ⁇ -1,4-glucan (degree of polymerization ⁇ ). Can also catalyze the reaction to synthesize The direction in which the reaction proceeds depends on the amount of substrate. In vivo, the amount of inorganic phosphate is large, so that glucan phosphorylase reacts in the direction of phosphorolysis. In the method of the present invention, the inorganic phosphoric acid is used for the decomposition of j8-l, 4-glucan by carophosphoric acid. Advances.
- ⁇ -1,4-glucan phosphorylase is considered to be universally present in various plants, animals and microorganisms that can store starch or glycogen.
- Examples of plants that produce ⁇ -1,4-glucan phosphorylase include algae, potatoes (also called potatoes), sweet potatoes (also called sweet potatoes), potatoes such as potatoes, taros, and cassava, cabbage, spinach, and the like. And cereals such as corn, rice, wheat, oats, rye, algae, and beans such as peas, soybeans, red beans, and quail beans.
- Examples of animals that produce ⁇ -1,4-glucan phosphorylase include mammals such as humans, egrets, rats, and pigs.
- Examples of the microorganisms that produce ⁇ -1,4-glucan phosphorylase include Thermus aqu aticus, Bacillus stearothermophilus, Demococcus raaioaurans ⁇ Tnermo coccus litoralis, Streptomyces coelicolor, Pyrococcus horikoshi ⁇ Myco bacterium tubercula e ausicato thermotous Meth anococcus Jannaschii, Pseudomonas aeruginosa ⁇ Chlamydia pneumoni ae, Chlorella vulgaris ⁇ Agrobacterium tumefaciens, Clostridium pasteu rianum, Klebsiella pneumoniae ⁇ Synecococcus sp., Synechocystis sp.
- Organisms producing ⁇ -1,4-glucan phosphorylase are not limited to these.
- the 4-glucan phosphorylase used in the present invention is derived from potato, Thermus a quaticus, and Bacillus stearothermophilus; more preferably, it is more preferably derived from Nya 7imo.
- the 4-glucan phosphorylase used in the present invention preferably has a high optimal reaction temperature.
- the 4-glucanphosphorylase having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- the ⁇ -1,4-glucan phosphorylase used in the present invention can be isolated directly from animals, plants, and microorganisms that exist in nature and produce 4-glucan phosphorylase as described above.
- the 4-glucan phosphorylase used in the present invention is a microorganism, such as a bacterium or a fungus, which has been genetically modified using a gene encoding 4-glucan phosphorylase isolated from these animals, plants or microorganisms. ).
- the ⁇ -1,4-glucan phosphorylase can be obtained from a genetically modified microorganism in the same manner as the above-mentioned 13-1,4-gnolecan phosphorylase.
- microorganism used in the genetic recombination e.g., bacteria, fungi etc.
- beta-1, 4 Dal cans phosphorylase ease of expression of the hydrolase, easy culture It can be easily selected in consideration of various conditions such as growth speed and safety.
- 4-glucan phosphorylase does not contain amylase as a contaminant, so that a gene (eg, a bacterium or a fungus) that does not produce amylase or has a low level and does not express force is genetically engineered. It is preferably used instead.
- ⁇ -1,4-glucanphosphorylase For the genetic modification of ⁇ -1,4-glucanphosphorylase, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis. 4-glucan phosphorylase produced using microorganisms (eg, bacteria, fungi, etc.) that do not produce amylase or express it at low levels is substantially free of amylase. Preferred for use in the method of the invention.
- the amount of ⁇ -1,4-glucan phosphorylase contained in the solution at the start of the reaction was determined by comparing the amount of j8-1,4-gnolecan in the solution at the start of the reaction.
- 0.05-1, 000 U / g ⁇ -1,4-glucan preferably about 0.1 l-500 U / g ⁇ -1,4-glucan, more preferably Is about 0.5-lOOUZg j8-1,4-glucan, particularly preferably about 110-80 UZg ⁇ -1,4-glucan, and most preferably about 10-50 UZg ⁇ -1,4-glucan.
- the weight of ⁇ -1,4-glucan phosphorylase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself may occur, but the glucan yield may decrease.
- the 4-glucan phosphorylase may be purified or unpurified.
- the 1,4-glucan phosphorylase may or may not be immobilized.
- ⁇ -1,4-glucan phosphorylase is immobilized.
- the method of immobilization may be a method known to those skilled in the art, such as a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a crosslinking method or an entrapping method (lattice type or microcapsule type).
- a method can be used.
- ⁇ -1,4-glucan phosphorylase is preferably immobilized on a carrier.
- ⁇ -1,4-glucan phosphorylase may be immobilized on the same carrier as 13-1,4-glucan phosphorylase, or immobilized on another carrier. V ,. It is preferable that they are fixed on the same carrier.
- the solution preferably further contains glucose isomerase.
- glucose isomerase By including glucose isomerase in the solution, glucose produced by the phosphorolysis of cellobiose can be converted to fructose. Glucose inhibits the power of cellobiose!] Since it inhibits the reaction in the direction of phosphorolysis, the inclusion of glucose isomerase in the solution can further accelerate the carophosphate decomposition of cellobiose, which is finally obtained ⁇ -Dalkan yield can be improved.
- the glucose isomerase that can be used in the production method of the present invention is an enzyme that can catalyze the interconversion between D-glucose and D-fructose.
- Glucose isomerase is called xylose isomerase because it can also catalyze the interconversion of D-xylose and D-xylulose.
- Glucose isomerase is present in microorganisms, animals and plants.
- microorganisms that produce glucose isomerase include Streptomyces rubiginosus, Streptomyces olivochromogenes, Streptomyces murmus, and Streptomvces violaceo.
- Lactobacillus brevis Lactobacillus brevis, Lactobacillus xylosus, Agrobacterium tumefaciens, Bacillus sp., Actinoplanes missouriensis and Paracolobacterium aerogenoides.
- animals producing glucose isomerase include Trypanosoma brucei.
- Glucose isomerase may be of plant origin. Organisms that produce glucose isomerase are not limited to these.
- the glucose isomerase that can be used in the present invention is preferably derived from Streptomyces rubiginosus or Bacillus sp., And more preferably derived from streptomyces rubiginosus.
- the glucose isomerase used in the present invention preferably has a high optimal reaction temperature.
- Glucose isomerase having a high optimal reaction temperature can be derived from, for example, a highly thermophilic bacterium.
- glucose isomerase that can be used in the present invention can be isolated directly from the above-mentioned naturally occurring organisms that produce glucose isomerase.
- the glucose isomerase that can be used in the present invention may also be isolated from microorganisms (eg, bacteria, fungi, etc.) that have been genetically modified using these biologically isolated genes encoding dalcos isomerase.
- microorganisms eg, bacteria, fungi, etc.
- Glucose isomerase can be obtained from a genetically modified microorganism, as in the case of the above 13-1,4-glucan phosphorylase.
- Microorganisms used for genetic recombination can be easily expressed with glucose isomerase, easily cultivated, and rapidly grown, similarly to the aforementioned ⁇ -1,4 dalcan phosphorylase. , Can be easily selected in consideration of various conditions such as safety. Since glucose isomerase preferably does not contain amylase as a contaminant, it is preferable to use a microorganism (eg, a bacterium or a fungus) that does not produce amylase or has a low level and does not express force, for genetic recombination.
- a microorganism eg, a bacterium or a fungus
- glucose isomerase For this purpose, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis.
- Glucose isomerase produced using a microorganism that does not produce amylase or has low levels and does not express force (eg, bacteria, fungi, etc.) is substantially free of amylase and is therefore used in the method of the present invention. Preferred to,.
- the amount of glucose isomerase contained in the solution at the start of the reaction was determined by comparing the amount of j8-1,4-gnolecan in the solution at the start of the reaction [typically about 0.01 U to 500 U / g ⁇ -1,4-gnolecan, preferably about 0.05 to 100 U / g ⁇ -1,4-gnolecan, more preferably about 0.1 -50 U / g j8-1,4-glucan Particularly preferred is about 0.5-lOUZg ⁇ -1,4-glucan, most preferably about 11-5 UZg ⁇ -1,4-glucan. If the weight of glucose isomerase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the yield of glucan may decrease.
- Glucose isomerase may be purified! Or unpurified! / ⁇ .
- the glucose isomerase may or may not be fixed.
- the glucose isomerase is preferably immobilized.
- the method of immobilization is well known to those skilled in the art, such as a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a cross-linking method or an entrapping method (lattice type or microcapsule type). Can be used.
- the glucose isomerase is preferably immobilized on a carrier.
- Glucose isomerase may also be immobilized on at least one of the ⁇ -1,4-glucan phosphorylase and ⁇ 1,4-glucan phosphorylase on the same carrier, or may be immobilized on another carrier. It may be done. It is preferable that both ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase are immobilized on the same carrier.
- the solution further contains glucose oxidase.
- glucose oxidase By containing glucose oxidase in the reaction solution, ⁇ -glucose naturally converted from ⁇ -glucose generated by the phosphorolysis of cellobiose can be converted to ⁇ -glucose. It can be converted to Luconau ⁇ rataton. Since ⁇ -glucose inhibits the reaction of cellobiose in the direction of carophosphate decomposition, the inclusion of glucose oxidase in the solution can further accelerate the cellophosphate decomposition of cellobiose, and the ⁇ -glucose finally obtained can be obtained. The yield of glucan can be improved.
- Glucose oxidase that can be used in the production method of the present invention is an enzyme that can catalyze the following reaction:
- Glucose oxidase is present in microorganisms and plants. Rows f of microorganisms that produce glucose oxidase include Aspergillus niger, Penicillium amagasaki ense, Penicillium notatum and Phanerochaete chrysosporium. Glucose oxidase may be of plant origin. Organisms that produce glucose oxidase are not limited to these!
- the glucose oxidase that can be used in the present invention is derived from Aspergillus niger or Penicillium amagasakiense; 0 is preferred, and more preferably is derived from Aspergillus niger.
- the glucose oxidase used in the present invention preferably has a high optimal reaction temperature. Glucose oxidase having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- Glucose oxidase that can be used in the present invention can be directly isolated from an organism that produces glucose oxidase that exists in nature as described above.
- Glucose oxidase that can be used in the present invention also isolates microorganisms (for example, bacteria, fungi, etc.) that have been genetically modified using a gene encoding glucosoxidase isolated from these organisms. May be.
- microorganisms for example, bacteria, fungi, etc.
- Glucose oxidase can be obtained from a genetically modified microorganism in the same manner as the above-mentioned ⁇ -1,4-glucan phosphorylase.
- Microorganisms used for genetic recombination can be easily expressed in glucose oxidase and easily cultured in the same manner as in the aforementioned ⁇ -1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as growth speed and safety. Since the darcosoxidase preferably does not contain amylase as a contaminant, it is preferable to use a microorganism (eg, a bacterium, a fungus, etc.) that does not produce amylase or has a low level and does not express force, for gene recombination. .
- a microorganism eg, a bacterium, a fungus, etc.
- a mesophilic bacterium such as Escherichia coli or Bacillus subtilis for the genetic recombination of glucose oxidase.
- Glucose oxidase produced using microorganisms that do not produce or produce low levels of amylase is substantially free of amylase, and thus is not suitable for use in the methods of the present invention. Preferred,.
- the amount of glucose oxidase contained in the solution at the start of the reaction is typically about 0.5-1, 1, OOOU /, relative to the amount of j8-1,4-gnorecan in the solution at the start of the reaction.
- g ⁇ -1,4 glucan preferably about 1 to 500 UZg ⁇ -1,4-glucan, more preferably about 5 to 400 U / g j8-1,4-glucan, and particularly preferably about 10 to 300 UZg j8 — 1,4-glucan, most preferably about 20-200 UZg ⁇ -1,4-glucan. If the weight of glucose oxidase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the glucan yield may decrease.
- Glucose oxidase may be purified! Or unpurified. Glucose oxidase may or may not be immobilized. Glucose oxidase is preferably immobilized. As methods for immobilization, methods known to those skilled in the art, such as a carrier binding method (for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method), a crosslinking method or an entrapping method (lattice type or microcapsule type), etc. Can be used. Glucose oxidase is preferably immobilized on a carrier.
- a carrier binding method for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method
- a crosslinking method or an entrapping method laattice type or microcapsule type
- Glucose oxidase may be immobilized on the same carrier as at least one of j8-1,4-glucan phosphorylase and ⁇ -1,4-glucan phosphorylase, or immobilized on another carrier. You may be josuled. It is preferable that both j8-l, 4-glucan phosphorylase and 4-gunolecan phosphorylase are immobilized on the same carrier. [0143] (8. Mutalotase)
- mutarotase is further contained in the solution.
- a-glucose and 13-glucose generated by phosphorolysis of cellobiose can be interconverted.
- OC-glucose and ⁇ -glucose are naturally interconverted without the addition of mutarotase, the interconversion is promoted by the removal of mutarotase, so the ⁇ -glucose generated by the reaction also reduces the solution power. Efficiency can be further improved.
- the concentration of ⁇ -glucose in the reaction solution can be decreased, and as a result, carophosphoric acid decomposition of cellobiose can be further promoted.
- the yield of the obtained ⁇ -glucan can be improved.
- the mutarotase that can be used in the production method of the present invention is an enzyme that can catalyze the interconversion between a-glucose and 13-glucose.
- Mutarotase is present in microorganisms, animals and plants.
- microorganisms that produce mutarotase include Penicillium notatum and Escherichia coli.
- animals producing mutarotase include pigs and Bos taurus.
- plants producing mutarotase include Capsicum frutescens.
- Organisms producing mutarotase are not limited to these.
- the mutarotase that can be used in the present invention is preferably derived from pig or Bos taurus, and more preferably derived from pig.
- the mutarotase used in the present invention preferably has a high optimal reaction temperature.
- a mutarotase having a high optimal reaction temperature can be derived, for example, from a highly thermophilic bacterium.
- the mutarotase that can be used in the present invention can be directly isolated from a naturally occurring mutarotase-producing organism as described above.
- the mutarotase that can be used in the present invention may be isolated from a microorganism (for example, a bacterium or a fungus) that has been genetically modified using a gene encoding mutarotase isolated from these organisms. .
- a microorganism for example, a bacterium or a fungus
- Mutarotase is a recombinant gene similar to the above-mentioned ⁇ -1,4-glucan phosphorylase. Obtained from the isolated microorganism.
- Microorganisms used for genetic recombination can be easily expressed with mutarotase, easily cultivated, and rapidly grown, similarly to the aforementioned ⁇ -1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as safety. Mutarotase preferably does not contain amylase as a contaminant, so do not produce amylase! Microorganisms (eg, bacteria, fungi, etc.) that have low or low levels of expression are preferably used for gene recombination.
- mutarotase For the gene recombination of mutarotase, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis. Mutarotase produced using a microorganism that does not produce amylase or that expresses only low levels (eg, bacteria, fungi, etc.) is substantially free of amylase, and therefore is not suitable for use in the methods of the present invention. preferable.
- the amount of mutarotase contained in the solution at the start of the reaction is determined by comparing the amount of ⁇ 1,4-gnolecan in the solution at the start of the reaction [typically about 0.01 U to 500 U / g. ⁇ -1,4-gnolecan, preferably about 0.1 Ol-lOUOU / g ⁇ -1,4-glucan, more preferably about 0.01-1-50UZ g j8-1,4-glucan, particularly preferably It is about 0.05-lOUZg j8-1,4-glucan, most preferably about 0.1-5U / g ⁇ -1,4-glucan. If the weight of mutarotase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the glucan yield may decrease.
- Mutarotase may be purified or unpurified! /.
- the mutarotase may or may not be immobilized.
- the mutarotase is preferably immobilized.
- Examples of the method for immobilization include a carrier binding method (for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method), a crosslinking method or an entrapping method (lattice type or microcapsule type), which are well known to those skilled in the art.
- a method can be used. It is preferable that mutarotase is immobilized on a carrier.
- Mutarotase may be immobilized on the same carrier as at least one of ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase, or may be immobilized on another carrier. It is preferable that both ⁇ -1,4-glucan phosphorylase and ⁇ -1,4-glucan phosphorylase are immobilized on the same carrier. [0154] (9. Catalase and peroxidase)
- the solution when glucose oxidase is contained in the solution, it is preferable that the solution further contains catalase or peroxidase.
- catalase or peroxidase By including catalase or peroxidase in the solution, hydrogen peroxide generated by the reaction catalyzed by glucose oxidase can be converted to oxygen and oxygen can be recycled. Therefore, by including glucose oxidase and catalase or peroxidase in the reaction solution, the concentration of glucose in the reaction solution can be reduced, and as a result, carophosphoric acid decomposition of mouth bioose can be further promoted. The yield of ⁇ -dalcan finally obtained can be improved.
- Catalase that can be used in the production method of the present invention is an enzyme that catalyzes a reaction to decompose hydrogen peroxide into oxygen and water.
- Catalase is present in microorganisms, animals and plants. Acetobacter peroxydans, Acholeplasma equifetale, Achole plasma hippikon, Acholeplasma laidlawu, Aspergillus niger, Penicillium janthinellum, Halobacterium halobium, Haloarcula marismortui, Esch erichia coli, Mycoplasma arthral smegmatis, Mycobacterium tuberculosis ⁇ Mycoplasma pulmon is, Mycoplasma sp., Bacillus stearothermophilus ⁇ Rhodobacter sphaer oides, Lactobacillus plantarum, Thermoleophilum album ⁇ Phanerochaet e chrysosporium, Saccharomyces cerevisiae, Keramona, and saccharomyces cerevisiae, and acidaeosa sera No.
- Examples of animals that produce catalase include Capra aeg agrus nircus, Bos taurus, Homo sapiens ⁇ Rattus norvegicus and Noto mastus lobatus (polychaete).
- Examples of plants that produce catalase include Gossypium hirsutum, Smapis aloa, 3 ⁇ 4pmacia oleracea, Nicotiana tabacum L., Nicotiana sylvestris, Euglena gracilis (algae) and Pisum sativum.
- Organisms producing catalase are not limited to these.
- Catalase that can be used in the present invention is preferably derived from Aspergillus niger, Bovine Liver (bovine liver) or Human Erythrocyte (human erythrocyte), and more preferably derived from Aspergillus niger.
- the catalase used in the present invention preferably has a high optimal reaction temperature.
- Catalase having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- Peroxidase that can be used in the production method of the present invention is an enzyme that catalyzes the oxidation of various organic substances using hydrogen peroxide as a hydrogen acceptor.
- Peroxidase is present in microorganisms, animals and plants.
- microorganisms that produce peroxidase include Pleurotus ostreatus, Halobacterium halobium, Haloarcula marismortui, Oprmus friesu, Phanerochaete chrysosporium, Mycobacterium smegmatis, Mycobacterium tuberculosis ⁇ Illum, Arthritis, Artichoke, Arthritis Kloeckera sp., Bacillus s tearothermophilus, Coprinus cinereus and Coprinus macrorhizus power S.
- microorganisms include bacteria and fungi.
- Examples of animals that produce peroxidase include Homo sapiens, Cards familiaris, Rattus no rvegicus, Sus scrofa, and Ovis aries.
- the f-row of plants that produce ⁇ norreoxidase include horseradish (horseradish), Armoracia rusticana, Armoraci a lapathifolia, Actmiaia chmensis, Citrus sinensis ⁇ Populus trichocarpa, Nicotiana sylvestris, Picea sitchensis Carr., Picea abies L.
- Organisms that produce peroxidase are not limited to these.
- the peroxidase which can be used in the present invention is preferably derived from horseradish and Bacillus stearot hermophilus, more preferably from horseradish.
- the peroxidase used in the present invention preferably has a high optimal reaction temperature.
- Peroxidase having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- the catalase or peroxidase that can be used in the present invention includes the natural The biological power to produce catalase or peroxidase, present in E. coli, can also be isolated directly.
- Catalase or peroxidase that can be used in the present invention is obtained by isolating the power of a microorganism (for example, a bacterium or a fungus) that has been genetically modified using a gene encoding catalase or peroxidase isolated from these organisms. Is also good.
- a microorganism for example, a bacterium or a fungus
- Catalase or peroxidase can be obtained from a genetically modified microorganism in the same manner as in the above-mentioned 131,4-glucan phosphorylase.
- Microorganisms used for genetic recombination can be easily expressed with catalase or peroxidase, easily cultivated, and grown at a high speed, similarly to the aforementioned ⁇ 1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as safety.
- Catalase or peroxidase preferably does not contain amylase as a contaminant, so that microorganisms (eg, bacteria, fungi, etc.) that do not produce amylase or do not express at low levels are gene-modified. Is preferably used.
- catalase or peroxidase For the gene recombination of catalase or peroxidase, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis. Catalase or peroxidase produced using microorganisms (eg, bacteria, fungi, etc.) that do not produce amylase or express at low levels is substantially free of amylase, and Preferred for use in the method described above.
- microorganisms eg, bacteria, fungi, etc.
- the amount of catalase or peroxidase contained in the solution at the start of the reaction is typically about 0.05-1, OOOU /, relative to j8-1,4-glucan in the solution at the start of the reaction.
- g j8-1,4-glucan preferably about 0.1-500 UZg j8-1,4-glucan, more preferably about 1.0-200 U / g ⁇ -1,4-glucan. If the weight of catalase or peroxidase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the glucan yield may decrease.
- Catalase or peroxidase may be purified or unpurified. Power tarase or peroxidase may be immobilized or non-immobilized. Yes. Catalase or peroxidase is preferably immobilized.
- the method of immobilization may be a method known to those skilled in the art, such as a carrier binding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a cross-linking method or an entrapment method (lattice type or microcapsule type). Can be used.
- Catalase or peroxidase is preferably immobilized on a carrier.
- Catalase or peroxidase may also be immobilized on at least one of j8-1,4 dalcan phosphorylase and 1,4-glucan phosphorylase, or immobilized on another carrier. May be. It is preferable that both ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase are immobilized on the same carrier.
- a debranching enzyme when the product is branched, such as when a starting material containing an ⁇ -1,6 darcoside bond is used, a debranching enzyme can be used as necessary.
- a debranching enzyme that can be used in the present invention is an enzyme that can cleave ⁇ -1,6 darcoside bonds.
- Debranching enzymes are isoamylase (EC 3.2.1.68), which works well on both amylopectin and glycogen, and at-dextrin end, which works on amylopectin, glycogen and pullulan—1,6-a Dalcosidase (also with pullulanase! /) (EC 3.2.1.41).
- Debranching enzymes are present in microorganisms and plants.
- microorganisms that produce a debranching enzyme include Saccharomyces cerevisiae, Chlamyaomonas sp., Bacillus brev is, Bacillus acidopullulyticus, Bacillus macerans, Bacillus stearothermo philus, Bacillus circulans, Thermus aquaticus, Kerbonias erum, Hermoniatus, Kleboniapus erumer, Hermonia espermum eudomonas amyloderamosa and the like.
- plants that produce a debranching enzyme include potato, sweet potato, corn, rice, wheat, oats, oats, sugar beet, and the like.
- Organisms that produce debranching enzymes are not limited to these
- Branching enzymes that can be used in the present invention include Klebsiella pneumoniae, Bacillus brevi s, Bacillus acidopullulyticus, Pseudomonas amyloderamosa, and more preferably Klebsiella pneumoniae ⁇ Pseudomonas amyloderamosa.
- the debranching enzyme used in the present invention preferably has a high optimal reaction temperature.
- a debranching enzyme having a high optimal reaction temperature can be derived, for example, from a highly thermophilic bacterium.
- the debranching enzyme that can be used in the present invention can be directly isolated from microorganisms and plants that exist in nature and produce the debranching enzyme as described above.
- the debranching enzyme that can be used in the present invention can be obtained from these microorganisms and microorganisms (for example, bacteria, fungi, etc.) that have been genetically modified using a gene encoding a plant-derived isolated debranching enzyme. May be separated.
- the debranching enzyme can be obtained from a genetically modified microorganism, as in the case of j8-1,4-glucan phosphorylase described above.
- Microorganisms used for genetic recombination can be easily expressed in a debranching enzyme, easily cultured, and rapidly grown, similarly to the aforementioned ⁇ -1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as safety.
- the debranching enzyme preferably does not contain amylase as a contaminant, so do not produce amylase!
- Microorganisms eg, bacteria, fungi, etc.
- that have low or low levels of expression are preferably used for gene recombination.
- a mesophilic bacterium such as Escherichia coli or Bacillus subtilis.
- Branching enzymes produced using microorganisms that do not produce amylase or that express it only at low levels e.g., bacteria, fungi, etc.
- the amount of the debranching enzyme contained in the solution at the start of the reaction was ⁇ 1,4-gnolecan in the solution at the start of the reaction [in contrast to a typical amount of about 0.05 to 1,000 U / g ⁇ -1,4-gnolecan, preferably about 0.1-500 U / g ⁇ -1,4-glucan, more preferably about 0.5-100 UZ g ⁇ -1,4-glucan. If the weight of the debranching enzyme is too large, the denatured enzyme may easily aggregate during the reaction. If the amount used is too small, the reaction itself will occur, , The yield of glucan may decrease.
- the debranching enzyme may be purified or unpurified.
- the debranching enzyme may or may not be immobilized.
- the debranching enzyme is preferably immobilized.
- Methods of immobilization include well-known methods to those skilled in the art, such as a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a crosslinking method, or an entrapping method (lattice type or microcapsule type). Can be used.
- the debranching enzyme is preferably immobilized on a carrier.
- the debranching enzyme may be immobilized on the same carrier as at least one of ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase, or may be immobilized on another carrier. It is preferable that both ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase are immobilized on the same carrier.
- a branching enzyme can be used if necessary.
- the branching enzyme which can be used in the present invention is obtained by adding a part of the ⁇ -1,4-glucan chain to the 6-position of a certain glucose residue in the D a-1,4-glucan chain.
- An enzyme that can be transferred to form a branch.Branchingenzyme is also called 1,4 ⁇ -glucan branching enzyme, branching enzyme or Q enzyme.
- Branchengensim is present in microorganisms, animals, and plants.
- microorganisms that produce branching enzymes include Bacillus stearothermophilus, Bacillus subtilis, Bacillus caldolyticus, Bacillus licneniformis, Bacillus amyloi iquefaciens, Bacillus coagulans, Bacillus caldovelox, Bacillus thermocausacus, Bacillus thermocaususus, Bacillus thermocausacusus sp., Streptomyces coelicolor, Aquifex aeolicus, Syne chosystis sp., E.
- branchesengenzyme examples include mammals such as humans, egrets, rats, and pigs.
- animals that produce blanchen genzam include algae, potatoes such as potatoes, sweet potatoes, yams, cassavas, potatoes such as spinach, and vegetables such as spinach.
- Grains such as sorghum, rice, wheat, oats, rye, algae, and beans such as peas, soybeans, red beans, and quail beans.
- Organisms that produce branchengenzyme are not limited to these.
- the branching genzyme that can be used in the present invention is preferably derived from potato, Bacillus stearoth ermophilus, Aquifex aeolicus, and more preferably derived from Bacillus stearother mophilus, Aquifex aeolicus.
- the branching enzyme used in the present invention preferably has a high optimum reaction temperature. Branching genzym having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- Branching genzym that can be used in the present invention can be directly isolated from microorganisms, animals, and plants that exist in nature and produce branching genzym as described above.
- Branching genzymes that can be used in the present invention include microorganisms that have been genetically modified using genes encoding branching genzymes isolated from these microorganisms, animals, and plants (for example, , Bacteria, fungi, etc.).
- branching genzyme can be obtained from a genetically modified microorganism.
- Microorganisms eg, bacteria, fungi, etc.
- used for genetic recombination can be easily expressed in branching genzym and easily cultured, similarly to the aforementioned ⁇ -1,4 dalcan phosphorylase.
- branching genzyme preferably does not contain amylase as a contaminant, it is preferable to use a microorganism (for example, a bacterium or a fungus) that does not produce amylase or has a low level and does not express force, for genetic recombination. It is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis for the genetic recombination of branchebenzyme.
- a microorganism for example, a bacterium or a fungus
- mesophilic bacterium such as Escherichia coli or Bacillus subtilis
- Branching genzymes produced using microorganisms that do not produce amylase or produce low levels and that do not express force eg, bacteria, fungi, etc.
- branching genzymes produced using microorganisms that do not produce amylase or produce low levels and that do not express force are substantially free of amylase, and therefore are not subject to the present invention. Preferred for use in the method.
- branching genzyme by genetic recombination can be performed in the same manner as in the above-mentioned ⁇ -1,4-glucan phosphorylase.
- the amount of branching genzyme contained in the solution at the start of the reaction is typically about 10-100, OOOU, relative to the amount of j8-1,4-gnolecan in the solution at the start of the reaction.
- / g ⁇ -1,4 glucan preferably about 100-50,000 U / g ⁇ -1,4-glucan, more preferably about 1,000-10,000 U / g ⁇ -1,4-glucan. If the weight of the branching enzyme is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the glucan yield may decrease.
- Branching enzym may be refined or unrefined! / ⁇ .
- the branching algorithm may or may not be fixed.
- the branching enzyme is fixed.
- methods for immobilization methods known to those skilled in the art, such as a carrier binding method (for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method), a crosslinking method or an entrapping method (lattice type or microcapsule type), etc. Can be used. It is preferable that the branching enzyme is fixed on the carrier.
- the branching enzym may also be immobilized on at least one of the same carriers as j8-l, 4-glucan phosphorylase and 4-glucan phosphorylase, or immobilized on another carrier. It may be done. It is preferable that both the -1,4-glucan phosphorylase and the 4-gunolecan phosphorylase are immobilized on the same carrier.
- 4-a dulkanotransferase when a cyclic structure is to be formed in the product, 4-a dulkanotransferase can be used as necessary.
- the 4-a-glucanotransferase that can be used in the present invention is also called disproportionate onetingenzyme, D enzyme, amyloid maltase, disproportionating enzyme, and the like. It is an enzyme that can catalyze a transfer reaction (heterogeneization reaction).
- 4-a-glucanotransferase is an enzyme that transfers a dalcosyl group or maltosyl or maltooligosyl unit from the non-reducing end of a donor molecule to the non-reducing end of an acceptor molecule.
- the enzymatic reaction leads to a heterogeneity in the degree of polymerization of the maltooligosaccharides initially given. If the donor molecule and the acceptor molecule are the same, an intramolecular rearrangement occurs, resulting in a product having a cyclic structure.
- 4-a dulcanotransferase is present in microorganisms and plants.
- plants that produce 4- ⁇ -glucanotransferase include potatoes such as potatoes, sweet potatoes, potatoes, cassava, cereals such as corn, rice and wheat, and beans such as peas and soybeans.
- potatoes such as potatoes, sweet potatoes, potatoes, cassava, cereals such as corn, rice and wheat, and beans such as peas and soybeans.
- Can be An organism that produces 4 ⁇ -dalcanotransferase is not limited to these.
- the 4-a-glucanotransferase that can be used in the present invention is preferably derived from potato, Thermus aquaticus, or Tnermococcus litralis; more preferably, derived from potato, Thermus aquaticus, or Thermus aquaticus. .
- the 4- ⁇ -glucanotransferase used in the present invention preferably has a high optimal reaction temperature.
- 4- ⁇ -Dalkanotransferase having a high optimal reaction temperature can be derived, for example, from highly thermophilic bacteria.
- the 4 ⁇ -dalcanotransferase that can be used in the present invention can be directly isolated from microorganisms and plants that exist in nature and produce 4a-dalcanotransferase as described above.
- the 4 ⁇ -dalcanotransferase that can be used in the present invention is a microorganism (for example, a bacterium, a bacterium, or the like) that has been genetically modified using a gene encoding a 4a-dalcanotransferase isolated from these microorganisms and plants. (Eg, fungi) may also be isolated.
- the 4- ⁇ -glucanotransferase can be obtained from a genetically modified microorganism in the same manner as the above 13-1,4-glucan phosphorylase.
- Microorganisms for example, bacteria, fungi, etc.
- Microorganisms used for genetic recombination can be easily expressed and cultured easily, as in the above-mentioned ⁇ -1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as growth speed and safety. It is preferable that the 4 ⁇ -dalcanotransferase does not contain amylase as a contaminant. Therefore, microorganisms that do not produce amylase or express at a low level and do not express force (e.g., For example, bacteria, fungi, etc.) are preferably used for gene recombination.
- the amount of 4a-darcanotransferase contained in the solution at the start of the reaction was determined to be j8-1,4-gnolecan [typically about 0.05 -1, OOOU / g ⁇ -1,4-glucan, preferably about 0.1 l-500U / g ⁇ -1,4-glucan, more preferably about 0.5-100U / g ⁇ -1,4-glucan is there. If the weight of 4- ⁇ -glucanotransferase is too large, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself occurs, but the glucan yield may decrease.
- the 4a darcanotransferase may be purified! Or unpurified.
- the 4a dalcanotransferase may or may not be immobilized.
- the 4 ⁇ -dalcanotransferase is preferably immobilized.
- Examples of the method of immobilization include a carrier binding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a bridging method or an entrapping method (lattice type or microcapsule type), which are well known to those skilled in the art. A method can be used. It is preferable that the 4 ⁇ -dalcanotransferase is immobilized on a carrier.
- the 4- ⁇ -dalcanotransferase may be immobilized on the same carrier as at least one of 13-1,4-glucan phosphorylase and ⁇ -1,4-glucan phosphorylase, or may be a different carrier. It may be immobilized on the top. It is preferable that both ⁇ -1,4-glucan phosphorylase and ⁇ -1,4-glucan phosphorylase are immobilized on the same carrier.
- Glycogen debranching genzaim that can be used in the present invention is an enzyme having two kinds of activities, 6-dalcosidase activity and 4a-dalcanotransferase activity. Glycogen debranchingenzyme's 4 ⁇ -dalcanotransferase activity gives a product with a cyclic structure.
- Glycogen debranchingenzymes are present in microorganisms and animals.
- microorganisms that produce glycogen debranching genzym include yeast and the like.
- animals that produce glycogen debranching genzym include mammals such as humans, egrets, rats, and pigs.
- Organisms that produce glycogen debranching genzaim are not limited to these.
- the glycogen debranching genzaim that can be used in the present invention is preferably derived from yeast.
- the glycogen debranching enzyme used in the present invention preferably has a high optimal reaction temperature.
- Glycogen debranching enzymes having a high optimum reaction temperature can be obtained, for example, by a protein engineering technique by excluding the modification of an enzyme that can act at a medium temperature.
- Glycogen debranching genzyme that can be used in the present invention can be directly isolated from microorganisms and animal creatures that produce glycogen debranching genzyme that exist in nature as described above.
- Glycogen debranching genzym that can be used in the present invention is a microorganism (for example, a microorganism that has been genetically modified using an isolated gene encoding glycogen debranching genzyme). , Bacteria, fungi, etc.) may also be isolated.
- Glycogen debranchingenzyme can be obtained from a genetically modified microorganism in the same manner as in the above-mentioned 13-1,4-glucan phosphorylase.
- Microorganisms used for genetic recombination can be easily expressed with glycogen debranched genzyme, easily cultured, as in the case of the above ⁇ 1,4 dalcan phosphorylase. It can be easily selected in consideration of various conditions such as growth speed and safety. Glycogen debranching genzyme preferably does not contain amylase as a contaminant, and therefore does not produce amylase or is expressed only at low levels. Microorganisms (eg, bacteria, fungi, etc.) ) Is preferably used for gene recombination.
- glycogen It is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis for zym gene recombination.
- Glycogen debranchingenzymes produced using microorganisms that do not produce amylase or that do not express at low levels are substantially free of amylase, and therefore, the method of the present invention. Preferred for use in
- the amount of glycogen debranched genzyme contained in the solution at the start of the reaction is typically about 0.01 with respect to j8-1,4-glucan in the solution at the start of the reaction. -5, OOOU / g ⁇ -1,4-glucan, preferably about 0.1-1, OOOU / g ⁇ -1,4-glucan, more preferably about 500 UZg ⁇ -1,4-glucan . If the glycogen debranching enzyme is too heavy, the denatured enzyme may easily aggregate during the reaction. If the amount is too small, the reaction itself may occur, but the glucan yield may decrease.
- Glycogen debranching enzym may be purified or unpurified.
- the glycogen debranching gene may or may not be fixed. It is preferable that the glycogen debranching enzyme is fixed.
- Methods of immobilization include well-known methods to those skilled in the art, such as a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a crosslinking method, or an entrapping method (lattice type or microcapsule type). Can be used. It is preferable that the glycogen debranching enzyme is fixed on the carrier.
- Glycogen debranching genzyme may also be immobilized on the same carrier as at least one of j8-1,4-glucan phosphorylase and ⁇ -1,4-glucan phosphorylase, or It may be fixed on a carrier.
- U preferably immobilized on the same carrier as both j8-l, 4-glucan phosphorylase and 4-glucan phosphorylase.
- the solvent used in the method of the present invention can be any solvent as long as it does not impair the enzyme activity of ⁇ -1,4-glucan phosphorylase and 4-glucan phosphorylase.
- the solvent does not need to completely dissolve the material used in the method of the present invention as long as the reaction for producing glucan can proceed.
- the enzyme when the enzyme is supported on a solid carrier, the enzyme need not be dissolved in the solvent. Further, it is not necessary that all the reaction materials such as / 3-1,4-glucan be dissolved, and it is sufficient that some of the materials are dissolved to such an extent that the reaction can proceed.
- a representative solvent is water.
- the solvent used in the preparation of j8-1,4-glucan phosphorylase or ⁇ -1,4-glucan phosphorylase is the cell disruption obtained with
- the water may be any of soft water, intermediate water and hard water.
- Hard water refers to water with a hardness of 20 ° or more
- intermediate water refers to water with a hardness of 10 ° or more and less than 20 °
- soft water refers to water with a hardness of less than 10 °.
- the water is preferably soft or intermediate water, more preferably softened.
- ⁇ -1,4-glucan In a solution containing ⁇ -1,4-glucan, primer, inorganic phosphate or glucose-1-phosphate, ⁇ -1,4-glucan phosphorylase and oi-1,4-glucan phosphorylase, j8-l, 4 Any other substance may be included as long as it does not interfere with the interaction between -glucan phosphorylase and j8-l, 4-glucan and the interaction between ⁇ -1,4-glucan phosphorylase and the primer.
- Such substances include buffers, components of microorganisms that produce ⁇ -1,4-glucan phosphorylase (eg, bacteria, fungi, etc.), microorganisms that produce ⁇ -1,4 dalcan phosphorylase (eg, bacteria , Fungi, etc.), salts, medium components and the like.
- the ⁇ -glucan of the present invention can be obtained by reacting a solution containing ⁇ -1,4-glucan, a primer, an inorganic phosphate or dalcose-monophosphate, ⁇ -1,4-glucan phosphorylase, and 4-gunolecan phosphorylase. Manufactured.
- FIG. 2 shows an outline of a reaction that occurs in the production method of the present invention.
- 8 1,4 Gurkha (D-1) and inorganic phosphoric acid are used to generate darco-monophosphate and j8-1,4-glucan (degree of polymerization n-1) using ⁇ -1,4-glucan phosphorylase.
- the produced glucose 1-phosphate (and glucose monophosphate in solution) is immediately transferred to an appropriate primer (degree of polymerization m) by 4-linkage by ⁇ -1,4-glucan phosphorylase. —Extended as glucan chains (degree of polymerization m + 1).
- the inorganic phosphate generated at that time is recycled again to j8-1,4-glucan phosphorylase reaction.
- FIG. 2 shows an outline of a reaction that occurs in the production method of the present invention when the first 13-1,4-glucan is cellobiose and the 13-1,4-glucan phosphorylase is cellobiose phosphorylase.
- Glucose-1 phosphate and glucose are produced from cellobiose (degree of polymerization 2) and inorganic phosphoric acid using cellobiose phosphorylase.
- the resulting glucose 1-phosphate (and glucose 1-phosphate added to the solution) is immediately transferred to the appropriate primer (degree of polymerization m) via ⁇ -1,4 bonds by 4-glucan phosphorylase, and the ⁇ -glucan chain (Degree of polymerization m + 1) is extended.
- the inorganic phosphate generated at that time is recycled again to the reaction of j8-l, 4-glucan phosphorylase.
- a solution is prepared.
- the solution may be, for example, solid j8-1,4-glucan, a primer, inorganic phosphate or glucose monophosphate, ⁇ -1,4-glucan phosphorylase, and 4-glucan phosphorylase in a suitable solvent. It can be prepared by adding.
- the solution may be a ⁇ -1,4-glucan, a primer, a source of phosphate such as inorganic phosphate or glucose-1-phosphate, a ⁇ -1,4-glucan phosphorylase, or an ⁇ -1,4-glucan. It may be prepared by mixing solutions each containing glucan phosphorylase.
- the solution may contain ⁇ -1,4-glucan, primers, a source of phosphate such as inorganic phosphate or glucose-1-phosphate, ⁇ -1,4-glucan phosphorylase, and some of the following: It may be prepared by mixing a solid containing other components with a solution containing the components. If necessary, any buffer may be added to the solution used in the production method of the present invention for the purpose of adjusting ⁇ as long as the enzyme reaction is not inhibited. The ⁇ of this solution does not unduly inhibit the enzymatic reaction. Any pH can be used. The pH value is preferably from about 6 to about 8, more preferably from about 6.5 to about 7.5. The pH can be set appropriately according to the optimum pH of the enzyme used in the reaction.
- the salt concentration of the solution can also be any salt concentration as long as it does not unduly inhibit the enzymatic reaction. The salt concentration is preferably 1. OmM-50 mM, more preferably 5 mM-30 mM.
- this solution contains glucose produced during the production of ⁇ -dalcan.
- glucose isomerase or glucose oxidase (and mutarotase) may be further added.
- catalase or peroxidase may be added to the solution.
- a microorganism such as yeast that removes glucose from a solution by assimilating glucose may be added.
- glucose-specific adsorption resin may be added. The method of adding an enzyme or a microorganism is preferable because glucose can be simultaneously removed while the reaction proceeds continuously. In this specification, “removing” includes reducing the amount of glucose in the reaction solution and eliminating glucose.
- an enzyme selected from the group consisting of a branching enzyme, branching enzym, 4a-glucanotransferase and glycogen debranching enzym is added to this solution. May be. These enzymes may be added at the beginning of the ⁇ -glucan synthesis reaction or may be added during the reaction, or may be added after the reaction is completed.
- the temperature of the solution is an arbitrary temperature as long as the effects of the present invention can be obtained, and is a temperature at which the added enzyme exhibits its activity.
- the temperature of the solution in this reaction step is preferably at least about 50% of the activity of at least one of j8-1,4-glucan phosphorylase and glucan phosphorylase contained in this solution before the reaction after a predetermined reaction time, preferably about 50% or more of both activities.
- it is a temperature at which about 80% or more of the activity remains. This temperature is preferably about 30 ° C
- the temperature is about 70 ° C, more preferably about 35 ° C to about 60 ° C.
- the reaction time can be set at an arbitrary time in consideration of the reaction temperature, the molecular weight of glucan produced by the reaction, and the residual activity of the enzyme. Reaction times are typically from about 1 hour to about 100 hours, more preferably from about 1 hour to about 72 hours, even more preferably from about 2 hours to about 36 hours, and most preferably from about 2 hours to about 24 hours. is there.
- Heating may be performed using any means, but it is preferable to perform heating while stirring so that heat is uniformly transmitted to the entire solution.
- the solution is stirred, for example, in a stainless steel reaction tank equipped with a warm water jacket and a stirrer.
- ⁇ -1,4-glucan force is cellobiose
- ⁇ -1,4-gnorecan phosphorylase is cellose biose phosphorylase
- an enzyme such as glucose isomerase is added, and ⁇ -
- the step of removing by-product glucose at the same time as the production of dalcan is preferably performed simultaneously with the production step.
- the step of removing glucose may be performed at a timing shifted from the ⁇ -glucan production step.
- the solution is subjected to a physical glucose removal method such as chromatographic fractionation and membrane fractionation, Thereafter, the reaction may be allowed to proceed again.
- the physical glucose removal method may be performed once or two or more times. When the reaction is carried out twice or more, for example, the reaction is allowed to proceed for 2 hours, glucose is removed, then the reaction is carried out again for 2 hours, glucose is removed, and then the reaction is carried out again for 2 hours. .
- the enzyme in the solution can be inactivated by heating the solution at 100 ° C for 10 minutes as needed.
- the subsequent step may be performed without performing the treatment for inactivating the enzyme.
- the solution may be stored as is or processed to isolate the glucan produced.
- ⁇ Purification method> The produced ⁇ -glucan can be purified if necessary.
- An example of an impurity that is removed by purification is glucose.
- Examples of the purification method of ⁇ -dalkan include a method using an organic solvent (TJ Schoch et al., J. American Chemical Society, 64, 2957 (1942)) and a method using an organic solvent.
- Examples of the organic solvent that can be used for purification using an organic solvent include acetone, n-amyl-anoreco nore, pentazonole, n-propinolenoreconole, n-hexinoleanoreconole, and 2-ethylinore.
- reaction solution is cooled to gel the ⁇ -glucan, the gelled ⁇ -glucan is recovered, and glucose is separated from the gelled ⁇ -glucan by water. Washing, freeze-thaw, filtration, etc .;
- ultrafiltration membranes that can be used for purification include a molecular weight cut off of about 1,000 to about 100,0.
- preferred ⁇ about 5,000 to about 50,000, more preferred ⁇ is about 10,000 to about 30,000 ultrafiltration membranes (UF membrane unit manufactured by Daicel).
- Examples of carriers that can be used for chromatography include a carrier for gel filtration chromatography, a carrier for ligand exchange chromatography, a carrier for ion exchange chromatography, and a carrier for hydrophobic chromatography.
- a mixture of 30 ⁇ l of a 40 mM cellobiose aqueous solution and 30 ⁇ l of a 40 mM sodium phosphate aqueous solution ( ⁇ 7.5) is added, and an appropriately diluted enzyme solution (sample) 601 is added to form a mixture of 1201. Let it start. The reaction is allowed to proceed by incubating the mixture at 37 ° C for 10 minutes, and then inactivating the enzyme by maintaining the mixture at 100 ° C for 10 minutes. Continue V. Add 780 ⁇ l of 1 M Tris-HCl buffer (pH 7.0) and 120 ⁇ l of a coloring reagent (Dalkose AR-II coloring reagent (manufactured by Wako Pure Chemical Industries)) to this mixture.
- a coloring reagent Dalkose AR-II coloring reagent (manufactured by Wako Pure Chemical Industries)
- One unit of cellobiose phosphorylase is defined as the amount of enzyme that produces 1 ⁇ mol of glucose per minute from 2 OmM cellobiose by the above method.
- the activity of producing 1 mol of inorganic phosphate per minute is defined as 1 unit of ⁇ -1,4-glucan phosphorylase.
- the yield of a-glucan according to the production method of the present invention was calculated based on what percentage of the number of moles of the initial cellobiose initially added was the molar power of glucose residues incorporated in the obtained a-glucan. After completion of the reaction, ethanol was added to a final concentration of 50% to precipitate a-glucan, and the supernatant was discarded. The a-glucan was washed twice with an appropriate amount of 50% ethanol, dried, and dried. After dissolving in water, the glucose concentration was measured by the phenol-sulfuric acid method to calculate the yield (moles) of a -dalcan. The yield was calculated by dividing the yield (number of moles) by the number of moles of cellobiose and multiplying by 100. The formula is shown below.
- a-glucan synthesized in the present invention After completely dissolving a-glucan synthesized in the present invention with 1N sodium hydroxide and neutralizing with an appropriate amount of hydrochloric acid, about 300 / zg of a-glucan is detected by a differential refractometer and multi-angle light scattering detection. Average molecular weight was determined by subjecting it to gel filtration chromatography using
- Shodex SB806M-HQ manufactured by Showa Denko
- DAWN-DSP multi-angle light scattering detector
- Shodex RI-71 differential refractometer
- Showa Denko were used in this order.
- the column was maintained at 40 ° C, and a 0.1 M sodium nitrate solution was used as an eluent at a flow rate of 1 mLZ minute.
- the obtained signals were collected using data analysis software (trade name: ASTRA, manufactured by Wyatt Technology) and analyzed using the software to determine the weight average molecular weight.
- Synthetic DNA primer 2 5, aaactcgagaattacttcaactttgtgagtcttt 3, (system IJ number 2)
- the region containing the CBP gene was amplified by performing PCR under the following conditions.
- the amplified gene along with a selectable marker gene Km 1 incorporated into an expression vector pET28a (STRATAGENE Co.) to obtain plasmid pET28a- CBP1.
- the seropioose phosphorylase gene was operably linked under the control of an isopropyl-1- ⁇ -D-thiogalatatopyranoside (IPTG) -inducible promoter.
- This plasmid was introduced into the Otsuki fungus BL21 (DE3) pLysS (manufactured by STRATAGENE) by the combi- nation cell method.
- This E. coli contains LB medium containing antibiotic kanamycin (1% tryptone (Difco), 0.5% yeast extract (Difco), 1% sodium salt, 1.5% agar) The plate was plated and cultured at 37 ° C. By selecting Escherichia coli grown on this plate, Escherichia coli into which the cellobiose phosphorylase gene derived from Clostridium thermocellum was introduced was obtained.
- the obtained Escherichia coli contained the cellobiose phosphorylase gene by analyzing the sequence of the introduced gene. In addition, it was confirmed by activity measurement that the obtained Escherichia coli expressed cellobiose phosphorylase.
- This Escherichia coli was inoculated into 1 liter of an LB medium (1% tryptone, 0.5% yeast extract (both manufactured by Difco), 1% sodium chloride salt) containing the antibiotic kanamycin, and shaken at 120 rpm.
- the cells were cultured with shaking at 37 ° C for 3 hours. Thereafter, IPTG was added to this medium to a concentration of 1. OmM, and cultured with shaking at 37 ° C for an additional 8 hours. Then, the culture was centrifuged at 5, OOO rpm for 5 minutes to collect E. coli cells.
- the obtained cells were suspended in 50 ml of a 50 mM phosphate buffer (pH 7.5) containing 1.4 mM of 2-mercaptoethanol. Was disrupted by sonication to obtain 50 ml of a disrupted cell suspension.
- the crushed liquid contained 132 U / ml cellobiose phosphorylase! / ⁇ .
- the cell lysate was heated at 55 ° C for 20 minutes. After heating, the mixture was centrifuged at 8,500 rpm for 20 minutes to remove insoluble proteins and the like to obtain a supernatant. The obtained supernatant was preliminarily equilibrated and then passed through His-Tag adsorbed resin Ni-NTA agarose (manufactured by QIAGEN) to adsorb cellobiose phosphorylase to this resin. This resin was washed with a buffer containing 300 mM sodium chloride, 20 mM imidazole and 1.4 mM 2-mercaptoethanol to remove impurities.
- the protein was eluted with a buffer containing 300 mM sodium chloride, 150 mM imidazole and 1.4 mM 2-mercaptoethanol to obtain a recombinant cellobiose phosphorylase enzyme solution.
- the potato ⁇ -1,4-glucan phosphorylase gene (Nakano et al., Journal of Biochemistry (Tokyo) 106 (1989) 691) was inserted into the expression vector pET3d (manufactured by STRATAGENE) together with the selectable marker gene Amp 1 ", and the plasmid pET was added.
- the glucan phosphorylase gene was operably linked under the control of an isopropyl-18-D-chiogalla-tatobilanoside (IPTG) -inducible promoter.
- IPTG isopropyl-18-D-chiogalla-tatobilanoside
- the E. coli was inoculated into 1 liter of an LB medium (1% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 1% sodium salt of sodium salt) containing the antibiotic ampicillin.
- the cells were cultured with shaking at 37 ° C for 3 hours while shaking. Then, add IPTG to 0.1 lmM and pyridoxine. Each medium was added to this medium so as to obtain ImM, and cultured with shaking at 22 ° C for another 20 hours. Next, this culture was centrifuged at 5, OOO rpm for 5 minutes to collect E. coli cells.
- the obtained cells were suspended in 50 ml of 20 mM Tris-hydrochloric acid buffer (pH 7.0) containing 0.05% Triton X-100, and then crushed by sonication to obtain 50 ml of a cell lysate.
- This crushed liquid contained 4.7 U / mg glucan phosphorylase.
- the disrupted cell suspension was heated at 55 ° C for 30 minutes. After heating, the mixture was centrifuged at 8,500 rpm for 20 minutes to remove insoluble proteins and the like to obtain a supernatant.
- the obtained supernatant (containing 125 mg of protein) was anion-exchanged resin Q—Sepharose which had been previously equilibrated using an equilibration buffer (20 mM phosphate buffer, pH 7.0). And glucan phosphorylase was adsorbed to the resin.
- the resin was washed with a buffer containing 200 mM sodium chloride to remove impurities. Subsequently, the protein was eluted with a buffer containing 300 mM sodium chloride, to give a recombinant glucan phosphorylase enzyme solution.
- Amylose synthesis was performed by incubating at 16 ° C. for 16 hours at 45 ° C. using the reaction mixture obtained at the start of the reaction shown in Table 1 below.
- Phosphoric acid was added as potassium dihydrogen phosphate monobasic disodium hydrogen phosphate buffer.
- the pH of the phosphate buffer is 7.0.
- CBP cellobiose phosphorylase
- the degree of polymerization of the synthesized amylose can be freely controlled by changing the primer concentration of the reaction solution.
- a low-molecular-weight amylose can be obtained by using a small amount of primer It was confirmed that a large amount of primers could be used in this case.
- Example 2-1 2-5 Amylose Synthesis at Various Cellobiose Phosphorylase Concentrations
- Phosphoric acid was added as potassium dihydrogen phosphate monobasic hydrogen phosphate buffer.
- the pH of the phosphate buffer is 7.0.
- Amylose synthesis was carried out by incubating for 16 hours at 45 ° C. using the reaction mixture obtained at the start of the reaction shown in Table 3 below.
- amylose yield was highest when the concentration of phosphoric acid was 15 mM to 30 mM, but in the range of 5 mM to 45 mM, the amylose yield did not change so much. It was found that efficient amylose synthesis could be performed.
- Amylose synthesis was carried out by incubating for 16 hours at 45 ° C. using the reaction mixture obtained at the start of the reaction shown in Table 4 below.
- Phosphoric acid was added as a buffer solution of potassium dihydrogen phosphate and disodium hydrogen phosphate.
- the ⁇ ⁇ of the phosphate buffer is 7.0.
- Example 5-1-5-4 Amylose synthesis using glucose isomerase or glucose oxidase, mutarotase and peroxidase
- Amylose synthesis was carried out by incubating for 16 hours at 45 ° C. using the reaction mixture at the start of the reaction shown in Table 5 below.
- amylose yield was dramatically improved by adding glucose isomerase (GI) or glucose oxidase (GOx) + mutarotase (MT) + peroxidase (POx) to the reaction system. I found out. In particular, when glucose oxidase (GOx) + mutarotase (MT) + peroxidase (POx) was added, the amylose yield was 64.8%, and when these enzymes were not added, 32.8%).
- GI glucose isomerase
- GOx glucose oxidase
- MT mutarotase
- POx peroxidase
- 0.3 g of cellobiose and 0.75 micromol of primer (G4) were dissolved in 10 ml of 30 mM phosphate buffer (pH 7.0), and the recombinant cellobiose obtained according to the preparation method described in 2.1 above was added thereto.
- a reaction solution was prepared by adding 1,500 U of branching genzyme derived from exaeolicus, and the reaction solution was incubated at 45 ° C. for 16 hours.
- Example 6 Whether or not the glucan synthesized in Example 6 has a branched structure and the average unit chain length of the synthesized darcan are described in H. Takata et al., Carbohydr. Res., 295, 91-101 (1996). Was determined according to the method described above. As a result, it was confirmed that the synthesized glucan had a branched structure and the average unit chain length was 11. As described above, it was found that glucans having a branched structure can be synthesized from cellobiose by further containing branching enzyme in addition to CBP and GP in the reaction solution. (Example 7: Synthesis of glucan having cyclic structure)
- Thermus aquaticus 4-a-glucanotransferase As the Thermus aquaticus 4-a-glucanotransferase, the only known DNA sequence of Thermus aquaticus 4-glucanotransferase was used, and the same as the ⁇ -1,4-glucan phosphorylase of 2.2 above. The one prepared by the method was used.
- the measured amount of cyclic dalcan was compared with the amount of cellobiose as a starting material, and the yield of cyclic dalcan was calculated to be 9.6%. Therefore, it was found that about 29% of the glucans obtained in Example 7 were cyclic glucans and about 71% were linear amylose. As described above, it was found that glucan having a cyclic structure can be synthesized from cellobiose by further including 4a-glucanotransferase in addition to CBP and GP in the reaction solution.
- Phosphoric acid was added as potassium dihydrogen phosphate monobasic hydrogen phosphate buffer.
- the pH of the phosphate buffer was 7.0.
- sucrose yield was increased by eliminating glucose in the reaction system using glucose oxidase, mutarotase, and peroxidase, but the yield was hardly increased.
- indigestible ⁇ 1,4-glucan (particularly, cellulose and its partially decomposed products) can be converted into edible foods.
- j8-l, 4-glucan which is a large amount of biomass on the earth, can be efficiently and inexpensively converted into 4-dalcan, thereby solving the food crisis problem and the garbage problem. Also greatly contributes to
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US10/596,243 US20070092949A1 (en) | 2003-12-12 | 2004-12-09 | Method of converting beta-1,4-glucan to alpha-glucan |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2010148407A (ja) * | 2008-12-24 | 2010-07-08 | Ezaki Glico Co Ltd | グルコースをα−1,4−グルカンに変換する方法 |
JP6321857B1 (ja) * | 2017-05-17 | 2018-05-09 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
JP6417060B1 (ja) * | 2018-02-20 | 2018-10-31 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
WO2022153771A1 (ja) * | 2021-01-12 | 2022-07-21 | 第一工業製薬株式会社 | セロオリゴ糖の製造方法 |
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JP4898135B2 (ja) * | 2005-04-13 | 2012-03-14 | 松谷化学工業株式会社 | セロビオースの精製方法及び製造方法 |
US20090074829A1 (en) * | 2006-03-14 | 2009-03-19 | Masao Tanihara | Novel Heparin Alternative Material and Method for Producing the Same |
GB201008573D0 (en) * | 2010-05-21 | 2010-07-07 | Univ Gent | Biocatalytic production of cellobiosides |
EP2665745A1 (en) * | 2011-01-21 | 2013-11-27 | The Board Of Trustees Of The UniversityOf Illinois | Enhanced fermentation of cellodextrins and beta-d-glucose |
CN102640858A (zh) * | 2012-04-13 | 2012-08-22 | 湖南农业大学 | 一种多功效饲用复合酶制剂 |
WO2016038142A1 (en) * | 2014-09-10 | 2016-03-17 | Pfeifer & Langen GmbH & Co. KG | Process for the enzymatic preparation of a product glucoside and of a co-product from an educt glucoside |
CN114517216B (zh) * | 2020-11-20 | 2024-06-14 | 中国科学院天津工业生物技术研究所 | 一种有机溶剂在体外合成可溶性直链淀粉中延长其聚合度的用途 |
CN114686544B (zh) * | 2020-12-30 | 2024-08-06 | 广东省科学院生物工程研究所 | 利用三水相体系自发调控水解生成特定分子量的α-葡聚糖的方法及其应用 |
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JP2001112496A (ja) * | 1999-10-20 | 2001-04-24 | Nippon Paper Industries Co Ltd | セロオリゴ糖の製造法 |
WO2002097107A1 (en) * | 2001-05-28 | 2002-12-05 | Ezaki Glico Co., Ltd. | Production method and preparation method of glucans |
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US6077695A (en) * | 1995-07-13 | 2000-06-20 | Bioflexin Ab | Method of producing derivatives of Glc-β 1-4Glc-N-acetyl |
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- 2004-12-09 CN CN2004800369720A patent/CN1894418B/zh active Active
- 2004-12-09 JP JP2005516181A patent/JP4318315B2/ja active Active
- 2004-12-09 US US10/596,243 patent/US20070092949A1/en not_active Abandoned
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JP2001112496A (ja) * | 1999-10-20 | 2001-04-24 | Nippon Paper Industries Co Ltd | セロオリゴ糖の製造法 |
WO2002097107A1 (en) * | 2001-05-28 | 2002-12-05 | Ezaki Glico Co., Ltd. | Production method and preparation method of glucans |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010148407A (ja) * | 2008-12-24 | 2010-07-08 | Ezaki Glico Co Ltd | グルコースをα−1,4−グルカンに変換する方法 |
JP6321857B1 (ja) * | 2017-05-17 | 2018-05-09 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
JP2018191566A (ja) * | 2017-05-17 | 2018-12-06 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
JP6417060B1 (ja) * | 2018-02-20 | 2018-10-31 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
JP2019140960A (ja) * | 2018-02-20 | 2019-08-29 | サンエイ糖化株式会社 | 糖カルボン酸の製造方法 |
WO2022153771A1 (ja) * | 2021-01-12 | 2022-07-21 | 第一工業製薬株式会社 | セロオリゴ糖の製造方法 |
Also Published As
Publication number | Publication date |
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CN1894418B (zh) | 2010-12-08 |
JP4318315B2 (ja) | 2009-08-19 |
CN1894418A (zh) | 2007-01-10 |
JPWO2005056811A1 (ja) | 2007-12-06 |
US20070092949A1 (en) | 2007-04-26 |
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