WO2009136601A1 - Α-1,4-glucan-grafted cellulose - Google Patents

Abstract

A graft polymer in which an α-1,4-glucan side chain is bonded to the main chain of cellulose: wherein the terminal carbon atom at the 1-position of the α-1,4-glucan side chain is covalently bonded via an –NH- group to the carbon atom at the 6-position in at least one glucose residue of the cellulose main chain; two hydrogen groups are bonded to the preceding carbon atom at the 1-position or one oxygen atom is bonded thereto; the preceding cellulose main chain has a degree of polymerization of 25 or higher; and the preceding α-1,4-glucan side chain has a degree of polymerization of 25 or higher.

Description

α-1,4 glucan grafted cellulose

The present invention relates to cellulose combined with α-1,4 glucan and a method for producing the same.

In the polymer material field, two types of polymers having different structures and / or functions are combined to develop polymers having different functions and / or physical properties. For example, a polymer obtained by grafting acrylic acid to starch is used as a superabsorbent polymer in the daily necessities field.

In addition, branched complex polysaccharides are known to play an important role in nature. These materials have a branched structure composed of different sugar units, and it is considered that such a structure affects the function of the material. On the other hand, natural polysaccharides such as starch, amylose, cellulose, and chitin are abundant in nature and are expected to be used as low environmental impact materials because they are biodegradable and recyclable carbon resources.

Cellulose is a polysaccharide in which many glucose residues are linked by β-1,4-linkages. Cellulose is insoluble in water, has high crystallinity, and has high mechanical strength, and is used for structural materials, films, fibers, and the like.

On the other hand, amylose is a polysaccharide in which many glucoses are bonded with α-1,4-bonds, and dissolves or swells in water to form an aqueous solution or hydrogel. Amylose has a characteristic function of forming a clathrate compound by taking a guest substance into a hollow portion of the amylose and forming an inclusion compound. It has been reported that the inclusion compound forming ability of amylose is greatly influenced by the polymerization degree of amylose, and is stronger as the polymerization degree of amylose is higher (Non-patent Document 1). Further, it is known that the branched polysaccharides amylopectin and glycogen in which a short α-1,4 glucan having a degree of polymerization of about 10 to 20 is linked by α-1,6 bonds have no inclusion function. Thus, in order to fully exhibit the inclusion function, amylose is preferably a completely linear α-1,4 glucan containing no branched structure, and the longer the degree of polymerization, the more preferable. On the other hand, amylose is also characterized by being easily degraded in the environment and in vivo.

In this field, for example, Patent Document 1 discloses a fiber made of a polymer inclusion compound in which a cyclic oligosaccharide is bound to a linear amino polysaccharide (for example, chitosan fiber). Furthermore, the synthesis of amylose-grafted chitin and amylose-grafted chitosan by a chemical-enzymatic method is reported in Non-Patent Document 2. Non-Patent Document 2 discloses that a graft polymer is produced by bonding a side chain to an NH ₂ group present in the structure of chitin.

However, since there are no NH ₂ groups in cellulose, hydroxyl groups must be used to produce the graft polymer. However, a very large number of hydroxyl groups are present in cellulose and cannot be selectively reacted like chitin. If an attempt is made to graft all the hydroxyl groups in cellulose, structural failure occurs and the reaction does not proceed. Also, amylose having a high degree of polymerization such as a degree of polymerization of 25 or more has low reactivity and cannot be grafted by chemical synthesis. For this reason, there is no report on a graft polymer in which an amylose side chain is grafted to a cellulose main chain.

JP 2002-327338 A

The present invention is intended to solve the above-mentioned problems, and graft polymers having α-1,4 glucan having a polymerization degree of 25 or more grafted to cellulose and having an inclusion function of α-1,4 glucan (that is, a graft polymer) , Α-1,4 glucan grafted cellulose).

As a result of intensive studies to solve the above-mentioned problems, the present inventors have carried out polymerization by covalently bonding a primer to cellulose and then extending the primer enzymatically to α-1,4 glucan. The inventors have found that α-1,4 glucan side chains having a degree of 25 or more can be covalently bonded to the cellulose main chain, and based on this, the present invention has been completed.

In order to obtain cellulose having amylose side chains using enzyme-catalyzed polymerization, it is necessary to introduce maltooligosaccharide or short chain amylose into cellulose. In one embodiment of the invention, maltooligosaccharides or short chain amylose are introduced by a reductive amination reaction. That is, a part of the hydroxyl group at the 6-position in the glucose residue in the cellulose main chain is converted to an amino group, and a malto-oligosaccharide or short-chain amylose is introduced by performing a reductive amination reaction on this amino group. To do. Thereafter, amylose-forming polymerization is performed from the maltooligosaccharide site or the short-chain amylose site to synthesize amylose-grafted cellulose. Other embodiments will be described in detail in the “Description of Embodiments” of this specification.

An example of a method for producing a graft polymer according to the present invention is shown in FIG. In this example, maltoheptaose is used as a primer and α-glucan phosphorylase is used as an enzyme. As shown in FIG. 1, in the present invention, an amino group is first introduced into cellulose by a chemical reaction to obtain an amino group-containing cellulose (cellulose having amino group). Subsequently, maltoheptaose is introduced into the amino group-containing cellulose by a chemical reaction to obtain maltoheptaose-grafted cellulose. In maltoheptaose grafted cellulose, a maltoheptaose chain is bonded via an amino group introduced into the cellulose. Using this maltoheptaose grafted cellulose, the maltoheptaose chain is elongated by an enzymatic reaction using α-glucan phosphorylase to form an amylose chain, thereby producing an amylose grafted cellulose (amylose). -Crafted cellulose) is obtained.

The present invention provides, for example:
(Item 1)
a graft polymer in which α-1,4 glucan side chains are bonded to a cellulose main chain,
The first carbon at the end of the α-1,4 glucan side chain is covalently bonded via the —NH— group to the 6th carbon of at least one glucose residue in the cellulose backbone;
Two hydrogen groups are bonded to the first carbon atom, or one oxygen atom is bonded;
The degree of polymerization of the cellulose main chain is 25 or more;
The degree of polymerization of the α-1,4 glucan side chain is 25 or more.
Graft polymer.

(Item 2)
Item 2. The graft polymer according to Item 1, wherein the α-1,4 glucan side chain is completely linear.

(Item 3)
The graft polymer has the following structure (I):

Where each R is —OH, —NH ₂ and the following structure (II) or (III):

Independently selected from the group consisting of: n is an integer from 25 to 20,000, m is an integer from 24 to 1000, and glucose residues in the cellulose main chain are β-1,4 bonded 3. The graft polymer according to item 1 or 2, which is bound by

(Item 4)
Item 4. The graft polymer according to any one of Items 1 to 3, wherein the degree of polymerization of the α-1,4 glucan side chain is 40 or more.

(Item 5)
Item 5. The graft polymer according to any one of Items 1 to 4, wherein the degree of polymerization of the α-1,4 glucan side chain is 50 or more.

(Item 6)
Item 6. The graft polymer according to any one of Items 1 to 5, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 10,000.

(Item 7)
Item 7. The item 1-6, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 50 per 100 glucose residues in the cellulose main chain. Graft polymer.

(Item 8)
The method for producing a graft polymer according to any one of items 1 to 7,
Replacing the OH group bonded to the 6-position carbon of at least one glucose residue in cellulose with an NH ₂ group to obtain an aminated cellulose;
By reacting the aminated cellulose with a primer, the 6-position carbon of at least one glucose residue in the aminated cellulose and the 1-position carbon of the end of the primer are covalently bonded via an —NH— group. Obtaining primer-bound cellulose;
By extending the primer by contacting the primer-bound cellulose, a substrate, and an α-1,4 glucan chain extender, an α-1,4 glucan side chain having a polymerization degree of 25 or more is formed, Including the step of obtaining the graft polymer according to any one of 1 to 7,
The primer is an α-1,4 glucan having a degree of polymerization of 3 to 20, or a reducing end oxide or lactonized product thereof,
The substrate is a substance that provides a glucose residue to the primer by the action of the enzyme;
The degree of polymerization of the α-1,4 glucan side chain is 25 or more;
In the α-1,4-glucan-bonded cellulose, a method in which two hydrogen groups are bonded to the 1-position carbon or one oxygen atom is bonded.

(Item 9)
Item 9. The method according to Item 8, wherein the degree of polymerization of the α-1,4 glucan side chain is 40 or more.

(Item 10)
Item 10. The method according to Item 8 or 9, wherein the degree of polymerization of the α-1,4 glucan side chain is 50 or more.

(Item 11)
Item 11. The method according to any one of Items 8 to 10, wherein the number of α-1,4 glucan bound to the cellulose is 1 to 10,000.

(Item 12)
Item 12. The item 8 to 11, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 50 per 100 glucose residues in the cellulose main chain. the method of.

(Item 13)
Item 13. The method according to any one of Items 8 to 12, further comprising the step of purifying the graft polymer.

(Item 14)
Obtaining the aminated cellulose comprises
Reacting cellulose with p-toluenesulfonyl chloride in the presence of triethylamine, lithium chloride and N, N-dimethylacetamide to obtain tosylated cellulose;
Reacting the tosylated cellulose with sodium azide in the presence of tetra-n-butylammonium iodide and dimethyl sulfoxide to give 6-azido-6-deoxycellulose; and the 6-azido-6 in dimethyl sulfoxide The method according to any one of items 8 to 13, which is carried out by reacting deoxycellulose with sodium borohydride to obtain an aminated cellulose.

(Item 15)
Item 15. The method according to any one of Items 8 to 14, wherein the α-1,4 glucan chain extender is α-glucan phosphorylase and the substrate is glucose-1-phosphate.

(Item 16)
Item 15. The item 8-14, wherein the α-1,4 glucan chain extender is α-glucan phosphorylase and sucrose phosphorylase, and the substrate is sucrose and inorganic phosphate or glucose-1-phosphate. the method of.

(Item 17)
Item 15. The method according to any one of Items 8 to 14, wherein the α-1,4 glucan chain extender is amylosucrase and the substrate is sucrose.

According to the present invention, α-1,4 glucan grafted cellulose (also referred to as amylose grafted cellulose) can be obtained. According to the present invention, cellulose or a cellulose product to which amylose function is imparted can be obtained. The amylose-grafted cellulose of the present invention is a material having both the performance of cellulose (for example, physical properties such as mechanical strength) and the performance of amylose (for example, inclusion function), and can be used for various applications.

FIG. 1 is a schematic view showing an example of a method for producing a graft polymer according to the present invention.

Hereinafter, the present invention will be described in detail.

(1. Cellulose)
As used herein, the term “cellulose” refers to a molecule in which a large number (for example, 25 or more) of D-glucopyranose are linked by β1 → 4 glucoside bonds. Each binding unit in cellulose is also referred to as a glucose residue. Cellulose may be represented by (C ₆ H ₁₀ O ₅ ) _n . Cellulose is the main polysaccharide of the plant that forms the framework of the plant cell wall, and is a substance that the plant photosynthesizes, accounting for about one-third of the plant. Cellulose is the most abundant carbohydrate on earth. Cellulose is usually a white, odorless solid and highly hygroscopic. Cellulose has a strong affinity for highly polar solvents and is soluble in sulfuric acid, copper ammonium solution, zinc chloride solution, and hydrochloric acid. Cellulose is insoluble in water, alcohol, ether and methanol. Cellulose has 40-50 molecules arranged in parallel to form microfibrils, which run through interspersed amorphous molecules to form a full-like micelle. It is hydrolyzed to glucose by acid and cellulase, and its intermediate products include cellobiose, cellotriose, cellototeraose and the like.

The number of glucose residues in one molecule of cellulose used in the present invention is preferably about 25 or more, more preferably about 30 or more, still more preferably about 35 or more, and particularly preferably about 40 or more. And most preferably about 50 or greater. The number of glucose residues in one molecule of cellulose used in the present invention is, for example, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800. More than about 900, about 1,000, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, etc. may be sufficient. The number of glucose residues in one molecule of cellulose used in the present invention is preferably about 30,000 or less, more preferably about 25,000 or less, still more preferably about 20,000 or less, Particularly preferred is about 15,000 or less, and most preferred is about 10,000 or less. The number of glucose residues in one molecule of cellulose used in the present invention is, for example, about 9,000 or less, about 8,000 or less, about 7,000 or less, about 6,000 or less, about 5,000 or less, About 4,000 or less, about 3,000 or less, about 2,000 or less, about 1,000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, etc. May be.

Generally, commercially available cellulose is a natural product, and is a mixture of cellulose having various molecular weights.

In the present invention, commercially available cellulose can be used.

(2. Aminated cellulose)
The method for producing a graft polymer of the present invention comprises a step of substituting an NH ₂ group for an OH group bonded to the 6-position carbon of at least one glucose residue in cellulose to obtain an aminated cellulose; And a primer, and the 6-position carbon of at least one glucose residue in the aminated cellulose and the 1-position carbon of the terminal of the primer are covalently bonded via a —NH— group, A step of obtaining bound cellulose; an α-1,4 glucan side chain having a polymerization degree of 25 or more by extending the primer by bringing the primer bound cellulose into contact with a substrate and an α-1,4 glucan chain extending enzyme. To obtain the graft polymer of the present invention. Here, first, a method for producing an aminated cellulose will be described below.

In addition, about the manufacturing method of aminated cellulose, what passes through various paths is well-known in the said field | area, and can also manufacture by paths other than the path | route described in this specification. For example, in addition to producing via a 6-tosylated cellulose derivative, a method for producing via a 6-oxidized cellulose derivative is known. Aminated cellulose can be produced by methods known in the art. See, for example, Carbohydrate Research, 340 (2005) 1403-1406 and Carbohydrate Research, 208 (1990) 83-191.

(3. Scheme 1 Synthesis of amino group-containing cellulose from cellulose)
A scheme of one embodiment of a method for producing an aminated cellulose (ie, amino group-containing cellulose) is shown below.

In one embodiment of the present invention, first, amino group-containing cellulose (Chemical Formula 3 in Scheme 1) is synthesized according to Scheme 1 above. In brief, cellulose is tosylate using tosyl chloride (p-toluenesulfonyl chloride) to obtain a p-toluenesulfonyl group-containing cellulose (cellulose having p-toluenesulfonyl group) 1.

In the present specification, as shown in Scheme 1 for the p-toluenesulfonyl group-containing cellulose 1, there are cases where it shows two structures separated by / in () _{n of the} chemical formula. This indicates that the structural unit of the polymer is one of two structures separated by /. The arrangement of these two structures, the frequency of existence, and the like vary depending on reaction conditions, reactants, etc., and thus cannot be expressed as a fixed chemical formula.

) Each process will be described in more detail.

(3.1 Tosylate)
Specifically, first, cellulose is tosylated to obtain a p-toluenesulfonyl group-containing cellulose (Chemical Formula 1 in Scheme 1). Typical conditions for tosylation are 24 hours at 10 ° C. with tosyl chloride in N, N-dimethylacetamide containing triethylamine and lithium chloride (LiCl). When the reaction is carried out under these conditions, the hydroxyl group at the 6-position is selectively tosylate and other hydroxyl groups are not tosylate. Cellulose, tosyl chloride, N, N-dimethylacetamide, triethylamine, and lithium chloride may be mixed at a time, but it is preferable to add tosyl chloride after preparing a cellulose solution in advance. Before tosylation, it is preferable to dissolve cellulose sufficiently in N, N-dimethylacetamide, then add lithium chloride to dissolve, add triethylamine and mix well, and then add tosyl chloride.

In order to increase the number or ratio of p-toluenesulfonyl groups introduced into cellulose, the amount of tosyl chloride may be increased, the reaction temperature may be increased, or the reaction time may be lengthened. In order to reduce the number or ratio of p-toluenesulfonyl groups introduced into cellulose, the amount of tosyl chloride may be reduced, the reaction temperature may be lowered, or the reaction time may be shortened. Adjustment of the number or proportion of p-toluenesulfonyl groups introduced into cellulose can be easily performed by those skilled in the art.

(3.2 Azidation)
Next, the p-toluenesulfonyl group-containing cellulose (Chemical Formula 1 in Scheme 1) obtained above is azidated to obtain cellulose azide group-containing cellulose (Chemical Formula 2 in Scheme 1). Typical conditions for the azidation of p-toluenesulfonyl group-containing cellulose 1 are dimethyl sulfoxide (DMSO) containing tetra-n-butylammonium iodide ((n-Bu) ₄ NI) and sodium azide (NaN ₃ ). ) For 72 hours at 70 ° C. The p-toluenesulfonyl group-containing cellulose, tetra-n-butylammonium iodide, sodium azide, and dimethyl sulfoxide may be mixed at a time.

(3.3 Reduction of azide group)
Next, an azide group-containing cellulose (Chemical Formula 2 in Scheme 1) is reduced to synthesize an amino group-containing cellulose (Chemical Formula 3 in Scheme 1). A typical condition for the reduction of the azide group-containing cellulose (Chemical Formula 2 in Scheme 1) is 72 hours at 60 ° C. in DMSO containing sodium borohydride (NaBH ₄ ). The azide group-containing cellulose (Chemical Formula 2 in Scheme 1), sodium borohydride, and DMSO may be mixed at once.

(4. Primer)
The primer used in the present invention refers to a molecule that acts as a starting material in the synthesis of α-1,4 glucan chains. In the method of the present invention, sugar units are sequentially bound to the primer by α-1,4-glucoside bonds, and an α-1,4 glucan chain having a length of about 25 residues or more is synthesized. Examples of primers include any saccharide to which a saccharide unit can be added by α-glucan phosphorylase.

The primer may be an α-1,4-glucan containing only an α-1,4-glucoside bond or may partially have an α-1,6-glucoside bond. One skilled in the art can readily select appropriate primers depending on the desired glucan. In the present invention, since it is preferable to synthesize cellulose grafted with linear amylose, α-1,4 glucan containing only α-1,4-glucoside bond, or a reducing end oxide or lactonized product thereof Is preferably used as a primer. In the present specification, a primer that is α-1,4 glucan may be referred to as an α-1,4 glucan primer, and a primer that is a reducing end oxide of α-1,4 glucan may be referred to as an oxidation primer. A primer that is a lactonized product of a glucose residue at the reducing end of α-1,4 glucan is sometimes referred to as a lactonized primer.

(4.1 α-1,4 glucan primer)
Examples of α-1,4 glucan primers include maltooligosaccharides and short chain amylose. The reducing end of α-1,4 glucan is neither oxidized nor lactonized and maintains an equilibrium state between the aldehyde state and the cyclic structure in an aqueous solution. The α-1,4 glucan primer contributes to reductive amination in the aldehyde state.

In the present specification, maltooligosaccharide refers to a substance produced by dehydration condensation of 2 to 10 glucoses and linked by α-1,4 bonds. The maltooligosaccharide preferably has 4 or more saccharide units, more preferably 5 or more saccharide units, and still more preferably 7 or more saccharide units. The number of sugar units is also called the degree of polymerization. The maltooligosaccharide preferably has 10 or fewer sugar units. The number of saccharide units of maltooligosaccharide may be, for example, 9 or less, 8 or less, 7 or less. The smaller the number of sugar units of maltooligosaccharide, the easier the adjustment, the lower the cost, and the easier the subsequent handling. If the maltooligosaccharide is too short, the enzyme cannot act efficiently, and therefore the sugar chain cannot be extended. Examples of malto-oligosaccharides include malto-oligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose, maltodekaose. The maltooligosaccharide is preferably maltotetraose, maltopentaose, maltohexaose or maltoheptaose. The maltooligosaccharide used in the present invention may be a pure single compound or a mixture of a plurality of maltooligosaccharides. The maltooligosaccharide may be a linear oligosaccharide or a branched oligosaccharide. Maltooligosaccharides can have a cyclic moiety in the molecule. In the present invention, a linear maltooligosaccharide is preferred.

In the present specification, short-chain amylose is a linear molecule composed of glucose units linked by α-1,4 bonds having a degree of polymerization of 11 or more and 20 or less. Short-chain amylose is obtained by completely decomposing amylopectin contained in natural starch with a branching enzyme (for example, isoamylase). When used as a primer in the present invention, the number of sugar units of short-chain amylose can be, for example, about 11 or more, about 12 or more, about 13 or more, and the like. When used as a primer in the present invention, the number of sugar units of short-chain amylose is, for example, about 20 or less, about 19 or less, about 18 or less, about 17 or less, about 16 or less, about 15 or less, about 14 or less, about 13 or less. , About 12 or less.

(4.2 Oxidation primer)
In the present specification, the “oxidation primer” refers to an aldehyde group at the reducing end of the primer that is oxidized to a carboxyl group. In the oxidation primer, it is preferable that portions other than the aldehyde group are not oxidized.

An oxidation primer can be produced by oxidizing the aldehyde group at the reducing end of the maltooligosaccharide and short-chain amylose described in 4.1 above. The oxidation of the aldehyde group at the reducing end of maltooligosaccharide (or short-chain amylose) can be carried out, for example, enzymatically or chemically. For example, oligosaccharide oxidase can be used for the enzymatic oxidation. For chemical oxidation, for example, bromine water or iodine can be used.

Examples of oligosaccharide oxidase include oligosaccharide oxidase derived from Acremonium genus described in JP-A-5-84074. When oligosaccharide oxidase is allowed to act, the reducing terminal glucose residue of the raw maltooligosaccharide (or short-chain amylose) is oxidized into a gluconic acid type. For example, the concentration of malto-oligosaccharide serving as a substrate is 1 to 20% (w / v), pH is 6 to 8, and this oxidase is added at 0.1 to 0.5 units at a temperature of 30 to 50 ° C. By performing the reaction for 10 hours, maltooligosaccharide can be converted to maltooligosaccharide acid.

The chemical oxidation of the aldehyde group at the reducing end of the raw maltooligosaccharide (or short-chain amylose) with bromine water or iodine can be performed, for example, as follows. When iodine is used, 1-10 wt% maltooligosaccharide aqueous solution and, for example, 5 wt% iodine-methanol solution are mixed in equal amounts, and 30% to 50 ° C. is added 4% potassium hydroxide to 1/5 to 1 / of the total volume. By dropping 3 drops, the raw maltooligosaccharide can be converted into maltooligosaccharide acid.

(4.3 Lactonization primer)
In the present specification, the “lactonized primer” refers to a lactonized glucose residue at the reducing end of the primer. In the lactonization primer, it is preferable that portions other than the glucose residue at the reducing end are not lactonized.

The lactonization primer can be obtained by removing counter ions from the solution containing the oxidation primer described in 4.2 above using an ion exchange resin, and collecting and drying the fractions containing the oxidation primer.

(5. Preparation method of primer-grafted aminated cellulose)
Any of the following methods can be selected as a method for making a primer-grafted cellulose by binding a primer to aminated cellulose.
(1) Reductive amination method: A method in which a Schiff base is formed from an amino group bonded to cellulose and the reducing end of a primer, and then this Schiff base is reductively aminated using a reducing agent. When this method is used, the primer is covalently bonded (grafted) to cellulose with the structure of the following structural formula (III).
(2) Carbodiimide coupling method: A method in which an amide bond is formed using carbodiimide between a carboxyl group of an oxidized primer obtained by oxidizing the reducing end of a primer and an amino group bonded to cellulose. When this method is used, the primer is covalently bonded (grafted) to cellulose with the structure of the following structural formula (II).
(3) Lactonization primer method: The lactonization primer obtained by dehydrating the oxidized primer obtained by oxidizing the reducing end of the primer and the aminated cellulose are heated in an anhydrous environment, and the lactonization primer -CO-O A method of forming an amide bond between the moiety and the amino group attached to the cellulose. In this case, the primer is covalently bonded (grafted) to cellulose with the structure of the following structural formula (II).

(5.1 Scheme 2 Synthesis of primer-grafted cellulose)
One embodiment of a method for synthesizing primer-grafted cellulose is shown in the following scheme.

In the said scheme 2, maltoheptaose is shown as an example of a primer and maltoheptaose grafted cellulose (Chemical formula 5 in Scheme 2) is shown as an example of a primer grafted cellulose. In brief, the introduction reaction of a primer (for example, maltoheptaose (Chemical Formula 4 in Scheme 2)) to an amino group-containing cellulose (Chemical Formula 3 in Scheme 2) is carried out using cyano. Performed by a reductive amination reaction using sodium trihydroborate (NaBH ₃ CN). When the obtained product was analyzed by ¹ H NMR spectrum, it was found that even if it was sufficiently washed with DMSO, which is a good solvent for maltoheptaose (Chemical Formula 4 in Scheme 2), a peak derived from the anomeric position of cellulose as the main chain A peak derived from the anomeric position of maltoheptaose (Chemical Formula 4 in Scheme 2) is observed. Therefore, it is confirmed that the structure of the product is maltoheptaose-grafted cellulose (Chemical Formula 5 in Scheme 2).

(5.2 Reductive amination)
Typical conditions for reductive amination are 3 days at room temperature (rt) in a methanolic acetic acid mixed solution (1: 1 mixed solution) containing sodium cyanotrihydroborate and a primer. Amino group-containing cellulose, sodium cyanotrihydroborate, triethylamine, primer, acetic acid, and methanol may be mixed at once, and after preparing an acetic acid ethanol mixed solution in advance, add others. May be. Any other mixing order may be used.

(5.3 Carbodiimide coupling)
Typical conditions for carbodiimide coupling include: an aqueous solution containing an amino group-containing cellulose, an oxidizing primer, carbodiimide (eg, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide chloride) and N-hydroxysuccinimide. In one day at room temperature. The amino group-containing cellulose, the oxidation primer, and the N-hydroxysuccinimide may be mixed at once, or other substances may be added after preparing the oxidation primer solution in advance. Any other mixing order may be used.

(5.4 Lactonization primer method)
A typical condition of the lactonization primer method is heating of ethylene glycol containing an amino group-containing cellulose and a lactonization primer at 70 ° C. for 6 hours. The amino group-containing cellulose, lactonized primer, and ethylene glycol may be mixed at once or in any other mixing order.

(6. Substrate and α-1,4 glucan chain extender)
In the present specification, the term “substrate” refers to a substance that is changed by being catalyzed by an enzyme. In the present specification, in some cases, the substrate concept may not include a primer. Preferably, the substrate is a compound that is catalyzed by an enzyme to extend the α-1,4 glucan chain relative to the primer.

In this field, various α-1,4 glucan chain elongation reactions performed in an aqueous solution are known. In the present invention, any substrate known in the art can be used as long as the α-1,4 glucan chain is elongated. The substrate is selected to be appropriate for the α-1,4 glucan chain extender used.

In this specification, the term “α-1,4 glucan chain extender” refers to an enzyme that extends an α-1,4 glucan chain relative to a primer and an enzyme involved in the enzyme.

Examples of α-1,4 glucan chain extension systems that can be used in the present invention include the following:
(1) α-Glucan phosphorylase (GP) (eg, derived from potato) is used to convert the glucosyl group of α-glucose-1-phosphate (alpha-glucose-1-phosphate) into a primer such as maltoheptaose. A method of synthesizing α-1,4-glucan chains by transfer;
(2) Method of synthesizing α-1,4-glucan chain by simultaneously acting sucrose phosphorylase and glucan phosphorylase using primer, sucrose and inorganic phosphate or glucose-1-phosphate as a substrate (hereinafter referred to as SP-GP method) (Waldmann, H. et al., Carbohydrate Research, 157 (1986) c4-c7; WO2002 / 097107). This method has the advantage that a linear glucan can be synthesized at a lower cost than other methods;
(3) A method of synthesizing an α-1,4-glucan chain by reacting amylosucrase using a primer and sucrose as a substrate.

Therefore, when the reaction system (1) is used, the α-1,4 glucan chain extender is α-glucan phosphorylase, and the substrate is a primer and glucose-1-phosphate.

When the reaction system (2) is used, the α-1,4 glucan chain extender is α-glucan phosphorylase and sucrose phosphorylase, and the substrate is primer, sucrose and inorganic phosphate or glucose-1-phosphate.

When the reaction system (3) is used, the α-1,4 glucan chain extender is amylosucrase, and the substrate is primer and sucrose.

(6.1 Inorganic phosphoric acid)
In this specification, inorganic phosphoric acid refers to a substance that can donate a phosphate substrate in the reaction of SP. Here, the phosphate substrate refers to a substance that is a raw material for the phosphate moiety of glucose-1-phosphate. In sucrose phosphorolysis catalyzed by sucrose phosphorylase, inorganic phosphate is thought to act as a substrate in the form of phosphate ions. Since this substrate is conventionally referred to as inorganic phosphoric acid in this field, this substrate is also referred to as inorganic phosphoric acid in this specification. Inorganic phosphoric acid includes phosphoric acid and inorganic salts of phosphoric acid. Usually, inorganic phosphoric acid is used in water containing cations such as alkali metal ions. In this case, since phosphoric acid, phosphate, and phosphate ions are in an equilibrium state, it is difficult to distinguish between phosphoric acid and phosphate. Therefore, for convenience, phosphoric acid and phosphate are collectively referred to as inorganic phosphoric acid. In the present invention, the inorganic phosphoric acid is preferably any metal salt of phosphoric acid, more preferably an alkali metal salt of phosphoric acid. Preferable specific examples of inorganic phosphoric acid include sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, phosphoric acid (H ₃ PO ₄ ), ammonium dihydrogen phosphate, diammonium hydrogen phosphate, and the like.

Only one type of inorganic phosphoric acid may be used, or a plurality of types may be used.

Inorganic phosphoric acid reacts, for example, a phosphoric acid condensate such as polyphosphoric acid (for example, pyrophosphoric acid, triphosphoric acid and tetraphosphoric acid) or a salt thereof decomposed by physical, chemical or enzymatic reaction. It can be provided by adding to the solution.

In this specification, glucose-1-phosphate refers to glucose-1-phosphate (C ₆ H ₁₃ O ₉ P) and salts thereof. Glucose-1-phosphate is preferably any metal salt of glucose-1-phosphate (C ₆ H ₁₃ O ₉ P) in the narrow sense, more preferably glucose-1-phosphate (C ₆ H ₁₃ O ₉ P) any alkali metal salt. Preferable specific examples of glucose-1-phosphate include glucose-1-phosphate disodium, glucose-1-dipotassium phosphate, glucose-1-phosphate (C ₆ H ₁₃ O ₉ P), and the like. It is done. In the present specification, glucose-1-phosphate not having a chemical formula in parentheses means glucose-1-phosphate in a broad sense, that is, glucose-1-phosphate in a narrow sense (C ₆ H ₁₃ O ₉ P) and its Indicates salt.

Glucose-1-phosphate may be used alone or in the SP-GP reaction system at the start of the reaction.

The sum of the molar concentration of inorganic phosphoric acid and the molar concentration of glucose-1-phosphate contained in the system of the present invention (preferably, an aqueous solvent phase mainly composed of water) is typically about 1 mM or more. Yes, preferably about 10 mM or more, more preferably about 20 mM or more. The sum of the molar concentration of inorganic phosphoric acid and the molar concentration of glucose-1-phosphate contained in the system of the present invention (preferably, an aqueous solvent phase containing water as a main component) is preferably about 1000 mM or less, Preferably it is about 500 mM or less, More preferably, it is about 250 mM or less. If the amount of inorganic phosphoric acid and glucose-1-phosphate is too large, the yield of glucan may decrease. If the amount used is too small, it may take time to synthesize glucan.

(6.2 Sucrose)
Sucrose is a disaccharide having a molecular weight of about 342, represented by C ₁₂ H ₂₂ O ₁₁ . Sucrose is present in every plant that has photosynthetic ability. Sucrose may be isolated from plants or chemically synthesized. In view of cost, it is preferable to isolate sucrose from plants. Examples of plants containing a large amount of sucrose include sugar cane and sugar beet. Sugar cane contains about 20% sucrose in the juice. Sugar beet contains about 10-15% sucrose in the juice. Sucrose may be provided at any purification stage from plant juice containing sucrose to purified sugar.

Sucrose used in the method of the present invention is preferably pure. However, any other contaminants may be included as long as α-1,4 glucan chain elongation is not inhibited.

(6.3 α-glucan phosphorylase (EC.2.4.1.1):
α-glucan phosphorylase is a general term for enzymes that catalyze the phosphorolysis of α-1,4-glucan, and is sometimes called glucan phosphorylase, phosphorylase, starch phosphorylase, glycogen phosphorylase, maltodextrin phosphorylase, and the like. α-glucan phosphorylase can also catalyze an α-1,4-glucan synthesis reaction that is the reverse reaction of phosphorolysis. Which direction the reaction proceeds depends on the amount of substrate. In vivo, since the amount of inorganic phosphate is large, α-glucan phosphorylase reacts in the direction of phosphorolysis. When the SP-GP method is used in the method of the present invention, inorganic phosphoric acid is used for phosphorolysis of sucrose, and since the amount of inorganic phosphoric acid contained in the reaction solution is small, α-1,4-glucan is used. The reaction proceeds in the direction of synthesis. Even when other systems are used, the amount of the substrate is adjusted so that the reaction proceeds in the direction of the synthesis of α-1,4-glucan.

Α-glucan phosphorylase is thought to be universally present in various plants, animals and microorganisms capable of storing starch or glycogen. When α-glucan phosphorylase is used in the present invention, this α-glucan phosphorylase may be derived from a plant, an animal or a microorganism, or may be derived from these by genetic engineering.

α-glucan phosphorylase is algae, potato (also called potato), sweet potato (also called sweet potato), yam, taro, cassava and other vegetables, cabbage, spinach and other vegetables, corn, rice, wheat, barley, rye, It may be derived from a selected plant selected from the group consisting of cereals such as millet, beans such as peas, soybeans, red beans, quail beans and the like.

Α-glucan phosphorylase may be derived from an animal selected from the group consisting of mammals such as humans, rabbits, rats, and pigs.

α- glucan phosphorylase, Thermus aquaticus, Bacillus stearothermophilus, Deinococcus radiodurans, Thermococcus litoralis, Streptomyces coelicolor, Pyrococcus horikoshi, Mycobacterium tuberculosis, Thermotoga maritima, Aquifex aeolicus, Methanococcus Jannaschii, Pseudomonas aeruginosa, Chlamydia pneumoniae, Chlorella vulgaris, Agrobacterium tumefaciens, C ostridium pasteurianum, Klebsiella pneumoniae, Synecococcus sp. Synechocystis sp. , E.C. E. coli, Neurospora crassa, Saccharomyces cerevisiae, Chlamydomonas sp. It may be derived from a microorganism selected from the group consisting of The organism producing glucan phosphorylase is not limited to these.

Α-glucan phosphorylase is preferably derived from potato, Thermus aquaticus, or Bacillus stearothermophilus, and more preferably from potato. α-glucan phosphorylase preferably has a high optimum reaction temperature. Α-glucan phosphorylase having a high optimal reaction temperature can be derived from, for example, a highly thermophilic bacterium. α-glucan phosphorylase may be purified or unpurified.

(6.4 Sucrose phosphorylase (EC 2.4.4.1.7))
As used herein, “sucrose phosphorylase” refers to any enzyme that undergoes phosphorolysis by transferring the α-glycosyl group of sucrose to a phosphate group. The reaction catalyzed by sucrose phosphorylase is shown by the following formula:

Sucrose phosphorylase is contained in various organisms in nature. Sucrose phosphorylase is a bacterium belonging to the genus Streptococcus (eg, Streptococcus thermophilus, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus mitos. Clostridium sp. , Pullularia pullulans, Acetobacter xylinum, Agrobacterium sp. Synecoccus sp. , E.C. coli, Listeria monocytogenes, Bifidobacterium adolecentis, Aspergillus niger, Monilia sitophila, Sclerotinea estrotiorum, and Chlamydomonas sp. May be derived from a bacterium selected from the group consisting of The organism from which sucrose phosphorylase is derived is not limited to these.

The sucrose phosphorylase can be derived from any organism that produces sucrose phosphorylase. Sucrose phosphorylase preferably has a certain degree of heat resistance. Sucrose phosphorylase is preferred as it has higher heat resistance when present alone. For example, when sucrose phosphorylase is heated at 55 ° C. for 30 minutes in the presence of 4% sucrose, it preferably retains 50% or more of the activity of sucrose phosphorylase before heating. The sucrose phosphorylase is preferably derived from a bacterium belonging to the genus Streptococcus, and more preferably from Streptococcus mutans, Streptococcus thermophilus, Streptococcus pneumoniae or Streptococcus mitis. Sucrose phosphorylase may be purified or unpurified.

(6.5 General description of α-1,4 glucan chain extender)
The α-1,4 glucan chain extender of the present invention as described above can be derived from any organism. In this specification, the phrase “derived from” an organism does not mean that the enzyme is directly isolated from the organism, but that the enzyme is obtained by utilizing the organism in some form. Say. For example, when a gene encoding the enzyme obtained from the organism is introduced into E. coli and the enzyme is isolated from the E. coli, the enzyme is said to be “derived” from the organism.

The α-1,4 glucan chain extender used in the present invention can be directly isolated from animals, plants, and microorganisms that produce α-1,4 glucan chain extender that exist in nature as described above.

The α-1,4 glucan chain extender used in the present invention is a microorganism genetically modified using a gene encoding an α-1,4 glucan chain extender isolated from these animals, plants or microorganisms ( For example, it may be isolated from bacteria, fungi and the like. The α-1,4 glucan chain extender can be obtained from a genetically modified microorganism. A method for producing sucrose phosphorylase and α-glucan phosphorylase is disclosed in, for example, WO2002 / 097107. Other α-1,4 glucan chain extenders can be similarly performed according to this description.

(7. Preparation method of α-1,4 glucan grafted cellulose from primer grafted cellulose)
In the following, by extending a primer by contacting primer-grafted cellulose (also referred to as primer-bound cellulose), a substrate and an α-1,4-glucan chain extender, α-1, A process of forming a 4-glucan side chain to obtain the amylose-grafted cellulose of the present invention (also referred to as a graft polymer) will be described.

(7.1 Scheme 3 Synthesis of amylose-grafted cellulose)

Next, the synthesis of amylose grafted cellulose (Chemical Formula 6 in Scheme 3) is shown in Scheme 3. In this scheme 3, the case where the reaction system (1) is used as the enzyme reaction system is shown. Briefly, acetate buffer in the presence of 100 equivalents of glucose-1-phosphate (G-1-P) per maltoheptaose unit of maltoheptaose grafted cellulose (Chemical Formula 5 in Scheme 3) Among them, enzyme-catalyzed polymerization with α-1,4 glucan chain extender (eg, α-glucan phosphorylase) is performed to synthesize amylose-grafted cellulose (Chemical Formula 6 in Scheme 3). The product is soluble in 1 mol / L sodium hydroxide aqueous solution, and the structure of the product obtained by ¹ H NMR spectrum, XRD measurement, and TG measurement is amylose-grafted cellulose (chemical formula 6 in Scheme 3). Is confirmed.

As described above, the enzyme reaction system is not limited to (1), any one of (1) to (3) can be used, and an enzyme and a substrate are appropriately selected according to the enzyme reaction system. Can be done.

(7.2 Extension of α-1,4 glucan chain)
When (1) is used as the enzyme reaction system, typical conditions for the extension of the α-1,4 glucan chain are primer grafted cellulose (eg, maltoheptaose grafted cellulose (Chemical Formula 5 in Scheme 3)). In the presence of 100 equivalents of glucose-1-phosphate (G-1-P) to a primer unit (eg, maltoheptaose unit), α-1,4 glucan chain extender (eg, α-glucan phosphorylase) at about 42 ° C. for about 7 hours. Primer-grafted cellulose, glucose-1-phosphate, α-1,4 glucan chain extender, and acetate buffer may be mixed at one time, or else after preparing a mixed ethanol / acetate solution in advance. May be added. Any other mixing order may be used.

When any of the reaction systems (1) to (3) is used as the enzyme reaction system, the amount of primer-grafted cellulose in the solution at the start of the α-1,4 glucan chain elongation reaction is preferably about 0. 0.1% by weight or more, more preferably about 0.2% by weight or more, further preferably about 0.3% by weight or more, particularly preferably about 0.4% by weight or more, most preferably About 0.5% by weight or more. The amount of primer-grafted cellulose in the solution at the start of the elongation reaction of α-1,4 glucan chain is preferably about 10% by weight or less, more preferably about 5% by weight or less, and still more preferably about It is 4% by weight or less, particularly preferably about 3% by weight or less, and most preferably about 2% by weight or less.

When the reaction system (1) is used as the enzyme reaction system, the concentration of glucose-1-phosphate in the system at the start of the extension reaction of α-1,4 glucan chain is preferably in the primer-grafted cellulose. About 40 times or more of the primer molar concentration, more preferably about 50 times or more, further preferably about 60 times or more, particularly preferably about 70 times or more, and most preferably about 80 times or more. . The concentration of inorganic phosphate or glucose-1-phosphate in the solution at the start of the elongation reaction of α-1,4 glucan chain is preferably about 1000 times or less of the primer molar concentration in the primer-grafted cellulose. More preferably, it is about 800 times or less, More preferably, it is about 600 times or less, Especially preferably, it is about 400 times or less, Most preferably, it is about 300 times or less.

When the reaction system of (1) is used as the enzyme reaction system, the amount of α-glucan phosphorylase contained in the system at the start of the reaction is preferably relative to glucose-1-phosphate in the solution at the start of the reaction. About 0.05 U / g glucose-1-phosphate or more, more preferably about 0.1 U / g glucose-1-phosphate or more, and more preferably about 0.5 U / g glucose-1-phosphate. That's it. The amount of α-glucan phosphorylase contained in the system at the start of the reaction is preferably about 1,000 U / g glucose-1-phosphate or less with respect to glucose-1-phosphate in the solution at the start of the reaction. More preferably about 500 U / g glucose-1-phosphate or less, still more preferably about 100 U / g glucose-1-phosphate or less. If the weight of α-glucan phosphorylase is too large, the enzyme denatured during the reaction may easily aggregate. If the amount used is too small, the yield of glucan may decrease.

When the reaction system of (2) is used as the enzyme reaction system, the molar concentration of sucrose contained in the system at the start of the reaction is preferably about 40 times or more the primer molar concentration in the primer-grafted cellulose, Preferably it is about 50 times or more, more preferably about 60 times or more. The concentration of sucrose is, for example, about 6 w / v% or more, about 7 w / v% or more, about 8 w / v% or more, about 9 w / v% or more, about 10 w / v% or more, about 15 w / v% or more, etc. There may be. The concentration of sucrose contained in the system of the present invention is about 1000 times or less, more preferably about 800 times or less, more preferably about 600 times or less the primer molar concentration in the primer-grafted cellulose. The concentration of sucrose is, for example, about 50 w / v% or less, about 40 w / v% or less, about 30 w / v% or less, about 20 w / v% or less, about 15 w / v% or less, about 10 w / v% or less, etc. There may be.

The sucrose concentration mentioned above is Weight / Volume, ie
(Weight of sucrose) × 100 / (volume of solution)
Calculate with If the weight of sucrose is too large, unreacted sucrose may precipitate during the reaction. If the amount of sucrose used is too small, the yield may decrease in a reaction at a high temperature.

When the reaction system of (2) is used as the enzyme reaction system, the total amount of inorganic phosphate or glucose-1-phosphate in the system at the start of the α-1,4 glucan chain elongation reaction is preferably Is about 1/40 or more of the molar concentration of sucrose, more preferably about 1/30 or more, further preferably about 1/25 or more, particularly preferably about 1/20 or more, most preferably About 1/15% by weight or more. The amount of inorganic phosphate or glucose-1-phosphate in the solution at the start of the elongation reaction of α-1,4 glucan chain is preferably about ½ or less of the molar concentration of sucrose as a total, More preferably, it is about 1/3 wt% or less, more preferably about 1/4 wt% or less, particularly preferably about 1/5 wt% or less, and most preferably about 1/6 wt% or less. is there.

When the reaction system (2) is used as the enzyme reaction system, the amount of α-glucan phosphorylase contained in the system at the start of the reaction is preferably about 0.05 U with respect to sucrose in the solution at the start of the reaction. / G sucrose or more, more preferably about 0.1 U / g sucrose or more, and still more preferably about 0.5 U / g sucrose or more. The amount of α-glucan phosphorylase contained in the system at the start of the reaction is preferably about 1,000 U / g sucrose or less, more preferably about 500 U / g sucrose, relative to the sucrose in the solution at the start of the reaction. Or less, more preferably about 100 U / g sucrose or less. If the weight of α-glucan phosphorylase is too large, the enzyme denatured during the reaction may easily aggregate. If the amount used is too small, the yield of glucan may decrease.

When the reaction system of (2) is used as the enzyme reaction system, the amount of sucrose phosphorylase contained in the system at the start of the reaction is preferably about 0.05 U / g with respect to sucrose in the solution at the start of the reaction. It is sucrose or more, more preferably about 0.1 U / g sucrose or more, and still more preferably about 0.5 U / g sucrose or more. The amount of sucrose phosphorylase contained in the system at the start of the reaction is preferably about 1,000 U / g sucrose or less, more preferably about 500 U / g sucrose or less, relative to the sucrose in the solution at the start of the reaction. More preferably about 100 U / g sucrose or less. If the weight of sucrose phosphorylase is too large, the enzyme denatured during the reaction may easily aggregate. If the amount used is too small, the yield of glucan may decrease.

When the reaction system of (3) is used as the enzyme reaction system, the amount of sucrose contained in the system at the start of the reaction is preferably about 1 w / v% or more, more preferably about 3 w / v% or more. More preferably about 5 w / v% or more. The concentration of sucrose is, for example, about 6 w / v% or more, about 7 w / v% or more, about 8 w / v% or more, about 9 w / v% or more, about 10 w / v% or more, about 15 w / v% or more, etc. There may be. The concentration of sucrose contained in the system of the present invention is preferably about 80 w / v% or less, more preferably about 70 w / v% or less, and still more preferably about 60 w / v% or less. The concentration of sucrose is, for example, about 50 w / v% or less, about 40 w / v% or less, about 30 w / v% or less, about 20 w / v% or less, about 15 w / v% or less, about 10 w / v% or less, etc. There may be.

When the reaction system (3) is used as the enzyme reaction system, the amount of amylosucrase contained in the system at the start of the reaction is preferably about 0.05 U / g with respect to sucrose in the solution at the start of the reaction. It is sucrose or more, more preferably about 0.1 U / g sucrose or more, and still more preferably about 0.5 U / g sucrose or more. The amount of amylosucrase contained in the system at the start of the reaction is preferably about 1,000 U / g sucrose or less, more preferably about 500 U / g sucrose or less, relative to the sucrose in the solution at the start of the reaction. More preferably about 100 U / g sucrose or less. If the weight of sucrose phosphorylase is too large, the enzyme denatured during the reaction may easily aggregate. If the amount used is too small, the yield of glucan may decrease.

When any of the reaction systems (1) to (3) is used as the enzyme reaction system, the temperature conditions for the elongation of the α-1,4 glucan chain include the optimum reaction temperature and heat resistance of the enzyme used. It is set appropriately in consideration. In the case of a normal enzyme having an optimum reaction temperature of about 37 ° C., the reaction temperature is preferably about 10 ° C. or more, more preferably about 20 ° C. or more, further preferably about 25 ° C. or more, Especially preferably, it is about 30 degreeC or more. The temperature condition during the extension of the α-1,4 glucan chain is preferably about 45 ° C. or less, more preferably about 42 ° C. or less, still more preferably about 40 ° C. or less, and particularly preferably about 38 ° C. It is below ℃. For example, in the case of a thermostable enzyme having an optimum reaction temperature of about 50 ° C., the reaction temperature is preferably about 30 ° C. or more, more preferably about 35 ° C. or more, and further preferably about 40 ° C. or more. Especially preferably, it is about 45 degreeC or more. The temperature condition during the extension of the α-1,4 glucan chain is preferably about 65 ° C. or less, more preferably about 60 ° C. or less, still more preferably about 55 ° C. or less, and particularly preferably about 53 ° C. It is below ℃. The reaction temperature is preferably within ± 10 ° C. of the optimum reaction temperature of the enzyme used, and more preferably within ± 5 ° C. If the reaction temperature in this step is too high, the enzyme may be deactivated and the α-1,4 glucan chain may not be extended. If the reaction temperature in this step is too low, the enzyme reaction may not proceed and the α-1,4 glucan chain may not be extended.

The heating may be performed by any means, but it is preferable to perform the heating while stirring in, for example, an organic solvent phase so that the heat is uniformly transferred to the entire reaction system. The solution is stirred in, for example, a stainless steel reaction tank equipped with a hot water jacket and a stirring device.

The reaction time is preferably about 0.5 hour or more, more preferably about 1 hour or more, further preferably about 3 hours or more, particularly preferably about 5 hours or more, and most preferably about 8 hours. It's over time. The reaction time is preferably about 72 hours or less, more preferably about 48 hours or less, further preferably about 36 hours or less, particularly preferably about 24 hours or less, and most preferably about 20 hours or less. It is. If the reaction time in this step is too long, the risk of microbial contamination increases. If the reaction time in this step is too short, the amount of enzyme required increases and the cost increases.

After completion of the reaction, the reaction system can be deactivated, for example, by heating at 100 ° C. for 10 minutes as necessary. Or you may perform a next process, without performing the process which inactivates an enzyme. The reaction system may be stored as is or may be processed to isolate the produced graft polymer.

Each α-1,4 glucan side chain is preferably extended until the degree of polymerization is about 25 or more. The degree of polymerization of each α-1,4 glucan side chain is controlled by the amount of substrate contained in the reaction system, the amount of primer introduced into cellulose, the length of reaction time, and the like. Generally, the greater the amount of substrate, the higher the degree of polymerization, and the lower the amount of substrate, the lower the degree of polymerization. The greater the amount of primer introduced into the cellulose, the lower the degree of polymerization, and the smaller the amount of primer, the higher the degree of polymerization. The longer the reaction time, the lower the degree of polymerization, and the shorter the reaction time, the higher the degree of polymerization.

The product obtained by the method of the present invention can be purified by methods known to those skilled in the art. Examples of the purification method include membrane fractionation using an ultrafiltration membrane, chromatography, filtration, and centrifugation.

The product obtained by the method of the present invention is obtained by conventional methods well known to those skilled in the art (for example, thin layer chromatography (TLC), NMR (nuclear magnetic resonance spectrum), HPLC (high performance liquid chromatography), melting point, mass spectrometry. (MS), elemental analysis, etc.) can be analyzed and / or checked for purity. The structure of the reaction product can be confirmed in detail by carrying out NMR (nuclear magnetic resonance spectrum) and MS after purifying the reaction product.

(8. Graft polymer of the present invention)
The graft polymer of the present invention is a graft polymer in which α-1,4 glucan side chains are bonded to a cellulose main chain, and α-1,4 glucan is attached to the 6-position carbon of at least one glucose residue in the cellulose main chain. The terminal 1-position carbon of the side chain is covalently bonded via the —NH— group; the 2-position carbon has two hydrogen groups or one oxygen atom bonded; The polymerization degree of the cellulose main chain is 25 or more; the polymerization degree of the α-1,4 glucan side chain is 25 or more.

Here, the bonds between glucose residues in the α-1,4 glucan side chain are mainly α-1,4 bonds. Of the bonds between glucose residues, 90% or more are preferably α-1,4 bonds, more preferably 95% or more are α-1,4 bonds, more preferably 98%. The above is more preferably an α-1,4 bond, more preferably 99% or more is an α-1,4 bond, and most preferably 99.5% or more is an α-1,4 bond. .

The α-1,4 glucan side chain is preferably completely linear. Here, “fully linear” means that the bonds between glucose residues in the α-1,4 glucan side chain are all α-1,4 bonds, and α-1 , 6 means that no bond is included.

The graft polymer of the present invention is also called α-1,4 glucan grafted cellulose, amylose grafted cellulose or the like.

The graft polymer of the present invention preferably has the following structure (I):

Wherein each R is independently selected from the group consisting of —OH, —NH ₂ and the following structure (II) or (III), wherein n is any integer from 25 to 20,000, m is an integer from 24 to 1000, and glucose residues in the cellulose main chain are linked by β-1,4 bonds:

.

When an α-1,4 glucan primer (that is, a primer whose reducing end is not oxidized or lactonized) is used as a primer in the production method of the present invention, it binds to the 6th carbon of cellulose via an —NH— group. A graft polymer in which two hydrogen groups are bonded to the 1-position carbon at the terminal of α-1,4 glucan is obtained. That is, a graft polymer in which the α-1,4 glucan side chain having the above structure III is bonded to the cellulose skeleton is obtained.

When an oxidation primer or a lactonization primer is used as a primer in the production method of the present invention, one per 1-position carbon of α-1,4 glucan bonded to 6-position carbon of cellulose via —NH— group. A graft polymer to which oxygen atoms are bonded is obtained. That is, a graft polymer having an α-1,4 glucan side chain having the above structure II bonded to a cellulose skeleton is obtained.

The number (ie, n) of glucose residues in one molecule of the cellulose main chain in the graft polymer of the present invention is preferably about 25 or more, more preferably about 30 or more, and further preferably about 35 or more. Particularly preferably about 40 or more, most preferably about 50 or more. For example, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800 or more, about 900 or more, about 1,000 or more, about 2,000 or more, It may be about 2,500 or more, about 3,000 or more, about 4,000 or more, about 5,000 or more, and the like. The number of glucose residues in one molecule of the cellulose main chain used in the present invention is preferably about 30,000 or less, more preferably about 25,000 or less, and further preferably about 20,000 or less. Particularly preferably about 15,000 or less, and most preferably about 10,000 or less. For example, about 9,000 or less, about 8,000 or less, about 7,000 or less, about 6,000 or less, about 5,000 or less, about 4,000 or less, about 3,000 or less, about 2,000 or less, It may be about 1,000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, and the like.

In the graft polymer of the present invention, the degree of polymerization of at least one α-1,4 glucan chain is preferably about 25 or more, more preferably about 30 or more, and further preferably about 40 or more. For example, it may be about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more. In the graft polymer of the present invention, the degree of polymerization of at least one α-1,4 glucan chain is preferably about 1,000 or less, more preferably about 800 or less, and further preferably about 500 or less. is there. For example, it may be about 400 or less, about 300 or less, about 200 or less, about 100 or less, about 90 or less, about 80 or less, about 70 or less, about 60 or less, about 50 or less. If the degree of polymerization of the α-1,4 glucan side chain is too large, steric hindrance may occur and the inclusion function may not be fully exhibited. If the degree of polymerization of the α-1,4 glucan side chain is too small, the desired inclusion function may not be obtained.

In the graft polymer of the present invention, it is particularly preferable that the number average degree of polymerization of α-1,4 glucan side chain bonded to the cellulose main chain is within the above-mentioned preferable range. In the graft polymer of the present invention, the proportion of α-1,4 glucan side chains satisfying the above preferred range among α-1,4 glucan side chains bonded to the cellulose main chain is preferably about 50. % Or more, more preferably about 60% or more, still more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more.

In the graft polymer of the present invention, the number of α-1,4 glucan side chains bonded to one cellulose molecule is preferably about 1 or more per several hundred glucose residues in the cellulose main chain. More preferably, it is about 2 or more, more preferably about 3 or more, particularly preferably about 4 or more, and most preferably about 5 or more. In the graft polymer of the present invention, the number of α-1,4 glucan side chains bonded to one cellulose molecule is preferably about 50 or less per several hundred glucose residues in the cellulose main chain. More preferably, it is about 45 or less, more preferably about 40 or less, particularly preferably about 35 or less, and most preferably about 30 or less.

(9. Use of graft polymer of the present invention)
Conventionally, cellulose has been used for various applications, and its use is known. The graft polymer of the present invention can be used in its various applications. That is, in a material in which cellulose is conventionally used, a part or all of the cellulose can be replaced with the graft polymer of the present invention.

More specifically, for example, the graft polymer of the present invention can be used for composite functional materials and devices such as filters having an environmental purification function, column fillers, and fragrance-containing fibers.

For example, the graft polymer of the present invention can be processed into a fiber form, and the resulting fiber can be processed into a desired form to obtain a final product.

Also, for example, when producing cellulose fibers, the graft polymer of the present invention is mixed into the raw material cellulose to produce graft polymer-containing fibers, and the fibers are processed into a desired form to obtain the desired product. You can also.

Also, for example, the fiber obtained from the graft polymer of the present invention can be mixed with cellulose fiber, and the resulting mixed fiber can be processed into a desired form to obtain a final product. Such a mixed fiber is a fiber provided with an inclusion function of amylose, and can be used for a water treatment filter having a function of removing environmental hormones and surfactants.

Also, for example, a product having a desired shape can be obtained by molding the graft polymer of the present invention. For example, the graft polymer of the present invention can be molded into a film to obtain a film product.

Furthermore, for example, the graft polymer of the present invention can be added to a desired product as an additive.

(Example)
The following examples illustrate the invention in more detail. The present invention is not limited only to the following examples.

(Synthesis of amino group-containing cellulose)
(1) Synthesis of tosylated cellulose 50 ml of N, N-dimethylacetamide and 0.5 g of cellulose (reagent grade; microcrystalline cellulose; number average degree of polymerization of about 300) were added to an eggplant type flask and stirred at 130 ° C. for 1 hour. Further, 4.2 g of lithium chloride was added, stirred for 30 minutes at 100 ° C., and allowed to stand until room temperature to dissolve. To this was added 0.5 ml of triethylamine, and after cooling to 10 ° C., 0.0412 g (0.4 equivalents with respect to glucose unit) of -p-toluenesulfonyl chloride was added and reacted at 10 ° C. for 24 hours. The reaction solution was reprecipitated in water, suction filtered, and dried under reduced pressure to obtain 0.56 g of tosylated cellulose.

(2) Synthesis of azido cellulose 25 ml of dimethyl sulfoxide, 0.5 g of tosylated cellulose, 0.027 g of tetra-n-butylammonium iodide and 0.468 g of sodium azide were added to an eggplant type flask and reacted at 70 ° C for 72 hours. I let you. The reaction solution was reprecipitated in water, filtered with suction, and dried under reduced pressure to obtain 0.463 g of 6-azido-6-deoxycellulose.

(3) Synthesis of Aminated Cellulose 45 ml of dimethyl sulfoxide, 0.450 g of 6-azido-6-deoxycellulose and 0.603 g of sodium borohydride were added to an eggplant type flask and reacted at 60 ° C. for 72 hours. The reaction solution was cooled to 0 ° C., and 1N hydrochloric acid was slowly added dropwise until no gas was generated in order to remove unreacted sodium borohydride, and then neutralized with a saturated aqueous sodium hydrogen carbonate solution. This was isolated by suction filtration and freeze-dried to obtain 0.444 g of 6-amino-6-deoxycellulose.

(Synthesis of maltoheptaose grafted cellulose by reductive amination method)
0.444 g of 6-amino-6-deoxycellulose obtained in Example 1, 20 ml of a mixed solution of acetic acid-methanol, 0.784 g of maltoheptaose and 0.237 g of sodium cyanotrihydroborate were added to the sample tube, and at room temperature. The reaction was performed for 3 days. This was put into methanol, subjected to suction filtration, and washed and washed with dimethyl sulfoxide in order to remove unreacted maltoheptaose. It dried under reduced pressure and obtained 0.478g of maltoheptaose grafted cellulose.

When the obtained product was analyzed by ¹ H NMR spectrum, a peak derived from the anomeric position of cellulose as the main chain and a peak derived from the anomeric position of maltoheptaose were observed. The product structure was confirmed to be maltoheptaose grafted cellulose.

(Synthesis of maltoheptaose grafted cellulose by carbodiimide coupling method)
(Synthesis of oxidized maltoheptaose)
Dissolve 1 g of maltoheptaose in 20 ml of water, add 25 ml of 5% (w / v) iodine-methanol solution, and drop 1 drop of 10 ml of 4% (w / v) potassium hydroxide-methanol solution while stirring at 40 ° C. Added dropwise. After stirring for 1 hour, 4% (w / v) potassium hydroxide-methanol solution was further added dropwise until the iodine color disappeared, and the reaction was terminated after stirring for 30 minutes. The reaction solution was cooled, the precipitate was filtered, dissolved in 45 ml of deionized water, and passed through Amberlite IR-120B (H + type). The target product was collected by flowing deionized water until the eluate had a pH of 7.0, and then dried under reduced pressure several times by a rotary evaporator. Dissolved in 45 ml of deionized water, an aqueous solution of maltoheptaose oxide (19 mg / ml) was obtained.

(Synthesis of maltoheptaose grafted cellulose)
0.42 g of 6-amino-6-deoxycellulose obtained in Example 1, 40 ml of an aqueous solution of oxidized maltoheptaose (19 mg / ml), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide chloride 13 g and N-hydroxysuccinimide 0.08 g were added to the sample tube and allowed to react at room temperature for 1 day. This was passed through a gel filtration column to remove unreacted maltoheptaose oxide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide chloride and N-hydroxysuccinimide. It dried under reduced pressure and obtained 0.41g of maltoheptaose grafted cellulose.

(Synthesis of maltoheptaose grafted cellulose by lactonization primer method)
(Synthesis of lactonized maltoheptaose)
2.0 g of maltoheptaose was dissolved in 3.0 ml of water, and a solution obtained by dissolving 1.33 g of iodine in 10 ml of methanol was added thereto, followed by stirring at 40 ° C. for 2 hours. Subsequently, 1.21 g of potassium hydroxide was dissolved in 35 ml of methanol, dropped dropwise and then stirred for 1 hour. After the reaction
Upon cooling, the resulting precipitate was isolated by decantation. In order to remove unreacted iodine and produced potassium iodide, 10 ml of water was added to obtain a solution, and 100 ml of methanol was added to obtain a precipitate. Decanted again, dissolved in water, and formed a precipitate with methanol. After decantation, the precipitate was dissolved in 9 ml of water and passed through an ion exchange column to recover the product. It dried under reduced pressure and obtained 1.2g of lactonized maltoheptaose. In ¹ H NMR of the product, peaks at the 2-position (4.47 ppm) and the 3-position (4.25 ppm) of the lactone ring were observed, confirming that the product was lactonized maltoheptaose.

(Synthesis of maltoheptaose grafted cellulose)
0.7 g of 6-amino-6-deoxycellulose obtained in Example 1, 1.2 g of lactonized maltoheptaose and 10 ml of ethylene glycol were added to a sample tube and reacted at 70 ° C. for 6 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and acetone was added under a nitrogen atmosphere. The precipitate was filtered and suction filtered while washing with acetone and hexane to remove ethylene glycol and unreacted lactonized maltoheptaose. Drying under reduced pressure gave 1.25 g of maltoheptaose grafted cellulose.

(Synthesis of amylose-grafted cellulose)
0.14 g of maltoheptaose grafted cellulose obtained in Example 2, acetate buffer 12.5 ml, glucose-1-sodium phosphate 0.38 g (100 equivalents with respect to maltoheptaose group), potato-derived α- 42 units of glucan phosphorylase was added to the sample tube and reacted at pH 6.2 and 42 ° C. for 7 hours. In order to remove impurities, the mixture was dialyzed with 1N aqueous sodium hydroxide solution, poured into methanol, and isolated by suction filtration. Drying under reduced pressure gave 0.19 g of amylose grafted cellulose.

From the ¹ H NMR of the product, a peak derived from the anomeric position of cellulose and a peak derived from the anomeric position of amylose were observed, and this product was obtained by grafting amylose having a polymerization degree of 49 to cellulose. confirmed. Elemental analysis confirmed that amylose was bound to cellulose at a rate of 1 per 28 glucose residues in the cellulose main chain. Unlike cellulose, the product was soluble in 1 mol / L sodium hydroxide aqueous solution. From these results, it was confirmed that the structure of the product was amylose grafted cellulose. Therefore, in this product, the 1-position carbon of the terminal of the α-1,4-glucan side chain is covalently bonded to the 6-position carbon of the glucose residue in the cellulose main chain via the —NH— group. Two hydrogen groups are bonded to the 1-position carbon, the number average degree of polymerization of the cellulose main chain is about 300, and amylose having a degree of polymerization of 49 per 28 glucose residues in the cellulose main chain. A graft polymer having a grafted structure.

(Production of film from α-1,4 glucan grafted cellulose)
When the reaction solution of Example 5 was placed in a glass container and allowed to stand, an opaque and swollen gel was obtained. Washing with water was performed to remove unreacted materials and the like. Cellulose could not form such a gel under the same conditions. The gel was then air dried to obtain an opaque solid. Furthermore, when water was added to the dried solid, it returned to an opaque and infiltrated gel. Moreover, when this gel was air-dried, an opaque solid substance was obtained again.

Further, when the reaction solution of Example 5 was spread thinly on a glass plate and allowed to stand, an opaque and swollen film was obtained. Washing with water was performed to remove unreacted materials and the like. When this film was naturally dried, an opaque and somewhat flexible film was obtained. Further, when water was added to the dried film, it returned to an opaque and swollen film. When this film was naturally dried, an opaque and somewhat flexible film was obtained again. When 1N NaOH aqueous solution was added to the dried film, this film was dissolved. Thus, α-1,4 glucan grafted cellulose, unlike cellulose, exhibited unique physical properties that have both amylose solubility and flexibility.

Furthermore, when the obtained gel and film were each added to 10 ml of a 5 mM iodine solution (KI · I ₂ : 0.024 M / 0.010 M), both were stained blue. Therefore, it was confirmed that the α-1,4 glucan grafted cellulose was provided with not only the physical properties of amylose but also the inclusion function of amylose.

As described above, the present invention has been exemplified using the preferred embodiment of the present invention, but the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

Cellulose is used in a wide range of industrial fields such as fiber, paper, non-woven fabric, fabric and filter. By utilizing the present invention, the function of amylose can be imparted to cellulose or cellulose products. For example, amylose can form inclusion complexes with various chemicals, and it can be used to design composite functional materials and devices such as filters and fragrance-containing fibers that have environmental purification functions. Become.

Claims (17)

1. a graft polymer in which α-1,4 glucan side chains are bonded to a cellulose main chain,
   The first carbon at the end of the α-1,4 glucan side chain is covalently bonded via the —NH— group to the 6th carbon of at least one glucose residue in the cellulose backbone;
   Two hydrogen groups are bonded to the first carbon atom, or one oxygen atom is bonded;
   The degree of polymerization of the cellulose main chain is 25 or more;
   The degree of polymerization of the α-1,4 glucan side chain is 25 or more.
   Graft polymer.
2. The graft polymer according to claim 1, wherein the α-1,4 glucan side chain is completely linear.
3. The graft polymer has the following structure (I):
   

   

   Where each R is —OH, —NH ₂ and the following structure (II) or (III):
   

   

   Independently selected from the group consisting of: n is an integer from 25 to 20,000, m is an integer from 24 to 1000, and glucose residues in the cellulose main chain are β-1,4 bonded The graft polymer according to claim 1, which is bound by:
4. The graft polymer according to claim 1, wherein the polymerization degree of the α-1,4 glucan side chain is 40 or more.
5. The graft polymer according to claim 1, wherein the polymerization degree of the α-1,4 glucan side chain is 50 or more.
6. The graft polymer according to claim 1, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 10,000.
7. The graft polymer according to claim 1, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 50 per 100 glucose residues in the cellulose main chain.
8. A method for producing the graft polymer according to claim 1,
   Replacing the OH group bonded to the 6-position carbon of at least one glucose residue in cellulose with an NH ₂ group to obtain an aminated cellulose;
   By reacting the aminated cellulose with a primer, the 6-position carbon of at least one glucose residue in the aminated cellulose and the 1-position carbon of the end of the primer are covalently bonded via an —NH— group. Obtaining primer-bound cellulose;
   By extending the primer by contacting the primer-bound cellulose, a substrate, and an α-1,4 glucan chain extender, an α-1,4 glucan side chain having a degree of polymerization of 25 or more is formed. Including the step of obtaining the graft polymer according to Item 1,
   The primer is an α-1,4 glucan having a degree of polymerization of 3 to 20, or a reducing end oxide or lactonized product thereof,
   The substrate is a substance that provides a glucose residue to the primer by the action of the enzyme;
   The degree of polymerization of the α-1,4 glucan side chain is 25 or more;
   In the α-1,4-glucan-bonded cellulose, a method in which two hydrogen groups are bonded to the 1-position carbon or one oxygen atom is bonded.
9. The method according to claim 8, wherein the polymerization degree of the α-1,4 glucan side chain is 40 or more.
10. The method according to claim 8, wherein the degree of polymerization of the α-1,4 glucan side chain is 50 or more.
11. The method according to claim 8, wherein the number of α-1,4 glucan bound to the cellulose is 1 to 10,000.
12. The method according to claim 8, wherein the number of α-1,4 glucan side chains bonded to the cellulose main chain is 1 to 50 per 100 glucose residues in the cellulose main chain.
13. The method according to claim 8, further comprising the step of purifying the graft polymer.
14. Obtaining the aminated cellulose comprises
    Reacting cellulose with p-toluenesulfonyl chloride in the presence of triethylamine, lithium chloride and N, N-dimethylacetamide to obtain tosylated cellulose;
    Reacting the tosylated cellulose with sodium azide in the presence of tetra-n-butylammonium iodide and dimethyl sulfoxide to give 6-azido-6-deoxycellulose; and the 6-azido-6 in dimethyl sulfoxide The process according to claim 8, which is carried out by reacting deoxycellulose with sodium borohydride to obtain aminated cellulose.
15. The method according to claim 8, wherein the α-1,4 glucan chain extender is α-glucan phosphorylase and the substrate is glucose-1-phosphate.
16. The method according to claim 8, wherein the α-1,4 glucan chain extender is α-glucan phosphorylase and sucrose phosphorylase, and the substrate is sucrose and inorganic phosphate or glucose-1-phosphate.
17. The method according to claim 8, wherein the α-1,4 glucan chain extender is amylosucrase and the substrate is sucrose.

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