WO2016140369A1 - Cellulose three-dimensional structure and production method therefor - Google Patents

Abstract

The purpose of the present invention is to provide a cellulose structure having novel characteristics. Specifically, the present invention pertains to synthesizing a cellulose three-dimensional structure having novel characteristics by using various polymers in the synthesis of a cellulose using cellodextrin phosphorylase (CDP).

Description

Cellulose three-dimensional structure and method for producing the same

The present invention relates to, for example, an artificially synthesized cellulose three-dimensional structure and a method for producing the same.

Cellulose is the most abundant organic polymer on earth. Cellulose is a material material attracting attention because of its high sustainability in addition to its resource. Cellulose obtained from natural products by mechanical treatment or chemical treatment is known to be in a fibrous form, and its use according to its structure and physical properties is being developed. In addition, cellulose nanocrystals (CNC) obtained by acid treatment of natural resources are expected to be used as new cellulose materials. CNC has an I-type crystal structure in which cellulose chains are arranged in parallel, and has attracted attention as a filler for composite materials because of its high aspect ratio, excellent mechanical strength, thermal stability, and the like.
On the other hand, it is known that crystalline cellulose can also be synthesized artificially (since cellulose obtained by artificial synthesis is generally an oligomer, such cellulose is called cellodextrin. All called cellulose). The following reaction shows the enzymatic synthesis of cellulose using the reverse reaction of cellodextrin phosphorylase (CDP).

As shown in the above reaction, a cellulose crystal (cellulose nanosheet having a nanosheet structure having a length of several μm, a width of several hundreds of nm, and a thickness of 4.5 nm is obtained by enzymatic synthesis utilizing a reverse reaction of CDP which is a phosphorolytic enzyme. CNS) is obtained (Non-patent Document 1). In the synthesis, α-D-glucose monophosphate (αG1P) is sequentially polymerized as a monomer with respect to D-(+)-glucose serving as a primer. Compared with the ability to recognize a substrate that CDP inherently hydrolyzes, D-(+)-glucose used for cellulose synthesis is difficult to recognize, but as a result, the polymerization reaction proceeds successfully. In addition, CNS has a more stable antiparallel chain type II crystal structure, unlike naturally occurring type I crystals. Therefore, the cellulose chains are arranged in antiparallel to the thickness direction in the CNS, and as a result, the reducing end and non-reducing end of cellulose are regularly exposed on the sheet surface. CNS is stably dispersed in pure water, but has a property of dissolving in an aqueous NaOH solution.

In the synthesis of cellulose using the above-mentioned cellodextrin phosphorylase (CDP), it is considered that by adding various polymers, the interaction between cellulose molecules is changed and a cellulose structure having novel properties can be synthesized. .
In view of these circumstances, an object of the present invention is to synthesize a cellulose structure having novel characteristics using various polymers in the synthesis of cellulose using cellodextrin phosphorylase (CDP). .
As a result of diligent research to solve the above problems, in the synthesis of cellulose using cellodextrin phosphorylase (CDP), by adding various polymers such as cellulose nanocrystals and water-soluble polymers, new characteristics can be obtained. The present inventors have found that a cellulose three-dimensional structure having the above can be synthesized and have completed the present invention.
That is, the present invention includes the following.
(1) The following formula (I):

(In the formula, n is 4 to 10)
The cellulose three-dimensional structure which contains the compound shown by these as a structural component.
(2) The cellulose three-dimensional structure according to (1), which contains a polymer.
(3) The cellulose three-dimensional structure according to (2), wherein the polymer is a cellulose nanocrystal or a water-soluble polymer.
(4) The cellulose three-dimensional structure according to (3), wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, dextran and polyvinylpyrrolidone.
(5) The three-dimensional cellulose structure according to (3) or (4), wherein the water-soluble polymer contained is removed.
(6) The cellulose three-dimensional structure according to any one of (1) to (5), wherein the cellulose three-dimensional structure has a three-dimensional network structure.
(7) The cellulose three-dimensional structure according to (6), wherein the three-dimensional network structure is sponge-like.
(8) A scaffold comprising the cellulose three-dimensional structure according to any one of (1) to (7).
(9) A film comprising the cellulose three-dimensional structure according to any one of (1) to (7).
(10) A separator including the cellulose three-dimensional structure according to any one of (1) to (7) or the film according to (9).
(11) A step of reacting α-D-glucose monophosphate and D-(+)-glucose as a primer with cellodextrin phosphorylase in the presence of a polymer, represented by the following formula (I):

(In the formula, n is 4 to 10)
The manufacturing method of the cellulose three-dimensional structure which contains the compound shown by these as a structural component.
(12) The method according to (11), wherein the polymer is a cellulose nanocrystal or a water-soluble polymer.
(13) The method according to (12), wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, dextran and polyvinylpyrrolidone.
(14) The method according to (12) or (13), further comprising a step of removing the water-soluble polymer from the cellulose three-dimensional structure.
(15) The method according to any one of (11) to (14), wherein the cellulose three-dimensional structure has a three-dimensional network structure.
(16) The method according to (15), wherein the three-dimensional network structure is sponge-like.
This specification includes the disclosure of Japanese Patent Application No. 2015-040735, which is the basis of the priority of the present application.

The photograph of the sample tube overturning test when the concentration of the maboya-derived cellulose nanocrystal (CNC) in the cellulose synthesis method 1 of Example 1 is changed (cellodextrin phosphorylase (CDP) is 0.20 U / mL) is shown. The photograph of the sample tube fall test when the CDP density | concentration in the synthesis method 1 of the cellulose of Example 1 is changed (Condition of maboya origin CNC is 0.10 w / v%) is shown. A scanning electron microscope (magnification of 20,000 times) of a uniformly sponged sample (conditions of maboya-derived CNC of 0.10 w / v% and CDP of 0.20 U / mL) in the cellulose synthesis method 1 of Example 1 ) Shows the observation results. The observation result of the AFM (atomic force microscope) of the uniformly sponged sample (conditions of maboya-derived CNC of 0.10 w / v% and CDP of 0.20 U / mL) in the cellulose synthesis method 1 of Example 1 Show. The analysis result by the total reflection infrared spectrophotometer of the sample sponge-formed uniformly in the synthesis method 1 of the cellulose of Example 1 is shown. The analysis result by the nuclear magnetic resonance spectroscopy apparatus of the cellulose sponge-formed uniformly in the cellulose synthesis method 1 of Example 1 is shown. MALDI-TOF-MS (Matrix-Assisted Laser Desorption) of Sample Sponge Uniformly Sponge in Cellulose Synthesis Method 1 of Example 1 (Condition of Maboya-derived CNC of 0.10 w / v% and CDP of 0.20 U / mL) An analysis result by an ionization time-of-flight mass spectrometer is shown. The numbers indicate the degree of polymerization of cellulose. The measurement result of the indentation fracture | rupture of the sample sponge-formed uniformly in the synthesis method 1 of the cellulose of Example 1 is shown. The photograph of the sample tube fall test when changing the tree origin CNC density | concentration in the synthesis method 1 of the cellulose of Example 1 (CDP is 0.20 U / mL conditions) is shown. The analysis result by the total reflection infrared spectrophotometer of the sample sponge-formed uniformly in the synthesis method 1 of the cellulose of Example 1 (Condition that wood origin CNC is 0.50 w / v%) is shown. Samples uniformly sponged in the cellulose synthesis method 1 of Example 1 (conditions where maboya-derived CNC is 0.10 w / v%, wood-derived CNC is 0.50 w / v%, CDP is 0.20 U / mL) The photograph after a sponge fracture | ruptures by pressing of a test stick | rod is shown. The photograph of the sample tube fall test when the polyethyleneglycol (PEG) density | concentration in the synthesis method 2 of the cellulose of Example 1 is changed (conditions where CDP is 0.20 U / mL) is shown. The photograph of the sample tube fall test when changing the CDP density | concentration in the synthesis method 2 of the cellulose of Example 1 (condition that PEG is 10 w / v%) is shown. Observation result of scanning electron microscope (left photo: magnification 5000 times) of the uniformly sponged sample (conditions in which PEG is 10 w / v% and CDP is 0.20 U / mL) in the cellulose synthesis method 2 of Example 1 Right photograph: 10000 times magnification). Sample tube inversion test of uniformly sponged sample (conditions of dextran (Dex), PEG or polyvinylpyrrolidone (PVP) 10 w / v%, CDP 0.20 U / mL) in cellulose synthesis method 2 of Example 1 And a result of scanning electron microscope observation (left photo) of a uniform sponged sample (PEG is 10 w / v%, CDP is 0.20 U / mL) and αG1P conversion rate (right graph) Indicates. The observation result of the scanning electron microscope of the sample (Dex is 10 w / v%, CDP is 0.20 U / mL) uniformly sponged in the cellulose synthesis method 2 of Example 1 is shown. The observation result of the scanning electron microscope of the sample (PVP is 10 w / v%, CDP is 0.20 U / mL) uniformly sponged in the cellulose synthesis method 2 of Example 1 is shown. The analysis result by the total reflection infrared spectrophotometer of the uniform sponge-formed sample (conditions of PEG of 10 w / v%, CDP of 0.20 or 0.40 U / mL) in the cellulose synthesis method 2 of Example 1 Show. The analysis result by the nuclear magnetic resonance spectrometer of the cellulose in the synthesis | combining method 2 of Example 1 of the sponge sponge uniformly (conditions of PEG is 10 w / v%, CDP is 0.20 or 0.40 U / mL) is shown. The chemical structure and the crystal structure analysis result of the uniformly sponged cellulose (Dex, PEG or PVP is 10 w / v%, CDP is 0.20 U / mL) in the cellulose synthesis method 2 of Example 1 are shown. The photograph of the sample tube fall test of the sponge-like cellulose structure (Dex is 10 w / v%, CDP is 0.20 U / mL) prepared by coexistence of Dex in the cellulose synthesis method 2 of Example 1 is shown. The photograph of the sample tube tipping test of the sponge-like cellulose structure prepared by the coexistence of Dex in the cellulose synthesis method 2 of Example 1 is shown. The measurement result of the indentation fracture | rupture of the sample sponge-formed uniformly in the synthesis method 2 of the cellulose of Example 1 is shown. Indentation of uniformly sponged sample (conditions of Dex = 10 w / v%, PEG = 10 w / v%, PVP = 10 w / v%, CDP = 0.20 U / mL) in cellulose synthesis method 2 of Example 1 The measurement result of a fracture is shown. The sponge was formed by pressing the test rod into the uniformly sponged sample (conditions in which PEG was 10 w / v%, Dex was 10 w / v%, and CDP was 0.20 U / mL) in the cellulose synthesis method 2 of Example 1. The photograph after a fracture is shown. The analysis result by the total reflection infrared spectrophotometer of the sample sponge-formed uniformly by the coexistence of Dex or PEG in the cellulose synthesis method 2 of Example 1 is shown. Sample prepared by coexistence of Dex in cellulose synthesis method 2 of Example 1 (Dex concentration is 10 w / v% with respect to CDP concentration of 0.20 or 0.40 U / mL, or Dex concentration of 2 to 20 w / NMR measurement of CDP concentration with respect to v% of 0.20 U / mL) and sample (Dex concentration with respect to CDP concentration of 0.05 to 0.40 U / mL is 10 w / v%, or The analysis results by IR measurement under the condition that the CDP concentration is 0.20 U / mL with respect to the Dex concentration of 2 to 20 w / v% are shown. Samples uniformly sponged due to coexistence of Dex in the cellulose synthesis method 2 of Example 1 (Dex concentration is 10 w / v% with respect to CDP concentration of 0.20 or 0.40 U / mL, or Dex concentration The analysis results by NMR measurement under the condition that the CDP concentration is 0.20 U / mL with respect to 2 to 20 w / v% are shown. The observation result of the AFM (atomic force microscope) of the sample sponge-formed uniformly (Dex is 10 w / v%, CDP is 0.20 U / mL) in the cellulose synthesis method 2 of Example 1 is shown. The observation result when the sponge-like cellulose structure in Example 1 is immersed in an enzyme aqueous solution is shown.

1. Cellulose three-dimensional structure according to the present invention The cellulose three-dimensional structure according to the present invention has the following formula (I):

(In the formula, n is 4 to 10)
Is contained as a constituent component. Unlike the type I cellulose structure in which naturally occurring cellulose chains are arranged in parallel, the cellulose three-dimensional structure according to the present invention has a more stable antiparallel chain type II cellulose structure.
The degree of polymerization of the compound of the formula (I) is, for example, 6 or more, 7 or more, preferably 8 or more (that is, n is 4 or more, 5 or more, preferably 6 or more in the compound of the formula (I)), and 12 or less, preferably 11 or less (that is, in the compound of formula (I), n is 10 or less, preferably 9 or less).
The cellulose three-dimensional structure according to the present invention has a three-dimensional network structure such as a sponge-like structure (ribbon-like cellulose network) due to the coexistence of polymers in the synthesis of cellulose using cellodextrin phosphorylase (CDP). It becomes the three-dimensional structure which has. In the three-dimensional structure, the voids in the network structure are connected to the outer surface (thus, the solvent (moisture) goes out). Accordingly, the three-dimensional structure has a structure in which the solvent (moisture) comes out on the outer surface while being collapsed (applying pressure), accompanied by the collapse of the structure corresponding to the pressure.
Due to the coexistence of the polymer during synthesis, the cellulose three-dimensional structure according to the present invention contains a polymer. Examples of the polymer include cellulose nanocrystals and water-soluble polymers. Cellulose nanocrystals are of natural origin such as maboya or wood. Cellulose nanocrystals derived from maboya or wood are described in J. Am. Araki et al., Colloids Surf. A: Physicochem. Eng. It can be produced according to the method described in Aspects, 1998, 142, 75-82. Examples of the water-soluble polymer include polyethylene glycol (for example, molecular weight: 15000 to 25000), dextran (for example, molecular weight: 90000 to 210000), and polyvinylpyrrolidone (for example, weight average molecular weight: ca. 29000).
In addition, after the production of the three-dimensional cellulose structure according to the present invention, the water-soluble polymer can be removed to obtain a three-dimensional cellulose structure composed of the compound of formula (I) alone.
2. Production method of cellulose three-dimensional structure according to the present invention The cellulose three-dimensional structure according to the present invention described above can be produced according to the reaction shown below.

Specifically, α-D-glucose monophosphate (αG1P) and D-(+)-glucose as a primer in the presence of a polymer are obtained by enzymatic synthesis utilizing the reverse reaction of cellodextrin phosphorylase (CDP). The cellulose three-dimensional structure according to the present invention can be produced by reacting with CDP. For D-(+)-glucose acting as a primer, αG1P is sequentially polymerized as a monomer.
αG1P and D-(+)-glucose may be commercially available products.
On the other hand, CDP is, for example, M.P. Krishnareddy et al. Appl. Glycosci. , 2002, 49, 1-8. Specifically, M.M. Krishnareddy et al. Appl. Glycosci. , 2002, 49, 1-8, CDP derived from Clostridium thermocellum YM4 strain can be prepared.
The enzyme amount of CDP is determined by, for example, incubating α-D-glucose monophosphate, D-(+)-cellobiose and CDP, quantifying the phosphate produced by CDP, and adding 1 μmol of phosphate per minute. The amount of enzyme released can be determined as 1U.
For example, 10 to 1000 mM (preferably 100 to 300 mM) αG1P, 10 to 200 mM (preferably 50 to 60 mM) D-(+)-glucose, polymer (for example, 0.001 to 1 w in the case of cellulose nanocrystals) / V%, preferably 0.02 to 0.50 w / v%; in the case of water-soluble polymers, 2 to 20 w / v%, 5 to 20 w / v%, preferably 10 w / v%), and 0.01 ~ 1.5 U / mL (preferably 0.05 to 0.50 U / mL) of CDP is added to 100 to 1000 mM (preferably 250 to 750 mM) of 2- [4- (2-hydroxyethyl) -1-piperazinyl] Mix in ethanesulfonic acid (HEPES) buffer (pH 7.0 to 8.0 (preferably pH 7.5)) and mix at 10 to 80 ° C. (preferably 20 to 60 ° C.) for 0.5 to 30 days ( Preferably 1-14 days Incubate and react. In this manner, the cellulose three-dimensional structure according to the present invention can be manufactured.
Moreover, the water-soluble polymer used at the time of manufacture can be removed from the manufactured cellulose three-dimensional structure. The removal of the water-soluble polymer can be performed, for example, by immersing the cellulose three-dimensional structure in water.
3. 3. Use of cellulose three-dimensional structure according to the present invention 3-1. Medical field 3-1-1. Cell culture scaffold The cellulose three-dimensional structure according to the present invention can be used as a scaffold for growing animal cells on its surface, for example.
The cells to be grown may be derived from animals, and examples include cells derived from mammals, reptiles, or insects. Further, primary cultured cells isolated from organs and tissues such as heart, liver, spleen and epidermis, subcultured established cells and tumor cells may be used. Furthermore, somatic stem cells such as mesenchymal stem cells (MSC), induced pluripotent stem cells, CHO cells, BHK cells, cell lines such as Vero cells, and the like may be used.
Examples of the medium used for cell culture include a basal medium in which low molecular weight compounds such as amino acids and vitamins are added to a balanced salt solution. In addition, a medium supplemented with serum or serum / tissue extract, hydrolyzate, growth factor, hormone, carrier protein, lipid, polyamic acid, trace element, vitamin, thickener, surfactant, cell adhesion factor, etc. It can also be used.
The cellulose three-dimensional structure can be formed into a thin film and cells can be cultured on the surface of the thin film. The culture efficiency can be increased by supplying the nutrient components also from the bottom surface of the thin film.
Cells grown in a monolayer on the cellulose three-dimensional structure can be detached and recovered with a cell release agent. As the cell release agent, a chelating agent for removing divalent cations such as ethylenediaminetetraacetic acid (EDTA), a protease such as trypsin for cell-matrix, cell-cell adhesion protein, or cellulase can be used. The cell can also be detached by decomposing and solubilizing part or all of the cellulose three-dimensional structure. In the latter method using no protease, the cell-cell adhesion does not peel off and does not affect the cells, so that a highly active suspension cell or cell sheet can be obtained.
The obtained cell sheet was laminated with the obtained cell sheet in the same manner, or the surface of the cell sheet was coated with an intercellular adhesion protein or the like, and further cultured, the cells were three-dimensionally thick. A cell mass can be obtained.
3-1-2. Scaffold for high-concentration cell culture The cellulose three-dimensional structure according to the present invention can be used as a scaffold for growing animal cells therein. The cellulose three-dimensional structure can be formed into a structure having voids of a size that allows cells to enter or larger, and the cells can be cultured in the structure.
The cells to be grown may be any cells as long as they are derived from animals, as described in Section 3-1-1 above. The medium used for the culture may be a basal medium, a serum-added medium, or a medium supplemented with other components, as described in Section 3-1-1 above.
By culturing the cells in the cellulose three-dimensional structure, cell damage due to shearing stress generated during suspension suspension culture does not occur, and the concentration of cells per culture solution can be increased.
In addition, if rCHO cells and the like that have been given the ability to produce biopharmaceuticals such as antibodies and proteins are cultured at a high concentration in the cellulose three-dimensional structure, the production amount of antibodies and proteins per batch will increase, Manufacturing cost can be reduced. Furthermore, after collecting the target product, a new medium is added and cultured again, or a medium containing the target product is continuously collected and a new medium is additionally added. You can get a product.
Furthermore, cells that have been cultured at a high concentration can be recovered by subjecting the three-dimensional cellulose structure used as a scaffold to cellulase treatment to release the cells without causing damage to the cells as occurs during protease treatment. be able to. Totipotent stem cells such as iPS cells and ES cells can be cultured in a large amount by this technique, and cells necessary for regenerative medicine and drug discovery industry can be supplied.
3-1-3. Three-dimensional structure scaffold The cellulose three-dimensional structure according to the present invention can be used as a scaffold for growing animal cells therein. The cellulose three-dimensional structure can be formed into a structure having voids of a size that allows cells to enter or larger, and the cells can be cultured in the structure.
The cells to be grown may be any cells as long as they are derived from animals, as described in Section 3-1-1 above. The medium used for the culture may be a basal medium, a serum-added medium, or a medium to which other components are added, as described in Section 3-1-1 above.
The cellulose three-dimensional structure can be formed into a shape suitable for a target tissue before, during or after cell culture and used for regeneration of a living tissue or organ. In the case of molding before cell culture, the cellulose three-dimensional structure can be reacted in a mold having a desired shape during the polymerization reaction, or can be formed into a desired shape by cutting, laminating, weaving, etc. after molding. .
The cellulose three-dimensional structure can be decomposed and solubilized by cellulase treatment without affecting the cells. Prior to transplantation into the organ, it may be subjected to cellulase treatment, and part or all may be decomposed and solubilized, or may be transplanted together with the scaffold without being subjected to cellulase treatment.
Furthermore, the three-dimensional structure in which the cells are grown is used for basic medical research such as elucidation of the mechanism of cancer metastasis, evaluation of the efficacy of anticancer drugs, etc., assuming that the shape and function of the tissue or organ are reproduced ex vivo. Alternatively, it can be used as a material for studying the effects of cosmetics, which have been prohibited from animal tests in recent years, on living tissues such as skin.
3-1-4. Transplant Material to Bone Defect Site The cellulose three-dimensional structure according to the present invention can also be used as a transplant material to an animal bone defect site and a periodontal tissue regeneration-inducing material. The cellulose three-dimensional structure can be easily molded into a desired shape and can be sterilized by high-pressure steam. The cellulose three-dimensional structure may be engrafted with cells before transplantation, or may be transplanted as it is.
3-2. Environment / Energy Field As described below, the three-dimensional cellulose structure according to the present invention includes a film (for example, a microporous film (application example: separator, adsorbent, biosensor, etc.), a dense film (application). For example, it can be used in the environment / energy field as a barrier film or the like)).
3-2-1. Application to Battery Separator The three-dimensional cellulose structure according to the present invention can be used as a separator for a secondary battery such as a lithium ion battery. By using it as a separator, it has higher heat resistance than conventional polyolefin separators. For example, when the battery overheats due to overcharging, etc., it prevents the separator from melting and causing an internal short circuit. And safety can be improved.
3-2-2. Adsorbent, concentration (biodegradable)
The cellulose three-dimensional structure according to the present invention is obtained by dispersing and supporting adsorbent fine particles such as silica, alumina, zeolite, Prussian blue, etc., toxic gases such as ammonia and aldehydes, harmful substances such as radioactive waste, It is possible to absorb and recover valuable metals.
In addition, since the cellulose three-dimensional structure can be easily decomposed using a degrading enzyme, it has an advantage that the substance collected by adsorption can be easily concentrated as necessary.
3-2-3. Biosensor The three-dimensional cellulose structure according to the present invention can be used as a biosensor by supporting an enzyme or an antibody. For example, if an enzyme such as glucose oxidase is supported, it can be used as a glucose sensor.
3-2-4. Gas barrier sheet (including structure)
By densely supporting the inorganic nanomaterial on the cellulose three-dimensional structure according to the present invention, for example, a gas barrier sheet that blocks gas such as water vapor can be obtained. Examples of the inorganic nanomaterial include nanoparticles such as silica or clay such as montmorillonite.
3-2-5. Heat-dissipating sheet It is possible to use as a heat-dissipating material by densely supporting a material having high thermal conductivity on the three-dimensional cellulose structure according to the present invention. Examples of the material having high thermal conductivity include carbon-based materials such as diamond, graphene, and carbon nanotubes, metal materials such as silver, copper, gold, and aluminum, and inorganic materials such as alumina, magnesia, and hexagonal boron nitride.
3-2-6. Thermal storage sheet It is possible to use as a thermal storage material by disperse | distributing and carrying the material which has thermal storage property in the cellulose three-dimensional structure which concerns on this invention. Examples of the heat storage material include latent heat storage materials such as erythritol, sodium acetate trihydrate, sodium sulfate decahydrate, paraffin, and Fe-Co alloy, and other sensible heat storage materials and chemical heat storage materials. The latent heat storage material uses the phase transition of the substance, and in the case of the heat storage material using the solid-liquid phase transition, it is necessary to devise so that no leakage occurs when it becomes liquid, such as petroleum resin There is a method of inclusion in the capsule.
3-2-7. Separation and purification substrate (column packing, electrophoresis)
Since the cellulose three-dimensional structure according to the present invention has a nano-sized space, it can be used as a separation / purification column or a packing for electrophoresis using the space.
EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
In the following examples, abbreviations indicate the following terms: CDP: cellodextrin phosphorylase, CNC: cellulose nanocrystal, PEG: polyethylene glycol, Dex: dextran, PVP: polyvinylpyrrolidone.

Synthesis of sponge-like cellulose structure Experimental method 1-1. Material α-D-glucose monophosphate disodium n hydrate (for biochemistry) was purchased from Wako Pure Chemical Industries, Ltd. D-(+)-glucose (special grade, biotechnology grade) and D-(+)-cellobiose (special grade) were purchased from Nacalai Tesque.
CDP is an M.I. Krishnareddy et al. Appl. Glycosci. , 2002, 49, 1-8.
Avicel was purchased from Sigma-Aldrich. Maboya-derived and Avicel-derived CNCs are Araki et al., Colloids Surf. A: Physicochem. Eng. Prepared by a method similar to that described in Aspects, 1998, 142, 75-82.
PEG (molecular weight 15000-25000) was purchased from Nacalai Tesque, and Dex (molecular weight 90000-210000) was purchased from Wako Pure Chemical Industries, Ltd. PVP has a weight average molecular weight of ca. The 29,000 one was purchased from Sigma-Aldrich.
2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (Good buffer) and 3- (N-morpholino) propanesulfonic acid (Biotechnology grade) were purchased from Nacalai Tesque.
A 4 mol / L aqueous sodium hydroxide solution was purchased from Nacalai Tesque.
Acetic acid (special reagent for spectrum) and sodium acetate (special grade) were purchased from Nacalai Tesque.
Cellulase was purchased from Sigma-Aldrich from Trichoderma violet.
As the ultrapure water, water purified by a Milli-Q system (Milli-Q Advantage A10, Millipore) was used.
40% sodium bicarbonate / heavy aqueous solution, glycerol (special grade), kanamycin sulfate (for biochemistry), isopropyl-β-D (−)-thiogalactopyranoside (for biochemistry), N, N, N ′, N'-tetramethylethylenediamine (special grade) was purchased from Wako Pure Chemical.
Acrylamide (for electrophoresis), N, N′-methylenebisacrylamide (for electrophoresis), sodium dodecyl sulfate (A.C.S. reagent), ammonium persulfate (for molecular biology, for electrophoresis), 2,5-dihydroxybenzoic acid (Matrix substantence for MALDI-MS), peptide standard used for calibration of heavy water, MALDI-TOF-MS measurement, and 1% aqueous trifluoroacetic acid and acetonitrile (ProteoMass ^™ Peptide and Protein MALDI-MS Calibration Kit) from Sigma-Aldri Purchased.
Carbon tape and dotite were purchased from Nissin EM.
Dithiothreitol (special reagent for molecular biology research), L (+)-ascorbic acid (biotechnology grade), hexaammonium heptamolybdate tetrahydrate (special grade), zinc acetate dihydrate (special grade), phosphorus Sodium dihydrogen acid dihydrate (special grade), sodium chlorite (chemical grade), sodium hydroxide (special grade), potassium hydroxide (special grade), imidazole (special reagent for molecular biology research), sodium chloride (molecule) Special reagent for biological research), sodium azide (special grade), tris (hydroxymethyl) aminomethane (special reagent for molecular biology research), 6M hydrochloric acid, 1-butanol (special grade), glycine (special grade for molecular biology research) Reagent), ethanol (biotechnology grade), ethanol (special reagent for spectrum), tert-butyl alcohol (special grade) are Naka It was purchased from Itesuku.
LB-BROTH LENNOX and LB-AGAR LENNOX were purchased from Funakoshi.
Ni-NTA agarose gel was purchased from QIAGEN.
1-2. Measurement of enzyme activity The activity of CDP was measured as follows. 50 mM 3- (N-morpholino) propanesulfonic acid buffer (pH 7.5) containing 10 mM α-D-glucose monophosphate, 10 mM D-(+)-cellobiose, and CDP diluted at a predetermined magnification. Incubated at 37 ° C. Phosphoric acid produced by CDP was quantified, and U / mL was defined as the enzyme activity when the amount of enzyme that liberates 1 μmol of phosphoric acid per minute was defined as 1 U. The dilution rate of CDP was determined such that the conversion rate of α-D-glucose monophosphate was 10% or less when the reaction time was 100 minutes.
1-3. Cellulose synthesis method 1: coexistence of CNC The monomer α-D-glucose monophosphate is 200 mM, the primer D-(+)-glucose is 50 mM, CDP is 0.01 to 0.40 U / mL, and the CNC is These were mixed in 500 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer (pH 7.5) so as to be 0.001 to 0.50 w / v%. The reaction was carried out at 60 ° C. for 3 days. The produced sponge-like cellulose structure was immersed in ultrapure water for 1 week and purified.
1-4. Cellulose synthesis method 2: Coexistence of water-soluble polymer Monomer α-D-glucose monophosphate is 200 mM, primer D-(+)-glucose is 50 mM, CDP is 0.05 to 0.40 U / mL. , PEG, Dex or PVP so that they are 2 to 20 w / v% or 5 to 20 w / v%, these are added with 500 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer. (PH 7.5) and mixed at 60 ° C. for 3 days. The produced sponge-like cellulose structure was immersed in ultrapure water for 1 week and purified.
1-5. Cellulose Evaluation Method The presence or absence of sponge formation was evaluated by a sample tube overturning test (determined that the sample tube did not flow when the sample tube was placed upside down).
The structure of the sponge-like cellulose structure was determined by freeze-drying the sponge-like cellulose structure and then using a scanning electron microscope (JSM-7500F, JEOL) and a total reflection infrared spectrophotometer (FT / IR-4100, JASCO). evaluated.
The structure and average degree of polymerization of cellulose were evaluated by a nuclear magnetic resonance spectrometer (DPX-300, Bruker) after freeze-drying the sponge-like cellulose structure and adding a 4% sodium bicarbonate / heavy aqueous solution. In addition, in the case of the cellulose obtained in Section 1-3, the coexisting CNC was removed by centrifugation after adding a 4% sodium bicarbonate / heavy aqueous solution.
A sponge having a diameter of 3 mm was pushed into the sponge-like cellulose at a speed of 1 mm / min by a universal small tester (AGS-X, Shimadzu Corp.) to break the sponge. At this time, the state of cellulose was evaluated from the state after the fracture.
In addition, the structure of the sponge-like cellulose structure includes AFM (atomic force microscope; SPM-9600, Shimadzu Corporation) and MALDI-TOF-MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer; AXIMA-performance, Shimadzu). Evaluation was performed by a manufacturing company).
1-6. Enzymatic degradation of sponge-like cellulose structure Enzymatic degradation of sponge-like cellulose was performed in a columnar shape having a thickness of 0.75 mm and a diameter of 8.5 mm in 50 mM acetic acid-sodium acetate buffer (pH 4.8) containing 3 mg / mL cellulase. This was evaluated by immersing the sponge-like cellulose and reacting at 50 ° C., and observing the change in size over time.
2. Result 2-1. Cellulose Synthesis Method 1: Coexistence of CNC FIG. 1 shows a photograph of a sample tube overturning test when the concentration of maboya-derived CNC was changed. Due to the coexistence of the CNC, all or part of the solution stopped flowing after the reaction. When the CNC was 0.01 w / v% or less, a part of the solution was spongy, but it was non-uniform. On the other hand, when the CNC was 0.02 w / v% or more, it was spongy uniformly.
FIG. 2 shows a photograph of the sample tube overturning test when the CDP concentration was changed. At a concentration of CDP of 0.025 U / mL or more, the solution stopped flowing after the reaction. When the concentration of CDP was 0.025 U / mL, a part of the solution was spongy, but nonuniform. On the other hand, when the concentration of CDP was 0.05 U / mL or more, it was sponged uniformly.
FIG. 3 shows an observation result of a uniform sponged sample with a scanning electron microscope (magnification of 20,000 times). A network structure (ribbon-like cellulose network) characteristic of the sponge-like cellulose structure was observed.
FIG. 4 shows the observation results of an AFM (atomic force microscope) of a uniformly sponged sample (conditions of maboya-derived CNC of 0.10 w / v% and CDP of 0.20 U / mL). The ribbon thickness characteristic of the sponge-like cellulose structure was found to be approximately 5.7 nm.
FIG. 5 shows the result of analysis of a uniform sponged sample by a total reflection infrared spectrophotometer. Peak derived from a type II cellulose is observed around 3488cm ^-1 and 3445cm ^-1, it was found to be sponge-like cellulosic structure including type II cellulose.
FIG. 6 shows the result of analysis of cellulose sponged uniformly by a nuclear magnetic resonance spectrometer. A cellulose-derived peak was observed in the vicinity of 3 to 5 ppm, and the average degree of polymerization determined from the proton ratio of the 1-position CH group of the glucose unit at the reducing end to the 1-position CH group of the other glucose unit was 9 to 10. .
FIG. 7 shows a MALDI-TOF-MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis) of a uniformly sponged sample (conditions of maboya-derived CNC of 0.10 w / v% and CDP of 0.20 U / mL). The result of the analysis is shown. The degree of polymerization was 6-12.
FIG. 8 shows the measurement results of the indentation fracture of the uniformly sponged sample. The strength increased as the CDP concentration and the concentration of maboya-derived CNC increased.
9A and 9B show a photograph of the sample tube overturning test when the tree-derived CNC concentration was changed (FIG. 9A) and an analysis result by a total reflection infrared spectrophotometer (FIG. 9B). Due to the coexistence of the CNC, all or part of the solution stopped flowing after the reaction. When the CNC was 0.10 w / v% or less, a part of the solution was spongy, but was non-uniform. On the other hand, when the CNC was at a concentration of 0.50 w / v%, it was spongy uniformly. As a result of analysis by the total reflection infrared spectrometer, a peak derived from a type II cellulose is observed around 3488cm ^-1 and 3445cm ^-1, it was found to be sponge-like cellulosic structure including type II cellulose.
FIG. 10 shows a photograph after the sponge is broken by pressing the test rod into the uniformly sponged sample. By pushing the test rod into the sample, the squeezed water (arrow part) was observed, and it was confirmed that the sample was sponge-like.
2-2. Cellulose Synthesis Method 2: Coexistence of Water-Soluble Polymers FIG. 11 shows a photograph of a sample tube tipping test when the PEG concentration is changed. At a concentration of 5 w / v% PEG, the solution flowed slowly. On the other hand, when the concentration of PEG was 10 w / v%, it was spongy uniformly. When the concentration of PEG was 20 w / v%, the solution was separated into two phases, and only the lower phase was spongy.
FIG. 12 shows a photograph of the sample tube overturning test when the CDP concentration was changed. At a concentration of CDP of 0.20 U / mL or more, the solution stopped flowing after the reaction and was uniformly spongy.
FIG. 13 shows the results of observation of a uniformly sponged sample with a scanning electron microscope (left photo: magnification 5000 times; right photo: magnification 10,000 times). A network-like structure (network) characteristic of the sponge-like cellulose structure was observed.
FIG. 14 shows a photograph of a sample tube overturning test of a uniformly sponged sample and an observation result of a scanning electron microscope of a uniformly sponged sample (condition that PEG is 10 w / v% and CDP is 0.20 U / mL). (Left photo) and αG1P conversion (right graph). Ribbon-like structures were seen in spongy samples prepared using PEG. In addition, the conversion rate of αG1P when no additive was added was about 35%, whereas when the water-soluble polymer was added, it was lower than that.
15 and 16 show the results of observation of a uniform sponged sample with a scanning electron microscope. A network structure (ribbon) was seen in spongy samples prepared using Dex or PVP.
FIG. 17 shows the analysis result of a uniformly spongy sample by a total reflection infrared spectrophotometer. Peak derived from a type II cellulose is observed around 3488cm ^-1 and 3445cm ^-1, it was found to be sponge-like cellulosic structure including type II cellulose.
FIG. 18 shows the result of analysis of cellulose sponged uniformly by a nuclear magnetic resonance spectrometer. A cellulose-derived peak was observed in the vicinity of 3 to 5 ppm, and the average degree of polymerization determined from the proton ratio of the 1-position CH group of the glucose unit at the reducing end to the 1-position CH group of the other glucose units was 9 to 11. .
FIG. 19 shows the chemical structure / crystal structure analysis results of uniformly sponged cellulose. Sponge cellulose was found to be composed of crystalline cellulose oligomers.
FIG. 20 shows a photograph of a sample tube overturning test of a sponge-like cellulose structure prepared by the coexistence of Dex. Due to the coexistence of Dex, the solution stopped flowing after the reaction and was similarly spongy.
FIG. 21 shows a photograph of a sample tube overturning test of a sponge-like cellulose structure prepared by the coexistence of Dex. Sponge was uniformly formed with a CDP concentration of 0.20 U / mL or more and a Dex concentration of 5 w / v% or more.
22 and 23 show the measurement results of the indentation breakage of the uniformly sponged sample. The strength increased as the concentration of CDP increased. Moreover, compared with sponge-like cellulose prepared by coexistence of PEG or PVP, the strength of sponge-like cellulose prepared by coexistence of Dex was higher.
FIG. 24 shows a photograph after the sponge was broken by pressing the test rod into the uniformly sponged sample. By squeezing the test rod into the sample, the squeezed water was observed, confirming that the sample was sponge-like.
FIG. 25 shows the result of analysis by a total reflection infrared spectrophotometer of a sample sponged uniformly by coexistence of Dex or PEG. Peak derived from a type II cellulose is observed around 3488cm ^-1 and 3445cm ^-1, it was found to be sponge-like cellulosic structure including type II cellulose.
FIG. 26 shows a sample prepared by the coexistence of Dex (the Dex concentration is 10 w / v% with respect to the CDP concentration of 0.20 or 0.40 U / mL, or the CDP with respect to the Dex concentration of 2 to 20 w / v%. NMR measurement under conditions where the concentration is 0.20 U / mL) and a sample (CDP concentration 0.05 to 0.40 U / mL, Dex concentration is 10 w / v%, or Dex concentration 2 to 20 w / The analysis result by IR measurement under the condition that the CDP concentration is 0.20 U / mL with respect to v% is shown.
FIG. 27 shows a sample prepared by coexistence of Dex (the Dex concentration is 10 w / v% with respect to the CDP concentration of 0.20 or 0.40 U / mL, or the CDP with respect to the Dex concentration of 2 to 20 w / v%. The analysis result by NMR measurement under the condition that the concentration is 0.20 U / mL is shown. A cellulose-derived peak is observed in the vicinity of 3-5 ppm, and the average degree of polymerization determined from the proton ratio of the 1-position CH group of the glucose unit at the reducing end to the 1-position CH group of the other glucose unit is 8-10. I understood. In FIG. 26, the sample having a CDP concentration of 0.05 U / mL and the sample having a Dex concentration of 2 w / v% were not spongy. In FIG. 27, the sample having a Dex concentration of 2 w / v% was not spongy.
FIG. 28 shows an AFM (atomic force microscope) observation result of a uniformly sponged sample (conditions where Dex is 10 w / v% and CDP is 0.20 U / mL). The thickness of the nanoribbon was approximately 5.4 nm, which was in good agreement with the length of the molecular chain having a polymerization degree of 9 (4.7 nm).
2-3. Enzymatic degradation of sponge-like cellulose structure FIG. 29 shows the observation results when the sponge-like cellulose structure is immersed in an aqueous enzyme solution. It was confirmed that the size of the sponge decreased with the passage of time and was degraded by the enzyme. After the enzyme treatment, the maboya-derived CNC-added sponge-like cellulose structure was almost degraded in 48 hours, and the PEG-added sponge-like cellulose structure was almost degraded in 3 hours.

ADVANTAGE OF THE INVENTION According to this invention, the cellulose three-dimensional structure useful in the medical field | areas, such as a scaffold, and environmental / energy fields, such as a separator for storage batteries, can be provided.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (16)

1. Formula (I):
   

   

   (In the formula, n is 4 to 10)
   The cellulose three-dimensional structure which contains the compound shown by these as a structural component.
2. The cellulose three-dimensional structure according to claim 1, comprising a polymer.
3. The cellulose three-dimensional structure according to claim 2, wherein the polymer is a cellulose nanocrystal or a water-soluble polymer.
4. The cellulose three-dimensional structure according to claim 3, wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, dextran and polyvinylpyrrolidone.
5. The cellulose three-dimensional structure according to claim 3 or 4, wherein the water-soluble polymer contained therein is removed.
6. The cellulose three-dimensional structure according to any one of claims 1 to 5, wherein the cellulose three-dimensional structure has a three-dimensional network structure.
7. The cellulose three-dimensional structure according to claim 6, wherein the three-dimensional network structure is sponge-like.
8. A scaffold comprising the cellulose three-dimensional structure according to any one of claims 1 to 7.
9. A film comprising the cellulose three-dimensional structure according to any one of claims 1 to 7.
10. A separator comprising the cellulose three-dimensional structure according to any one of claims 1 to 7 or the film according to claim 9.
11. A step of reacting α-D-glucose monophosphate and D-(+)-glucose as a primer with cellodextrin phosphorylase in the presence of a polymer, represented by the following formula (I):
    

    

    (In the formula, n is 4 to 10)
    The manufacturing method of the cellulose three-dimensional structure which contains the compound shown by these as a structural component.
12. The method according to claim 11, wherein the polymer is a cellulose nanocrystal or a water-soluble polymer.
13. The method according to claim 12, wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, dextran and polyvinylpyrrolidone.
14. The method according to claim 12 or 13, further comprising a step of removing the water-soluble polymer from the cellulose three-dimensional structure.
15. The method according to any one of claims 11 to 14, wherein the cellulose three-dimensional structure has a three-dimensional network structure.
16. The method according to claim 15, wherein the three-dimensional network structure is sponge-like.

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