JP2018174871A - Nanoribbon structure comprising cellulose oligomer having amino group, and production method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To synthesize a cellulose structure having novel properties by using, as a primer, a glucose having an amino group.SOLUTION: The present invention provides a cellulose structure containing as a constituent a compound represented by a formula (I) in the figure, where m is from 4 to 14.SELECTED DRAWING: Figure 1

Description

  The present invention relates to, for example, artificially synthesized cellulose structures and methods for producing and using the same.

  Cellulose is the most abundant organic polymer on earth. Cellulose is an attractive material because it has high sustainability in addition to its resources. Cellulose obtained from a natural product by mechanical treatment or chemical treatment is known to be in a fibrous form, and utilization according to its structure and physical properties is 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 is attracting 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 be artificially synthesized (the cellulose obtained by artificial synthesis is generally an oligomer, so such cellulose is called cellodextrin. Here, without distinction). All called cellulose). The following reaction is an enzyme synthesis of cellulose utilizing the reverse reaction of a phospholytic enzyme, cellodextrin phosphorylase (CDP), which is α to D-(+)-glucose which works as a primer (starting material) Glucose-1-phosphate (αG1P) is polymerized sequentially as a monomer.

（図中、ｎはセルロースの重合度を示す。）

(In the figure, n represents the degree of polymerization of cellulose.)

By the above reaction, a cellulose crystal (cellulose nanosheet; CNS) having a nanosheet structure with a length of several μm, a width of several hundred nm, and a thickness of 4.5 nm is obtained (Non-patent Document 1). Furthermore, some of the present inventors, in the enzymatic synthesis of cellulose using the above CDP, C _2-5 as a primer _- glucose having branched alkyl group be straight-chain alkyl group or a C _2-5 main chain By using a derivative, it is possible to produce a cellulose nanostructure having a three-dimensional network structure containing a solvent such as a sponge-like structure (Patent Document 1), and when cellulose is similarly synthesized in the presence of a water-soluble polymer, It has been found that a two-dimensional crystal is further grown to form a nanoribbon-like network structure (Patent Document 2). The inventors of the present invention have further found that in the above enzyme synthesis of cellulose using CDP, a nanoribbon-like network structure is also formed by using a glucose derivative having a hydrophilic substituent having a repeating unit as a primer. (Patent Document 3).

International application number PCT / JP2015 / 071047 International Application Number PCT / JP2016 / 057253 Patent Application No. 2016-118227

M. Hiraishi et al. , Carbohydr. Res. , 2009, Vol. 344, pp. 2468-2473

  An object of the present invention is to synthesize a cellulose structure having novel properties by using glucose having an amino group as a primer.

  Cellulose structure comprising a cellulose derivative when glucose having an amino group is used as a primer in the synthesis of cellulose using CDP which is bound to the primer while dephosphorylating α-glucose-1-phosphate (αG1P) to the primer Found that enzyme synthesis can be performed in one step. In addition, the obtained cellulose structure was capable of adsorbing a protein which is an enzyme while maintaining the activity.

［式中、ｍは、４〜１４である］
で示される化合物を構成成分とするセルロース構造体を包含する。 The present invention provides the following formula (I):

[Wherein, m is 4 to 14]
And a cellulose structure having as a component the compound represented by

の化合物とを反応させる工程を含む、次式（Ｉ）：

［式中、ｍは、４〜１４である］
で示される化合物を構成成分とするセルロース構造体の製造方法を包含する。 The present invention further provides α-glucose 1-phosphate and the following formula (II) as a primer:

Reacting the compound with a compound of the following formula (I):

[Wherein, m is 4 to 14]
And a method for producing a cellulose structure comprising the compound represented by

  According to the present invention, it is possible to provide a cellulose-only structure useful in the medical field such as scaffolding and in the environment / energy field such as film.

The result of the inversion inversion test of the cellulose structure (FIG. 1 left) which has an amino group by this invention is shown (FIG. 1 right). ¹ shows the ¹ H NMR spectrum of cellulose structures with amino groups according to the invention. In the figure, n shows the polymerization degree of the cellulose structure which has an amino group. Fig. 6 shows a MALDI-TOF-MS spectrum of a cellulose structure having an amino group according to the present invention. Figure 2 shows a WAXD chart of a cellulose structure with amino groups according to the invention. Fig. 6 shows ATR / FTIR spectra of a cellulose structure having an amino group according to the present invention. Fig. 6 shows an AFM image of a cellulose structure having an amino group according to the present invention. Scale bars indicate 1 μm. Figure 2 shows a TEM image of a cellulose structure with amino groups according to the invention. The result of having evaluated the adsorption characteristic of serum albumin (BSA) to the cellulose structure (Primary amino group (+)) which has an amino group by this invention and the cellulose structure (-) which does not have an amino group is shown. In the graph of (a), the horizontal axis indicates the initial concentration of BSA (μM), and the vertical axis indicates the concentration of BSA (μM) in the supernatant after reaching adsorption equilibrium. In the graph of (b), the horizontal axis indicates the initial concentration (μM) of BSA, and the vertical axis indicates the adsorption amount (ngcm ⁻² ) of BSA per unit area of the cellulose structure after reaching adsorption equilibrium. Figure 2 shows an AFM image of the structure after mixing and incubation of the cellulose structure with amino groups according to the invention with BSA at an initial concentration of 5 μM. The result of having evaluated the adsorption characteristic of three proteins (fibrinogen, transferrin, IgG) to the cellulose structure which has an amino group by this invention is shown. In the graph of (a), the horizontal axis indicates the initial concentration (μM) of fibrinogen (upper), transferrin (middle) or IgG (lower), and the vertical axis indicates the respective proteins in the supernatant after reaching adsorption equilibrium. Indicates the concentration (μM) of The graph of (b) shows the adsorption amount of protein per unit area (longitudinal axis, ng cm ⁻² ) to the cellulose structure having the amino group of fibrinogen (upper), transferrin (middle) or IgG (lower), the equilibrium concentration Plotted against and fitted by a Langmuir-type adsorption isotherm. The result of having evaluated the adsorption characteristic of three proteins (cytochrome C, lysozyme, and trypsin) to the cellulose structure which has an amino group by this invention is shown. The horizontal axis shows the initial concentration (μM) of cytochrome C (left), lysozyme (middle) or trypsin (right), and the vertical axis shows the concentration (μM) of each protein in the supernatant after reaching adsorption equilibrium. Show. Figure 2 shows the change in zeta potential of a cellulose structure with an amino group according to the invention due to BSA adsorption. After mixing and incubating the initial concentration of BSA at 0, 1, 2, 3, 5, 10, and 20 μM and the cellulose structure having an amino group according to the present invention and after reaching adsorption equilibrium and removing unadsorbed BSA The zeta potential is being measured. The horizontal axis shows zeta potential (mV), and the vertical axis shows intensity. 6 shows the change in the amount of BSA adsorbed onto a cellulose structure having an amino group according to the present invention due to the change in salt concentration. Cellulose structure having an amino group calculated after reaching the adsorption equilibrium by mixing and incubating a cellulose structure having an amino group according to the present invention and BSA with an initial concentration of 5 μM at a salt concentration (mM) indicated by the middle axis of the graph. The amount of adsorption of BSA (ng cm ^-2 ) per unit area of the body is shown on the vertical axis. 6 shows the evaluation of the activity of β-Gal adsorbed to a cellulose structure having an amino group according to the present invention. A mixture of cellulose having a final concentration of 0.05% amino group and β-Gal having a final concentration of 5 nM is allowed to stand at 25 ° C. for 1 hour, and then separated into a supernatant and a precipitate, and the respective β-Gal activities Is shown. The relative activity when the activity of liberated β-Gal (nanoribbon (-)) is 100% is shown on the vertical axis. The result of having evaluated the cytotoxicity of the cellulose structure which has an amino group by this invention is shown. HeLa cells for 72 hours in the presence of 0% (w / v), 0.001% (w / v), 0.01% (w / v), 0.05% (w / v) cellulose structures After culture, live cells (green) and dead cells (red) were respectively stained and imaged with a fluorescence microscope. Scale bar indicates 100 μm. The result of having evaluated the cytotoxicity of the cellulose structure which has an amino group by this invention is shown. HeLa cells for 1 day in the presence of 0% (w / v), 0.001% (w / v), 0.01% (w / v) and 0.05% (w / v) cellulose structures Or, the survival rate (%) after 3 days of culture is shown.

［式中、ｍは、４〜１４である］
で示される化合物を構成成分として含有するものである。 1. The cellulose structure according to the present invention is
The following formula (II):

[Wherein, m is 4 to 14]
It contains the compound shown by these as a component.

  The average degree of polymerization of the compound represented by the above-mentioned formula (I) is 10, and the average degree of polymerization of cellulose oligomers (Average DP to 10) when enzyme synthesis is performed using D-glucose as a primer (T. Serizawa et al., Polym J., 2016, 48, 539-544). Moreover, the average degree of polymerization of the said compound is calculated with 10, and is comparable as the case where the above-mentioned D-glucose is used as a primer.

  The degree of polymerization of the compound of the formula (I) is, for example, 6 or more, preferably 7 or more (that is, in the compound of the formula (I), n is 4 or more, preferably 5 or more) and 16 or less, preferably 15 Or less (that is, in the compound of formula (I), n is 14 or less, preferably 13 or less).

  Unlike the type I cellulose structure in which naturally derived cellulose chains are arranged in parallel, the cellulose structure according to the present invention forms a cellulose type II crystal form which is thermodynamically stable. This is similar to the crystal form formed by the cellulose oligomer when D-glucose is used as a primer, and the 2-aminoethyl group at the end of the cellulose chain does not affect the crystal form. The cellulose derivative having a 2-aminoethyl group is aligned in the film thickness direction of the nanoribbon to form lamellar crystals, and the amino group at the end of the cellulose chain is exposed on the surface of the nanoribbon.

  The cellulose structure according to the present invention has a ribbon-like structure (nanoribbon). The nanoribbon does not form a hydrogel and is obtained as a precipitate.

  In the specification, "hydrogel" refers to a polymeric substance which is insoluble in liquid, contains water internally, and has a three-dimensional structure and a swollen body thereof. On the other hand, the term "precipitate" as used herein refers to a liquid-free internal structure that is insoluble in liquid. An example of a precipitate is T.I. Serizawa et al., Polym. J. , 2016, 48, 539-544, etc.

  The cellulose structure according to the present invention adsorbs proteins. The protein to be adsorbed is adsorbed in a monolayer, and adsorbed on both sides of the structure in a sandwich-like manner. Electrostatic interaction contributes to this adsorption.

  The cellulose structure according to the present invention can adsorb a protein enzyme while maintaining its activity.

  The cellulose construct according to the present invention does not show significant cytotoxicity in in vitro cell culture environment.

2. The manufacturing method of the cellulose structure concerning the present invention.
The cellulose structure according to the present invention described above can be produced according to the reaction shown below.

（図中、ｎはアミノ基をもつセルロース誘導体の重合度を示す。）

(In the figure, n represents the degree of polymerization of the cellulose derivative having an amino group.)

  This is an enzyme synthesis utilizing the reverse reaction of CDP, and the cellulose structure according to the present invention is produced by reacting 2-aminoethyl-β-D-glucoside as a primer and αG1P as a substrate with CDP. Can be manufactured. ΑG1P is sequentially polymerized as a monomer to 2-aminoethyl-β-D-glucoside as a primer.

  The concentration of CDP used in this reaction may be 0.1 U / ml or more, and may be 0.2 U / ml or more or 0.4 U / ml.

  αG1P may be commercially available.

  2-aminoethyl-β-D-glucoside is, for example, Rao et al., Chem. Commun. , 2013, 49, 10808-10810. The compound can be synthesized with reference to the method described in, but not limited thereto. Specifically, O-tetraacetyl-2-bromoethyl-β-D-glucoside is synthesized from O-pentaacetyl-β-D-glucoside and 2-bromoethanol, and O-pentaacetyl-2-bromoethyl-β Synthesis of O-pentaacetyl-2-azidoethyl-β-D-glucoside from D-glucoside and sodium azide, deacetylation with sodium methoxide, and then reduction of the azido group to give 2-aminoethyl-β Although -D-glucoside can be obtained, it is not limited to this.

  On the other hand, CDP is, for example, M.I. Krishnareddy et al. Appl. Glycosci. Although it can prepare according to the method as described in, 49, 1-8 in 2002, it is not limited to this. Specifically, M.I. Krishnareddy et al. Appl. Glycosci. According to J. Chem., 2002, 49, 1-8, CDP derived from Clostridium thermocellum strain YM4 can be prepared, but it is not limited thereto.

  The enzyme amount of CDP can be determined, for example, based on the enzyme activity. In this case, for example, by incubating αG1P with D-(+)-cellobiose and CDP, the phosphate produced by CDP can be quantified, and the amount of enzyme releasing 1 μmol of phosphate per minute can be 1 U. .

  For example, 10 to 1000 mM (preferably 100 to 300 mM) αG1P, 10 to 200 mM (preferably 50 to 60 mM) 2-aminoethyl-β-D-glucoside of the formula (II), and 0.1 U / mL or more ( Or 0.2 U / mL or more, preferably 0.4 U / mL or more), and 100 to 1000 mM (preferably 250 to 750 mM) of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane sulfone Mixed in acid (HEPES) buffer (pH 7.0-8.0 (preferably pH 7.5)) for 30 minutes to 30 days (preferably, at 10-80 ° C. (preferably 20-60 ° C.) The cellulose structure according to the present invention can be produced by incubating and reacting for 1 hour to 14 days.

3. Applications of Cellulose Structure According to the Present Invention 3-1. Medical field 3-1-1. Cell Culture Scaffold Material The cellulose structure according to the present invention can be used as a scaffold material for growing animal cells on its surface. The cellulose structure according to the present invention is particularly useful for this use because it does not show significant cytotoxicity.

  The cells to be grown may be of animal origin and include, for example, cells of mammalian, reptile, amphibian or insect origin. Further, it may be primary cultured cells isolated from organs or tissues such as heart, liver, spleen, epidermis, etc., or subcultured established cell lines or tumor cells. Furthermore, somatic stem cells such as mesenchymal stem cells (MSC), induced pluripotent stem cells, cell lines such as CHO cells, BHK cells, Vero cells, etc. 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. Furthermore, a medium to which serum or serum / tissue extract, hydrolyzate, growth factor, hormone, carrier protein, lipid, polyamic acid, trace element, vitamin, thickener, surfactant, cell adhesion factor, etc. is added It can also be used.

  Since the cellulose structure according to the present invention can be adsorbed while maintaining its activity, the additive can be used by being adsorbed to the cellulose structure.

  The cellulose 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 enhanced by supplying the nutrient component also from the bottom of the thin film.

  Cells grown in the form of a monolayer on the cellulose structure can be exfoliated and recovered by a cell exfoliation agent. Cell release agents can be chelating agents for removing divalent cations such as ethylenediaminetetraacetic acid (EDTA), proteases such as trypsin for cells / substrates, cell / cell adhesion proteins, and cellulases. It is also possible to detach cells by degrading and solubilizing part or all of the cellulose structure. In the latter method which does not use a protease, the cell-cell adhesion is not exfoliated and has no influence on the cells, whereby a highly active suspended cell or cell sheet can be obtained.

  The obtained cell sheet was laminated with the cell sheet obtained in the same manner, or the surface of the cell sheet was coated with an intercellular adhesion protein or the like, and the cells were cultured to obtain a three-dimensional thickness. Cell masses can be obtained.

3-1-2. Scaffold for High Concentration Cell Culture The cellulose structure according to the present invention can be used as a scaffold for growing animal cells therein. The cellulose structure can be formed into a structure having a void size as large as or larger than the cell can enter, and the cells can be cultured in the structure. The cellulose structure according to the present invention is particularly useful for this use because it does not show significant cytotoxicity.

  The cells to be grown may be any cells of animal origin as described in Section 3-1-1 above. The culture medium used for culture may be a basal medium, a serum-added culture medium or a culture medium to which other components are added as described in Section 3-1-1 above. Furthermore, those additives may be adsorbed to the cellulose structure and used as described in Section 3-1-1 above.

  By culturing the cells in the cellulose structure, damage to the cells due to the shear stress of agitation that occurs in suspension suspension culture does not occur, and it is possible to increase the concentration of cells per culture solution.

  In addition, if rCHO cells or the like to which biopharmaceuticals such as antibodies and proteins have been added are cultured at high concentrations on the cellulose structure, the amount of antibodies and proteins produced per batch increases, and the cost of manufacturing biopharmaceuticals is increased. A reduction is possible. Furthermore, after collecting the target product, a new medium is added and culture is performed again, or the medium containing the target product is collected continuously, and new culture medium is additionally added to perform batch continuous system or continuous system. Products can be obtained.

  Furthermore, by subjecting the cellulose structure used as a scaffold to a cellulase treatment, the cells cultured at high concentration can be released and recovered without causing damage to the cells as occurs in the protease treatment. it can. The pluripotent stem cells such as iPS cells and ES cells can be cultured in large amounts by this method, and cells necessary for the regenerative medicine and drug discovery industries can be supplied.

3-1-3. Three-Dimensional Structure Scaffold Material The cellulose structure according to the present invention can be used as a scaffold material for growing animal cells inside. The cellulose structure can be formed into a structure having a void size as large as or larger than the cell can enter, and the cells can be cultured in the structure. The cellulose structure according to the present invention is particularly useful for this use because it does not show significant cytotoxicity.

  The cells to be grown may be any cells of animal origin as described in Section 3-1-1 above. The medium used for culture may be a basal medium, a serum-supplemented medium, or a medium to which other components are added, as described in Section 3-1-1 above. Furthermore, those additives may be adsorbed to the cellulose structure and used as described in Section 3-1-1 above.

  Before, during, or after cell culture, the cellulose structure can be formed into a shape adapted to the target tissue and used for regeneration of a living tissue or organ. In the case of molding before cell culture, reaction can be carried out with a mold of a target shape at the time of polymerization reaction of the cellulose structure, or can be made into a target shape by cutting, laminating, knitting, etc. after molding.

  The cellulose structure can be degraded and solubilized by cellulase treatment without affecting cells. Prior to transplantation into an organ, it may be subjected to cellulase treatment, partially or completely degraded and solubilized, or may be transplanted together with the scaffold without being subjected to cellulase treatment.

  Furthermore, three-dimensional structures in which cells are grown are for ex vivo (ex vivo) reproduction of the shape or function of a tissue or an organ, for basic medical research such as elucidation of the mechanism of cancer metastasis, efficacy evaluation of anticancer drugs, etc. Alternatively, it can be used as a material for examining the effect on skin and other biological tissues of cosmetics for which animal testing has been banned in recent years.

3-1-4. Implantation material to bone defect site The cellulose structure according to the present invention can also be used as an implant material to bone defect site of animal and regeneration induction material of periodontal tissue. The cellulose structure can be easily shaped and processed as desired, and can also be sterilized by high pressure steam. The cellulose construct may engraft cells before transplantation or may be transplanted as it is.

3-2. Environment / Energy Field As described below, the cellulose structure according to the present invention is a film (for example, a microporous film (application example: separator, adsorbent, biosensor etc.), a dense film (application example: It can be used in the field of environment and energy as a barrier film etc.). The cellulose structure according to the present invention is particularly useful as a protein adsorbent because it can adsorb proteins. In addition, since proteins can be adsorbed while maintaining their activity, they can also be used as highly functional films having the function of proteins to be adsorbed.

3-2-1. Battery Separator Application The cellulose structure according to the present invention can also be used, for example, as a separator for secondary batteries such as lithium ion batteries. By using it as a separator, it has higher heat resistance than conventional polyolefin separators, so that it is possible to prevent the separator from melting and causing an internal short circuit, for example, when the battery abnormally generates heat due to overcharging or the like. It is possible to improve safety. Also, due to the presence of a hydrophilic substituent, it is easier to hold polar solvents generally used in batteries, stable ion conductivity, that is, maintenance of battery input / output characteristics can be expected, and life can be improved. It is possible.

3-2-2. Adsorbent, Concentrated (Biodegradable)
The cellulose structure according to the present invention disperses and carries adsorbent fine particles such as silica, alumina, zeolite, Prussian blue etc., toxic gases such as ammonia, aldehyde etc., harmful substances such as radioactive wastes, valuable in seawater. It is possible to adsorb and recover metals and the like.

  Moreover, since the said cellulose structure is easily decomposable using a decomposing enzyme, it also has the feature that the substance which adsorbed and recovered can be easily condensed as needed.

3-2-3. Biosensor The cellulose structure according to the present invention can be used as a biosensor by carrying an enzyme or an antibody. For example, if an enzyme such as glucose oxidase is supported, it can be used as a glucose sensor, and if a hydrophilic substituent is used, for example, if ion conduction characteristics such as sodium ion are observed, the sodium ion concentration in blood It is possible to use as a sensor which measures etc. The cellulose structure according to the present invention is particularly useful in this application because it can adsorb proteins while maintaining its activity.

3-2-4. Gas barrier sheet (including structure)
By densely supporting the inorganic nanomaterial on the cellulose structure according to the present invention, for example, a gas barrier sheet that shuts off a gas such as water vapor can be obtained. As an inorganic nanomaterial, nanoparticles, such as a silica, or clays, such as montmorillonite, are mentioned. Since these inorganic nanomaterials and hydrophilic substituents have high affinity and interact strongly, it is possible to form a composite material in which the inorganic nanomaterials are firmly incorporated and to improve barrier properties.

3-2-5. Heat Dissipation Sheet The material having high thermal conductivity can be densely supported on the cellulose structure according to the present invention, whereby the heat dissipating material can be used. 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. In addition, it is possible to soften the sheet surface by the hydrophilic substituent, and by improving the adhesion to the heat generating body and reducing the thermal interface resistance, it is possible to improve the heat dissipation.

3-2-6. Heat storage sheet It is possible to use as a heat storage material by dispersing and supporting a material having a heat storage property in the cellulose structure according to the present 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, as well as sensible heat storage materials, chemical heat storage materials, and the like. The latent heat storage material utilizes the phase transition of the substance, and in the case of a heat storage material utilizing solid-liquid phase transition, it is necessary to devise so that no leakage occurs when it becomes liquid, for example, petroleum resin etc. There is a method of including it in the capsule of

3-2-7. Substrate for separation and purification (column packing material, electrophoresis)
Since the cellulose structure according to the present invention has nano-sized spaces, it can be used as a separation and purification column or a filler for electrophoresis using the spaces.

  Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples.

（図中、ｎはアミノ基をもつセルロース誘導体の重合度を示す。）

(In the figure, n represents the degree of polymerization of the cellulose derivative having an amino group.)

1. Experimental method 1-1. Reagents LB-BROTH LENNOX and LB-AGAR LENNOX were purchased from Funakoshi.

Glycerol, kanamycin sulfate, isopropyl-β-D (-)-thiogalactopyranoside (Isopropyl-β-D (-)-thiogalactopyranoside: IPTG), N, N, N ', N'-tetramethylethylenediamine (N , N, N', N'-Tetramethyl ethylenediamine: TEMED), dithiothreitol (dithiothreitol: DTT), 1- butanol, Arufaji1P disodium hydrate, 40% sodium hydroxide heavy solution, molecular sieves 4A, Dowex ^TM 50WX4 100-200 mesh strongly acidic cation exchange resin, bovine serum albumin (BSA, 98%, protease free), Immunoglobulin G (bovine-derived, IgG, for immunochemistry), Lanceferin (human-derived, for cell culture), streptomycin sulfate, 10 × Dulbeccox Phosphate Buffered Saline (D-PBS (−), for cell culture), Dulbecco's Modified Eagle Medium (D-MEM), β- D-galactosidase and p-nitrophenyl-β-D-galactopyranoside were purchased from Wako Pure Chemical Industries.

  Dichloromethane, ethyl acetate, silica gel 60 N (40-100 μm, spherical, neutral) were purchased from Kanto Chemical.

  O-pentaacetyl-β-D-glucoside, 2-bromoethanol, anhydrous hydrazine, boron trifluoride diethyl ether, p-anisaldehyde was purchased from Tokyo Kasei.

  Nickel-nitrilotriacetic acid (Ni-NTA) agarose gel was purchased from QIAGEN.

  Protein Molecular Weight Marker (Broad) was purchased from Takara Bio.

Acrylamide, N-N'-methylenebisacrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), heavy water, 2,5-dihydroxybenzoic acid (DHBA) ^{, ProteoMass TM Bradykinin fragment 1-7 MALDI-} MS Standard _{^{(Bradykinin), ProteoMass TM P 14}} R MALDI-MS Standard ^{_{(P 14 R), ProteoMass TM}} ACTH fragment 18-39 MALDI-MS Standard (ACTH), trifluoroacetic acid ( TFA), acetonitrile, lysozyme (Niwato) Egg-derived,> 90%), trypsin (pig pancreas-derived, BioReagent grade), cytochrome C (horse-heart-derived,> 99%), fibrinogen (bovine plasma-derived, Type I-S), Penicillin-streptomycin solution stabilized, Trypsin EDTA (10 ×) was purchased from Sigma-Aldrich.

  The Nisshin EM Stainer was purchased from Nisshin EM Co., Ltd.

  Fetal bovine serum (FBS) for cell culture was obtained from BioWest.

The Callstain ^R Double Staining Kit and Cell Counting KIT-8 were obtained from Dojindo Laboratories.

  Other reagents were purchased from Nacalai Tesque unless otherwise noted, and reagents of special grade or higher were used.

  Ultra pure water was supplied by Milli-Q system (Milli-Q Advantage A-10, Merck Millipore) and pure water was supplied by TANK 60 Lite PE (Merck Millipore).

  The dry dichloromethane used the dichloromethane refine | purified by Glass Contour organic-solvent purification apparatus (Nikko Hansen).

1-2. Preparation of CDP and Evaluation of Enzyme Activity Krishnareddy et al. Appl. Glycosci. , 2002, 49, 1-8. It prepared by the method similar to the method as described in.

1-2-1. Preparation of Reagent (1) Sterilized purified water 500 mL of ultrapure water was collected in a 500 mL wide-mouth medium bottle, autoclaved (121 ° C., 20 minutes, BS-245, TOMY) and stored at room temperature.

(2) 50 mg / mL kanamycin stock solution 1.5 g of kanamycin sulfate was dissolved in sterile purified water and made up to 30 mL. The prepared solution was filter-sterilized with a polyvinylidene fluoride (PVDF) 0.22 μm filter in a clean bench, and aliquoted into 1.7 mL tubes and stored at -20 ° C.

(3) 10 mM IPTG stock solution 71.5 mg of IPTG was dissolved in sterile purified water and made up to 30 mL. The prepared solution was filter-sterilized with a PVDF 0.22 μm filter in a clean bench. 1 mL aliquots were placed in 1.7 mL tubes and stored at -20 ° C.

(4) LB-AGAR / Kanamycin plate 2.8 g of LB-AGAR LENNOX was collected in a 250 mL wide-mouth medium bottle, and 80 mL of ultrapure water was added to dissolve and autoclave. After cooling at room temperature to 60 ° C. or less, 80 μL of 50 mg / mL kanamycin stock solution was added and mixed in a clean bench. The aliquots were poured into sterile petri dishes and allowed to solidify at room temperature and then stored at 4 ° C. Used within 1 month after preparation.

(5) 50% glycerol solution After adding 50 mL of glycerol and 50 mL of ultrapure water to a 250 mL wide-mouthed medium bottle and mixing, the solution was autoclaved and stored at room temperature.

(6) LB medium In a 500 mL wide-mouthed medium bottle, 10 g of LB-BROTH LENNOX was dissolved in 500 mL of pure water, autoclaved and stored at room temperature.

(7) MOPS-Na buffer solution (20 mM, pH 7.5)
4.18 g of 3-morpholinopropane sulfonic acid (MOPS) was dissolved in about 600 mL of ultrapure water. The pH was adjusted to 7.5 with a 4N aqueous solution of sodium hydroxide, and the solution was then adjusted to 1 L. The solution was sterilized by filtration through a PVDF 0.10 μm filter and stored at 4 ° C.

(8) 70% ethanol (biotechnology grade)
After 140 mL of ethanol (biotechnology grade) and 60 mL of ultrapure water were collected in a 250 mL wide-mouth medium bottle and mixed, they were stored at room temperature.

(9) Washing buffer (20 mM MOPS, 300 mM NaCl, 10 mM imidazole, pH 7.5)
2.09 g of MOPS, 8.77 g of sodium chloride and 340 mg of imidazole were dissolved in about 350 mL of ultrapure water. The pH was adjusted to 7.5 with a 4N aqueous solution of sodium hydroxide, and then the volume was increased to 500 mL. The solution was sterilized by filtration through a PVDF 0.10 μm filter and stored at 4 ° C.

(10) Elution buffer (20 mM MOPS, 300 mM NaCl, 250 mM imidazole, pH 7.5)
2.09 g of MOPS, 8.77 g of sodium chloride and 8.51 g of imidazole were dissolved in about 350 mL of ultrapure water. The pH was adjusted to 7.5 with a 4N aqueous solution of sodium hydroxide, and then the volume was increased to 500 mL. The solution was sterilized by filtration through a PVDF 0.10 μm filter and stored at 4 ° C.

(11) 5% (w / v) sodium azide stock solution Weigh 500 mg of sodium azide with a plastic spatula, add MOPS-Na buffer solution (20 mM, pH 7.5) and dissolve to a total volume of 10 mL It was stored at 4 ° C. in the dark.

(12) 30% Acrylamide / Bis Mixed Solution Since acrylamide is a deleterious substance, careful attention was paid to its preparation. 29.2 g of acrylamide and 0.8 g of N-N'-methylenebisacrylamide were dissolved in about 70 mL of ultrapure water with stirring. The whole was weighed up to 100 mL and stored in the dark at 4 ° C.

(13) Tris-HCl buffer (1.5 M, pH 8.8)
18.17 g of tris (hydroxymethyl) aminomethane (Tris) was dissolved in about 80 mL of ultrapure water with stirring. After adjusting to pH 8.8 with 6 N hydrochloric acid, the total volume was adjusted to 100 mL and stored at 4 ° C.

(14) Tris-HCl buffer (0.5 M, pH 6.8)
6.06 g of Tris was dissolved in about 80 mL of ultrapure water with stirring. After adjusting to pH 6.8 with 6 N hydrochloric acid, the total volume was adjusted to 100 mL and stored at 4 ° C.

(15) 10% SDS aqueous solution 10 g of sodium dodecyl sulfate (SDS) was dissolved in about 90 mL of ultrapure water with stirring. The whole was weighed up to 100 mL and stored at room temperature.

(16) 10% APS aqueous solution 0.1 g of ammonium persulfate (APS) was dissolved in about 1 mL of ultrapure water and stored at 4 ° C.

(17) Saturated 1-Butanol 1-Butanol and ultrapure water were mixed in a ratio of 2: 1 and stored at room temperature.

(18) 5 × running buffer (125 mM Tris, 960 mM glycine, 0.5% SDS)
15.1 g of Tris, 72.1 g of glycine and 5.0 g of SDS were dissolved in ultrapure water so that the total amount was 1 L, and stored at 4 ° C. When used, it was diluted 5-fold with ultrapure water. The electrophoresis buffer used once was recovered and used again, and the number of times of use was up to twice.

(19) 5 x Loading buffer (200 mM Tris, 0.05% bromophenol blue, 10% SDS, 50% glycerol)
2.42 g of Tris, 10.0 g of SDS, and 50 mg of bromophenol blue were dissolved by adding 50 mL of glycerol and about 40 mL of ultrapure water. The pH was adjusted to 6.8 with 6N hydrochloric acid and the volume was increased to 100 mL. 1 mL aliquots were placed in 1.7 mL tubes and stored at 4 ° C.

(20) 1 MDTT Solution About 150 mg of dithiothreitol (DTT) was dissolved by adding about 1 mL of ultrapure water to 1 M, and stored at -20 ° C.

(21) Fixative solution After mixing 50 mL of acetic acid and 200 mL of ethanol, it was measured to 500 mL with ultrapure water and stored at room temperature.

(22) Decolorizing solution After mixing 40 mL of acetic acid and 125 mL of ethanol, the mixture was made up to 500 mL with ultrapure water and stored at room temperature.

(23) Staining solution 0.25 g of Coomassie Brilliant Blue R-250 was added to 100 mL of the decolorizing solution, stirred and dissolved, and kept in the dark at room temperature.

1-2-2. Culture of E. coli and expression of CDP (1) Preparation of E. coli BL21-CDP Glycerol Stock The Escherichia coli BL21-Gold (DE3) strain (E. coli BL21-CDP) having a plasmid containing the gene of CDP, which was heat-sterilized with a gas burner and allowed to cool, was used. The glycerol stock was inoculated on LB-AGAR / kanamycin plates and cultured at 37 ° C. for 8-12 hours. A single colony was inoculated from a single colony with a heated platinum loop into a mixed solution of 5 μL of 50 mg / mL kanamycin stock solution and 5 mL of LB medium added to a dry heat-sterilized test tube in advance. The shaking culture (200 rpm) was carried out at 37 ° C. in a shaking incubator (BR-23FP, TAITEC) until the OD ₆₆₀ reached 0.6-0.8. Thereafter, 800 μL of the culture solution was collected in a 1.7 mL tube, and 100 μL of 50% glycerol solution was added and mixed. It was frozen in liquid nitrogen and stored at -80 ° C.

(2) E. Preparation of E. coli BL21-CDP Master Plate The heat-sterilized with a gas burner and allowed to cool was used to make E. coli. From a master plate within 1 month after glycerol stock or preparation of E. coli BL21-CDP, the cells were inoculated on LB-AGAR / kanamycin plates and cultured at 37 ° C. for 8 to 12 hours. When inoculated from the master plate, it was stored at 4 ° C. as it was. When inoculated from a glycerol stock, the cells were further inoculated on LB-AGAR / kanamycin plates, cultured at 37 ° C for 8-12 hours, and stored at 4 ° C.

(3) E. Preparation of E. coli BL21-CDP pre-culture solution 3 mL of LB medium was collected in a dry heat sterilized test tube, and 3 μL of 50 mg / mL kanamycin stock solution was added. Heat sterilized and allowed to cool using platinum ear, E. It inoculated from the single colony of E. coli BL21-CDP master plate. The shaking culture (reciprocal, 200 rpm) was carried out at 37 ° C. for 10-12 hours using a shaking incubator.

(4) E. Cultivation of E. coli BL21-CDP and expression of CDP 6 g of LB-BROTH LENNOX was respectively weighed in two 1 L-baffled flasks, 300 mL of pure water was added, and the mixture was left to cool under autoclaving. After adding 300 μL of 50 mg / mL kanamycin stock solution to each and mixing, E. 300 μL of E. coli BL21-CDP preculture solution was added. After shaking culture (rotation, 600 rpm) using a shaking incubator at 30 ° C and confirming that the OD ₆₆₀ reached 0.55-0.60, 3 mL of 10 mM IPTG stock solution is added respectively and 20 hours at 25 ° C Shake culture (rotation, 200 rpm) was used to express CDP.

1-2-3. His tag purification and buffer substitution (1) Extraction of CDP by disruption of E. coli BL21-CDP E. coli expressing CDP prepared in 1-2-2 (2). Approximately 50 mL aliquots of the E. coli BL21-CDP suspension are equally aliquoted into 12 × 50 mL tubes and centrifuged (3500 rpm, 20) using a centrifuge (centrifuge: EX-126, TOMY, rotor: 3850-04P, TOMY) 4 minutes). The supernatant was removed by decantation and the precipitated E. coli. MOPS-Na buffer solution (20 mM, pH 7.5) was added to each of the E. coli BL21-CDP pellets, and each was made up to about 15 mL and suspended by shaking vigorously by hand. E. E. coli BL21-CDP suspension was dispensed in about 45 mL aliquots into four 100 mL freestanding tubes and sonicated using a probe-type ultrasonic wave irradiator (Sonifier 250, BRANSON) while in an ice bath. E. coli BL21-CDP was disrupted. The ultrasonic irradiation was performed for two cycles (one cycle of 5 seconds of ultrasonic irradiation and 25 seconds of repeated intervals) under the conditions of power: 200 W (dial 10) and duty cycle: 30%. E. Since the E. coli BL21-CDP disruption solution contains protease and the like in addition to CDP, in order to prevent the degradation of CDP by them, E. coli is subsequently used. The E. coli lysate was always in an ice bath. E. in four 100 mL tubes. E. coli BL21-CDP lysate was transferred to a 50 mL tube and centrifuged (3500 rpm,> 20 minutes, 4 ° C.). E. precipitated. The supernatant was collected by decantation into a 50 mL tube, taking care not to allow the E. coli pellet to scatter.

(2) Purification of CDP by Ni-NTA Affinity Column In order to avoid contamination, the tip of the micropipette used in the subsequent purification operation and the 1.7 mL tube were used after being autoclaved and dried.
9.8 mL of a Ni-NTA agarose gel (GE Healthcare) dispersion was loaded on a plastic column 1.8 cm in diameter and 10 cm in length which was previously sterilized with 70% ethanol and replaced with ultrapure water. The NI-NTA agarose gel was sterilized and washed by pouring the inside of the column with 70% ethanol prepared in Section 1-2-1 (8). This operation was repeated twice. 3 mL of MOPS-Na buffer solution (20 mM, pH 7.5) was applied and flowed, and another 3 mL was applied. The air was removed by redispersing the Ni-NTA agarose gel by stirring with a spatula or pipetting with a micropipette. Thereafter, it was replaced four times with 5 mL of MOPS-Na buffer (20 mM, pH 7.5).

  E. The supernatant of the E. coli BL21-CDP lysate was applied to the column, and the solution flowing out of the column was collected and used as a flow-through solution. 3-5 mL of wash buffer was applied and replaced, and another 3-5 mL was applied. The Ni-NTA agarose gel was redispersed by pipetting to make the surface of the gel layer uniform, and then the applied washing buffer was flushed. Furthermore, the operation of applying and replacing the washing buffer 5 mL was repeated, and a total of 30 mL or more was applied. All the solution passed when the washing buffer was applied was collected and used as a washing solution. Thereafter, 3-5 mL of elution buffer was applied, and 1 mL aliquots were collected in 1.7 mL tubes and collected as an eluate. This operation was repeated and a total of 25 mL of elution buffer was applied. The absorbance of each fraction was measured by a micro ultraviolet visible spectrophotometer (NanoDrop 2000c, Thermo Scientific), and elution of the protein was confirmed using the absorbance at 280 nm as an index. At this time, ultrapure water was used for background measurement. If protein elution was not complete, the elution buffer was applied to the column until the absorbance at 280 nm disappeared. Among the collected eluates, fractions with high absorbance were collected and used as CDP solution.

(3) Buffer substitution of CDP solution Buffer substitution of CDP solution was carried out using a PD10 column (Sephadex G-25, GE Healthcare) according to the attached instruction. The required number of PD10 columns was used, depending on the volume of the CDP solution. The PD10 column was fixed to a 50 mL tube using the attached adapter. It was filled with 70% ethanol (biotechnology grade) and centrifuged (1000 g, 2 minutes, 4 ° C.) using a centrifuge (MX-305, TOMY). This operation was repeated twice to sterilize and wash the column. Fill with 0.02% sodium azide / 20 mM MOPS-Na buffer solution prepared by diluting 250% of 5% (w / v) sodium azide stock solution with MOPS-Na buffer (20 mM, pH 7.5), The PD10 column was replaced with buffer solution by repeating the procedure of standing still and flowing the solution four times. Furthermore, 0.02% sodium azide / 20 mM MOPS-Na buffer was added and centrifuged.

  The PD10 column was transferred to a new 50 mL tube, and CDP solution was applied at 1.75 to 2.5 mL per PD10 column so as not to break the slope of Sephadex by centrifugation. It fixed to the centrifuge at the same angle as the time of the above-mentioned centrifugation, and centrifuged. The eluted solution was collected and used as a CDP stock solution, and the absorbance at 280 nm of the CDP stock solution was measured by NanoDrop 2000c, and aliquoted into 1.7 mL tubes and stored at 4 ° C. When using the CDP stock solution, the necessary amount was dispensed as appropriate in the clean bench.

  The column was washed by adding 30 mL of MOPS-Na buffer (20 mM, pH 7.5) per column and leaving to stand. Next, the operation of filling with ultrapure water and centrifuging was repeated twice. Similarly, it was washed twice with 50% aqueous ethanol. The PD-10 column was filled with 50% aqueous ethanol and stored at 4 ° C.

1-2-4. Confirmation of purification of CDP by polyacrylamide gel electrophoresis (SDS-PAGE) (1) Preparation of 10% acrylamide gel 3.33 mL of 30% acrylamide / bis mixed solution in a 50 mL tube, Tris-HCl buffer (1.5 M, pH 8.8) 2.5 mL and ultra pure water 4.01 mL were added, and suctioned by an aspirator and degassing while being ultrasonically irradiated. 100 μL of a 10% SDS aqueous solution, 50 μL of a 10% APS aqueous solution, and 10 μL of TEMED were added and mixed by pipetting so as not to foam. 3.5 mL of the mixed solution was added to the gap (0.75 mm) of two glass plates, 900 μL of saturated 1-butanol was gently overlaid, and polymerization was allowed to stand at room temperature for 1 hour for polymerization. Saturated 1-butanol was removed using a micropipette and then washed with ultrapure water to form a separated gel.

  In a 50 mL tube, add 0.44 mL of 30% acrylamide / bis mixed solution, 0.44 mL of Tris-HCl buffer (0.5 M, pH 6.8), 2.03 mL of ultrapure water, and aspirate with ultrasonication while aspirating. Was degassed. 33 μL of 10% SDS aqueous solution, 16.7 μL of 10% APS aqueous solution and 1.65 μL of TEMED were added and mixed by pipetting so as not to foam. The solution was sufficiently added onto the separating gel, and a 10-well comb was carefully set so as not to contain air, and allowed to stand at room temperature for 1 hour for polymerization. The resulting gel was sandwiched with a glass plate with Kimwipe sufficiently moistened and further wrapped in a wrap and stored at 4 ° C. The gel was used within one month after preparation.

(2) Evaluation by electrophoresis 5 .mu.L of Protein Molecular Weight Marker (Broad), 2 .mu.L of 1 M DTT solution, 20 .mu.L of 5.times. Loading buffer, and 173 .mu.L of sterile purified water are added to a 1.7 mL tube, mixed and used as a marker at -20.degree. Saved with. The CDP stock solution and 1 μL of the flow-through solution and washing solution recovered in the purification of CDP were mixed with 2 μL of 5 × Loading buffer, 1 μL of 1 M DTT, and 6 μL of ultrapure water. These and 10 μL of the marker which had been thawed at room temperature were heat-treated at 105 ° C., and spun down with a tabletop microcentrifuge (R5-AQBD02, Recenttec). The polyacrylamide gel prepared in this paragraph (1) was set in a vessel for electrophoresis (Mini-Protoian Tetra cell, BIO RAD), 500 mL of 5 × running buffer diluted 5-fold with ultrapure water was poured, and the comb was removed. The electrophoresis sample was loaded into each well, and was electrophoresed at a voltage of 150 V for about 40 minutes using an electrophoresis apparatus (Mini-Protoian Tetra system, BIO RAD). The migration was stopped when the band reached about 1 cm above the lower end of the gel, the gel was removed from the glass plate, immersed in a fixing solution, shielded from light, and shaken with a shaker (Wave-PR, TAITECH) for 30 minutes. The fixing solution was removed, the staining solution was added to immerse the gel, and shaking was performed for 60 minutes in the dark. The staining solution was recovered, and the decolorizing solution was added to immerse the gel, and shaken in the dark for 30 minutes. The operation of removing the decolorizing solution, adding a new decolorizing solution and shaking was repeated until the protein band contained in the marker was clearly visible. The gel was washed with ultrapure water, photographed by Gel Doc EZ Imager (BIO RAD), and analyzed by the attached software.

1-2-5. Enzyme activity measurement of CDP The activity of CDP was measured as follows. A 3-morpholinopropane sulfonic acid buffer (50 mM, pH 7.5) containing αG1P 50 mM, D-(+)-cellobiose 50 mM, and CDP diluted with a predetermined ratio was incubated at 37 ° C. Phosphoric acid generated by CDP was quantified, and U / mL when the amount of enzyme releasing 1 μmol of phosphoric acid per minute was defined as 1 U was determined as enzyme activity. The dilution rate of CDP was determined so that the conversion of αG1P was 10% or less at 100 minutes of reaction time.

1-3. Synthesis of 2-aminoethyl-β-D-glucoside 2-aminoethyl-β-D-glucoside was prepared according to Rao et al., Chem. Commun. , 2013, 49, 10808-10810. It synthesize | combined referring to the method as described in.
All the following reactions were carried out while confirming the progress of the reaction by p-anisaldehyde TLC color reagent or ¹ H NMR measurement (JEOL Model-ECP 400 (JEOL, magnetic field strength: 400 MHz)). The p-anisaldehyde TLC color reagent is prepared by mixing 13 mL of p-anisaldehyde, 5 mL of acetic acid and 478 mL of ethanol under ice-cooling, 18 mL of concentrated sulfuric acid is dropped with stirring using a magnetic stirrer, and used until 4 ° C. until use Saved with.

1-3-1. Synthesis of O-tetraacetyl-2-bromoethyl-β-D-glucoside: Dissolve O-pentaacetyl-β-D-glucoside (1.0 equivalent) and 2-bromoethanol (1.2 equivalents) in dry dichloromethane I did. Powdered molecular sieve 4A previously activated with a heater at 350 ° C. as a dehydrating agent was added, and stirred at room temperature using a magnetic stirrer under an argon atmosphere for 1 hour. To the reaction solution cooled in ice, boron trifluoride diethyl ether (5.0 equivalent) was added dropwise, and the reaction solution was returned to room temperature, stirred overnight, and subjected to O-glycosylation reaction. The reaction solution was added dropwise to ice-cold saturated aqueous sodium hydrogen carbonate solution / ethyl acetate (50% (v / v)) to quench the reaction. The disappearance of O-pentaacetyl-β-D-glucoside, which is a raw material, was confirmed by TLC using a p-anisaldehyde coloring reagent, followed by ice-cold saturated aqueous sodium hydrogen carbonate solution / ethyl acetate (50% (v / v The reaction solution was dropped to) to stop the reaction. The reaction solution was transferred to a separatory funnel, shaken vigorously, and allowed to stand until the solution separated into two layers, and the upper oil layer was recovered. The lower aqueous layer was transferred to a new separatory funnel, extracted with ethyl acetate, and the operation of recovering the oil layer was repeated twice. The collected oil layer was again transferred to a separatory funnel, saturated brine was added, the mixture was vigorously shaken, and water dissolved in the oil layer was removed. The aqueous layer was drained and anhydrous magnesium sulfate was added to the oil layer as a desiccant. Magnesium sulfate was removed using a glass filter, and the organic solvent was distilled off using a rotary evaporator. Here, in the TLC test, it was found that the spots of the starting material, O-pentaacetyl-β-D-glucoside, and the target substance overlap, and removal of the starting material by silica gel column chromatography is difficult. In order to enable purification of the desired product, it was decided to selectively hydrolyze the raw material of the residue. The crude product obtained was dissolved in N, N-dimethylformamide (DMF), hydrazine anhydride (0.15 eq) and acetic acid (0.15 eq) were added and stirred overnight at room temperature. After stirring, the reaction solution was added dropwise to ice-cold hydrochloric acid (3 N) / ethyl acetate (50% (v / v)) to stop the reaction. Hydrochloric acid (3 N) was used as the aqueous layer in order to remove DMF used as the solvent to the aqueous layer by the extraction operation. Thereafter, extraction was performed using ethyl acetate in the same manner as the above-described operation, and the oil layer was dehydrated with saturated brine and anhydrous magnesium sulfate. Magnesium sulfate was removed, and the organic solvent was distilled off with a rotary evaporator to obtain a white powder. At this time, when the obtained crude product is in the form of oil, it is suggested that DMF can not be sufficiently removed by liquid separation operation. If DMF remains, it can not be used for purification by silica gel column chromatography, so hydrochloric acid (3 N) / ethyl acetate (50% (v / v)) is added again, and a series of extraction, dehydration, concentration I did the operation. The obtained white powder is dissolved in a small amount of dichloromethane and applied to column chromatography, and the developing solvent is acetone / hexane (25% (v / v)) to obtain a white powder of O-pentan in a yield of 51%. Acetyl-2-bromoethyl-β-D-glucoside was obtained.

1-3-2. Synthesis of O-tetraacetyl-2-azidoethyl-β-D-glucoside O-pentaacetyl-2-bromoethyl-β-D-glucoside (1 equivalent) was dissolved in DMF and ice-cooled to obtain sodium azide (3 Equal volume was added and it stirred at room temperature for 3 hours. After stirring, the reaction solution was added dropwise to ice-cold water / ethyl acetate (50% (v / v)) to stop the reaction. Under acidic conditions, water was used as the aqueous layer in order to remove the explosive sodium azide into the aqueous layer by the extraction operation. Extraction was carried out using ethyl acetate in the same manner as in the operation of section 1-3-1, and the oil layer was recovered. Here, in order to remove DMF used as a solvent to the aqueous layer by the extraction operation, the oil layer was dropped and mixed with ice-cold hydrochloric acid (3 N, approximately 1/3 volume of the oil layer). Extraction was performed using ethyl acetate in the same manner as the operation in section 1-3-1, and the oil layer was dehydrated with saturated brine and anhydrous magnesium sulfate. Magnesium sulfate was removed, and the organic solvent was distilled off with a rotary evaporator to obtain a white powder. The obtained white powder is dissolved in a small amount of dichloromethane and applied to column chromatography, and the developing solvent is acetone / hexane (25% (v / v)), and the white powder of O-pentan is obtained in a yield of 81%. Acetyl-2-azidoethyl-β-D-glucoside was obtained.

1-3-3. Synthesis of 2-aminoethyl-.beta.-D-glucoside O-pentaacetyl-2-azidoethyl-.beta.-D-glucoside is dissolved in methanol / tetrahydrofuran (THF) (1/1), and the reaction solution is ice-cooled to catalyze An amount of sodium methoxide (0.1 equivalent) was added. The reaction solution was returned to room temperature and stirred for 4 hours using a magnetic stirrer, and then Dowex cation exchange resin was added to stop the reaction. The reaction solution was filtered using filter paper loaded with celite, and the organic solvent was distilled off by a rotary evaporator. A catalyst cartridge filled with 10% palladium / carbon was placed in a flow hydrogenation reaction system (H-Cube, ThalesNano), and the crude product dissolved in methanol was flowed into H-Cub (hydrogen pressure: 50 bar, 55 ° C. The azide group was reduced by 1 mL / min). The flowed solution was ice-cooled, 4 M hydrochloric acid / dioxane (1.5 equivalents) was added dropwise, and the organic solvent was distilled off by a rotary evaporator. The obtained product was dissolved in methanol, and 2-propanol was dropped to obtain 2-aminoethyl-β-D-glucoside as a white hydrochloride.

1-4. Enzymatic synthesis of nanoribbons composed of cellulose oligomers having amino groups 1-4-1. Enzymatic synthesis by CDP 23.8 g of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) were dissolved in about 60 mL of ultrapure water. The pH was adjusted to 7.5 with a 4N aqueous solution of sodium hydroxide, then the volume was increased to 100 mL and filter-sterilized with a PVDF 0.22 μm filter. This was made into HEPES buffer solution (1 M, pH 7.5), and was stored at 4 ° C.
A predetermined amount of αG1P disodium hydrate and 2-aminoethyl-β-D-glucoside were weighed, and dissolved in ultrapure water to 1 M.

  Add HEPES buffer (1 M, pH 7.5), 1 M αG1 P aqueous solution, 1 M 2-aminoethyl-β-D-glucoside aqueous solution to 4 mL tube at 500 mM, 200 mM and 50 mM concentrations at the time of reaction, respectively. The amount of stock solution was taken into consideration, and it was measured with ultrapure water. It aspirated and deaerated with an aspirator while ultrasonication. An appropriate amount of CDP stock solution was added to a final concentration of 0.2 U / mL and mixed by pipetting so as not to generate air bubbles. Aliquots were aliquoted into 1.7 mL tubes or 1 mL mighty vials and incubated at 60 ° C. for 3 days.

1-4-2. Purification of Enzymatically Synthesized Product The product prepared in section 1-4-1 was purified with ultrapure water to be used for various analyses. The reaction solution was added with ultrapure water, dispersed by pipetting, and centrifuged (15000 rpm,> 10 minutes, 4 ° C.) to remove the supernatant. The precipitate was dispersed again by adding ultrapure water, and the operation of centrifuging was repeated to purify the solution until the substitution rate of the solution became 99.999% or more. The purified dispersion was heated in a heat block at 100 ° C. for 10 minutes to deactivate the remaining CDP.

When used for measurement by proton nuclear magnetic resonance ( ¹ H NMR) method, total reflection infrared spectroscopy (ATR / FTIR) method, wide-angle X-ray diffraction (WAXD) method, 1.7 mL tube of water dispersion after purification is used Aliquots and lyophilized for 24 hours. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) measurement, atomic force microscope (AFM) observation, transmission electron microscope (TEM) observation, when used for evaluation of protein adsorption characteristics The purified dispersion was diluted with ultrapure water and used without lyophilization.

1-5. Characterization of Cellulose Having Amino Group Generated 1-5-1. Product Inversion Test The product prepared in 1 mL mighty vials was used to evaluate hydrogelation by a inversion test. The vial was turned upside down and allowed to stand, and it was determined that hydrogel was produced when no product flow was observed.

1-5-2. Measurement of product yield The sample tube previously dried at 105 ° C. for 24 hours, allowed to cool in a desiccator, was weighed, the purified aqueous dispersion was added and dried at 105 ° C. for 24 hours, and then allowed to cool in a desiccator Weighed. The yield of the product was calculated from the difference in weight before and after drying. This operation was performed twice at the same time, and the yield of the product was calculated from their average value.

1-5-3. Evaluation of Chemical Structure and Crystal Structure of Product (1) Measurement of ¹ H NMR Spectrum of Product 50 μL of 40% aqueous sodium hydroxide solution and 450 μL of heavy water were mixed to prepare 4% aqueous sodium hydroxide solution. 15 mg or more of the lyophilized product was dissolved in 500 μL of 4% aqueous sodium bicarbonate solution. The solution was transferred to an NMR tube and measured by JEOL Model-ECP 400 (JEOL, magnetic field strength: 400 MHz). The number of integrations was 128 times. The chemical shift of water was calibrated as a reference (4.79 ppm).

(2) MALDI-TOF-MS measurement of product The standard sample used for calibration of mass to charge ratio is Bradykin fragment 1-7 aqueous solution (10 nmol / mL Bradykinin fragment 1-7, 0.05% trifluoroacetic acid (TFA), 50 % Acetonitrile, P ₁₄ R aqueous solution (10 nmol / mL P ₁₄ R, 0.1% TFA), ACTH fragment 18-39 aqueous solution (10 nmol / mL ACTH fragment 18-39, 0.1% TFA) 5 μL and 10 mg /, respectively The mixture was prepared by mixing 5 μL of an aqueous DHBA solution, 1 μL of a 1.0% aqueous TFA solution, and 4 μL of acetonitrile in a 1.7 mL tube.

  The sample for measurement of the product was 6 μL of an aqueous dispersion of the product at 0.05% (w / v), 6 μL of 10 mg / mL aqueous DHBA solution, 15 μL of acetonitrile, 3 μL of an aqueous 1% trifluoroacetic acid solution KOH / methanol (3. Prepared by mixing in 1 mL mighty vials washed with 3% (w / v)).

1 μL of each of the standard sample and the measurement sample of the product was mounted on a sample plate, and the operation of air-drying was repeated five times. It vacuum-dried for 1 hour or more, and measured by MALDI-TOF-MS (AXIMA-performance, Shimadzu Corp.). Measurement conditions are: Mode: Liner (positive), Mass Range: 1.0-3000.0, Max Leaser Rap Rate: 10, power: 100-115, profiles: 100, shots: 2, Ion Gate (Da): Blank 500 , Pulsed Extraction optimized at (Da): 1500.0.
The MS spectrum obtained by the measurement was processed under the conditions of Smoothing method: Gaussian, Smoothing filter width: 19 and Baseline filter width: 1000.

  The average degree of polymerization was calculated using the area ratio of the strongest peak among the proton, sodium or potassium ion adducts of the product.

(3) WAXD Measurement of Product The freeze-dried product was mounted in the recess of a silicon nonreflective sample plate, and the surface was leveled and placed on a tabletop powder X-ray diffractometer (MiniFlex 600, RIGAKU) for measurement. Measurement conditions are as follows: Detector: D / teX Ultra, X-ray output: 40 kV and 15 mA, filter: Kβ, scan speed: 2.0000 deg / min, step width: 0.0200 deg, scan axis: 2θ / θ, scan range: 5.0000 to 7.000 deg, incident slit: 1.250 deg.

(4) Measurement of ATR / FTIR spectrum of product The lyophilized product is placed on a diamond cell at the stage of total reflection infrared spectrophotometer (FT / IR-4100 type A, JASCO) using a spatula and attached The sample was pressed against the diamond cell by a pressure tip of and measured. Measurement conditions, measurement wavelength: 350-7800Cm ^-1, resolution: ^{2 cm -1,} the number of integrations: was 100.

1-5-4. Microscopic observation of the product (1) AFM observation of the product Diluted so that the concentration of the product is 0.001% (w / v) and mounted 15 μL on a mica substrate of which the surface was peeled 5 times with cellophane tape at 600 rpm Spin-coated for 30 minutes. It dried in a desiccator for 12 hours or more, and observed with AFM using a scanning probe microscope (SPM-9600, Shimadzu Corp.). The measurement conditions were: tapping mode, operation speed: 0.5-1 Hz, number of pixels: 512 × 512, Z range: × 1, operation mode: constant force. Ten structures were observed from each sample, the height of 9 points was calculated for each observed structure, and all of them were averaged to calculate the height of the structure.

(2) TEM observation of the product A small amount of ethanol for disinfection was dropped on a petri dish made of plastic to adhere the parafilm. 15 μL of the sample diluted with ultrapure water so that the nanoribbon concentration was 0.001% (w / v) was mounted on parafilm. The drops were loaded with collodion supporting membrane and allowed to interact for 1 hour. A grid was vertically applied to the filter paper to remove moisture. 15 μL of ultrapure water was mounted on parafilm, and the collodion supporting membrane was placed for 10 seconds for washing. Again, a grid was vertically applied to the filter paper to remove water. 15 μL of Nisshin EM Stainer (stock solution) was dropped onto Parafilm, and the surface was put on the microgrid of the collodion supporting film so as to be in contact with the staining solution for 30 minutes for staining, and dried overnight with a desiccator. The Nisshin EM Stainer used a glass syringe from a reagent bottle, collected an appropriate amount in a 1.7 mL tube while replacing with nitrogen, and used it within one week. The prepared sample was observed by TEM (H7560 Zero. A, manufactured by Hitachi, Ltd.). The observation conditions are electron gun: thermionic emission W-hairpin filament, acceleration voltage: 100 kV, holder: side entry type uniaxial tilt, resolution: 0.204 nm (lattice image), spot size: 0.8 to 4 μm (HC Mode), 0.6 to 2 μm (HR mode), and the number of CCD images: 1024 × 1024 pixels.

1-6 Evaluation of adsorption characteristics of various proteins on cellulose nanoribbon having amino group 1-6-1 Preparation of reagent (1) 10 mM HEPES solution (pH 7.4)
119 mg of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) was dissolved in a predetermined amount of ultrapure water. The pH was adjusted to pH 7.4 with 4N aqueous NaOH solution, and made up to 50 mL, and stored at 4 ° C.

(2) 10 mM HEPES 1.5 M NaCl solution (pH 7.4)
143 mg of HEPES and 5.26 g of NaCl were dissolved in predetermined amounts of ultrapure water. The pH was adjusted to pH 7.4 with 4N aqueous NaOH solution, and the volume was raised to 60 mL and stored at 4 ° C.

(3) Preparation of Various Protein Stock Solutions Various proteins (BSA, fibrinogen, transferrin, cytochrome C, lysozyme, trypsin) were weighed and dissolved in 10 mM HEPES solution (pH 7.4) to prepare a protein stock solution. The concentrations of various protein stock solutions were BSA: 50 μM, fibrinogen: 7 μM, transferrin: 50 μM, cytochrome C: 250 μM, lysozyme: 250 μM, trypsin: 250 μM, and used within one day of preparation.

Since IgG was purchased in the solution state (94 μM IgG, 10 mM phosphoric acid, 150 mM NaCl, pH 7.2), it was used by dialysis and replaced with 10 mM HEPES solution (pH 7.4). It added 10 mM HEPES solution (pH 7.4) 1.5 mL and micro magnetic stirrer chip 2mL ^{tube, Slide-A-Lyzer TM Dialysis} Cassette (MWCO: 10kDa, ThermoFisher) was installed. 300 μL of the purchased IgG solution was placed in a Slide-A-Lyzer cassette and stirred by a magnetic stirrer at 4 ° C. The 10 mM HEPES solution (pH 7.4) was replaced a total of seven times every 1-2 hours to make the solution substitution rate 99.999% or more. In addition, IgG diluted the solution at the time of purchase to prepare a calibration curve, and the concentration after dialysis was determined using the molar absorption coefficient calculated from the calibration curve.

(4) Preparation of Cellulose Nanoribbon Dispersion Containing Amino Group (0.5% (w / v), 10 mM HEPES, pH 7.4) Cellulose Nanoribbon with Amino Group Purified by Ultrapure Water in Section 1-4-2. A predetermined amount of the dispersion (the concentration was determined by absolute drying) was aliquoted into a 1.7 mL tube, and centrifuged at 15000 rpm and 4 ° C. for 5 minutes or more. The supernatant was removed, 10 mM HEPES (pH 7.4) was added to re-disperse the precipitate, and centrifugation was repeated five times or more to make the solution substitution rate 99.999%. 10 mM HEPES (pH 7.4) was added to achieve a nanoribbon concentration of 0.5% (w / v), redispersed, and stored at 4 ° C.

(5) Preparation of two-dimensional nanocrystal dispersion (0.5% (w / v), 10 mM HEPES, pH 7.4) Using D-glucose as a primer, enzymatic synthesis under the same conditions as in section 1-4-1. Two-dimensional nanocrystal dispersion (T. Serizawa et al., Polym. J., 2016, 48, 539-544) prepared in the same manner as in section 1-6-1 (4) by centrifugation and redispersion to 10 mM HEPES ( pH 7.4) and stored at 4 ° C.

(6) D-MEM (containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin)
The final concentration of fetal bovine serum (FBS), 10000 U / mL penicillin, 10 ng / mL streptomycin was 10%, 100 U / mL, and 100 μg / mL, respectively, mixed with 500 mL of D-MEM, and stored at 4 ° C.

(7) Preparation of Calibration Curves of Various Proteins Various protein stock solutions are serially diluted with 10 mM HEPES solution (pH 7.4), and 80 μL aliquots are taken in a micro quartz cell with an optical path length of 10 mm for ultraviolet-visible (UV-Vis) spectroscopy It measured by the photometer (V-550 or V-670, JASCO). The measurement conditions were: scan speed: 400 nm / min, bandwidth: 0-5 nm, data acquisition interval: 0.5 nm, response: Fast. The measurement wavelength was 200 to 400 nm when BSA, fibrinogen, transferrin, IgG, lysozyme, and trypsin were used, and 200 to 600 nm when cytochrome C was used. The samples were prepared three times independently, and a calibration curve was prepared by plotting the absorbance at 280 nm. The molar absorption coefficient calculated from the weight is respectively BSA: ε ₂₈₀ = 3.96 × 10 ⁴ , fibrinogen: ε ₂₈₀ = 3.07 × 10 ⁵ , transferrin: ε ₂₈₀ = 8.16 × 10 ⁴ , IgG: ε ₂₈₀ = 2.04 × 10 ^5, cytochrome _{C: ε 280 = 1.56 × 10} 4, lysozyme ε ₂₈₀ = 3.38 × 10 ^4, was trypsin ε ₂₈₀ = 2.84 × 10 ^4.

(8) Adsorption experiments of various proteins on cellulose nanoribbons having amino groups Dispersions of cellulose nanoribbons having amino groups (0.5% (w / v), 10 mM HEPES, pH 7.4) or two-dimensional nanocrystal dispersions (0) Add 12 μL of .5% (w / v), 10 mM HEPES, pH 7.4 to the PCR tube, and consider the amount of protein stock solution to be added. 10 mM HEPES solution (pH 7.4) or 10 mM HEPES 1.5 M NaCl solution (pH 7) Measured by .4). A predetermined amount of protein stock solution was added, mixed by pipetting, and allowed to stand at 25 ° C. for 1 hour. After centrifuging at 15000 rpm and 25 ° C. for 10 minutes, 90 μL of the supernatant was aliquoted into a new PCR tube. The absorbance at 280 nm of the supernatant was measured with a UV-Vis spectrophotometer, and the protein concentration of the supernatant was calculated using the calibration curve prepared in section 1-6-1 (7). The samples were independently prepared three times and were similarly measured. From the initial protein concentration, the amount of protein adsorbed onto the nanoribbon was calculated by subtracting the protein concentration of the supernatant.

Using the area of the upper and lower surfaces of the nanoribbon calculated from the lattice constant of cellulose type II (65 Å ² / cellulose chain, M. Hiraishi et al., Carbohydr. Res., 2009, 344, 2468-2473), per unit area per nanoribbon The amount of protein (A / ngcm- ² ) was calculated. The adsorbed amount of protein per unit area (A / ng cm ⁻² ) was plotted against the protein concentration of the supernatant ([protein] / μM). Assuming that the protein concentration of the supernatant is the concentration at which the adsorption has reached equilibrium (Equilibrium concentration), and assuming the adsorption of Langmuir type, the apparent adsorption constant (K _app / M ⁻¹ ) of the protein to the nanoribbon from the following equation and the saturation The adsorption amount ( _Amax / ngcm- ² ) was calculated.

(9) AFM observation After mixing nanoribbons and proteins in the same manner as in section 1-6-1 (8) and leaving them to stand for 1 hour, using ultrapure water so that the nanoribbon concentration becomes 0.001% (w / v) Diluted. After that, spin casting was performed on a mica substrate in the same manner as in section 1-5-4 (1), and AFM observation was performed.

(10) Zeta Potential Measurement The nanoribbon and the protein were mixed and allowed to stand for 1 hour in the same manner as in section 1-6-1 (8), and then centrifuged at 15000 rpm and 25 ° C. for 10 minutes. 90 μL of the supernatant was removed, and 90 μL of 10 mM HEPES (pH 7.4) was added to redisperse the precipitate. Similarly, after centrifugation and removal of 90 μL of the supernatant, the supernatant is dispersed in a 1.7 mL tube so that the nanoribbon concentration becomes 0.01% (w / v) with 10 mM HEPES (pH 7.4), and remains in the supernatant Protein to be less than 1%. 750 μL of the nanoribbon dispersion was aliquoted into a dedicated cell and measured with a Zetasizer Nano (Zetasizer Nano ZSP, Malvern Instruments Ltd). Measurement conditions were temperature: 25 ° C., equilibration time: 300 seconds, and Run: 10-100 times.

1-7 Use of cellulose nanoribbon having amino group as enzyme immobilization support 1-7-1 Preparation of reagent (1) 200 mM sodium phosphate buffer (pH 7.3)
2.68 g of disodium hydrogen phosphate heptahydrate (Na ₂ HPO ₄ · 7 H ₂ O) and 1.56 g of sodium dihydrogen phosphate dihydrate (NaH ₂ PO ₄ · 2 H ₂ O) in 50 mL of ultrapure water Were each dissolved in 38.5 mL of Na ₂ HPO ₄ solution and 11.5 mL of NaH ₂ PO ₄ solution were mixed and stored at 4 ° C.

(2) 30 mM aqueous solution of magnesium chloride 61 mg of magnesium chloride hexahydrate was weighed, dissolved in 10 mL of ultrapure water, and stored at 4 ° C.

(3) 3.36 M 2-mercaptoethanol aqueous solution It dissolved in ultrapure water so that it might become 10 mL of 2-mercapto ethanol, and stored at 4 ° C.

(4) 50 mM sodium phosphate, 1 mM magnesium chloride buffer (pH 7.3)
7.5 mL of 200 mM sodium phosphate buffer (pH 7.3), 1 mL of a 30 mM aqueous solution of magnesium chloride, and 21.5 mL of ultrapure water were mixed and stored at 4 ° C.

(5) Preparation of stock solution of β-D-galactosidase (β-Gal) Dissolve β-D-galactosidase (β-Gal) in a predetermined amount of 50 mM sodium phosphate 1 mM magnesium chloride buffer (pH 7.3) Stored at ° C.

(6) 50 mM sodium phosphate, 116 mM 2-mercaptoethanol, 1 mM aqueous solution of magnesium chloride 200 mM sodium phosphate buffer (pH 7.3), 7.5 mL of aqueous solution of 30 mM magnesium chloride, 1 mL of 3.36 M aqueous solution of 2-mercaptoethanol 19.5 mL of pure water was mixed and stored at 4 ° C.

(7) 1 mM p-nitrophenyl-β-D-galactopyranoside (PNPG) solution (50 mM sodium phosphate, 116 mM 2-mercaptoethanol, 1 mM magnesium chloride)
PNPG was dissolved in predetermined amounts of 50 mM sodium phosphate, 116 mM 2-mercaptoethanol, 1 mM magnesium chloride aqueous solution, and used within 1 day of preparation.

(8) Preparation of Cellulose Nanoribbon Dispersion (0.5% (w / v), 50 mM Sodium Phosphate, 1 mM Magnesium Chloride Buffer, pH 7.3) Having an Amino Group Using Ultrapure Water in Section 1-4-2 A predetermined amount of a cellulose nanoribbon dispersion having a purified amino group (the concentration was determined by absolute drying) was dispensed into a 1.7 mL tube, and centrifuged at 15000 rpm and 4 ° C. for 5 minutes or more. Remove the supernatant, add 50 mM sodium phosphate, 1 mM magnesium chloride buffer (pH 7.3), re-disperse the precipitate, repeat the operation of centrifuging 5 times or more, and the solution substitution rate is 99.999%. It was made to become. 50 mM sodium phosphate, 1 mM magnesium chloride buffer (pH 7.3) was added so that the nanoribbon concentration was 0.5% (w / v), redispersed, and stored at 4 ° C.

1-7-2 Activity evaluation of β-Gal adsorbed on cellulose nanoribbon having amino group 18 μL of cellulose nanoribbon dispersion (0.5% (w / v)) having amino group and sodium phosphate 1 mM magnesium chloride buffer ( pH 7.3) 144 μL were mixed in a PCR tube and 18 μL of 50 nM aqueous β-Gal solution was added (final concentration Cellulose nanoribbon with amino group: 0.05% (w / v), β-Gal: 5 nM) After standing at 25 ° C. for 1 hour, it was centrifuged at 15000 rpm and 25 ° C. for 10 minutes. 162 μL of the supernatant was collected, and 162 μL of 50 mM sodium phosphate 1 mM magnesium chloride buffer (pH 7.3) was added to redisperse the precipitate. Similarly, after centrifugation and removal of the supernatant twice, redispersion is carried out with 50 mM sodium phosphate 1 mM magnesium chloride buffer (pH 7.3) so that the nanoribbon concentration becomes 0.05% (w / v). The amount of .beta.-Gal remaining in the supernatant was controlled to be 0.1% or less. To the polystyrene 96-well plate (Thermo) was added 50 μL of the cellulose nanoribbon solution having amino groups which had been repeatedly centrifuged and redispersed or the separated supernatant, and 50 μL of 1 mM PNPG solution was added and incubated at 37 ° C. for 30 minutes. The p-nitrophenol produced by hydrolysis of PNPG was quantified by measuring the absorbance at 405 nm using a plate reader (Model 680, Bio-rad). The activity of the β-Gal adsorbed on the nanoribbon was evaluated by comparing with the amount of p-nitrophenol produced when the nanoribbon was not added. The samples were independently prepared six times and were similarly measured.

Cell culture in the presence of cellulose nanoribbons having 1-8 amino groups HeLa cells are 10 cm in D-MEM (containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin) at 37 ° C., 5% CO ₂ The cells were cultured in a dish and cultured and passaged to about 80% confluence. Passaging was carried out by the following procedure: The medium was removed, 10 mL D-PBS was added to the dish and lightly stirred. D-PBS was removed, 1 mL of a trypsin solution was added, spread on the front, and incubated at 37 ° C., 5% CO ₂ for 3 to 5 minutes. About 9 mL of D-MEM was added, transferred to a 15 mL tube, and centrifuged at 500 rpm for 3 minutes. The supernatant was removed, D-MEM was added and suspended, 1/8 to 1/10 was added to the next dish, and the mixture was lightly stirred to reciprocate, and cultured under conditions of 37 ° C., 5% CO ₂ .

The number of cells not used at the time of passaging was counted, and the cells had an amino group at 5000 cells / mL. Cellulose nanoribbon (autoclaved (121 ° C, 20 minutes, BS-245 TOMY)) was 0, 0.001. , 0.01 and 0.05% (w / v) were mixed. 200 μL of each was seeded on a cell culture polystyrene (TCPS) 96-well plate (TPP) and incubated at 37 ° C., 5% CO ₂ for 1 day or 3 days. Non-adherent cells were removed by removing the supernatant, and the cells were washed with D-PBS when subjected to fluorescence microscopy. Living cells and dead cells were respectively stained with Calcein AM and Propidium iodide according to the manual of Callstain ^R Double Staining Kit, and observed with a fluorescence microscope (ZOE fluorescent imager, Bio-rad). When quantifying viable cells, wash with D-MEM instead of D-PBS, add CCK mixed with D-MEM according to the manual of Cell counting kit-8 (CCK), 37 ° C, 5% CO ₂ Incubated for 60 minutes under conditions. The absorbance at 450 nm was measured by a plate reader (Model 680, Bio-rad). The cytotoxicity of the nanoribbon was evaluated by comparison with the number of viable cells when the nanoribbon was not added.

2. Results and Discussion 2-1. Enzymatic synthesis of cellulose having an amino group 2-1-1. Inverted inversion test of product A glucose derivative (2-aminoethyl-β-D-glucoside) having an amino group introduced through an ethyl group was used as a primer and applied to an enzyme synthesis system by CDP. As a result, the solution after the reaction became cloudy, suggesting that the enzyme reaction was progressing and that 2-aminoethyl-β-D-glucoside was recognized by CDP. As a result of evaluating the hydrogelation of the product by the inversion and inversion test, the solution runs down, and the case where D-glucose is used as a primer (T. Serizawa et al., Polym. J., 2016, 48, 539-544) It was found that the product did not form a hydrogel and was obtained as a precipitate (FIG. 1).

2-1-2. For evaluation (1) 1 ^H NMR spectrum of cellulose chemical structure of the product to be dissolved in aqueous sodium hydroxide if the low molecular weight having a degree of polymerization of 15 or less, can be ^{1 H} NMR measurement by dissolving the sodium hydrogen hydroxide heavy solution Is known (A. Isogai, Cellulose, 1997, 4, 99-107). Therefore, as a result of dissolving the product in 4% sodium hydroxide / heavy water solution and performing ¹ H NMR measurement, cellulose oligomers already reported for any chain length (M. Hiraishi et al., Carbohydr. Res., 2009, 344, L.A. Flugge et al., J. Am. Chem. Soc., 1999, 121, 7228-7238, H. Sugiyama et al., J. Mol. Struct., 2000, 556, 173-177, B. Xiong et al., Cellulose, 2013, 20, 613-621, M. U. Roslund et al., Carbohydr. Res., 2008, 343, 101-112) and ethyl groups (δ ~ 2.6, 3.4) Shows a peak attributed to ̃3.5), and a terminal 2-aminoethyl group It has become clear that the cellulose derivative having the can be enzymatically synthesized (FIG. 2). Subsequently, the integral value of the hydrogen at the anomeric position (δ ̃4.2) of the reducing end of the cellulose derivative having a 2-aminoethyl group and the other anomeric position (δ ̃4.3) is applied to the following formula, The degree of polymerization was calculated.

  As a result, the average degree of polymerization was calculated to be 10, and it was found that it was about the same as the average degree of polymerization of the cellulose oligomer (-10) when D-glucose was used as a primer under the same conditions (T. Serizawa et al., Polym J., 2016, 48, 539-544).

(2) MALDI-TOF-MS spectrum As a result of identifying the chemical structure of the product by MALDI-TOF-MS measurement, three peak groups having an interval of 162 which is the mass number of Gluco − It was done (Figure 3). The three peak groups correspond to the mass to charge ratio of proton, sodium or potassium ion adducts of cellulose derivatives having an amino group, respectively, and it was supported that cellulose derivatives having a 2-aminoethyl group could be synthesized. Also, the distribution of the degree of polymerization was 6 to 16, and it was found that it had a molecular weight distribution as in the case of using D-glucose as a primer. As a result of calculating the average degree of polymerization from the integral ratio of the peaks of the MS spectrum, it was calculated to be 8.7 and showed a value about 1 lower than the average degree of polymerization calculated from ¹ H NMR measurement. Since only ionized molecules were detected by MALDI-TOF-MS measurement, it was suggested that the cellulose derivative having a high degree of polymerization of 2-aminoethyl group was not sufficiently ionized under the measured conditions. The present inventors have since been used to date ^{1 H} NMR measurements for the calculation of the average degree of polymerization of the cellulose oligomers prepared by enzymatic synthesis, the use of the average polymerization degree calculated by also ^{1 H} NMR measurement in this embodiment And

(3) Conversion of αG1P The conversion of αG1P, a monomer, was calculated using the yield of the product calculated by the absolute drying method and the average degree of polymerization calculated from ¹ H NMR measurement. When -D-glucoside was used as a primer, it was about 20%. When D-glucose is used as a primer under the same conditions, the conversion is about 35% (T. Serizawa et al., Polym. J., 2016, 48, 539-544), 2-aminoethyl-.beta.-D -When glucoside was used as a primer, it was low, and it was suggested that the terminal 2-aminoethyl group reduces the recognition by CDP.

2-1-3. Evaluation of Crystal Structure of Product (1) WAXD chart In order to clarify the crystal form formed by a cellulose derivative having a 2-aminoethyl group, the result of measurement by WAXD shows 2θ = 12.3, 20.0, 22. It showed a sharp diffraction peak at 1 ° (FIG. 4). These diffraction peaks were assigned to the 1-10, 110 and 020 planes of cellulose type II crystal (M. Hiraishi et al., Carbohydr. Res., 2009, 344, 2468-2473), respectively. This was similar to the crystal form formed by the cellulose oligomer when D-glucose was used as a primer, and it was found that the 2-aminoethyl group at the end of the cellulose chain did not affect the crystal form.

(2) ATR / FTIR spectrum As a result of analyzing a crystal form formed by a cellulose derivative having a 2-aminoethyl group by ATR / FTIR method, two sharp peaks derived from OH stretching vibration characteristic of cellulose type II crystal (Q. Wu et al., Carbohydr. Polym., 2015, 133, 438-447) was shown near 3442 and 3490 cm- ¹ (Figure 5). Similar to the case where D-glucose is used as a primer (T. Serizawa et al., Polym. J., 2016, 48, 539-544), the cellulose II is also used as a primer when 2-aminoethyl-β-D-glucoside is used as a primer It was supported to form mold crystals.

2-1-4. Aggregated Structure of Cellulose (1) AFM Image In order to clarify the aggregate structure formed by a cellulose derivative having a 2-aminoethyl group, the product was spin-casted on a mica substrate and observed by AFM. As a result, a well-dispersed ribbon-like structure (nanoribbon) was observed (FIG. 6). As a result of evaluating the thickness of the nanoribbon, it is calculated to be 5.3 ± 0.3 nm, and a molecular chain length of 5.2 nm of cellodecaose which forms a cellulose type II crystal (M. Hiraishi et al., Carbohydr. Res., 2009, 344) , 2468-2473). Therefore, similar to two-dimensional nanocrystals when D-glucose is used as a primer, a cellulose derivative having a 2-aminoethyl group is aligned in the film thickness direction of the nanoribbon to form lamellar crystals, and It is believed that the amino group is exposed on the surface of the nanoribbon.

  Focusing on the aspect ratio of the generated nanoribbon, when D-glucose is used as a primer (M. Hiraishi et al., Carbohydr. Res., 2009, 344, 2468-2473, T. Serizawa et al., Polym. J., 2016 The contribution of the terminal 2-aminoethyl group suggested that the crystals grow more anisotropically, as compared with the year 48, 539-544). Since a nanoribbon is also formed when a glucose derivative having an ethyl group is used as a primer (Y. Yataka et al., Langmuir, 2016, 32, 10120-10125), the terminal ethyl group contributes to anisotropic crystal growth. It is thought that it is doing. When a glucose derivative having a 2-aminoethyl group is used as a primer, nanoribbons are formed as a precipitate, whereas when a glucose derivative having an ethyl group is used as a primer, a hydrogel composed of nanoribbons is formed. (Y. Yataka et al., Langmuir, 2016, 32, 10120-10125). Thus, it was found that the contribution of the terminal amino group greatly changes the aggregate structure of the product on a macro scale.

(2) TEM image In order to clarify the aggregate structure formed by a cellulose derivative having a 2-aminoethyl group, the product is adsorbed to a collodion supporting film and observed by TEM, and a ribbon-like structure (nanoribbon) is observed. (FIG. 7), it was supported that the nanoribbon is generated also from the TEM observation.

2-2. Adsorption Properties of Various Proteins to Cellulose Nanoribbons Having Amino Groups In order to clarify the availability of cellulose nanoribbons having amino groups to biomaterials, the adsorption properties of various proteins (Table 1) on nanoribbons were systematically evaluated.

Serum albumin (BSA), the most abundant protein in plasma, was mixed with the nanoribbon at a concentration of 1-20 μM and incubated. Then, the mixed solution of nanoribbon and BSA was centrifuged to quantify the BSA concentration of the supernatant. As a result of plotting the BSA concentration (Equilibrium concentration) of the supernatant that reached adsorption equilibrium against the initial concentration of BSA (Initial concentration), the BSA concentration of the supernatant was clearly reduced (FIG. 8 a, primary amino group) (+)). Thus, it was found that BSA was adsorbed to the nanoribbon. On the other hand, when two-dimensional nanocrystals having no amino group and BSA are mixed, the BSA concentration in the supernatant hardly changes (Fig. 8a, (-)), and BSA hardly adsorbs on two-dimensional nanocrystals. I understood it. Thus, it was suggested that the amino groups on the surface of the nanoribbon contribute to the adsorption of BSA. The isoelectric point of BSA is known to be 4.6 (S. Brewer et al., Langmuir, 2005, 21, 9303-9307) (Table 1), and the effect of BSA under experimental conditions (pH 7.4) The charge is considered negative. On the other hand, the pKa of the primary alkylamine is about 10 (M. John, McMurray Organic Chemistry (bottom) 8th edition, Tokyo Kagaku Dojin, 2013), and the amino group on the nanoribbon surface is the experimental condition (pH 7.4) Is believed to be protonated. Therefore, it is considered that the electrostatic interaction between the amino group on the nanoribbon surface and BSA is mainly contributing to the adsorption. It was suggested that BSA was not detected in the supernatant until the initial concentration of BSA was 3 μM (therefore, fitting with the Langmuir adsorption isotherm was not possible), and that BSA was strongly adsorbed to the nanoribbon. Using the area (65 Å ² / cellulose chain, M. Hiraishi et al., Carbohydr. Res., 2009, 344, 2468-2473.) Of the nanoribbon calculated from the lattice constant of cellulose type II, per unit area The adsorption amount of BSA was calculated and plotted against the initial concentration of BSA (FIG. 8b). As a result, it was suggested that the adsorption amount of BSA per unit area increases with the increase of the initial concentration of BSA, and the adsorption amount of BSA reaches saturation (250 to 300 ng cm ⁻² ) at an initial concentration of about 5 μM. Here, in order to evaluate the saturated adsorption amount of BSA, an equilateral triangular prism (one side: 8.4 nm, height: 3.15 nm, C. Rocker et al., Nat. Nanotechnol., 2009) used as a shape model of BSA. , 4, 577-580.), And assuming an adsorption on a single layer with the equilateral triangle face or side facing, the ideal amount of adsorption per unit area was calculated (Table 2). As a result, it was calculated to be 360 ± 410 ng cm ⁻² , which was in good agreement with the saturated adsorption amount (250-300 ng cm ⁻² ) of BSA per unit area to the nanoribbon obtained by the experiment. Therefore, it was speculated that BSA was adsorbed to the nanoribbon as a single layer.

  In order to directly confirm the adsorption of BSA on the nanoribbon, the initial concentration of BSA was mixed with the nanoribbon at 5 μM, and after incubation, it was spin cast on a mica substrate and observed by AFM (FIG. 9). As a result, as in the case of mixing with BSA (FIG. 6), a ribbon-like structure (nanoribbon) was observed, but the surface was apparently rough. The observed thickness of the nanoribbon was 12.6 ± 1.6 nm, which was described above as the thickness (5.3 ± 0.3 nm) of the nanoribbon before BSA adsorption calculated in section 2-1-4 (1) It was in good agreement with the sum of the thickness (3.15 × 2 nm) of two layers of an equilateral triangular prism model of BSA. Therefore, it is suggested from the AFM observation that BSA is adsorbed in a monolayer like sandwich on both sides of the nanoribbon.

Furthermore, the adsorption properties of other representative serum proteins (fibrinogen, transferrin, IgG) to nanoribbons were evaluated. As in the case of BSA, various serum proteins were mixed with the nanoribbon and the concentration of supernatant was plotted against the initial concentration (Figure 10a). As a result, it was found that the protein concentration of the supernatant decreased and was adsorbed to the nanoribbon. The isoelectric points of fibrinogen, transferrin and IgG are 5.6 (M. De et al., Nat. Chem., 2009, 1, 461-465.) And 5.6 (M. De et al., Nat. Chem., 2009, respectively. 1, 461-465.), 6.8-7.4 (H. Orelma et al., Biomacromolecules, 201 12, 4311-4318.) (Table 1), as in the case of BSA, negatively charged. It is suggested that the electrostatic interaction between the protein and the amino group on the surface of the nanoribbon contributes to the adsorption. Therefore, in order to calculate the apparent adsorption constant (K _app / M ^-1 ) and the saturated adsorption amount (A _max / ng cm ^-2 ) of various proteins to nanoribbon, the adsorption amount of protein per unit area reaches the adsorption equilibrium The concentration was plotted against the concentration of the supernatant, and fitted with a Langmuir-type adsorption isotherm (Equation 1, FIG. 10 b). As a result, all of the apparent adsorption constants are in the order of 10 ⁵ -10 ⁷ M ^-1 (Table 2), and the past reports on the adsorption constants of serum proteins based on electrostatic interaction (S. Brewer et al., Langmuir, 2005) , 21, 9303-9307., D. Li et al., Chem. Mater., 2006, 18, 6403-6413). The apparent adsorption constants are different depending on the type of protein, and it is considered that not only the electrostatic interaction but also the isoelectric point and the size and shape of the protein contribute to the adsorption. Moreover, the saturated adsorption amount of fibrinogen, transferrin, and IgG was computed with 832 ngcm ^{< -2} >, 178 ngcm ^{< -2} >, 515 ngcm ^{< -2} >, respectively. Here, rectangular parallelepipeds (Fibrinogen: 5 × 5 × 47 (A. Sethuraman et al., Langmuir, 2004, 20, 7779-7788.), Transferrin: 4.2 × 5 × 7 (X. Jiang) as shape models of various proteins. Et al, J. R. Soc. Interface, 2010, 7, S5-S13), IgG: 6.5 × 7.7 × 10 (L. Harris. Biochemistry, 1997, 36, 1581-1597.)). The ideal adsorption amount per unit area when single layer adsorption was carried out with each of narrow area (end-on) or wide area (side on) was calculated (Table 2). As a result, the ideal saturated adsorption amount assuming single layer adsorption and the saturated adsorption amount obtained by the experiment were comparable. Thus, as in the case of BSA, it was suggested that any serum protein adsorbs to the nanoribbon in a monolayer.

  Therefore, in order to clarify that electrostatic interaction contributes to the adsorption of proteins to nanoribbons, proteins with an isoelectricity of 10 or more that are considered to be positively charged under experimental conditions (pH 7.4) (cytochrome The adsorption properties of C, lysozyme, trypsin (Table 1)) were evaluated (FIG. 11). As a result, it was found that cytochrome C, lysozyme and trypsin did not adsorb to the nanoribbon even if they were adsorbed at initial concentrations as high as 200 μM. Thus, it was supported that electrostatic interaction contributes to adsorption.

  The zeta potential of the nanoribbon was assessed before and after protein adsorption (Figure 12). BSA and nanoribbons were mixed in the same manner as described above, and BSA adsorbed to the nanoribbons was removed by centrifugation and redispersion to measure zeta potential. As a result, it was found that the zeta potential (+24 mV) of the nanoribbon before BSA adsorption was negatively shifted with the increase of the BSA concentration. Therefore, it was supported that electrostatic interaction also contributes to adsorption from zeta potential measurement. In addition, the negative shift of the zeta potential reached saturation at a BSA concentration of 5 μM (-15 mV), and was consistent with the concentration dependence of the above-described adsorption data. Furthermore, it was found that the adsorption amount of BSA on nanoribbons decreased with the increase of salt concentration (NaCl) (FIG. 13), and it was supported that the electrostatic interaction certainly contributed to the adsorption.

2-3 Activity of Enzyme Adsorbed to Cellulose Nanoribbon with Amino Group To clarify the availability of cellulose nanoribbon with amino group as the immobilization support of enzyme, the enzyme activity of β-Gal adsorbed to nanoribbon is evaluated did. The mixed solution of nanoribbon and β-Gal was centrifuged, and the enzyme activity (nanoribbon (+), supernatant) of the supernatant was measured. As a result, free β-Gal (nanoribbon (-)) when no nanoribbon was added Β-Gal (isoelectric point: 4.1, M. Whitesides. Langmuir, 2003, 19, 1861-1872. Was found to be adsorbed on the nanoribbon. The enzyme activity of β-Gal (nanoribbon (+), precipitate) adsorbed to the nanoribbon was found to be similar to the free β-Gal (nanoribbon (−)) within the error range (FIG. 14). That is, it became clear that (beta) -Gal adsorb | sucks to a nanoribbon, maintaining the activity, and it can use nanoribbon as an immobilization support of an enzyme.

2-4. Cellulose nanoribbon having an amino group exhibits cytotoxicity To evaluate the cytotoxicity of an amino group containing cellulose nanoribbon, cells were cultured in the presence of the nanoribbon. HeLa cells were cultured at a nanoribbon concentration of 0-0.05% (w / v) for 3 days, live cells and dead cells were stained with different fluorescent dyes and observed under a fluorescence microscope (FIG. 15). As a result, many cells survived at any nanoribbon concentration, and the ratio of live cells to dead cells did not change significantly regardless of the nanoribbon concentration. Furthermore, the cells were cultured in the presence of nanoribbons. HeLa cells were cultured at a nanoribbon concentration of 0-0.05% (w / v) for 1 day or 3 days, and then the number of living cells was quantified using a cell quantification kit. Cell viability did not change significantly compared to the absence of nanoribbons (FIG. 16). Thus, it has become clear that in an in vitro cell culture environment, cellulose nanoribbons having an amino group do not exhibit significant cytotoxicity.

Claims (10)

The following equation (I):

[Wherein, m is 4 to 14]
The cellulose structure containing the compound shown by these as a component.   The cellulose structure according to claim 1 having a nanoribbon structure.   The cellulose structure according to claim 1 or 2, which is a precipitate.   The adsorption agent of the protein containing the cellulose structure of any one of Claims 1-3.   An immobilized carrier for a protein comprising the cellulose structure according to any one of claims 1 to 3.   The immobilization carrier according to claim 5, wherein the protein is an enzyme.   The scaffold of the cell containing the cellulose structure of any one of Claims 1-3. α-D-glucose monophosphate and the following formula (III) as a primer:

Reacting a glucose derivative represented by the following formula with cellodextrin phosphorylase:

[Wherein, m is 4 to 14]
The manufacturing method of the cellulose structure which contains the compound shown by these as a component.   The method according to claim 8, wherein the reaction is carried out at 60 ° C for 3 days.   The method according to claim 8 or 9, wherein the concentration of the cellodextrin phosphorylase in the reaction solution is 0.2 U / mL or more.

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